HYAL-2–WWOX–SMAD4 Signaling in Cell Death and Anticancer Response

PubMed Central

Hsu, Li-Jin; Chiang, Ming-Fu; Sze, Chun-I; Su, Wan-Pei; Yap, Ye Vone; Lee, I-Ting; Kuo, Hsiang-Ling; Chang, Nan-Shan

2016-01-01

Hyaluronidase HYAL-2 is a membrane-anchored protein and also localizes, in part, in the lysosome. Recent study from animal models revealed that both HYAL-1 and HYAL-2 are essential for the metabolism of hyaluronan (HA). Hyal-2 deficiency is associated with chronic thrombotic microangiopathy with hemolytic anemia in mice due to over accumulation of high molecular size HA. HYAL-2 is essential for platelet generation. Membrane HYAL-2 degrades HA bound by co-receptor CD44. Also, in a non-canonical signal pathway, HYAL-2 serves as a receptor for transforming growth factor beta (TGF-β) to signal with downstream tumor suppressors WWOX and SMAD4 to control gene transcription. When SMAD4 responsive element is overly driven by the HYAL-2–WWOX–SMAD4 signaling complex, cell death occurs. When rats are subjected to traumatic brain injury, over accumulation of a HYAL-2–WWOX complex occurs in the nucleus to cause neuronal death. HA induces the signaling of HYAL-2–WWOX–SMAD4 and relocation of the signaling complex to the nucleus. If the signaling complex is overexpressed, bubbling cell death occurs in WWOX-expressing cells. In addition, a small synthetic peptide Zfra (zinc finger-like protein that regulates apoptosis) binds membrane HYAL-2 of non-T/non-B spleen HYAL-2+ CD3− CD19− Z lymphocytes and activates the cells to generate memory anticancer response against many types of cancer cells in vivo. Whether the HYAL-2–WWOX–SMAD4 signaling complex is involved is discussed. In this review and opinion article, we have updated the current knowledge of HA, HYAL-2 and WWOX, HYAL-2–WWOX–SMAD4 signaling, bubbling cell death, and Z cell activation for memory anticancer response. PMID:27999774

Transforming Growth Factor β Suppresses Peroxisome Proliferator–Activated Receptor γ Expression via Both SMAD Binding and Novel TGF-β Inhibitory Elements

PubMed Central

Lakshmi, Sowmya P.; Reddy, Aravind T.; Reddy, Raju C.

2017-01-01

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other’s expression, and such PPARγ downregulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here we show that TGF-β induces association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive corepressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. PMID:28100650

A Poised Chromatin Platform for TGF-[beta] Access to Master Regulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xi, Qiaoran; Wang, Zhanxin; Zaromytidou, Alexia-Ileana

2012-02-07

Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-{beta} signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compactingmore » factor HP1, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.« less

A role for human MUC4 mucin gene, the ErbB2 ligand, as a target of TGF-beta in pancreatic carcinogenesis.

PubMed

Jonckheere, Nicolas; Perrais, Michaël; Mariette, Christophe; Batra, Surinder K; Aubert, Jean-Pierre; Pigny, Pascal; Van Seuningen, Isabelle

2004-07-29

MUC4: encodes a large transmembrane mucin that is overexpressed in pancreatic adenocarcinomas. The molecular mechanisms responsible for that altered pattern of expression are unknown. TGF-beta, a pleiotropic cytokine, regulates numerous genes involved in pancreatic carcinogenesis via activation of the Smads proteins and MUC4 promoter is rich in Smad-binding elements. Our aim was to study whether the regulation of MUC4 expression by TGF-beta in pancreatic cancer cells was strictly dependent on Smad4 activity. Three pancreatic cancer cell lines, CAPAN-1 (MUC4+/Smad4-), CAPAN-2 (MUC4+/Smad4+) and PANC-1 (MUC4-/Smad4+), were used. By RT-PCR, transfection assays and immunohistochemistry, we show that (i) both MUC4 mRNA and apomucin expression are upregulated by TGF-beta, (ii) Smad2 positively cooperates with Smad4 to activate the promoter, (iii) activation of Smad4 by exogenous TGF-beta induces Smad4 binding to the promoter, (iv) Smad7 and c-ski both inhibit activation by Smad4. When Smad4 is mutated and inactive, TGF-beta activates MUC4 expression via MAPK, PI3K and PKA signaling pathways. Absence of expression in PANC-1 cells is due to histone deacetylation. Altogether, these results indicate that upregulation of MUC4 by TGF-beta is restricted to well-differentiated pancreatic cancer cells, and point out a novel mechanism for TGF-beta as a key molecule in targeting MUC4 overexpression in pancreatic adenocarcinomas.

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein*

PubMed Central

Yang, Huibin; Li, Gangyong; Wu, Jing-Jiang; Wang, Lidong; Uhler, Michael; Simeone, Diane M.

2013-01-01

Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290–300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281–285 and 320–329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo. PMID:23362281

Transforming growth factor β suppresses peroxisome proliferator-activated receptor γ expression via both SMAD binding and novel TGF-β inhibitory elements.

PubMed

Lakshmi, Sowmya P; Reddy, Aravind T; Reddy, Raju C

2017-04-24

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other's expression, and such PPARγ down-regulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here, we show that TGF-β induces the association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive co-repressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated the partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

PubMed

Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

2000-11-10

In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells. Copyright 2000 Academic Press.

Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection.

PubMed

de la Fuente, Cynthia; Pinkham, Chelsea; Dabbagh, Deemah; Beitzel, Brett; Garrison, Aura; Palacios, Gustavo; Hodge, Kimberley Alex; Petricoin, Emanuel F; Schmaljohn, Connie; Campbell, Catherine E; Narayanan, Aarthi; Kehn-Hall, Kylene

2018-01-01

Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.

Human T-cell leukemia virus type I oncoprotein Tax represses Smad-dependent transforming growth factor beta signaling through interaction with CREB-binding protein/p300.

PubMed

Mori, N; Morishita, M; Tsukazaki, T; Giam, C Z; Kumatori, A; Tanaka, Y; Yamamoto, N

2001-04-01

Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)

Opposite Smad and chicken ovalbumin upstream promoter transcription factor inputs in the regulation of the collagen VII gene promoter by transforming growth factor-beta.

PubMed

Calonge, María Julia; Seoane, Joan; Massagué, Joan

2004-05-28

A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-beta (TGF-beta). The gene, COL7A1, is activated by TGF-beta via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-beta may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-beta-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-beta-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-beta-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-beta addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-beta signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs.

c-Jun N-terminal kinase 1 promotes transforming growth factor-β1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3.

PubMed

Velden, Jos L J van der; Alcorn, John F; Guala, Amy S; Badura, Elsbeth C H L; Janssen-Heininger, Yvonne M W

2011-04-01

Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT.

Effects of birth trauma and estrogen on urethral elastic fibers and elastin expression.

PubMed

Lin, Guiting; Ning, Hongxiu; Wang, Guifang; Banie, Lia; Lue, Tom F; Lin, Ching-Shwun

2010-10-01

To investigate the effects of birth trauma and estrogen on urethral elastic fibers and elastin expression. Pregnant rats were subjected to sham operation (Delivery-only), DVDO (delivery, vaginal distension and ovariectomy), or DVDO + E₂ (estrogen). At 2, 4, 8, or 12 weeks, their urethras were harvested for elastic fiber staining and reverse transcription-polymerase chain reaction analysis. Urethral cells were treated with transforming growth factor- β1 (TGFβ1) and/or estrogen and analyzed for elastin mRNA expression. Urethral cells were also examined for the activities of Smad1- and Smad3/4-responsive elements in response to TGFβ1 and estrogen. At 8 weeks post-treatment, the urethras of DVDO rats had fewer and shorter elastic fibers when compared with Delivery-only rats, and those of DVDO + E₂ rats had fewer and shorter elastic fibers when compared with DVDO rats. Elastin mRNA was expressed at low levels in Delivery-only rats and at increasingly higher levels in DVDO rats at 2, 4, and 8 weeks but at sharply lower levels in DVDO + E₂ rats when compared with DVDO rats at 8 weeks. Urethral cells expressed increasingly higher levels of elastin mRNA in response to increasing concentrations of TGFβ1 up to 1 ng/mL. At this TGFβ1 concentration, urethral cells expressed significantly lower levels of elastin mRNA when treated with estrogen before or after TGFβ1 treatment. Both Smad1- and Smad3/4-responsive elements were activated by TGFβ1 and such activation was suppressed by estrogen. Birth trauma appears to activate urethral elastin expression via TGFβ1 signaling. Estrogen interferes with this signaling, resulting in improper assembly of elastic fibers. Copyright © 2010 Elsevier Inc. All rights reserved.

RACK1 binds to Smad3 to modulate transforming growth factor-beta1-stimulated alpha2(I) collagen transcription in renal tubular epithelial cells.

PubMed

Okano, Kazuhiro; Schnaper, H William; Bomsztyk, Karol; Hayashida, Tomoko

2006-09-08

Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter.

Smad7 Protein Induces Interferon Regulatory Factor 1-dependent Transcriptional Activation of Caspase 8 to Restore Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Apoptosis

PubMed Central

Hong, Suntaek; Kim, Hye-Youn; Kim, Jooyoung; Ha, Huyen Trang; Kim, Young-Mi; Bae, Eunjin; Kim, Tae Hyung; Lee, Kang Choon; Kim, Seong-Jin

2013-01-01

Smad7 has been known as a negative regulator for the transforming growth factor-β (TGF-β) signaling pathway through feedback regulation. However, Smad7 has been suspected to have other biological roles through the regulation of gene transcription. By screening differentially regulated genes, we found that the caspase 8 gene was highly up-regulated in Smad7-expressing cells. Smad7 was able to activate the caspase 8 promoter through recruitment of the interferon regulatory factor 1 (IRF1) transcription factor to the interferon-stimulated response element (ISRE) site. Interaction of Smad7 on the caspase 8 promoter was confirmed with electrophoretic mobility shift assay and chromatin immunoprecipitation experiment. Interestingly, Smad7 did not directly interact with the ISRE site, but it increased the binding activity of IRF1 with ISRE. These results support that Smad7 recruits IRF1 protein on the caspase 8 promoter and functions as a transcriptional coactivator. To confirm the biological significance of caspase 8 up-regulation, we tested tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cell death assay in breast cancer cells. Smad7 in apoptosis-resistant MCF7 cells markedly sensitized the cells to TRAIL-induced cell death by restoring the caspase cascade. Furthermore, restoration of caspase 8-mediated apoptosis pathway repressed the tumor growth in the xenograft model. In conclusion, we suggest a novel role for Smad7 as a transcriptional coactivator for caspase 8 through the interaction with IRF1 in regulation of the cell death pathway. PMID:23255602

SMAD1 and SMAD5 Expression Is Coordinately Regulated by FLI1 and GATA2 during Endothelial Development.

PubMed

Marks-Bluth, Jonathon; Khanna, Anchit; Chandrakanthan, Vashe; Thoms, Julie; Bee, Thomas; Eich, Christina; Kang, Young Chan; Knezevic, Kathy; Qiao, Qiao; Fitch, Simon; Oxburgh, Leif; Ottersbach, Katrin; Dzierzak, Elaine; de Bruijn, Marella F T R; Pimanda, John E

2015-06-01

The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1+63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortas in vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cells in vitro. In contrast, CD31(+) cKit(-) endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reduced Smad1 but not Smad5 transcript levels. This is suggestive of a degree of in vivo selection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Smad2 and Smad3 have differential sensitivity in relaying TGFβ signaling and inversely regulate early lineage specification

PubMed Central

Liu, Ling; Liu, Xu; Ren, Xudong; Tian, Yue; Chen, Zhenyu; Xu, Xiangjie; Du, Yanhua; Jiang, Cizhong; Fang, Yujiang; Liu, Zhongliang; Fan, Beibei; Zhang, Quanbin; Jin, Guohua; Yang, Xiao; Zhang, Xiaoqing

2016-01-01

The transforming growth factor beta (TGFβ) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFβ signals. Here, we show that Smad3 is an insensitive TGFβ transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFβ signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFβ signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFβ/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity and differential transcriptional activity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFβ signaling and have distinct roles in regulating early developmental events. PMID:26905010

Smad2 and Smad3 have differential sensitivity in relaying TGFβ signaling and inversely regulate early lineage specification.

PubMed

Liu, Ling; Liu, Xu; Ren, Xudong; Tian, Yue; Chen, Zhenyu; Xu, Xiangjie; Du, Yanhua; Jiang, Cizhong; Fang, Yujiang; Liu, Zhongliang; Fan, Beibei; Zhang, Quanbin; Jin, Guohua; Yang, Xiao; Zhang, Xiaoqing

2016-02-24

The transforming growth factor beta (TGFβ) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFβ signals. Here, we show that Smad3 is an insensitive TGFβ transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFβ signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFβ signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFβ/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity and differential transcriptional activity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFβ signaling and have distinct roles in regulating early developmental events.

Requirement for the SnoN oncoprotein in transforming growth factor beta-induced oncogenic transformation of fibroblast cells.

PubMed

Zhu, Qingwei; Pearson-White, Sonia; Luo, Kunxin

2005-12-01

Transforming growth factor beta (TGF-beta) was originally identified by virtue of its ability to induce transformation of the AKR-2B and NRK fibroblasts but was later found to be a potent inhibitor of the growth of epithelial, endothelial, and lymphoid cells. Although the growth-inhibitory pathway of TGF-beta mediated by the Smad proteins is well studied, the signaling pathway leading to the transforming activity of TGF-beta in fibroblasts is not well understood. Here we show that SnoN, a member of the Ski family of oncoproteins, is required for TGF-beta-induced proliferation and transformation of AKR-2B and NRK fibroblasts. TGF-beta induces upregulation of snoN expression in both epithelial cells and fibroblasts through a common Smad-dependent mechanism. However, a strong and prolonged activation of snoN transcription that lasts for 8 to 24 h is detected only in these two fibroblast lines. This prolonged induction is mediated by Smad2 and appears to play an important role in the transformation of both AKR-2B and NRK cells. Reduction of snoN expression by small interfering RNA or shortening of the duration of snoN induction by a pharmacological inhibitor impaired TGF-beta-induced anchorage-independent growth of AKR-2B cells. Interestingly, Smad2 and Smad3 play opposite roles in regulating snoN expression in both fibroblasts and epithelial cells. The Smad2/Smad4 complex activates snoN transcription by direct binding to the TGF-beta-responsive element in the snoN promoter, while the Smad3/Smad4 complex inhibits it through a novel Smad inhibitory site. Mutations of Smad4 that render it defective in heterodimerization with Smad3, which are found in many human cancers, convert the activity of Smad3 on the snoN promoter from inhibitory to stimulatory, resulting in increased snoN expression in cancer cells. Thus, we demonstrate a novel role of SnoN in the transforming activity of TGF-beta in fibroblasts and also uncovered a mechanism for the elevated SnoN expression in some human cancer cells.

Ferulic acid suppresses activation of hepatic stellate cells through ERK1/2 and Smad signaling pathways in vitro.

PubMed

Xu, Tianjiao; Pan, Zhi; Dong, Miaoxian; Yu, Chunlei; Niu, Yingcai

2015-01-01

Hepatic stellate cells (HSCs) are the primary source of matrix components in hepatic fibrosis. Ferulic acid (FA) has antifibrotic potential in renal and cardiac disease. However, whether FA comprises inhibitive effects of HSCs activation remains to be clarified. This study aims at evaluating the hypothesis that FA inhibits extracellular matrix (ECM)-related gene expression by the interruption of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) or/and Smad signaling pathways in HSC-T6. Our results indicated that FA significantly inhibited both viability and activation of HSC-T6 cells in vitro. In addition, we demonstrated, for the first time, that FA dramatically inhibited the expression of α1(I) collagen (Col-I) and fibronectin at levels of transcription and translation. Moreover, FA treatment inhibited Smad transcriptional activity, as evaluated by transient transfection with a plasmid construction containing SMAD response element and the luciferase reporter gene. Furthermore, FA inhibition of HSCs activation involved in both focal adhesion kinase (FAK)-dependent ERK1/2 and Smad signaling pathways with independent manner. Blocking transforming growth factor-β by a neutralizing antibody caused a marked reduction in both ERK1/2 and Smad signaling. These results support FA as an effective therapeutic agent for the prevention and treatment of hepatic fibrosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6

PubMed Central

Mouillesseaux, Kevin P.; Wiley, David S.; Saunders, Lauren M.; Wylie, Lyndsay A.; Kushner, Erich J.; Chong, Diana C.; Citrin, Kathryn M.; Barber, Andrew T.; Park, Youngsook; Kim, Jun-Dae; Samsa, Leigh Ann; Kim, Jongmin; Liu, Jiandong; Jin, Suk-Won; Bautch, Victoria L.

2016-01-01

Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation. PMID:27834400

SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy.

PubMed

Wang, Feng; Xia, Xiaojun; Yang, Chunying; Shen, Jianliang; Mai, Junhua; Kim, Han-Cheon; Kirui, Dickson; Kang, Ya'an; Fleming, Jason B; Koay, Eugene J; Mitra, Sankar; Ferrari, Mauro; Shen, Haifa

2018-03-30

Understanding the mechanism of radioresistance could help develop strategies to improve therapeutic response of patients with PDAC. The SMAD4 gene is frequently mutated in pancreatic cancer. In this study, we investigated the role of SMAD4 deficiency in pancreatic cancer cells' response to radiotherapy. We downregulated SMAD4 expression with SMAD4 siRNA or SMAD4 shRNA and overexpressed SMAD4 in SMAD4 mutant pancreatic cancer cells followed by clonogenic survival assay to evaluate their effects on cell radioresistance. To study the mechanism of radioresistance, the effects of SMAD4 loss on reactive oxygen species (ROS) and autophagy were determined by Flow Cytometry and immunoblot analysis, respectively. Furthermore, we measured radioresistance by clonogenic survival assay after treatment with autophagy inhibitor (Chloroquine) and ROS inhibitor (N-acetyl-L-cysteine) in SMAD4 -depleted pancreatic cancer cells. Finally, the effects of SMAD4 on radioresistance were also confirmed in an orthotopic tumor model derived from SMAD4 -depleted Panc-1 cells. SMAD4 -depleted pancreatic cancer cells were more resistant to radiotherapy based on clonogenic survival assay. Overexpression of wild type SMAD4 in SMAD4 -mutant cells rescued their radiosensitivity. Radioresistance mediated by SMAD4 depletion was associated with persistently higher levels of ROS and radiation-induced autophagy. Finally, SMAD4 depletion induced in vivo radioresistance in Panc-1-derived orthotopic tumor model ( P = 0.038). More interestingly, we observed that the protein level of SMAD4 is inversely correlated with autophagy in orthotopic tumor tissue samples. Our results demonstrate that defective SMAD4 is responsible for radioresistance in pancreatic cancer through induction of ROS and increased level of radiation-induced autophagy. Copyright ©2018, American Association for Cancer Research.

STAT3 selectively interacts with Smad3 to antagonize TGF-β signaling

PubMed Central

Wang, Gaohang; Yu, Yi; Sun, Chuang; Liu, Ting; Liang, Tingbo; Zhan, Lixing; Lin, Xia; Feng, Xin-Hua

2015-01-01

Smad and STAT proteins are critical signal transducers and transcription factors in controlling cell growth and tumorigenesis. Here we report that the STAT3 signaling pathway attenuates TGF-β-induced responses through a direct Smad3-STAT3 interplay. Activated STAT3 blunts TGF-β-mediated signaling. Depletion of STAT3 promotes TGF-β-mediated transcriptional and physiological responses, including cell cycle arrest, apoptosis and epithelial-to-mesenchymal transition. STAT3 directly interacts with Smad3 in vivo and in vitro, resulting in attenuation of the Smad3-Smad4 complex formation and suppression of DNA-binding ability of Smad3. The N-terminal region of DNA-binding domain of STAT3 is responsible for the STAT3-Smad3 interaction and also indispensable for STAT3-mediated inhibition of TGF-β signaling. Thus, our finding illustrates a direct crosstalk between the STAT3 and Smad3 signaling pathways that may contribute to tumor development and inflammation. PMID:26616859

Pathway Model of the Kinetics of the TGFbeta Antagonist Smad7 and Cross-Talk with the ATM and WNT Pathways

NASA Technical Reports Server (NTRS)

Carra, Claudio; Wang, Minli; Huff, Janice L.; Hada, Megumi; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

Signal transduction controls cellular and tissue responses to radiation. Transforming growth factor beta (TGFbeta) is an important regulator of cell growth and differentiation and tissue homeostasis, and is often dis-regulated in tumor formation. Mathematical models of signal transduction pathways can be used to elucidate how signal transduction varies with radiation quality, and dose and dose-rate. Furthermore, modeling of tissue specific responses can be considered through mechanistic based modeling. We developed a mathematical model of the negative feedback regulation by Smad7 in TGFbeta-Smad signaling and are exploring possible connections to the WNT/beta -catenin, and ATM/ATF2 signaling pathways. A pathway model of TGFbeta-Smad signaling that includes Smad7 kinetics based on data in the scientific literature is described. Kinetic terms included are TGFbeta/Smad transcriptional regulation of Smad7 through the Smad3-Smad4 complex, Smad7-Smurf1 translocation from nucleus to cytoplasm, and Smad7 negative feedback regulation of the TGFO receptor through direct binding to the TGFO receptor complex. The negative feedback controls operating in this pathway suggests non-linear responses in signal transduction, which are described mathematically. We then explored possibilities for cross-talk mediated by Smad7 between DNA damage responses mediated by ATM, and with the WNT pathway and consider the design of experiments to test model driven hypothesis. Numerical comparisons of the mathematical model to experiments and representative predictions are described.

Hydrophobic patches on SMAD2 and SMAD3 determine selective binding to cofactors.

PubMed

Miyazono, Ken-Ichi; Moriwaki, Saho; Ito, Tomoko; Kurisaki, Akira; Asashima, Makoto; Tanokura, Masaru

2018-03-27

The transforming growth factor-β (TGF-β) superfamily of cytokines regulates various biological processes, including cell proliferation, immune responses, autophagy, and senescence. Dysregulation of TGF-β signaling causes various diseases, such as cancer and fibrosis. SMAD2 and SMAD3 are core transcription factors involved in TGF-β signaling, and they form heterotrimeric complexes with SMAD4 (SMAD2-SMAD2-SMAD4, SMAD3-SMAD3-SMAD4, and SMAD2-SMAD3-SMAD4) in response to TGF-β signaling. These heterotrimeric complexes interact with cofactors to control the expression of TGF-β-dependent genes. SMAD2 and SMAD3 may promote or repress target genes depending on whether they form complexes with other transcription factors, coactivators, or corepressors; therefore, the selection of specific cofactors is critical for the appropriate activity of these transcription factors. To reveal the structural basis by which SMAD2 and SMAD3 select cofactors, we determined the crystal structures of SMAD3 in complex with the transcription factor FOXH1 and SMAD2 in complex with the transcriptional corepressor SKI. The structures of the complexes show that the MAD homology 2 (MH2) domains of SMAD2 and SMAD3 have multiple hydrophobic patches on their surfaces. The cofactors tether to various subsets of these patches to interact with SMAD2 and SMAD3 in a cooperative or competitive manner to control the output of TGF-β signaling. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary

PubMed Central

Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K.

2011-01-01

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. PMID:21664424

The BMP pathway acts to directly regulate Tbx20 in the developing heart

PubMed Central

Mandel, Elizabeth M.; Kaltenbrun, Erin; Callis, Thomas E.; Zeng, Xin-Xin I.; Marques, Sara R.; Yelon, Deborah; Wang, Da-Zhi; Conlon, Frank L.

2010-01-01

TBX20 has been shown to be essential for vertebrate heart development. Mutations within the TBX20 coding region are associated with human congenital heart disease, and the loss of Tbx20 in a wide variety of model systems leads to cardiac defects and eventually heart failure. Despite the crucial role of TBX20 in a range of cardiac cellular processes, the signal transduction pathways that act upstream of Tbx20 remain unknown. Here, we have identified and characterized a conserved 334 bp Tbx20 cardiac regulatory element that is directly activated by the BMP/SMAD1 signaling pathway. We demonstrate that this element is both necessary and sufficient to drive cardiac-specific expression of Tbx20 in Xenopus, and that blocking SMAD1 signaling in vivo specifically abolishes transcription of Tbx20, but not that of other cardiac factors, such as Tbx5 and MHC, in the developing heart. We further demonstrate that activation of Tbx20 by SMAD1 is mediated by a set of novel, non-canonical, high-affinity SMAD-binding sites located within this regulatory element and that phospho-SMAD1 directly binds a non-canonical SMAD1 site in vivo. Finally, we show that these non-canonical sites are necessary and sufficient for Tbx20 expression in Xenopus, and that reporter constructs containing these sites are expressed in a cardiac-specific manner in zebrafish and mouse. Collectively, our findings define Tbx20 as a direct transcriptional target of the BMP/SMAD1 signaling pathway during cardiac maturation. PMID:20460370

Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

PubMed Central

Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael

2011-01-01

Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses. PMID:21949838

Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.

PubMed

Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael

2011-01-01

Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses.

Smad3 linker phosphorylation attenuates Smad3 transcriptional activity and TGF-β1/Smad3-induced epithelial-mesenchymal transition in renal epithelial cells.

PubMed

Bae, Eunjin; Kim, Seong-Jin; Hong, Suntaek; Liu, Fang; Ooshima, Akira

2012-10-26

Transforming growth factor-β1 (TGF-β1) has a distinct role in renal fibrosis associated with epithelial-mesenchymal transition (EMT) of the renal tubules and synthesis of extracellular matrix. Smad3 plays an essential role in fibrosis initiated by EMT. Phosphorylation of Smad3 in the C-terminal SSXS motif by type I TGF-β receptor kinase is essential for mediating TGF-β response. Smad3 activity is also regulated by phosphorylation in the linker region. However, the functional role of Smad3 linker phosphorylation is not well characterized. We now show that Smad3 EPSM mutant, which mutated the four phosphorylation sites in the linker region, markedly enhanced TGF-β1-induced EMT of Smad3-deficient primary renal tubular epithelial cells, whereas Smad3 3S-A mutant, which mutated the C-terminal phosphorylation sites, was unable to induce EMT in response to TGF-β1. Furthermore, immunoblotting and RT-PCR analysis showed a marked induction of fibrogenic gene expression with a significant reduction in E-cadherin in HK2 human renal epithelial cells expressing Smad3 EPSM. TGF-β1 could not induce the expression of α-SMA, vimentin, fibronectin and PAI-1 or reduce the expression of E-cadherin in HK2 cells expressing Smad3 3S-A in response to TGF-β1. Our results suggest that Smad3 linker phosphorylation has a negative regulatory role on Smad3 transcriptional activity and TGF-β1/Smad3-induced renal EMT. Elucidation of mechanism regulating the Smad3 linker phosphorylation can provide a new strategy to control renal fibrosis. Copyright © 2012 Elsevier Inc. All rights reserved.

Reduced Smad4 expression and DNA topoisomerase inhibitor chemosensitivity in non-small cell lung cancer.

PubMed

Ziemke, Michael; Patil, Tejas; Nolan, Kyle; Tippimanchai, Darinee; Malkoski, Stephen P

2017-07-01

Smad4 is a tumor suppressor that transduces transforming growth factor beta signaling and regulates genomic stability. We previously found that Smad4 knockdown in vitro inhibited DNA repair and increased sensitivity to DNA topoisomerase inhibitors. In this study, we assessed the association between reduced Smad4 expression and DNA topoisomerase inhibitor sensitivity in human non-small cell lung cancer (NSCLC) patients and evaluated the relationship between genomic alterations of Smad4 and molecular alterations in DNA repair molecules. We retrospectively identified NSCLC patients who received etoposide or gemcitabine. Chemotherapeutic response was quantified by RECIST 1.1 criteria and Smad4 expression was assessed by immunohistochemistry. Relationships between Smad4 mutation and DNA repair molecule mutations were evaluated using publically available datasets. We identified 28 individuals who received 30 treatments with gemcitabine or etoposide containing regimens for NSCLC. Reduced Smad4 expression was seen in 13/28 patients and was not associated with significant differences in clinical or pathologic parameters. Patients with reduced Smad4 expression had a larger response to DNA topoisomerase inhibitor containing regimens then patients with high Smad4 expression (-25.7% vs. -6.8% in lesion size, p=0.03); this relationship was more pronounced with gemcitabine containing regimens. The overall treatment response was higher in patients with reduced Smad4 expression (8/14 vs 2/16 p=0.02). Analysis of data from The Cancer Genome Atlas revealed that Smad4 mutation or homozygous loss was mutually exclusive with genomic alterations in DNA repair molecules. Reduced Smad4 expression may predict responsiveness to regimens that contain DNA topoisomerase inhibitors. That Smad4 signaling alterations are mutually exclusive with alterations in DNA repair machinery is consistent with an important role of Smad4 in regulating DNA repair. Copyright © 2017 Elsevier B.V. All rights reserved.

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

PubMed

Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K

2011-08-06

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. Copyright © 2011 Elsevier Ltd. All rights reserved.

Combined Biology and Bioinformatics Approaches to Breast Cancer

DTIC Science & Technology

2006-04-01

In these experiments, LMO4 interacted with the MH1 and linker domains of Smad3 ; no interaction was found with the MH2 domain (Figure 4b). Figure 2...transcriptional response to TGFb by interacting with Smad proteins, and that both the MH1 and linker domains of Smad3 participate in the interaction. LMO4 can...LMO4 interacts with the MH1 and linker regions of Smad proteins. (a) Full-length, 35S-labeled Smad2, Smad3 , Smad4, Smad5, and Smad8 were incubated with

Pokemon (FBI-1) interacts with Smad4 to repress TGF-β-induced transcriptional responses.

PubMed

Yang, Yutao; Cui, Jiajun; Xue, Feng; Zhang, Chuanfu; Mei, Zhu; Wang, Yue; Bi, Mingjun; Shan, Dapeng; Meredith, Alex; Li, Hui; Xu, Zhi-Qing David

2015-03-01

Pokemon, an important proto-oncoprotein, is a transcriptional repressor that belongs to the POK (POZ and Krüppel) family. Smad4, a key component of TGF-β pathway, plays an essential role in TGF-β-induced transcriptional responses. In this study, we show that Pokemon can interact directly with Smad4 both in vitro and in vivo. Overexpression of Pokemon decreases TGF-β-induced transcriptional activities, whereas knockdown of Pokemon increases these activities. Interestingly, Pokemon does not affect activation of Smad2/3, formation of Smads complex, or DNA binding activity of Smad4. TGF-β1 treatment increases the interaction between Pokemon and Smad4, and also enhances the recruitment of Pokemon to Smad4-DNA complex. In addition, we also find that Pokemon recruits HDAC1 to Smad4 complex but decreases the interaction between Smad4 and p300/CBP. Taken together, all these data suggest that Pokemon is a new partner of Smad4 and plays a negative role in TGF-β pathway. Copyright © 2014. Published by Elsevier B.V.

The Smad3 linker region contains a transcriptional activation domain

PubMed Central

2004-01-01

Transforming growth factor-β (TGF-β)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-β/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-β transcriptional activation responses, although it can be phosphorylated by the TGF-β receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-β. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control. PMID:15588252

The Smad3 linker region contains a transcriptional activation domain.

PubMed

Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

2005-02-15

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells.

PubMed

Bae, Eunjin; Sato, Misako; Kim, Ran-Ju; Kwak, Mi-Kyung; Naka, Kazuhito; Gim, Jungsoo; Kadota, Mitsutaka; Tang, Binwu; Flanders, Kathleen C; Kim, Tae-Aug; Leem, Sun-Hee; Park, Taesung; Liu, Fang; Wakefield, Lalage M; Kim, Seong-Jin; Ooshima, Akira

2014-11-01

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer. ©2014 American Association for Cancer Research.

A negative feedback control of transforming growth factor-beta signaling by glycogen synthase kinase 3-mediated Smad3 linker phosphorylation at Ser-204.

PubMed

Millet, Caroline; Yamashita, Motozo; Heller, Mary; Yu, Li-Rong; Veenstra, Timothy D; Zhang, Ying E

2009-07-24

Through the action of its membrane-bound type I receptor, transforming growth factor-beta (TGF-beta) elicits a wide range of cellular responses that regulate cell proliferation, differentiation, and apo ptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. Here, we show that TGF-beta induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and glycogen synthase kinase 3 is responsible for phosphorylation at one of these sites, namely Ser-204. Alanine substitution at Ser-204 and/or the neighboring Ser-208, the priming site for glycogen synthase kinase 3 in vivo activity, strengthened the affinity of Smad3 to CREB-binding protein, suggesting that linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of the Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-beta.

Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical Wnt signaling and causes an osteogenesis-imperfecta-like phenotype

PubMed Central

Salazar, Valerie S.; Zarkadis, Nicholas; Huang, Lisa; Norris, Jin; Grimston, Susan K.; Mbalaviele, Gabriel; Civitelli, Roberto

2013-01-01

Summary To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-β–BMP pathway, in Osx1+ cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of β-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt. PMID:24006258

Transcriptional Profiling Identifies Functional Interactions of TGFβ and PPARβ/δ Signaling

PubMed Central

Kaddatz, Kerstin; Adhikary, Till; Finkernagel, Florian; Meissner, Wolfgang; Müller-Brüsselbach, Sabine; Müller, Rolf

2010-01-01

Peroxisome proliferator-activated receptors (PPARs) not only play a key role in regulating metabolic pathways but also modulate inflammatory processes, pointing to a functional interaction between PPAR and cytokine signaling pathways. In this study, we show by genome-wide transcriptional profiling that PPARβ/δ and transforming growth factor-β (TGFβ) pathways functionally interact in human myofibroblasts and that a subset of these genes is cooperatively activated by TGFβ and PPARβ/δ. Using the angiopoietin-like 4 (ANGPTL4) gene as a model, we demonstrate that two enhancer regions cooperate to mediate the observed synergistic response. A TGFβ-responsive enhancer located ∼8 kb upstream of the transcriptional start site is regulated by a mechanism involving SMAD3, ETS1, RUNX, and AP-1 transcription factors that interact with multiple contiguous binding sites. A second enhancer (PPAR-E) consisting of three juxtaposed PPAR response elements is located in the third intron ∼3.5 kb downstream of the transcriptional start site. The PPAR-E is strongly activated by all three PPAR subtypes, with a novel type of PPAR response element motif playing a central role. Although the PPAR-E is not regulated by TGFβ, it interacts with SMAD3, ETS1, RUNX2, and AP-1 in vivo, providing a possible mechanistic explanation for the observed synergism. PMID:20595396

Arginine methylation of Smad7 by PRMT1 in TGF-β-induced epithelial-mesenchymal transition and epithelial stem cell generation.

PubMed

Katsuno, Yoko; Qin, Jian; Oses-Prieto, Juan A; Wang, Hongjun; Jackson-Weaver, Olan; Zhang, Tingwei; Lamouille, Samy; Wu, Jian; Burlingame, A L L; Xu, Jian; Derynck, Rik

2018-06-15

The epithelial-to-mesenchymal transdifferentiation (EMT) is crucial for tissue differentiation in development, and drives essential steps in cancer and fibrosis. EMT is accompanied by reprogramming of gene expression, and has been associated with the epithelial stem cell state in normal and carcinoma cells. The cytokine TGF-β drives this program in cooperation with other signaling pathways and through TGF-β-activated Smad3 as major effector. TGF-β-induced Smad3 activation is inhibited by Smad7 and to a lesser extent by Smad6, and Smad6 and Smad7 both inhibit Smad1 and Smad5 activation in response to the TGF-β-related bone morphogenetic proteins (BMPs). We previously reported that, in response to BMP, the protein arginine methyltransferase PRMT1 methylates Smad6 at the BMP receptor complex, thereby promoting its dissociation from the receptors and enabling BMP-induced Smad1 and Smad5 activation. We now provide evidence that PRMT1 also facilitates TGF-β signaling by methylating Smad7, which complements Smad6 methylation. We found that PRMT1 is required for TGF-β-induced Smad3 activation, through a mechanism similar to that of BMP-induced Smad6 methylation, and thus promotes the TGF-β-induced EMT and epithelial stem cell generation. This critical mechanism positions PRMT1 as an essential mediator of TGF-β signaling that controls the EMT and epithelial cell stemness through Smad7 methylation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling.

PubMed

Wu, Jia Wei; Krawitz, Ariel R; Chai, Jijie; Li, Wenyu; Zhang, Fangjiu; Luo, Kunxin; Shi, Yigong

2002-11-01

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.

Sorting nexin 9 differentiates ligand-activated Smad3 from Smad2 for nuclear import and transforming growth factor β signaling

PubMed Central

Wilkes, Mark C.; Repellin, Claire E.; Kang, Jeong-Han; Andrianifahanana, Mahefatiana; Yin, Xueqian; Leof, Edward B.

2015-01-01

Transforming growth factor β (TGFβ) is a pleiotropic protein secreted from essentially all cell types and primary tissues. While TGFβ’s actions reflect the activity of a number of signaling networks, the primary mediator of TGFβ responses are the Smad proteins. Following receptor activation, these cytoplasmic proteins form hetero-oligomeric complexes that translocate to the nucleus and affect gene transcription. Here, through biological, biochemical, and immunofluorescence approaches, sorting nexin 9 (SNX9) is identified as being required for Smad3-dependent responses. SNX9 interacts with phosphorylated (p) Smad3 independent of Smad2 or Smad4 and promotes more rapid nuclear delivery than that observed independent of ligand. Although SNX9 does not bind nucleoporins Nup153 or Nup214 or some β importins (Imp7 or Impβ), it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates nuclear translocation of pSmad3 but not SNX9. PMID:26337383

Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development.

PubMed

Yan, Jianyun; Li, Jun; Hu, Jun; Zhang, Lu; Wei, Chengguo; Sultana, Nishat; Cai, Xiaoqiang; Zhang, Weijia; Cai, Chen-Leng

2018-06-15

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre ( Tbx18 Cre /+ ) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 ( Smad4 f/f ) in the limbs of mice. We found that the Smad4 -deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan , in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 ( Runx2 ), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4 -deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia. © 2018 Yan et al.

LIM mineralization protein-1 potentiates bone morphogenetic protein responsiveness via a novel interaction with Smurf1 resulting in decreased ubiquitination of Smads.

PubMed

Sangadala, Sreedhara; Boden, Scott D; Viggeswarapu, Manjula; Liu, Yunshan; Titus, Louisa

2006-06-23

Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway.

Breast Tumorigenesis: Interaction of Two Signaling Pathways- -TGF- -beta versus Estrogen Receptor.

DTIC Science & Technology

1997-08-01

on the functional role of Smad3 and Smad4 as tumor suppressors in mediating the TGF-B signal in transactivating downstream target genes. We have...extended our analysis of the biological activity of the Smad proteins in TGF-B signaling by studying the nuclear activity of Smad2, Smad3 and Sliad4...groups using in vitro phosphorylation assays. Taken together these data suggest that Smad2 and Smad3 are inducibly phosphorylated in response to TGF-P3 and

Smad phospho-isoforms direct context-dependent TGF-β signaling.

PubMed

Matsuzaki, Koichi

2013-08-01

Better understanding of TGF-β signaling has deepened our appreciation of normal epithelial cell homeostasis and its dysfunction in such human disorders as cancer and fibrosis. Smad proteins, which convey signals from TGF-β receptors to the nucleus, possess intermediate linker regions connecting Mad homology domains. Membrane-bound, cytoplasmic, and nuclear protein kinases differentially phosphorylate Smad2 and Smad3 to create C-tail (C), the linker (L), or dually (L/C) phosphorylated (p, phospho-) isoforms. According to domain-specific phosphorylation, distinct transcriptional responses, and selective metabolism, Smad phospho-isoform pathways can be grouped into 4 types: cytostatic pSmad3C signaling, mitogenic pSmad3L (Ser-213) signaling, invasive/fibrogenic pSmad2L (Ser-245/250/255)/C or pSmad3L (Ser-204)/C signaling, and mitogenic/migratory pSmad2/3L (Thr-220/179)/C signaling. We outline how responses to TGF-β change through the multiple Smad phospho-isoforms as normal epithelial cells mature from stem cells through progenitors to differentiated cells, and further reflect upon how constitutive Ras-activating mutants favor the Smad phospho-isoform pathway promoting tumor progression. Finally, clinical analyses of reversible Smad phospho-isoform signaling during human carcinogenesis could assess effectiveness of interventions aimed at reducing human cancer risk. Spatiotemporally separate, functionally different Smad phospho-isoforms have been identified in specific cells and tissues, answering long-standing questions about context-dependent TGF-β signaling. Copyright © 2013 Elsevier Ltd. All rights reserved.

The oncoprotein Ski acts as an antagonist of transforming growth factor-beta signaling by suppressing Smad2 phosphorylation.

PubMed

Prunier, Celine; Pessah, Marcia; Ferrand, Nathalie; Seo, Su Ryeon; Howe, Philip; Atfi, Azeddine

2003-07-11

The phosphorylation of Smad2 and Smad3 by the transforming growth factor (TGF)-beta-activated receptor kinases and their subsequent heterodimerization with Smad4 and translocation to the nucleus form the basis for a model how Smad proteins work to transmit TGF-beta signals. The transcriptional activity of Smad2-Smad4 or Smad3-Smad4 complexes can be limited by the corepressor Ski, which is believed to interact with Smad complexes on TGF-beta-responsive promoters and represses their ability to activate TGF-beta target genes by assembling on DNA a repressor complex containing histone deacetylase. Here we show that Ski can block TGF-beta signaling by interfering with the phosphorylation of Smad2 and Smad3 by the activated TGF-beta type I receptor. Furthermore, we demonstrate that overexpression of Ski induces the assembly of Smad2-Smad4 and Smad3-Smad4 complexes independent of TGF-beta signaling. The ability of Ski to engage Smad proteins in nonproductive complexes provides new insights into the molecular mechanism used by Ski for disabling TGF-beta signaling.

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling.

PubMed

Gao, Sheng; Alarcón, Claudio; Sapkota, Gopal; Rahman, Sadia; Chen, Pan-Yu; Goerner, Nina; Macias, Maria J; Erdjument-Bromage, Hediye; Tempst, Paul; Massagué, Joan

2009-11-13

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here, we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction.

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase.

PubMed

Qu, Hongxia; Zheng, Liduan; Jiao, Wanju; Mei, Hong; Li, Dan; Song, Huajie; Fang, Erhu; Wang, Xiaojing; Li, Shiwang; Huang, Kai; Tong, Qiangsong

2016-09-06

Heparanase (HPSE) is the only endo-β-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression.

Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

PubMed

Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

2009-04-10

Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

Selective Small Molecule Compounds Increase BMP-2 Responsiveness by Inhibiting Smurf1-mediated Smad1/5 Degradation

PubMed Central

Cao, Yu; Wang, Cheng; Zhang, Xueli; Xing, Guichun; Lu, Kefeng; Gu, Yongqing; He, Fuchu; Zhang, Lingqiang

2014-01-01

The ubiquitin ligase Smad ubiquitination regulatory factor-1 (Smurf1) negatively regulates bone morphogenetic protein (BMP) pathway by ubiquitinating certain signal components for degradation. Thus, it can be an eligible pharmacological target for increasing BMP signal responsiveness. We established a strategy to discover small molecule compounds that block the WW1 domain of Smurf1 from interacting with Smad1/5 by structure based virtual screening, molecular experimental examination and cytological efficacy evaluation. Our selected hits could reserve the protein level of Smad1/5 from degradation by interrupting Smurf1-Smad1/5 interaction and inhibiting Smurf1 mediated ubiquitination of Smad1/5. Further, these compounds increased BMP-2 signal responsiveness and the expression of certain downstream genes, enhanced the osteoblastic activity of myoblasts and osteoblasts. Our work indicates targeting Smurf1 for inhibition could be an accessible strategy to discover BMP-sensitizers that might be applied in future clinical treatments of bone disorders such as osteopenia. PMID:24828823

Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

PubMed Central

Yoon, Jeong-Hwan; Jung, Su Myung; Park, Seok Hee; Kato, Mitsuyasu; Yamashita, Tadashi; Lee, In-Kyu; Sudo, Katsuko; Nakae, Susumu; Han, Jin Soo; Kim, Ok-Hee; Oh, Byung-Chul; Sumida, Takayuki; Kuroda, Masahiko; Ju, Ji-Hyeon; Jung, Kyeong Cheon; Park, Seong Hoe; Kim, Dae-Kee; Mamura, Mizuko

2013-01-01

Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation. PMID:24127404

Unique players in the BMP pathway: Small C-terminal domain phosphatases dephosphorylate Smad1 to attenuate BMP signaling

PubMed Central

Knockaert, Marie; Sapkota, Gopal; Alarcón, Claudio; Massagué, Joan; Brivanlou, Ali H.

2006-01-01

Smad transcription factors are key signal transducers for the TGF-β/bone morphogenetic protein (BMP) family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-β and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. Here we provide functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes. PMID:16882717

Postnatal ablation of osteoblast Smad4 enhances proliferative responses to canonical Wnt signaling through interactions with β-catenin

PubMed Central

Salazar, Valerie S.; Zarkadis, Nicholas; Huang, Lisa; Watkins, Marcus; Kading, Jacqueline; Bonar, Sheri; Norris, Jin; Mbalaviele, Gabriel; Civitelli, Roberto

2013-01-01

Summary Canonical Wnt (cWnt) signaling through β-catenin regulates osteoblast proliferation and differentiation to enhance bone formation. We previously reported that osteogenic action of β-catenin is dependent on BMP signaling. Here, we further examined interactions between cWnt and BMP in bone. In osteoprogenitors stimulated with BMP2, β-catenin localizes to the nucleus, physically interacts with Smad4, and is recruited to DNA-binding transcription complexes containing Smad4, R-Smad1/5 and TCF4. Furthermore, Tcf/Lef-dependent transcription, Ccnd1 expression and proliferation all increase when Smad4, 1 or 5 levels are low, whereas TCF/Lef activities decrease when Smad4 expression is high. The ability of Smad4 to antagonize transcription of Ccnd1 is dependent on DNA-binding activity but Smad4-dependent transcription is not required. In mice, conditional deletion of Smad4 in osterix+ cells increases mitosis of cells on trabecular bone surfaces as well as in primary osteoblast cultures from adult bone marrow and neonatal calvaria. By contrast, ablation of Smad4 delays differentiation and matrix mineralization by primary osteoblasts in response to Wnt3a, indicating that loss of Smad4 perturbs the balance between proliferation and differentiation in osteoprogenitors. We propose that Smad4 and Tcf/Lef transcription complexes compete for β-catenin, thus restraining cWnt-dependent proliferative signals while favoring the matrix synthesizing activity of osteoblasts. PMID:24101723

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase

PubMed Central

Qu, Hongxia; Zheng, Liduan; Jiao, Wanju; Mei, Hong; Li, Dan; Song, Huajie; Fang, Erhu; Wang, Xiaojing; Li, Shiwang; Huang, Kai; Tong, Qiangsong

2016-01-01

Heparanase (HPSE) is the only endo-β-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression. PMID:27595937

Fussel-15, a novel Ski/Sno homolog protein, antagonizes BMP signaling.

PubMed

Arndt, Stephanie; Poser, Ina; Moser, Markus; Bosserhoff, Anja-Katrin

2007-04-01

The Ski family of nuclear oncoproteins represses transforming growth factor-beta (TGF-beta) signaling through inhibition of transcriptional activity of Smad proteins. In this study, we identified a novel gene, fussel-15 (functional smad suppressing element on chromosome 15) with high homology to the recently discovered Fussel-18 protein. Both, Fussel-15 and Fussel-18, share important structural features, significant homology and similar genomic organization with the homolog Ski family members, Ski and SnoN. Unlike Ski and SnoN, which are ubiquitously expressed in human tissues, Fussel-15 expression, like Fussel-18, is much more restricted in its expression and is principally found in the nervous system of mouse and humans. Interestingly, Fussel-15 expression is even more restricted in adulthood to Purkinje cells of human cerebellum. In contrast to Fussel-18 that interacts with Smad 2, Smad3 and Smad4 and has an inhibitory activity on TGF-beta signaling, Fussel-15 interacts with Smad1, Smad2 and Smad3 molecules and suppresses mainly BMP signaling pathway but has only minor effects on TGF-beta signaling. This new protein expands the family of Ski/Sno proto-oncoproteins and represents a novel molecular regulator of BMP signaling.

The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

PubMed

Qu, Xinli; Jiang, Mengjie; Sun, Yu Bo Yang; Jiang, Xiaoyun; Fu, Ping; Ren, Yi; Wang, Die; Dai, Lie; Caruana, Georgina; Bertram, John F; Nikolic-Paterson, David J; Li, Jinhua

2015-12-01

Transforming growth factor-β1 (TGF-β1)/Smad signaling has a central role in the pathogenesis of renal fibrosis. Smad3 and Smad4 are pro-fibrotic, while Smad2 is anti-fibrotic. However, these Smads form heterogeneous complexes, the functions of which are poorly understood. Here we studied Smad complex function in renal fibrosis using the mouse model of unilateral ureteric obstruction. Mice heterozygous for Smad3/4 (Smad3/4 +/- ) exhibited substantial protection from renal fibrosis through day 7 of obstruction, whereas Smad2/3 +/- and Smad2/4 +/- mice showed only modest protection. Formation of Smad3/Smad4/CDK9 complexes was an early event following obstruction in wild-type mice, which involved nuclear phosphorylation of the linker regions of Smad3. Significantly, Smad3 or Smad4 deficiency decreased the formation of Smad4/CDK9 or Smad3/CDK9 complex, Smad3 linker phosphorylation, and fibrosis but at different degrees. In vitro, TGF-β1 stimulation of collagen I promoter activity involved formation of Smad3/Smad4/CDK9 complexes, and overexpression of each component gave additive increases in collagen promoter activity. Co-administration of a CDK9 inhibitor and Smad3-specific inhibition achieved better protection from TGF-β1-induced fibrotic response in vitro and renal interstitial fibrosis in vivo. Thus formation of Smad3/Smad4/CDK9 complex drives renal fibrosis during ureteral obstruction. Formation of this complex represents a novel target for antifibrotic therapies.

Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

PubMed

Sapkota, Gopal; Knockaert, Marie; Alarcón, Claudio; Montalvo, Ermelinda; Brivanlou, Ali H; Massagué, Joan

2006-12-29

Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.

Interaction with Smad4 is indispensable for suppression of BMP signaling by c-Ski.

PubMed

Takeda, Masafumi; Mizuide, Masafumi; Oka, Masako; Watabe, Tetsuro; Inoue, Hirofumi; Suzuki, Hiroyuki; Fujita, Toshiro; Imamura, Takeshi; Miyazono, Kohei; Miyazawa, Keiji

2004-03-01

c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5. Through binding to Smad proteins, c-Ski suppresses signaling of transforming growth factor-beta (TGF-beta) as well as bone morphogenetic proteins (BMPs). In the present study, we found that a mutant of c-Ski, termed c-Ski (ARPG) inhibited TGF-beta/activin signaling but not BMP signaling. Selectivity was confirmed in luciferase reporter assays and by determination of cellular responses in mammalian cells (BMP-induced osteoblastic differentiation of C2C12 cells and TGF-beta-induced epithelial-to-mesenchymal transdifferentiation of NMuMG cells) and Xenopus embryos. The ARPG mutant recruited histone deacetylases 1 (HDAC1) to the Smad3-Smad4 complex but not to the Smad1/5-Smad4 complex. c-Ski (ARPG) was unable to interact with Smad4, and the selective loss of suppression of BMP signaling by c-Ski (ARPG) was attributed to the lack of Smad4 binding. We also found that c-Ski interacted with Smad3 or Smad4 without disrupting Smad3-Smad4 heteromer formation. c-Ski (ARPG) would be useful for selectively suppressing TGF-beta/activin signaling.

Interaction with Smad4 Is Indispensable for Suppression of BMP Signaling by c-Ski

PubMed Central

Takeda, Masafumi; Mizuide, Masafumi; Oka, Masako; Watabe, Tetsuro; Inoue, Hirofumi; Suzuki, Hiroyuki; Fujita, Toshiro; Imamura, Takeshi; Miyazono, Kohei; Miyazawa, Keiji

2004-01-01

c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5. Through binding to Smad proteins, c-Ski suppresses signaling of transforming growth factor-β (TGF-β) as well as bone morphogenetic proteins (BMPs). In the present study, we found that a mutant of c-Ski, termed c-Ski (ARPG) inhibited TGF-β/activin signaling but not BMP signaling. Selectivity was confirmed in luciferase reporter assays and by determination of cellular responses in mammalian cells (BMP-induced osteoblastic differentiation of C2C12 cells and TGF-β–induced epithelial-to-mesenchymal transdifferentiation of NMuMG cells) and Xenopus embryos. The ARPG mutant recruited histone deacetylases 1 (HDAC1) to the Smad3-Smad4 complex but not to the Smad1/5-Smad4 complex. c-Ski (ARPG) was unable to interact with Smad4, and the selective loss of suppression of BMP signaling by c-Ski (ARPG) was attributed to the lack of Smad4 binding. We also found that c-Ski interacted with Smad3 or Smad4 without disrupting Smad3-Smad4 heteromer formation. c-Ski (ARPG) would be useful for selectively suppressing TGF-β/activin signaling. PMID:14699069

Bone morphogenetic protein 2 activates Smad6 gene transcription through bone-specific transcription factor Runx2.

PubMed

Wang, Qing; Wei, Xiaochao; Zhu, Tianhui; Zhang, Ming; Shen, Run; Xing, Lianping; O'Keefe, Regis J; Chen, Di

2007-04-06

BMP-2 plays an essential role in osteoblast and chondrocyte differentiation, but its signaling mechanism has not been fully defined. In the present studies, we investigated the mechanism through which BMP-2 activates the Smad6 gene. A -2006/+45 Smad6 promoter-luciferase construct was generated along with deletions and Runx2 binding site mutations to examine the role of Smad1 and Runx2 signaling following BMP-2 stimulation in osteoblasts. Transfection of Runx2 or treatment with BMP-2-stimulated promoter activity of the -2006/+45 and -1191/+45 reporters but not the -829/+45 and -374/+45 reporters. No Smad1/5 binding site is present in the -1191/-829 region of the Smad6 promoter. Mutation of the OSE2-a site (-1036/-1031) completely abolished the stimulatory effect of Runx2 as well as BMP-2 on the -2006/+45 and -1191/+45 Smad6 reporters. Gel shift and chromatin immunoprecipitation (ChIP) assays showed that Runx2 binds the OSE2-a element. ChIP assays demonstrated that Smad1 also interacts with the OSE2-a site at the Smad6 promoter through Runx2. The protein degradation of Runx2 is mediated by the E3 ubiquitin ligase Smurf1. In the present studies, we found that Smurf1 binds the OSE2-a site through Runx2 and inhibits Smad6 gene transcription. Treatment with BMP-2 and transfection of Smad1 abolished Smurf1 binding to the OSE2 site. These results show that Smad1 binding excludes Smurf1 interaction with the OSE2 site and promotes Smad6 gene transcription.

Serine-204 in the Linker Region of Smad3 Mediates the Collagen-I Response to TGF-β in a Cell Phenotype-Specific Manner

PubMed Central

Browne, James A.; Liu, Xiaoying; Schnaper, H. William; Hayashida, Tomoko

2013-01-01

Regulation of TGF-β1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-β-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-β are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-β-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-β stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake. Renal epithelial cells (HKC) respond differently from MEF or mesangial cells; blocking ERK neither changed TGF-β-stimulated S204 phosphorylation nor prevented Smad3-mediated COL1A2 promoter activity in HKC. Furthermore, re-expression of wild type-Smad3 or the S204A-Smad3 mutant in Smad3-knockdown HKC reconstituted Smad3-mediated COL1A2 promoter activity. Collectively, these data suggest that Serine-204 phosphorylation in the Smad3LR is a critical event by which ERK enhances Smad3-mediated COL1A2 promoter activity in mesenchymal cells. PMID:24080014

Serine-204 in the linker region of Smad3 mediates the collagen-I response to TGF-β in a cell phenotype-specific manner.

PubMed

Browne, J A; Liu, X; Schnaper, H W; Hayashida, T

2013-11-15

Regulation of TGF-β1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-β-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-β are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-β-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-β stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake. Renal epithelial cells (HKC) respond differently from MEF or mesangial cells; blocking ERK neither changed TGF-β-stimulated S204 phosphorylation nor prevented Smad3-mediated COL1A2 promoter activity in HKC. Furthermore, re-expression of wild type-Smad3 or the S204A-Smad3 mutant in Smad3-knockdown HKC reconstituted Smad3-mediated COL1A2 promoter activity. Collectively, these data suggest that Serine-204 phosphorylation in the Smad3LR is a critical event by which ERK enhances Smad3-mediated COL1A2 promoter activity in mesenchymal cells. Copyright © 2013 Elsevier Inc. All rights reserved.

TGF-β/SMAD Pathway and Its Regulation in Hepatic Fibrosis

PubMed Central

Xu, Fengyun; Liu, Changwei; Zhou, Dandan; Zhang, Lei

2016-01-01

Transforming growth factor-beta1 (TGF-β1), a key member in the TGF-β superfamily, plays a critical role in the development of hepatic fibrosis. Its expression is consistently elevated in affected organs, which correlates with increased extracellular matrix deposition. SMAD proteins have been studied extensively as pivotal intracellular effectors of TGF-β1, acting as transcription factors. In the context of hepatic fibrosis, SMAD3 and SMAD4 are pro-fibrotic, whereas SMAD2 and SMAD7 are protective. Deletion of SMAD3 inhibits type I collagen expression and blocks epithelial-myofibroblast transition. In contrast, disruption of SMAD2 upregulates type I collagen expression. SMAD4 plays an essential role in fibrosis disease by enhancing SMAD3 responsive promoter activity, whereas SMAD7 negatively mediates SMAD3-induced fibrogenesis. Accumulating evidence suggests that divergent miRNAs participate in the liver fibrotic process, which partially regulates members of the TGF-β/SMAD signaling pathway. In this review, we focus on the TGF-β/SMAD and other relative signaling pathways, and discussed the role and molecular mechanisms of TGF-β/SMAD in the pathogenesis of hepatic fibrosis. Moreover, we address the possibility of novel therapeutic approaches to hepatic fibrosis by targeting to TGF-β/SMAD signaling. PMID:26747705

SMAD4 and NF1 mutations as potential biomarkers for poor prognosis to cetuximab-based therapy in Chinese metastatic colorectal cancer patients.

PubMed

Mei, Zhu; Shao, Yang W; Lin, Peinan; Cai, Xiaomin; Wang, Biao; Ding, Yan; Ma, Xiangyuan; Wu, Xue; Xia, Yewei; Zhu, Dongqin; Shu, Yongqian; Fu, Zan; Gu, Yanhong

2018-04-27

Cetuximab, an anti-EGFR monoclonal antibody, is used in combination with chemotherapy in clinic to enhance the outcome in metastatic colorectal cancer (mCRC) patients with only ~ 20% response rate. To date only activating mutations in KRAS and NRAS have been identified as poor prognosis biomarkers in cetuximab-based treatment, which makes an urgent need for identification of novel prognosis biomarkers to precisely predict patients' response in order to maximize the benefit. In this study, we analysed the mutation profiles of 33 Chinese mCRC patients using comprehensive next-generation sequencing (NGS) targeting 416 cancer-relevant genes before cetuximab treatment. Upon receiving cetuximab-based therapy, patients were evaluated for drug response, and the progression-free survival (PFS) was monitored. The association of specific genetic alterations and cetuximab efficacy was analyzed. Patients carrying SMAD4 mutations (SMAD4 mut , n = 8) or NF1 mutations (NF1 mut , n = 4) had significantly shorter PFS comparing to those carrying wildtype SMAD4 (SMAD4 wt , n = 25) (P = 0.0081) or wildtype NF1 (NF1 wt , n = 29) (P = 0.0028), respectively. None of the SMAD4 mut or NF1 mut patients showed response to cetuximab when assessed at 12-week post-treatment. Interestingly, two patients carrying both SMAD4 mut and NF1 mut showed the shortest PFS among all the patients. Our results demonstrated that SMAD4 and NF1 mutations can serve as potential biomarkers for poor prognosis to cetuximab-based therapy in Chinese mCRC patients.

Linking Smads and transcriptional activation.

PubMed

Inman, Gareth J

2005-02-15

TGF-beta1 (transforming growth factor-beta1) is the prototypical member of a large family of pleiotropic cytokines that regulate diverse biological processes during development and adult tissue homoeostasis. TGF-beta signals via membrane bound serine/threonine kinase receptors which transmit their signals via the intracellular signalling molecules Smad2, Smad3 and Smad4. These Smads contain conserved MH1 and MH2 domains separated by a flexible linker domain. Smad2 and Smad3 act as kinase substrates for the receptors, and, following phosphorylation, they form complexes with Smad4 and translocate to the nucleus. These Smad complexes regulate gene expression and ultimately determine the biological response to TGF-beta. In this issue of the Biochemical Journal, Wang et al. have shown that, like Smad4, the linker domain of Smad3 contains a Smad transcriptional activation domain. This is capable of recruiting the p300 transcriptional co-activator and is required for Smad3-dependent transcriptional activation. This study raises interesting questions about the nature and regulation of Smad-regulated gene activation and elevates the status of the linker domain to rival that of the much-lauded MH1 and MH2 domains.

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis.

PubMed

Conery, Andrew R; Cao, Yanna; Thompson, E Aubrey; Townsend, Courtney M; Ko, Tien C; Luo, Kunxin

2004-04-01

Transforming growth factor beta (TGF-beta) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-beta is specified. Smad proteins are critical mediators of TGF-beta signalling. After stimulation by TGF-beta, Smad2 and Smad3 become phosphorylated by the activated TGF-beta receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-beta target genes. Here we report that the sensitivity to TGF-beta induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-beta induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-beta. These findings identify an important determinant of sensitivity to TGF-beta-induced apoptosis that involves crosstalk between the TGF-beta and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.

Aberrant hypertrophy in Smad3-deficient murine chondrocytes is rescued by restoring transforming growth factor beta-activated kinase 1/activating transcription factor 2 signaling: a potential clinical implication for osteoarthritis.

PubMed

Li, Tian-Fang; Gao, Lin; Sheu, Tzong-Jen; Sampson, Erik R; Flick, Lisa M; Konttinen, Yrjö T; Chen, Di; Schwarz, Edward M; Zuscik, Michael J; Jonason, Jennifer H; O'Keefe, Regis J

2010-08-01

To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor beta (TGFbeta) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. Joint disease in Smad3-knockout (Smad3(-/-)) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3(-/-) mice. In Smad3(-/-) mice, an end-stage OA phenotype gradually developed. TGFbeta-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3(-/-) mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3(-/-) cells restored the normal p38 response to TGFbeta. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3(-/-) chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms alpha, beta, and gamma, but not delta. Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3-phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.

STAT3-mediated SMAD3 activation underlies Oncostatin M-induced Senescence

PubMed Central

Junk, Damian J.; Cipriano, Rocky; Jackson, Mark W.

2017-01-01

ABSTRACT Cytokines in the developing tumor microenvironment (TME) can drive transformation and subsequent progression toward metastasis. Elevated levels of the Interleukin-6 (IL-6) family cytokine Oncostatin M (OSM) in the breast TME correlate with aggressive, metastatic cancers, increased tumor recurrence, and poor patient prognosis. Paradoxically, OSM engages a tumor-suppressive, Signal Transducer and Activator of Transcription 3 (STAT3)-dependent senescence response in normal and non-transformed human mammary epithelial cells (HMEC). Here, we identify a novel link between OSM-activated STAT3 signaling and the Transforming Growth Factor-β (TGF-β) signaling pathway that engages senescence in HMEC. Inhibition of functional TGF-β/SMAD signaling by expressing a dominant-negative TGF-β receptor, treating with a TGF-β receptor inhibitor, or suppressing SMAD3 expression using a SMAD3-shRNA prevented OSM-induced senescence. OSM promoted a protein complex involving activated-STAT3 and SMAD3, induced the nuclear localization of SMAD3, and enhanced SMAD3-mediated transcription responsible for senescence. In contrast, expression of MYC (c-MYC) from a constitutive promoter abrogated senescence and strikingly, cooperated with OSM to promote a transformed phenotype, epithelial-mesenchymal transition (EMT), and invasiveness. Our findings suggest that a novel STAT3/SMAD3-signaling axis is required for OSM-mediated senescence that is coopted during the transformation process to confer aggressive cancer cell properties. Understanding how developing cancer cells bypass OSM/STAT3/SMAD3-mediated senescence may help identify novel targets for future “pro-senescence” therapies aiming to reengage this hidden tumor-suppressive response. PMID:27892764

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

PubMed

Zhao, Bryan M; Hoffmann, F Michael

2006-09-01

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

Impact of T-cell-specific Smad4 deficiency on the development of autoimmune diabetes in NOD mice

PubMed Central

Kim, Donghee; Lee, Song Mi; Jun, Hee-Sook

2017-01-01

Type 1 diabetes results from autoimmune-mediated pancreatic beta-cell destruction and transforming growth factor-beta (TGF-β) is known to play a preventive role in type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we investigated the role of Smad4, a key molecule for Smad-dependent TGF-β signaling, in T cells of NOD mice in the pathogenesis of autoimmune diabetes. We generated T-cell-specific Smad4 knockout (Smad4 tKO) NOD mice and assessed the pathological and immunological changes. Smad4 tKO showed earlier onset and increased incidence of diabetes than wild type (WT) NOD mice. Pathological features such as insulitis, anti-glutamic acid decarboxylase auto-antibody levels and serum IFN-γ levels were significantly increased in Smad4 tKO compared with WT NOD mice. Proportion and number of activated/memory CD4+ T cell were significantly increased in pancreatic lymph nodes of Smad4 tKO compared with WT NOD mice. However, the proportion and function of regulatory T cells was not different. Effector CD4+ T cells from Smad4 tKO were more resistant to suppression by regulatory T cells than effector cells from WT NOD mice. The proliferative potential of effector T cells from Smad4 tKO was significantly elevated compared with WT NOD mice, and activation of sterol regulatory element binding protein-1c (SREBP-1c) in T cells of Smad4 tKO NOD mice was correlated with this proliferative activity. We conclude that Smad4 deletion in T cells of NOD mice accelerated the development of autoimmune diabetes and increased the incidence of the disease by dysregulation of T cell activation at least in part via SREBP-1c activation. PMID:27686408

Impact of T-cell-specific Smad4 deficiency on the development of autoimmune diabetes in NOD mice.

PubMed

Kim, Donghee; Lee, Song Mi; Jun, Hee-Sook

2017-03-01

Type 1 diabetes results from autoimmune-mediated pancreatic beta-cell destruction and transforming growth factor-beta (TGF-β) is known to play a preventive role in type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we investigated the role of Smad4, a key molecule for Smad-dependent TGF-β signaling, in T cells of NOD mice in the pathogenesis of autoimmune diabetes. We generated T-cell-specific Smad4 knockout (Smad4 tKO) NOD mice and assessed the pathological and immunological changes. Smad4 tKO showed earlier onset and increased incidence of diabetes than wild type (WT) NOD mice. Pathological features such as insulitis, anti-glutamic acid decarboxylase auto-antibody levels and serum IFN-γ levels were significantly increased in Smad4 tKO compared with WT NOD mice. Proportion and number of activated/memory CD4 + T cell were significantly increased in pancreatic lymph nodes of Smad4 tKO compared with WT NOD mice. However, the proportion and function of regulatory T cells was not different. Effector CD4 + T cells from Smad4 tKO were more resistant to suppression by regulatory T cells than effector cells from WT NOD mice. The proliferative potential of effector T cells from Smad4 tKO was significantly elevated compared with WT NOD mice, and activation of sterol regulatory element binding protein-1c (SREBP-1c) in T cells of Smad4 tKO NOD mice was correlated with this proliferative activity. We conclude that Smad4 deletion in T cells of NOD mice accelerated the development of autoimmune diabetes and increased the incidence of the disease by dysregulation of T cell activation at least in part via SREBP-1c activation.

Zinc Finger Protein 451 Is a Novel Smad Corepressor in Transforming Growth Factor-β Signaling*

PubMed Central

Feng, Yili; Wu, Hongxing; Xu, Yongxian; Zhang, Zhengmao; Liu, Ting; Lin, Xia; Feng, Xin-Hua

2014-01-01

ZNF451 is a transcriptional cofactor localized to promyelocytic leukemia bodies. Here, we present evidence demonstrating that ZNF451 physically interacts with Smad3/4 and functionally inhibits TGF-β signaling. Increased expression of ZNF451 attenuates TGF-β-induced growth inhibitory and gene transcriptional responses, whereas depletion of ZNF451 enhances TGF-β responses. Mechanistically, ZNF451 blocks the ability of Smad3/4 to recruit p300 in response to TGF-β, which causes reduction of histone H3K9 acetylation on the promoters of TGF-β target genes. Taken together, ZNF451 acts as a transcriptional corepressor for Smad3/4 and negatively regulates TGF-β signaling. PMID:24324267

APC and Smad7 link TGFβ type I receptors to the microtubule system to promote cell migration

PubMed Central

Ekman, Maria; Mu, Yabing; Lee, So Young; Edlund, Sofia; Kozakai, Takaharu; Thakur, Noopur; Tran, Hoanh; Qian, Jiang; Groeden, Joanna; Heldin, Carl-Henrik; Landström, Maréne

2012-01-01

Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3β (GSK-3β). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-β (TGFβ) and is known to inhibit various TGFβ-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen–activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFβ stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3β kinases to facilitate local TGFβ/p38–dependent inactivation of GSK-3β, accumulation of β-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7–APC complex links the TGFβ type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFβ. PMID:22496417

SOCS3 promotes TLR4 response in macrophages by feedback inhibiting TGF-beta1/Smad3 signaling.

PubMed

Liu, Xia; Zhang, Yongliang; Yu, Yizhi; Yang, Xiao; Cao, Xuetao

2008-03-01

Endogenous transforming growth factor-beta1 (TGF-beta1) plays an important role in the negative regulation of toll-like receptor (TLR) signaling in a feedback manner. Suppressors of cytokine signaling 3 (SOCS3) has been shown to be induced by TGF-beta1 in osteoclast/macrophage, while the reports on the role of SOCS3 in regulating TLR4 signaling were controversial. The functional relationship between SOCS3 and TGF-beta1/Smad3 pathway in TLR4 response also remains unclear. In this study, we demonstrate that LPS-induced endogenous TGF-beta1 contributes to the inducible SOCS3 expression in macrophages. SOCS3 silencing could markedly decrease the LPS-induced production of TNF-alpha and IL-6 in macrophages. Interestingly, less decrease of LPS-induced TNF-alpha, IL-6 by SOCS3 silencing was observed in Smad3 null macrophages. Furthermore, we found SOCS3 could interact with Smad3, and inhibit Smad3 nuclear translocation and transcriptional activity. Therefore, our data demonstrate that SOCS3 is a positive regulator of TLR4 response by feedback inhibiting endogenous TGF-beta1/Smad3 signaling, thus outlining a new feedback regulatory manner for TLR4 response in macrophages.

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland

PubMed Central

2010-01-01

Introduction Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ)/Smad pathway as an example. Methods We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ. Results IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium. PMID:20942910

Sumoylation of Smad3 stimulates its nuclear export during PIASy-mediated suppression of TGF-{beta} signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imoto, Seiyu; Ohbayashi, Norihiko; Ikeda, Osamu

2008-05-30

Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-{beta} (TGF-{beta})-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-{beta} signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-{beta} signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-{beta}-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression ofmore » Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-{beta}/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.« less

SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells.

PubMed

Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

2016-10-25

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells

PubMed Central

Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H.; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

2016-01-01

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner. PMID:27655713

Src is a major signaling component for CTGF induction by TGF-β1 in osteoblasts

PubMed Central

X, Zhang; JA, Arnott; S, Rehman; WG, DeLong; A, Sanjay; FF, Safadi; SN, Popoff

2010-01-01

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta 1(TGF-β1) where it acts as a downstream mediator of TGF-β1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk and Smad signaling for CTGF induction by TGF-β1 in osteoblasts, however the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-β1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-β1. Additionally, inhibiting Src activation prevented Erk activation, Smad 2 & 3 activation and nuclear translocation by TGF-β1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway through directly mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059 it inhibited TGF-β1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) on the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. This data demonstrates that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-β1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. PMID:20432467

Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3

PubMed Central

Tang, Liu-Ya; Yamashita, Motozo; Coussens, Nathan P; Tang, Yi; Wang, Xiangchun; Li, Cuiling; Deng, Chu-Xia; Cheng, Steven Y; Zhang, Ying E

2011-01-01

TGF-β signalling is regulated by post-translational modifications of Smad proteins to translate quantitative difference in ligand concentration into proportional transcriptional output. Previous studies in cell culture systems suggested that Smad ubiquitination regulatory factors (Smurfs) act in this regulation by targeting Smads for proteasomal degradation, but whether this mechanism operates under physiological conditions is not clear. Here, we generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse embryonic fibroblasts and dermal fibroblasts, we show that TGF-β-mediated, Smad-dependent transcriptional responses are elevated in the absence of Smurf2. Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data support a model in which Smurf2 negatively regulates TGF-β signalling by attenuating the activity of Smad3 rather than promoting its degradation. PMID:22045334

Requirements of transcription factor Smad-dependent and -independent TGF-β signaling to control discrete T-cell functions.

PubMed

Gu, Ai-Di; Wang, Yunqi; Lin, Lin; Zhang, Song S; Wan, Yisong Y

2012-01-17

TGF-β modulates immune response by suppressing non-regulatory T (Treg) function and promoting Treg function. The question of whether TGF-β achieves distinct effects on non-Treg and Treg cells through discrete signaling pathways remains outstanding. In this study, we investigated the requirements of Smad-dependent and -independent TGF-β signaling for T-cell function. Smad2 and Smad3 double deficiency in T cells led to lethal inflammatory disorder in mice. Non-Treg cells were spontaneously activated and produced effector cytokines in vivo on deletion of both Smad2 and Smad3. In addition, TGF-β failed to suppress T helper differentiation efficiently and to promote induced Treg generation of non-Treg cells lacking both Smad2 and Smad3, suggesting that Smad-dependent signaling is obligatory to mediate TGF-β function in non-Treg cells. Unexpectedly, however, the development, homeostasis, and function of Treg cells remained intact in the absence of Smad2 and Smad3, suggesting that the Smad-independent pathway is important for Treg function. Indeed, Treg-specific deletion of TGF-β-activated kinase 1 led to failed Treg homeostasis and lethal immune disorder in mice. Therefore, Smad-dependent and -independent TGF-β signaling discretely controls non-Treg and Treg function to modulate immune tolerance and immune homeostasis.

Functional characteristics of a novel SMAD4 mutation from thoracic aortic aneurysms (TAA).

PubMed

Wu, Lifei

2017-09-10

SMAD4 is as an essential mediator of the transforming growth factor β (TGF-β) signaling pathway, and dysregulated TGF-β signaling is linked with thoracic aortic aneurysms (TAAs). In this study, we functionally characterized the Smad4 S271N mutation (the mutation c. 812G>A in Smad4 results in the amino acid substitution Ser271Asn) that was isolated from TAA individuals. We first constructed wild-type human Smad4 and Smad4 S271N plasmids. These constructs were then transiently transfected into HEK293T cells, and subsequent real-time PCR and western blotting demonstrated that wild-type Smad4 and Smad4 S271N were successfully expressed in 293T cells. We found that HEK293T cells overexpressing Smad4 S271N showed a strong increase in both cytoplasmic and nuclear Smad4 protein levels in response to TGF-β1. Although TGF-β signaling was the same in wild-type Smad4- and Smad4 S271N-transfected cells following TGF-β1 exposure, interestingly, we observed that transient Smad4 S271N expression in HEK293T cells caused a significant basal activation of TGF-β signaling. These results indicated that Smad4 may not directly induce TAA; rather it may contribute to TAA in combination with other risk factors. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.

PubMed

Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D

2014-01-01

Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquitination and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding assays in vitro. The activities of various wild-type and mutant LMP-1 proteins were evaluated using a BMP-responsive luciferase reporter and alkaline phosphatase assay in mouse myoblastic cells that were differentiated toward the osteoblastic phenotype. Finally, to strengthen physiological relevance of LMP-1 and Jab1 interaction, we showed that overexpression of LMP-1 caused nuclear accumulation of Smad4 upon BMP treatment which is reflective of increased Smad signaling in cells.

Differential regulation of Smad3 and of the type II transforming growth factor-β receptor in mitosis: implications for signaling.

PubMed

Hirschhorn, Tal; Barizilay, Lior; Smorodinsky, Nechama I; Ehrlich, Marcelo

2012-01-01

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis.

Differential Regulation of Smad3 and of the Type II Transforming Growth Factor-β Receptor in Mitosis: Implications for Signaling

PubMed Central

Hirschhorn, Tal; Barizilay, Lior; Smorodinsky, Nechama I.; Ehrlich, Marcelo

2012-01-01

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis. PMID:22927969

Cross-talk between Smad and p38 MAPK signalling in transforming growth factor {beta} signal transduction in human glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dziembowska, Magdalena; Danilkiewicz, Malgorzata; Wesolowska, Aleksandra

2007-03-23

Transforming growth factor-beta (TGF-{beta}) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-{beta} by growth inhibition, but retain responsiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-{beta}1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-{beta} receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2more » and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-{beta}1-induced signalling.« less

Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling.

PubMed

Guo, Xing; Ramirez, Alejandro; Waddell, David S; Li, Zhizhong; Liu, Xuedong; Wang, Xiao-Fan

2008-01-01

The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.

p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts.

PubMed

Furukawa, Fukiko; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yoshida, Katsunori; Sugano, Yasushi; Yamagata, Hideo; Matsushita, Masanori; Seki, Toshihito; Inagaki, Yutaka; Nishizawa, Mikio; Fujisawa, Junichi; Inoue, Kyoichi

2003-10-01

Hepatic stellate cells (HSCs) spontaneously transdifferentiate into myofibroblast (MFB)-phenotype on plastic dishes. This response recapitulates the features of activation in vivo. Transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by MFBs. In quiescent HSCs, TGF-beta signaling involves TGF-beta type I receptor (TbetaRI)-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. The middle linker regions of Smad2 and Smad3 also are phosphorylated by mitogen-activated protein kinase (MAPK). This study elucidates the change of Smad3-mediated signals during the transdifferentiation process. By using antibodies highly specific to the phosphorylated C-terminal region and the phosphorylated linker region of Smad3, we found that TGF-beta-dependent Smad3 phosphorylation at the C-terminal region decreased, but that the phosphorylation at the linker region increased in the process of transdifferentiation. TGF-beta activated the p38 MAPK pathway, further leading to Smad3 phosphorylation at the linker region in the cultured MFBs, irrespective of Smad2. The phosphorylation promoted hetero-complex formation and nuclear translocation of Smad3 and Smad4. Once combined with TbetaRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to plasminogen activator inhibitor-type I promoter could enhance the transcription. In addition, Smad3 phosphorylation mediated by the activated TbetaRI was impaired severely in MFBs during chronic liver injury, whereas Smad3 phosphorylation at the linker region was remarkably induced by p38 MAPK pathway. In conclusion, p38 MAPK-dependent Smad3 phosphorylation promoted extracellular matrix production in MFBs both in vitro and in vivo.

Phospho-control of TGF-β superfamily signaling

PubMed Central

Wrighton, Katharine H; Lin, Xia; Feng, Xin-Hua

2010-01-01

Members of the transforming growth factor-β (TGF-β) family control a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. Thus, aberrant TGF-β signaling can play a key role in the pathogenesis of several diseases, including cancer. TGF-β signaling pathways are activated by a short phospho-cascade, from receptor phosphorylation to the subsequent phosphorylation and activation of downstream signal transducers called R-Smads. R-Smad phosphorylation state determines Smad complex assembly/disassembly, nuclear import/export, transcriptional activity and stability, and is thus the most critical event in TGF-β signaling. Dephosphorylation of R-Smads by specific phosphatases prevents or terminates TGF-β signaling, highlighting the need to consider Smad (de)phosphorylation as a tightly controlled and dynamic event. This article illustrates the essential roles of reversible phosphorylation in controlling the strength and duration of TGF-β signaling and the ensuing physiological responses. PMID:19114991

Pin1 promotes transforming growth factor-beta-induced migration and invasion.

PubMed

Matsuura, Isao; Chiang, Keng-Nan; Lai, Chen-Yu; He, Dongming; Wang, Guannan; Ramkumar, Romila; Uchida, Takafumi; Ryo, Akihide; Lu, Kunping; Liu, Fang

2010-01-15

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation.

PubMed

Park, Sujin; Yang, Kyung-Min; Park, Yuna; Hong, Eunji; Hong, Chang Pyo; Park, Jinah; Pang, Kyoungwha; Lee, Jihee; Park, Bora; Lee, Siyoung; An, Haein; Kwak, Mi-Kyung; Kim, Junil; Kang, Jin Muk; Kim, Pyunggang; Xiao, Yang; Nie, Guangjun; Ooshima, Akira; Kim, Seong-Jin

2018-03-01

Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2 , SNAI1 , and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B , CTGF , and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B , CTGF , and JUNB genes in various cancers.

Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in the cpk mouse, a model of autosomal recessive polycystic kidney disease.

PubMed

Hama, Taketsugu; Nakanishi, Koichi; Sato, Masashi; Mukaiyama, Hironobu; Togawa, Hiroko; Shima, Yuko; Miyajima, Masayasu; Nozu, Kandai; Nagao, Shizuko; Takahashi, Hisahide; Sako, Mayumi; Iijima, Kazumoto; Yoshikawa, Norishige; Suzuki, Hiroyuki

2017-12-01

Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-β/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-β/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-β, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/ cpk double-mutant mice revealed amelioration of cpk abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-β/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention. Copyright © 2017 the American Physiological Society.

Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis.

PubMed

Halder, Sunil K; Beauchamp, R Daniel; Datta, Pran K

2005-07-01

Smad proteins play a key role in the intracellular signaling of the transforming growth factor beta (TGF-beta) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-beta family signaling and is upregulated in several cancers. To determine the effect of Smad7-mediated blockade of TGF-beta signaling, we have stably expressed Smad7 in a TGF-beta-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-beta-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-beta-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-beta and enhances TGF-beta-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-beta-induced growth inhibition by preventing TGF-beta-induced G1 arrest. Smad7 inhibits TGF-beta-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21(Cip1). As a result, Smad7 inhibits TGF-beta-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-beta-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumor-suppressive actions of TGF-beta that might result in increased tumorigenicity.

Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-β signalling pathway

PubMed Central

Zhang, Yan; Fan, Kai-Ji; Sun, Qiang; Chen, Ai-Zhong; Shen, Wen-Long; Zhao, Zhi-Hu; Zheng, Xiao-Fei; Yang, Xiao

2012-01-01

The transforming growth factor-β (TGF-β) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-β signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-β transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3′-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-β to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-β signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4. PMID:22821565

Myostatin induces insulin resistance via Casitas B-lineage lymphoma b (Cblb)-mediated degradation of insulin receptor substrate 1 (IRS1) protein in response to high calorie diet intake.

PubMed

Bonala, Sabeera; Lokireddy, Sudarsanareddy; McFarlane, Craig; Patnam, Sreekanth; Sharma, Mridula; Kambadur, Ravi

2014-03-14

To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat-mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner.

Heat shock transcription factor 1 protects against pressure overload-induced cardiac fibrosis via Smad3.

PubMed

Zhou, Ning; Ye, Yong; Wang, Xingxu; Ma, Ben; Wu, Jian; Li, Lei; Wang, Lin; Wang, Dao Wen; Zou, Yunzeng

2017-04-01

Fibrotic cardiac muscle exhibits high stiffness and low compliance which are major risk factors of heart failure. Although heat shock transcription factor 1 (HSF1) was identified as an intrinsic cardioprotective factor, the role that HSF1 plays in cardiac fibrosis remains unclear. Our study aims to investigate the role of HSF1 in pressure overload-induced cardiac fibrosis and the underlying mechanism. HSF1 phosphorylation was significantly downregulated in transverse aortic constriction (TAC)-treated mouse hearts and mechanically stretched cardiac fibroblasts (cFBs). HSF1 transgenic (TG) mice, HSF1 deficient heterozygote (KO) mice, and their wild-type littermates were subjected to sham or TAC surgery for 4 weeks. HSF1 overexpression significantly attenuated pressure overload-induced cardiac fibrosis and dysfunction. Conversely, HSF1 KO mice showed deteriorated fibrotic response and cardiac dysfunction upon TAC. Moreover, we uncovered that overexpression of HSF1 protected against fibrotic response of cFBs to pressure overload. Mechanistically, we observed that the phosphorylation and the nuclear distribution of the Smad family member 3 (Smad3) were significantly decreased in HSF1-overexpressing mouse hearts, while being greatly increased in HSF1 KO mouse hearts upon TAC, compared to the control hearts, respectively. Similar alteration of Smad3 phosphorylation and nuclear distribution were found in isolated mouse cardiac fibroblasts and mechanically stretched cFBs. Constitutively active Smad3 blocked the anti-fibrotic effect of HSF1 in cFBs. Furthermore, we found a direct binding of phosphorylated HSF1 and Smad3, which can be suppressed by mechanical stress. In conclusion, the present study demonstrated for the first time that HSF1 acts as a novel negative regulator of cardiac fibrosis by blocking Smad3 activation. HSF1 activity is decreased in fibrotic hearts. HSF1 overexpression attenuates pressure overload-induced cardiac fibrosis and dysfunction. Deficiency of HSF1 deteriorates fibrotic response and cardiac dysfunction upon TAC. HSF1 inhibits phosphorylation and nuclear distribution of Smad3 via direct binding to Smad3. Active Smad3 blocks the anti-fibrotic effect of HSF1.

The transforming activity of Ski and SnoN is dependent on their ability to repress the activity of Smad proteins.

PubMed

He, Jun; Tegen, Sarah B; Krawitz, Ariel R; Martin, G Steven; Luo, Kunxin

2003-08-15

The regulation of cell growth and differentiation by transforming growth factor-beta (TGF-beta) is mediated by the Smad proteins. In the nucleus, the Smad proteins are negatively regulated by two closely related nuclear proto-oncoproteins, Ski and SnoN. When overexpressed, Ski and SnoN induce oncogenic transformation of chicken embryo fibroblasts. However, the mechanism of transformation by Ski and SnoN has not been defined. We have previously reported that Ski and SnoN interact directly with Smad2, Smad3, and Smad4 and repress their ability to activate TGF-beta target genes through multiple mechanisms. Because Smad proteins are tumor suppressors, we hypothesized that the ability of Ski and SnoN to inactivate Smad function may be responsible for their transforming activity. Here, we show that the receptor regulated Smad proteins (Smad2 and Smad3) and common mediator Smad (Smad4) bind to different regions in Ski and SnoN. Mutation of both regions, but not each region alone, markedly impaired the ability of Ski and SnoN to repress TGF-beta-induced transcriptional activation and cell cycle arrest. Moreover, when expressed in chicken embryo fibroblasts, mutant Ski or SnoN defective in binding to the Smad proteins failed to induce oncogenic transformation. These results suggest that the ability of Ski and SnoN to repress the growth inhibitory function of the Smad proteins is required for their transforming activity. This may account for the resistance to TGF-beta-induced growth arrest in some human cancer cell lines that express high levels of Ski or SnoN.

Phosphorylated Smad2 and Smad3 signaling: Shifting between tumor suppression and fibro-carcinogenesis in chronic hepatitis C.

PubMed

Yamaguchi, Takashi; Matsuzaki, Koichi; Inokuchi, Ryosuke; Kawamura, Rinako; Yoshida, Katsunori; Murata, Miki; Fujisawa, Junichi; Fukushima, Nobuyoshi; Sata, Michio; Kage, Masayoshi; Nakashima, Osamu; Tamori, Akihiro; Kawada, Norifumi; Tsuneyama, Koichi; Dooley, Steven; Seki, Toshihito; Okazaki, Kazuichi

2013-12-01

Insight into hepatic fibrogenesis and carcinogenesis (fibro-carcinogenesis) caused by hepatitis C virus (HCV) infection has come from recent analyses of transforming growth factor (TGF)-β signaling. TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create C-terminally (C), linker (L) or dually (L/C) phosphorylated (p) isoforms. This study aimed to elucidate how HCV infection affected hepatic fibro-carcinogenesis, particularly via phospho-Smad signaling. We first studied phospho-Smad2/3 positivity of 100 patients in different stages of HCV-related chronic liver disease. To examine changes in phospho-Smad2/3 after HCV clearance, we analyzed 32 paired liver biopsy samples obtained before and after sustained virological response (SVR), dividing patients into two groups: 20 patients not developing hepatocellular carcinoma (HCC) after attaining SVR (non-HCC group), and 12 patients who developed HCC despite SVR (HCC group). Hepatocytic tumor-suppressive pSmad3C signaling shifted to carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling as liver diseases progressed. In the non-HCC group, 13 patients (65%) displayed fibrotic regression and inflammation reduction after SVR. Interestingly, SVR restored cytostatic pSmad3C signaling in hepatocytes, while eliminating prior carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling. In the HCC group, seven patients (58%) displayed unchanged or even progressed fibrosis despite smoothened inflammatory activity, reflecting persistently high numbers of hepatocytes with pSmad3L- and pSmad2L/C-signaling and low pSmad3C-signaling. HCV clearance limits fibrosis and reduces HCC incidence by switching inflammation-dependent phospho-Smad signaling from fibro-carcinogenesis to tumor suppression. However, progression to HCC would occur in severely fibrotic livers if an inflammation-independent fibro-carcinogenic process has already begun before HCV clearance. © 2013 The Japan Society of Hepatology.

3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

PubMed

Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

2007-04-20

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

Sox5 is a DNA binding co-factor for BMP R-Smads that directs target specificity during patterning of the early ectoderm

PubMed Central

Nordin, Kara; LaBonne, Carole

2014-01-01

SUMMARY The SoxD factor, Sox5, is expressed in ectodermal cells at times and places where BMP signaling is active, including the cells of the animal hemisphere at blastula stages, and the neural plate border (NPB) and neural crest (NC) at neurula stages. Sox5 is required for proper ectoderm development, and deficient embryos display patterning defects characteristic of perturbations of BMP signaling, including loss of neural crest and epidermis and expansion of the neural plate. We show that Sox5 is essential for activation of BMP target genes in embryos and explants, that it physically interacts with BMP R-Smads, and that it is essential for recruitment of Smad1/4 to BMP regulatory elements. Our findings identify Sox5 as the long sought DNA binding partner for BMP R-Smads essential to plasticity and pattern in the early ectoderm. PMID:25453832

Evolution history of duplicated smad3 genes in teleost: insights from Japanese flounder, Paralichthys olivaceus

PubMed Central

Du, Xinxin; Liu, Yuezhong; Liu, Jinxiang; Zhang, Quanqi

2016-01-01

Following the two rounds of whole-genome duplication (WGD) during deuterosome evolution, a third genome duplication occurred in the ray-fined fish lineage and is considered to be responsible for the teleost-specific lineage diversification and regulation mechanisms. As a receptor-regulated SMAD (R-SMAD), the function of SMAD3 was widely studied in mammals. However, limited information of its role or putative paralogs is available in ray-finned fishes. In this study, two SMAD3 paralogs were first identified in the transcriptome and genome of Japanese flounder (Paralichthys olivaceus). We also explored SMAD3 duplication in other selected species. Following identification, genomic structure, phylogenetic reconstruction, and synteny analyses performed by MrBayes and online bioinformatic tools confirmed that smad3a/3b most likely originated from the teleost-specific WGD. Additionally, selection pressure analysis and expression pattern of the two genes performed by PAML and quantitative real-time PCR (qRT-PCR) revealed evidence of subfunctionalization of the two SMAD3 paralogs in teleost. Our results indicate that two SMAD3 genes originate from teleost-specific WGD, remain transcriptionally active, and may have likely undergone subfunctionalization. This study provides novel insights to the evolution fates of smad3a/3b and draws attentions to future function analysis of SMAD3 gene family. PMID:27703851

Novel regulation of Smad3 oligomerization and DNA binding by its linker domain.

PubMed

Vasilaki, Eleftheria; Siderakis, Manos; Papakosta, Paraskevi; Skourti-Stathaki, Konstantina; Mavridou, Sofia; Kardassis, Dimitris

2009-09-08

Smad proteins are key effectors of the transforming growth factor beta (TGFbeta) signaling pathway in mammalian cells. Smads are composed of two highly structured and conserved domains called Mad homology 1 (MH1) and 2 (MH2), which are linked together by a nonconserved linker region. The recent identification of phosphorylation sites and binding sites for ubiquitin ligases in the linker regions of TGFbeta and bone morphogenetic protein (BMP) receptor-regulated Smads suggested that the linker may contribute to the regulation of Smad function by facilitating cross-talks with other signaling pathways. In the present study, we have generated and characterized novel Smad3 mutants bearing individual substitutions of conserved and nonconserved amino acid residues within a previously described transcriptionally active linker fragment. Our analysis showed that the conserved linker amino acids glutamine 222 and proline 229 play important roles in Smad functions such as homo- and hetero-oligomerization, nuclear accumulation in response to TGFbeta stimulation, and DNA binding. Furthermore, a Smad3 mutant bearing a substitution of the nonconserved amino acid asparagine 218 to alanine displayed enhanced transactivation potential relative to wild type Smad3. Finally, Smad3 P229A inhibited TGFbeta signaling when overexpressed in mammalian cells. In conclusion, our data are in line with previous studies supporting an important regulatory role of the linker region of Smads in their function as key transducers of TGFbeta signaling.

Inactivation of Smad5 in Endothelial Cells and Smooth Muscle Cells Demonstrates that Smad5 Is Required for Cardiac Homeostasis

PubMed Central

Umans, Lieve; Cox, Luk; Tjwa, Marc; Bito, Virginie; Vermeire, Liesbeth; Laperre, Kjell; Sipido, Karin; Moons, Lieve; Huylebroeck, Danny; Zwijsen, An

2007-01-01

Smads are intracellular signaling proteins that transduce signals elicited by members of the transforming growth factor (TGF)-β superfamily. Smad5 and Smad1 are highly homologous, and they mediate primarily bone morphogenetic protein (Bmp) signals. We used the Cre-loxP system and Sm22-Cre and Tie-1-Cre mice to study the function of Smad5 in the developing blood vessel wall. Analysis of embryos demonstrated that deletion of Smad5 in endothelial or smooth muscle cells resulted in a normal organization of embryonic and extra-embryonic vasculature. Angiogenic assays performed in adult mice revealed that mutant mice display a comparable angiogenic and vascular remodeling response to control mice. In Sm22-Cre;Smad5fl/− mice, Smad5 is also deleted in cardiomyocytes. Echocardiographic analysis on those 9-month-old female mice demonstrated larger left ventricle internal diameters and decreased fractional shortening compared with control littermates without signs of cardiac hypertrophy. The decreased cardiac contractility was associated with a decreased performance in a treadmill experiment. In isolated cardiomyocytes, fractional shortening was significantly reduced compared with control cells. These data demonstrate that restricted deletion of Smad5 in the blood vessel wall results in viable mice. However, loss of Smad5 in cardiomyocytes leads to a mild heart defect. PMID:17456754

Inactivation of Smad5 in endothelial cells and smooth muscle cells demonstrates that Smad5 is required for cardiac homeostasis.

PubMed

Umans, Lieve; Cox, Luk; Tjwa, Marc; Bito, Virginie; Vermeire, Liesbeth; Laperre, Kjell; Sipido, Karin; Moons, Lieve; Huylebroeck, Danny; Zwijsen, An

2007-05-01

Smads are intracellular signaling proteins that transduce signals elicited by members of the transforming growth factor (TGF)-beta superfamily. Smad5 and Smad1 are highly homologous, and they mediate primarily bone morphogenetic protein (Bmp) signals. We used the Cre-loxP system and Sm22-Cre and Tie-1-Cre mice to study the function of Smad5 in the developing blood vessel wall. Analysis of embryos demonstrated that deletion of Smad5 in endothelial or smooth muscle cells resulted in a normal organization of embryonic and extra-embryonic vasculature. Angiogenic assays performed in adult mice revealed that mutant mice display a comparable angiogenic and vascular remodeling response to control mice. In Sm22-Cre; Smad5(fl/-) mice, Smad5 is also deleted in cardiomyocytes. Echocardiographic analysis on those 9-month-old female mice demonstrated larger left ventricle internal diameters and decreased fractional shortening compared with control littermates without signs of cardiac hypertrophy. The decreased cardiac contractility was associated with a decreased performance in a treadmill experiment. In isolated cardiomyocytes, fractional shortening was significantly reduced compared with control cells. These data demonstrate that restricted deletion of Smad5 in the blood vessel wall results in viable mice. However, loss of Smad5 in cardiomyocytes leads to a mild heart defect.

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation

PubMed Central

Park, Sujin; Yang, Kyung-Min; Park, Yuna; Hong, Eunji; Hong, Chang Pyo; Park, Jinah; Pang, Kyoungwha; Lee, Jihee; Park, Bora; Lee, Siyoung; An, Haein; Kwak, Mi-Kyung; Kim, Junil; Kang, Jin Muk; Kim, Pyunggang; Xiao, Yang; Nie, Guangjun; Ooshima, Akira

2018-01-01

Background Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. Methods We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. Results In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. Conclusions These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers. PMID:29629343

Negative regulation of BMP signaling by the ski oncoprotein.

PubMed

Luo, Kunxin

2003-01-01

The bone morphogenetic proteins (BMPs) play important roles in the regulation of multiple aspects of vertebrate development. BMPs signal through the cell surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. The activity of this signal pathway can be modulated both by extracellular factors that regulate the binding of BMPs to the receptor and by intracellular proteins that interact with the Smad proteins. We have shown that Ski is an important negative regulator of the Smad proteins. Ski can bind to the BMP-Smad protein complexes in response to BMP and repress their ability to activate BMP target genes through disruption of a functional Smad complex and through recruitment of transcriptional co-repressors. The antagonism of BMP signaling by Ski results in neural specification in Xenopus embryos and inhibition of osteoblast differentiation in mouse bone-marrow stromal progenitor cells. This ability to modulate BMP signaling by Ski may play an important role in the regulation of craniofacial, neuronal, and skeletal muscle development.

Molecular interaction between Smurf1 WW2 domain and PPXY motifs of Smad1, Smad5, and Smad6--modeling and analysis.

PubMed

Sangadala, Sreedhara; Metpally, Raghu Prasad Rao; Reddy, Boojala Vijay B

2007-08-01

The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction. One of the important regulatory mechanisms by which the bone-inducing activity of the bone morphogenetic protein (BMP) signaling is modulated involves ubiquitin-mediated proteasomal degradation. The BMP induced receptor signal is transmitted intracellularly by phosphorylation of Smad proteins by the activated receptor I. The phosphorylated Smads 1, 5, and 8 (R-Smads) oligomerize with the co-Smad (Smad4). The complex, thus, formed translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. R-Smads contain PPXY motif in the linker region that interacts with Smad ubiquitin regulatory factor 1 (Smurf1), an E3 ubiquitin ligase that catalyzes ubiquitination of target proteins for proteasomal degradation. Smurf1 contains a HECT domain, a C2 domain, and 2 WW domains (WW1, WW2). The PPXY motif in target proteins and its interaction with Smurf1 may form the basis for regulation of steady-state levels of Smads in controlling BMP-responsiveness of cells. Here, we present a homology-based model of the Smurf1 WW2 domain and the target octa-peptides containing PPXY motif of Smurf1-interacting Smads. We carried out docking of Smurf1 WW2 domain with the PPXY motifs of Smad1, Smad5, and Smad6 and identified the key amino acid residues involved in interaction. Furthermore, we present experimental evidence that WW2 domain of Smurf1 does indeed interact with the Smad proteins and that the deletion of WW2 domain of Smurf1 results in loss of its binding to Smads using the purified recombinant proteins. Finally, we also present data confirming that the deletion of WW2 domain in Smurf1 abolishes its ubiquitination activity on Smad1 in an in vitro ubiquitination assay. It shows that the interaction between the WW domain and Smad PPXY motif is a key step in Smurf1-mediated ubiquitination of its natural targets such as Smad1, Smad5, and Smad6. This work facilitates further strategies to unravel the biological function of such interactions and help in designing effective mimetic compounds that either mimic or disrupt the specific interaction.

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

PubMed

Wu, Kongming; Yang, Ying; Wang, Chenguang; Davoli, Maria A; D'Amico, Mark; Li, Anping; Cveklova, Kveta; Kozmik, Zbynek; Lisanti, Michael P; Russell, Robert G; Cvekl, Ales; Pestell, Richard G

2003-12-19

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Smad ubiquitination regulatory factor-2 in the fibrotic kidney: regulation, target specificity, and functional implication.

PubMed

Tan, Ruoyun; He, Weichun; Lin, Xia; Kiss, Lawrence P; Liu, Youhua

2008-05-01

Smad ubiquitination regulatory factor-2 (Smurf2) is an E3 ubiqutin ligase that plays a pivotal role in regulating TGF-beta signaling via selectively targeting key components of the Smad pathway for degradation. In this study, we have investigated the regulation of Smurf2 expression, its target specificity, and the functional implication of its induction in the fibrotic kidney. Immunohistochemical staining revealed that Smurf2 was upregulated specifically in renal tubules of kidney biopsies from patients with various nephropathies. In vitro, Smurf2 mRNA and protein were induced in human proximal tubular epithelial cells (HKC-8) upon TGF-beta1 stimulation. Ectopic expression of Smurf2 was sufficient to reduce the steady-state levels of Smad2, but not Smad1, Smad3, Smad4, and Smad7, in HKC-8 cells. Interestingly, Smurf2 was also able to downregulate the Smad transcriptional corepressors Ski, SnoN, and TG-interacting factor. Inhibition of the proteasomal pathway prevented Smurf2-mediated downregulation of Smad2 and Smad corepressors. Functionally, overexpression of Smurf2 enhanced the transcription of the TGF-beta-responsive promoter and augmented TGF-beta1-mediated E-cadherin suppression, as well as fibronectin and type I collagen induction in HKC-8 cells. These results indicate that Smurf2 specifically targets both positive and negative Smad regulators for destruction in tubular epithelial cells, thereby providing a complex fine-tuning of TGF-beta signaling. It appears that dysregulation of Smurf2 could contribute to an aberrant TGF-beta/Smad signaling in the pathogenesis of kidney fibrosis.

Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan Xinjuan; Dai Yujie; Li Xing

2011-08-01

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3more » phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.« less

Ligand-activated PPARδ upregulates α-smooth muscle actin expression in human dermal fibroblasts: A potential role for PPARδ in wound healing.

PubMed

Ham, Sun Ah; Hwang, Jung Seok; Yoo, Taesik; Lee, Won Jin; Paek, Kyung Shin; Oh, Jae-Wook; Park, Chan-Kyu; Kim, Jin-Hoi; Do, Jung Tae; Kim, Jae-Hwan; Seo, Han Geuk

2015-12-01

The phenotypic changes that accompany differentiation of resident fibroblasts into myofibroblasts are important aspects of the wound healing process. Recent studies showed that peroxisome proliferator-activated receptor (PPAR) δ plays a critical role in wound healing. To determine whether the nuclear receptor PPARδ can modulate the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. These studies were undertaken in primary HDFs using Western blot analyses, small interfering (si)RNA-mediated gene silencing, reporter gene assays, chromatin immunoprecipitation (ChIP), migration assays, collagen gel contraction assays, and real-time PCR. Activation of PPARδ by GW501516, a specific ligand of PPARδ, specifically upregulated the myofibroblast marker α-smooth muscle actin (α-SMA) in a time- and concentration-dependent manner. This induction was significantly inhibited by the presence of siRNA against PPARδ, indicating that PPARδ is involved in myofibroblast transdifferentiation of HDFs. Ligand-activated PPARδ increased α-SMA promoter activity in a dual mode by directly binding a direct repeat-1 (DR1) site in the α-SMA promoter, and by inducing expression of transforming growth factor (TGF)-β, whose downstream effector Smad3 interacts with a Smad-binding element (SBE) in another region of the promoter. Mutations in these cis-elements totally abrogated transcriptional activation of the α-SMA gene by the PPARδ ligand; thus both sites represent novel types of PPARδ response elements. GW501516-activated PPARδ also increased the migration and contractile properties of HDFs, as demonstrated by Transwell and collagen lattice contraction assays, respectively. In addition, PPARδ-mediated upregulation of α-SMA was correlated with elevated expression of myofibroblast markers such as collagen I and fibronectin, with a concomitant reduction in expression of the epithelial marker E-cadherin. PPARδ plays pivotal roles in wound healing by promoting fibroblast-to-myofibroblast differentiation via TGF-β/Smad3 signaling. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Smad4 restricts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration.

PubMed

Paris, Nicole D; Soroka, Andrew; Klose, Alanna; Liu, Wenxuan; Chakkalakal, Joe V

2016-11-18

Skeletal muscle regenerative potential declines with age, in part due to deficiencies in resident stem cells (satellite cells, SCs) and derived myogenic progenitors (MPs); however, the factors responsible for this decline remain obscure. TGFβ superfamily signaling is an inhibitor of myogenic differentiation, with elevated activity in aged skeletal muscle. Surprisingly, we find reduced expression of Smad4 , the downstream cofactor for canonical TGFβ superfamily signaling, and the target Id1 in aged SCs and MPs during regeneration. Specific deletion of Smad4 in adult mouse SCs led to increased propensity for terminal myogenic commitment connected to impaired proliferative potential. Furthermore, SC-specific Smad4 disruption compromised adult skeletal muscle regeneration. Finally, loss of Smad4 in aged SCs did not promote aged skeletal muscle regeneration. Therefore, SC-specific reduction of Smad4 is a feature of aged regenerating skeletal muscle and Smad4 is a critical regulator of SC and MP amplification during skeletal muscle regeneration.

Iron regulation of hepcidin despite attenuated Smad1,5,8 signaling in mice without transferrin receptor 2 or Hfe

PubMed Central

Corradini, Elena; Rozier, Molly; Meynard, Delphine; Odhiambo, Adam; Lin, Herbert Y.; Feng, Qi; Migas, Mary C.; Britton, Robert S.; Babitt, Jodie L.; Fleming, Robert E.

2011-01-01

Background & Aims HFE and transferrin receptor 2 (TFR2) are each necessary for the normal relationship between body iron status and liver hepcidin expression. In murine Hfe and Tfr2 knockout models of hereditary hemochromatosis (HH), signal transduction to hepcidin via the bone morphogenetic protein 6 (Bmp6)/Smad1,5,8 pathway is attenuated. We examined the effect of dietary iron on regulation of hepcidin expression via the Bmp6/Smad1,5,8 pathway using mice with targeted disruption of Tfr2, Hfe, or both genes. Methods Hepatic iron concentrations and mRNA expression of Bmp6 and hepcidin were compared with wild-type mice in each of the HH models on standard or iron-loading diets. Liver phospho-Smad (P-Smad)1,5,8 and Id1 mRNA levels were measured as markers of Bmp/Smad signaling. Results While Bmp6 expression was increased, liver hepcidin and Id1 expression were decreased in each of the HH models compared with wild-type mice. Each of the HH models also demonstrated attenuated P-Smad1,5,8 levels relative to liver iron status. Mice with combined Hfe/Tfr2 disruption were most affected. Dietary iron loading increased hepcidin and Id1 expression in each of the HH models. Compared with wild-type mice, HH mice demonstrated attenuated (Hfe knockout) or no increases in P-Smad1,5,8 levels in response to dietary iron loading. Conclusions These observations demonstrate that Tfr2 and Hfe are each required for normal signaling of iron status to hepcidin via Bmp6/Smad1,5,8 pathway. Mice with combined loss of Hfe and Tfr2 up-regulate hepcidin in response to dietary iron loading without increases in liver BMP6 mRNA or steady-state P-Smad1,5,8 levels. PMID:21745449

SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy.

PubMed

Goldstein, Jeffery A; Kelly, Sean M; LoPresti, Peter P; Heydemann, Ahlke; Earley, Judy U; Ferguson, Edwin L; Wolf, Matthew J; McNally, Elizabeth M

2011-03-01

Loss-of-function mutations in the genes encoding dystrophin and the associated membrane proteins, the sarcoglycans, produce muscular dystrophy and cardiomyopathy. The dystrophin complex provides stability to the plasma membrane of striated muscle during muscle contraction. Increased SMAD signaling due to activation of the transforming growth factor-β (TGFβ) pathway has been described in muscular dystrophy; however, it is not known whether this canonical TGFβ signaling is pathogenic in the muscle itself. Drosophila deleted for the γ/δ-sarcoglycan gene (Sgcd) develop progressive muscle and heart dysfunction and serve as a model for the human disorder. We used dad-lacZ flies to demonstrate the signature of TGFβ activation in response to exercise-induced injury in Sgcd null flies, finding that those muscle nuclei immediately adjacent to muscle injury demonstrate high-level TGFβ signaling. To determine the pathogenic nature of this signaling, we found that partial reduction of the co-SMAD Medea, homologous to SMAD4, or the r-SMAD, Smox, corrected both heart and muscle dysfunction in Sgcd mutants. Reduction in the r-SMAD, MAD, restored muscle function but interestingly not heart function in Sgcd mutants, consistent with a role for activin but not bone morphogenic protein signaling in cardiac dysfunction. Mammalian sarcoglycan null muscle was also found to exhibit exercise-induced SMAD signaling. These data demonstrate that hyperactivation of SMAD signaling occurs in response to repetitive injury in muscle and heart. Reduction of this pathway is sufficient to restore cardiac and muscle function and is therefore a target for therapeutic reduction.

SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy

PubMed Central

Goldstein, Jeffery A.; Kelly, Sean M.; LoPresti, Peter P.; Heydemann, Ahlke; Earley, Judy U.; Ferguson, Edwin L.; Wolf, Matthew J.; McNally, Elizabeth M.

2011-01-01

Loss-of-function mutations in the genes encoding dystrophin and the associated membrane proteins, the sarcoglycans, produce muscular dystrophy and cardiomyopathy. The dystrophin complex provides stability to the plasma membrane of striated muscle during muscle contraction. Increased SMAD signaling due to activation of the transforming growth factor-β (TGFβ) pathway has been described in muscular dystrophy; however, it is not known whether this canonical TGFβ signaling is pathogenic in the muscle itself. Drosophila deleted for the γ/δ-sarcoglycan gene (Sgcd) develop progressive muscle and heart dysfunction and serve as a model for the human disorder. We used dad-lacZ flies to demonstrate the signature of TGFβ activation in response to exercise-induced injury in Sgcd null flies, finding that those muscle nuclei immediately adjacent to muscle injury demonstrate high-level TGFβ signaling. To determine the pathogenic nature of this signaling, we found that partial reduction of the co-SMAD Medea, homologous to SMAD4, or the r-SMAD, Smox, corrected both heart and muscle dysfunction in Sgcd mutants. Reduction in the r-SMAD, MAD, restored muscle function but interestingly not heart function in Sgcd mutants, consistent with a role for activin but not bone morphogenic protein signaling in cardiac dysfunction. Mammalian sarcoglycan null muscle was also found to exhibit exercise-induced SMAD signaling. These data demonstrate that hyperactivation of SMAD signaling occurs in response to repetitive injury in muscle and heart. Reduction of this pathway is sufficient to restore cardiac and muscle function and is therefore a target for therapeutic reduction. PMID:21138941

Cyclic AMP Enhances TGFβ Responses of Breast Cancer Cells by Upregulating TGFβ Receptor I Expression

PubMed Central

Oerlecke, Ilka; Bauer, Elke; Dittmer, Angela; Leyh, Benjamin; Dittmer, Jürgen

2013-01-01

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected. PMID:23349840

Molecular Interaction Between Smurfl WW2 Domain and PPXY Motifs of Smadl, Smad5, and Smad6-Modeling and Analysis.

PubMed

Sangadala, Sreedhara; Rao Metpally, Raghu Prasad; B Reddy, Boojala Vijay

2007-08-01

Abstract The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction. One of the important regulatory mechanisms by which the bone-inducing activity of the bone morphogenetic protein (BMP) signaling is modulated involves ubiquitin-mediated proteasomal degradation. The BMP induced receptor signal is transmitted intracellularly by phosphorylation of Smad proteins by the activated receptor I. The phosphorylated Smads 1, 5, and 8 (R-Smads) oligomerize with the co-Smad (Smad4). The complex, thus, formed translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. R-Smads contain PPXY motif in the linker region that interacts with Smad ubiquitin regulatory factor 1 (Smurf1), an E3 ubiquitin ligase that catalyzes ubiquitination of target proteins for proteasomal degradation. Smurf1 contains a HECT domain, a C2 domain, and 2 WW domains (WW1, WW2). The PPXY motif in target proteins and its interaction with Smurf1 may form the basis for regulation of steady-state levels of Smads in controlling BMP-responsiveness of cells. Here, we present a homology-based model of the Smurf1 WW2 domain and the target octa-peptides containing PPXY motif of Smurf1- interacting Smads. We carried out docking of Smurf1 WW2 domain with the PPXY motifs of Smadl, Smad5, and Smad6 and identified the key amino acid residues involved in interaction. Furthermore, we present experimental evidence that WW2 domain of Smurf1 does indeed interact with the Smad proteins and that the deletion of WW2 domain of Smurf1 results in loss of its binding to Smads using the purified recombinant proteins. Finally, we also present data confirming that the deletion of WW2 domain in Smurf1 abolishes its ubiquitination activity on Smad1 in an in vitro ubiquitination assay. It shows that the interaction between the WW domain and Smad PPXY motif is a key step in Smurf1-mediated ubiquitination of its natural targets such as Smad1, Smad5, and Smad6. This work facilitates further strategies to unravel the biological function of such interactions and help in designing effective mimetic compounds that either mimic or disrupt the specific interaction.

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

PubMed

Hayashida, Tomoko; Decaestecker, Mark; Schnaper, H William

2003-08-01

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.

Regulation of CCN2/CTGF Expression in the Nucleus Pulposus of the Intervertebral Disc: Role of Smad and AP1 Signaling

PubMed Central

Tran, Cassie M.; Markova, Dessislava; Smith, Harvey E.; Susarla, Bala; Ponnappan, Ravi Kumar; Anderson, D Greg; Symes, Aviva; Shapiro, Irving M.; Risbud, Makarand V.

2011-01-01

Objective To investigate TGFβ regulation of CTGF expression in cells of the nucleus pulposus. Methods Real Time RT-PCR and Western blot analysis was used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effect of Smad2/3/7 and AP1on TGFβ mediated CTGF promoter activity. Results CTGF expression was lower in the neonatal disc compared with the skeletally mature rat disc. An increase in CTGF expression and promoter activity was observed in nucleus pulposus cells after TGFβ treatment. Deletion analysis indicated that promoter constructs lacking smad and AP1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3-MH2 domain alone increased CTGF activity. Further evidence of Smad3 and AP1 involvement was seen when DN-Smad3, SiRNA-Smad3, smad7 and DN-AP1 suppressed TGFβ mediated activation of the CTGF promoter. When either Smad3 or AP1 sites were mutated, CTGF promoter induction by TGFβ was suppressed. We also observed a decrease in expression of CTGF in discs of Smad3 null mice compared to the wild type. Analysis of human nucleus pulposus indicated a trend of increasing CTGF and TGFβ expression in the degenerate state. Conclusion TGFβ, through Smad3 and AP1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerate disc. PMID:20222112

Valproic acid inhibits epithelial‑mesenchymal transition in renal cell carcinoma by decreasing SMAD4 expression.

PubMed

Mao, Shaowei; Lu, Guoliang; Lan, Xiaopeng; Yuan, Chuanwei; Jiang, Wei; Chen, Yougen; Jin, Xunbo; Xia, Qinghua

2017-11-01

Renal cell carcinoma (RCC) is the most common malignancy in urogenital neoplasms worldwide. According to previous studies, valproic acid (VPA), an anticonvulsant drug, can suppress tumor metastasis and decrease the expression level of Mothers against decapentaplegic homolog 4 (SMAD4) and therefore may inhibit epithelial‑mesenchymal transition (EMT), which is responsible for cancer metastasis. However, the association between VPA, EMT and SMAD4 in RCC metastasis remains obscure. In the present study, it was demonstrated that in the RCC cell lines 786‑O and Caki‑1 treated with VPA, the neural (N)‑cadherin, vimentin and SMAD4 protein and mRNA levels were decreased, accompanied with an increase in expression of epithelial (E)‑cadherin. Silencing SMAD4 expression decreased the expression of EMT markers, including N‑cadherin and simultaneously upregulated E‑cadherin in RCC cell lines. SMAD4 overexpression counteracted the VPA‑mediated EMT‑inhibitory effect (P<0.05). The present study demonstrates that VPA inhibited EMT in RCC cells via altering SMAD4 expression. In addition, immunohistochemical staining demonstrated that transforming growth factor‑β (TGF‑β) and low expression of SMAD4 was associated with a lower Fuhrman grade and low expression of transcription intermediary factor 1‑γ was associated with a higher tumor Fuhrman grade (P<0.05), Therefore, based on the regulatory effect of SMAD4 on EMT‑associated transcription factors, SMAD4 which can form a SMAD3/SMAD4 complex induced by TGF‑β, could be a potential anticancer drug target inhibiting tumor invasion and metastasis in RCC.

Anterior visceral endoderm SMAD4 signaling specifies anterior embryonic patterning and head induction in mice.

PubMed

Li, Cuiling; Li, Yi-Ping; Fu, Xin-Yuan; Deng, Chu-Xia

2010-09-27

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4(Co/Co);TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4(Co/Co);TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction.

Anterior Visceral Endoderm SMAD4 Signaling Specifies Anterior Embryonic Patterning and Head Induction in Mice

PubMed Central

Li, Cuiling; Li, Yi-Ping; Fu, Xin-Yuan; Deng, Chu-Xia

2010-01-01

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4Co/Co;TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4Co/Co;TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction. PMID:20941375

Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer

PubMed Central

Bardeesy, Nabeel; Cheng, Kuang-hung; Berger, Justin H.; Chu, Gerald C.; Pahler, Jessica; Olson, Peter; Hezel, Aram F.; Horner, James; Lauwers, Gregory Y.; Hanahan, Douglas; DePinho, Ronald A.

2006-01-01

SMAD4 is inactivated in the majority of pancreatic ductal adenocarcinomas (PDAC) with concurrent mutational inactivation of the INK4A/ARF tumor suppressor locus and activation of the KRAS oncogene. Here, using genetically engineered mice, we determined the impact of SMAD4 deficiency on the development of the pancreas and on the initiation and/or progression of PDAC—alone or in combination with PDAC-relevant mutations. Selective SMAD4 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRASG12D allele, SMAD4 deficiency enabled rapid progression of KRASG12D-initiated neoplasms. While KRASG12D alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to carcinoma, the combination of KRASG12D and SMAD4 deficiency resulted in the rapid development of tumors resembling intraductal papillary mucinous neoplasia (IPMN), a precursor to PDAC in humans. SMAD4 deficiency also accelerated PDAC development of KRASG12D INK4A/ARF heterozygous mice and altered the tumor phenotype; while tumors with intact SMAD4 frequently exhibited epithelial-to-mesenchymal transition (EMT), PDAC null for SMAD4 retained a differentiated histopathology with increased expression of epithelial markers. SMAD4 status in PDAC cell lines was associated with differential responses to transforming growth factor-β (TGF-β) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-β-induced proliferation and migration. These results provide genetic confirmation that SMAD4 is a PDAC tumor suppressor, functioning to block the progression of KRASG12D-initiated neoplasms, whereas in a subset of advanced tumors, intact SMAD4 facilitates EMT and TGF-β-dependent growth. PMID:17114584

Protective role of Smad6 in inflammation-induced valvular cell calcification

PubMed Central

Li, Xin; Lim, Jina J.; Lu, Jinxiu; Pedego, Taylor M.; Demer, Linda; Tintut, Yin

2016-01-01

Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I-Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I-Smads regulate inflammation-induced calcific vasculopathy is not clear. Therefore, we investigated the role of I-Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF-a) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle alpha actin, and negative for an endothelial cell marker. TNF-a upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF-a significantly upregulated bone morphogenetic protein 2 (BMP-2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF-a-induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF-a further augmented ALP activity, expression of BMP-2, Wnt- and redox-regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF-a induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP-2 signaling inhibitor, Smad6. PMID:25864564

Protective Role of Smad6 in Inflammation-Induced Valvular Cell Calcification.

PubMed

Li, Xin; Lim, Jina; Lu, Jinxiu; Pedego, Taylor M; Demer, Linda; Tintut, Yin

2015-10-01

Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I-Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I-Smads regulate inflammation-induced calcific vasculopathy is not clear. Therefore, we investigated the role of I-Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF-α) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle α actin, and negative for an endothelial cell marker. TNF-α upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF-α significantly upregulated bone morphogenetic protein 2 (BMP-2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF-α-induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF-α further augmented ALP activity, expression of BMP-2, Wnt- and redox-regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF-α induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP-2 signaling inhibitor, Smad6. © 2015 Wiley Periodicals, Inc.

Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.

PubMed

Stroschein, S L; Wang, W; Zhou, S; Zhou, Q; Luo, K

1999-10-22

Smad proteins mediate transforming growth factor-beta (TGF-beta) signaling to regulate cell growth and differentiation. The SnoN oncoprotein was found to interact with Smad2 and Smad4 and to repress their abilities to activate transcription through recruitment of the transcriptional corepressor N-CoR. Immediately after TGF-beta stimulation, SnoN is rapidly degraded by the nuclear accumulation of Smad3, allowing the activation of TGF-beta target genes. By 2 hours, TGF-beta induces a marked increase in SnoN expression, resulting in termination of Smad-mediated transactivation. Thus, SnoN maintains the repressed state of TGF-beta-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-beta signaling.

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease.

PubMed

Turner, Adam W; Martinuk, Amy; Silva, Anada; Lau, Paulina; Nikpay, Majid; Eriksson, Per; Folkersen, Lasse; Perisic, Ljubica; Hedin, Ulf; Soubeyrand, Sebastien; McPherson, Ruth

2016-05-01

A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-β pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease-associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D', 0.97; r(2), 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. The coronary artery disease-associated rs17293632C>T single nucleotide polymorphism represents a novel functional cis-acting element at the SMAD3 locus. The protective (T) allele of rs17293632 disrupts a consensus AP-1 binding site in a SMAD3 intron 1 enhancer, reduces enhancer activity and SMAD3 expression, altering human arterial smooth muscle cell proliferation. © 2016 American Heart Association, Inc.

Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-beta signaling.

PubMed

Rodriguez, C; Huang, L J; Son, J K; McKee, A; Xiao, Z; Lodish, H F

2001-08-10

Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-beta (TGF-beta) signaling pathway. We infected a TGF-beta-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-beta. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-beta-induced promoter (p15), it up-regulates a TGF-beta-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-beta. Our results suggest that BF-1 is a general inhibitor of TGF-beta signaling and as such may play a key role during brain development.

Dynamic Regulation of Tgf-B Signaling by Tif1γ: A Computational Approach

PubMed Central

Andrieux, Geoffroy; Fattet, Laurent; Le Borgne, Michel; Rimokh, Ruth; Théret, Nathalie

2012-01-01

TIF1γ (Transcriptional Intermediary Factor 1 γ) has been implicated in Smad-dependent signaling by Transforming Growth Factor beta (TGF-β). Paradoxically, TIF1γ functions both as a transcriptional repressor or as an alternative transcription factor that promotes TGF-β signaling. Using ordinary differential-equation models, we have investigated the effect of TIF1γ on the dynamics of TGF-β signaling. An integrative model that includes the formation of transient TIF1γ-Smad2-Smad4 ternary complexes is the only one that can account for TGF-β signaling compatible with the different observations reported for TIF1γ. In addition, our model predicts that varying TIF1γ/Smad4 ratios play a critical role in the modulation of the transcriptional signal induced by TGF-β, especially for short stimulation times that mediate higher threshold responses. Chromatin immunoprecipitation analyses and quantification of the expression of TGF-β target genes as a function TIF1γ/Smad4 ratios fully validate this hypothesis. Our integrative model, which successfully unifies the seemingly opposite roles of TIF1γ, also reveals how changing TIF1γ/Smad4 ratios affect the cellular response to stimulation by TGF-β, accounting for a highly graded determination of cell fate. PMID:22461896

PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3.

PubMed

Remy, Ingrid; Montmarquette, Annie; Michnick, Stephen W

2004-04-01

Transforming growth factor beta (TGF-beta) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-beta pathway with other signalling cascades that control the same cellular processes may modulate TGF-beta responses. Here we report the discovery of a new functional link between TGF-beta and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-beta stimulation, placing PKB-Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-beta-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-beta-mediated pathways.

Disruption of Smad4 Expression in T Cells Leads to IgA Nephropathy-Like Manifestations

PubMed Central

Yamashita, Michifumi; Choi, Sung Hee; Tomino, Yasuhiko; Letterio, John J.; Emancipator, Steven N.

2013-01-01

The link between glomerular IgA nephropathy (IgAN) and T helper 2 (Th2) response has been implicated, however, the mechanisms are poorly defined because of the lack of an appropriate model. Here we report a novel murine model characterized by lineage-restricted deletion of the gene encoding MAD homologue 4 (Smad4) in T cells (Smad4co/co;Lck-cre). Loss of Smad4 expression in T cells results in overproduction of Th2 cytokines and high serum IgA levels. We found that Smad4co/co;Lck-cre mice exhibited massive glomerular IgA deposition, increased albumin creatinine ratio, aberrant glycosylated IgA, IgA complexed with IgG1 and IgG2a, and polymeric IgA, all known features of IgAN in humans. Furthermore, we examined the β1, 4-galactosyltransferases (β4GalT) enzyme which is involved in the synthesis of glycosylated murine IgA, and we found reduced β4GalT2 and β4GalT4 mRNA levels in B cells. These findings indicate that Smad4co/co;Lck-cre mice could be a useful model for studying the mechanisms between IgAN and Th2 response, and further, disruption of Smad4-dependent signaling in T cells may play an important role in the pathogenesis of human IgAN and contributing to a Th2 T cell phenotype. PMID:24223846

SMAD regulatory networks construct a balanced immune system.

PubMed

Malhotra, Nidhi; Kang, Joonsoo

2013-05-01

A balanced immune response requires combating infectious assaults while striving to maintain quiescence towards the self. One of the central players in this process is the pleiotropic cytokine transforming growth factor-β (TGF-β), whose deficiency results in spontaneous systemic autoimmunity in mice. The dominant function of TGF-β is to regulate the peripheral immune homeostasis, particularly in the microbe-rich and antigen-rich environment of the gut. To maintain intestinal integrity, the epithelial cells, myeloid cells and lymphocytes that inhabit the gut secrete TGF-β, which acts in both paracrine and autocrine fashions to activate its signal transducers, the SMAD transcription factors. The SMAD pathway regulates the production of IgA by B cells, maintains the protective mucosal barrier and promotes the balanced differentiation of CD4(+) T cells into inflammatory T helper type 17 cells and suppressive FOXP3(+) T regulatory cells. While encounters with pathogenic microbes activate SMAD proteins to evoke a protective inflammatory immune response, SMAD activation and synergism with immunoregulatory factors such as the vitamin A metabolite retinoic acid enforce immunosuppression toward commensal microbes and innocuous food antigens. Such complementary context-dependent functions of TGF-β are achieved by the co-operation of SMAD proteins with distinct dominant transcription activators and accessory chromatin modifiers. This review highlights recent advances in unravelling the molecular basis for the multi-faceted functions of TGF-β in the gut that are dictacted by fluid orchestrations of SMADs and their myriad partners. © 2013 Blackwell Publishing Ltd.

The methyltransferase SET9 regulates TGFB1 activation of renal fibroblasts via interaction with SMAD3.

PubMed

Shuttleworth, Victoria G; Gaughan, Luke; Nawafa, Lotfia; Mooney, Caitlin A; Cobb, Steven L; Sheerin, Neil S; Logan, Ian R

2018-01-08

Chronic kidney disease (CKD) is a global socioeconomic problem. It is characterised by the presence of differentiated myofibroblasts, which cause tissue fibrosis in response to TGFB1, leading to renal failure. Here, we define a novel interaction between the SET9 lysine methyltransferase (also known as SETD7) and SMAD3, the principal mediator of TGFB1 signalling in myofibroblasts. We show that SET9-deficient fibroblasts exhibit globally altered gene expression profiles in response to TGFB1, whilst overexpression of SET9 enhances SMAD3 transcriptional activity. We also show that SET9 facilitates nuclear import of SMAD3 and controls SMAD3 protein degradation via ubiquitylation. On a cellular level, we demonstrate that SET9 is broadly required for the effects of TGFB1 in diseased primary renal fibroblasts; SET9 promotes fibroblast migration into wounds, expression of extracellular matrix proteins, collagen contractility and myofibroblast differentiation. Finally, we demonstrate that SET9 is recruited to the α-smooth muscle actin gene in response to TGFB1, providing a mechanism by which SET9 regulates myofibroblast contractility and differentiation. Together with previous studies, we make the case for SET9 inhibition in the treatment of progressive CKD. © 2018. Published by The Company of Biologists Ltd.

Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways

PubMed Central

2011-01-01

Background Bone morphogenetic protein (BMP)7 evokes both inductive and axon orienting responses in dorsal interneurons (dI neurons) in the developing spinal cord. These events occur sequentially during the development of spinal neurons but in these and other cell types such inductive and acute chemotactic responses occur concurrently, highlighting the requirement for divergent intracellular signaling. Both type I and type II BMP receptor subtypes have been implicated selectively in orienting responses but it remains unclear how, in a given cell, divergence occurs. We have examined the mechanisms by which disparate BMP7 activities are generated in dorsal spinal neurons. Results We show that widely different threshold concentrations of BMP7 are required to elicit the divergent inductive and axon orienting responses. Type I BMP receptor kinase activity is required for activation of pSmad signaling and induction of dI character by BMP7, a high threshold response. In contrast, neither type I BMP receptor kinase activity nor Smad1/5/8 phosphorylation is involved in the low threshold orienting responses of dI axons to BMP7. Instead, BMP7-evoked axonal repulsion and growth cone collapse are dependent on phosphoinositide-3-kinase (PI3K) activation, plausibly through type II receptor signaling. BMP7 stimulates PI3K-dependent signaling in dI neurons. BMP6, which evokes neural induction but does not have orienting activity, activates Smad signaling but does not stimulate PI3K. Conclusions Divergent signaling through pSmad-dependent and PI3K-dependent (Smad-independent) mechanisms mediates the inductive and orienting responses of dI neurons to BMP7. A model is proposed whereby selective engagement of BMP receptor subunits underlies choice of signaling pathway. PMID:22085733

High SMAD7 and p-SMAD2,3 expression is associated with environmental enteropathy in children.

PubMed

Syed, Sana; Dinallo, Vincenzo; Iqbal, Najeeha T; Di Iorio, Laura; Di Fusco, Davide; Guleria, Shan; Amadi, Beatrice C; Sadiq, Kamran; Moskaluk, Christopher; Ali, S Asad; Kelly, Paul; Monteleone, Giovanni

2018-02-01

Enteropathies such as Crohn's disease are associated with enteric inflammation characterized by impaired TGF-β signaling, decreased expression of phosphorylated (p)-SMAD2,3 and increased expression of SMAD7 (an inhibitor of SMAD3 phosphorylation). Environmental enteropathy (EE) is an acquired inflammatory disease of the small intestine (SI), which is associated with linear growth disruption, cognitive deficits, and reduced oral vaccine responsiveness in children <5 y in resource-poor countries. We aimed to characterize EE inflammatory pathways by determining SMAD7 and p-SMAD2,3 levels (using Western blotting) in EE duodenal biopsies (N = 19 children, 7 from Pakistan, 12 from Zambia) and comparing these with healthy controls (Ctl) and celiac disease (CD) patients from Italy. Densitometric analysis of immunoblots showed that EE SI biopsies expressed higher levels of both SMAD7 (mean±SD in arbitrary units [a.u.], Ctl = 0.47±0.20 a.u., EE = 1.13±0.25 a.u., p-value = 0.03) and p-SMAD2,3 (mean±SD, Ctl = 0.38±0.14 a.u., EE = 0.60±0.10 a.u., p-value = 0.03). Immunohistochemistry showed that, in EE, SMAD7 is expressed in both the epithelium and in mononuclear cells of the lamina propria (LP). In contrast, p-SMAD3 in EE is expressed much more prominently in epithelial cells than in the LP. The high SMAD7 immunoreactivity and lack of p-SMAD3 expression in the LP suggests defective TGF-β signaling in the LP in EE similar to a previously reported SMAD7-mediated inflammatory pathway in refractory CD and Crohn's disease. However, Western blot densitometry showed elevated p-SMAD2,3 levels in EE, possibly suggesting a different inflammatory pathway than Crohn's disease but more likely reflecting cumulative protein expression from across all compartments of the mucosa as opposed to the LP alone. Further studies are needed to substantiate these preliminary results and to illustrate the relationship between SMAD proteins, TGF-β signaling, and inflammatory cytokine production, all of which may be potential therapeutic targets.

SMAD4 Loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells

PubMed Central

2014-01-01

Background SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized. Methods The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down. Results Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment. Conclusions This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects. PMID:24625091

SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.

PubMed

Chen, Yu-Wen; Hsiao, Pi-Jung; Weng, Ching-Chieh; Kuo, Kung-Kai; Kuo, Tzu-Lei; Wu, Deng-Chyang; Hung, Wen-Chun; Cheng, Kuang-Hung

2014-03-14

SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized. The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down. Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment. This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation.

PubMed

Suh, Ji Ho; Huang, Jiansheng; Park, Yun-Yong; Seong, Hyun-A; Kim, Dongwook; Shong, Minho; Ha, Hyunjung; Lee, In-Kyu; Lee, Keesook; Wang, Li; Choi, Hueng-Sik

2006-12-22

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

Differential Phosphorylation of Smad1 Integrates BMP and Neurotrophin Pathways through Erk/Dusp in Axon Development

PubMed Central

Finelli, Mattéa J.; Murphy, Kevin J.; Chen, Lei; Zou, Hongyan

2013-01-01

SUMMARY Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2—two key neurotrophin effectors. Specifically, BMPs signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2, and constituting a negative feedback loop to prevent axon overgrowth. Together, BMP and neurotrophin pathways are integrated in a tightly regulated signaling network with balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. PMID:23665221

Ski represses BMP signaling in Xenopus and mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

kluo@lbl.gov

2001-05-16

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells bymore » directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-{beta} family members.« less

The role of TGF-β/SMAD4 signaling in cancer.

PubMed

Zhao, Ming; Mishra, Lopa; Deng, Chu-Xia

2018-01-01

Transforming growth factor β (TGF-β) signaling pathway plays important roles in many biological processes, including cell growth, differentiation, apoptosis, migration, as well as cancer initiation and progression. SMAD4, which serves as the central mediator of TGF-β signaling, is specifically inactivated in over half of pancreatic duct adenocarcinoma, and varying degrees in many other types of cancers. In the past two decades, multiple studies have revealed that SMAD4 loss on its own does not initiate tumor formation, but can promote tumor progression initiated by other genes, such as KRAS activation in pancreatic duct adenocarcinoma and APC inactivation in colorectal cancer. In other cases, such as skin cancer, loss of SMAD4 plays an important initiating role by disrupting DNA damage response and repair mechanisms and enhance genomic instability, suggesting its distinct roles in different types of tumors. This review lists SMAD4 mutations in various types of cancer and summarizes recent advances on SMAD4 with focuses on the function, signaling pathway, and the possibility of SMAD4 as a prognostic indicator.

Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells

PubMed Central

Wang, Wei; Mariani, Francesca V.; Harland, Richard M.; Luo, Kunxin

2000-01-01

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-β family members. PMID:11121043

Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells.

PubMed

Wang, W; Mariani, F V; Harland, R M; Luo, K

2000-12-19

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-beta family members.

Smad4 restricts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration

PubMed Central

Paris, Nicole D; Soroka, Andrew; Klose, Alanna; Liu, Wenxuan; Chakkalakal, Joe V

2016-01-01

Skeletal muscle regenerative potential declines with age, in part due to deficiencies in resident stem cells (satellite cells, SCs) and derived myogenic progenitors (MPs); however, the factors responsible for this decline remain obscure. TGFβ superfamily signaling is an inhibitor of myogenic differentiation, with elevated activity in aged skeletal muscle. Surprisingly, we find reduced expression of Smad4, the downstream cofactor for canonical TGFβ superfamily signaling, and the target Id1 in aged SCs and MPs during regeneration. Specific deletion of Smad4 in adult mouse SCs led to increased propensity for terminal myogenic commitment connected to impaired proliferative potential. Furthermore, SC-specific Smad4 disruption compromised adult skeletal muscle regeneration. Finally, loss of Smad4 in aged SCs did not promote aged skeletal muscle regeneration. Therefore, SC-specific reduction of Smad4 is a feature of aged regenerating skeletal muscle and Smad4 is a critical regulator of SC and MP amplification during skeletal muscle regeneration. DOI: http://dx.doi.org/10.7554/eLife.19484.001 PMID:27855784

The role of TGF-β/SMAD4 signaling in cancer

PubMed Central

Zhao, Ming; Mishra, Lopa; Deng, Chu-Xia

2018-01-01

Transforming growth factor β (TGF-β) signaling pathway plays important roles in many biological processes, including cell growth, differentiation, apoptosis, migration, as well as cancer initiation and progression. SMAD4, which serves as the central mediator of TGF-β signaling, is specifically inactivated in over half of pancreatic duct adenocarcinoma, and varying degrees in many other types of cancers. In the past two decades, multiple studies have revealed that SMAD4 loss on its own does not initiate tumor formation, but can promote tumor progression initiated by other genes, such as KRAS activation in pancreatic duct adenocarcinoma and APC inactivation in colorectal cancer. In other cases, such as skin cancer, loss of SMAD4 plays an important initiating role by disrupting DNA damage response and repair mechanisms and enhance genomic instability, suggesting its distinct roles in different types of tumors. This review lists SMAD4 mutations in various types of cancer and summarizes recent advances on SMAD4 with focuses on the function, signaling pathway, and the possibility of SMAD4 as a prognostic indicator. PMID:29483830

Smad7 enables STAT3 activation and promotes pluripotency independent of TGF-β signaling

PubMed Central

Yu, Yi; Gu, Shuchen; Li, Wenjian; Sun, Chuang; Chen, Fenfang; Xiao, Mu; Wang, Lei; Xu, Dewei; Li, Ye; Ding, Chen; Xia, Zongping; Li, Yi; Ye, Sheng; Xu, Pinglong; Zhao, Bin; Qin, Jun; Chen, Ye-Guang; Lin, Xia; Feng, Xin-Hua

2017-01-01

Smad7 is a negative feedback product of TGF-β superfamily signaling and fine tunes a plethora of pleiotropic responses induced by TGF-β ligands. However, its noncanonical functions independent of TGF-β signaling remain to be elucidated. Here, we show that Smad7 activates signal transducers and activators of transcription 3 (STAT3) signaling in maintaining mouse embryonic stem cell pluripotency in a manner independent of the TGF-β receptors, yet dependent on the leukemia inhibitory factor (LIF) coreceptor glycoprotein 130 (gp130). Smad7 directly binds to the intracellular domain of gp130 and disrupts the SHP2–gp130 or SOCS3–gp130 complex, thereby amplifying STAT3 activation. Consequently, Smad7 facilitates LIF-mediated self-renewal of mouse ESCs and is also critical for induced pluripotent stem cell reprogramming. This finding illustrates an uncovered role of the Smad7–STAT3 interplay in maintaining cell pluripotency and also implicates a mechanism involving Smad7 underlying cytokine-dependent regulation of cancer and inflammation. PMID:28874583

miR-26b-5p regulates hypoxia-induced phenotypic switching of vascular smooth muscle cells via the TGF-β/Smad4 signaling pathway.

PubMed

Ruan, Changwu; Lu, Jide; Wang, Hairong; Ge, Zhiru; Zhang, Chenjun; Xu, Maochun

2017-06-01

Hypoxia contributes to the phenotypic switch of vascular smooth muscle cells (VSMCs). Various microRNAs (miRNAs) participate in this process as post‑transcriptional regulators, however the mechanism remains unclear. In the present study, mouse VSMCs (mVSMCs) harvested from aortas were cultured in normoxic and hypoxic conditions, and the mRNA levels of miR-26b-5p, desmin, H‑caldesmon and smoothelin were quantified using reverse transcription‑quantitative polymerase chain reaction. Following treatment with a miR‑26b‑5p antagonist (agomir) or non‑targeting control (scramble), the cell areas of normoxic and hypoxic mVSMCs were analyzed by immunofluorescence staining. In addition, the protein expression levels of collagen Iα, Smad2/phosphorylated (p)‑Smad2, Smad3/p‑Smad3 and Smad4 were determined by western blotting. Potential miRNA26b‑5p binding sequences in the 3'‑untranslated region (UTR) of Smad4 were investigated, and the distribution of Smad4 in mVSMCs was visualized using immunofluorescence methods. Hypoxic mVSMCs exhibited a significant downregulation miR‑26b‑5p, upregulation of hypoxia inducible factor‑1α mRNA and suppression of desmin, H‑caldesmon and smoothelin mRNA levels. Additionally, miR‑26b‑5p agomir reduced the cell area and decreased collagen Iα expression levels in hypoxic mVSMCs compared with normoxic mVSMCs transfected with agomir, and the area was comparable with those of normoxic mVSMCs transfected with agomir or scramble. Furthermore, miR‑26b‑5p suppressed Smad4 expression in hypoxic mVSMCs, but did not change the expression levels of Smad2 and Smad3, p‑Smad2 and p‑Smad3, however p‑Smad2 and p‑Smad3 levels were upregulated in response to hypoxic stimuli. Additionally, the miR‑26b‑5p agomir caused weak immunoreactivity with Smad4 in hypoxic mVSMCs. The binding motif of miR‑26b‑5p in the Smad4 3'‑UTR was identified as UACUUGA at position 978-984. These findings suggest that miR‑26b‑5p regulates hypoxia‑induced phenotypic switching of VSMCs via the transforming growth factor β/Smad4 signaling pathway.

Association between Altered Expression and Genetic Variations of Transforming Growth Factor β-Smad Pathway with Chronic Myeloid Leukemia.

PubMed

Shokeen, Yogender; Sharma, Neeta Raj; Vats, Abhishek; Dinand, Veronique; Beg, Mirza Adil; Sanskaran, Satish; Minhas, Sachin; Jauhri, Mayank; Hariharan, Arun K; Taneja, Vibha; Aggarwal, Shyam

2018-01-01

Background: Chronic myeloid leukemia (CML) is a hematological disorder caused by fusion of BCR and ABL genes. BCR-ABL dependent and independent pathways play equally important role in CML. TGFβ-Smad pathway, an important BCR -ABL independent pathway, has scarce data in CML. Present study investigate the association between TGFβ-Smad pathway and CML. Materials and Methods: Sixty-four CML patients and age matched healthy controls (n=63) were enrolled in this study. Patients were segregated into responder and resistant groups depending on their response to Imatinib mesylate (IM). TGFβ1 serum levels were evaluated by ELISA and transcript levels of TGFβ1 receptors, SMAD4 and SMAD7 were evaluated by Real-Time PCR. Sequencing of exons and exon-intron boundaries of study genes was performed using Next Generation Sequencing (NGS) in 20 CML patients. Statistical analysis was performed using SPSS version 16.0. Results: TGFβ1 serum levels were significantly elevated ( p = 0.02) and TGFβR2 and SMAD4 were significantly down-regulated ( p = 0.012 and p = 0.043 respectively) in the patients. c.69A>G in TGFβ1 , c.1024+24G>A in TGFβR1 and g.46474746C>T in SMAD7 were the most important genetic variants observed with their presence in 10/20, 8/20 and 7/20 patients respectively. In addition, TGFβR1 transcript levels were reduced in CML patients with c.69A>G mutation. None of the genes differed significantly in terms of expression or genetic variants between responder and resistant patient groups. Conclusion: Our findings demonstrate the role of differential expression and genetic variants of TGFβ-Smad pathway in CML. Decreased TGFβR2 and SMAD4 levels observed in the present study may be responsible for reduced tumor suppressive effects of this pathway in CML.

Transforming growth factor-β1 induces expression of human coagulation factor XII via Smad3 and JNK signaling pathways in human lung fibroblasts.

PubMed

Jablonska, Ewa; Markart, Philipp; Zakrzewicz, Dariusz; Preissner, Klaus T; Wygrecka, Malgorzata

2010-04-09

Coagulation factor XII (FXII) is a liver-derived serine protease involved in fibrinolysis, coagulation, and inflammation. The regulation of FXII expression is largely unknown. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that has been linked to several pathological processes, including tissue fibrosis by modulating procoagulant and fibrinolytic activities. This study investigated whether TGF-beta1 may regulate FXII expression in human lung fibroblasts. Treatment of human lung fibroblasts with TGF-beta1 resulted in a time-dependent increase in FXII production, activation of p44/42, p38, JNK, and Akt, and phosphorylation and translocation into the nucleus of Smad3. However, TGF-beta1-induced FXII expression was repressed only by the JNK inhibitor and JNK and Smad3 antisense oligonucleotides but not by MEK, p38, or phosphoinositide 3-kinase blockers. JNK inhibition had no effect on TGF-beta1-induced Smad3 phosphorylation, association with Smad4, and its translocation into the nucleus but strongly suppressed Smad3-DNA complex formation. FXII promoter analysis revealed that the -299/+1 region was sufficient for TGF-beta1 to induce FXII expression. Sequence analysis of this region detected a potential Smad-binding element at position -272/-269 (SBE-(-272/-269)). Chromatin immunoprecipitation and streptavidin pulldown assays demonstrated TGF-beta1-dependent Smad3 binding to SBE-(-272/-269). Mutation or deletion of SBE-(-272/-269) substantially reduced TGF-beta1-mediated activation of the FXII promoter. Clinical relevance was demonstrated by elevated FXII levels and its co-localization with fibroblasts in the lungs of patients with acute respiratory distress syndrome. Our results show that JNK/Smad3 pathway plays a critical role in TGF-beta1-induced FXII expression in human lung fibroblasts and implicate its possible involvement in pathological conditions characterized by elevated TGF-beta1 levels.

SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-β signaling.

PubMed

Cortez, Victor S; Ulland, Tyler K; Cervantes-Barragan, Luisa; Bando, Jennifer K; Robinette, Michelle L; Wang, Qianli; White, Andrew J; Gilfillan, Susan; Cella, Marina; Colonna, Marco

2017-09-01

Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of 'imprinting' by cytokines of the TGF-β family. We studied mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-β family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-β signaling mediated by the cytokine receptor TGFβR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-β. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-β signaling in NK cells.

Smad Signaling Dynamics: Insights from a Parsimonious Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiley, H. S.; Shankaran, Harish

2008-09-09

The molecular mechanisms that transmit information from cell surface receptors to the nucleus are exceedingly complex; thus, much effort has been expended in developing computational models to understand these processes. A recent study on modeling the nuclear-cytoplasmic shuttling of Smad2-Smad4 complexes in response to transforming growth factor β (TGF-β) receptor activation has provided substantial insight into how this signaling network translates the degree of TGF-β receptor activation (input) into the amount of nuclear Smad2-Smad4 complexes (output). The study addressed this question by combining a simple, mechanistic model with targeted experiments, an approach that proved particularly powerful for exploring the fundamentalmore » properties of a complex signaling network. The mathematical model revealed that Smad nuclear-cytoplasmic dynamics enables a proportional, but time-delayed coupling between the input and the output. As a result, the output can faithfully track gradual changes in the input, while the rapid input fluctuations that constitute signaling noise are dampened out.« less

SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-β signaling

PubMed Central

Cortez, Victor S; Ulland, Tyler K; Cervantes-Barragan, Luisa; Bando, Jennifer K; Robinette, Michelle L; Wang, Qianli; White, Andrew J; Gilfillan, Susan; Cella, Marina; Colonna, Marco

2017-01-01

Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of ‘imprinting’ by cytokines of the TGF-β family. We examined mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-β family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-β signaling mediated by the cytokine receptor TGF-βR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-β. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-β signaling in NK cells. PMID:28759002

Loss of c-myc repression coincides with ovarian cancer resistance to transforming growth factor beta growth arrest independent of transforming growth factor beta/Smad signaling.

PubMed

Baldwin, Rae Lynn; Tran, Hang; Karlan, Beth Y

2003-03-15

Many epithelial carcinomas, including ovarian, are refractory to the antiproliferative effects of transforming growth factor (TGF) beta. In some cancers, TGF-beta resistance has been linked to TGF-beta receptor II (TbetaR-II) and Smad4 mutations; however, in ovarian cancer, the mechanism of resistance remains unclear. Primary ovarian epithelial cell cultures were used as a model system to determine the mechanisms of TGF-beta resistance. To simulate in vivo responses to TGF-beta, primary cultures derived from normal human ovarian surface epithelium (HOSE) and from ovarian carcinomas (CSOC) were grown on collagen I gel, the predominant matrix molecule in the ovarian tumor milieu. When treated with 5 ng/ml TGF-beta for 72 h, HOSE (n = 11) proliferation was inhibited by 20 +/- 21% on average. In contrast, CSOC (n = 10) proliferation was stimulated 5 +/- 10% in response to TGF-beta (a statistically significant difference in response when compared with HOSE; P = 0.001). To dissect the TGF-beta/Smad signaling pathway we used a quantitative RNase protection assay (RPA) for measuring mRNA levels of TGF-beta pathway components in 20 HOSE and 20 CSOC cultures. Basal mRNA levels of TGF-beta receptors I and II, downstream signaling components Smad2, 3, 4, 6, 7, and the transcriptional corepressors Ski and SnoN did not show a statistically significant difference between HOSE and CSOC, and cannot explain their differential susceptibility to TGF-beta-induced cell cycle arrest. To assess functional differences of the TGF-beta pathway in TGF-beta-sensitive HOSE and TGF-beta-resistant CSOC, we measured Smad2/4 and 3/4 complex induction after TGF-beta treatment. HOSE and CSOC showed equivalent Smad2/4 and 3/4 complex induction after TGF-beta exposure for 0, 0.5, 2, and 4 h. It has been proposed that SnoN and Ski are corepressors of the TGF-beta/Smad pathway and undergo TGF-beta-induced degradation followed by reinduction of SnoN mRNA. However, our data show equivalent SnoN degradation in HOSE and CSOC, and equivalent SnoN mRNA induction after TGF-beta treatment. Surprising, TGF-beta-induced Ski degradation was not observed in HOSE or CSOC, suggesting that Ski may not function as a TGF-beta/Smad corepressor in ovarian epithelial cells. These data implied that the TGF-beta/Smad pathway remains functional in CSOC, although CSOC cells are resistant to antimitogenic TGF-beta effects. CSOC resistance to TGF-beta coincided with the loss of c-myc down-regulation. These data suggest that TGF-beta/Smad signaling is blocked downstream of Smad complex formation or that an alternate signaling pathway other than TGF-beta/Smad may transmit TGF-beta-induced cell cycle arrest in the ovarian epithelium.

Smad4 deletion in blood vessel endothelial cells promotes ovarian cancer metastasis.

PubMed

Yang, Jie; Wang, Ya; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

2017-05-01

SMAD4 is a critical co-smad in signal transduction pathways activated in response to transforming growth factor-β (TGF-β)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumors highlighted it as a tumor-suppressor gene. Herein, we report that loss of SMAD4 expression in vascular endothelial cells promotes ovarian cancer invasion. SiRNA transfer of this gene in the HUVEC reduced SMAD4 protein expression and function. Although it reduced the vessel endothelial cell tubule formation in vitro and in vivo, it did not affect the tumor growth significantly in vivo. However, it weakened the barrier integrity in endothelial cells and increased vessel permeability and the ovarian cancer liver metastasis. We documented reduced angiogenesis and increased invasion histologically and by intravital microscopy, and gained mechanistic insight at the messenger and gene level. Finally, we found a negative reciprocal regulation between SMAD4 and FYN. FYN is one of the Src family kinases (SFK), activation of which can cause dissociation of cell-cell junctions and adhesion, resulting in paracellular hypermeability. Upon SMAD4 deletion, we detected high expression levels of FYN in vessel endothelial cells, suggesting the mechanism of the ovarian tumor cells cross the endothelial barrier and transform to an invasive phenotype.

Effects of c-Jun N-terminal kinase on Activin A/Smads signaling in PC12 cell suffered from oxygen-glucose deprivation.

PubMed

Wang, J Q; Xu, Z H; Liang, W Z; He, J T; Cui, Y; Liu, H Y; Xue, L X; Shi, W; Shao, Y K; Mang, J; Xu, Z X

2016-02-29

Activin A (Act A), a member of transforming growth factor-β (TGF-β) superfamily, is an early gene in response to cerebral ischemia. Growing evidences confirm the neuroprotective effect of Act A in ischemic injury through Act A/Smads signal activation. In this process, regulation networks are involved in modulating the outcomes of Smads signaling. Among these regulators, crosstalk between c-Jun N-terminal kinase (JNK) and Smads signaling has been found in the TGF-β induced epithelial-mesenchymal transition. However, in neural ischemia, the speculative regulation between JNK and Act A/Smads signaling pathways has not been clarified. To explore this issue, an Oxygen Glucose Deprivation (OGD) model was introduced to nerve-like PC12 cells. We found that JNK signal activation occurred at the early time of OGD injury (1 h). Act A administration suppressed JNK phosphorylation. In addition, JNK inhibition could elevate the strength of Smads signaling and attenuate neural apoptosis after OGD injury. Our results indicated a negative regulation effect of JNK on Smads signaling in ischemic injury. Taken together, JNK, as a critical site for neural apoptosis and negative regulator for Act A/Smads signaling, was presumed to be a molecular therapeutic target for ischemia.

Insulin-like growth factor-I increases bone sialoprotein (BSP) expression through fibroblast growth factor-2 response element and homeodomain protein-binding site in the proximal promoter of the BSP gene.

PubMed

Nakayama, Youhei; Nakajima, Yu; Kato, Naoko; Takai, Hideki; Kim, Dong-Soon; Arai, Masato; Mezawa, Masaru; Araki, Shouta; Sodek, Jaro; Ogata, Yorimasa

2006-08-01

Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.

GDF11 Modulates Ca2+-Dependent Smad2/3 Signaling to Prevent Cardiomyocyte Hypertrophy.

PubMed

Duran, Javier; Troncoso, Mayarling Francisca; Lagos, Daniel; Ramos, Sebastian; Marin, Gabriel; Estrada, Manuel

2018-05-18

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-β family, has been shown to act as a negative regulator in cardiac hypertrophy. Ca 2+ signaling modulates cardiomyocyte growth; however, the role of Ca 2+ -dependent mechanisms in mediating the effects of GDF11 remains elusive. Here, we found that GDF11 induced intracellular Ca 2+ increases in neonatal rat cardiomyocytes and that this response was blocked by chelating the intracellular Ca 2+ with BAPTA-AM or by pretreatment with inhibitors of the inositol 1,4,5-trisphosphate (IP₃) pathway. Moreover, GDF11 increased the phosphorylation levels and luciferase activity of Smad2/3 in a concentration-dependent manner, and the inhibition of IP₃-dependent Ca 2+ release abolished GDF11-induced Smad2/3 activity. To assess whether GDF11 exerted antihypertrophic effects by modulating Ca 2+ signaling, cardiomyocytes were exposed to hypertrophic agents (100 nM testosterone or 50 μM phenylephrine) for 24 h. Both treatments increased cardiomyocyte size and [³H]-leucine incorporation, and these responses were significantly blunted by pretreatment with GDF11 over 24 h. Moreover, downregulation of Smad2 and Smad3 with siRNA was accompanied by inhibition of the antihypertrophic effects of GDF11. These results suggest that GDF11 modulates Ca 2+ signaling and the Smad2/3 pathway to prevent cardiomyocyte hypertrophy.

Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-beta signaling.

PubMed

Wrighton, Katharine H; Willis, Danielle; Long, Jianyin; Liu, Fang; Lin, Xia; Feng, Xin-Hua

2006-12-15

Transforming growth factor-beta (TGF-beta) controls a diverse set of cellular processes, and its canonical signaling is mediated via TGF-beta-induced phosphorylation of receptor-activated Smads (2 and 3) at the C-terminal SXS motif. We recently discovered that PPM1A can dephosphorylate Smad2/3 at the C-terminal SXS motif, implicating a critical role for phosphatases in regulating TGF-beta signaling. Smad2/3 activity is also regulated by phosphorylation in the linker region (and N terminus) by a variety of intracellular kinases, making it a critical platform for cross-talk between TGF-beta and other signaling pathways. Using a functional genomic approach, we identified the small C-terminal domain phosphatase 1 (SCP1) as a specific phosphatase for Smad2/3 dephosphorylation in the linker and N terminus. A catalytically inactive SCP1 mutant (dnSCP1) had no effect on Smad2/3 phosphorylation in vitro or in vivo. Of the other FCP/SCP family members SCP2 and SCP3, but not FCP1, could also dephosphorylate Smad2/3 in the linker/N terminus. Depletion of SCP1/2/3 enhanced Smad2/3 linker phosphorylation. SCP1 increased TGF-beta-induced transcriptional activity in agreement with the idea that phosphorylation in the Smad2/3 linker must be removed for a full transcriptional response. SCP1 overexpression also counteracts the inhibitory effect of epidermal growth factor on TGF-beta-induced p15 expression. Taken together, this work identifies the first example of a Smad2/3 linker phosphatase(s) and reveals an important new substrate for SCPs.

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

PubMed

Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2004-09-23

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Smad phosphoisoform signaling specificity: the right place at the right time.

PubMed

Matsuzaki, Koichi

2011-11-01

Transforming growth factor (TGF)-β antagonizes mitogenic Ras signaling during epithelial regeneration, but TGF-β and Ras act synergistically in driving tumor progression. Insights into these apparently contradictory effects have come from recent detailed analyses of the TGF-β signaling process. Here, we summarize the different modes of TGF-β/Ras signaling in normal epithelium and neoplasms and show how perturbation of TGF-β signaling by Ras may contribute to a shift from tumor-suppressive to protumorigenic TGF-β activity during tumor progression. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β Type I receptor and Ras-associated kinases differentially phosphorylate Smad2 and Smad3 to create C-terminally (C), linker (L) or dually (L/C) phosphorylated (p) isoforms. In epithelial homeostasis, TGF-β-mediated pSmad3C signaling opposes proliferative responses induced by mitogenic signals. During carcinogenesis, activation of cytoplasmic Ras-associated kinases including mitogen-activated protein kinase confers a selective advantage on benign tumors by shifting Smad3 signaling from a tumor-suppressive pSmad3C to an oncogenic pSmad3L pathway, leading to carcinoma in situ. Finally, at the edges of advanced carcinomas invading adjacent tissues, nuclear Ras-associated kinases such as cyclin-dependent kinases, together with cytoplasmic kinases, alter TGF-β signals to more invasive and proliferative pSmad2L/C and pSmad3L/C signaling. Taken together, TGF-β signaling specificity arises from spatiotemporal dynamics of Smad phosphoisoforms. Based on these findings, we have reason to hope that pharmacologic inhibition of linker phosphorylation might suppress progression to human advanced carcinomas by switching from protumorigenic to tumor-suppressive TGF-β signaling.

Smad4 inactivation promotes malignancy and drug resistance of colon cancer

PubMed Central

Papageorgis, Panagiotis; Cheng, Kuanghung; Ozturk, Sait; Gong, Yi; Lambert, Arthur W.; Abdolmaleky, Hamid M.; Zhou, Jin-Rong; Thiagalingam, Sam

2010-01-01

SMAD4 is localized to chromosome 18q21, a frequent site for loss of heterozygosity (LOH) in advanced stage colon cancers. Although Smad4 is regarded as a signaling mediator of the TGFβ signaling pathway, its role as a major suppressor of colorectal cancer progression and the molecular events underlying this phenomenon, remain elusive. Here, we describe the establishment and use of colon cancer cell line model systems to dissect the functional roles of TGFβ and Smad4 inactivation in the manifestation of a malignant phenotype. We found that loss of SMAD4 and retention of intact TGFβ receptors could synergistically increase the levels of VEGF, a major pro-angiogenic factor. Pharmacological inhibition studies suggest that overactivation of the TGFβ-induced MEK-Erk and p38-MAPK auxiliary pathways are involved in the induction of VEGF expression in SMAD4 null cells. Overall, SMAD4 deficiency was responsible for the enhanced migration of colon cancer cells with a corresponding increase in MMP9, enhanced hypoxia-induced GLUT1 expression, increased aerobic glycolysis and resistance to 5′-fluoruracil-mediated apoptosis. Interestingly, Smad4 specifically interacts with HIF1α under hypoxic conditions providing a molecular basis for the differential regulation of target genes to suppress a malignant phenotype. In summary, our results define a molecular mechanism that explains how loss of the tumor suppressor Smad4 promotes colorectal cancer progression. These findings are also consistent with targeting TGFβ-induced auxiliary pathways, such as MEK-ERK, p38-MAPK and the glycolytic cascade, in SMAD4-deficient tumors as attractive strategies for therapeutic intervention. PMID:21245094

β-2 spectrin is involved in hepatocyte proliferation through the interaction of TGFβ/Smad and PI3K/AKT signalling.

PubMed

Wang, Zhijun; Song, Yuhu; Tu, Wei; He, Xingxing; Lin, Jusheng; Liu, Fang

2012-08-01

Transforming growth factor (TGF) β signalling pathway plays a crucial role in liver regeneration following partial hepatectomy in mice. Evidence demonstrated that β-2 Spectrin is involved in TGFβ/Smad signalling pathway as a Smad3/4 adaptor protein. The aim of this study was to explore the role of β-2 Spectrin in hepatocyte proliferation. β-2 Spectrin expression was evaluated in mice receiving partial hepatectomy. The effect of siRNA against β-2 Spectrin on hepatocyte proliferation was determined. The interaction between TGFβ/Smad and PI3K/Akt signalling was investigated. Hepatic β-2 Spectrin decreased dramatically 2 days after 70% hepatectomy in mice. In AML-12 cells, hepatocyte proliferation was inhibited after the stimulation of TGF β1 and a reduction in β-2 Spectrin mediated by siRNA resulted in increase in proliferative response. Confocal results revealed that β-2 Spectrin represented a key regulator in TGFβ/Smad signalling through controlling Smad3/4 subcellular localization. Moreover, Alternation of Akt phosphorylation led to the change in subcellular localization of Smad2, 3, 4 and β-2 Spectrin, A reduction in Smad2, 3 and 4 mediated by siRNA resulted in the induction of pAkt expression. These findings reveal that β-2 Spectrin plays a crucial role in hepatocyte proliferation, which contributes to liver regeneration following hepatectomy in mice. In addition, PI3K/Akt is involved in TGFβ/Smad signalling pathway through the interaction with Smad proteins and β-2 Spectrin. © 2012 John Wiley & Sons A/S.

Galangin enhances TGF-β1-mediated growth inhibition by suppressing phosphorylation of threonine 179 residue in Smad3 linker region.

PubMed

Kwak, Mi-Kyung; Yang, Kyung-Min; Park, Jinah; Lee, Siyoung; Park, Yuna; Hong, Eunji; Sun, Eun Jin; An, Haein; Park, Sujin; Pang, Kyoungwha; Lee, Jihee; Kang, Jin Muk; Kim, Pyunggang; Ooshima, Akira; Kim, Seong-Jin

2017-12-16

Smad3 linker phosphorylation is a candidate target for several kinases that play important roles in cancer cell initiation, proliferation and progression. Also, Smad3 is an essential intracellular mediator of TGF-β1-induced transcriptional responses during carcinogenesis. Therefore, it is highly advantageous to identify and develop inhibitors targeting Smad3 linker phosphorylation for the treatment of cancers. Galangin (3,5,7-trihydroxyflavone) has been known to be an active flavonoid showing a cytotoxic effect on several cancer cells. However, the mechanism of action of galangin in various cancers remains unclear, and there has been no report concerning regulation of Smad3 phosphorylation by galangin. In the present study, we show that galangin significantly induced apoptosis and inhibited cell proliferation in the presence of TGF-β1 in both human prostate and pancreatic cancer cell lines. Particularly, galangin effectively inhibits phosphorylation of the Thr-179 site at Smad3 linker region through suppression of CDK4 phosphorylation. Thus, galangin can be a promising candidate as a selective inhibitor to suppress phosphorylation of Smad3 linker region. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential phosphorylation of Smad1 integrates BMP and neurotrophin pathways through Erk/Dusp in axon development.

PubMed

Finelli, Mattéa J; Murphy, Kevin J; Chen, Lei; Zou, Hongyan

2013-05-30

Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2-two key neurotrophin effectors. Specifically, bone morphogenetic proteins (BMPs) signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2 and constituting a negative-feedback loop for the prevention of axon overgrowth. Together, the BMP and neurotrophin pathways form a tightly regulated signaling network with a balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

The type I BMP receptors, Bmpr1a and Acvr1, activate multiple signaling pathways to regulate lens formation

PubMed Central

Rajagopal, Ramya; Huang, Jie; Dattilo, Lisa K.; Kaartinen, Vesa; Mishina, Yuji; Deng, Chu-Xia; Umans, Lieve; Zwijsen, An; Roberts, Anita B.; Beebe, David C.

2009-01-01

BMPs play multiple roles in development and BMP signaling is essential for lens formation. However, the mechanisms by which BMP receptors function in vertebrate development are incompletely understood. To determine the downstream effectors of BMP signaling and their functions in the ectoderm that will form the lens, we deleted the genes encoding the type I BMP receptors, Bmpr1a and Acvr1, and the canonical transducers of BMP signaling, Smad4, Smad1 and Smad5. Bmpr1a and Acvr1 regulated cell survival and proliferation, respectively. Absence of both receptors interfered with the expression of proteins involved in normal lens development and prevented lens formation, demonstrating that BMPs induce lens formation by acting directly on the prospective lens ectoderm. Remarkably, the canonical Smad signaling pathway was not needed for most of these processes. Lens formation, placode cell proliferation, the expression of FoxE3, a lens-specific transcription factor, and the lens protein, αA-crystallin were regulated by BMP receptors in a Smad-independent manner. Placode cell survival was promoted by R-Smad signaling, but in a manner that did not involve Smad4. Of the responses tested, only maintaining a high level of Sox2 protein, a transcription factor expressed early in placode formation, required the canonical Smad pathway. A key function of Smad-independent BMP receptor signaling may be reorganization of actin cytoskeleton to drive lens invagination. PMID:19733164

Radiofrequency ablation for locally advanced pancreatic cancer: SMAD4 analysis segregates a responsive subgroup of patients.

PubMed

Paiella, Salvatore; Malleo, Giuseppe; Cataldo, Ivana; Gasparini, Clizia; De Pastena, Matteo; De Marchi, Giulia; Marchegiani, Giovanni; Rusev, Borislav; Scarpa, Aldo; Girelli, Roberto; Giardino, Alessandro; Frigerio, Isabella; D'Onofrio, Mirko; Secchettin, Erica; Bassi, Claudio; Salvia, Roberto

2018-03-01

SMAD4 mutational status correlates with pancreatic ductal adenocarcinoma (PDAC) failure pattern. We investigated in a subset of locally advanced patients submitted to radiofrequency ablation (RFA) whether the assessment of SMAD4 status is a useful way to select the patients. Clinical, radiological, and follow-up details of patients submitted to RFA for locally advanced pancreatic cancer (LAPC), in whom cytohistological material was available at our institution, were retrospectively retrieved. SMAD4 expression was evaluated by immunohistochemistry (IHC) and considered "negative" or "positive." The survival analysis was conducted using Kaplan-Meier and Cox proportional hazards models. The study population consisted of 30 patients. Thirteen patients (43.3%) received RFA upfront, whereas 17 (56.7%) after induction treatments. SMAD4 was mutant in 18 out of 30 patients (60%). The overall estimated post-RFA disease-specific survival (DSS) was 15 months (95% CI 11.64-18.35). The estimated post-RFA DSS of patients with wild-type and mutant SMAD4 was 22 and 12 months, respectively (log-rank p < 0.05). At the multivariate analysis, SMAD4 was the only independent predictor of survival (p = 0.05). The pattern of failure was not associated with SMAD4 status (p = 0.4). Within patients undergoing RFA for LAPC, SMAD4 analysis could segregate a subgroup of subjects with improved survival, who likely benefited from tumor ablation.

Ski co-repressor complexes maintain the basal repressed state of the TGF-beta target gene, SMAD7, via HDAC3 and PRMT5.

PubMed

Tabata, Takanori; Kokura, Kenji; Ten Dijke, Peter; Ishii, Shunsuke

2009-01-01

The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-beta-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-beta target gene, in TGF-beta-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-beta-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.

CEA SMAD 2016 Digitizer Evaluation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merchant, Bion J.

Sandia National Laboratories has tested and evaluated an updated SMAD digitizer, developed by the French Alternative Energies and Atomic Energy Commission (CEA). The SMAD digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self-noise, dynamic range, system noise, response, passband, and timing. The SMAD digitizers have been updated since their last evaluation by Sandia to improve their performance when recording at a sample rate of 20 Hz for infrasound applications and 100 Hzmore » for hydro-acoustic seismic stations. This evaluation focuses primarily on the 20 Hz and 100 Hz sample rates. The SMAD digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test- Ban-Treaty Organization (CTBTO).« less

Selenium modulates MMP2 expression through the TGFβ1/Smad signalling pathway in human umbilical vein endothelial cells and rabbits following lipid disturbance.

PubMed

Xu, Chenggui; Lu, Guihua; Li, Qinglang; Zhang, Juhong; Huang, Zhibin; Gao, Xiuren

2017-07-01

A high-fat diet is a major risk factor for coronary heart diseases. Matrix metalloprotease (MMP) expression is changed in many cardiovascular diseases. Selenium, which is an important trace element in animals, has a close relationship with cardiovascular diseases. The TGFβ1/Smad signalling pathway is ubiquitous in diverse tissues and cells, and it is also associated with the occurrence and development of cardiovascular diseases. Therefore, in this study, we aimed to determine selenium's effect on lipid metabolism, atherosclerotic plaque formation, and MMP2 expression, as well as the underlying functional mechanism. In vivo tests: 24 male New Zealand white rabbits were randomly divided into 4 groups: regular diet, high-fat diet, high-fat diet+selenium and regular diet+selenium groups. The high-fat diet induced the lipid disturbances of rabbits at week 12. Selenium supplementation lowered total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels (p<0.01). Selenium supplementation also suppressed MMP2 over-expression in thoracic aortas. In vitro tests: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of selenium or ox-LDL. Ox-LDL promoted MMP2 expression by increasing TGFβ1, pSmad2, pSmad3 and Smad3 expression (p<0.01). Selenium attenuated MMP2 over-expression by regulating the TGFβ1/Smad signalling pathway. Selenium suppressed high-fat diet-induced MMP2 over-expression in vivo by improving lipid metabolism. In vitro, selenium attenuated MMP2 over-expression through the TGFβ1/Smad signalling pathway. Copyright © 2017 Elsevier GmbH. All rights reserved.

Brain cytoplasmic RNA 1 suppresses smooth muscle differentiation and vascular development in mice.

PubMed

Wang, Yung-Chun; Chuang, Ya-Hui; Shao, Qiang; Chen, Jian-Fu; Chen, Shi-You

2018-04-13

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-β/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-β-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-β-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo , BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Testosterone Administration Inhibits Hepcidin Transcription and is Associated with Increased Iron Incorporation into Red Blood Cells

PubMed Central

Guo, Wen; Bachman, Eric; Li, Michelle; Roy, Cindy N.; Blusztajn, Jerzy; Wong, Siu; Chan, Stephen Y.; Serra, Carlo; Jasuja, Ravi; Travison, Thomas G.; Muckenthaler, Martina U.; Nemeth, Elizabeta; Bhasin, Shalender

2013-01-01

Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown. Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferring saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound 58Fe, the amount of 58Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to BMP-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. Conclusion: Testosterone inhibits hepcidin transcription through its interaction with BMP-Smad signaling. Testosterone administration is associated with increased iron incorporation into red blood cells. PMID:23399021

Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

PubMed Central

Di Pasquale, Elisa; Brivanlou, Ali H.

2009-01-01

Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua

2008-03-10

Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen genemore » expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.« less

Association of SMAD4 mutation with patient demographics, tumor characteristics, and clinical outcomes in colorectal cancer.

PubMed

Mehrvarz Sarshekeh, Amir; Advani, Shailesh; Overman, Michael J; Manyam, Ganiraju; Kee, Bryan K; Fogelman, David R; Dasari, Arvind; Raghav, Kanwal; Vilar, Eduardo; Manuel, Shanequa; Shureiqi, Imad; Wolff, Robert A; Patel, Keyur P; Luthra, Raja; Shaw, Kenna; Eng, Cathy; Maru, Dipen M; Routbort, Mark J; Meric-Bernstam, Funda; Kopetz, Scott

2017-01-01

SMAD4 is an essential mediator in the transforming growth factor-β pathway. Sporadic mutations of SMAD4 are present in 2.1-20.0% of colorectal cancers (CRCs) but data are limited. In this study, we aimed to evaluate clinicopathologic characteristics, prognosis, and clinical outcome associated with this mutation in CRC cases. Data for patients with metastatic or unresectable CRC who underwent genotyping for SMAD4 mutation and received treatment at The University of Texas MD Anderson Cancer Center from 2000 to 2014 were reviewed. Their tumors were sequenced using a hotspot panel predicted to cover 80% of the reported SMAD4 mutations, and further targeted resequencing that included full-length SMAD4 was performed on mutated tumors using a HiSeq sequencing system. Using The Cancer Genome Atlas data on CRC, the characteristics of SMAD4 and transforming growth factor-β pathway mutations were evaluated according to different consensus molecular subtypes of CRC. Among 734 patients with CRC, 90 (12%) had SMAD4 mutations according to hotspot testing. SMAD4 mutation was associated with colon cancer more so than with rectal cancer (odds ratio 2.85; p<0.001), female sex (odds ratio 1.71; p = 0.02), and shorter overall survival than in wild-type SMAD4 cases (median, 29 months versus 56 months; hazard ratio 2.08; p<0.001 [log-rank test]). SMAD4 mutation was not associated with age, stage at presentation, colonic location, distant metastasis, or tumor grade. A subset of patients with metastatic CRC (n = 44) wild-type for KRAS, NRAS, and BRAF who received anti-epidermal growth factor receptor therapy with mutated SMAD4 (n = 13) had shorter progression-free survival duration than did patients wild-type for SMAD4 (n = 31) (median, 111 days versus 180 days; p = 0.003 [log-rank test]). Full-length sequencing confirmed that missense mutations at R361 and P356 in the MH2 domain were the most common SMAD4 alterations. In The Cancer Genome Atlas data, SMAD4 mutation frequently occurred with KRAS, NRAS, and BRAF mutations and was more common in patients with the consensus molecular subtype 3 of CRC than in those with the other 3 subtypes. This is one of the largest retrospective studies to date characterizing SMAD4 mutations in CRC patients and demonstrates the prognostic role and lack of response of CRC to anti-epidermal growth factor receptor therapy. Further studies are required to validate these findings and the role of SMAD4 mutation in CRC.

Association of SMAD4 mutation with patient demographics, tumor characteristics, and clinical outcomes in colorectal cancer

PubMed Central

Sarshekeh, Amir Mehrvarz; Advani, Shailesh; Overman, Michael J.; Manyam, Ganiraju; Kee, Bryan K.; Fogelman, David R.; Dasari, Arvind; Raghav, Kanwal; Vilar, Eduardo; Manuel, Shanequa; Shureiqi, Imad; Wolff, Robert A.; Patel, Keyur P.; Luthra, Raja; Shaw, Kenna; Eng, Cathy; Maru, Dipen M.; Routbort, Mark J.; Meric-Bernstam, Funda

2017-01-01

SMAD4 is an essential mediator in the transforming growth factor-β pathway. Sporadic mutations of SMAD4 are present in 2.1–20.0% of colorectal cancers (CRCs) but data are limited. In this study, we aimed to evaluate clinicopathologic characteristics, prognosis, and clinical outcome associated with this mutation in CRC cases. Data for patients with metastatic or unresectable CRC who underwent genotyping for SMAD4 mutation and received treatment at The University of Texas MD Anderson Cancer Center from 2000 to 2014 were reviewed. Their tumors were sequenced using a hotspot panel predicted to cover 80% of the reported SMAD4 mutations, and further targeted resequencing that included full-length SMAD4 was performed on mutated tumors using a HiSeq sequencing system. Using The Cancer Genome Atlas data on CRC, the characteristics of SMAD4 and transforming growth factor-β pathway mutations were evaluated according to different consensus molecular subtypes of CRC. Among 734 patients with CRC, 90 (12%) had SMAD4 mutations according to hotspot testing. SMAD4 mutation was associated with colon cancer more so than with rectal cancer (odds ratio 2.85; p<0.001), female sex (odds ratio 1.71; p = 0.02), and shorter overall survival than in wild-type SMAD4 cases (median, 29 months versus 56 months; hazard ratio 2.08; p<0.001 [log-rank test]). SMAD4 mutation was not associated with age, stage at presentation, colonic location, distant metastasis, or tumor grade. A subset of patients with metastatic CRC (n = 44) wild-type for KRAS, NRAS, and BRAF who received anti-epidermal growth factor receptor therapy with mutated SMAD4 (n = 13) had shorter progression-free survival duration than did patients wild-type for SMAD4 (n = 31) (median, 111 days versus 180 days; p = 0.003 [log-rank test]). Full-length sequencing confirmed that missense mutations at R361 and P356 in the MH2 domain were the most common SMAD4 alterations. In The Cancer Genome Atlas data, SMAD4 mutation frequently occurred with KRAS, NRAS, and BRAF mutations and was more common in patients with the consensus molecular subtype 3 of CRC than in those with the other 3 subtypes. This is one of the largest retrospective studies to date characterizing SMAD4 mutations in CRC patients and demonstrates the prognostic role and lack of response of CRC to anti-epidermal growth factor receptor therapy. Further studies are required to validate these findings and the role of SMAD4 mutation in CRC. PMID:28267766

Down-Regulation of MicroRNA-210 Confers Sensitivity towards 1’S-1’-Acetoxychavicol Acetate (ACA) in Cervical Cancer Cells by Targeting SMAD4

PubMed Central

Phuah, Neoh Hun; Azmi, Mohamad Nurul; Awang, Khalijah; Nagoor, Noor Hasima

2017-01-01

MicroRNAs (miRNAs) are short non-coding RNAs that regulate genes posttranscriptionally. Past studies have reported that miR-210 is up-regulated in many cancers including cervical cancer, and plays a pleiotropic role in carcinogenesis. However, its role in regulating response towards anti-cancer agents has not been fully elucidated. We have previously reported that the natural compound 1’S-1’-acetoxychavicol acetate (ACA) is able to induce cytotoxicity in various cancer cells including cervical cancer cells. Hence, this study aims to investigate the mechanistic role of miR-210 in regulating response towards ACA in cervical cancer cells. In the present study, we found that ACA down-regulated miR-210 expression in cervical cancer cells, and suppression of miR-210 expression enhanced sensitivity towards ACA by inhibiting cell proliferation and promoting apoptosis. Western blot analysis showed increased expression of mothers against decapentaplegic homolog 4 (SMAD4), which was predicted as a target of miR-210 by target prediction programs, following treatment with ACA. Luciferase reporter assay confirmed that miR-210 binds to sequences in 3′UTR of SMAD4. Furthermore, decreased in SMAD4 protein expression was observed when miR-210 was overexpressed. Conversely, SMAD4 protein expression increased when miR-210 expression was suppressed. Lastly, we demonstrated that overexpression of SMAD4 augmented the anti-proliferative and apoptosis-inducing effects of ACA. Taken together, our results demonstrated that down-regulation of miR-210 conferred sensitivity towards ACA in cervical cancer cells by targeting SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer. PMID:28401751

Down-Regulation of MicroRNA-210 Confers Sensitivity towards 1'S-1'-Acetoxychavicol Acetate (ACA) in Cervical Cancer Cells by Targeting SMAD4.

PubMed

Phuah, Neoh Hun; Azmi, Mohamad Nurul; Awang, Khalijah; Nagoor, Noor Hasima

2017-04-01

MicroRNAs (miRNAs) are short non-coding RNAs that regulate genes posttranscriptionally. Past studies have reported that miR-210 is up-regulated in many cancers including cervical cancer, and plays a pleiotropic role in carcinogenesis. However, its role in regulating response towards anti-cancer agents has not been fully elucidated. We have previously reported that the natural compound 1'S-1'-acetoxychavicol acetate (ACA) is able to induce cytotoxicity in various cancer cells including cervical cancer cells. Hence, this study aims to investigate the mechanistic role of miR-210 in regulating response towards ACA in cervical cancer cells. In the present study, we found that ACA down-regulated miR-210 expression in cervical cancer cells, and suppression of miR-210 expression enhanced sensitivity towards ACA by inhibiting cell proliferation and promoting apoptosis. Western blot analysis showed increased expression of mothers against decapentaplegic homolog 4 (SMAD4), which was predicted as a target of miR-210 by target prediction programs, following treatment with ACA. Luciferase reporter assay confirmed that miR-210 binds to sequences in 3'UTR of SMAD4. Furthermore, decreased in SMAD4 protein expression was observed when miR-210 was overexpressed. Conversely, SMAD4 protein expression increased when miR-210 expression was suppressed. Lastly, we demonstrated that overexpression of SMAD4 augmented the anti-proliferative and apoptosis-inducing effects of ACA. Taken together, our results demonstrated that down-regulation of miR-210 conferred sensitivity towards ACA in cervical cancer cells by targeting SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer.

Smad7 mediates inhibition of Saos2 osteosarcoma cell differentiation by NF{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eliseev, Roman A.; Schwarz, Edward M.; Zuscik, Michael J.

2006-01-01

The transcription factor NF{kappa}B is constitutively activated in various tumor cells where it promotes proliferation and represses apoptosis. The bone morphogenetic proteins (BMPs) delay cell proliferation and promote differentiation and apoptosis of bone cells through activation of Smad downstream effectors and via Smad-independent mechanisms. Thus, NF{kappa}B and BMP pathways play opposing roles in regulating osteoblastic cell fate. Here, we show that in osteosarcoma Saos2 osteoblasts, NF{kappa}B regulates the activity of the BMP/Smad signaling. Inhibition of NF{kappa}B by overexpression of mI{kappa}B leads to the induction of osteoblast differentiation. Saos2 cells overexpressing mI{kappa}B (Saos2-mI{kappa}B) exhibit higher expression of osteoblast phenotypic genes suchmore » as alkaline phosphatase, Runx2 and osteocalcin and are more responsive to BMP2 in comparison to wild-type cells (Saos2-wt) or empty vector infected controls (Saos2-EV). Furthermore, BMP-2 signaling and Smad phosphorylation are significantly increased in Saos2-mI{kappa}B cells in comparison to Saos2-EV cells. Inhibition of NF{kappa}B signaling in Saos2-mI{kappa}B cells is associated with decreased expression of the BMP signaling inhibitor Smad7. While gain of Smad7 function in Saos2-mI{kappa}B cells results in inhibition of BMP signaling, anti-sense knockdown of Smad7 in Saos2-EV cells leads to upregulation of BMP signaling. We therefore conclude that in osteosarcoma Saos2 cells, NF{kappa}B represses BMP/Smad signaling and BMP2-induced differentiation through Smad7.« less

Disruption of the Smad7 gene enhances CCI4-dependent liver damage and fibrogenesis in mice

PubMed Central

Hamzavi, Jafar; Ehnert, Sabrina; Godoy, Patricio; Ciuclan, Loredana; Weng, Honglei; Mertens, Peter R; Heuchel, Rainer; Dooley, Steven

2008-01-01

Transforming growth factor-β (TGF-β) signalling is induced in liver as a consequence of damage and contributes to wound healing with transient activation, whereas it mediates fibrogenesis with long-term up-regulation in chronic disease. Smad-dependent TGF-β effects are blunted by antagonistic Smad7, which is transcriptionally activated as an immediate early response upon initiation of TGF-β signalling in most cell types, thereby providing negative feedback regulation. Smad7 can be induced by other cytokines, e.g. IFN-γ, leading to a crosstalk of these signalling pathways. Here we report on a novel mouse strain, denoted S7ΔE1, with a deletion of exon I from the endogenous smad7 gene. The mice were viable and exhibited normal adult liver architecture. To obtain insight into Smad7-depend-ent protective effects, chronic liver damage was induced in mice by carbon tetrachloride (CCI4) administration. Subsequent treatment, elevated serum liver enzymes indicated enhanced liver damage in mice lacking functional Smad7. CCI4-dependent Smad2 phosphoryla-tion was pronounced in S7ΔE1 mice and accompanied by increased numbers of α-smooth muscle actin positive ‘activated’ HSCs. There was evidence for matrix accumulation, with elevated collagen deposition as assessed morphometrically in Sirius red stained tissue and confirmed with higher levels of hydroxyproline in S7ΔE1 mice. In addition, the number of CD43 positive infiltrating lymphocytes as well as of apoptotic hepatocytes was increased. Studies with primary hepatocytes from S7ΔE1 and wild-type mice indicate that in the absence of functional Smad7 protein, hepatocytes are more sensitive for TGF-β effects resulting in enhanced cell death. Furthermore, S7ΔE1 hepatocytes display increased oxidative stress and cell damage in response to CCI4, as measured by reactive oxygen species production, glutathione depletion, lactate dehydrogenase release and lipid peroxidation. Using an ALK-5 inhibitor all investigated CCI4 effects on hepatocytes were blunted, confirming participation of TGF-β signalling. We conclude that Smad7 mediates a protective effect from adverse TGF-β signalling in damaged liver, re-iterating its negative regulatory loop on signalling. PMID:18266971

Smurf2 negatively modulates RIG-I-dependent antiviral response by targeting VISA/MAVS for ubiquitination and degradation.

PubMed

Pan, Yu; Li, Rui; Meng, Jun-Ling; Mao, He-Ting; Zhang, Yu; Zhang, Jun

2014-05-15

VISA (also known as MAVS, Cardif, IPS-1) is the essential adaptor protein for virus-induced activation of IFN regulatory factors 3 and 7 and production of type I IFNs. Understanding the regulatory mechanisms for VISA will provide detailed insights into the positive or negative regulation of innate immune responses. In this study, we identified Smad ubiquitin regulatory factor (Smurf) 2, one of the Smad ubiquitin regulator factor proteins, as an important negative regulator of virus-triggered type I IFN signaling, which targets at the VISA level. Overexpression of Smurf2 inhibits virus-induced IFN-β and IFN-stimulated response element activation. The E3 ligase defective mutant Smurf2/C716A loses the ability to suppress virus-induced type I IFN signaling, suggesting that the negative regulation is dependent on the ubiquitin E3 ligase activity of Smurf2. Further studies demonstrated that Smurf2 interacted with VISA and targeted VISA for K48-linked ubiquitination, which promoted the degradation of VISA. Consistently, knockout or knockdown of Smurf2 expression therefore promoted antiviral signaling, which was correlated with the increase in protein stability of VISA. Our findings suggest that Smurf2 is an important nonredundant negative regulator of virus-triggered type I IFN signaling by targeting VISA for K48-linked ubiquitination and degradation.

SMAD4 Defect Causes Auditory Neuropathy Via Specialized Disruption of Cochlear Ribbon Synapses in Mice.

PubMed

Liu, Ke; Ji, Fei; Yang, Guan; Hou, Zhaohui; Sun, Jianhe; Wang, Xiaoyu; Guo, Weiwei; Sun, Wei; Yang, Weiyan; Yang, Xiao; Yang, Shiming

2016-10-01

More than 100 genes have been associated with deafness. However, SMAD4 is rarely considered a contributor to deafness in humans, except for its well-defined role in cell differentiation and regeneration. Here, we report that a SMAD4 defect in mice can cause auditory neuropathy, which was defined as a mysterious hearing and speech perception disorder in human for which the genetic background remains unclear. Our study showed that a SMAD4 defect induces failed formation of cochlear ribbon synapse during the earlier stage of auditory development in mice. Further investigation found that there are nearly normal morphology of outer hair cells (OHCs) and post-synapse spiral ganglion nerves (SGNs) in SMAD4 conditional knockout mice (cKO); however, a preserved distortion product of otoacoustic emission (DPOAE) and cochlear microphonic (CM) still can be evoked in cKO mice. Moreover, a partial restoration of hearing detected by electric auditory brainstem response (eABR) has been obtained in the cKO mice using electrode stimuli toward auditory nerves. Additionally, the ribbon synapses in retina are not affected by this SMAD4 defect. Thus, our findings suggest that this SMAD4 defect causes auditory neuropathy via specialized disruption of cochlear ribbon synapses.

Non-Smad signaling pathways.

PubMed

Mu, Yabing; Gudey, Shyam Kumar; Landström, Maréne

2012-01-01

Transforming growth factor-beta (TGFβ) is a key regulator of cell fate during embryogenesis and has also emerged as a potent driver of the epithelial-mesenchymal transition during tumor progression. TGFβ signals are transduced by transmembrane type I and type II serine/threonine kinase receptors (TβRI and TβRII, respectively). The activated TβR complex phosphorylates Smad2 and Smad3, converting them into transcriptional regulators that complex with Smad4. TGFβ also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Ubiquitin ligase tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGFβ-associated kinase 1 (TAK1) have recently been shown to be crucial for the activation of the p38 and JNK MAPK pathways. Other TGFβ-induced non-Smad signaling pathways include the phosphoinositide 3-kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway. Signals induced by TGFβ are tightly regulated and specified by post-translational modifications of the signaling components, since they dictate the subcellular localization, activity, and duration of the signal. In this review, we discuss recent findings in the field of TGFβ-induced responses by non-Smad signaling pathways.

Differential regulations of fibronectin and laminin in Smad2 activation in vascular endothelial cells in response to disturbed flow.

PubMed

Yang, Tung-Lin; Lee, Pei-Ling; Lee, Ding-Yu; Wang, Wei-Li; Wei, Shu-Yi; Lee, Chih-I; Chiu, Jeng-Jiann

2018-01-02

Atherosclerosis occurs in arterial curvatures and branches, where the flow is disturbed with low and oscillatory shear stress (OSS). The remodeling and alterations of extracellular matrices (ECMs) and their composition is the critical step in atherogenesis. In this study, we investigated the effects of different ECM proteins on the regulation of mechanotransduction in vascular endothelial cells (ECs) in response to OSS. Through the experiments ranging from in vitro cell culture studies on effects of OSS on molecular signaling to in vivo examinations on clinical specimens from patients with coronary artery disease (CAD), we elucidated the roles of integrins and different ECMs, i.e., fibronectin (FN) and laminin (LM), in transforming growth factor (TGF)-β receptor (TβR)-mediated Smad2 activation and nuclear factor-κB (NF-κB) signaling in ECs in response to OSS and hence atherogenesis. OSS at 0.5±12 dynes/cm 2 induces sustained increases in the association of types I and II TβRs with β1 and β3 integrins in ECs grown on FN, but it only transient increases in ECs grown on LM. OSS induces a sustained activation of Smad2 in ECs on FN, but only a transient activation of Smad2 in ECs on LM. OSS-activation of Smad2 in ECs on FN regulates downstream NF-κB signaling and pro-inflammatory gene expression through the activation of β1 integrin and its association with TβRs. In contrast, OSS induces transient activations of β1 and β3 integrins in ECs on LM, which associate with type I TβR to regulate Smad2 phosphorylation, resulting in transient induction of NF-κB and pro-inflammatory gene expression. In vivo investigations on diseased human coronary arteries from CAD patients revealed that Smad2 is highly activated in ECs of atherosclerotic lesions, which is accompanied by the concomitant increase of FN rather than LM in the EC layer and neointimal region of atherosclerotic lesions. Our findings provide new insights into the mechanisms of how OSS regulates Smad2 signaling and pro-inflammatory genes through the complex signaling networks of integrins, TβRs, and ECMs, thus illustrating the molecular basis of regional pro-inflammatory activation within disturbed flow regions in the arterial tree.

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furukawa, Kiyoshi; Sato, Toru; Katsuno, Tatsuro, E-mail: katsuno@faculty.chiba-u.jp

2011-02-25

Research highlights: {yields} Smad3{sup -/-} mice showed an increased number of proliferating epithelial cells in colonic crypts. {yields} Proliferating epithelial cells showed activated Wnt/{beta}-catenin pathway. {yields} Smad3{sup -/-} mice also showed intermingling of proliferating cells with differentiated cells. {yields} Loss of EphB receptor expression was observed in the colonic crypts of Smad3{sup -/-} mice. {yields} Loss of EphB receptor expression is likely responsible for cell intermingling. -- Abstract: Deficiency of Smad3, an intracellular mediator of TGF-{beta}, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonicmore » epithelial cell proliferation and crypt formation. Smad3{sup ex8/ex8} C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3{sup -/-} mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3{sup -/-} mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear {beta}-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3{sup -/-} mice in accordance with nuclear {beta}-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3{sup -/-} mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3{sup -/-} mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a 'proliferative zone' at the bottom of colonic crypts in the normal colon.« less

Characterization of a novel transcriptionally active domain in the transforming growth factor beta-regulated Smad3 protein.

PubMed

Prokova, Vassiliki; Mavridou, Sofia; Papakosta, Paraskevi; Kardassis, Dimitris

2005-01-01

Transforming growth factor beta (TGFbeta) regulates transcriptional responses via activation of cytoplasmic effector proteins termed Smads. Following their phosphorylation by the type I TGFbeta receptor, Smads form oligomers and translocate to the nucleus where they activate the transcription of TGFbeta target genes in cooperation with nuclear cofactors and coactivators. In the present study, we have undertaken a deletion analysis of human Smad3 protein in order to characterize domains that are essential for transcriptional activation in mammalian cells. With this analysis, we showed that Smad3 contains two domains with transcriptional activation function: the MH2 domain and a second middle domain that includes the linker region and the first two beta strands of the MH2 domain. Using a protein-protein interaction assay based on biotinylation in vivo, we were able to show that a Smad3 protein bearing an internal deletion in the middle transactivation domain is characterized by normal oligomerization and receptor activation properties. However, this mutant has reduced transactivation capacity on synthetic or natural promoters and is unable to interact physically and functionally with the histone acetyltransferase p/CAF. The loss of interaction with p/CAF or other coactivators could account, at least in part, for the reduced transactivation capacity of this Smad3 mutant. Our data support an essential role of the previously uncharacterized middle region of Smad3 for nuclear functions, such as transcriptional activation and interaction with coactivators.

Klotho down-regulates Egr-1 by inhibiting TGF-β1/Smad3 signaling in high glucose treated human mesangial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yang; Department of Geriatrics, Zhu Jiang Hospital, Southern Medical University, Guangzhou, Guangdong; Hu, Fang

Diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease worldwide and is associated with glomerular mesangial cell (MC) proliferation and excessive extracellular matrix (ECM) production. Klotho can attenuate renal fibrosis in part by inhibiting TGF-β1/Smad3 signaling in DKD. Early growth response factor 1 (Egr-1) has been shown to play a key role in renal fibrosis in part by facilitating the formation of a positive feedback loop involving TGF-β1. However, whether Klotho down-regulates Egr-1 by inhibiting TGF-β1/Smad3 signaling in DKD is unclear. In the present study, we assessed human MCs that were incubated under high-glucose conditions tomore » mimic diabetes. Then, we transfected the cells with Klotho plasmid or siRNA to overexpress or knock down Klotho gene and protein expression. Klotho, Egr-1, fibronectin (FN), collagen type I (Col I), Smad3 and phosphorylated Smad3 (p-Smad3) gene and protein expression levels were determined by RT-qPCR and western blotting respectively. High glucose time-dependently down-regulated Klotho mRNA and protein expression in cultured human MCs. pcDNA3.1-Klotho transfection-mediated Klotho overexpression down-regulated Egr-1, FN and Col I expression and the p-Smad3/Smad3 ratio in human MCs. Conversely, siRNA-mediated Klotho silencing up-regulated Egr-1, FN, and Col I expression and the p-Smad3/Smad3 ratio. Moreover, the effects of si-Klotho on Egr-1 expression were abolished by the TGF-β1 inhibitor SB-431542. Klotho overexpression can prevent mesangial ECM production in high-glucose-treated human MCs, an effect that has been partially attributed to Egr-1 down-regulation facilitated by TGF-β1/Smad3 signaling inhibition. - Highlights: • High glucose time-dependently down-regulated Klotho mRNA and protein expression in cultured human MCs. • Klotho overexpression down-regulated Egr-1 and prevented mesangial ECM production in high-glucose-treated human MCs. • Klotho down-regulated Egr-1 by inhibiting TGF-β1/Smad3 signaling in high-glucose-treated human MCs.« less

WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors.

PubMed

Motamedi, Fariba Jian; Badro, Danielle A; Clarkson, Michael; Lecca, M Rita; Bradford, Stephen T; Buske, Fabian A; Saar, Kathrin; Hübner, Norbert; Brändli, André W; Schedl, Andreas

2014-07-17

Kidney organogenesis requires the tight control of proliferation, differentiation and apoptosis of renal progenitor cells. How the balance between these cellular decisions is achieved remains elusive. The Wilms' tumour suppressor Wt1 is required for progenitor survival, but the molecular cause for renal agenesis in mutants is poorly understood. Here we demonstrate that lack of Wt1 abolishes fibroblast growth factor (FGF) and induces BMP/pSMAD signalling within the metanephric mesenchyme. Addition of recombinant FGFs or inhibition of pSMAD signalling rescues progenitor cell apoptosis induced by the loss of Wt1. We further show that recombinant BMP4, but not BMP7, induces an apoptotic response within the early kidney that can be suppressed by simultaneous addition of FGFs. These data reveal a hitherto unknown sensitivity of early renal progenitors to pSMAD signalling, establishes FGF and pSMAD signalling as antagonistic forces in early kidney development and places WT1 as a key regulator of pro-survival FGF signalling pathway genes.

TGF-{beta} receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Droguett, Rebeca; Cabello-Verrugio, Claudio; Santander, Cristian

2010-09-10

Skeletal muscle differentiation is strongly inhibited by transforming growth factor type {beta} (TGF-{beta}), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-{beta}-receptors (TGF-{beta}-Rs) during skeletal muscle differentiation. We found a decrease of TGF-{beta} signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-{beta}. No changemore » in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-{beta}-R type I (TGF-{beta}-RI) and type II (TGF-{beta}-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-{beta}-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-{beta}-RII lacking the cytoplasmic domain. The TGF-{beta}-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-{beta}-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-{beta} receptors independent of Smad proteins are essential for skeletal muscle differentiation.« less

Endothelial cells respond to the direction of mechanical stimuli through SMAD signaling to regulate coronary artery size.

PubMed

Poduri, Aruna; Chang, Andrew H; Raftrey, Brian; Rhee, Siyeon; Van, Mike; Red-Horse, Kristy

2017-09-15

How mechanotransduction intersects with chemical and transcriptional factors to shape organogenesis is an important question in developmental biology. This is particularly relevant to the cardiovascular system, which uses mechanical signals from flowing blood to stimulate cytoskeletal and transcriptional responses that form a highly efficient vascular network. Using this system, artery size and structure are tightly regulated, but the underlying mechanisms are poorly understood. Here, we demonstrate that deletion of Smad4 increased the diameter of coronary arteries during mouse embryonic development, a phenotype that followed the initiation of blood flow. At the same time, the BMP signal transducers SMAD1/5/8 were activated in developing coronary arteries. In a culture model of blood flow-induced shear stress, human coronary artery endothelial cells failed to align when either BMPs were inhibited or SMAD4 was depleted. In contrast to control cells, SMAD4- deficient cells did not migrate against the direction of shear stress and increased proliferation rates specifically under flow. Similar alterations were seen in coronary arteries in vivo Thus, endothelial cells perceive the direction of blood flow and respond through SMAD signaling to regulate artery size. © 2017. Published by The Company of Biologists Ltd.

CD147-induced cell proliferation is associated with Smad4 signal inhibition.

PubMed

Qin, Hui; Rasul, Azhar; Li, Xin; Masood, Muqaddas; Yang, Guang; Wang, Na; Wei, Wei; He, Xi; Watanabe, Nobumoto; Li, Jiang; Li, Xiaomeng

2017-09-15

CD147 is a multifunctional trans-membrane glycoprotein, which is highly expressed in many cancers. However, the mechanism by which CD147 modulates cell proliferation is not fully understood. The aim of this study is to investigate the role of CD147 in cell proliferation associated with the TGF-β/Smad4 signaling pathway. Here, we used cell viability and clone formation assays in LNCaP prostate cancer cells to demonstrate that CD147 promotes cell proliferation. The luciferase assay and western blotting show that silencing CD147 using shRNA enhances transcription and expression of p21 WAF1 . Using immunofluorescence and nuclear-cytoplasmic separation, we show that this is primarily attributed to transport of Smad4 from the cytoplasm to nucleus. Other assays (GST pull-down, co-immunoprecipitation and immunofluorescence) demonstrate that Smad4 is a new interaction partner of CD147, with the Smad4 MH2 domain and CD147 intracellular domain (CD147-ICD) being involved in the interaction. Furthermore, we report that a phosphoserine (pSer) in CD147 (pSer252) is responsible for this interaction and inhibition of the Smad4/p21 WAF1 signal that promotes cell proliferation. Our results provide a novel molecular mechanism for CD147-induced cell proliferation associated with Smad4 signal inhibition. Copyright © 2017. Published by Elsevier Inc.

TGF-β Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

PubMed Central

Gingery, Anne; Bradley, Elizabeth W.; Pederson, Larry; Ruan, Ming; Horwood, Nikki J.; Oursler, Merry Jo

2008-01-01

To better understand the roles of TGF-β in bone metabolism, we investigated osteoclast survival in response TGF-β and found that TGF-β inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-β receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-β treatment. Since osteoclast survival involves MEK, AKT, and NFκB activation, we examined TGF-β effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IκB, and NFκB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFκB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFκB repressed TGF-β-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-β-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclXL expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-β-induced NFκB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-β to support of osteoclast survival. PMID:18586026

The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region.

PubMed

Jeon, Woo-Kwang; Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Young K; Lim, Seunghwan; Kim, Jae-Hong; Letterio, John J; Liu, Fang; Kim, Seong-Jin; Kim, Byung-Chul

2015-12-08

Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-β1 (TGF-β1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-β1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-β1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-β1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-β1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth.

The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region

PubMed Central

Park, Seong Ji; Jo, Eun Ji; Lee, Young K.; Lim, Seunghwan; Kim, Jae-Hong; Letterio, John J.; Liu, Fang; Kim, Seong-Jin; Kim, Byung-Chul

2015-01-01

Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-β1 (TGF-β1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-β1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-β1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-β1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-β1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth. PMID:26497676

Transforming growth factor β-induced expression of chondroitin sulfate proteoglycans is mediated through non-Smad signaling pathways.

PubMed

Jahan, Naima; Hannila, Sari S

2015-01-01

The expression of chondroitin sulfate proteoglycans (CSPGs) by reactive astrocytes is a major factor contributing to glial scarring and regenerative failure after spinal cord injury, but the molecular mechanisms underlying CSPG expression remain largely undefined. One contributing factor is transforming growth factor β (TGFβ), which is upregulated after injury and has been shown to induce expression of CSPGs in vitro. TGFβ typically mediates its effects through the Smad2/3 signaling pathway, and it has been suggested that this pathway is responsible for CSPG expression. However, there is evidence that TGFβ can also activate non-Smad signaling pathways. In this study, we report that TGFβ-induced expression of three different CSPGs--neurocan, brevican, and aggrecan--is mediated through non-Smad signaling pathways. We observed significant increases in TGFβ-induced expression of neurocan, brevican, and aggrecan following siRNA knockdown of Smad2 or Smad4, which indicates that Smad signaling is not required for the expression of these CSPGs. In addition, we show that neurocan, aggrecan, and brevican levels are significantly reduced when TGFβ is administered in the presence of either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin, but not the MEK1/2 inhibitor U0126. This suggests that TGFβ mediates this effect through non-Smad-dependent activation of the PI3K-Akt-mTOR signaling pathway, and targeting this pathway may therefore be an effective means of reducing CSPG expression in the injured CNS. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

PubMed

Dzieran, Johanna; Fabian, Jasmin; Feng, Teng; Coulouarn, Cédric; Ilkavets, Iryna; Kyselova, Anastasia; Breuhahn, Kai; Dooley, Steven; Meindl-Beinker, Nadja M

2013-01-01

Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

Amelotin gene expression is temporarily being upregulated at the initiation of apoptosis induced by TGFβ1 in mouse gingival epithelial cells.

PubMed

Nakayama, Yohei; Matsui, Sari; Noda, Keisuke; Yamazaki, Mizuho; Iwai, Yasunobu; Matsumura, Hiroyoshi; Izawa, Takashi; Tanaka, Eiji; Ganss, Bernhard; Ogata, Yorimasa

2016-10-01

Amelotin (AMTN) is expressed and secreted by ameloblasts in the maturation stage of amelogenesis and persist with low levels in the junctional epithelium (JE) of erupted teeth. The purpose of this study is to investigate the transcriptional regulation of the AMTN gene by transforming growth factor beta1 (TGFβ1) in gingival epithelial (GE1) cells in the apoptosis phase. Apoptosis was evaluated by the fragmentation of chromosomal DNA and TUNEL staining. A real-time PCR was carried out to examine the AMTN mRNA levels induced by TGFβ1 and Smad3 overexpression. Transient transfection analyses were completed using the various lengths of mouse AMTN gene promoter constructs with or without TGFβ1. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the Smad3 bindings to the AMTN gene promoter by TGFβ1. TGFβ1-induced apoptosis in GE1 cells were detected at 24 and 48 h by DNA fragmentation and TUNEL staining. AMTN mRNA levels increased at 6 h and reached maximum at 24 h in GE1 cells. Luciferase activities of the mouse AMTN gene promoter constructs were induced by TGFβ1. The results of the ChIP assays showed that there was an increase in Smad3 binding to Smad-binding element (SBE)#1 and SBE#2 after stimulation by TGFβ1. Immunohistochemical localization of AMTN was detected in the JE, and the AMTN protein levels in Smad3-deficient mice were decreased compared with wild-type mice. AMTN mRNA levels were induced at the initiation of apoptosis by TGFβ1, which mediated through the Smad3 bindings to SBEs in the mouse AMTN gene promoter.

Metastic Progression of Breast Cancer by Allelic Loss on Chromosome 18q21

DTIC Science & Technology

2006-03-01

Smad5 Smad2 Smad3 Smad8 Smad6 Samd7 MH1 Linker MH2 S1 S2 S3 Smad4 Smad1, Smad5 Samd2, Smad3 ,Smad8 Smad6,Smad7 ?? ?? A. B. Figure 1...homologous amino acid sequences at their N- and C- terminal regions (MH1 and MH2 respectively), which are separated by a highly divergent linker region...cancers (Figure 2). NB 2 3 4 5 6 7 8 9 10 11 12 13 14 Smad8α Smad8β Smad8γ Smad3α Smad3 β NB 1 2 3 4 5

[The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

PubMed

Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

2003-09-01

To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone formation.

SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFβ-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells

PubMed Central

de Kroon, Laurie M. G.; Narcisi, Roberto; van den Akker, Guus G. H.; Vitters, Elly L.; Blaney Davidson, Esmeralda N.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

2017-01-01

To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-β (TGFβ) receptor ALK5 is crucial for chondrogenesis induced by TGFβ. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFβ-induced chondrogenesis. Activation of TGFβ signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFβ signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFβ-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFβ-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFβ-induced chondrogenesis of human BMSCs. PMID:28240243

SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFβ-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

PubMed

de Kroon, Laurie M G; Narcisi, Roberto; van den Akker, Guus G H; Vitters, Elly L; Blaney Davidson, Esmeralda N; van Osch, Gerjo J V M; van der Kraan, Peter M

2017-02-27

To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-β (TGFβ) receptor ALK5 is crucial for chondrogenesis induced by TGFβ. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFβ-induced chondrogenesis. Activation of TGFβ signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFβ signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFβ-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFβ-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFβ-induced chondrogenesis of human BMSCs.

Smad Acetylation: A New Level of Regulation in TGF-Beta Signaling

DTIC Science & Technology

2007-07-01

of Smad2 and Smad3 , resulting in their oligomerization with the common mediator Smad4 (10-11). This Smad2/ Smad3 /Smad4 complex can then translocate...Smad2 and Smad3 , enabling oligomerization with Smad4 and translocation of the entire Smad complex into the nucleus. Once in the nucleus, the...performed prior to DOD funding determined that Smad2 but not Smad3 is efficiently acetylated in a p300 depend manner both in in vivo and in vitro models

Imaging colon cancer development in mice: IL-6 deficiency prevents adenoma in azoxymethane-treated Smad3 knockouts

NASA Astrophysics Data System (ADS)

Harpel, Kaitlin; Leung, Sarah; Faith Rice, Photini; Jones, Mykella; Barton, Jennifer K.; Bommireddy, Ramireddy

2016-02-01

The development of colorectal cancer in the azoxymethane-induced mouse model can be observed by using a miniaturized optical coherence tomography (OCT) imaging system. This system is uniquely capable of tracking disease development over time, allowing for the monitoring of morphological changes in the distal colon due to tumor development and the presence of lymphoid aggregates. By using genetically engineered mouse models deficient in Interleukin 6 (IL-6) and Smad family member 3 (Smad3), the role of inflammation on tumor development and the immune system can be elucidated. Smad3 knockout mice develop inflammatory response, wasting, and colitis associated cancer while deficiency of proinflammatory cytokine IL-6 confers resistance to tumorigenesis. We present pilot data showing that the Smad3 knockout group had the highest tumor burden, highest spleen weight, and lowest thymus weight. The IL-6 deficiency in Smad3 knockout mice prevented tumor development, splenomegaly, and thymic atrophy. This finding suggests that agents that inhibit IL-6 (e.g. anti-IL-6 antibody, non-steroidal anti-inflammatory drugs [NSAIDs], etc.) could be used as novel therapeutic agents to prevent disease progression and increase the efficacy of anti-cancer agents. OCT can also be useful for initiating early therapy and assessing the benefit of combination therapy targeting inflammation.

BMP suppresses PTEN expression via RAS/ERK signaling.

PubMed

Beck, Stayce E; Carethers, John M

2007-08-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

SKI promotes Smad3 linker phosphorylations associated with the tumor-promoting trait of TGFbeta.

PubMed

Lin, Qiushi; Chen, Dahu; Timchenko, Nikolai A; Medrano, Estela E

2010-05-01

The transcriptional co-regulator SKI is a potent inhibitor of TGFbeta-growth inhibitory signals. SKI binds to receptor-activated Smads in the nucleus, forming repressor complexes containing HDACs, mSin3, NCoR, and other protein partners. Alternatively, SKI binds to activated Smads in the cytoplasm, preventing their nuclear translocation. SKI is necessary for anchorage-independent growth of melanoma cells in vitro, and most important, for human melanoma xenograft growth in vivo. We recently identified a novel role of SKI in TGFbeta signaling. SKI promotes the switch of Smad3 from repressor of proliferation to activator of oncogenesis by facilitating phosphorylations in the linker domain. High levels of endogenous SKI are required by the tumor promoting trait of TGFbeta to induce expression of the plasminogen-activator inhibitor-1 (PAI-1), sustained expression of C-Myc and for aborting upregulation of p21(Waf-1). Here we discuss how SKI diversifies and amplifies its functions by associating with multiple protein partners and by promoting Smad3 linker phosphorylation(s) in response to TGFbeta signaling in melanoma cells.

Identification and expression of Smads associated with TGF-beta/activin/nodal signaling pathways in the rainbow trout (Oncorhynuchus mykiss)

USDA-ARS?s Scientific Manuscript database

The Smad proteins are essential components of the TGF-beta/activin/nodal family signaling pathway. We report the identification and characterization of transcripts representing 3 receptor Smads (Smad2a, Smad2b, Smad3), 2 common Smads (Smad4a, Smad4b) and one inhibitory Smad (Smad7). Phylogenetic an...

Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-{gamma}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Asish K; Wei, Jun; Wu, Minghua

2008-09-19

Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI),more » and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.« less

Plasma Gelsolin Induced Glomerular Fibrosis via the TGF-β1/Smads Signal Transduction Pathway in IgA Nephropathy

PubMed Central

Zhang, Lei; Han, Changsong; Ye, Fei; He, Yan; Jin, Yinji; Wang, Tianzhen; Wu, Yiqi; Jiang, Yang; Zhang, Fengmin; Jin, Xiaoming

2017-01-01

Glomerular fibrosis has been shown to be closely related to the progression and prognosis of IgA nephropathy (IgAN). However, mechanism underlying IgAN glomerular fibrosis remains unclear. Recently, our study showed that plasma gelsolin (pGSN) was decreased in the serum of an IgAN mouse model and that pGSN deposition was found in the glomeruli. Another cytokine, TGF-β1, which is closely related to glomerular fibrosis, was also found to be highly expressed in the glomeruli. In the present study, we report that pGSN induces glomerular fibrosis through the TGF-β1/Smads signal transduction pathway. This is supported by the following findings: human mesangial cells (HMCs) show remarkable morphological changes and proliferation in response to co-stimulation with pGSN and polymeric IgA1 (pIgA1) from IgAN patients compared to other controls. Moreover, ELISA assays showed that more TGF-β1 secretion was found in HMCs supernatants in the co-stimulation group. Further experiments showed increased TGF-β1, Smad3, p-Smad2/3, Smad4, and collagen 1 and decreased Smad7 expression in the co-stimulation group. Our present study implied that the synergistic effect of pGSN and pIgA induced glomerular fibrosis via the TGF-β1/Smads signal transduction pathway. This might be a potential mechanism for the glomerular fibrosis observed in IgAN patients. PMID:28208683

Transforming growth factor β-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression.

PubMed

Stephen, Tom L; Rutkowski, Melanie R; Allegrezza, Michael J; Perales-Puchalt, Alfredo; Tesone, Amelia J; Svoronos, Nikolaos; Nguyen, Jenny M; Sarmin, Fahmida; Borowsky, Mark E; Tchou, Julia; Conejo-Garcia, Jose R

2014-09-18

Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⁺ T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⁺ T cells in response to microenvironmental transforming growth factor-β (TGF-β), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-β-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-β signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Antipsychotics activate the TGFβ pathway effector SMAD3

PubMed Central

Cohen, T.; Sundaresh, S.; Levine, F.

2014-01-01

Although effective in treating an array of neurological disorders, antipsychotics are associated with deleterious metabolic side effects. Through high-throughput screening, we previously identified phenothiazine antipsychotics as modulators of the human insulin promoter. Here, we extended our initial finding to structurally diverse typical and atypical antipsychotics. We then identified the TGFβ pathway as being involved in the effect of antipsychotics on the insulin promoter, finding that antipsychotics activated SMAD3, a downstream effector of the TGFβ pathway, through a receptor distinct from the TGFβ receptor family and known neurotransmitter receptor targets of antipsychotics. Of note, antipsychotics that do not cause metabolic side effects did not activate SMAD3. In vivo relevance was demonstrated by reanalysis of gene expression data from human brains treated with antipsychotics, which showed altered expression of SMAD3 responsive genes. This work raises the possibility that antipsychotics could be designed that retain beneficial CNS activity while lacking deleterious metabolic side effects. PMID:22290122

Transcription factor PREP1 induces EMT and metastasis by controlling the TGF-β–SMAD3 pathway in non-small cell lung adenocarcinoma

PubMed Central

Risolino, Maurizio; Mandia, Nadia; Iavarone, Francescopaolo; Dardaei, Leila; Longobardi, Elena; Fernandez, Serena; Talotta, Francesco; Bianchi, Fabrizio; Pisati, Federica; Spaggiari, Lorenzo; Harter, Patrick N.; Mittelbronn, Michel; Schulte, Dorothea; Incoronato, Mariarosaria; Di Fiore, Pier Paolo; Blasi, Francesco; Verde, Pasquale

2014-01-01

Pre–B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial–mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-β. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-β. PREP1 modulates the cellular sensitivity to TGF-β by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-β pathway, EMT, and metastasis in NSCLC. PMID:25157139

Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription

PubMed Central

Faura Tellez, Grissel; Vandepoele, Karl; Brouwer, Uilke; Koning, Henk; Elderman, Robin M.; Hackett, Tillie-Louise; Willemse, Brigitte W. M.; Holloway, John; Van Roy, Frans; Koppelman, Gerard H.

2015-01-01

Genetic studies have identified Protocadherin-1 (PCDH1) and Mothers against decapentaplegic homolog-3 (SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-β1 (TGF-β1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o− (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-β1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-β1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with PCDH1-siRNA. We confirm PCDH1 and SMAD3 interactions by Y2H and by co-IP in HEK293T cells overexpressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-β-induced activation of a SMAD3-driven reporter was reduced by exogenous PCDH1 in BEAS2B cells, whereas it was increased by siRNA-mediated knockdown of endogenous PCDH1 in 16HBE cells. Overexpression of PCDH1 suppressed expression of TGF-β target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-β-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-β signaling in bronchial epithelial cells. We propose that PCDH1 and SMAD3 act in a single pathway in asthma susceptibility that affects sensitivity of the airway epithelium to TGF-β. PMID:26209277

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes.

PubMed

Wang, Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-beta activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-beta enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-beta type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smad complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.

Predicting Bone Metastatic Potential of Prostate Cancer via Computational Modeling of TGF-Beta Signaling

DTIC Science & Technology

2008-05-01

receptor trafficking. Smad phosphorylation Although the receptors for TGF-β signal through both Smad2 and Smad3 proteins in epithelial cells, we...select Smad2 to represent the R-Smads, because the two are virtually identical kinetically; furthermore, Smad2 is ~12-fold more abundant than Smad3 (11...the type of R-Smads. For example, regarding nuclear import, it has been proposed that Smad2, Smad3 , and Smad4 enter the nucleus by direct

Identification of the Downstream Promoter Targets of Smad Tumor Suppressors in Human Breast Cancer Cells

DTIC Science & Technology

2004-10-01

signaling mediator Smad2, Smad3 and Smad4 which form oligomeric complexes and migrate into nucleus to function as transcription factors to modulate... Smad3 and Smad4. 2. Identification of the downstream promoter targets of Smad3 or Smad4 in breast cancer cells. 3. Identify Smad4 regulated downstream...Development of a novel chromatin immunoprecipitation assay (CHIPS) using a TAP-TAG system to isolate in vivo binding targets of Smad3 and Smad4

Function of the two Xenopus smad4s in early frog development.

PubMed

Chang, Chenbei; Brivanlou, Ali H; Harland, Richard M

2006-10-13

Signals from the transforming growth factor beta family members are transmitted in the cell through specific receptor-activated Smads and a common partner Smad4. Two Smad4 genes (alpha and beta/10, or smad4 and smad4.2) have been isolated from Xenopus, and conflicting data are reported for Smad4beta/10 actions in mesodermal and neural induction. To further understand the functions of the Smad4s in early frog development, we analyzed their activities in detail. We report that Smad10 is a mutant form of Smad4beta that harbors a missense mutation of a conserved arginine to histidine in the MH1 domain. The mutation results in enhanced association of Smad10 with the nuclear transcription corepressor Ski and leads to its neural inducing activity through inhibition of bone morphogenetic protein (BMP) signaling. In contrast to Smad10, both Smad4alpha and Smad4beta enhanced BMP signals in ectodermal explants. Using antisense morpholino oligonucleotides (MOs) to knockdown endogenous Smad4 protein levels, we discovered that Smad4beta was required for both activin- and BMP-mediated mesodermal induction in animal caps, whereas Smad4alpha affected only the BMP signals. Neither Smad4 was involved directly in neural induction. Expression of Smad4beta-MO in early frog embryos resulted in reduction of mesodermal markers and defects in axial structures, which were rescued by either Smad4alpha or Smad4beta. Smad4alpha-MO induced only minor deficiency at late stages. As Smad4beta, but not Smad4alpha, is expressed at high levels maternally and during early gastrulation, our data suggest that although Smad4alpha and Smad4beta may have similar activities, they are differentially utilized during frog embryogenesis, with only Smad4beta being essential for mesoderm induction.

The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians.

PubMed

Molina, M Dolores; Saló, Emili; Cebrià, Francesc

2007-11-01

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.

Smad7 Protein Interacts with Receptor-regulated Smads (R-Smads) to Inhibit Transforming Growth Factor-β (TGF-β)/Smad Signaling.

PubMed

Yan, Xiaohua; Liao, Hongwei; Cheng, Minzhang; Shi, Xiaojing; Lin, Xia; Feng, Xin-Hua; Chen, Ye-Guang

2016-01-01

TGF-β is a pleiotropic cytokine that regulates a wide range of cellular actions and pathophysiological processes. TGF-β signaling is spatiotemporally fine-tuned. As a key negative regulator of TGF-β signaling, Smad7 exerts its inhibitory effects by blocking receptor activity, inducing receptor degradation or interfering with Smad-DNA binding. However, the functions and the molecular mechanisms underlying the actions of Smad7 in TGF-β signaling are still not fully understood. In this study we report a novel mechanism whereby Smad7 antagonizes TGF-β signaling at the Smad level. Smad7 oligomerized with R-Smad proteins upon TGF-β signaling and directly inhibited R-Smad activity, as assessed by Gal4-luciferase reporter assays. Mechanistically, Smad7 competes with Smad4 to associate with R-Smads and recruits the E3 ubiquitin ligase NEDD4L to activated R-Smads, leading to their polyubiquitination and proteasomal degradation. Similar to the R-Smad-Smad4 oligomerization, the interaction between R-Smads and Smad7 is mediated by their mad homology 2 (MH2) domains. A positive-charged basic region including the L3/β8 loop-strand module and adjacent amino acids in the MH2 domain of Smad7 is essential for the interaction. These results shed new light on the regulation of TGF-β signaling by Smad7. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of a novel Drosophila SMAD on the X chromosome.

PubMed

Henderson, K D; Andrew, D J

1998-11-09

TGF-beta signaling from the cell surface to the nucleus is mediated by the SMAD family of proteins, which have been grouped into three classes based upon sequence identity and function. Receptor-regulated, or pathway-restricted, SMADs (R-SMADs) are phosphorylated by ligand-specific serine/threonine kinase receptors. Phosphorylated R-SMADs oligomerize with the coactivating, or shared, SMAD (Co-SMAD) mediator and translocate to the nucleus where the complex directs transcription of downstream target genes. Inhibitory SMADs (I-SMADs) block receptor-mediated phosphorylation of R-SMADs. In Drosophila, one member of each class of SMAD has been reported: MAD, an R-SMAD, MEDEA, a Co-SMAD, and DAD, an I-SMAD. Here, we report the first identification of a novel Drosophila R-SMAD, which we have named Smox for Smad on X. We have localized the Smox gene to a specific interval on the X chromosome and shown that Smox is transcribed throughout development. Copyright 1998 Academic Press.

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-{beta} activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-{beta} enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-{beta} type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smadmore » complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.« less

Combined Biology and Bioinformatics Approaches to Breast Cancer

DTIC Science & Technology

2005-04-01

interacted with the MH 1 and linker domains in both Smad3 and Smad4; no interaction was found with the MH2 domain (Fig. 7C). These data suggest that LMO4...LMO4 interacts with these Smads. GST protein-protein interaction assays showed that LMO4 binds to the MH1 and linker domain of Smad 1, Smad3 and Smad4...by facilitating the nuclear translocation and DNA-binding of a complex composed of an R-Smad (Smad2 and/or Smad3 ) and the co-Smad, Smad4 (10). To

Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis.

PubMed

Sharifat, Narges; Mohammad Zadeh, Ghorban; Ghaffari, Mohammad-Ali; Dayati, Parisa; Kamato, Danielle; Little, Peter J; Babaahmadi-Rezaei, Hossein

2017-01-01

G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4-6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ET A and ET B receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis. © 2016 Royal Pharmaceutical Society.

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast.

PubMed

Haider, S; Kunihs, V; Fiala, C; Pollheimer, J; Knöfler, M

2017-09-01

TGF-β superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts. Expression and localisation of the canonical TGF-β targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium. Smad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs. The lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-β could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cooperative Assembly of Co-Smad4 MH1 with R-Smad1/3 MH1 on DNA: A Molecular Dynamics Simulation Study

PubMed Central

Wang, Guihong; Li, Chaoqun; Wang, Yan; Chen, Guangju

2013-01-01

Background Smads, the homologs of Sma and MAD proteins, play a key role in gene expression regulation in the transforming growth factor-β (TGF-β) signaling pathway. Recent experimental studies have revealed that Smad4/R-Smad heterodimers bound on DNA are energetically more favorable than homodimeric R-Smad/R-Smad complexes bound on DNA, which indicates that Smad4 might act as binding vehicle to cooperatively assemble with activated R-Smads on DNA in the nucleus. However, the details of interaction mechanism for cooperative recruitment of Smad4 protein to R-Smad proteins on DNA, and allosteric communication between the Smad4-DNA and R-Smad-DNA interfaces via DNA mediating are not yet clear so far. Methodology In the present work, we have constructed a series of Smadn+DNA+Smadn (n = 1, 3, 4) models and carried out molecular dynamics simulations, free energy calculations and DNA dynamics analysis for them to study the interaction properties of Smadn (n = 1, 3, 4) with DNA molecule. Results The results revealed that the binding of Smad4 protein to DNA molecule facilitates energetically the formation of the heteromeric Smad4+DNA+Smad1/3 complex by increasing the affinity of Smad1/3 with DNA molecule. Further investigations through the residue/base motion correlation and DNA dynamics analyses predicted that the binding of Smad4 protein to DNA molecule in the heteromeric Smad4+DNA+Smad1/3 model induces an allosteric communication from the Smad4-DNA interface to Smad1/Smad3-DNA interface via DNA base-pair helical motions, surface conformation changes and new hydrogen bond formations. The present work theoretically explains the mechanism of cooperative recruitment of Smad4 protein to Smad1/3 protein via DNA-mediated indirect readout mode in the nucleus. PMID:23326519

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

PubMed

Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

2007-04-13

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

Sustained activation of SMAD3/SMAD4 by FOXM1 promotes TGF-β–dependent cancer metastasis

PubMed Central

Xue, Jianfei; Lin, Xia; Chiu, Wen-Tai; Chen, Yao-Hui; Yu, Guanzhen; Liu, Mingguang; Feng, Xin-Hua; Sawaya, Raymond; Medema, René H.; Hung, Mien-Chie; Huang, Suyun

2014-01-01

A key feature of TGF-β signaling activation in cancer cells is the sustained activation of SMAD complexes in the nucleus; however, the drivers of SMAD activation are poorly defined. Here, using human and mouse breast cancer cell lines, we found that oncogene forkhead box M1 (FOXM1) interacts with SMAD3 to sustain activation of the SMAD3/SMAD4 complex in the nucleus. FOXM1 prevented the E3 ubiquitin-protein ligase transcriptional intermediary factor 1 γ (TIF1γ) from binding SMAD3 and monoubiquitinating SMAD4, which stabilized the SMAD3/SMAD4 complex. Loss of FOXM1 abolished TGF-β–induced SMAD3/SMAD4 formation. Moreover, the interaction of FOXM1 and SMAD3 promoted TGF-β/SMAD3–mediated transcriptional activity and target gene expression. We found that FOXM1/SMAD3 interaction was required for TGF-β–induced breast cancer invasion, which was the result of SMAD3/SMAD4-dependent upregulation of the transcription factor SLUG. Importantly, the function of FOXM1 in TGF-β–induced invasion was not dependent on FOXM1’s transcriptional activity. Knockdown of SMAD3 diminished FOXM1-induced metastasis. Furthermore, FOXM1 levels correlated with activated TGF-β signaling and metastasis in human breast cancer specimens. Together, our data indicate that FOXM1 promotes breast cancer metastasis by increasing nuclear retention of SMAD3 and identify crosstalk between FOXM1 and TGF-β/SMAD3 pathways. This study highlights the critical interaction of FOXM1 and SMAD3 for controlling TGF-β signaling during metastasis. PMID:24382352

Smad3 phosphoisoform-mediated signaling during sporadic human colorectal carcinogenesis.

PubMed

Matsuzaki, K

2006-06-01

Transforming growth factor-beta (TGF-beta) signaling occurring during human colorectal carcinogenesis involves a shift in TGF-beta function, reducing the cytokine's antiproliferative effect, while increasing actions that promote invasion and metastasis. TGF-beta signaling involves phosphorylation of Smad3 at serine residues 208 and 213 in the linker region and serine residues 423 and 425 in the C-terminal region. Exogenous TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). Either pSmad3C or pSmad3L oligomerizes with Smad4, and translocates into nuclei. While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells in vivo, JNK/pSmad3L-mediated signaling promotes tumor cell invasion and extracellular matrix synthesis by activated mesenchymal cells. Furthermore, hepatocyte growth factor signaling interacts with TGF-beta to activate the JNK/pSmad3L pathway, accelerating nuclear transport of cytoplasmic pSmad3L. This reduces accessibility of unphosphorylated Smad3 to membrane-anchored TbetaRI, preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and antiproliferative effects of TGF-beta on epithelial cells. As neoplasia progresses from normal colorectal epithelium through adenoma to invasive adenocarcinoma with distant metastasis, nuclear pSmad3L gradually increases while pSmad3C decreases. The shift from TbetaRI/pSmad3C-mediated to JNK/pSmad3L-mediated signaling is a major mechanism orchestrating a complex transition of TGF-beta signaling during sporadic human colorectal carcinogenesis. This review summarizes the recent understanding of Smad3 phosphoisoform-mediated signaling, particularly 'cross-talk' between Smad3 and JNK pathways that cooperatively promote oncogenic activities. Understanding of these actions should help to develop more effective therapy against human colorectal cancer, involving inhibition of JNK/pSmad3L pathway.

Disruption of transforming growth factor-beta signaling by curcumin induces gene expression of peroxisome proliferator-activated receptor-gamma in rat hepatic stellate cells.

PubMed

Zheng, Shizhong; Chen, Anping

2007-01-01

Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-beta (TGF-beta) and a dramatic reduction in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-gamma in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-gamma activation suppressed gene expression of TGF-beta receptors in activated HSC, leading to the interruption of TGF-beta signaling. This observation supported our assumption of an antagonistic relationship between PPAR-gamma activation and TGF-beta signaling in HSC. In this study, we further hypothesize that TGF-beta signaling might negatively regulate gene expression of PPAR-gamma in activated HSC. The present report demonstrates that exogenous TGF-beta1 inhibits gene expression of PPAR-gamma in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-beta signaling. Transfection assays further indicate that blocking TGF-beta signaling by dominant negative type II TGF-beta receptor increases the promoter activity of PPAR-gamma gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-gamma gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-gamma gene promoter and TGF-beta signaling. Taken together, these results demonstrate that the interruption of TGF-beta signaling by curcumin induces gene expression of PPAR-gamma in activated HSC in vitro. Our studies provide novel insights into the molecular mechanisms of curcumin in the induction of PPAR-gamma gene expression and in the inhibition of HSC activation.

Smad Acetylation: A New Level of Regulation in TGF-Beta Signaling

DTIC Science & Technology

2005-07-01

Our lab has determined that Smad2, but not Smad3 , can be acetylated by the acetyltransferase protein p300 in vivo and in vitro. The residues...terminal of Smad2 and Smad3 , allowing oligomerization with the common mediator Smad4 [9-10]. The Smad2/3/4 complex then translocates to the nucleus where...Smad2, but not Smad3 , could be acetylated in a p300 dependent manner. Both in vivo and in vitro data support the conclusion that only Smad2 could be

Smad2 and Smad3 phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer.

PubMed

Matsuzaki, Koichi; Kitano, Chiaki; Murata, Miki; Sekimoto, Go; Yoshida, Katsunori; Uemura, Yoshiko; Seki, Toshihito; Taketani, Shigeru; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2009-07-01

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.

Canonical TGF-β Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

PubMed

Nakashima, Hideyuki; Tsujimura, Keita; Irie, Koichiro; Ishizu, Masataka; Pan, Miao; Kameda, Tomonori; Nakashima, Kinichi

2018-05-16

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases. SIGNIFICANCE STATEMENT Canonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our results suggest that proper control of TGF-β/Smads/CRMP2 signaling pathways is critical for the precise execution of neuronal morphogenesis, whose impairment eventually results in neurodevelopmental disorders. Copyright © 2018 the authors 0270-6474/18/384791-20$15.00/0.

The Smad4/PTEN Expression Pattern Predicts Clinical Outcomes in Colorectal Adenocarcinoma.

PubMed

Chung, Yumin; Wi, Young Chan; Kim, Yeseul; Bang, Seong Sik; Yang, Jung-Ho; Jang, Kiseok; Min, Kyueng-Whan; Paik, Seung Sam

2018-01-01

Smad4 and PTEN are prognostic indicators for various tumor types. Smad4 regulates tumor suppression, whereas PTEN inhibits cell proliferation. We analyzed and compared the performance of Smad4 and PTEN for predicting the prognosis of patients with colorectal adenocarcinoma. Combined expression patterns based on Smad4+/- and PTEN+/- status were evaluated by immunostaining using a tissue microarray of colorectal adenocarcinoma. The relationships between the protein expression and clinicopathological variables were analyzed. Smad4-/PTEN- status was most frequently observed in metastatic adenocarcinoma, followed by primary adenocarcinoma and tubular adenoma (p<.001). When Smad4-/PTEN- and Smad4+/PTEN+ groups were compared, Smad4-/PTEN- status was associated with high N stage (p=.018) and defective mismatch repair proteins (p=.006). Significant differences in diseasefree survival and overall survival were observed among the three groups (Smad4+/PTEN+, Smad4-/PTEN+ or Smad4+/PTEN-, and Smad4-/PTEN-) (all p<.05). Concurrent loss of Smad4 and PTEN may lead to more aggressive disease and poor prognosis in patients with colorectal adenocarcinoma compared to the loss of Smad4 or PTEN alone.

Spatio-Temporal Distribution of Smads and Role of Smads/TGF-β/BMP-4 in the Regulation of Mouse Bladder Organogenesis

PubMed Central

Islam, Syed S.; Mokhtari, Reza Bayat; Kumar, Sushil; Maalouf, Joe; Arab, Sara; Yeger, Herman; Farhat, Walid A.

2013-01-01

Although Shh, TGF-β and BMP-4 regulate radial patterning of the bladder mesenchyme and smooth muscle differentiation, it is not known what transcription factors, local environmental cues or signaling cascades mediate bladder smooth muscle differentiation. We investigated the expression patterns of signaling mediated by Smad2 and Smad3 in the mouse embryonic bladder from E12.5 to E16.5 by using qRT-PCR, in situ hybridization and antibodies specifically recognizing individual Smad proteins. The role of Smad2 and Smad3 during smooth muscle formation was examined by disrupting the Smad2/3 signaling pathway using TβR1 inhibitor SB-431542 in organ culture system. qRT-PCR results showed that R-Smads, Co-Smad and I-Smads were all expressed during bladder development. RNA ISH for BMP-4 and immunostaining of TGF-β1 showed that BMP-4 and TGF-β1 were expressed in the transitional epithelium, lamina propia and muscularis mucosa. Smad1, Smad5 and Smad8 were first expressed in the bladder epithelium and continued to be expressed in the transitional epithelium, muscularis mesenchyme and lamina propia as the bladder developed. Smad2, Smad3 and Smad4 were first detected in the bladder epithelium and subsequently were expressed in the muscularis mesenchyme and lamina propia. Smad6 and Smad7 showed overlapping expression with R-Smads, which are critical for bladder development. In bladder explants (E12.5 to E16.5) culture, Smad2 and Smad3 were found localized within the nuclei, suggesting critical transcriptional regulatory effects during bladder development. E12.5 to E16.5 bladders were cultured with and without TβR1 inhibitor SB-431542 and assessed by qRT-PCR and immunofluorescence. After three days in culture in SB-431542, α-SMA, Smad2 and Smad3 expressions were significantly decreased compared with controls, however, with no significant changes in the expression of smooth muscle myosin heavy chain (SM-Myh. Based on the Smad expression patterns, we suggest that individual or combinations of Smads may be necessary during mouse bladder organogenesis and may be critical mediators for bladder smooth muscle differentiation. PMID:23620745

Magnolol Attenuates Concanavalin A-induced Hepatic Fibrosis, Inhibits CD4+ T Helper 17 (Th17) Cell Differentiation and Suppresses Hepatic Stellate Cell Activation: Blockade of Smad3/Smad4 Signalling.

PubMed

Zhang, Hongjun; Ju, Baoling; Zhang, Xiaoli; Zhu, Yanfei; Nie, Ying; Xu, Yuanhong; Lei, Qiuxia

2017-06-01

Magnolol is a pharmacological biphenolic compound extracted from Chinese herb Magnolia officinalis, which displays anti-inflammatory and antioxidant effects. This study was aimed at exploring the potential effect of magnolol on immune-related liver fibrosis. Herein, BALB/c mice were injected with concanavalin A (ConA, 8 mg/kg/week) up to 6 weeks to establish hepatic fibrosis, and magnolol (10, 20, 30 mg/kg/day) was given to these mice orally throughout the whole experiment. We found that magnolol preserved liver function and attenuated liver fibrotic injury in vivo. In response to ConA stimulation, the CD4 + T cells preferred to polarizing towards CD4 + T helper 17 (Th17) cells in liver. Magnolol was observed to inhibit Th17 cell differentiation in ConA-treated liver in addition to suppressing interleukin (IL)-17A generation. Hepatic stellate cells were activated in fibrotic liver as demonstrated by increased alpha smooth muscle actin (α-SMA) and desmin. More transforming growth factor (TGF)-β1 and activin A were secreted into the serum. Magnolol suppressed this abnormal HSC activation. Furthermore, the phosphorylation of Smad3 in its linker area (Thr179, Ser 204/208/213) was inhibited by magnolol. In vitro, the recombinant IL-17A plus TGF-β1 or activin A induced activation of human LX2 HSCs and promoted their collagen production. Smad3/Smad4 signalling pathway was activated in LX2 cells exposed to the fibrotic stimuli, as illustrated by the up-regulated phospho-Smad3 and the enhanced interaction between Smad3 and Smad4. These alterations were suppressed by magnolol. Collectively, our study reveals a novel antifibrotic effect of magnolol on Th17 cell-mediated fibrosis. © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

E-Cadherin Antagonizes Transforming Growth Factor β1 Gene Induction in Hepatic Stellate Cells by Inhibiting RhoA–Dependent Smad3 Phosphorylation

PubMed Central

Cho, Il Je; Kim, Young Woo; Han, Chang Yeob; Kim, Eun Hyun; Anderson, Richard A.; Lee, Young Sok; Lee, Chang Ho; Hwang, Se Jin; Kim, Sang Geon

2011-01-01

Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fibrosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECAD’s potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1-inducible or Smad3-inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Conclusion Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFβ1 and its target genes, and facilitates liver fibrosis. PMID:20890948

E-cadherin antagonizes transforming growth factor β1 gene induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation.

PubMed

Cho, Il Je; Kim, Young Woo; Han, Chang Yeob; Kim, Eun Hyun; Anderson, Richard A; Lee, Young Sok; Lee, Chang Ho; Hwang, Se Jin; Kim, Sang Geon

2010-12-01

Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fibrosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECAD's potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1-inducible or Smad3-inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFβ1 and its target genes, and facilitates liver fibrosis. Copyright © 2010 American Association for the Study of Liver Diseases.

PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki

2014-07-18

Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylationmore » of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.« less

The prognostic significance of Smad3, Smad4, Smad3 phosphoisoform expression in esophageal squamous cell carcinoma.

PubMed

Cho, Soo Youn; Ha, Sang Yun; Huang, Song-Mei; Kim, Jeong Hoon; Kang, Myung Soo; Yoo, Hae-Yong; Kim, Hyeon-ho; Park, Cheol-Keun; Um, Sung-Hee; Kim, Kyung-Hee; Kim, Seok-Hyung

2014-11-01

Smad3 functions as an integrator of diverse signaling, including transforming growth factor β signaling and the function of Smad3 is complexly regulated by differential phosphorylation at various sites of Smad3. Despite the importance of Smad3 and its various phosphoisoforms, their prognostic significance has rarely been studied. In this study, we demonstrated the prognostic significance of Smad3, its phosphoisoforms, and Smad4 expression by immunohistochemistry in 126 esophageal squamous cell carcinomas. The phosphoisoforms of Smad3 studied in this article included phosphorylation at C-terminal (pSmad3C)(Ser(423/425)) and phosphorylation at the linker region (pSmad3L)(Ser(213)). High expression of Smad3 was associated with shorter overall survival. Co-existence of high expression of pSmad3L(S213) and low expression of pSmad3C(S423/425) were associated with advanced N stage and an independent prognostic factor for overall [hazard ratio (HR) 2.03, 95 % confidence interval (CI) (1.10-3.75), p = 0.023] and disease-free survival [HR 2.41, 95 % CI (1.32-4.39), p = 0.004]. In conclusion, co-existence of high pSmad3L(Ser(213)) expression and low pSmad3C(Ser(423/425)) expression can be considered as immunohistochemical biomarkers for predicting prognosis as well as future therapeutic targets. In addition, our results of combinatory effect of differential phosphorylation of Smad3 on prognosis suggest the mode of action of Smad3 might be logically determined by its phosphorylation pattern.

Follicle-stimulating hormone synthesis and fertility are intact in mice lacking SMAD3 DNA binding activity and SMAD2 in gonadotrope cells

PubMed Central

Fortin, Jérôme; Boehm, Ulrich; Weinstein, Michael B.; Graff, Jonathan M.; Bernard, Daniel J.

2014-01-01

The activin/inhibin system regulates follicle-stimulating hormone (FSH) synthesis and release by pituitary gonadotrope cells in mammals. In vitro cell line data suggest that activins stimulate FSH β-subunit (Fshb) transcription via complexes containing the receptor-regulated SMAD proteins SMAD2 and SMAD3. Here, we used a Cre-loxP approach to determine the necessity for SMAD2 and/or SMAD3 in FSH synthesis in vivo. Surprisingly, mice with conditional mutations in both Smad2 and Smad3 specifically in gonadotrope cells are fertile and produce FSH at quantitatively normal levels. Notably, however, we discovered that the recombined Smad3 allele produces a transcript that encodes the entirety of the SMAD3 C-terminal Mad homology 2 (MH2) domain. This protein behaves similarly to full-length SMAD3 in Fshb transcriptional assays. As the truncated protein lacks the N-terminal Mad homology 1 (MH1) domain, these results show that SMAD3 DNA-binding activity as well as SMAD2 are dispensable for normal FSH synthesis in vivo. Furthermore, the observation that deletion of proximal exons does not remove all SMAD3 function may facilitate interpretation of divergent phenotypes previously described in different Smad3 knockout mouse lines.—Fortin, J., Boehm, U., Weinstein, M. B., Graff, J. M., Bernard, D. J. Follicle-stimulating hormone synthesis and fertility are intact in mice lacking SMAD3 DNA binding activity and SMAD2 in gonadotrope cells. PMID:24308975

Differential role of Sloan–Kettering Institute (Ski) protein in Nodal and transforming growth factor-beta (TGF-β)-induced Smad signaling in prostate cancer cells

PubMed Central

Khan, Shafiq A.

2012-01-01

Transforming growth factor-beta (TGF-β) signaling pathways contain both tumor suppressor and tumor promoting activities. We have demonstrated that Nodal, another member of the TGF-β superfamily, and its receptors are expressed in prostate cancer cells. Nodal and TGF-β exerted similar biological effects on prostate cells; both inhibited proliferation in WPE, RWPE1 and DU145 cells, whereas neither had any effect on the proliferation of LNCaP or PC3 cells. Interestingly, Nodal and TGF-β induced migration in PC3 cells, but not in DU145 cells. TGF-β induced predominantly phosphorylation of Smad3, whereas Nodal induced phosphorylation of only Smad2. We also determined the expression and differential role of Ski, a corepressor of Smad2/3, in Nodal and TGF-β signaling in prostate cancer cells. Similar levels of Ski mRNA were found in several established prostate cell lines; however, high levels of Ski protein were only detected in prostate cancer cells and prostate cancer tissue samples. Exogenous Nodal and TGF-β had no effects on Ski mRNA levels. On the other hand, TGF-β induced a rapid degradation of Ski protein mediated by the proteasomal pathway, whereas Nodal had no effect on Ski protein. Reduced Ski levels correlated with increased basal and TGF-β-induced Smad2/3 phosphorylation. Knockdown of endogenous Ski reduced proliferation in DU145 cells and enhanced migration of PC3 cells. We conclude that high levels of Ski expression in prostate cancer cells may be responsible for repression of TGF-β and Smad3 signaling, but Ski protein levels do not influence Nodal and Smad2 signaling. PMID:22843506

Direct interaction of Ski with either Smad3 or Smad4 is necessary and sufficient for Ski-mediated repression of transforming growth factor-beta signaling.

PubMed

Ueki, Nobuhide; Hayman, Michael J

2003-08-29

The oncoprotein Ski represses transforming growth factor (TGF)-beta signaling in an N-CoR-independent manner. However, the molecular mechanism(s) underlying this event has not been elucidated. Here, we identify an additional domain in Ski that mediates interaction with Smad3 which is important for this repression. This domain is distinct from the previously reported N-terminal Smad3 binding domain in Ski. Individual alanine substitution of several residues in the domain significantly affected Ski-Smad3 interaction. Furthermore, combined mutations within this domain, together with those in the previously identified Smad3 binding domain, can completely abolish the interaction of Ski with Smad3, while mutation in each domain alone retained partial interaction. By introducing those mutations that abolish direct interaction with Smad3 or Smad4 individually, or in combination, we show that interaction of Ski with either Smad3 or Smad4 is sufficient for Ski-mediated repression of TGF-beta signaling. Furthermore our results clearly demonstrate that Ski does not disrupt Smad3-Smad4 heteromer formation, and recruitment of Ski to the Smad3/4 complex through binding to either Smad3 or Smad4 is both necessary and sufficient for repression.

The Smad4/PTEN Expression Pattern Predicts Clinical Outcomes in Colorectal Adenocarcinoma

PubMed Central

Chung, Yumin; Wi, Young Chan; Kim, Yeseul; Bang, Seong Sik; Yang, Jung-Ho; Jang, Kiseok; Min, Kyueng-Whan; Paik, Seung Sam

2018-01-01

Background Smad4 and PTEN are prognostic indicators for various tumor types. Smad4 regulates tumor suppression, whereas PTEN inhibits cell proliferation. We analyzed and compared the performance of Smad4 and PTEN for predicting the prognosis of patients with colorectal adenocarcinoma. Methods Combined expression patterns based on Smad4+/– and PTEN+/– status were evaluated by immunostaining using a tissue microarray of colorectal adenocarcinoma. The relationships between the protein expression and clinicopathological variables were analyzed. Results Smad4–/PTEN– status was most frequently observed in metastatic adenocarcinoma, followed by primary adenocarcinoma and tubular adenoma (p<.001). When Smad4–/PTEN– and Smad4+/PTEN+ groups were compared, Smad4–/PTEN– status was associated with high N stage (p=.018) and defective mismatch repair proteins (p=.006). Significant differences in diseasefree survival and overall survival were observed among the three groups (Smad4+/PTEN+, Smad4–/PTEN+ or Smad4+/PTEN–, and Smad4–/PTEN–) (all p<.05). Conclusions Concurrent loss of Smad4 and PTEN may lead to more aggressive disease and poor prognosis in patients with colorectal adenocarcinoma compared to the loss of Smad4 or PTEN alone. PMID:29056035

SMAD4 is a potential prognostic marker in human breast carcinomas

PubMed Central

Liu, Nan-nan; Xi, Yue; Callaghan, Michael U.; Fribley, Andrew; Moore-Smith, Lakisha; Zimmerman, Jacquelyn W.; Pasche, Boris

2014-01-01

SMAD4 is a downstream mediator of transforming growth factor beta. While its tumor suppressor function has been investigated as a prognostic biomarker in several human malignancies, its role as a prognostic marker in breast carcinoma is still undefined. We investigated SMAD4 expression in breast carcinoma samples of different histologic grades to evaluate the association between SMAD4 and outcome in breast cancer. We also investigated the role of SMAD4 expression status in MDA-MB-468 breast cancer cells in responding to TGF-β stimulation. SMAD4 expression was assessed in 53 breast ductal carcinoma samples and in the surrounding normal tissue from 50 of the samples using immunohistochemistry, Western blot, and real-time PCR. TGF-β-SMAD and non-SMAD signaling was assessed by Western blot in MDA-MB-468 cells with and without SMAD4 restoration. SMAD4 expression was reduced in ductal breast carcinoma as compared to surrounding uninvolved ductal breast epithelia (p <0.05). SMAD4 expression levels decreased from Grade 1 to Grade 3 ductal breast carcinoma as assessed by immunohistochemistry (p <0.05). Results were recapitulated by tissue array. In addition, immunohistochemistry results were further confirmed at the protein and mRNA level. We then found that non-SMAD MEK/MAPK signaling was significantly different between SMAD4 expressing MDA-MB-468 cells and SMAD4-null MDA-MB-468 cells. This is the first study indicating that SMAD4 plays a key role in shifting MAPK signaling. Further, we have demonstrated that SMAD4 has a potential role in the development of breast carcinoma and SMAD4 was a potential prognostic marker of breast carcinoma. Our findings further support the role of SMAD4 in breast carcinoma development. In addition, we observed an inverse relationship between SMAD4 levels and breast carcinoma histological grade. Our finding indicated that SMAD4 expression level in breast cancer cells played a role in responding non-SMAD signaling but not the canonic SMAD signaling. Further mechanistic studies are necessary to establish the role of SMAD4 in breast carcinoma prognosis and potential specific targeting. PMID:23975369

Role of Smad signaling in kidney disease.

PubMed

Zhang, Yanhua; Wang, Songyan; Liu, Shengmao; Li, Chunguang; Wang, Ji

2015-12-01

Smads are the key intermediates of canonical transforming growth factor-beta (TGF-β) signaling. These intermediates are divided into three distinct subgroups based on their role in TGF-β family signal transduction: Receptor-regulated Smads (R-Smads) 1, 2, 3, 5 and 8, common Smad4, and inhibitory Smads6 and 7. TGF-β signaling through Smad pathway involves phosphorylation, ubiquitination, sumoylation, acetylation, and protein-protein interactions with mitogen-activated protein kinases, PI3K-Akt/PKB, and Wnt/GSK-3. Several studies have suggested that upregulation or downregulation of TGF-β/Smad signaling pathways may be a pathogenic mechanism in the progression of chronic kidney disease. Smad2 and 3 are the two major downstream R-Smads in TGF-β-mediated renal fibrosis, while Smad7 also controls renal inflammation. In this review, we characterize the role of Smads in kidney disease, describe the molecular mechanisms, and discuss the potential of Smads as a therapeutic target in chronic kidney disease.

Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.

PubMed

Pierreux, C E; Nicolás, F J; Hill, C S

2000-12-01

Smad4 plays a pivotal role in all transforming growth factor beta (TGF-beta) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-beta signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-beta signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-beta signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.

Molecular mechanism of the negative regulation of Smad1/5 protein by carboxyl terminus of Hsc70-interacting protein (CHIP).

PubMed

Wang, Le; Liu, Yi-Tong; Hao, Rui; Chen, Lei; Chang, Zhijie; Wang, Hong-Rui; Wang, Zhi-Xin; Wu, Jia-Wei

2011-05-06

The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.

Transforming Growth Factor β-Independent Shuttling of Smad4 between the Cytoplasm and Nucleus

PubMed Central

Pierreux, Christophe E.; Nicolás, Francisco J.; Hill, Caroline S.

2000-01-01

Smad4 plays a pivotal role in all transforming growth factor β (TGF-β) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-β signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-β signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-β signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm. PMID:11074002

TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji

The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, theymore » soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. -- Highlights: •TGF-β1 pretreatment accelerates γ-radiation-induced DNA damage response. •TGF-β1-accelerated DNA damage response is dependent on Smad signaling and DNA Ligase IV. •TGF-β1 pretreatment protects epithelial cells from γ-radiation in vivo.« less

Expression of VEGF-D, SMAD4, and SMAD7 and Their Relationship with Lymphangiogenesis and Prognosis in Colon Cancer.

PubMed

Su, Fei; Li, Xuemei; You, Kai; Chen, Mingwei; Xiao, Jianbing; Zhang, Yafang; Ma, Jing; Liu, Baoquan

2016-12-01

The vascular endothelial growth factor (VEGF) and TGF-β1 pathways play important roles in cancer. However, few studies have evaluated the expression and roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, and the conclusions remain controversial. To clarify the roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, we examined their expression and evaluated correlations with lymphangiogenesis, prognosis, and chemotherapeutic outcome. The expression of VEGF-D, SMAD4, and SMAD7 was immunohistochemically examined in 251 primary colon cancer samples obtained from the Harbin Medical University. The expression of VEGF-D, SMAD4, and SMAD7 was identified in 71.7, 41.0, and 69.7 % of samples, respectively. Positive expression of VEGF-D and SMAD7 and lost expression of SMAD4 were significantly correlated with lymph node metastasis and high lymphatic vessel density. VEGF-D and SMAD7 were found to be independent indicators of prognosis and chemotherapy outcome, and positive expression of either VEGF-D or SMAD7 was associated with significantly shorter overall survival and disease-free survival (OS and DFS) than negative expression in all 251 patients (P < 0.001 for OS and DFS) and patients following chemotherapy (P < 0.001 for OS and DFS). VEGF-D, SMAD4, and SMAD7 were involved in lymphangiogenesis and lymph node metastasis. VEGF-D and SMAD7 can serve as predictors of prognosis and chemotherapeutic outcome in colon cancer.

fussel (fuss)--A negative regulator of BMP signaling in Drosophila melanogaster.

PubMed

Fischer, Susanne; Bayersdorfer, Florian; Harant, Eva; Reng, Renate; Arndt, Stephanie; Bosserhoff, Anja-Katrin; Schneuwly, Stephan

2012-01-01

The TGF-β/BMP signaling cascades control a wide range of developmental and physiological functions in vertebrates and invertebrates. In Drosophila melanogaster, members of this pathway can be divided into a Bone Morphogenic Protein (BMP) and an Activin-ß (Act-ß) branch, where Decapentaplegic (Dpp), a member of the BMP family has been most intensively studied. They differ in ligands, receptors and transmitting proteins, but also share some components, such as the Co-Smad Medea (Med). The essential role of Med is to form a complex with one of the two activating Smads, mothers against decapentaplegic (Mad) or dSmad, and to translocate together to the nucleus where they can function as transcriptional regulators of downstream target genes. This signaling cascade underlies different mechanisms of negative regulation, which can be exerted by inhibitory Smads, such as daughters against decapentaplegic (dad), but also by the Ski-Sno family. In this work we identified and functionally analyzed a new member of the Ski/Sno-family, fussel (fuss), the Drosophila homolog of the human functional suppressing element 15 (fussel-15). fuss codes for two differentially spliced transcripts with a neuronal expression pattern. The proteins are characterized by a Ski-Sno and a SAND homology domain. Overexpression studies and genetic interaction experiments clearly reveal an interaction of fuss with members of the BMP pathway, leading to a strong repression of BMP-signaling. The protein interacts directly with Medea and seems to reprogram the Smad pathway through its influence upon the formation of functional Mad/Medea complexes. This leads amongst others to a repression of downstream target genes of the Dpp pathway, such as optomotor blind (omb). Taken together we could show that fuss exerts a pivotal role as an antagonist of BMP signaling in Drosophila melanogaster.

Oncogenic Smad3 signaling induced by chronic inflammation is an early event in ulcerative colitis-associated carcinogenesis.

PubMed

Kawamata, Seiji; Matsuzaki, Koichi; Murata, Miki; Seki, Toshihito; Matsuoka, Katsuyoshi; Iwao, Yasushi; Hibi, Toshifumi; Okazaki, Kazuichi

2011-03-01

Both chronic inflammation and somatic mutations likely contribute to the pathogenesis of ulcerative colitis (UC)-associated dysplasia and cancer. On the other hand, both tumor suppression and oncogenesis can result from transforming growth factor (TGF)-β signaling. TGF-β type I receptor (TβRI) and Ras-associated kinases differentially phosphorylate a mediator, Smad3, to become C-terminally phosphorylated Smad3 (pSmad3C), linker phosphorylated Smad3 (pSmad3L), and both C-terminally and linker phosphorylated Smad3 (pSmad3L/C). The pSmad3C/p21(WAF1) pathway transmits a cytostatic TGF-β signal, while pSmad3L and pSmad3L/C promote cell proliferation by upregulating c-Myc oncoprotein. The purpose of this study was to clarify the alteration of Smad3 signaling during UC-associated carcinogenesis. By immunostaining and immunofluorescence, we compared pSmad3C-, pSmad3L-, and pSmad3L/C-mediated signaling in colorectal specimens representing colitis, dysplasia, or cancer from eight UC patients with signaling in normal colonic crypts. We also investigated p53 expression and mutations of p53 and K-ras genes. We further sought functional meaning of the phosphorylated Smad3-mediated signaling in vitro. As enterocytes in normal crypts migrated upward toward the lumen, cytostatic pSmad3C/p21(WAF1) tended to increase, while pSmad3L/c-Myc shown by progenitor cells gradually decreased. Colitis specimens showed prominence of pSmad3L/C/c-Myc, mediated by TGF-β and tumor necrosis factor (TNF)-α, in all enterocyte nuclei throughout entire crypts. In proportion with increases in frequency of p53 and K-ras mutations during progression from dysplasia to cancer, the oncogenic pSmad3L/c-Myc pathway came to be dominant with suppression of the pSmad3C/p21(WAF1) pathway. Oncogenic Smad3 signaling, altered by chronic inflammation and eventually somatic mutations, promotes UC-associated neoplastic progression by upregulating growth-related protein. Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins.

PubMed

Nakano, Ayako; Koinuma, Daizo; Miyazawa, Keiji; Uchida, Takafumi; Saitoh, Masao; Kawabata, Masahiro; Hanai, Jun-ichi; Akiyama, Hirotada; Abe, Masahiro; Miyazono, Kohei; Matsumoto, Toshio; Imamura, Takeshi

2009-03-06

Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

Smad 1/5 and Smad 4 Expression Are Important for Osteoclast Differentiation

PubMed Central

Tasca, Amy; Stemig, Melissa; Broege, Aaron; Huang, Brandon; Davydova, Julia; Zwijsen, An; Umans, Lieve; Jensen, Eric D.; Gopalakrishnan, Raj; Mansky, Kim C.

2015-01-01

To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP’s effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity. PMID:25711193

Inhibition of BMP signaling overcomes acquired resistance to cetuximab in oral squamous cell carcinomas.

PubMed

Yin, Jinlong; Jung, Ji-Eun; Choi, Sun Il; Kim, Sung Soo; Oh, Young Taek; Kim, Tae-Hoon; Choi, Eunji; Lee, Sun Joo; Kim, Hana; Kim, Eun Ok; Lee, Yu Sun; Chang, Hee Jin; Park, Joo Yong; Kim, Yeejeong; Yun, Tak; Heo, Kyun; Kim, Youn-Jae; Kim, Hyunggee; Kim, Yun-Hee; Park, Jong Bae; Choi, Sung Weon

2018-02-01

Despite expressing high levels of the epidermal growth factor receptor (EGFR), a majority of oral squamous cell carcinoma (OSCC) patients show limited response to cetuximab and ultimately develop drug resistance. However, mechanism underlying cetuximab resistance in OSCC is not clearly understood. Here, using a mouse orthotopic xenograft model of OSCC, we show that bone morphogenic protein-7-phosphorylated Smad-1, -5, -8 (BMP7-p-Smad1/5/8) signaling contributes to cetuximab resistance. Tumor cells isolated from the recurrent cetuximab-resistant xenograft models exhibited low EGFR expression but extremely high levels of p-Smad1/5/8. Treatment with the bone morphogenic protein receptor type 1 (BMPRI) inhibitor, DMH1 significantly reduced cetuximab-resistant OSCC tumor growth, and combined treatment of DMH1 and cetuximab remarkably reduced relapsed tumor growth in vivo. Importantly, p-Smad1/5/8 level was elevated in cetuximab-resistant patients and this correlated with poor prognosis. Collectively, our results indicate that the BMP7-p-Smad1/5/8 signaling is a key pathway to acquired cetuximab resistance, and demonstrate that combination therapy of cetuximab and a BMP signaling inhibitor as potentially a new therapeutic strategy for overcoming acquired resistance to cetuximab in OSCC. Copyright © 2017 Elsevier B.V. All rights reserved.

Galectin-7 promotes proliferation and Th1/2 cells polarization toward Th1 in activated CD4+ T cells by inhibiting The TGFβ/Smad3 pathway.

PubMed

Luo, Zhenlong; Ji, Yudong; Tian, Dean; Zhang, Yong; Chang, Sheng; Yang, Chao; Zhou, Hongmin; Chen, Zhonghua Klaus

2018-06-08

Galectin-7 (Gal-7) has been associated with cell proliferation and apoptosis. It is known that Gal-7 antagonises TGFβ-mediated effects in hepatocytes by interacting with Smad3. Previously, we have demonstrated that Gal-7 is related to CD4+ T cells responses; nevertheless, its effect and functional mechanism on CD4+ T cells responses remain unclear. The murine CD4+ T cells were respectively cultured with Gal-7, anti-CD3/CD28 mAbs, or with anti-CD3/CD28 mAbs & Gal-7. The effects of Gal-7 on proliferation and the phenotypic changes in CD4+ T cells were assessed by flow cytometry. The cytokines from CD4+ T cells were analysed by quantitative real-time PCR. Subcellular localisation and expression of Smad3 were determined by immunofluorescence staining and Western blot, respectively. Gal-7 enhanced the proliferation of activated CD4+ T cells in a dose- and β-galactoside-dependent manner. Additionally, Gal-7 treatment did not change the ratio of Th2 cells in activated CD4+ T cells, while it increased the ratio of Th1 cells. Gal-7 also induced activated CD4+ T cells to produce a higher level of IFN-γ and TNF-α and a lower level of IL-10. Moreover, Gal-7 treatment significantly accelerated nuclear export of Smad3 in activated CD4+ T cells. These results revealed a novel role of Gal-7 in promoting proliferation and Th1/2 cells polarization toward Th1 in activated CD4+ T cells by inhibiting the TGFβ/Smad3 pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.

Trastuzumab inhibits pituitary tumor growth modulating the TGFB/Smad2/3 pathway.

PubMed

Petiti, Juan Pablo; Sosa, Liliana Del Valle; Picech, Florencia; Moyano Crespo, Gabriela Deisi; Arevalo Rojas, Jean Zander; Pérez, Pablo Anibal; Guido, Carolina Beatriz; Leimgruber, Carolina; Sabatino, María Eugenia; García, Pedro Emilio; Bengió, Verónica; Papalini, Francisco Roque; Estario, Paula; Bernhardt, Maria Celina; Villarreal, Marcos; Gutiérrez, Silvina; De Paul, Ana Lucía; Mukdsi, Jorge Humberto; Torres, Alicia I

2018-06-06

In pituitary adenomas, early recurrences and resistance to conventional pharmacotherapies are common, but the mechanisms involved are still not understood. The high expression of epidermal growth factor receptor 2 (HER2)/extracellular signal-regulated kinase (ERK1/2) signal observed in human pituitary adenomas, together with the low levels of the antimitogenic transforming growth factor beta receptor 2 (TBR2), encouraged us to evaluate the effect of the specific HER2 inhibition with trastuzumab on experimental pituitary tumor cell growth and its effect on the antiproliferative response to TGFB1. Trastuzumab decreased the pituitary tumor growth as well as the expression of ERK1/2 and the cell cycle regulators cyclin D1 and CDK4. The HER2/ERK1/2 pathway is an attractive therapeutic target, but its intricate relations with other signaling modulators still need to be unraveled. Thus, we investigated possible cross-talk with TGFB signaling, which has not yet been studied in pituitary tumors. In tumoral GH3 cells, co-incubation with trastuzumab and TGFB1 significantly decreased cell proliferation, an effect accompanied by a reduction in ERK1/2 phosphorylation, an increase of SMAD2/3 activation. In addition, through immunoprecipitation assays, a diminution of SMAD2/3-ERK1/2 and an increase SMAD2/3-TGFBR1 interactions were observed when cells were co-incubated with Trastuzumab and TGFB1. These findings indicate that blocking HER2 by trastuzumab inhibited pituitary tumor growth and modulated HER2/ERK1/2 signaling and consequently the anti-mitogenic TGFB1/TBRs/SMADs cascade. The imbalance between HER2 and TGFBRs expression observed in human adenomas and the response to trastuzumab on experimental tumor growth, may make the HER2/ERK1/2 pathway an attractive target for future pituitary adenoma therapy.

The Roles of TGF-Beta and TGF-Beta Signaling Receptors in Breast Carcinogenesis.

DTIC Science & Technology

1997-07-01

phosphorylation of these molecules in a normal mammary epithelial cell line. Subsequently, we have focused on the functional role of Smad3 and Smad4 as...serine residues in the C-terminal portion of Smadl and Smad2, though the corresponding highly conserved sites in Smad3 and Smad5 most likely serve the...far, it appears that Smad2 and Smad3 , which share 92% sequence identity, are likely mediators for the TGF-B signal, whereas Smadl and Smad5, which

SnoN Stabilizes the SMAD3/SMAD4 Protein Complex

PubMed Central

Walldén, Karin; Nyman, Tomas; Hällberg, B. Martin

2017-01-01

TGF-β signaling regulates cellular processes such as proliferation, differentiation and apoptosis through activation of SMAD transcription factors that are in turn modulated by members of the Ski-SnoN family. In this process, Ski has been shown to negatively modulate TGF-β signaling by disrupting active R-SMAD/Co-SMAD heteromers. Here, we show that the related regulator SnoN forms a stable complex with the R-SMAD (SMAD3) and the Co-SMAD (SMAD4). To rationalize this stabilization at the molecular level, we determined the crystal structure of a complex between the SAND domain of SnoN and the MH2-domain of SMAD4. This structure shows a binding mode that is compatible with simultaneous coordination of R-SMADs. Our results show that SnoN, and SMAD heteromers can form a joint structural core for the binding of other transcription modulators. The results are of fundamental importance for our understanding of the molecular mechanisms behind the modulation of TGF-β signaling. PMID:28397834

SnoN Stabilizes the SMAD3/SMAD4 Protein Complex.

PubMed

Walldén, Karin; Nyman, Tomas; Hällberg, B Martin

2017-04-11

TGF-β signaling regulates cellular processes such as proliferation, differentiation and apoptosis through activation of SMAD transcription factors that are in turn modulated by members of the Ski-SnoN family. In this process, Ski has been shown to negatively modulate TGF-β signaling by disrupting active R-SMAD/Co-SMAD heteromers. Here, we show that the related regulator SnoN forms a stable complex with the R-SMAD (SMAD3) and the Co-SMAD (SMAD4). To rationalize this stabilization at the molecular level, we determined the crystal structure of a complex between the SAND domain of SnoN and the MH2-domain of SMAD4. This structure shows a binding mode that is compatible with simultaneous coordination of R-SMADs. Our results show that SnoN, and SMAD heteromers can form a joint structural core for the binding of other transcription modulators. The results are of fundamental importance for our understanding of the molecular mechanisms behind the modulation of TGF-β signaling.

Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-beta signaling from tumor suppression to oncogenesis in early chronic hepatitis B.

PubMed

Murata, Miki; Matsuzaki, Koichi; Yoshida, Katsunori; Sekimoto, Go; Tahashi, Yoshiya; Mori, Shigeo; Uemura, Yoshiko; Sakaida, Noriko; Fujisawa, Junichi; Seki, Toshihito; Kobayashi, Kazuki; Yokote, Koutaro; Koike, Kazuhiko; Okazaki, Kazuichi

2009-04-01

Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor beta (TGF-beta) signaling involves both tumor suppression and oncogenesis. TGF-beta activates TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3-mediated signaling between tumor suppression and oncogenesis in HBx-expressing hepatocytes indicated that TbetaRI-dependent pSmad3C transmitted a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L- and pSmad3C-mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx-activated JNK/pSmad3L/c-Myc oncogenic pathway was enhanced, while the TbetaRI/pSmad3C/p21(WAF1) tumor-suppressive pathway was impaired as human and mouse HBx-associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor-suppressive pSmad3C developed HCC within 12 years. HBx shifts hepatocytic TGF-beta signaling from the tumor-suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV-infected liver specimens should prove clinically useful for predicting risk of HCC.

Genetic Variants in HSD17B3, SMAD3, and IPO11 Impact Circulating Lipids in Response to Fenofibrate in Individuals With Type 2 Diabetes.

PubMed

Rotroff, Daniel M; Pijut, Sonja S; Marvel, Skylar W; Jack, John R; Havener, Tammy M; Pujol, Aurora; Schluter, Agatha; Graf, Gregory A; Ginsberg, Henry N; Shah, Hetal S; Gao, He; Morieri, Mario-Luca; Doria, Alessandro; Mychaleckyi, Josyf C; McLeod, Howard L; Buse, John B; Wagner, Michael J; Motsinger-Reif, Alison A

2018-04-01

Individuals with type 2 diabetes (T2D) and dyslipidemia are at an increased risk of cardiovascular disease. Fibrates are a class of drugs prescribed to treat dyslipidemia, but variation in response has been observed. To evaluate common and rare genetic variants that impact lipid responses to fenofibrate in statin-treated patients with T2D, we examined lipid changes in response to fenofibrate therapy using a genomewide association study (GWAS). Associations were followed-up using gene expression studies in mice. Common variants in SMAD3 and IPO11 were marginally associated with lipid changes in black subjects (P < 5 × 10 -6 ). Rare variant and gene expression changes were assessed using a false discovery rate approach. AKR7A3 and HSD17B13 were associated with lipid changes in white subjects (q < 0.2). Mice fed fenofibrate displayed reductions in Hsd17b13 gene expression (q < 0.1). Associations of variants in SMAD3, IPO11, and HSD17B13, with gene expression changes in mice indicate that transforming growth factor-beta (TGF-β) and NRF2 signaling pathways may influence fenofibrate effects on dyslipidemia in patients with T2D. © 2017 American Society for Clinical Pharmacology and Therapeutics.

Silibinin inhibits the fibrotic responses induced by cigarette smoke via suppression of TGF-β1/Smad 2/3 signaling.

PubMed

Ko, Je-Won; Shin, Na-Rae; Park, Sung-Hyeuk; Lee, In-Chul; Ryu, Jung-Min; Kim, Ha-Jung; Cho, Young-Kwon; Kim, Jong-Choon; Shin, In-Sik

2017-08-01

Cigarette smoke (CS) is generally accepted as a major contributor to chronic obstructive pulmonary disease (COPD) which is characterized by chronic inflammation, fibrotic response, and airway obstruction. In this study, we investigated the preventive effects of silibinin, an active constitute of silymarin on CS and lipopolysaccharide (LPS) exposure-induced fibrotic response. Mice were exposed to CS for 1 h per day (8 cigarettes per day) for 4 weeks. On day 12 and 26, mice were treated with LPS intranasally. Silibinin (10 or 20 mg/kg) was administered orally 1 h before CS exposure. Silibinin markedly decreased the inflammatory cell count in the bronchoalveolar lavage fluid, and reduced levels of proinflammatory mediators. Silibinin suppressed CS + LPS-induced collagen deposition in lung tissue, as evidenced via immunohistochemistry and Masson's trichrome stain. Additionally, silibinin effectively inhibited CS + LPS-mediated expression of transforming growth factor-β1 (TGF-β1) and Smad 2/3 phosphorylation. Taken together, our data indicate that silibinin effectively inhibits the fibrotic response induced by CS + LPS exposure, possibly via suppression of TGF-β1/Smad 2/3 signaling, which results in reduced collagen deposition. These findings suggest that silibinin has therapeutic potential for the treatment of COPD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Two short segments of Smad3 are important for specific interaction of Smad3 with c-Ski and SnoN.

PubMed

Mizuide, Masafumi; Hara, Takane; Furuya, Toshio; Takeda, Masafumi; Kusanagi, Kiyoshi; Inada, Yuri; Mori, Masatomo; Imamura, Takeshi; Miyazawa, Keiji; Miyazono, Kohei

2003-01-03

c-Ski and SnoN are transcriptional co-repressors that inhibit transforming growth factor-beta signaling through interaction with Smad proteins. Among receptor-regulated Smads, c-Ski and SnoN bind more strongly to Smad2 and Smad3 than to Smad1. Here, we show that c-Ski and SnoN bind to the "SE" sequence in the C-terminal MH2 domain of Smad3, which is exposed on the N-terminal upper side of the toroidal structure of the MH2 oligomer. The "QPSMT" sequence, located in the vicinity of SE, supports the interaction with c-Ski and SnoN. Sequences similar to SE and QPSMT are found in Smad2, but not in Smad1. The N-terminal MH1 domain and linker region of Smad3 protrude from the N-terminal upper side of the MH2 oligomer toroid. Smurf2 induces ubiquitin-dependent degradation of SnoN, since it appears to be located close to SnoN through binding to the linker region of Smad2. In contrast, transcription factors Mixer and FoxH3 (FAST1) bind to the bottom side of the Smad3 MH2 toroid; therefore, c-Ski does not affect the interaction of Smads with these transcription factors. Our findings thus demonstrate the stoichiometry of how multiple molecules can associate with the Smad oligomers and how the Smad-interacting proteins functionally interact with each other.

Transcription factor specificity protein 1 modulates TGFβ1/Smad signaling to negatively regulate SIGIRR expression by human M1 macrophages stimulated with substance P.

PubMed

Yamaguchi, Rui; Sakamoto, Arisa; Yamaguchi, Reona; Haraguchi, Misa; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

2018-08-01

The stimuli inducing expression of single immunoglobulin IL-1-related receptor (SIGIRR) and the relevant regulatory mechanisms are not well defined. Transforming growth factor β1 (TGFβ1) delays internalization of neurokinin-1 receptor (NK1R) and subsequently enhances cellular signaling. This study investigated the effect of TGFβ1 on SIGIRR protein production by human M1 macrophages in response to stimulation with substance P (SP). SP caused upregulation of SIGIRR expression in a concentration-dependent manner, whereas aprepitant (an NK1R inhibitor) blunted this response. Silencing p38γMAPK or TAK-1 partially attenuated the response to SP stimulation, while TGFβ1/2/3 siRNA dramatically diminished it. SP induced much greater SIGIRR protein production than either lipopolysaccharide (a TLR4 agonist) or resiquimod (a TLR7/8 agonist). Unexpectedly, silencing of transcription factor specificity protein 1 (Sp1) led to significant upregulation of SIGIRR expression after SP stimulation, while KLF2 siRNA only partially enhanced it and Fli-1 siRNA reduced it. SP also upregulated TGFβ1 expression, along with a corresponding increase of SIGIRR protein, whereas silencing TGFβ1/2/3 blunted these responses. Sp1 siRNA or mithramycin (a gene-selective Sp1 inhibitor) significantly enhanced the expression of TGFβ1 and SIGIRR by macrophages after SP stimulation. Importantly, this effect of Sp1 siRNA on TGFβ1 and SIGIRR was blunted by siRNA for Smad2, Smad3, or Smad4, but not by TAK-1 siRNA. Next, we investigated the influence of transcription factor cross-talk on SIGIRR expression in response to SP. Co-transfection of macrophages with Sp1 siRNA and C/EBPβ or TIF1β siRNA attenuated the upregulation of SIGIRR by SP, while a combination of Sp1 siRNA and Fli-1 siRNA dramatically diminished it. In conclusion, TGFβ1 may be an intermediary between SP/NK1R activation and SIGIRR expression in Sp1 siRNA-transfected macrophages. In addition, Sp1 modulates TGFβ1/Smad signaling and negatively regulates SIGIRR protein production by macrophages after SP stimulation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors

PubMed Central

Kalra, Sean; Cleaver, Timothy G.; Merrick, Daniel; Wang, Xiao-Jing; Malkoski, Stephen P.

2015-01-01

Non-small cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer, however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC datasets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms including mutation, homozygous deletion, and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4−/− mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage while Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4 deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4 deficient tumors can be exploited by specific therapeutic strategies. PMID:25893305

Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH(2)-terminal kinase during human colorectal carcinogenesis.

PubMed

Yamagata, Hideo; Matsuzaki, Koichi; Mori, Shigeo; Yoshida, Katsunori; Tahashi, Yoshiya; Furukawa, Fukiko; Sekimoto, Go; Watanabe, Toshihiko; Uemura, Yoshiko; Sakaida, Noriko; Yoshioka, Kazuhiko; Kamiyama, Yasuo; Seki, Toshihito; Okazaki, Kazuichi

2005-01-01

Conversion of normal epithelial cells to tumors is associated with a shift in transforming growth factor-beta (TGF-beta) function: reduction of tumor suppressor activity and increase of oncogenic activity. However, specific mechanisms of this functional alteration during human colorectal carcinogenesis remain to be elucidated. TGF-beta signaling involves Smad2/3 phosphorylated at linker regions (pSmad2/3L) and COOH-terminal regions (pSmad2/3C). Using antibodies specific to each phosphorylation site, we herein showed that Smad2 and Smad3 were phosphorylated at COOH-terminal regions but not at linker regions in normal colorectal epithelial cells and that pSmad2/3C were located predominantly in their nuclei. However, the linker regions of Smad2 and Smad3 were phosphorylated in 31 sporadic colorectal adenocarcinomas. In particular, late-stage invasive and metastatic cancers typically showed a high degree of phosphorylation of Smad2/3L. Their extent of phosphorylation in 11 adenomas was intermediate between those in normal epithelial cells and adenocarcinomas. Whereas pSmad2L remained in the cytoplasm, pSmad3L was located exclusively in the nuclei of Ki-67-immunoreactive adenocarcinomas. In contrast, pSmad3C gradually decreased as the tumor stage progressed. Activated c-Jun NH(2)-terminal kinase in cancers could directly phosphorylate Smad2/3L. Although Mad homology 2 region sequencing in the Smad4 gene revealed a G/A substitution at codon 361 in one adenocarcinoma, the mutation did not correlate with phosphorylation. No mutations in the type II TGF-beta receptor and Smad2 genes were observed in the tumors. In conclusion, pSmad3C, which favors tumor suppressor activity of TGF-beta, was found to decrease, whereas c-Jun NH(2)-terminal kinase tended to induce the phosphorylation of Smad2/3L in human colorectal adenoma-carcinoma sequence.

Smad3 allostery links TGF-β receptor kinase activation to transcriptional control

PubMed Central

Qin, Bin Y.; Lam, Suvana S.; Correia, John J.; Lin, Kai

2002-01-01

Smad3 transduces the signals of TGF-βs, coupling transmembrane receptor kinase activation to transcriptional control. The membrane-associated molecule SARA (Smad Anchor for Receptor Activation) recruits Smad3 for phosphorylation by the receptor kinase. Upon phosphorylation, Smad3 dissociates from SARA and enters the nucleus, in which its transcriptional activity can be repressed by Ski. Here, we show that SARA and Ski recognize specifically the monomeric and trimeric forms of Smad3, respectively. Thus, trimerization of Smad3, induced by phosphorylation, simultaneously activates the TGF-β signal by driving Smad3 dissociation from SARA and sets up the negative feedback mechanism by Ski. Structural models of the Smad3/SARA/receptor kinase complex and Smad3/Ski complex provide insights into the molecular basis of regulation. PMID:12154125

Mucin1 shifts Smad3 signaling from the tumor-suppressive pSmad3C/p21(WAF1) pathway to the oncogenic pSmad3L/c-Myc pathway by activating JNK in human hepatocellular carcinoma cells.

PubMed

Li, Qiongshu; Liu, Guomu; Yuan, Hongyan; Wang, Juan; Guo, Yingying; Chen, Tanxiu; Zhai, Ruiping; Shao, Dan; Ni, Weihua; Tai, Guixiang

2015-02-28

Mucin1 (MUC1) is a transmembrane glycoprotein that acts as an oncogene in human hepatic tumorigenesis. Hepatocellular carcinoma (HCC) cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor beta (TGF-β) together with stimulation of its oncogenic activity as in MUC1 expressing HCC cells; however, molecular mechanisms remain largely unknown. Type I TGF-β receptor (TβRI) and c-Jun NH2-terminal kinase (JNK) differentially phosphorylate Smad3 mediator to create 2 phosphorylated forms: COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Here, we report that MUC1 overexpression in HCC cell lines suppresses TβRI-mediated pSmad3C signaling which involves growth inhibition by up-regulating p21(WAF1). Instead, MUC1 directly activates JNK to stimulate oncogenic pSmad3L signaling, which fosters cell proliferation by up-regulating c-Myc. Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo. In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients. Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

Nodal enhances the activity of FoxO3a and its synergistic interaction with Smads to regulate cyclin G2 transcription in ovarian cancer cells.

PubMed

Fu, G; Peng, C

2011-09-15

Nodal, a member of the transforming growth factor-β superfamily, has been recently shown to suppress cell proliferation and to stimulate the expression of cyclin G2 (CCNG2) in human epithelial ovarian cancer cells. However, the precise mechanisms underlying these events are not fully understood. In this study, we investigated the transcriptional regulation of CCNG2 by the Nodal signaling pathway. In ovarian cancer cells, overexpression of Nodal or its receptors, activin receptor-like kinase 7 (ALK7) or ALK4, resulted in an increase in the CCNG2 promoter activity. Several putative Forkhead box class O (FoxO)3a-binding sites are present in the human CCNG2 promoter and overexpression of FoxO3a enhanced the CCNG2 promoter activity. The functional FoxO3a-binding element (FBE) was mapped to a proximal region located between -398 and -380 bp (FBE1) through deletion and mutation analyses, as well as chromatin immunoprecipitation (IP) assay. Interestingly, mutation of the FBE1 not only abolished the effect of FoxO3a, but also blocked Nodal-induced CCNG2 transcription. Nodal stimulated FoxO3a mRNA and protein expression through the canonical Smad pathway and suppressed FoxO3a inactivation by inhibiting AKT activity. Silencing of FoxO3a using small interfering RNA significantly reduced the effect of Nodal on the CCNG2 promoter activity. On the other hand, overexpression of Smad2 and Smad3 enhanced the FoxO3a-induced CCNG2 promoter activity whereas knockdown of Smad4 blocked the activity of FoxO3a. Furthermore, IP assays revealed that FoxO3a formed complexes with Smad proteins and that Nodal enhanced the binding of FoxO3a to the CCNG2 promoter. Finally, silencing of FoxO3a reversed the inhibitory effect of Nodal on cell proliferation. Taken together, these findings demonstrated that Nodal signaling promotes CCNG2 transcription by upregulating FoxO3a expression, inhibiting FoxO3a phosphorylation and enhancing its synergistic interaction with Smads. These results also suggest that FoxO3a is an important mediator of Nodal signaling in ovarian cancer cells.

Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells

PubMed Central

Cushing, Melinda C.; Mariner, Peter D.; Liao, Jo-Tsu; Sims, Evan A.; Anseth, Kristi S.

2008-01-01

This study aimed to identify signaling pathways that oppose connective tissue fibrosis in the aortic valve. Using valvular interstitial cells (VICs) isolated from porcine aortic valve leaflets, we show that basic fibroblast growth factor (FGF-2) effectively blocks transforming growth factor-β1 (TGF-β1)-mediated myofibroblast activation. FGF-2 prevents the induction of α-smooth muscle actin (αSMA) expression and the exit of VICs from the cell cycle, both of which are hallmarks of myofibroblast activation. By blocking the activity of the Smad transcription factors that serve as the downstream nuclear effectors of TGF-β1, FGF-2 treatment inhibits fibrosis in VICs. Using an exogenous Smad-responsive transcriptional promoter reporter, we show that Smad activity is repressed by FGF-2, likely an effect of the fact that FGF-2 treatment prevents the nuclear localization of Smads in these cells. This appears to be a direct effect of FGF signaling through mitogen-activated protein kinase (MAPK) cascades as the treatment of VICs with the MAPK/extracellular regulated kinase (MEK) inhibitor U0126 acted to induce fibrosis and blocked the ability of FGF-2 to inhibit TGF-β1 signaling. Furthermore, FGF-2 treatment of VICs blocks the development of pathological contractile and calcifying phenotypes, suggesting that these pathways may be utilized in the engineering of effective treatments for valvular disease.—Cushing, M. C., Mariner, P. D., Liao, J. T., Sims, E. A., Anseth, K. S. Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells. PMID:18218921

Compound Astragalus and Salvia miltiorrhiza extracts suppress hepatocarcinogenesis by modulating transforming growth factor-β/Smad signaling.

PubMed

Hu, Xiangpeng; Rui, Wenjuan; Wu, Chao; He, Shufang; Jiang, Jiemei; Zhang, Xiaoxiang; Yang, Yan

2014-06-01

Previous studies showed Compound Astragalus and Salvia miltiorrhiza extract (CASE), extract from Astragalus membranaceus and Salvia miltiorhiza, significantly suppresses hepatocellular carcinoma (HCC) in rats induced by diethylinitrosamine (DEN), and in vitro experiments further demonstrated that CASE's anti-HepG2 cell invasion is associated with transforming growth factor-β (TGF-β). We hypothesized that CASE's suppression of HCC is modulated by TGF-β/Smad signaling, and we conducted this in vivo study to test this hypothesis. Rats were divided into the normal control, the DEN group, and three CASE (60, 120, and 240 mg/kg) treatment groups. The expression of phosphorylation(p) Smad both at C-terminal and linker region, plasminogen activator inhibitor 1, and Smad4 and Smad7 of liver tissues were measured and compared across the five groups. The positive staining of pSmad2L and pSmad3L increased both in hepatoma nodule areas and adjacent relatively normal liver tissues in rats treated with DEN, while the positive staining of pSmad2C and pSmad3C increased only in relatively normal liver tissues adjacent to hepatoma tissues. The elevated expression of pSmad2C, pSmad2L, pSmad3L, Smad4, and plasminogen activator inhibitor 1 proteins were suppressed by CASE in a dose-dependent manner. CASE treatment also significantly reduced the intranuclear amounts of pSmad2L and pSmad3L, and upregulated the elevation of pSmad3C positive cells and protein expression in a dose-dependent manner. The results suggest that CASE significantly suppresses HCC progression by mediating TGF-β/Smad signaling, especially by modulating Smad3 phosphorylation both at the C-terminal and linker region. © 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

Zinc affects miR-548n, SMAD4, SMAD5 expression in HepG2 hepatocyte and HEp-2 lung cell lines.

PubMed

Grider, Arthur; Lewis, Richard D; Laing, Emma M; Bakre, Abhijeet A; Tripp, Ralph A

2015-12-01

MicroRNAs affect disease progression and nutrient status. miR-548n increased 57 % in Zn supplemented plasma from adolescent females (ages 9 to 13 years). The purpose of this study was to determine the effects of Zn concentration in cell culture on the expression of miR-548n, SMAD4 and SMAD5 in hepatocyte (HepG2) and lung epithelium (HEp-2) cell lines. Cells were incubated for 48 h in media containing 10 % Chelex 100-treated FBS (0 μM Zn), or with 15 or 50 μM Zn, before isolation of total RNA and cDNA. Expression of miR-548n, SMAD4 and SMAD5 was measured by qPCR. The ΔΔCT method was used to calculate the fold-change, and 15 µM expression levels were used as reference values. HepG2 miR-548n expression decreased 5-fold, and SMAD4 expression increased 4-fold in the absence of Zn, while HEp-2 miR-548n expression increased 10.5-fold, and SMAD5 expression increased 20-fold in the absence of Zn. HEp-2 miR-548n expression increased 23-fold, while SMAD4 expression decreased twofold, in 50 μM Zn-treated cells. However, SMAD4 and SMAD5 expression was not correlated. These data indicate that miR-548n expression is in part regulated by Zn in a cell-specific manner. SMAD4 and SMAD5 are genes in the TGF-β/BMP signaling pathway, and SMAD5 is a putative target for miR-548n; Zn participates in regulating this pathway through controlling SMAD4 and SMAD5 expression. However, SMAD5 expression may be more sensitive to Zn than to miR-548n since SMAD5 expression was not inversely correlated with miR-548n expression.

PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1

NASA Astrophysics Data System (ADS)

Saijilafu; Hur, Eun-Mi; Liu, Chang-Mei; Jiao, Zhongxian; Xu, Wen-Lin; Zhou, Feng-Quan

2013-10-01

In contrast to neurons in the central nervous system, mature neurons in the mammalian peripheral nervous system (PNS) can regenerate axons after injury, in part, by enhancing intrinsic growth competence. However, the signalling pathways that enhance the growth potential and induce spontaneous axon regeneration remain poorly understood. Here we reveal that phosphatidylinositol 3-kinase (PI3K) signalling is activated in response to peripheral axotomy and that PI3K pathway is required for sensory axon regeneration. Moreover, we show that glycogen synthase kinase 3 (GSK3), rather than mammalian target of rapamycin, mediates PI3K-dependent augmentation of the growth potential in the PNS. Furthermore, we show that PI3K-GSK3 signal is conveyed by the induction of a transcription factor Smad1 and that acute depletion of Smad1 in adult mice prevents axon regeneration in vivo. Together, these results suggest PI3K-GSK3-Smad1 signalling as a central module for promoting sensory axon regeneration in the mammalian nervous system.

Nuclear Function of Smad7 Promotes Myogenesis▿

PubMed Central

Miyake, Tetsuaki; Alli, Nezeka S.; McDermott, John C.

2010-01-01

In the “canonical” view of transforming growth factor β (TGF-β) signaling, Smad7 plays an inhibitory role. While Smad7 represses Smad3 activation by TGF-β, it does not reverse the inhibitory effect of TGF-β on myogenesis, suggesting a different function in myogenic cells. We previously reported a promyogenic role of Smad7 mediated by an interaction with MyoD. Based on this association, we hypothesized a possible nuclear function of Smad7 independent of its role at the level of the receptor. We therefore engineered a chimera of Smad7 with a nuclear localization signal (NLS), which serves to prevent and therefore bypass binding to the TGF-β receptor while concomitantly constitutively localizing Smad7 to the nucleus. This Smad7-NLS did not repress Smad3 activation by TGF-β but did retain its ability to enhance myogenic gene activation and phenotypic myogenesis, indicating that the nuclear, receptor-independent function of Smad7 is sufficient to promote myogenesis. Furthermore, Smad7 physically interacts with MyoD and antagonizes the repressive effects of active MEK on MyoD. Reporter and myogenic conversion assays indicate a pivotal regulation of MyoD transcriptional properties by the balance between Smad7 and active MEK. Thus, Smad7 has a nuclear coactivator function that is independent of TGF-β signaling and necessary to promote myogenic differentiation. PMID:19995910

Decreased levels of active SMAD2 correlate with poor prognosis in gastric cancer.

PubMed

Wu, Yijun; Li, Qi; Zhou, Xinhui; Yu, Jiren; Mu, Yunchuan; Munker, Stefan; Xu, Chengfu; Shen, Zhe; Müllenbach, Roman; Liu, Yan; Li, Li; Gretz, Norbert; Zieker, Derek; Li, Jun; Matsuzaki, Kouichi; Li, Youming; Dooley, Steven; Weng, Honglei

2012-01-01

TGF-β plays a dual role in the progression of human cancer. During the early stages of carcinogenesis, TGF-β functions as a tumor suppressor. During the late stages of tumor development, however, TGF-β can promote tumor growth and metastasis. A shift in Smad2/3 phosphorylation from the carboxy terminus to linker sites is a key event determining biological function of TGF-β in colorectal and hepatocellular carcinoma. In the present study, we investigated the potential role of differential Smad2/3 phosphorylation in gastric adenocarcinoma. Immunohistochemical staining with anti-P-Smad2/3C and P-Smad2/3L antibodies was performed on 130 paraffin-embedded gastric adenocarcinoma specimens. The relationship between P-Smad2/3C and P-Smad2/3L immunohistochemical score and clinicopathologic characteristics of patients was analyzed. Real time PCR was used to measure mRNA expression of Smad2 and Smad3 in cancer and surrounding non-tumor tissue. No significant P-Smad2L and/or P-Smad3L positive staining was detected in the majority of specimens (positive staining in 18/130 samples). Positive P-Smad2/3L staining was not associated with a decrease in carboxyterminal phosphorylation staining. Loss of P-Smad2C remarkably correlated with depth of tumor infiltration and poor differentiation of cancer cells in patients with gastric cancer. No correlation was detectable between P-Smad3C and clinicopathologic characteristics of gastric adenocarcinoma. However, co-staining analysis revealed that P-Smad3C co-localised with α-SMA and collagen I in gastric cancer cells, indicating a potential link between P-Smad3C and epithelial-to-mesenchymal transition of cancer. Real time PCR demonstrated reduced mRNA expression of Smad2 in gastric cancer when compared with surrounding non-tumor tissue in 15/16 patients. Loss of P-Smad2C tightly correlated with cancer invasion and poor differentiation in gastric cancer. Contrary to colorectal and hepatocellular carcinoma, canonical carboxy-terminal phosphorylation, but not linker phosphorylation, of Smad2 is critical for gastric cancer.

Combination immunohistochemistry for SMAD4 and Runt-related transcription factor 3 may identify a favorable prognostic subgroup of pancreatic ductal adenocarcinomas.

PubMed

Lee, Yangkyu; Lee, Hyejung; Park, Hyunjin; Kim, Jin-Won; Hwang, Jin-Hyeok; Kim, Jaihwan; Yoon, Yoo-Seok; Han, Ho-Seong; Kim, Haeryoung

2017-09-29

SMAD4/DPC4 mutations have been associated with aggressive behavior in pancreatic ductal adenocarcinomas (PDAC), and it has recently been suggested that RUNX3 expression combined with SMAD4 status may predict the metastatic potential of PDACs. We evaluated the prognostic utility of SMAD4/RUNX3 status in human PDACs by immunohistochemistry. Immunohistochemical stains were performed for SMAD4 and RUNX3 on 210 surgically resected PDACs, and the results were correlated with the clinicopathological features. Loss of SMAD4 expression was associated with poor overall survival (OS) ( p = 0.015) and progression-free survival (PFS) ( p = 0.044). Nuclear RUNX3 expression was associated with decreased OS ( p = 0.010) and PFS ( p = 0.009), and more frequent in poorly differentiated PDACs ( p = 0.037). On combining RUNX3/SMAD4 status, RUNX3-/SMAD4+ PDACs demonstrated longer OS ( p = 0.008, median time; RUNX3-/SMAD4+ 34 months, others 17 months) and PFS ( p = 0.009, median time; RUNX3-/SMAD4+ 29 months, others 8 months) compared to RUNX3+/SMAD4+ and SMAD4- groups; RUNX3-/SMAD4+ was a significant independent predictive factor for both OS [ p = 0.025, HR 1.842 (95% CI 1.079-3.143)] and PFS [ p = 0.020, HR 1.850 (95% CI 1.100-3.113)]. SMAD4-positivity with RUNX3-negativity was a significant independent predictive factor for favorable OS and PFS in PDAC. This is the first and large clinicopathological study of RUNX3/SMAD4 expression status in human PDAC. Combination immunohistochemistry for SMAD4 and RUNX3 may help identify a favorable prognostic subgroup of PDAC.

Combination immunohistochemistry for SMAD4 and Runt-related transcription factor 3 may identify a favorable prognostic subgroup of pancreatic ductal adenocarcinomas

PubMed Central

Lee, Yangkyu; Lee, Hyejung; Park, Hyunjin; Kim, Jin-Won; Hwang, Jin-Hyeok; Kim, Jaihwan; Yoon, Yoo-Seok; Han, Ho-Seong; Kim, Haeryoung

2017-01-01

Purposes SMAD4/DPC4 mutations have been associated with aggressive behavior in pancreatic ductal adenocarcinomas (PDAC), and it has recently been suggested that RUNX3 expression combined with SMAD4 status may predict the metastatic potential of PDACs. We evaluated the prognostic utility of SMAD4/RUNX3 status in human PDACs by immunohistochemistry. Materials and Methods Immunohistochemical stains were performed for SMAD4 and RUNX3 on 210 surgically resected PDACs, and the results were correlated with the clinicopathological features. Results Loss of SMAD4 expression was associated with poor overall survival (OS) (p = 0.015) and progression-free survival (PFS) (p = 0.044). Nuclear RUNX3 expression was associated with decreased OS (p = 0.010) and PFS (p = 0.009), and more frequent in poorly differentiated PDACs (p = 0.037). On combining RUNX3/SMAD4 status, RUNX3-/SMAD4+ PDACs demonstrated longer OS (p = 0.008, median time; RUNX3-/SMAD4+ 34 months, others 17 months) and PFS (p = 0.009, median time; RUNX3-/SMAD4+ 29 months, others 8 months) compared to RUNX3+/SMAD4+ and SMAD4- groups; RUNX3-/SMAD4+ was a significant independent predictive factor for both OS [p = 0.025, HR 1.842 (95% CI 1.079-3.143)] and PFS [p = 0.020, HR 1.850 (95% CI 1.100-3.113)]. Conclusions SMAD4-positivity with RUNX3-negativity was a significant independent predictive factor for favorable OS and PFS in PDAC. This is the first and large clinicopathological study of RUNX3/SMAD4 expression status in human PDAC. Combination immunohistochemistry for SMAD4 and RUNX3 may help identify a favorable prognostic subgroup of PDAC. PMID:29100342

Smad4 Loss Synergizes with TGFα Overexpression in Promoting Pancreatic Metaplasia, PanIN Development, and Fibrosis

PubMed Central

Garcia-Carracedo, Dario; Yu, Chih-Chieh; Akhavan, Nathan; Fine, Stuart A.; Schönleben, Frank; Maehara, Naoki; Karg, Dillon C.; Xie, Chuangao; Qiu, Wanglong; Fine, Robert L.; Remotti, Helen E.; Su, Gloria H.

2015-01-01

Aims While overexpression of TGFα has been reported in human pancreatic ductal adenocarcinoma (PDAC), mice with overexpressed TGFα develop premalignant pancreatic acinar-to-ductal metaplasia (ADM) but not PDAC. TGF-β signaling pathway is pivotal to the development of PDAC and tissue fibrosis. Here we sought to investigate the interplay between TGFα and TGF-β signaling in pancreatic tumorigenesis and fibrosis, namely via Smad4 inactivation. Methods The MT-TGFα mouse was crossed with a new Smad4 conditional knock-out mouse (Smad4flox/flox;p48-Cre or S4) to generate Smad4flox/flox;MT-TGFα;p48-Cre (STP). After TGFα overexpression was induced with zinc sulfate water for eight months, the pancreata of the STP, MT-TGFα, and S4 mice were examined for tumor development and fibrotic responses. PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC). Qualitative analysis of fibrosis was analyzed by Trichrome Masson and Sirius Red staining, while vimentin was used for quantification. Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR. Results Our STP mice exhibited advanced ADM, increased fibrosis, increased numbers of PanIN lesions, overexpression of chronic pancreatitis-related marker Muc6, and elevated expression of desmoplasia-associated marker Col1A1, compared to the MT-TGFα mice. The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas. The phenotype of the STP mice represents a transient state from ADMs to PanINs, closely mimicking the interface area seen in human chronic pancreatitis associated with PDAC. Conclusion We have documented a novel mouse model, the STP mice, which displayed histologic presentations reminiscent to those of human chronic pancreatitis with signs of early tumorigenesis. The STP mice could be a suitable animal model for interrogating the transition of chronic pancreatitis to pancreatic cancer. PMID:25803032

Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

PubMed

González-Sastre, Alejandro; Molina, Ma Dolores; Saló, Emili

2012-01-01

Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea.

Evidence Supporting a Functional Requirement of SMAD4 for Bovine Preimplantation Embryonic Development: A Potential Link to Embryotrophic Actions of Follistatin1

PubMed Central

Lee, Kyung-Bon; Zhang, Kun; Folger, Joseph K.; Knott, Jason G.; Smith, George W.

2014-01-01

ABSTRACT Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent. PMID:25031360

Constitutive Smad linker phosphorylation in melanoma: a mechanism of resistance to transforming growth factor-β-mediated growth inhibition.

PubMed

Cohen-Solal, Karine A; Merrigan, Kim T; Chan, Joseph L-K; Goydos, James S; Chen, Wenjin; Foran, David J; Liu, Fang; Lasfar, Ahmed; Reiss, Michael

2011-06-01

Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition. 2011 John Wiley & Sons A/S.

Reversible Smad-dependent signaling between tumor suppression and oncogenesis.

PubMed

Sekimoto, Go; Matsuzaki, Koichi; Yoshida, Katsunori; Mori, Shigeo; Murata, Miki; Seki, Toshihito; Matsui, Hirofumi; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2007-06-01

Cancer cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor-beta (TGF-beta) together with stimulation of its oncogenic activity as in Ras-transformed cells; however, molecular mechanisms remain largely unknown. TGF-beta activates both its type I receptor (TbetaRI) and c-Jun NH2-terminal kinase (JNK), which phosphorylate Smad2 and Smad3 at the COOH-terminal (pSmad2/3C) and linker regions (pSmad2/3L). Here, we report that Ras transformation suppresses TbetaRI-mediated pSmad3C signaling, which involves growth inhibition by down-regulating c-Myc. Instead, hyperactive Ras constitutively stimulates JNK-mediated pSmad2/3L signaling, which fosters tumor invasion by up-regulating plasminogen activator inhibitor-1 and matrix metalloproteinase-1 (MMP-1), MMP-2, and MMP-9. Conversely, selective blockade of linker phosphorylation by a mutant Smad3 lacking JNK-dependent phosphorylation sites results in preserved tumor-suppressive function via pSmad3C in Ras-transformed cells while eliminating pSmad2/3L-mediated invasive capacity. Thus, specific inhibition of the JNK/pSmad2/3L pathway should suppress cancer progression by shifting Smad-dependent signaling from oncogenesis to tumor suppression.

Constitutive Smad linker phosphorylation in melanoma: A mechanism of resistance to Transforming Growth Factor-β-mediated growth inhibition

PubMed Central

Cohen-Solal, Karine A.; Merrigan, Kim T.; Chan, Joseph L.-K.; Goydos, James S.; Chen, Wenjin; Foran, David J.; Liu, Fang; Lasfar, Ahmed; Reiss, Michael

2011-01-01

SUMMARY Melanoma cells are resistant to Transforming Growth Factor-β (TGFβ)-induced cell cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and in tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15INK4B and p21WAF1, as compared with cells transfected with wild-type Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared to wild-type Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition. PMID:21477078

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hama, Kouji; Ohnishi, Hirohide; Aoki, Hiroyoshi

2006-02-17

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-{beta}{sub 1} inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-{beta}{sub 1}-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-{beta}{sub 1}-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSCmore » proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-{beta}{sub 1}-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.« less

MIR-27a regulates the TGF-β signaling pathway by targeting SMAD2 and SMAD4 in lung cancer.

PubMed

Chae, Dong-Kyu; Ban, Eunmi; Yoo, Young Sook; Kim, Eunice EunKyeong; Baik, Ja-Hyun; Song, Eun Joo

2017-08-01

The transforming growth factor-β (TGF-β) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-β signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-β-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy. © 2017 Wiley Periodicals, Inc.

Transcription Factor FoxO1 Is Essential for Enamel Biomineralization

PubMed Central

Poché, Ross A.; Sharma, Ramaswamy; Garcia, Monica D.; Wada, Aya M.; Nolte, Mark J.; Udan, Ryan S.; Paik, Ji-Hye; DePinho, Ronald A.; Bartlett, John D.; Dickinson, Mary E.

2012-01-01

The Transforming growth factor β (Tgf-β) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-β signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta. PMID:22291941

Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

PubMed

Li, Xuemei; Li, Xinlei; Lv, Xiaohong; Xiao, Jianbing; Liu, Baoquan; Zhang, Yafang

2017-09-01

Smad4 is a critical factor in the TGF-β pathway and is involved in tumor progression and metastasis, but the role of Smad4 in colon cancer cells is unclear. The aim of this study is to explore the effect and the underlying mechanism of Smad4 on the growth, migration and apoptosis of colon cancer cells as well as vascular endothelial growth factor (VEGF)-A and VEGF-C secreted by these cells. In this study, we showed that Smad4, VEGF-A, and VEGF-C are independent prognostic factors of colon cancer, and Smad4 expression was negatively correlated with VEGF-A and -C in samples. We found that Smad4 mRNA and protein levels in colon cancer cells, particularly in HCT-116 cells, were significantly lower than those in the human intestinal epithelial cell line (HIEC). Smad4 overexpression promoted tumor cell apoptosis, inhibited VEGF-A and -C expression in vitro and in vivo, but had no effect on cell proliferation and migration. Tail vein injection of the virus inhibited xenograft growth in nude mice. Importantly, we also demonstrated that Smad4 could increase the phosphorylation level of Smad3, but not Smad2, which may be one of the mechanisms underlying these effects of Smad4 in colon cancer. Therefore, Smad4 may be a new target for the treatment of colon cancer. Anat Rec, 300:1560-1569, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Interleukin 1 β-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFβ signaling in primary human mesenchymal stem cells.

PubMed

van den Akker, Guus G; van Beuningen, Henk M; Vitters, Elly L; Koenders, Marije I; van de Loo, Fons A; van Lent, Peter L; Blaney Davidson, Esmeralda N; van der Kraan, Peter M

2017-12-01

Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications. Co-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA 12 -luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA 12 -luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1β-induced SMAD2 linker modifications and CAGA 12 -luciferase activity. TGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1β-induced TGFβ signal modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Kaempferol Suppresses Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

PubMed

Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

2015-07-01

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-1791

PubMed Central

Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

2015-01-01

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non–small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1–induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1–mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1–mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1–induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1–mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1–induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1–induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway.

PubMed

Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

2013-06-14

Hydrogen peroxide (H2O2) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H2O2 are less understood. Here we report an important mechanism for antagonistic effects of H2O2 on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H2O2 (0.05-0.2 mM) completely blocked TGF-β1-mediated induction of p15(INK4B) expression and increase of its promoter activity. Interestingly, H2O2 selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H2O2 increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H2O2 on TGF-β1-induced increase of p15(INK4B)-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H2O2 as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H2O2 antagonizes the cytostatic function of TGF-β1. Copyright © 2013 Elsevier Inc. All rights reserved.

IGF-binding proteins mediate TGF-beta 1-induced apoptosis in bovine mammary epithelial BME-UV1 cells.

PubMed

Gajewska, Małgorzata; Motyl, Tomasz

2004-10-01

TGF-beta 1 is an antiproliferative and apoptogenic factor for mammary epithelial cells (MEC) acting in an auto/paracrine manner and thus considered an important local regulator of mammary tissue involution. However, the apoptogenic signaling pathway induced by this cytokine in bovine MEC remains obscure. The present study was focused on identification of molecules involved in apoptogenic signaling of transforming growth factor-beta 1 (TGF-beta 1) in the model of bovine mammary epithelial cell line (BME-UV1). Laser scanning cytometry (LSC), Western blot and electrophoretic mobility shift assay (EMSA) were used for analysis of expression and activity of TGF-beta 1-related signaling molecules. The earliest response occurring within 1-2 h after TGF-beta 1 administration was an induction and activation of R-Smads (Smad2 and Smad3) and Co-Smad (Smad4). An evident formation of Smad-DNA complexes began from 2nd hour after MEC exposure to TGF-beta 1. Similarly to Smads, proteins of AP1 complex: phosphorylated c-Jun and JunD appeared to be early reactive molecules; however, an increase in their expression was detected only in cytosolic fraction. In the next step, an increase of IGF binding protein-3 (IGFBP-3) and IGFBP-4 expression was observed from 6th hour followed by a decrease in the activity of protein kinase B (PKB/Akt), which occurred after 24 h of MEC exposure to TGF-beta 1. The decrease in PKB/Akt activity coincided in time with the decline of phosphorylated Bad expression (inactive form). Present study supported additional evidence that stimulation of insulin-like growth factor I (IGF-I) was associated with complete abrogation of TGF-beta 1-induced activation of Bad and Bax and in the consequence protection against apoptosis. In conclusion, apoptotic effect of TGF-beta 1 in bovine MEC is mediated by IGFBPs and occurs through IGF-I sequestration, resulting in inhibition of PKB/Akt-dependent survival pathway.

Structural basis for the versatile interactions of Smad7 with regulator WW domains in TGF-β pathways

PubMed Central

Aragón, Eric; Goerner, Nina; Xi, Qiaoran; Gomes, Tiago; Gao, Sheng; Massagué, Joan; Macias, Maria J.

2012-01-01

Summary TGF-β and BMP signaling is mediated by Smads 1–5 (R-Smads and Co-Smads) and inhibited by Smad7, a major hub of regulation of TGF-β and BMP receptors by negative feedback and antagonistic signals. The transcription coactivator YAP and the E3 ubiquitin ligases Smurf1/2 and Nedd4L target R-Smads for activation or degradation, respectively. Pairs of WW domain in these regulators bind PY motifs and adjacent CDK/MAPK and GSK3 phosphorylation sites in R-Smads in a selective and regulated manner. In contrast, here we show that Smad7 binds YAP, Smurf1, Smurf2 and Nedd4L constitutively, the binding involving a PY motif in Smad7 and no phosphorylation. We also provide a structural basis for how regulators that use WW domain pairs for selective interactions with R-Smads, resort to one single versatile WW domain for binding Smad7 to centralize regulation in the TGF-β and BMP pathways. PMID:22921829

Reversible Human TGF-β Signal Shifting between Tumor Suppression and Fibro-Carcinogenesis: Implications of Smad Phospho-Isoforms for Hepatic Epithelial-Mesenchymal Transitions.

PubMed

Yoshida, Katsunori; Murata, Miki; Yamaguchi, Takashi; Matsuzaki, Koichi; Okazaki, Kazuichi

2016-01-12

Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are observed during both physiological liver wound healing and the pathological fibrotic/carcinogenic (fibro-carcinogenetic) process. TGF-β and pro-inflammatory cytokine are considered to be the major factors accelerating liver fibrosis and promoting liver carcinogenesis. Smads, consisting of intermediate linker regions connecting Mad homology domains, act as the intracellular mediators of the TGF-β signal transduction pathway. As the TGF-β receptors, c-Jun N-terminal kinase and cyclin-dependent kinase, differentially phosphorylate Smad2/3, we have generated numerous antibodies against linker (L) and C-terminal (C) phosphorylation sites in Smad2/3 and identified four types of phosphorylated forms: cytostatic COOH-terminally-phosphorylated Smad3 (pSmad3C), mitogenic pSmad3L (Ser-213) signaling, fibrogenic pSmad2L (Ser-245/250/255)/C signaling and migratory pSmad2/3L (Thr-220/179)/C signaling. After acute liver injury, TGF-β upregulates pSmad3C signaling and terminates pSmad3L (Ser-213)-mediated hepatocyte proliferation. TGF-β and pro-inflammatory cytokines cooperatively enhance collagen synthesis by upregulating pSmad2L (Thr-220)/C and pSmad3L (Thr-179)/C pathways in activated hepatic stellate cells. During chronic liver injuries, hepatocytes persistently affected by TGF-β and pro-inflammatory cytokines eventually become pre-neoplastic hepatocytes. Both myofibroblasts and pre-neoplastic hepatocyte exhibit the same carcinogenic (mitogenic) pSmad3L (Ser-213) and fibrogenic pSmad2L (Ser-245/250/255)/C signaling, with acquisition of fibro-carcinogenic properties and increasing risk of hepatocellular carcinoma (HCC). Firstly, we review phospho-Smad-isoform signalings in epithelial and mesenchymal cells in physiological and pathological conditions and then consider Smad linker phosphorylation as a potential target for pathological EMT during human fibro-carcinogenesis, because human Smad phospho-isoform signals can reverse from fibro-carcinogenesis to tumor-suppression in a process of MET after therapy.

TGF-β induces fascin expression in gastric cancer via phosphorylation of smad3 linker area.

PubMed

Li, Liling; Cao, Fang; Liu, Baoan; Luo, Xiaojuan; Ma, Xin; Hu, Zhongliang

2015-01-01

Fascin is an actin-bundling protein critical for tumor invasion. TGF-β could induce fascin expression in gastric cancer cells. In this study, we attempted to explore the role of p-smad3L in the expression of fascin induced by TGF-β in gastric cancer cells. Pseudopodia were evaluated by immunofluorescence. Fascin expression was detected by RT-PCR and western blot. Smad3 siRNA was used to repress the endogenous smad3. The phosphorylations of smad3 linker region at sites s204, s208 and s213 were detected by western blot. The fascin promoter reporter activity was measured by dual luciferase assay. TGF-β could increase the formation of pseudopodia and the expression of fascin in gastric cancer cells. Smad3 depletion abrogated the expression of fascin induced by TGF-β. The phosphorylation of smad3 linker region at serine 204, 208 and 213 was enhanced in gastric cancer cells after TGF-β treatment. The fascin promoter reporter activity was significantly enhanced with TGF-β treatment in both wild-type Smad3 group and Smad3EPSM group (P<0.05). Furthermore, the fascin promoter reporter activity in the wild-type Smad3 transfectant cells was significantly higher than that in Smad3EPSM cells (P<0.05). fascin expression induced by TGF-β depends on smad3, at least in part, depends on smad3 linker phosphorylation.

Different modifications of phosphorylated Smad3C and Smad3L through TGF-β after spinal cord injury in mice.

PubMed

Joko, Masahiro; Osuka, Koji; Usuda, Nobuteru; Atsuzawa, Kimie; Aoyama, Masahiro; Takayasu, Masakazu

2013-08-09

Transforming growth factor-β (TGF-β) is an anti-inflammatory cytokine and is expressed in the injured spinal cord. TGF-β signals through receptors to activate Smad proteins, which translocate into the nucleus. In the present study, we investigated the chronological alterations and cellular locations of the TGF-β/Smad signaling pathway following spinal cord injury (SCI) in mice. ELISA analysis showed that the concentration of interleukin-6 (IL-6) in injured spinal cords significantly increases immediately after SCI, while the concentration of TGF-β gradually increased after SCI, peaked at 2 days, and then gradually decreased. Immunohistochemical studies revealed that Smad3 was mainly expressed in neurons of the spinal cord. Phosphorylated Smad3 at the C-terminus (p-Smad3C) was stained within the motor neurons in the anterior horn, while phosphorylated Smad3 at the linker regions (p-Smad3L) was expressed in astrocytes within gray matter. These findings suggest that SCI induces gradual increases in TGF-β and induces different activation of p-Smad3C and p-Smad3L. Phosphorylated Smad3C might be involved in neuronal degeneration after SCI, and p-Smad3L may play a role in glial scar formation by astrocytes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway.

PubMed

Li, Ren-Feng; Shang, Yu; Liu, Di; Ren, Ze-Song; Chang, Zhijie; Sui, Sen-Fang

2007-11-30

Smad1, a downstream regulator of the bone morphogenetic protein (BMP) receptors, is tightly regulated by the ubiquitin-proteasomal degradation system. To dissect the mechanisms that underlie the regulation of Smad1, it is important to investigate the specific ubiquitination site(s) in Smad1. Here we report that the alpha-NH(2) group of the N terminus and the epsilon-NH(2) groups of internal lysine residues 116, 118 and 269 (K116, K118 and K269) of Smad1 are ubiquitin acceptor sites mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). The in vitro degradation assay indicates that ubiquitination at the N terminus partially contributes to the degradation of Smad1. Furthermore, we demonstrate that the ubiquitination level of pseudo-phosphorylated Smad1 by CHIP is stronger than that of wild-type Smad1 and can be strongly inhibited by a phosphorylated tail of Smad1, PIS(pS)V(pS). Third, our results indicate that Hsp70 facilitates CHIP-mediated poly-ubiquitination of Smad1 whereas it attenuates CHIP-meditated mono-ubiquitination of Smad1. Finally, consistent with the in vitro observation, we show that CHIP preferentially mediates the degradation of phospho-Smad1/5 in vivo. Taken together, these results provide us a hint that CHIP might preferentially regulate phosphorylated Smad1 and thus the BMP signaling.

Temporal Control of Transforming Growth Factor (TGF) - Betal Expression on Mammary Cell Multistep Transformation

DTIC Science & Technology

2000-10-01

phosphorylation of Smad2 tumors, EMT appears to be initiated by TGF-P produced and Smad3 at specific Erk consensus sites in the linker by peritumoral host...1243-1252. linker region of Smad2 and Smad3 , which, in turn, inhibit Smad accumula- Inhibition of autocrine TGF-j signaling, by expression of dominant...mediated mostly by TGF-P1 and TGF-j2 are potent immunosuppressants the receptor specific Smad2 and Smad3 proteins [47,48], [73]. Thus, elevated levels

Aging and oxidative stress reduce the response of human articular chondrocytes to insulin-like growth factor 1 and osteogenic protein 1.

PubMed

Loeser, Richard F; Gandhi, Uma; Long, David L; Yin, Weihong; Chubinskaya, Susan

2014-08-01

To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). Chondrocytes isolated from normal articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50-100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis, and immunoblotting of cell lysates was performed to analyze cell signaling. Confocal microscopy was performed to measure nuclear translocation of Smad4. Chondrocytes isolated from the articular cartilage of tissue donors ranging in age from 24 years to 81 years demonstrated an age-related decline in proteoglycan synthesis stimulated by IGF-1 and IGF-1 plus OP-1. Induction of oxidative stress inhibited both IGF-1- and OP-1-stimulated proteoglycan synthesis. Signaling studies showed that oxidative stress inhibited IGF-1-stimulated Akt phosphorylation while increasing phosphorylation of ERK, and that these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation, which resulted in phosphorylation of Smad1 at the Ser(206) inhibitory site and reduced nuclear accumulation of Smad1. Oxidative stress also modestly reduced OP-1-stimulated nuclear translocation of Smad4. These results demonstrate an age-related reduction in the response of human chondrocytes to IGF-1 and OP-1, which are 2 important anabolic factors in cartilage, and suggest that oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling. Copyright © 2014 by the American College of Rheumatology.

Interaction of microRNA-21/145 and Smad3 domain-specific phosphorylation in hepatocellular carcinoma

PubMed Central

Wang, Ji Yu; Fang, Meng; Boye, Alex; Wu, Chao; Wu, Jia Jun; Ma, Ying; Hou, Shu; Kan, Yue; Yang, Yan

2017-01-01

MicroRNAs 21 and 145 exhibit inverse expression in Hepatocellular carcinoma (HCC), but how they relate to Smad3 C-terminal and Link region phosphorylation (pSmad3C and pSmad3L) downstream of TGF-β/MAPK signaling, remains inconclusive. Our results suggest microRNA-145 targets Smad3 in HepG2 cells. Decreased tumor volume and increased apoptosis were produced in both microRNA-21 antagomir and microRNA-145 agomir groups compared to controls. Inhibition of TβRI and MAPK (ERK, JNK, and p38) activation respectively produced decreased microRNA-21 but increased microRNA-145 expression. Correspondingly, the expression level of pSmad3C obviously increased while pSmad3L decreased in microRNA-145 agomir-group and the expression of pSmad3C/3L were not markedly changed but pERK, pJNK, pp38 decreased in microRNA-21 antagomir-group compared to controls. On the other hand, microRNA-145 and 21 increased respectively in xenografts of HepG2 cells transfected with Smad3 EPSM and 3S-A plasmid, and this correlated with the overexpression of pSmad3C and pSmad3L respectively compared to control. To conclude, microRNA-21 promotes tumor progression in a MAPK-dependent manner while microRNA-145 suppresses it via domain-specific phosphorylation of Smad3 in HCC. Meanwhile, increased pSmad3C/3L lead to the up-regulation of microRNA-145/21 respectively. The interaction between pSmad3C/3L and microRNA-145/21 regulates HCC progression and the switch of pSmad3C/3L may serve as an important target for HCC therapy. PMID:29156696

Nuclear Transport and Accumulation of Smad Proteins Studied by Single-Molecule Microscopy.

PubMed

Li, Yichen; Luo, Wangxi; Yang, Weidong

2018-05-08

Nuclear translocation of stimulated Smad heterocomplexes is a critical step in the signal transduction of transforming growth factor β (TGF-β) from transmembrane receptors into the nucleus. Specifically, normal nuclear accumulation of Smad2/Smad4 heterocomplexes induced by TGF-β1 is involved in carcinogenesis. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy and Förster resonance energy transfer technique, we tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complexes in live cells, with a high single-molecule localization precision of 2 ms and <20 nm. Our single-molecule Förster resonance energy transfer data have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, we found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, we believe that the determined TGF-β1-dependent transport configurations and efficiencies for the basal-state Smad or stimulated Smad heterocomplexes elucidate the basic molecular mechanism to understand their nuclear transport and accumulation. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model.

PubMed

Hara, Shintaro; Morita, Reiko; Ogawa, Takashi; Segawa, Risa; Takimoto, Norifumi; Suzuki, Kazuhiko; Hamadate, Naobumi; Hayashi, Shim-Mo; Odachi, Ayano; Ogiwara, Isao; Shibusawa, Sakae; Yoshida, Toshinori; Shibutani, Makoto

2014-08-01

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-β/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of Ki-67(+) proliferating cells within GST-P(+) foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P(+) foci (area) to 48.9-49.4% and Ki-67(+) cells to 55.5-61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN(-) foci, phospho-Akt substrate(+) foci, phospho-Smad3(-) foci and Smad4(-) foci in GST-P(+) foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN(-) foci in GST-P(+) foci to 59.8-72.2% and Smad4(-) foci to 62.4-71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate(+) foci in GST-P(+) foci to 75.2-75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-β/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Infection of Hepatocytes With HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Up-regulating SMAD6 and SMAD7.

PubMed

Zhang, Fang; Sodroski, Catherine; Cha, Helen; Li, Qisheng; Liang, T Jake

2017-01-01

The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor β signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6 and another inhibitory SMAD (SMAD7) promote HCV infection in human hepatoma cells and hepatocytes. We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with Japanese fulminant hepatitis-1 (JFH1) HCV cell culture system (HCVcc). We then measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. We examined HCV entry in HepG2/microRNA (miR) 122/CD81 cells, which support entry and replication of HCV, were transfected these cells with small interfering RNAs targeting inhibitory SMADs to analyze gene expression profiles. Uptake of labeled low-density lipoprotein (LDL) and cholesterol was measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 messenger RNAs (mRNAs). Small interfering RNA knockdown of SMAD6 blocked the binding and infection of hepatoma cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased HCV infection. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells after SMAD6 knockdown. SMAD6 knockdown also reduced transcription of genes encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR, and the scavenger receptor class B member 1 in hepatocytes; knockdown of SMAD6 also inhibited cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased the expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. In addition, HCV infection of cells increased the expression of SMAD6, which required the activity of nuclear factor-κB, but not transforming growth factor β. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. In studies of hepatoma cell lines and primary human hepatocytes, we found that infection with HCV leads to activation of nuclear factor-κB, resulting in increased expression of SMAD6 and SMAD7. Up-regulation of SMAD6 and SMAD7 induces the expression of HSPGs, such as SDC1, as well as LDLR, very LDLR, and the scavenger receptor class B member 1, which promote HCV entry and propagation, as well as cellular uptake of cholesterol and lipoprotein. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Linker phosphorylation of Smad3 promotes fibro-carcinogenesis in chronic viral hepatitis of hepatocellular carcinoma.

PubMed

Murata, Miki; Yoshida, Katsunori; Yamaguchi, Takashi; Matsuzaki, Koichi

2014-11-07

Epidemiological and clinical data point to a close association between chronic hepatitis B virus infection or chronic hepatitis C virus infection and development of hepatocellular carcinoma (HCC). HCC develops over several decades and is associated with fibrosis. This sequence suggests that persistent viral infection and chronic inflammation can synergistically induce liver fibrosis and hepatocarcinogenesis. The transforming growth factor-β (TGF-β) signaling pathway plays a pivotal role in diverse cellular processes and contributes to hepatic fibro-carcinogenesis under inflammatory microenvironments during chronic liver diseases. The biological activities of TGF-β are initiated by the binding of the ligand to TGF-β receptors, which phosphorylate Smad proteins. TGF-β type I receptor activates Smad3 to create COOH-terminally phosphorylated Smad3 (pSmad3C), while pro-inflammatory cytokine-activated kinases phosphorylates Smad3 to create the linker phosphorylated Smad3 (pSmad3L). During chronic liver disease progression, virus components, together with pro-inflammatory cytokines and somatic mutations, convert the Smad3 signal from tumor-suppressive pSmad3C to fibro-carcinogenic pSmad3L pathways, accelerating liver fibrosis and increasing the risk of HCC. The understanding of Smad3 phosphorylation profiles may provide new opportunities for effective chemoprevention and personalized therapy for patients with hepatitis virus-related HCC in the future.

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling.

PubMed

Zhou, Fangfang; Xie, Feng; Jin, Ke; Zhang, Zhengkui; Clerici, Marcello; Gao, Rui; van Dinther, Maarten; Sixma, Titia K; Huang, Huizhe; Zhang, Long; Ten Dijke, Peter

2017-06-01

SMAD4 is a common intracellular effector for TGF-β family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-β family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity. © 2017 The Authors.

Hindlimb unloading-induced muscle atrophy and phenotype transition is attenuated in Smad3+/- mice

NASA Astrophysics Data System (ADS)

Chen, X. P.; Zhang, P.; Liu, S. H.; Wang, F.; Ge, X.; Wu, Y.; Fan, M.

Currently it has been well defined that the microgravity-induced muscle disuse characterized by atrophy and slow-to-fast phenotype transition of the postural muscles such as soleus muscle but the basic mechanism underlying the atrophy and phenotype transition of soleus muscle is still unclear To investigate the developmental mechanisms of muscle atrophy and its phenotype transition under microgravity the soleus muscle of Smad3 and Smad3 - mice after 14 days hindlimb unloading was examined Using histology and immunohistochemistry assay we found that the soleus muscle volume and fiber number appeared a remarkable increases in Smad3 - mice compared to those in Smad3 control In addition Western blot analysis showed that the expression level of myosin heavy chain MHC -slow myofiber specific protein in soleus muscle was visibly higher in Smad3 - mice than in Smad3 mice In contrast the expression level of MHC-fast myofiber specific protein in soleus muscle was visibly lower in Smad3 - mice than in Smad3 mice Furthermore RT-PCR revealed that the expression of Smad3 and myogenic regulatory factor MRF mRNA was inversely regulated Finally we determined that either Smad3 mRNA or Smad3 protein were selectively distributed in quiescent satellite cells in vivo and in reserve cells in vitro Therefore our findings suggested that Smad3 might be a key transcriptional factor for soleus muscle atrophy and slow-to-fast phenotype transition of the slow muscle under microgravity In the future an agent that regulates Smad3 expression may be used to prevent

Regulation of renal fibrosis by Smad3 Thr388 phosphorylation.

PubMed

Qu, Xinli; Li, Xueling; Zheng, Yaowu; Ren, Yi; Puelles, Victor G; Caruana, Georgina; Nikolic-Paterson, David J; Li, Jinhua

2014-04-01

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease. Copyright © 2014. Published by Elsevier Inc.

MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii

PubMed Central

Sahni, Abha; Narra, Hema P.; Patel, Jignesh; Sahni, Sanjeev K.

2017-01-01

MicroRNAs (miRNAs) mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R. rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01) and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01). Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs). Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections. PMID:28698491

Bmp signaling regulates a dose-dependent transcriptional program to control facial skeletal development.

PubMed

Bonilla-Claudio, Margarita; Wang, Jun; Bai, Yan; Klysik, Elzbieta; Selever, Jennifer; Martin, James F

2012-02-01

We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in cranial neural crest (CNC). Conditional Bmp4 overexpression, using a tetracycline-regulated Bmp4 gain-of-function allele, resulted in facial skeletal changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4-induced genes (BIG) composed predominantly of transcriptional regulators that control self-renewal, osteoblast differentiation and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4 and Bmp7, resulted in complete or partial loss of multiple CNC-derived skeletal elements, revealing a crucial requirement for Bmp signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss-of-function mutants, indicating Bmp-regulated target genes are modulated by Bmp dose. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45γ and Gata3, that was bound by Smad1/5 in the developing mandible, revealing direct Smad-mediated regulation. These data support the hypothesis that Bmp signaling regulates craniofacial skeletal development by balancing self-renewal and differentiation pathways in CNC progenitors.

Selective deletion of Smad4 in postnatal germ cells does not affect spermatogenesis or fertility in mice.

PubMed

Hao, Xiao-Xia; Chen, Su-Ren; Tang, Ji-Xin; Li, Jian; Cheng, Jin-Mei; Jin, Cheng; Wang, Xiu-Xia; Liu, Yi-Xun

2016-07-01

SMAD4 is the central component of canonical signaling in the transforming growth factor beta (TGFβ) superfamily. Loss of Smad4 in Sertoli cells affects the expansion of the fetal testis cords, whereas selective deletion of Smad4 in Leydig cells alone does not appreciably alter fetal or adult testis development. Loss of Smad4 in Sertoli and Leydig cells, on the other hand, leads to testicular dysgenesis, and tumor formation in mice. Within the murine testes, Smad4 is also expressed in germ cells of the seminiferous tubules. We therefore, crossed Ngn3-Cre or Stra8-Cre transgenic mice with Smad4-flox mice to generate conditional knockout animals in which Smad4 was specifically deleted in postnatal germ cells to further uncover cell type-specific requirement of Smad4. Unexpectedly, these germ-cell-knockout mice were fertile and did not exhibit any detectable abnormalities in spermatogenesis, indicating that Smad4 is not required for the production of sperm; instead, these data indicate a cell type-specific requirement of Smad4 primarily during testis development. Mol. Reprod. Dev. 83: 615-623, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Aberrant phosphorylation of SMAD4 Thr277-mediated USP9x-SMAD4 interaction by free fatty acids promotes breast cancer metastasis

PubMed Central

Wu, Yong; Yu, Xiaoting; Yi, Xianghua; Wu, Ke; Dwabe, Sami; Atefi, Mohammad; Elshimali, Yahya; Kemp, Kevin T.; Bhat, Kruttika; Haro, Jesse; Sarkissyan, Marianna; Vadgama, Jaydutt V

2017-01-01

Obesity increases the risk of distant metastatic recurrence and reduces breast cancer (BC) survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGF-β transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4-USP9x interaction, SMAD4 nuclear retention, and stimulates TGF-β /SMAD3-mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining SMAD4 nuclear retention. FFA further facilitated TGF-β-induced ERK activation, SMAD4 phosphorylation and nuclear retention, promoting TGF-β-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. Additionally, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGF-β signaling and metastasis in overweight/obese patient BC specimens. Altogether, we demonstrate the vital interaction of USP9x and SMAD4 for governing TGF-β signaling and dyslipidemia-induced, aberrant TGF-β activation during BC metastasis. PMID:28115363

TGF-β induces fascin expression in gastric cancer via phosphorylation of smad3 linker area

PubMed Central

Li, Liling; Cao, Fang; Liu, Baoan; Luo, Xiaojuan; Ma, Xin; Hu, Zhongliang

2015-01-01

Background: Fascin is an actin-bundling protein critical for tumor invasion. TGF-β could induce fascin expression in gastric cancer cells. In this study, we attempted to explore the role of p-smad3L in the expression of fascin induced by TGF-β in gastric cancer cells. Methods: Pseudopodia were evaluated by immunofluorescence. Fascin expression was detected by RT-PCR and western blot. Smad3 siRNA was used to repress the endogenous smad3. The phosphorylations of smad3 linker region at sites s204, s208 and s213 were detected by western blot. The fascin promoter reporter activity was measured by dual luciferase assay. Results: TGF-β could increase the formation of pseudopodia and the expression of fascin in gastric cancer cells. Smad3 depletion abrogated the expression of fascin induced by TGF-β. The phosphorylation of smad3 linker region at serine 204, 208 and 213 was enhanced in gastric cancer cells after TGF-β treatment. The fascin promoter reporter activity was significantly enhanced with TGF-β treatment in both wild-type Smad3 group and Smad3EPSM group (P<0.05). Furthermore, the fascin promoter reporter activity in the wild-type Smad3 transfectant cells was significantly higher than that in Smad3EPSM cells (P<0.05). Conclusions: fascin expression induced by TGF-β depends on smad3, at least in part, depends on smad3 linker phosphorylation. PMID:26269751

Sma- and Mad-related protein 7 (Smad7) is required for embryonic eye development in the mouse.

PubMed

Zhang, Rui; Huang, Heng; Cao, Peijuan; Wang, Zhenzhen; Chen, Yan; Pan, Yi

2013-04-12

Smad7 is an intracellular inhibitory protein that antagonizes the signaling of TGF-β family members. Deletion of Smad7 in the mouse leads to an abnormality in heart development. However, whether Smad7 has a functional role in the development of other organs has been elusive. Here we present evidence that Smad7 imparts a role to eye development in the mouse. Smad7 is expressed in both the lens and retina in the developing embryonic eye. Depletion of Smad7 caused various degrees of coloboma and microphthalmia with alterations in cell apoptosis and proliferation in eyes. Smad7 was implicated in lens differentiation but was not required for the induction of the lens placode. The development of the periocular mesenchyme was retarded with the down-regulation of Bmp7 and Pitx2 in mutant mice. Retinal spatial patterning was affected by Smad7 deletion and was accompanied by altered bone morphogenetic protein (BMP) signaling. At late gestation stages, TGF-β signaling was up-regulated in the differentiating retina. Smad7 mutant mice displayed an expanded optic disc with increasing of sonic hedgehog (SHH) signaling. Furthermore, loss of Smad7 led to a temporal change in retinal neurogenesis. In conclusion, our study suggests that Smad7 is essential for eye development. In addition, our data indicate that alterations in the signaling of BMP, TGF-β, and SHH likely underlie the defects in eye development caused by Smad7 deletion.

PfSMAD4 plays a role in biomineralization and can transduce bone morphogenetic protein-2 signals in the pearl oyster Pinctada fucata.

PubMed

Zhao, Mi; Shi, Yu; He, Maoxian; Huang, Xiande; Wang, Qi

2016-04-26

Mollusca is the second largest phylum in nature. The shell of molluscs is a remarkable example of a natural composite biomaterial. Biomineralization and how it affects mollusks is a popular research topic. The BMP-2 signaling pathway plays a canonical role in biomineralization. SMAD4 is an intracellular transmitter in the BMP signaling pathway in mammals, and some genomic data show SMAD4's involvement in BMP signaling in invertebrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested. In this study, we identified a SMAD4 gene (hereafter designated PfSMAD4) in pearl oyster Pinctada fucata. Bioinformatics analysis of PfSMAD4 showed high identity with its orthologs. PfSMAD4 was located in the cytoplasm in immunofluorescence assays and analyses of PfSMAD4 mRNA in tissues and developmental stages showed high expression in ovaries and D-shaped larvae. An RNA interference experiment, performed by PfSMAD4 double-stranded RNA (dsRNA) injection, demonstrated inhibition not only of nacre growth but also organic sheet formation with a decrease in PfSMAD4 expression. A knockdown experiment using PfBMP2 dsRNA showed decreased PfBMP2 and PfSMAD4 mRNA and irregular crystallization of the nacreous layer using scanning electron microscopy. In co-transfection experiments, PfBMP2-transactivated reporter constructs contained PfSMAD4 promoter sequences. Our results suggest that PfSMAD4 plays a role in biomineralization and can transduce BMP signals in P. fucata. Our data provides important clues about the molecular mechanisms that regulate biomineralization in pearl oyster.

SMAD Signaling Is Required for Structural Integrity of the Female Reproductive Tract and Uterine Function During Early Pregnancy in Mice1

PubMed Central

Rodriguez, Amanda; Tripurani, Swamy K.; Burton, Jason C.; Clementi, Caterina; Larina, Irina; Pangas, Stephanie A.

2016-01-01

Pregnancy is a complex physiological process tightly controlled by the interplay among hormones, morphogens, transcription factors, and signaling pathways. Although recent studies using genetically engineered mouse models have revealed that ligands and receptors of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) signaling pathways are essential for multiple reproductive events during pregnancy, the functional role of SMAD transcription factors, which serve as the canonical signaling platform for the TGFbeta/BMP pathways, in the oviduct and uterus is undefined. Here, we used a mouse model containing triple conditional deletion of the BMP receptor signaling Smads (Smad1 and Smad5) and Smad4, the central mediator of both TGFbeta and BMP signaling, to investigate the role of the SMADs in reproductive tract structure and function in cells from the Amhr2 lineage. Unlike the respective single- or double-knockouts, female Smad1flox/flox Smad5flox/flox Smad4flox/flox Amhr2cre/+conditional knockout (i.e., Smad1/5/4-Amhr2-cre KO) mice are sterile. We discovered that Smad1/5/4-Amhr2-cre KO females have malformed oviducts that subsequently develop oviductal diverticuli. These oviducts showed dysregulation of multiple genes essential for oviduct and smooth muscle development. In addition, uteri from Smad1/5/4-Amhr2-cre KO females exhibit multiple defects in stroma, epithelium, and smooth muscle layers and fail to assemble a closed uterine lumen upon embryo implantation, with defective uterine decidualization that led to pregnancy loss at early to mid-gestation. Taken together, our study uncovers a new role for the SMAD transcription factors in maintaining the structural and functional integrity of oviduct and uterus, required for establishment and maintenance of pregnancy. PMID:27335065

Loss of DPC4/SMAD4 expression in primary gastrointestinal neuroendocrine tumors is associated with cancer-related death after resection.

PubMed

Roland, Christina L; Starker, Lee F; Kang, Y; Chatterjee, Deyali; Estrella, Jeannelyn; Rashid, Asif; Katz, Matthew H; Aloia, Thomas A; Lee, Jeffrey E; Dasari, Arvind; Yao, James C; Fleming, Jason B

2017-03-01

Gastrointestinal neuroendocrine tumors have frequent loss of DPC4/SMAD4 expression, a known tumor suppressor. The impact of SMAD4 loss on gastrointestinal neuroendocrine tumors aggressiveness or cancer-related patient outcomes is not defined. We examined the expression of SMAD4 in resected gastrointestinal neuroendocrine tumors and its impact on oncologic outcomes. Patients who underwent complete curative operative resection of gastrointestinal neuroendocrine tumors were identified retrospectively (n = 38). Immunohistochemical staining for SMAD4 expression was scored by a blinded pathologist and correlated with clinicopathologic features and oncologic outcomes. Twenty-nine percent of the gastrointestinal neuroendocrine tumors were SMAD4-negative and 71% SMAD4-positive. Median overall survival was 155 months (95% confidence interval, 102-208 months). Loss of SMAD4 was associated with both decreased median disease-free survival (28 months; 95% confidence interval, 16-40) months compared with 223 months (95% confidence interval, 3-443 months) for SMAD4-positive patients (P = .03) and decreased median disease-specific survival (SMAD4: 137 [95% confidence interval, 81-194] months versus SMAD4-positive: 204 [95% confidence interval, 143-264] months; P = .04). This translated into a decrease in median overall survival (SMAD4-negative: 125 (95% confidence interval, 51-214) months versus SMAD4-positive: 185 (95% confidence interval, 138-232) months; P = .02). Consistent with the known biology of the DPC4/SMAD4 gene, an absence of its protein expression in primary gastrointestinal neuroendocrine tumors was negatively associated with outcomes after curative operative resection. Copyright © 2016 Elsevier Inc. All rights reserved.

The Role of SncN and Ski in Mammary Epithelial Cell Transformation

DTIC Science & Technology

2005-07-01

cellular activities through the Smad proteins. Upon phosphorylation by the active TGFβ receptor kinases, Smad2 and Smad3 oligomerize with Smad4, translocate...sequence in sense and antisense orientation with an intervening linker . Primer pairs were designed to generate single-strand overhangs upon annealing...nuclear protein, based on studies of ectopically expressed SnoN and endogenous SnoN in cancer cell lines. In the nucleus, SnoN binds to Smad2, Smad3 , and

Linker phosphorylation of Smad3 promotes fibro-carcinogenesis in chronic viral hepatitis of hepatocellular carcinoma

PubMed Central

Murata, Miki; Yoshida, Katsunori; Yamaguchi, Takashi; Matsuzaki, Koichi

2014-01-01

Epidemiological and clinical data point to a close association between chronic hepatitis B virus infection or chronic hepatitis C virus infection and development of hepatocellular carcinoma (HCC). HCC develops over several decades and is associated with fibrosis. This sequence suggests that persistent viral infection and chronic inflammation can synergistically induce liver fibrosis and hepatocarcinogenesis. The transforming growth factor-β (TGF-β) signaling pathway plays a pivotal role in diverse cellular processes and contributes to hepatic fibro-carcinogenesis under inflammatory microenvironments during chronic liver diseases. The biological activities of TGF-β are initiated by the binding of the ligand to TGF-β receptors, which phosphorylate Smad proteins. TGF-β type I receptor activates Smad3 to create COOH-terminally phosphorylated Smad3 (pSmad3C), while pro-inflammatory cytokine-activated kinases phosphorylates Smad3 to create the linker phosphorylated Smad3 (pSmad3L). During chronic liver disease progression, virus components, together with pro-inflammatory cytokines and somatic mutations, convert the Smad3 signal from tumor-suppressive pSmad3C to fibro-carcinogenic pSmad3L pathways, accelerating liver fibrosis and increasing the risk of HCC. The understanding of Smad3 phosphorylation profiles may provide new opportunities for effective chemoprevention and personalized therapy for patients with hepatitis virus-related HCC in the future. PMID:25386050

Focal adhesion molecule Kindlin-1 mediates activation of TGF-β signaling by interacting with TGF-βRI, SARA and Smad3 in colorectal cancer cells

PubMed Central

Wang, Yunling; Yang, Mingzi; Gao, Jianchao; Wei, Xiaofan; Fang, Weigang; Zhan, Jun; Zhang, Hongquan

2016-01-01

Kindlin-1, an integrin-interacting protein, has been implicated in TGF-β/Smad3 signaling. However, the molecular mechanism underlying Kindlin-1 regulation of TGF-β/Smad3 signaling remains elusive. Here, we reported that Kindlin-1 is an important mediator of TGF-β/Smad3 signaling by showing that Kindlin-1 physically interacts with TGF-β receptor I (TβRI), Smad anchor for receptor activation (SARA) and Smad3. Kindlin-1 is required for the interaction of Smad3 with TβRI, Smad3 phosphorylation, nuclear translocation, and finally the activation of TGF-β/Smad3 signaling pathway. Functionally, Kindlin-1 promoted colorectal cancer (CRC) cell proliferation in vitro and tumor growth in vivo, and was also required for CRC cell migration and invasion via an epithelial to mesenchymal transition. Kindlin-1 was found to be increased with the CRC progression from stages I to IV. Importantly, raised expression level of Kindlin-1 correlates with poor outcome in CRC patients. Taken together, we demonstrated that Kindlin-1 promotes CRC progression by recruiting SARA and Smad3 to TβRI and thereby activates TGF-β/Smad3 signaling. Thus, Kindlin-1 is a novel regulator of TGF-β/Smad3 signaling and may also be a potential target for CRC therapeutics. PMID:27776350

Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

PubMed Central

Horiki, Mitsuru; Imamura, Takeshi; Okamoto, Mina; Hayashi, Makoto; Murai, Junko; Myoui, Akira; Ochi, Takahiro; Miyazono, Kohei; Yoshikawa, Hideki; Tsumaki, Noriyuki

2004-01-01

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo. PMID:15123739

SMAD4 is Involved in the Development of Endotoxin Tolerance in Microglia.

PubMed

Liu, Xiaorong; Qin, Yongwei; Dai, Aihua; Zhang, Yu; Xue, Huaqing; Ni, Haidan; Han, Lijian; Zhu, Liang; Yuan, Debin; Tao, Tao; Cao, Maohong

2016-07-01

Initial exposure of macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Smad4 plays important roles in the induction of LPS tolerance. However, the function of Smad4 in microglia remains unknown. Here we show that expression of Smad4 was highly up-regulated in LPS-tolerized mouse cerebral cortex. Smad4 was mostly colocalized with microglia, rarely with neurons. Using a microglia cell line, BV2, we find that LPS activates endogenous Smad4, inducing its migration into the nucleus and increasing its expression. Smad4 significantly suppressed TLR-triggered production of proinflammatory cytokines (IL-6), increased anti-inflammatory cytokine in LPS-tolerized microglia. Moreover, IL-6 concentrations in culture supernatants after second LPS challenge are higher in SMAD4 small interfering RNA (siRNA) BV2 cells than control siRNA BV2 cells, indicating failure to induce tolerance in absence of Smad4 signaling. In our study, we conclude that both in vivo and in vitro, Smad4 signaling is required for maximal induction of endotoxin tolerance.

Smad4 inhibits cell migration via suppression of JNK activity in human pancreatic carcinoma PANC-1 cells.

PubMed

Zhang, Xueying; Cao, Junxia; Pei, Yujun; Zhang, Jiyan; Wang, Qingyang

2016-05-01

Smad4 is a common Smad and is a key downstream regulator of the transforming growth factor-β signaling pathway, in which Smad4 often acts as a potent tumor suppressor and functions in a highly context-dependent manner, particularly in pancreatic cancer. However, little is known regarding whether Smad4 regulates other signaling pathways involved in pancreatic cancer. The present study demonstrated that Smad4 downregulates c-Jun N-terminal kinase (JNK) activity using a Smad4 loss-of-function or gain-of-function analysis. Additionally, stable overexpression of Smad4 clearly affected the migration of human pancreatic epithelioid carcinoma PANC-1 cells, but did not affect cell growth. In addition, the present study revealed that upregulation of mitogen-activated protein kinase phosphatase-1 is required for the reduction of JNK activity by Smad4, leading to a decrease in vascular endothelial growth factor expression and inhibiting cell migration. Overall, the present findings indicate that Smad4 may suppress JNK activation and inhibit the tumor characteristics of pancreatic cancer cells.

Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma.

PubMed

Matsuzaki, Koichi; Murata, Miki; Yoshida, Katsunori; Sekimoto, Go; Uemura, Yoshiko; Sakaida, Noriko; Kaibori, Masaki; Kamiyama, Yasuo; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi; Seki, Toshihito

2007-07-01

Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Whereas the TbetaRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21(WAF1) transcription, JNK/pSmad3L-mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI-1). We studied the domain-specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV-infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI-1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21(WAF1) decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro-inflammatory cytokine interleukin-1beta stimulated the pSmad3L/PAI-1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF-beta-dependent tumor-suppressive activity by the pSmad3C/p21(WAF1) pathway. These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF-beta signaling from tumor-suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC.

Smad4 in T cells plays a protective role in the development of autoimmune Sjögren's syndrome in the nonobese diabetic mouse.

PubMed

Kim, Donghee; Kim, Jae Young; Jun, Hee-Sook

2016-12-06

We investigated the role of Smad4, a signaling molecule of the TGF-beta pathway, in T cells on the pathology of Sjögren's syndrome (SS) in nonobese diabetic (NOD) mice, an animal model of SS. T cell-specific Smad4-deleted (Smad4fl/fl,CD4-Cre; Smad4 tKO) NOD mice had accelerated development of SS compared with wild-type (Smad4+/+,CD4-Cre; WT) NOD mice, including increased lymphocyte infiltration into exocrine glands, decreased tear and saliva production, and increased levels of autoantibodies at 12 weeks of age. Activated/memory T cells and cytokine (IFN-γ, IL-17)-producing T cells were increased in Smad4 tKO NOD mice, however the proportion and function of regulatory T (Treg) cells were not different between Smad4 tKO and WT NOD mice. Effector T (Teff) cells from Smad4 tKO NOD mice were less sensitive than WT Teff cells to suppression by Treg cells. Th17 differentiation capability of Teff cells was similar between Smad4 tKO and WT NOD mice, but IL-17 expression was increased under inducible Treg skewing conditions in T cells from Smad4 tKO NOD mice. Our results demonstrate that disruption of the Smad4 pathway in T cells of NOD mice increases Teff cell activation resulting in upregulation of Th17 cells, indicating that Smad4 in T cells has a protective role in the development of SS in NOD mice.

SMAD4 feedback regulates the canonical TGF-β signaling pathway to control granulosa cell apoptosis.

PubMed

Du, Xing; Pan, Zengxiang; Li, Qiqi; Liu, Honglin; Li, Qifa

2018-02-02

Canonical TGF-β signals are transduced from the cell surface to the cytoplasm, and then translocated into the nucleus, a process that involves ligands (TGF-β1), receptors (TGFBR2/1), receptor-activated SMADs (SMAD2/3), and the common SMAD (SMAD4). Here we provide evidence that SMAD4, a core component of the canonical TGF-β signaling pathway, regulates the canonical TGF-β signaling pathway in porcine granulosa cells (GCs) through a feedback mechanism. Genome-wide analysis and qRT-PCR revealed that SMAD4 affected miRNA biogenesis in GCs. Interestingly, TGFBR2, the type II receptor of the canonical TGF-β signaling pathway, was downregulated in SMAD4-silenced GCs and found to be a common target of SMAD4-inhibited miRNAs. miR-425, the most significantly elevated miRNA in SMAD4-silenced GCs, mediated the SMAD4 feedback regulation of the TGF-β signaling pathway. This was accomplished through a direct interaction between the transcription factor SMAD4 and the miR-425 promoter, and a direct interaction between miR-425 and the TGFBR2 3'-UTR. Furthermore, miR-425 enhanced GC apoptosis by targeting TGFBR2 and the canonical TGF-β signaling pathway, which was rescued by SMAD4 and TGF-β1. Overall, our findings demonstrate that a positive feedback mechanism exists within the canonical TGF-β signaling pathway. This study also provides new insights into mechanism underlying the canonical TGF-β signaling pathway, which regulates GC function and follicular development.

Smad4 in T cells plays a protective role in the development of autoimmune Sjögren's syndrome in the nonobese diabetic mouse

PubMed Central

Kim, Donghee; Kim, Jae Young; Jun, Hee-Sook

2016-01-01

We investigated the role of Smad4, a signaling molecule of the TGF-beta pathway, in T cells on the pathology of Sjögren's syndrome (SS) in nonobese diabetic (NOD) mice, an animal model of SS. T cell-specific Smad4-deleted (Smad4fl/fl,CD4-Cre; Smad4 tKO) NOD mice had accelerated development of SS compared with wild-type (Smad4+/+,CD4-Cre; WT) NOD mice, including increased lymphocyte infiltration into exocrine glands, decreased tear and saliva production, and increased levels of autoantibodies at 12 weeks of age. Activated/memory T cells and cytokine (IFN-γ, IL-17)-producing T cells were increased in Smad4 tKO NOD mice, however the proportion and function of regulatory T (Treg) cells were not different between Smad4 tKO and WT NOD mice. Effector T (Teff) cells from Smad4 tKO NOD mice were less sensitive than WT Teff cells to suppression by Treg cells. Th17 differentiation capability of Teff cells was similar between Smad4 tKO and WT NOD mice, but IL-17 expression was increased under inducible Treg skewing conditions in T cells from Smad4 tKO NOD mice. Our results demonstrate that disruption of the Smad4 pathway in T cells of NOD mice increases Teff cell activation resulting in upregulation of Th17 cells, indicating that Smad4 in T cells has a protective role in the development of SS in NOD mice. PMID:27880731

Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji

2013-06-14

Highlights: •H{sub 2}O{sub 2} inhibits TGF-β1-induced cell cycle arrest. •H{sub 2}O{sub 2} induces Smad3 linker phosphorylation through Akt-ERK1/2 pathway. •H{sub 2}O{sub 2}-mediated suppression of TGF-β signal requires Smad3 linker phosphorylation. •This is a first report about interplay between H{sub 2}O{sub 2} and growth inhibition pathway. -- Abstract: Hydrogen peroxide (H{sub 2}O{sub 2}) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H{sub 2}O{sub 2} are less understood. Here we report an important mechanism for antagonistic effectsmore » of H{sub 2}O{sub 2} on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H{sub 2}O{sub 2} (0.05–0.2 mM) completely blocked TGF-β1-mediated induction of p15{sup INK4B} expression and increase of its promoter activity. Interestingly, H{sub 2}O{sub 2} selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H{sub 2}O{sub 2} increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H{sub 2}O{sub 2} on TGF-β1-induced increase of p15{sup INK4B}-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H{sub 2}O{sub 2} as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H{sub 2}O{sub 2} antagonizes the cytostatic function of TGF-β1.« less

Expression Pattern of Smad4/GATA3 as a Predictor of Survival in Invasive Ductal Carcinoma of the Breast.

PubMed

Min, Kyueng-Whan; Kim, Dong-Hoon; Do, Sung-Im; Chae, Seoung Wan; Kim, Kyungeun; Sohn, Jin Hee; Lee, Hyun Joo; Do, In-Gu; Pyo, Jung-Soo; Kim, Yuil; Kim, Dong Hyun; Yang, Jung-Ho; Lee, Sang-Jo; Oh, Young Ha; Oh, Sukjoong; Choi, Seon Hyeong; Park, Yong Lai; Park, Chan Heun; Kim, Eun-Kyung; Kwon, Mi Jung; Seo, Jinwon

2017-01-01

Smad4 and GATA3 proteins are known prognostic markers in various cancers. Smad4 is a mediator linked to both tumour suppression and progression. GATA3 is a regulator of development and morphogenesis of the mammary gland. We assessed and compared the predictive performance of Smad4 and GATA3 for clinical outcomes in patients with breast cancer. The combined expression pattern based on Smad4+/- and GATA3+/- was evaluated by immunostaining using breast cancer tissue microarray, and the relationships between protein expression and clinicopathological variables were analysed. Smad4 expression was only associated with an ill-defined tumour border, whereas GATA3 was associated with several good prognostic factors. On analysis of combined markers, there was a significant difference in the expression of fascin (an important factor for cancer invasiveness) between the Smad4+/GATA3- and Smad4-/GATA3+ groups. Smad4+/GATA3- was correlated with worse clinicopathological parameters, relapse-free survival (RFS), and overall survival (OS), compared to Smad4-/GATA3+. Combined markers of Smad4/GATA3 showed a superior performance compared to single markers for predicting RFS and OS in patients with breast cancer. © 2017 S. Karger AG, Basel.

Aberrant Phosphorylation of SMAD4 Thr277-Mediated USP9x-SMAD4 Interaction by Free Fatty Acids Promotes Breast Cancer Metastasis.

PubMed

Wu, Yong; Yu, Xiaoting; Yi, Xianghua; Wu, Ke; Dwabe, Sami; Atefi, Mohammad; Elshimali, Yahya; Kemp, Kevin T; Bhat, Kruttika; Haro, Jesse; Sarkissyan, Marianna; Vadgama, Jaydutt V

2017-03-15

Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGFβ transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4-USP9x interaction, SMAD4 nuclear retention, and stimulates TGFβ/SMAD3-mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining the SMAD4 nuclear retention. FFA further facilitated TGFβ-induced ERK activation, SMAD4 phosphorylation, and nuclear retention, promoting TGFβ-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. In addition, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGFβ signaling and metastasis in overweight/obese patient breast cancer specimens. Altogether, we demonstrate the vital interaction of USP9x and SMAD4 for governing TGFβ signaling and dyslipidemia-induced aberrant TGFβ activation during breast cancer metastasis. Cancer Res; 77(6); 1383-94. ©2017 AACR . ©2017 American Association for Cancer Research.

Microglial SMAD4 regulated by microRNA-146a promotes migration of microglia which support tumor progression in a glioma environment

PubMed Central

Karthikeyan, Aparna; Gupta, Neelima; Tang, Carol; Mallilankaraman, Karthik; Silambarasan, Maskomani; Shi, Meng; Lu, Lei; Ang, Beng Ti; Ling, Eng-Ang; Dheen, S. Thameem

2018-01-01

Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-β (TGFβ) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGFβ level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell line-based model increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGFβ signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability.

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation.

PubMed

Li, Ping; Liu, Ping; Peng, Yan; Zhang, Zhuo-Hang; Li, Xiao-Ming; Xiong, Ren-Ping; Chen, Xing; Zhao, Yan; Ning, Ya-Lei; Yang, Nan; Zhang, Bo; Zhou, Yuan-Guo

2018-06-27

Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-β1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-β1 concentrations, but the mechanism by which low TGF-β1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-β1 concentrations not only promoted c-Ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-β1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-β1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-β1/Smad pathway.

Involvement of Smad3 pathway in atrial fibrosis induced by elevated hydrostatic pressure.

PubMed

Wei, Wei; Rao, Fang; Liu, Fangzhou; Xue, Yumei; Deng, Chunyu; Wang, Zhaoyu; Zhu, Jiening; Yang, Hui; Li, Xin; Zhang, Mengzhen; Fu, Yongheng; Zhu, Wensi; Shan, Zhixin; Wu, Shulin

2018-06-01

Hypertension is a main risk factor for atrial fibrillation, but the direct effects of hydrostatic pressure on the atrial fibrosis are still unknown. The present study investigated whether hydrostatic pressure is responsible for atrial fibrosis, and addressed a potential role of the Smad pathway in this pathology. Biochemical assays were used to study regulation and expression of fibrotic factors in spontaneously hypertensive rats (SHRs) and Wistar rats, and in cardiac fibroblasts (CFs) cultured under standard (0 mmHg) and elevated (20, 40 mmHg) hydrostatic pressure. Levels of atrial fibrosis and protein expression of fibrotic factors Col-1A1/-3A1, TGF-β1, and MMP-2 in SHRs' left atrial tissues were higher than those in Wistar rats. Exposure to elevated pressure was associated with the proliferation of CFs. The protein expression of Col-1A1/-3A1, TGF-β1, and MMP-2 in CFs was also up-regulated in a pressure-dependent manner. The proliferation of CFs and increased expressions of fibrotic markers induced by elevated hydrostatic pressure could be reversed by the Smad3 inhibitor naringenin. The activation of Smad3 pathway was also stimulated by elevated hydrostatic pressure. These results demonstrate that CF secretory function and proliferation can be up-regulated by exposure to elevated pressure, and that Smad3 may modulate CF activation induced by high hydrostatic pressure. © 2017 Wiley Periodicals, Inc.

Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation.

PubMed

Yoon, Jeong-Hwan; Sudo, Katsuko; Kuroda, Masahiko; Kato, Mitsuyasu; Lee, In-Kyu; Han, Jin Soo; Nakae, Susumu; Imamura, Takeshi; Kim, Juryun; Ju, Ji Hyeon; Kim, Dae-Kee; Matsuzaki, Koichi; Weinstein, Michael; Matsumoto, Isao; Sumida, Takayuki; Mamura, Mizuko

2015-07-21

Transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

Valproic acid (VPA) inhibits the epithelial-mesenchymal transition in prostate carcinoma via the dual suppression of SMAD4.

PubMed

Lan, Xiaopeng; Lu, Guoliang; Yuan, Chuanwei; Mao, Shaowei; Jiang, Wei; Chen, Yougen; Jin, Xunbo; Xia, Qinghua

2016-01-01

The epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. Previous studies have reported that valproic acid (VPA) suppresses prostate carcinoma (PCa) cell metastasis and down-regulates SMAD4 protein levels, which is the key molecule in TGF-β-induced EMT. However, the correlation between VPA and the EMT in PCa remains uncertain. Markers of the EMT in PCa cells and xenografts were molecularly assessed after VPA treatment. The expression and mono-ubiquitination of SMAD4 were also analyzed. After transfection with plasmids that express SMAD4 or short hairpin RNA for SMAD4 down-regulation, markers of EMT were examined to confirm whether VPA inhibits the EMT of PCa cells through the suppression of SMAD4. VPA induced the increase in E-cadherin (p < 0.05), and the decrease in N-cadherin (p < 0.05) and Vimentin (p < 0.05), in PCa cells and xenografts. SMAD4 mRNA and protein levels were repressed by VPA (p < 0.05), whereas the level of mono-ubiquitinated SMAD4 was increased (p < 0.05). SMAD4 knockdown significantly increased E-cadherin expression in PC3 cells, but SMAD4 over-expression abolished the VPA-mediated EMT-inhibitory effect. VPA inhibits the EMT in PCa cells via the inhibition of SMAD4 expression and the mono-ubiquitination of SMAD4. VPA could serve as a promising agent in PCa treatment, with new strategies based on its diverse effects on posttranscriptional regulation.

Releasing Ski-Smad4 mediated suppression is essential to license Th17 differentiation

PubMed Central

Zhang, Song; Takaku, Motoki; Zou, Liyun; Gu, Ai-di; Chou, Wei-chun; Zhang, Ge; Wu, Bing; Kong, Qing; Thomas, Seddon Y.; Serody, Jonathan S.; Chen, Xian; Xu, Xiaojiang; Wade, Paul A.; Cook, Donald N.; Ting, Jenny P.; Wan, Yisong Y.

2017-01-01

Th17 cells are critically involved in host defense, inflammation, and autoimmunity1–5. TGF-β is instrumental in Th17 differentiation by cooperating with IL-66,7. Yet, the mechanism of how TGF-β enables Th17 differentiation remains elusive. Here we reveal that TGF-β licenses Th17 differentiation by releasing Ski-Smad4-complex suppressed RORγt expression. We found serendipitously that, unlike wild-type T cells, Smad4-deficient T cells differentiated into Th17 cells in the absence of TGF-β signaling in a RORγt-dependent manner. Ectopic Smad4 expression suppressed the RORγt expression and Th17 differentiation of Smad4-deficient T cells. Unexpectedly however, TGF-β neutralized Smad4 mediated suppression without affecting Smad4 binding to Rorc locus. Proteomic analysis revealed that Smad4 interacted with Ski, a transcriptional repressor degraded upon TGF-β stimulation. Ski controlled the histone acetylation/de-acetylation of Rorc locus and Th17 differentiation via Smad4 because ectopic Ski expression inhibited H3K9Ac of Rorc locus, Rorc expression and Th17 differentiation in a Smad4-dependent manner. Therefore, TGF-β-induced disruption of Ski releases Ski-Smad4 complex imposed suppression of RORγt to license Th17 differentiation. This study reveals a critical mechanism by which TGF-β controls Th17 differentiation and uncovers Ski-Smad4 axis as a potential therapeutic target for treating Th17 related diseases. PMID:29072299

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PubMed

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2017-03-01

Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

PubMed

Funaba, Masayuki; Ikeda, Teruo; Murakami, Masaru; Ogawa, Kenji; Tsuchida, Kunihiro; Sugino, Hiromu; Abe, Matanobu

2003-12-26

Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction.

PubMed

Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei

2011-07-01

Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability.

PubMed

Moon, Young Jae; Yun, Chi-Young; Choi, Hwajung; Ka, Sun-O; Kim, Jung Ryul; Park, Byung-Hyun; Cho, Eui-Sic

2016-09-02

Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-β signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4Δ(Os) mice, in which Smad4 was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4Δ(Os) mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5'-bromo-2'deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4Δ(Os) mice. Apoptosis in isolated calvaria cells from Smad4Δ(Os) mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4Δ(Os) mice. Conversely, osteoblast cells overexpressing Smad4 showed increased apoptosis. In an apoptosis induction model of Smad4Δ(Os) mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.

Mechanisms of action of acetaldehyde in the up-regulation of the human α2(I) collagen gene in hepatic stellate cells: key roles of Ski, SMAD3, SMAD4, and SMAD7.

PubMed

Reyes-Gordillo, Karina; Shah, Ruchi; Arellanes-Robledo, Jaime; Hernández-Nazara, Zamira; Rincón-Sánchez, Ana Rosa; Inagaki, Yutaka; Rojkind, Marcos; Lakshman, M Raj

2014-05-01

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The correlation between pulmonary fibrosis and methylation of peripheral Smad3 in cases of pigeon breeder's lung in a Chinese Uygur population.

PubMed

Wu, Chao; Ding, Wei; Li, Qifeng; Wang, Wenyi; Deng, Mingqin; Jin, Rong; Pang, Baosen; Yang, Xiaohong

2017-06-27

Smad3 is a key protein in the transforming growth factor-beta (TGF-β)/Smad signaling pathway, which is involved in fibrosis in many organs. We investigated the relationship between Smad3 gene methylation and pulmonary fibrosis in pigeon breeder's lung (PBL). Twenty Uygur PBL patients with pulmonary fibrosis in Kashi between October 2015 and March 2016 were enrolled. Twenty PBL-free pigeon breeders and 20 healthy non-pigeon breeders enrolled during the same period constituted the negative and normal control groups, respectively. Participants' data and peripheral blood samples were collected, and three Smad3 CpG loci were examined. Distributions of CpG_2 and CpG_4 methylation rates did not differ across groups, whereas distributions of CpG_3 methylation rates were significantly different among the three groups. The CpG_3 methylation rate was significantly lower in the patient group than in the negative control group. Smad3 mRNA expression was significantly higher in the patient group than in the negative control group but did not differ between the two control groups. TGF-βlevels were significantly higher in the patient group than in either control group (both P<0.01). Smad3 gene methylation and Smad3 mRNA expression were negatively correlated, with a correlation coefficient of -0.84. The number of pigeons bred during the preceding three months was positively correlated with Smad3 mRNA expression, with a correlation coefficient of 0.77. Smad3 gene hypomethylation might promote pulmonary fibrosis in Uygur PBL patients via increased Smad3 mRNA expression. Smad3 methylation, Smad3 mRNA expression and TGF-β level were correlated with the number of pigeons bred by patients.

Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

PubMed

Jeong, Jaemin; Conboy, Michael J; Conboy, Irina M

2013-08-01

To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice. Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses. The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-β receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-β1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-β receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-β receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGFβ receptor/pSmad3 signaling. The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-β receptor signaling.

Participation of an abnormality in the transforming growth factor-beta signaling pathway in resistance of malignant glioma cells to growth inhibition induced by that factor.

PubMed

Zhang, Lei; Sato, Eiji; Amagasaki, Kenichi; Nakao, Atsuhito; Naganuma, Hirofumi

2006-07-01

Malignant glioma cells secrete and activate transforming growth factor-beta (TGFbeta) and are resistant to growth inhibition by that factor. Nevertheless, the mechanism underlying this effect remains poorly understood. In this study, the mechanism of the resistance to growth inhibition induced by TGFbeta was investigated. The authors examined the expression of downstream components of the TGFbeta receptor, including Smad2, Smad3, Smad4, and Smad7, and the effect of TGFbeta1 treatment on the phosphorylation of Smad2 and the nuclear translocation of Smad2 and Smad3 by using 10 glioma cell lines and the A549 cell line, which is sensitive to TGFbeta-mediated growth inhibition. The expression of two transcriptional corepressor proteins, SnoN and Ski, and the effect of TGFbeta1 treatment on the expression of the SnoN protein and the cell cycle regulators p21, p15, cyclin-dependent kinase-4 (CDK4), and cyclin D1 were also examined. Expression of the Smad2 and Smad3 proteins was lower in the glioma cell lines than in the A549 cell line and in normal astrocytes. In particular, Smad3 expression was low or very low in nine of the 10 malignant glioma cell lines. Expression of Smad4 was low in four glioma cell lines, and expression of the Smad7 protein was similar when compared with protein expression in the A549 cell line and in normal astrocytes. The levels of Smad2 phosphorylation after TGFbeta1 treatment were lower in glioma cell lines than in the A549 cell line, except for one glioma cell line. Seven of the 10 glioma cell lines exhibited lower levels of nuclear translocation of Smad2 and Smad3, and two cell lines that expressed very low levels of Smad3 protein showed no nuclear translocation. All glioma cell lines expressed the SnoN protein and its expression was unaltered by treatment with TGFbeta1. Three glioma cell lines expressed high levels of the Ski protein. The expression of the p21(cip1), p15(INK4B), CDK4, and cyclin D1 proteins was not altered by TGFbeta1, treatment, except in one cell line that displayed a slight increase in p21 protein. Overall, the expression of the Smad2 and Smad3 proteins was low in the glioma cell lines, the phosphorylation and nuclear translocation of Smad2 and Smad3 were impaired, and the TGFbeta receptor signal did not affect the expression of the SnoN, p21, p15, cyclin D1, and CDK4 proteins. These results suggest that the ability to resist TGFbeta-mediated growth inhibition in malignant glioma cells is due to abnormalities in the TGFbeta signaling pathway.

Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo- and hetero-trimerization.

PubMed

Correia, J J; Chacko, B M; Lam, S S; Lin, K

2001-02-06

SMAD proteins are known to oligomerize and hetero-associate during their activation and translocation to the nucleus for transcriptional control. Analytical ultracentrifuge studies on Smad3 and Smad4 protein constructs are presented to clarify the model of homo- and hetero-oligomerization and the role of phosphorylation in the activation process. These constructs all exhibit a tendency to form disulfide cross-linked aggregates, primarily dimers, and a strong reducing agent, TCEP, was found to be required to determine the best estimates for reversible association models and equilibrium constants. A Smad4 construct, S4AF, consisting of the middle linker (L) domain and the C-terminal (C) domain, is shown to be a monomer, while a Smad3 construct, S3LC, consisting of the LC domains, is shown to form a trimer with an affinity K(3) = (1.2-3.1) x 10(9) M(-2). A Smad3 construct that mimics phosphorylation at the C-terminal target sequence, S3LC(3E), has 17--35-fold enhanced ability to form trimer over that of the wild-type construct, S3LC. S4AF associates with either S3LC or S3LC(3E) to form a hetero-trimer. In each case, the hetero-trimer is favored over the formation of the homo-trimer. Despite high sequence homology between Smad3 and Smad4, a chimeric Smad4 construct with an engineered Smad3 C-terminal pseudo-phosphorylation sequence, S4AF(3E), shows no tendency to form trimer. This suggests a Smad4-specific sequence insert inhibits homo-trimer formation, or other domains or sequences in S3LC are required in addition to the target sequence to mediate the formation of trimer. These results represent a direct molecular measure of the importance of hetero-trimerization and phosphorylation in the TGF-beta-activated Smad protein signal transduction process.

SMAD3 Negatively Regulates Serum Irisin and Skeletal Muscle FNDC5 and Peroxisome Proliferator-activated Receptor γ Coactivator 1-α (PGC-1α) during Exercise*

PubMed Central

Tiano, Joseph P.; Springer, Danielle A.; Rane, Sushil G.

2015-01-01

Beige adipose cells are a distinct and inducible type of thermogenic fat cell that express the mitochondrial uncoupling protein-1 and thus represent a powerful target for treating obesity. Mice lacking the TGF-β effector protein SMAD3 are protected against diet-induced obesity because of browning of their white adipose tissue (WAT), leading to increased whole body energy expenditure. However, the role SMAD3 plays in WAT browning is not clearly understood. Irisin is an exercise-induced skeletal muscle hormone that induces WAT browning similar to that observed in SMAD3-deficient mice. Together, these observations suggested that SMAD3 may negatively regulate irisin production and/or secretion from skeletal muscle. To address this question, we used wild-type and SMAD3 knock-out (Smad3−/−) mice subjected to an exercise regime and C2C12 myotubes treated with TGF-β, a TGF-β receptor 1 pharmacological inhibitor, adenovirus expressing constitutively active SMAD3, or siRNA against SMAD3. We find that in Smad3−/− mice, exercise increases serum irisin and skeletal muscle FNDC5 (irisin precursor) and its upstream activator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) to a greater extent than in wild-type mice. In C2C12 myotubes, TGF-β suppresses FNDC5 and PGC-1α mRNA and protein levels via SMAD3 and promotes SMAD3 binding to the FNDC5 and PGC-1α promoters. These data establish that SMAD3 suppresses FNDC5 and PGC-1α in skeletal muscle cells. These findings shed light on the poorly understood regulation of irisin/FNDC5 by demonstrating a novel association between irisin and SMAD3 signaling in skeletal muscle. PMID:25648888

High p-Smad2 expression in stromal fibroblasts predicts poor survival in patients with clinical stage I to IIIA non-small cell lung cancer.

PubMed

Chen, Yongbing; Xing, Pengfei; Chen, Yuanyuan; Zou, Li; Zhang, Yongsheng; Li, Feng; Lu, Xueguan

2014-11-05

Increasing evidence indicates that the TGFβ/Smad signaling pathway plays a prominent role in tumor initiation, progression, and metastasis. Therefore, we investigate the expression of p-Smad2 in surgical resection specimens from non-small cell lung cancer, and evaluate the prognostic significance of p-Smad2 expression in stromal fibroblasts and cancer cells for patients with clinical stage I to IIIA non-small cell lung cancer. The immunohistochemical expression of p-Smad2 was evaluated in 78 formalin-fixed paraffin-embedded surgical resection specimens from clinical stage I to IIIA non-small cell lung cancer. Correlations between p-Smad2 expression and clinicopathologic characteristics were determined by Chi-square test. The prognostic significance of p-Smad2 expression in stromal fibroblasts and cancer cells with regard to overall survival was determined by Kaplan-Meier. There were 38.5% (30/78) and 92.3% (72/78) patients with high p-Smad2 expression in stromal fibroblasts and cancer cells, respectively. There was a positive correlation between the p-Smad2 expression level in stromal fibroblasts and the p-Smad2 expression level in cancer cells (χ2=4.176, P=0.045). No significant correlation of p-Smad2 expression in stromal fibroblasts or cancer cells with any of clinicopathologic characteristics was found. The 3-year overall survival rates with low and high p-Smad2 expression in stromal fibroblasts were 53.7% and 37.7%, respectively (χ2=3.86, P=0.049). No significant association was found between low and high p-Smad2 expression in cancer cells with respect to overall survival, respectively (χ2=0.34, P=0.562). The results suggested that high p-Smad2 expression in stromal fibroblasts predicted poor survival in patients with clinical stage I to IIIA non-small cell lung cancer.

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ichimura, Ryohei, E-mail: red0828@hotmail.co.j; Mizukami, Sayaka, E-mail: non_sugar_life@hotmail.co.j; Takahashi, Miwa, E-mail: mtakahashi@nihs.go.j

2010-08-01

To clarify the involvement of signaling of transforming growth factor (TGF)-{beta} during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6 weeks after starting promotion) and late-stage promotion (57 weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 - identified as Smad3 by later immunoblot analysis - increased in the subpopulation of GST-P{sup +} foci, while Smad4, a nuclear transporter ofmore » Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P{sup +} lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P{sup +} foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P{sup -} foci induced by promotion with agonists of peroxisome proliferator-activated receptor-{alpha} did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction of GST-P{sup +} proliferative lesions.« less

miR-24 and miR-122 Negatively Regulate the Transforming Growth Factor-β/Smad Signaling Pathway in Skeletal Muscle Fibrosis.

PubMed

Sun, Yaying; Wang, Hui; Li, Yan; Liu, Shaohua; Chen, Jiwu; Ying, Hao

2018-06-01

Fibrosis is common after skeletal muscle injury, undermining tissue regeneration and function. The mechanism underlying skeletal muscle fibrosis remains unveiled. Transforming growth factor-β/Smad signaling pathway is supposed to play a pivotal role. However, how microRNAs interact with transforming growth factor-β/Smad-related muscle fibrosis remains unclear. We showed that microRNA (miR)-24-3p and miR-122-5p declined in skeletal muscle fibrosis, which was a consequence of transforming growth factor-β. Upregulating Smad4 suppressed two microRNAs, whereas inhibiting Smad4 elevated microRNAs. Luciferase reporter assay and chromatin immunoprecipitation confirmed that Smad4 directly inhibited two microRNAs. On the other hand, overexpression of these two miRs retarded fibrotic process. We further identified that Smad2 was a direct target of miR-24-3p, whereas miR-122-5p targeted transforming growth factor-β receptor-II. Both targets were important participants in transforming growth factor-β/Smad signaling. Taken together, a positive feedback loop in transforming growth factor-β/Smad4 signaling pathway in skeletal muscle fibrosis was identified. Transforming growth factor-β/Smad axis could be downregulated by microRNAs. This effect, however, was suppressed by Smad4, the downstream of transforming growth factor-β. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ying, E-mail: yingliu@doheny.org; Sun Yet-sen University, Zhongshan Ophthalmic Center, State Key Ophthalmic Laboratory, Guangzhou 510060; Kawai, Kirio

Research highlights: {yields} Inactivation of Smad4 caused disruption in the development of the anterior segment. {yields} Inactivation of Smad4 failed to disrupt early lens development. {yields} Smad4 controlled lens cell cycle and cell death processes. {yields} Smad4 may regulate actin stress fiber assembly and eyelid epithelial movement. -- Abstract: Purpose: Signaling by members of the TGF{beta} superfamily of molecules is essential for embryonic development and homeostasis. Smad4, a key intracellular mediator in TGF{beta} signaling, forms transcriptional activator complexes with Activin-, BMP-, and TGF{beta}-restricted Smad proteins. However, the functional role of Smad4 in controlling different visual system compartments has not beenmore » fully investigated. Methods: Using the Pax6 promoter-driven Cre transgenic, smad4 was conditionally inactivated in the lens, cornea and ectoderm of the eyelids. Standard histological and molecular analytical approaches were employed to reveal morphological and cellular changes. Results: Inactivation of Smad4 in the lens led to microphthalmia and cataract formation in addition to the persistent adhesion of the retina to the lens and the iris to the cornea. Inactivation of Smad4 from the ectoderm of the eyelid and cornea caused disruption to eyelid fusion and proper development of the corneal epithelium and corneal stroma. Conclusions: Smad4 is required for the development and maintenance of the lens in addition to the proper development of the cornea, eyelids, and retina.« less

Glycyrrhetinic acid alleviates radiation-induced lung injury in mice

PubMed Central

Chen, Jinmei; Zhang, Weijian; Zhang, Lurong; Zhang, Jiemin; Chen, Xiuying; Yang, Meichun; Chen, Ting; Hong, Jinsheng

2017-01-01

Radiation-induced lung injury (RILI) is a common complication of thoracic radiotherapy, but efficacious therapy for RILI is lacking. This study ascertained whether glycyrrhetinic acid (GA; a functional hydrolyzed product of glycyrrhizic acid, which is extracted from herb licorice) can protect against RILI and investigated its relationship to the transforming growth factor (TGF)-β1/Smads signaling pathway. C57BL/6 mice were divided into four groups: a control group, a GA group and two irradiation (IR) groups. IR groups were exposed to a single fraction of X-rays (12 Gy) to the thorax and administered normal saline (IR + NS group) or GA (IR + GA group). Two days and 17 days after irradiation, histologic analyses were performed to assess the degree of lung injury, and the expression of TGF-β1, Smad2, Smad3 and Smad7 was recorded. GA administration mitigated the histologic changes of lung injury 2 days and 17 days after irradiation. Protein and mRNA expression of TGF-β1, Smad2 and Smad3, and the mRNA level of Smad7, in lung tissue were significantly elevated after irradiation. GA decreased expression of TGF-β1, Smad2 and Smad3 in lung tissue, but did not increase Smad7 expression. GA can protect against early-stage RILI. This protective effect may be associated with inhibition of the TGF-β1/Smads signaling pathway. PMID:27672101

Overexpression of activin-A and -B in malignant mesothelioma – Attenuated Smad3 signaling responses and ERK activation promote cell migration and invasive growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tamminen, Jenni A.; Yin, Miao; Transplantation Laboratory, Haartman Institute, University of Helsinki

Activin-A and activin-B, members of the TGF-β superfamily, are regulators of reproductive functions, inflammation and wound healing. These dimeric molecules regulate various cellular activities such as proliferation, migration and suvival. Malignant mesothelioma is an asbestos exposure related tumor affecting mainly pleura and it usually has a dismal prognosis. Here, we demonstrate that both activin-A and -B are abundantly expressed in mesothelioma tumor tissue as well as in cultured primary and established mesothelioma cells. Migratory and invasive mesothelioma cells were also found to have attenuated activation of the Smad2/3 pathway in response to activins. Migration and invasive growth of the cellsmore » in three-dimentional matrix was prevented by inhibition of activin activity using a soluble activin receptor 2B (sActR2B-Fc). This was associated with decreased ERK activity. Furthermore, migration and invasive growth was significantly inhibited by blocking ERK phosphorylation. Mesothelioma tumors are locally invasive and our results clearly suggest that acivins have a tumor-promoting function in mesothelioma through increasing expression and switching from canonical Smad3 pathway to non-canonical ERK pathway signaling. Blocking activin activity offers a new therapeutic approach for inhibition of mesothelioma invasive growth. - Highlights: • Activin-A and activin-B are highly expressed in mesothelioma. • Mesothelioma cell migration and invasive growth can be blocked with sActR2B. • Activin induced Smad3 activity is attenuated in invasive mesothelioma cells. • Activins induce ERK activity in mesothelioma cells.« less

Hepatitis B virus X promotes hepatocellular carcinoma development via nuclear protein 1 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bak, Yesol; Shin, Hye-jun; Bak, In seon

Hepatocellular carcinoma (HCC) is one of the most common malignancies and chronic hepatitis B virus (HBV) infection is a major risk factor for HCC. Hepatitis B virus X (HBx) protein relates to trigger oncogenesis. HBx has oncogenic properties with a hyperproliferative response to HCC. Nuclear protein 1 (NUPR1) is a stress-response protein, frequently upregulated in several cancers. Recent data revealed that NUPR1 is involved in tumor progression, but its function in HCC is not revealed yet. Here we report HBx can induce NUPR1 in patients, mice, and HCC cell lines. In an HBx transgenic mouse model, we found that HBxmore » overexpression upregulates NUPR1 expression consistently with tumor progression. Further, in cultured HBV positive cells, HBx knockdown induces downregulation of NUPR1. Smad4 is a representative transcription factor, regulated by HBx, and we showed that HBx upregulates NUPR1 by Smad4 dependent way. We found that NUPR1 can inhibit cell death and induce vasculogenic mimicry in HCC cell lines. Moreover, NUPR1 silencing in HepG2-HBx showed reduced cell motility. These results suggest that HBx can modulate NUPR1 expression through the Smad4 pathway and NUPR1 has a role in hepatocellular carcinoma progression. - Highlights: • NUPR1 is overexpressed in HBx transgenic mouse and HCC patients. • NUPR1 inactivation hampers the HBx induced growth, VM formation, and migration of HepG2 cells in vitro. • NUPR1 has a role for survival of HCC and mechanistically NUPR1 is activated by HBx-Smad4 axis.« less

Chlorogenic Acid Inhibits Liver Fibrosis by Blocking the miR-21-Regulated TGF-β1/Smad7 Signaling Pathway in Vitro and in Vivo.

PubMed

Yang, Fan; Luo, Lei; Zhu, Zhi-De; Zhou, Xuan; Wang, Yao; Xue, Juan; Zhang, Juan; Cai, Xin; Chen, Zhi-Lin; Ma, Qian; Chen, Yun-Fei; Wang, Yu-Jie; Luo, Ying-Ying; Liu, Pan; Zhao, Lei

2017-01-01

Aims: Chlorogenic acid (CGA) is a phenolic acid that has a wide range of pharmacological effects. However, the protective effects and mechanisms of CGA on liver fibrosis are not clear. This study explored the effects of CGA on miR-21-regulated TGF-β1/Smad7 liver fibrosis in the hepatic stellate LX2 cell line and in CCl4-induced liver fibrosis in Sprague-Dawley rats. Methods: The mRNA expression of miR-21, Smad7, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and transforming growth factor-β1 (TGF-β1) and the protein levels of Smad2, p-Smad2, Smad3, p-Smad3, Smad2/3, p-Smad2/3, Smad7, CTGF, α-SMA, TIMP-1, MMP-9 and TGF-β1 were assayed in LX2 cells and liver tissue. The effects of CGA after miR-21 knockdown or overexpression were analyzed in LX2 cells. The liver tissue and serum were collected for histopathological examination, immunohistochemistry (IHC) and ELISA. Results: The mRNA expression of miR-21, CTGF, α-SMA, TIMP-1, and TGF-β1 and the protein expression of p-Smad2, p-Smad3, p-Smad2/3, CTGF, α-SMA, TIMP-1, and TGF-β1 were inhibited by CGA both in vitro and in vivo . Meanwhile, CGA elevated the mRNA and protein expression of Smad7 and MMP-9. After miR-21 knockdown and overexpression, the downstream molecules also changed accordingly. CGA also lessened the degree of liver fibrosis in the pathological manifestation and reduced α-SMA and collagen I expression in liver tissue and TGF-β1 in serum. Conclusion: CGA might relieve liver fibrosis through the miR-21-regulated TGF-β1/Smad7 signaling pathway, which suggests that CGA might be a new anti-fibrosis agent that improves liver fibrosis.

Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis.

PubMed

Sakao, Masayuki; Sakaguchi, Yutaku; Suzuki, Ryo; Takahashi, Yu; Kishimoto, Masanobu; Fukui, Toshiro; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.

Up-regulated transcriptional repressors SnoN and Ski bind Smad proteins to antagonize transforming growth factor-beta signals during liver regeneration.

PubMed

Macias-Silva, Marina; Li, Wei; Leu, Julia I; Crissey, Mary Ann S; Taub, Rebecca

2002-08-09

Transforming growth factor-beta (TGF-beta) functions as an antiproliferative factor for hepatocytes. However, for unexplained reasons, hepatocytes become resistant to TGF-beta signals and can proliferate despite the presence of TGF-beta during liver regeneration. TGF-beta is up-regulated during liver regeneration, although it is not known whether it is active or latent. TGF-beta activity may be examined by assessing Smad activation, a downstream signaling pathway. Smad pathway activation during liver regeneration induced by partial hepatectomy or CC4 injury was examined by assessing the levels of phospho-Smad2 and Smad2-Smad4 complexes. We found that Smad proteins were slightly activated in quiescent liver, but that their activation was further enhanced in regenerating liver. Interestingly, TGF-beta/Smad pathway inhibitors (SnoN and Ski) were up-regulated during regeneration, and notably, SnoN was induced mainly in hepatocytes. SnoN and Ski are transcriptional repressors that may render some cells resistant to TGF-beta via binding Smad proteins. Complexes between SnoN, Ski, and the activated Smad proteins were detected from 2 to 120 h during the major proliferative phase in regenerating liver. Inhibitory complexes decreased after liver mass restitution (5-15 days), suggesting that persistently activated Smad proteins might participate in returning the liver to a quiescent state. Our data show that active TGF-beta/Smad signals are present during regeneration and suggest that SnoN/Ski induction might explain hepatocyte resistance to TGF-beta during the proliferative phase.

Synergistic action of Smad4 and Pten in suppressing pancreatic ductal adenocarcinoma formation in mice.

PubMed

Xu, X; Ehdaie, B; Ohara, N; Yoshino, T; Deng, C-X

2010-02-04

Mutations of SMAD4/DPC4 are found in about 60% of human invasive pancreatic ductal adenocarcinomas (PDACs); yet, the manner in which SMAD4 deficiency enhances tumorigenesis remains elusive. Using a Cre-LoxP approach, we generated a mutant mouse carrying a targeted deletion of Smad4 in the pancreas. We showed that the absence of Smad4 alone did not trigger pancreas tumor formation; however, it increased the expression of an inactivated form of Pten, suggesting a role of Pten in preventing Smad4-/- cells from undergoing malignancy. To investigate this, we disrupted both Pten and Smad4. We showed that Pten deficiency initiated widespread premalignant lesions, and a low tumor incidence that was significantly accelerated by Smad4-deficiency. The absence of Smad4 in a Pten-mutant background enhanced cell proliferation and triggered transdifferentiation from acinar, centroacinar and islet cells, accompanied by activation of Notch1 signaling. We showed that all tumors developed in the Smad4/Pten-mutant pancreas exhibited high levels of pAKT and mTOR, and that about 50 and 83% of human pancreatic cancers examined showed increased pAKT and pmTOR, respectively. Besides the similarity in gene expression, the pAKT and/or pmTOR-positive human PDACs and mouse pancreatic tumors also shared some histopathological similarities. These observations indicate that Smad4/Pten-mutant mice mimic the tumor progression of human pancreatic cancers that are driven by activation of the AKT-mTOR pathway, and uncovered a synergistic action of Smad4 and Pten in repressing pancreatic tumorigenesis.

Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation.

PubMed

Bornstein, Sophia; White, Ruth; Malkoski, Stephen; Oka, Masako; Han, Gangwen; Cleaver, Timothy; Reh, Douglas; Andersen, Peter; Gross, Neil; Olson, Susan; Deng, Chuxia; Lu, Shi-Long; Wang, Xiao-Jing

2009-11-01

Smad4 is a central mediator of TGF-beta signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4-/- mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4-/- mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4-/- mice exhibited severe inflammation, which was associated with increased expression of TGF-beta1 and activated Smad3. We present what we believe to be the first single gene-knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

Upregulation of SMAD4 by MZF1 inhibits migration of human gastric cancer cells.

PubMed

Lee, Jin-Hee; Kim, Sung-Su; Lee, Hun Seok; Hong, Sungyoul; Rajasekaran, Nirmal; Wang, Li-Hui; Choi, Joon-Seok; Shin, Young Kee

2017-01-01

SMAD4 is a tumor suppressor that is frequently inactivated in many types of cancer. The role of abnormal expression of SMAD4 has been reported in developmental processes and the progression of various human cancers. The expression level of SMAD4 has been related to the survival rate in gastric cancer patients. However, the molecular mechanism underlying transcriptional regulation of SMAD4 remains largely unknown. In the present study, we characterized the promoter region of SMAD4 and identified myeloid zinc finger 1 (MZF1), as a putative transcription factor. MZF1 directly bound to a core region of the SMAD4 promoter and stimulated transcriptional activity. We also found that the expression of MZF1 influences the migration ability of gastric adenocarcinoma cells. Collectively, our results showed that MZF1 has a role in cellular migration of gastric cancer cells via promoting an increase in intracellular SMAD4 levels. This study might provide new evidence for the molecular basis of the tumor suppressive effect of the MZF1-SMAD4 axis, a new therapeutic target in advanced human gastric cancer.

PTEN Regulates Beta-Catenin in Androgen Signaling: Implication in Prostate Cancer Progression

DTIC Science & Technology

2007-03-01

Smad3 and Smad4 proteins contain a number of functional domains, including MH1, MH2, and the linker region (2). It appears that theMH2 domain is involved...domains or Smad4 linker region. These data demonstrate that the MH2 domains of Smad3 and Smad4 are involved in the interaction with hZimp10. hZimp10...length (FL), MH1 domain (MH1, 1–146 amino acids), linker domain (L, 147–308 amino acids), and MH2 domain (MH2, 309 –553 amino acids) and GST- Smad3 full

Smad4 is required for the development of cardiac and skeletal muscle in zebrafish.

PubMed

Yang, Jie; Wang, Junnai; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Jiang, Lijun; Wei, Juncheng; Ma, Quanfu; Wu, Mingfu; Ye, Shuangmei; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

Transforming growth factor-beta (TGF-beta) regulates cellular functions and plays key roles in development and carcinogenesis. Smad4 is the central intracellular mediator of TGF-beta signaling and plays crucial roles in tissue regeneration, cell differentiation, embryonic development, regulation of the immune system and tumor progression. To clarify the role of smad4 in development, we examined both the pattern of smad4 expression in zebrafish embryos and the effect of smad4 suppression on embryonic development using smad4-specific antisense morpholino-oligonucleotides. We show that smad4 is expressed in zebrafish embryos at all developmental stages examined and that embryonic knockdown of smad4 results in pericardial edema, decreased heartbeat and defects in the trunk structure. Additionally, these phenotypes were associated with abnormal expression of the two heart-chamber markers, cmlc2 and vmhc, as well as abnormal expression of three makers of myogenic terminal differentiation, mylz2, smyhc1 and mck. Furthermore, a notable increase in apoptosis was apparent in the smad4 knockdown embryos, while no obvious reduction in cell proliferation was observed. Collectively, these data suggest that smad4 plays an important role in heart and skeletal muscle development. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity.

PubMed

Chong, P Andrew; Lin, Hong; Wrana, Jeffrey L; Forman-Kay, Julie D

2010-10-26

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching.

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity

PubMed Central

Chong, P. Andrew; Lin, Hong; Wrana, Jeffrey L.; Forman-Kay, Julie D.

2010-01-01

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching. PMID:20937913

Novel tumor suppressive function of Smad4 in serum starvation-induced cell death through PAK1–PUMA pathway

PubMed Central

Lee, S-H; Jung, Y-S; Chung, J-Y; Oh, A Y; Lee, S-J; Choi, D H; Jang, S M; Jang, K-S; Paik, S S; Ha, N-C; Park, B-J

2011-01-01

DPC4 (deleted in pancreatic cancer 4)/Smad4 is an essential factor in transforming growth factor (TGF)-β signaling and is also known as a frequently mutated tumor suppressor gene in human pancreatic and colon cancer. However, considering the fact that TGF-β can contribute to cancer progression through transcriptional target genes, such as Snail, MMPs, and epithelial–mesenchymal transition (EMT)-related genes, loss of Smad4 in human cancer would be required for obtaining the TGF-β signaling-independent advantage, which should be essential for cancer cell survival. Here, we provide the evidences about novel role of Smad4, serum-deprivation-induced apoptosis. Elimination of serum can obviously increase the Smad4 expression and induces the cell death by p53-independent PUMA induction. Instead, Smad4-deficient cells show the resistance to serum starvation. Induced Smad4 suppresses the PAK1, which promotes the PUMA destabilization. We also found that Siah-1 and pVHL are involved in PAK1 destabilization and PUMA stabilization. In fact, Smad4-expressed cancer tissues not only show the elevated expression of PAK1, but also support our hypothesis that Smad4 induces PUMA-mediated cell death through PAK1 suppression. Our results strongly suggest that loss of Smad4 renders the resistance to serum-deprivation-induced cell death, which is the TGF-β-independent tumor suppressive role of Smad4. PMID:22130069

Loss of Smad4 in colorectal cancer induces resistance to 5-fluorouracil through activating Akt pathway.

PubMed

Zhang, B; Zhang, B; Chen, X; Bae, S; Singh, K; Washington, M K; Datta, P K

2014-02-18

Higher frequency of Smad4 inactivation or loss of expression is observed in metastasis of colorectal cancer (CRC) leading to unfavourable survival and contributes to chemoresistance. However, the molecular mechanism of how Smad4 regulates chemosensitivity of CRC is unknown. We evaluated how the loss of Smad4 in CRC enhanced chemoresistance to 5-fluorouracil (5-FU) using two CRC cell lines in vitro and in vivo. Immunoblotting with cell and tumour lysates and immunohistochemical analyses with tissue microarray were performed. Knockdown or loss of Smad4 induced tumorigenicity, migration, invasion, angiogenesis, metastasis, and 5-FU resistance. Smad4 expression in mouse tumours regulated cell-cycle regulatory proteins leading to Rb phosphorylation. Loss of Smad4 activated Akt pathway that resulted in upregulation of anti-apoptotic proteins, Bcl-2 and Bcl-w, and Survivin. Suppression of phosphatidylinositol-3-kinase (PI3K)/Akt pathway by LY294002 restored chemosensitivity of Smad4-deficient cells to 5-FU. Vascular endothelial growth factor-induced angiogenesis in Smad4-deficient cells might also lead to chemoresistance. Low levels of Smad4 expression in CRC tissues correlated with higher levels of Bcl-2 and Bcl-w and with poor overall survival as observed in immunohistochemical staining of tissue microarrays. Loss of Smad4 in CRC patients induces resistance to 5-FU-based therapy through activation of Akt pathway and inhibitors of this pathway may sensitise these patients to 5-FU.

Loss of Smad4 in colorectal cancer induces resistance to 5-fluorouracil through activating Akt pathway

PubMed Central

Zhang, B; Zhang, B; Chen, X; Bae, S; Singh, K; Washington, M K; Datta, P K

2014-01-01

Background: Higher frequency of Smad4 inactivation or loss of expression is observed in metastasis of colorectal cancer (CRC) leading to unfavourable survival and contributes to chemoresistance. However, the molecular mechanism of how Smad4 regulates chemosensitivity of CRC is unknown. Methods: We evaluated how the loss of Smad4 in CRC enhanced chemoresistance to 5-fluorouracil (5-FU) using two CRC cell lines in vitro and in vivo. Immunoblotting with cell and tumour lysates and immunohistochemical analyses with tissue microarray were performed. Results: Knockdown or loss of Smad4 induced tumorigenicity, migration, invasion, angiogenesis, metastasis, and 5-FU resistance. Smad4 expression in mouse tumours regulated cell-cycle regulatory proteins leading to Rb phosphorylation. Loss of Smad4 activated Akt pathway that resulted in upregulation of anti-apoptotic proteins, Bcl-2 and Bcl-w, and Survivin. Suppression of phosphatidylinositol-3-kinase (PI3K)/Akt pathway by LY294002 restored chemosensitivity of Smad4-deficient cells to 5-FU. Vascular endothelial growth factor-induced angiogenesis in Smad4-deficient cells might also lead to chemoresistance. Low levels of Smad4 expression in CRC tissues correlated with higher levels of Bcl-2 and Bcl-w and with poor overall survival as observed in immunohistochemical staining of tissue microarrays. Conclusion: Loss of Smad4 in CRC patients induces resistance to 5-FU-based therapy through activation of Akt pathway and inhibitors of this pathway may sensitise these patients to 5-FU. PMID:24384683

Osthole inhibits the expressions of collagen I and III through Smad signaling pathway after treatment with TGF-β1 in mouse cardiac fibroblasts.

PubMed

Liu, Jin-Cheng; Wang, Feng; Xie, Mei-Lin; Cheng, Zong-Qi; Qin, Qiong; Chen, Lin; Chen, Rong

2017-02-01

Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-β1 (TGF-β1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-β1. The mouse CFs stimulated with TGF-β1 were cultured and treated with osthole 1.25-5μg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-β receptor I (TβRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively. After treatment with TGF-β1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TβRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole. The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-β1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

PubMed

Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

2005-04-01

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells.

PubMed

Matsuzaki, Koichi

2012-01-01

Hepatocellular carcinoma (HCC) usually arises from hepatic fibrosis caused by chronic inflammation. In chronic liver damage, hepatic stellate cells undergo progressive activation to myofibroblasts (MFB), which are important extracellular-matrix-producing mesenchymal cells. Concomitantly, perturbation of transforming growth factor (TGF)-β signaling by pro-inflammatory cytokines in the epithelial cells of the liver (hepatocytes) promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into fibro-carcinogenic effects on chronically damaged hepatocytes have come from recent detailed analyses of the TGF-β signaling process. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create phosphoisoforms phosphorylated at the COOH-terminal, linker, or both (L/C) regions. After acute liver injury, TGF-β-mediated pSmad3C signaling terminates hepatocytic proliferation induced by the pro-inflammatory cytokine-mediated mitogenic pSmad3L pathway; TGF-β and pro-inflammatory cytokines synergistically enhance collagen synthesis by activated hepatic stellate cells via pSmad2L/C and pSmad3L/C pathways. During chronic liver disease progression, pre-neoplastic hepatocytes persistently affected by TGF-β together with pro-inflammatory cytokines come to exhibit the same carcinogenic (mitogenic) pSmad3L and fibrogenic pSmad2L/C signaling as do MFB, thereby accelerating liver fibrosis while increasing risk of HCC. This review of Smad phosphoisoform-mediated signals examines similarities and differences between epithelial and mesenchymal cells in acute and chronic liver injuries and considers Smad linker phosphorylation as a potential target for the chemoprevention of fibro-carcinogenesis.

Overlapping spectra of SMAD4 mutations in juvenile polyposis (JP) and JP-HHT syndrome.

PubMed

Gallione, Carol; Aylsworth, Arthur S; Beis, Jill; Berk, Terri; Bernhardt, Barbara; Clark, Robin D; Clericuzio, Carol; Danesino, Cesare; Drautz, Joanne; Fahl, Jeffrey; Fan, Zheng; Faughnan, Marie E; Ganguly, Arupa; Garvie, John; Henderson, Katharine; Kini, Usha; Leedom, Tracey; Ludman, Mark; Lux, Andreas; Maisenbacher, Melissa; Mazzucco, Sara; Olivieri, Carla; Ploos van Amstel, Johannes K; Prigoda-Lee, Nadia; Pyeritz, Reed E; Reardon, Willie; Vandezande, Kirk; Waldman, J Deane; White, Robert I; Williams, Charles A; Marchuk, Douglas A

2010-02-01

Juvenile polyposis (JP) and hereditary hemorrhagic telangiectasia (HHT) are clinically distinct diseases caused by mutations in SMAD4 and BMPR1A (for JP) and endoglin and ALK1 (for HHT). Recently, a combined syndrome of JP-HHT was described that is also caused by mutations in SMAD4. Although both JP and JP-HHT are caused by SMAD4 mutations, a possible genotype:phenotype correlation was noted as all of the SMAD4 mutations in the JP-HHT patients were clustered in the COOH-terminal MH2 domain of the protein. If valid, this correlation would provide a molecular explanation for the phenotypic differences, as well as a pre-symptomatic diagnostic test to distinguish patients at risk for the overlapping but different clinical features of the disorders. In this study, we collected 19 new JP-HHT patients from which we identified 15 additional SMAD4 mutations. We also reviewed the literature for other reports of JP patients with HHT symptoms with confirmed SMAD4 mutations. Our combined results show that although the SMAD4 mutations in JP-HHT patients do show a tendency to cluster in the MH2 domain, mutations in other parts of the gene also cause the combined syndrome. Thus, any mutation in SMAD4 can cause JP-HHT. Any JP patient with a SMAD4 mutation is, therefore, at risk for the visceral manifestations of HHT and any HHT patient with SMAD4 mutation is at risk for early onset gastrointestinal cancer. In conclusion, a patient who tests positive for any SMAD4 mutation must be considered at risk for the combined syndrome of JP-HHT and monitored accordingly. Copyright 2010 Wiley-Liss, Inc.

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed

PubMed Central

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-01-01

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed. PMID:27845895

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

PubMed

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-03-21

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways.

PubMed

Strand, Douglas W; Liang, Yao-Yun; Yang, Feng; Barron, David A; Ressler, Steven J; Schauer, Isaiah G; Feng, Xin-Hua; Rowley, David R

2014-01-01

Transforming Growth Factor-β (TGF-β) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-β is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-β regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-β1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-β1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-β1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-β1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-β action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention.

PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis.

PubMed

Basso, Daniela; Gnatta, Elisa; Padoan, Andrea; Fogar, Paola; Furlanello, Sara; Aita, Ada; Bozzato, Dania; Zambon, Carlo-Federico; Arrigoni, Giorgio; Frasson, Chiara; Franchin, Cinzia; Moz, Stefania; Brefort, Thomas; Laufer, Thomas; Navaglia, Filippo; Pedrazzoli, Sergio; Basso, Giuseppe; Plebani, Mario

2017-10-17

Tumor genetics and escape from immune surveillance concur in the poor prognosis of PDAC. In this study an experimental model was set up to verify whether SMAD4 , deleted in about 55% PDAC and associated with poor prognosis, is involved in determining immunosuppression through Exosomes (Exo). Potential mechanisms and mediators underlying SMAD4 -dependent immunosuppression were evaluated by studying intracellular calcium (Fluo-4), Exo-miRNAs (microarray) and Exo-proteins (SILAC). Two PDAC cell lines expressing (BxPC3- SMAD4 +) or not-expressing (BxPC3) SMAD4 were used to prepare Exo-enriched conditioned media, employed in experiments with blood donors PBMCs. Exo expanded myeloid derived suppressor cells (gMDSC and mMDSC, flow cytometry) and altered intracellular calcium fluxes in an SMAD4 dependent manner. BxPC3- SMAD4 +, but mainly BxPC3 Exo, increased calcium fluxes of PBMCs ( p = 0.007) and this increased intracellular calcium trafficking characterized mMDSCs. The analysis of de-regulated Exo-miRNAs and transfection experiments revealed hsa-miR-494-3p and has-miR-1260a as potential mediators of SMAD4- associated de-regulated calcium fluxes. Eleven main biological processes were identified by the analysis of SMAD4 -associated de-regulated Exo-proteins, including translation, cell adhesion, cell signaling and glycolysis. A reverse Warburg effect was observed by treating PBMCs with PDAC-derived Exo: BxPC3 Exo induced a higher glucose consumption and lactate production than BxPC3- SMAD4 + Exo. PDAC-derived Exo from cells with , but mainly from those without SMAD4 expression, create an immunosuppressive myeloid cell background by increasing calcium fluxes and glycolysis through the transfer of SMAD4 -related differentially expressed miRNAs and proteins.

Blocking of SMAD4 expression by shRNA effectively inhibits fibrogenesis of human hepatic stellate cells.

PubMed

Khanizadeh, Sayyad; Ravanshad, Mehrdad; Hosseini, SeyedYounes; Davoodian, Parivash; Nejati Zadeh, Azim; Sarvari, Jamal

2015-01-01

In this study, to clarify the SMAD4 blocking impact on fibrosis process, we investigated its down-regulation by shRNA on activated human LX-2 cell, in vitro. Liver fibrosis is a critical consequence of chronic damage to the liver that can progress toward advanced diseases, liver cirrhosis and hepatocellular carcinoma (HCC). Different SMAD proteins play as major mediators in the fibrogenesis activity of hepatic stellate cells through TGF-β pathways, but the extent of SMAD4 as a co-SMAD protein remained less clear. vector expressing verified shRNA targeting human SMAD4 gene was transfected into LX-2 cells. The GFP expressing plasmid was transfected in the same manner as a control group while leptin treated cells were employed as positive controls. Subsequently, total RNA was extracted and real-time PCR was performed to measure the mRNA levels of SMAD4, COL-1A1, α-SMA, TGF-β and TIMP-1. Furthermore, trypan blue exclusion was performed to test the effect of plasmid transfection and SMAD4 shutting-down on cellular viability. The results indicated that the expression of SMAD4was down-regulated following shRNA transfection intoLX-2 cells (P<0.001). The gene expression analysis of fibrotic genes in LX-2 cells showed that SMAD4 blocking by shRNA significantly reduced the expression level of fibrotic genes when compared to control plasmids (P<0.001). Vector expressing SMAD4-shRNA induced no significant cytotoxic or proliferative effects on LX-2 cells as determined by viability assay (P<0.05). The results of this study suggested that knockdown of SMAD4 expression in stellate cell can control the progression of fibrogenesis through TGF-β pathway blocking.

Reversing SKI-SMAD4-mediated suppression is essential for TH17 cell differentiation.

PubMed

Zhang, Song; Takaku, Motoki; Zou, Liyun; Gu, Ai-di; Chou, Wei-Chun; Zhang, Ge; Wu, Bing; Kong, Qing; Thomas, Seddon Y; Serody, Jonathan S; Chen, Xian; Xu, Xiaojiang; Wade, Paul A; Cook, Donald N; Ting, Jenny P Y; Wan, Yisong Y

2017-11-02

T helper 17 (T H 17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor β (TGFβ) is instrumental in T H 17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables T H 17 cell differentiation remains elusive. Here we reveal that TGFβ enables T H 17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into T H 17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and T H 17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and T H 17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and T H 17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable T H 17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls T H 17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating T H 17-related diseases.

PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis

PubMed Central

Basso, Daniela; Gnatta, Elisa; Padoan, Andrea; Fogar, Paola; Furlanello, Sara; Aita, Ada; Bozzato, Dania; Zambon, Carlo-Federico; Arrigoni, Giorgio; Frasson, Chiara; Franchin, Cinzia; Moz, Stefania; Brefort, Thomas; Laufer, Thomas; Navaglia, Filippo; Pedrazzoli, Sergio; Basso, Giuseppe; Plebani, Mario

2017-01-01

Tumor genetics and escape from immune surveillance concur in the poor prognosis of PDAC. In this study an experimental model was set up to verify whether SMAD4, deleted in about 55% PDAC and associated with poor prognosis, is involved in determining immunosuppression through Exosomes (Exo). Potential mechanisms and mediators underlying SMAD4-dependent immunosuppression were evaluated by studying intracellular calcium (Fluo-4), Exo-miRNAs (microarray) and Exo-proteins (SILAC). Two PDAC cell lines expressing (BxPC3-SMAD4+) or not-expressing (BxPC3) SMAD4 were used to prepare Exo-enriched conditioned media, employed in experiments with blood donors PBMCs. Exo expanded myeloid derived suppressor cells (gMDSC and mMDSC, flow cytometry) and altered intracellular calcium fluxes in an SMAD4 dependent manner. BxPC3-SMAD4+, but mainly BxPC3 Exo, increased calcium fluxes of PBMCs (p = 0.007) and this increased intracellular calcium trafficking characterized mMDSCs. The analysis of de-regulated Exo-miRNAs and transfection experiments revealed hsa-miR-494-3p and has-miR-1260a as potential mediators of SMAD4-associated de-regulated calcium fluxes. Eleven main biological processes were identified by the analysis of SMAD4-associated de-regulated Exo-proteins, including translation, cell adhesion, cell signaling and glycolysis. A reverse Warburg effect was observed by treating PBMCs with PDAC-derived Exo: BxPC3 Exo induced a higher glucose consumption and lactate production than BxPC3-SMAD4+ Exo. Conclusion: PDAC-derived Exo from cells with, but mainly from those without SMAD4 expression, create an immunosuppressive myeloid cell background by increasing calcium fluxes and glycolysis through the transfer of SMAD4-related differentially expressed miRNAs and proteins. PMID:29156694

FSP1-specific SMAD2 knockout in renal tubular, endothelial, and interstitial cells reduces fibrosis and epithelial-to-mesenchymal transition in murine STZ-induced diabetic nephropathy.

PubMed

Loeffler, Ivonne; Liebisch, Marita; Allert, Stefanie; Kunisch, Elke; Kinne, Raimund W; Wolf, Gunter

2018-04-01

Extracellular matrix deposition during tubulointerstitial fibrosis (TIF), a central pathological process in patients with diabetic nephropathy (DN), is driven by locally activated, disease-relevant myofibroblasts. Myofibroblasts can arise from various cellular sources, e.g., tubular epithelial cells via a process named epithelial-to-mesenchymal transition (EMT). Transforming growth factor beta 1 (TGF-β1) and its downstream Smad signaling play a critical role in both TIF and EMT. Whereas Smad3 is one central mediator, the role of the other prominently expressed variant, Smad2, is not completely understood. In this study, we sought to analyze the role of renal Smad2 in the development of TIF and EMT during streptozotocin-induced DN by using a fibroblast-specific protein 1 (FSP1)-promotor-driven SMAD2 knockout mouse model with decreased tubular, endothelial, and interstitial Smad2 expression. In contrast to wild-type diabetic mice, diabetic SMAD2 knockout mice showed the following features: (1) significantly reduced DN and TIF (shown by KIM1 expression; periodic acid Schiff staining; collagen I and III, fibronectin, and connective tissue growth factor deposition); (2) significantly reduced tubular EMT-like changes (e.g., altered Snail1, E-cadherin, matrix metalloproteinase 2, and vimentin deposition); and (3) significantly decreased expression of myofibroblast markers (α-smooth muscle actin, FSP1). As one mechanism for the protection against diabetes-induced TIF and EMT, decreased Smad3 protein levels and, as a possible consequence, reduced TGF-β1 levels were observed in diabetic SMAD2 knockout mice. Our findings thus support the important role of Smad2 for pro-fibrotic TGF-β/Smad3 signaling in experimental DN.

Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation

PubMed Central

Bornstein, Sophia; White, Ruth; Malkoski, Stephen; Oka, Masako; Han, Gangwen; Cleaver, Timothy; Reh, Douglas; Andersen, Peter; Gross, Neil; Olson, Susan; Deng, Chuxia; Lu, Shi-Long; Wang, Xiao-Jing

2009-01-01

Smad4 is a central mediator of TGF-β signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4–/– mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited severe inflammation, which was associated with increased expression of TGF-β1 and activated Smad3. We present what we believe to be the first single gene–knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation. PMID:19841536

Inactivation of Smad4 in gastric carcinomas.

PubMed

Powell, S M; Harper, J C; Hamilton, S R; Robinson, C R; Cummings, O W

1997-10-01

Allelic loss of chromosome 18q has been noted in intestinal type gastric adenocarcinomas. Smad4 is a gene located at 18q that was recently cloned in humans and found to be significantly altered in pancreatic cancers. We sought to determine whether Smad4 genetic alterations played a significant role in gastric tumorigenesis by studying 35 gastric adenocarcinomas of all histopathological types and pathological stages. Microdissected specimens were used for mutational analysis of Smad4 at the nucleotide level, including the entire coding region and intron/exon boundaries. Allelic imbalance was also analyzed at the Smad4 locus using two nearby microsatellite markers. One case of apparent biallelic inactivation of Smad4 was found in our study of 35 gastric carcinomas. A nonsense point mutation at codon 334 was demonstrated, which, similar to other Smad4 mutations, is predicted to truncate the conserved COOH-terminal domain of this protein. This Smad4 C to T transition mutation was proven to be somatically acquired. Allelic loss was also noted on chromosome 18q at a marker near Smad4 in this mutated gastric cancer, apparently producing complete inactivation of Smad4 in this tumor. Significant 18q allelic loss (56% of 34 informative cases) was noted in our gastric carcinomas using microsatellite markers near the Smad4 locus, regardless of histological subtype or pathological stage. Additionally, three cases of microsatellite instability were observed. Thus, Smad4 inactivation was noted in our gastric carcinomas; however, this event was rare. The frequent loss of chromosomal arm 18q observed in gastric cancers suggests the presence of other tumor suppressor genes in this region that are involved in gastric tumorigenesis. Further studies are needed to identify these other targets of inactivation during gastric cancer development.

LOXL4 Is Induced by Transforming Growth Factor β1 through Smad and JunB/Fra2 and Contributes to Vascular Matrix Remodeling

PubMed Central

Busnadiego, Oscar; González-Santamaría, José; Lagares, David; Guinea-Viniegra, Juan; Pichol-Thievend, Cathy; Muller, Laurent

2013-01-01

Transforming growth factor β1 (TGF-β1) is a pleiotropic factor involved in the regulation of extracellular matrix (ECM) synthesis and remodeling. In search for novel genes mediating the action of TGF-β1 on vascular ECM, we identified the member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like 4 (LOXL4), as a direct target of TGF-β1 in aortic endothelial cells, and we dissected the molecular mechanism of its induction. Deletion mapping and mutagenesis analysis of the LOXL4 promoter demonstrated the absolute requirement of a distal enhancer containing an activator protein 1 (AP-1) site and a Smad binding element for TGF-β1 to induce LOXL4 expression. Functional cooperation between Smad proteins and the AP-1 complex composed of JunB/Fra2 accounted for the action of TGF-β1, which involved the extracellular signal-regulated kinase (ERK)-dependent phosphorylation of Fra2. We furthermore provide evidence that LOXL4 was extracellularly secreted and significantly contributed to ECM deposition and assembly. These results suggest that TGF-β1-dependent expression of LOXL4 plays a role in vascular ECM homeostasis, contributing to vascular processes associated with ECM remodeling and fibrosis. PMID:23572561

Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

PubMed

Abushahba, Walid; Olabisi, Oyenike O; Jeong, Byeong-Seon; Boregowda, Rajeev K; Wen, Yu; Liu, Fang; Goydos, James S; Lasfar, Ahmed; Cohen-Solal, Karine A

2012-01-01

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.

Non-Canonical Smads Phosphorylation Induced by the Glutamate Release Inhibitor, Riluzole, through GSK3 Activation in Melanoma

PubMed Central

Jeong, Byeong-Seon; Boregowda, Rajeev K.; Wen, Yu; Liu, Fang; Goydos, James S.; Lasfar, Ahmed; Cohen-Solal, Karine A.

2012-01-01

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas. PMID:23077590

SMAD4 gene mutation predicts poor prognosis in patients undergoing resection for colorectal liver metastases.

PubMed

Mizuno, Takashi; Cloyd, Jordan M; Vicente, Diego; Omichi, Kiyohiko; Chun, Yun Shin; Kopetz, Scott E; Maru, Dipen; Conrad, Claudius; Tzeng, Ching-Wei D; Wei, Steven H; Aloia, Thomas A; Vauthey, Jean-Nicolas

2018-05-01

Dorsophilia protein, mothers against decapentaplegic homolog 4 (SMAD4) is a key mediator in the transforming growth factor (TGF)-β signaling pathway and SMAD4 gene mutations are thought to play a critical role in colorectal cancer (CRC) progression. However, little is known about its influence on survival in patients undergoing resection for colorectal liver metastases (CLM). Between 2005 and 2015, all patients with known SMAD4 mutation status who underwent resection of CLM were identified. Patients with SMAD4 mutation were compared to those with SMAD4 wild type. Next, the prognostic value of SMAD4 mutation was validated in a separate cohort of patients with synchronous stage IV CRC who underwent systemic therapy alone. Of 278 patients, 37 (13%) were SMAD4 mutant while 241 (87%) were wild type. Overall survival (OS) after hepatic resection was worse in SMAD4-mutant patients compared to SMAD4 wild type (OS rate at 3 years, 62% vs. 82%; P < 0.0001). Independent predictors for worse OS were poor differentiation (hazard ratio [HR] 2.586; P = 0.007), multiple tumors (HR 1.970; P = 0.01), diameter greater than 3 cm (HR 1.752; P = 0.017), R1 margin status (HR 2.452; P = 0.014), RAS mutation (HR 2.044; P = 0.002), and SMAD4 mutation (HR 2.773; P < 0.0001). Among 237 patients in the validation cohort, SMAD4-mutations were significantly associated with worse 3-year OS rate (22% vs. 38%; P = 0.012) and was an independent predictor for worse OS (HR, 1.647; P = 0.032). SMAD4 mutation is independently associated with worse outcomes among patients undergoing resection of CLM. Copyright © 2018 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

Smad4 sensitizes colorectal cancer to 5-fluorouracil through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade.

PubMed

Zhang, Binhao; Leng, Chao; Wu, Chao; Zhang, Zhanguo; Dou, Lei; Luo, Xin; Zhang, Bixiang; Chen, Xiaoping

2016-03-01

5-Fluorouracil (5-FU), a cell cycle-specific antimetabolite, is one of the most commonly used chemotherapeutic agents for colorectal cancer (CRC). Yet, resistance to 5-FU-based chemotherapy is still an obstacle to the treatment of this malignancy. Mutation or loss of Smad4 in CRC is pivotal for chemoresistance. However, the mechanism by which Smad4 regulates the chemosensitivity of CRC remains unclear. In the present study, we investigated the role of Smad4 in the chemosensitivity of CRC to 5-FU, and whether Smad4-regulated cell cycle arrest is involved in 5-FU chemoresistance. We used Smad4-expressing CT26 and Smad4-null SW620 cell lines as experimental models, by knockdown or transgenic overexpression. Cells or tumors were treated with 5-FU to determine chemosensitivity by cell growth, tumorigenicity assay and a mouse model. Cell cycle distribution was examined with flow cytometric analysis, and cell cycle-related proteins were examined by western blotting. Smad4 deficiency in CT26 and SW620 cells induced chemoresistance to 5-FU both in vitro and in vivo. Smad4 deficiency attenuated G1 or G2 cell cycle arrest by activating the PI3K/Akt/CDC2/survivin pathway. The PI3K inhibitor, LY294002, reversed the activation of the Akt/CDC2/survivin cascade in the Smad4-deficient cells, while it had little effect on cells with high Smad4 expression. In conclusion, we discovered a novel mechanism mediated by Smad4 to trigger 5-FU chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade. The present study also implies that LY294002 has potential therapeutic value to reverse the chemosensitivity of CRC with low Smad4 expression.

Smad4 Loss in Esophageal Adenocarcinoma Is Associated With an Increased Propensity for Disease Recurrence and Poor Survival

PubMed Central

Singhi, Aatur D.; Foxwell, Tyler J.; Nason, Katie; Cressman, Kristi L.; McGrath, Kevin M.; Sun, Weijing; Bahary, Nathan; Zeh, Herbert J.; Levy, Ryan M.; Luketich, James D.; Davison, Jon M.

2015-01-01

Previously regarded as a rare neoplasm, the incidence of esophageal adenocarcinoma has risen rapidly in recent decades. It is often discovered late in the disease process and has a dismal prognosis. Current prognostic markers including clinical, radiographic, and histopathologic findings have limited utility and do not consider the biology of this deadly disease. Genome-wide analyses have identified SMAD4 inactivation in a subset of tumors. Although Smad4 has been extensively studied in other gastrointestinal malignancies, its role in esophageal adenocarcinoma remains to be defined. Herein, we show, in a large cohort of esophageal adenocarcinomas, Smad4 loss by immunohistochemistry in 21 of 205 (10%) tumors and that Smad4 loss correlated with increased postoperative recurrence (P=0.040). Further, patients whose tumors lacked Smad4 had shorter time to recurrence (TTR) (P=0.007) and poor overall survival (OS) (P=0.011). The median TTR and OS of patients with Smad4-negative tumors was 13 and 16 months, respectively, as compared with 23 and 22 months, respectively, among patients with Smad4-positive tumors. In multivariate analyses, Smad4 loss was a prognostic factor for both TTR and OS, independent of histologic grade, lymphovascular invasion, perineural invasion, tumor stage, and lymph node status. Considering Smad4 loss correlated with postoperative locoregional and/or distant metastases, Smad4 was also assessed in a separate cohort of 5 locoregional recurrences and 43 metastatic esophageal adenocarcinomas. In contrast to primary tumors, a higher prevalence of Smad4 loss was observed in metastatic disease (44% vs. 10%). In summary, loss of Smad4 protein expression is an independent prognostic factor for TTR and OS that correlates with increased propensity for disease recurrence and poor survival in patients with esophageal adenocarcinoma after surgical resection. PMID:25634752

Smad4-Mediated Signaling Inhibits Intestinal Neoplasia by Inhibiting Expression of β-Catenin

PubMed Central

Freeman, Tanner J.; Smith, J. Joshua; Chen, Xi; Washington, M. Kay; Roland, Joseph T.; Means, Anna L.; Eschrich, Steven A.; Yeatman, Timothy J.; Deane, Natasha G.; Beauchamp, R. Daniel

2012-01-01

Background & Aims Mutational inactivation of APC is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. CRC progression also involves inactivation of signaling via transforming growth factor (TGF)β and bone morphenogenic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 loss on levels of β-catenin mRNA and Wnt signaling. Methods We used microarray analysis to associate levels of Smad4 and β-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APCΔ1638/+) and (APCΔ1638/+)x(K19CreERT2Smad4lox/lox) mice using laser-capture microdissection. Results In human CRC samples, reduced levels of Smad4 correlated with increased levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the β-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice with heterozygous disruption of Apc(APCΔ1638/+), Smad4-deficient intestinal adenomas had increased levels of β-catenin mRNA and expression of Wnt target genes, compared with adenomas from APCΔ1638/+mice that expressed Smad4. Conclusions Transcription of β-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among TGF-β, BMP, and Wnt signaling pathways in CRC progression. PMID:22115830

Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation

PubMed Central

Yoon, Jeong-Hwan; Sudo, Katsuko; Kuroda, Masahiko; Kato, Mitsuyasu; Lee, In-Kyu; Han, Jin Soo; Nakae, Susumu; Imamura, Takeshi; Kim, Juryun; Ju, Ji Hyeon; Kim, Dae-Kee; Matsuzaki, Koichi; Weinstein, Michael; Matsumoto, Isao; Sumida, Takayuki; Mamura, Mizuko

2015-01-01

Transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4+ T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad–STAT3 signalling network in TH17 differentiation. PMID:26194464

Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal.

PubMed

Fuentealba, Luis C; Eivers, Edward; Ikeda, Atsushi; Hurtado, Cecilia; Kuroda, Hiroki; Pera, Edgar M; De Robertis, Edward M

2007-11-30

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

Structural Basis of Intracellular TGF-β Signaling: Receptors and Smads.

PubMed

Chaikuad, Apirat; Bullock, Alex N

2016-11-01

Stimulation of the transforming growth factor β (TGF-β) family receptors activates an intracellular phosphorylation-dependent signaling cascade that culminates in Smad transcriptional activation and turnover. Structural studies have identified a number of allosteric mechanisms that control the localization, conformation, and oligomeric state of the receptors and Smads. Such mechanisms dictate the ordered binding of substrate and adaptor proteins that determine the directionality of the signaling process. Activation of the pathway has been illustrated by the various structures of the receptor-activated Smads (R-Smads) with SARA, Smad4, and YAP, respectively, whereas mechanisms of down-regulation have been elucidated by the structural complexes of FKBP12, Ski, and Smurf1. Interesting parallels have emerged between the R-Smads and the Forkhead-associated (FHA) and interferon regulatory factor (IRF)-associated domains, as well as the Hippo pathway. However, important questions remain as to the mechanism of Smad-independent signaling. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension.

PubMed

Drake, Kylie M; Dunmore, Benjamin J; McNelly, Lauren N; Morrell, Nicholas W; Aldred, Micheala A

2013-09-01

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Thrombospondin-1 is a novel negative regulator of liver regeneration after partial hepatectomy through transforming growth factor-beta1 activation in mice.

PubMed

Hayashi, Hiromitsu; Sakai, Keiko; Baba, Hideo; Sakai, Takao

2012-05-01

The matricellular protein, thrombospondin-1 (TSP-1), is prominently expressed during tissue repair. TSP-1 binds to matrix components, proteases, cytokines, and growth factors and activates intracellular signals through its multiple domains. TSP-1 converts latent transforming growth factor-beta1 (TGF-β1) complexes into their biologically active form. TGF-β plays significant roles in cell-cycle regulation, modulation of differentiation, and induction of apoptosis. Although TGF-β1 is a major inhibitor of proliferation in cultured hepatocytes, the functional requirement of TGF-β1 during liver regeneration remains to be defined in vivo. We generated a TSP-1-deficient mouse model of a partial hepatectomy (PH) and explored TSP-1 induction, progression of liver regeneration, and TGF-β-mediated signaling during the repair process after hepatectomy. We show here that TSP-1-mediated TGF-β1 activation plays an important role in suppressing hepatocyte proliferation. TSP-1 expression was induced in endothelial cells (ECs) as an immediate early gene in response to PH. TSP-1 deficiency resulted in significantly reduced TGF-β/Smad signaling and accelerated hepatocyte proliferation through down-regulation of p21 protein expression. TSP-1 induced in ECs by reactive oxygen species (ROS) modulated TGF-β/Smad signaling and proliferation in hepatocytes in vitro, suggesting that the immediately and transiently produced ROS in the regenerating liver were the responsible factor for TSP-1 induction. We have identified TSP-1 as an inhibitory element in regulating liver regeneration by TGF-β1 activation. Our work defines TSP-1 as a novel immediate early gene that could be a potential therapeutic target to accelerate liver regeneration. Copyright © 2011 American Association for the Study of Liver Diseases.

Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor- suppression in rat hepatocellular carcinoma.

PubMed

Nagata, Hiromitsu; Hatano, Etsuro; Tada, Masaharu; Murata, Miki; Kitamura, Koji; Asechi, Hiroyuki; Narita, Masato; Yanagida, Atsuko; Tamaki, Nobuyuki; Yagi, Shintaro; Ikai, Iwao; Matsuzaki, Koichi; Uemoto, Shinji

2009-06-01

Transforming growth factor beta (TGF-beta) signaling involves both tumor-suppression and oncogenesis. TGF-beta activates the TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TbetaRI-dependent pSmad3C transmits a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promotes carcinogenesis in human chronic liver disorders. The aim of this study is to elucidate how SP600125, a JNK inhibitor, affected rat hepatocellular carcinoma (HCC) development, while focusing on the domain-specific phosphorylation of Smad3. The rats received subcutaneous injections of either SP600125 or vehicle 11 times weekly together with 100 ppm N-diethylnitrosamine (DEN) administration for 56 days and were sacrificed in order to evaluate HCC development 28 days after the last DEN administration. The number of tumor nodules greater than 3 mm in diameter and the liver weight/body weight ratio were significantly lower in the SP600125-treated rats than those in the vehicle-treated rats (7.9 +/- 0.8 versus 17.7 +/- 0.9: P < 0.001; 6.3 +/- 1.2 versus 7.1 +/- 0.2%: P < 0.05). SP600125 significantly prolonged the median survival time in rats with DEN-induced HCC (113 versus 97 days: log-rank P = 0.0018). JNK/pSmad3L/c-Myc was enhanced in the rat hepatocytes exposed to DEN. However, TbetaRI/pSmad3C/p21(WAF1) was impaired as DEN-induced HCC developed and progressed. The specific inhibition of JNK activity by SP600125 suppressed pSmad3L/c-Myc in the damaged hepatocytes and enhanced pSmad3C/p21(WAF1), acting as a tumor suppressor in normal hepatocytes. Administration of SP600125 to DEN-treated rats shifted hepatocytic Smad3-mediated signal from oncogenesis to tumor suppression, thus suggesting that JNK could be a therapeutic target of human HCC development and progression.

Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

PubMed Central

Sun, Yu Bo Yang; Qu, Xinli; Li, Xueling; Nikolic-Paterson, David J.; Li, Jinhua

2013-01-01

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation. PMID:24391884

Endothelial dysfunction exacerbates renal interstitial fibrosis through enhancing fibroblast Smad3 linker phosphorylation in the mouse obstructed kidney.

PubMed

Sun, Yu Bo Yang; Qu, Xinli; Li, Xueling; Nikolic-Paterson, David J; Li, Jinhua

2013-01-01

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.

Activation of Bmp2-Smad1 Signal and Its Regulation by Coordinated Alteration of H3K27 Trimethylation in Ras-Induced Senescence

PubMed Central

Kaneda, Atsushi; Fujita, Takanori; Anai, Motonobu; Yamamoto, Shogo; Nagae, Genta; Morikawa, Masato; Tsuji, Shingo; Oshima, Masanobu; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence. PMID:22072987

Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

NASA Astrophysics Data System (ADS)

Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

2016-09-01

Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

PubMed Central

Aragón, Eric; Goerner, Nina; Zaromytidou, Alexia-Ileana; Xi, Qiaoran; Escobedo, Albert; Massagué, Joan; Macias, Maria J.

2011-01-01

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events—Smad activation first and destruction later—and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers. PMID:21685363

Intratumoral Heterogeneity of SMAD4 Immunohistochemical Expression and Its Role in Prediction of Recurrence Pattern in Patients with Resectable Pancreatic Cancer.

PubMed

Pokataev, Ilya; Kudaibergenova, Asel; Artemyeva, Anna; Popova, Anna; Rumyantsev, Alexey; Podluzhny, Danil; Kudashkin, Nikolay; Fedyanin, Mikhail; Tryakin, Alexey; Tjulandin, Sergey

2018-04-20

The aim of our study was to evaluate consistency of SMAD4 expression in different tumor areas and its correlation with recurrence pattern in patients after resection for pancreatic cancer (PC). Records of patients who underwent resection for nonmetastatic PC between 2001 and 2015 were analyzed. Formalin-fixed, paraffin-embedded tissue sections from different areas of primary tumor and lymph node metastases were analyzed immunohistochemically (IHC) for SMAD4 expression using TMA technology. SMAD4 expression was assessed in 356 tissue sections obtained from 91 patients. SMAD4 expression was positive in all assessed tumor slides only in 7 of 26 patients (26.9%). There were 54 recurrences (9 locoregional, 41 distant, and 4 both local and distant) with median follow-up of 21.7 months. There was no correlation between SMAD4 expression and locoregional recurrence pattern (p = 0.30). SMAD4 status influenced neither distant recurrence-free survival (p = 0.99) nor overall survival (p = 0.13). Different areas inside primary tumor and lymph node metastases express SMAD4 heterogeneously. SMAD4 IHC expression is not a biomarker of the recurrence pattern after surgical resection for PC.

Vascular deficiency of Smad4 causes arteriovenous malformations: a mouse model of Hereditary Hemorrhagic Telangiectasia.

PubMed

Crist, Angela M; Lee, Amanda R; Patel, Nehal R; Westhoff, Dawn E; Meadows, Stryder M

2018-05-01

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). Mutations in TGFβ pathway members ALK1, ENG and SMAD4 lead to HHT. However, a Smad4 mouse model of HHT does not currently exist. We aimed to create and characterize a Smad4 endothelial cell (EC)-specific, inducible knockout mouse (Smad4 f/f ;Cdh5-Cre ERT2 ) that could be used to study AVM development in HHT. We found that postnatal ablation of Smad4 caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery-vein gene expression are associated with Smad4 deficiency in the vasculature. Furthermore, we show that depletion of Smad4 leads to decreased Vegfr2 expression, and concurrent loss of endothelial Smad4 and Vegfr2 in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFβ and VEGF signaling pathways in AVM pathogenesis.

Lactobacillus acidophilus attenuates Salmonella-induced intestinal inflammation via TGF-β signaling.

PubMed

Huang, I-Fei; Lin, I-Chun; Liu, Pei-Feng; Cheng, Ming-Fang; Liu, Yen-Chen; Hsieh, Yao-Dung; Chen, Jih-Jung; Chen, Chun-Lin; Chang, Hsueh-Wei; Shu, Chih-Wen

2015-10-07

Salmonella is a common intestinal pathogen that causes acute and chronic inflammatory response. Probiotics reduce inflammatory cytokine production and serve as beneficial commensal microorganisms in the human gastrointestinal tract. TGF-β (transforming growth factor β)/SMAD and NF-κB signaling play important roles in inflammation in intestinal cells. However, the involvement of the signaling in regulating inflammation between Salmonella and probiotics is not fully understood. L. acidophilus and prebiotic inulin were used to treat human intestinal Caco-2 cells prior to infection with Salmonella. The cells were harvested to examine the cytokines and MIR21 expression with immunoblotting and real-time PCR. NF-κB and SMAD3/4 reporter vectors were transfected into cells to monitor inflammation and TGF-β1 signaling, respectively. In this study, we showed that the probiotic L. acidophilus decreased Salmonella-induced NF-κB activation in human intestinal Caco-2 cells. Expression of the inflammatory cytokines, TNF-α and IL-8, in L. acidophilus-pretreated cells was also significantly lower than that in cells infected with Salmonella alone. Moreover, TGF-β1 and MIR21 expression was elevated in cells pretreated with L. acidophilus or synbiotic, a combination of inulin and L. acidophilus, compared to that in untreated cells or cells infected with S. typhimurium alone. By contrast, expression of SMAD7, a target of MIR21, was accordingly reduced in cells treated with L. acidophilus or synbiotics. Consistent with TGF-β1/MIR21 and SMAD7 expression, SMAD3/4 transcriptional activity was significantly higher in the cells treated with L. acidophilus or synbiotics. Furthermore, TGF-β1 antibody antagonized the SMAD3/4 and NF-κB transcriptional activity modulated by L. acidophilus in intestinal cells. Our results suggest that the TGF-β1/MIR21 signaling pathway may be involved in the suppressive effects of L. acidophilus on inflammation caused by S. typhimurium in intestinal Caco-2 cells.

Decorin alleviated chronic hydrocephalus via inhibiting TGF-β1/Smad/CTGF pathway after subarachnoid hemorrhage in rats.

PubMed

Yan, Hui; Chen, Yujie; Li, Lingyong; Jiang, Jiaode; Wu, Guangyong; Zuo, Yuchun; Zhang, John H; Feng, Hua; Yan, Xiaoxin; Liu, Fei

2016-01-01

Chronic hydrocephalus is one of the severe complications after subarachnoid hemorrhage (SAH). However, there is no efficient treatment for the prevention of chronic hydrocephalus, partially due to poor understanding of underlying pathogenesis, subarachnoid fibrosis. Transforming growth factor-β1(TGF-β1) is a potent fibrogenic factor implicated in wide range of fibrotic diseases. To investigate whether decorin, a natural antagonist for TGF-β1, protects against subarachnoid fibrosis and chronic hydrocephalus after SAH, two-hemorrhage-injection SAH model was conducted in 6-week-old rats. Recombinant human decorin(rhDecorin) (30ug/2ul) was administered before blood injection and on the 10th day after SAH. TGF-β1, p-Smad2/3, connective tissue growth factor (CTGF), collagen I and pro-collagen I c-terminal propeptide were assessed via western blotting, enzyme-linked immunosorbent assay, radioimmunoassay and immunofluorescence. And neurobehavioral tests and Morris water maze were employed to evaluate long-term neurological functions after SAH. We found that SAH induced heightened activation of TGF-β1/Smad/CTGF axis, presenting as a two peak response of TGF-β1 in cerebrospinal fluid, elevation of TGF-β1, p-Smad2/3, CTGF, collagen I in brain parenchyma and pro-collagen I c-terminal propeptide in cerebrospinal fluid, and increased lateral ventricle index. rhDecorin treatment effectively inhibited up-regulation of TGF-β1, p-Smad2/3, CTGF, collagen I and pro-collagen I c-terminal propeptide after SAH. Moreover, rhDecorin treatment significantly reduced lateral ventricular index and incidence of chronic hydrocephalus after SAH. Importantly, rhDecorin improved neurocognitive deficits after SAH. In conclusion, rhDecorin suppresses extracellular matrix accumulation and following subarachnoid fibrosis via inhibiting TGF-β1/Smad/CTGF pathway, preventing development of hydrocephalus and attenuating long-term neurocognitive defects after SAH. Copyright © 2015 Elsevier B.V. All rights reserved.

Cooperativity of E-cadherin and Smad4 loss to promote diffuse-type gastric adenocarcinoma and metastasis.

PubMed

Park, Jun Won; Jang, Seok Hoon; Park, Dong Min; Lim, Na Jung; Deng, Chuxia; Kim, Dae Yong; Green, Jeffrey E; Kim, Hark Kyun

2014-08-01

Loss of E-cadherin (CDH1), Smad4, and p53 has been shown to play an integral role in gastric, intestinal, and breast cancer formation. Compound conditional knockout mice for Smad4, p53, and E-cadherin were generated to define and compare the roles of these genes in gastric, intestinal, and breast cancer development by crossing with Pdx-1-Cre, Villin-Cre, and MMTV-Cre transgenic mice. Interestingly, gastric adenocarcinoma was significantly more frequent in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice than in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(+/+) mice, demonstrating that Cdh1 heterozygosity accelerates the development and progression of gastric adenocarcinoma, in combination with loss of Smad4 and p53. Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice developed gastric adenocarcinomas without E-cadherin expression. However, intestinal and mammary adenocarcinomas with the same genetic background retained E-cadherin expression and were phenotypically similar to mice with both wild-type Cdh1 alleles. Lung metastases were identified in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, but not in the other genotypes. Nuclear β-catenin accumulation was identified at the invasive tumor front of gastric adenocarcinomas arising in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. This phenotype was less prominent in mice with intact E-cadherin or Smad4, indicating that the inhibition of β-catenin signaling by E-cadherin or Smad4 downregulates signaling pathways involved in metastases in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. Knockdown of β-catenin significantly inhibited the migratory activity of Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) cell lines. Thus, loss of E-cadherin and Smad4 cooperates with p53 loss to promote the development and metastatic progression of gastric adenocarcinomas, with similarities to human gastric adenocarcinoma. This study demonstrates that inhibition of β-catenin is a converging node for the antimetastatic signaling pathways driven by E-cadherin and Smad4 in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, providing novel insights into mechanisms for gastric cancer metastasis. ©2014 American Association for Cancer Research.

Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

PubMed

Hough, Chris; Radu, Maria; Doré, Jules J E

2012-01-01

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Megakaryocytic Smad4 Regulates Platelet Function through Syk and ROCK2 Expression.

PubMed

Wang, Yanhua; Jiang, Lirong; Mo, Xi; Lan, Yu; Yang, Xiao; Liu, Xinyi; Zhang, Jian; Zhu, Li; Liu, Junling; Wu, Xiaolin

2017-09-01

Smad4, a key transcription factor in the transforming growth factor- β signaling pathway, is involved in a variety of cell physiologic and pathologic processes. Here, we characterized megakaryocyte/platelet-specific Smad4 deficiency in mice to elucidate its effect on platelet function. We found that megakaryocyte/platelet-specific loss of Smad4 caused mild thrombocytopenia and significantly extended first occlusion time and tail bleeding time in mice. Smad4-deficient platelets showed reduced agonist-induced platelet aggregation. Further studies showed that a severe defect was seen in integrin α IIb β 3 -mediated bidirectional (inside-out and outside-in) signaling in Smad4-deficient platelets, as evidenced by reduced fibrinogen binding and α -granule secretion, suppressed platelet spreading and clot retraction. Microarray analysis showed that the expression levels of multiple genes were altered in Smad4-deficient platelets. Among these genes, spleen tyrosine kinase (Syk) and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) were downregulated several times as confirmed by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Further research showed that Smad4 directly regulates ROCK2 transcription but indirectly regulates Syk. Megakaryocyte/platelet-specific Smad4 deficiency caused decreased expression levels of Syk and ROCK2 in platelets. These results suggest potential links among Smad4 deficiency, attenuated Syk, and ROCK2 expression and defective platelet activation. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.

2001-09-11

Smad proteins mediate transforming growth factor-b signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGFb signaling that functions to maintain the repressed state of TGFb target genes in the absence of ligand. Upon TGFb stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGFb target genes. Here we show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase promoting complex (APC) and the UbcH5 family of ubiquitin conjugating enzymes. Smad3 and to a lesser extent, Smad2,more » interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box-dependent manner. In addition to the destruction box, efficient degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGFb signaling. Our studies elucidate an important pathway for the degradation of SnoN and reveal a novel role of the APC in regulation of TGFb signaling.« less

Loss of Smad4 in Sertoli and Leydig Cells Leads to Testicular Dysgenesis and Hemorrhagic Tumor Formation in Mice1

PubMed Central

Archambeault, Denise R.; Yao, Humphrey Hung-Chang

2014-01-01

ABSTRACT As the central component of canonical TGFbeta superfamily signaling, SMAD4 is a critical regulator of organ development, patterning, tumorigenesis, and many other biological processes. Because numerous TGFbeta superfamily ligands are expressed in developing testes, there may exist specific requirements for SMAD4 in individual testicular cell types. Previously, we reported that expansion of the fetal testis cords requires expression of SMAD4 by the Sertoli cell lineage. To further uncover the role of Smad4 in murine testes, we produced conditional knockout mice lacking Smad4 in either Leydig cells or in both Sertoli and Leydig cells simultaneously. Loss of Smad4 concomitantly in Sertoli and Leydig cells led to underdevelopment of the testis cords during fetal life and mild testicular dysgenesis in young adulthood (decreased testis size, partially dysgenic seminiferous tubules, and low sperm production). When the Sertoli/Leydig cell Smad4 conditional knockout mice aged (56- to 62-wk old), the testis phenotypes became exacerbated with the appearance of hemorrhagic tumors, Leydig cell adenomas, and a complete loss of spermatogenesis. In contrast, loss of Smad4 in Leydig cells alone did not appreciably alter fetal and adult testis development. Our findings support a cell type-specific requirement of Smad4 in testis development and suppression of testicular tumors. PMID:24501173

Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Si, Lina; Shi, Jin; Gao, Wenqun

2014-07-18

Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part bymore » increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.« less

Two sides of the story? Smad4 loss in pancreatic cancer versus head-and-neck cancer

PubMed Central

Malkoski, Stephen P.; Wang, Xiao-Jing

2012-01-01

TGFβ signaling Smads (Smad2, 3, and 4) were suspected tumor suppressors soon after their discovery. Nearly two decades of research confirmed this role and revealed other divergent and cancer-specific functions including paradoxical tumor promotion effects. Although Smad4 is the most potent tumor suppressor, its functions are highly context-specific as exemplified by pancreatic cancer and head-and-neck cancer: in pancreatic cancer, Smad4 loss cannot initiate tumor formation but promotes metastases while in head-and-neck cancer Smad4 loss promotes cancer progression but also initiates tumor formation, likely through effects on genomic instability. The differing consequences of impaired Smad signaling in human cancers and the molecular mechanisms that underpin these differences will have important implications for the design and application of novel targeted therapies. PMID:22321641

Smad3 deficiency protects mice from obesity-induced podocyte injury that precedes insulin resistance.

PubMed

Sun, Yu B Y; Qu, Xinli; Howard, Victor; Dai, Lie; Jiang, Xiaoyun; Ren, Yi; Fu, Ping; Puelles, Victor G; Nikolic-Paterson, David J; Caruana, Georgina; Bertram, John F; Sleeman, Mark W; Li, Jinhua

2015-08-01

Signaling by TGF-β/Smad3 plays a key role in renal fibrosis. As obesity is one of the major risk factors of chronic and end-stage renal disease, we studied the role of Smad3 signaling in the pathogenesis of obesity-related renal disease. After switching to a high fat diet, the onset of Smad3 C-terminal phosphorylation, increase in albuminuria, and the early stages of peripheral and renal insulin resistance occurred at 1 day, and 4 and 8 weeks, respectively, in C57BL/6 mice. The loss of synaptopodin, a functional marker of podocytes, and phosphorylation of the Smad3 linker region (T179 and S213) appeared after 4 weeks of the high fat diet. This suggests a temporal pattern of Smad3 signaling activation leading to kidney injury and subsequent insulin resistance in the development of obesity-related renal disease. In vivo, Smad3 knockout attenuated the high fat diet-induced proteinuria, renal fibrosis, overall podocyte injury, and mitochondrial dysfunction in podocytes. In vitro palmitate caused a rapid activation of Smad3 in 30 min, loss of synaptopodin in 2 days, and impaired insulin signaling in 3 days in isolated mouse podocytes. Blockade of either Smad3 phosphorylation by SIS3 (a Smad3 inhibitor) or T179 phosphorylation by flavopiridol (a CDK9 inhibitor) prevented the palmitate-induced loss of synaptopodin and mitochondrial function in podocytes. Thus, Smad3 signaling plays essential roles in obesity-related renal disease and may be a novel therapeutic target.

Involvement of Smad3 phosphoisoform-mediated signaling in the development of colonic cancer in IL-10-deficient mice.

PubMed

Hachimine, Daisaku; Uchida, Kazushige; Asada, Masanori; Nishio, Akiyoshi; Kawamata, Seiji; Sekimoto, Go; Murata, Miki; Yamagata, Hideo; Yoshida, Katsunori; Mori, Shigeo; Tahashi, Yoshiya; Matsuzaki, Koichi; Okazaki, Kazuichi

2008-06-01

Chronic inflammation predisposes to cancer. Transforming growth factor (TGF)-beta, a multifunctional protein, suppresses the growth of normal colonic epithelial cells, whereas it stimulates the proliferation of cancer cells. Interleukin (IL)-10-deficient mice, which develop colitis and colorectal cancer, show an increased level of plasma TGF-beta. Although TGF-beta may be a key molecule in the development of colon cancer arising from chronic colitis in IL-10-deficient mice, the role of TGF-beta still remains unclear. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which converts the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). We studied C57BL/6-IL-10-deficient mice (n=18) at 4 to 32 weeks of age. We investigated histology, and pSmad2/3L, pSmad2/3C, and p53 by immunohistochemistry. pSmad3L staining was detected in the cancer cells in all 10 mice with colonic cancer and in the epithelial cells in 7 of 12 mice with colonic dysplasia, but not in the normal or colitic mice. pSmad3c was detected without any significant difference between stages. p53 was weakly stained in a few cancer cells in 5 out of 10 mice. Smad3L signaling plays an important role in the carcinogenesis of chronic colitis in IL-10-deficient mice.

Glycyrrhetinic acid alleviates radiation-induced lung injury in mice.

PubMed

Chen, Jinmei; Zhang, Weijian; Zhang, Lurong; Zhang, Jiemin; Chen, Xiuying; Yang, Meichun; Chen, Ting; Hong, Jinsheng

2017-01-01

Radiation-induced lung injury (RILI) is a common complication of thoracic radiotherapy, but efficacious therapy for RILI is lacking. This study ascertained whether glycyrrhetinic acid (GA; a functional hydrolyzed product of glycyrrhizic acid, which is extracted from herb licorice) can protect against RILI and investigated its relationship to the transforming growth factor (TGF)-β1/Smads signaling pathway. C57BL/6 mice were divided into four groups: a control group, a GA group and two irradiation (IR) groups. IR groups were exposed to a single fraction of X-rays (12 Gy) to the thorax and administered normal saline (IR + NS group) or GA (IR + GA group). Two days and 17 days after irradiation, histologic analyses were performed to assess the degree of lung injury, and the expression of TGF-β1, Smad2, Smad3 and Smad7 was recorded. GA administration mitigated the histologic changes of lung injury 2 days and 17 days after irradiation. Protein and mRNA expression of TGF-β1, Smad2 and Smad3, and the mRNA level of Smad7, in lung tissue were significantly elevated after irradiation. GA decreased expression of TGF-β1, Smad2 and Smad3 in lung tissue, but did not increase Smad7 expression. GA can protect against early-stage RILI. This protective effect may be associated with inhibition of the TGF-β1/Smads signaling pathway. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

SUMO-Specific Cysteine Protease 1 Promotes Epithelial Mesenchymal Transition of Prostate Cancer Cells via Regulating SMAD4 deSUMOylation.

PubMed

Zhang, Xiaoyan; Wang, Hao; Wang, Hua; Xiao, Fengjun; Seth, Prem; Xu, Weidong; Jia, Qinghua; Wu, Chutse; Yang, Yuefeng; Wang, Lisheng

2017-04-12

In advanced prostate cancer, small ubiquitin-like modifier (SUMO)-specific cysteine protease 1 (SENP1) is up-regulated. However, the role of SENP1 in regulating deSUMOylation of TGF-β/SMADs signaling is unknown. In this study, we developed a lentiviral vector, PLKO.1-shSENP1, to silence SENP1 in prostate cancer cells with high metastatic characteristics (PC3M). Likewise, we also created an adenovirus vector, Ad5/F11p-SENP1 to over-express SENP1 in prostate cancer cells with low metastatic potential (LNCaP). We showed that silencing of SENP1 promoted cellular apoptosis, and inhibited proliferation and migration of PC3M cells. Moreover, SENP1 silencing increased the SMAD4 expression at protein level, up-regulated E-cadherin and down-regulated Vimentin expression, indicating the inhibition of epithelial mesenchymal transition (EMT). Furthermore, SMAD4 interference abolished SENP1-mediated up-regulation of E-cadherin, suggesting that SENP1 regulated E-cadherin expression via SMAD4. SENP1 over-expression in LNCaP cells reduced SMAD4 protein, and promoted EMT via decreasing E-cadherin and increasing Vimentin. Moreover, down-regulation of SMAD4 and E-cadherin were blocked, after transfection with two SUMOylation sites mutated SMAD4, suggesting that SENP1 might reduce SMAD4 levels to regulate E-cadherin expression via deSUMOylation of SMAD4. In conclusion, SENP1 deSUMOylated SMAD4 to promote EMT via up-regulating E-cadherin in prostate cancer cells. Therefore, SENP1 is a potential target for treatment of advanced prostate cancer.

MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Jun; Zhang, Shuyu; Zhou, Yingqi

2011-03-25

Research highlights: {yields} We identify miR-421 as a novel potential regulator of DPC4/Smad4. {yields} The expression levels of miR-421 and DPC4/Smad4 are inversely correlated in human clinical specimens of pancreatic cancer. {yields} Overexpression of miR-421 represses the reporter activities driven by the 3'-UTR of DPC4/Smad4 and DPC4/Smad4 protein level in pancreatic cancer cell. {yields} Ectopic expression of miR-421 promotes the proliferation and colony formation of pancreatic cancer cell. -- Abstract: MicroRNAs (miRNAs) have emerged as important regulators in the development of pancreatic cancer and may be a valuable therapeutic application. DPC4/Smad4 is a critical tumor suppressor involved in the progressionmore » of pancreatic cancer, but few studies have been conducted to determine its relationship with miRNAs. In this study, we identify miR-421 as a potential regulator of DPC4/Smad4. We find that in human clinical specimens of pancreatic cancer miR-421 is aberrantly upregulated while DPC4/Smad4 is strongly repressed, and their levels of expression are inversely correlated. Moreover, ectopic expression of miR-421 significantly decreases DPC4/Smad4 protein level in pancreatic cancer cell lines and simultaneously promotes cell proliferation and colony formation in vitro. Our findings identify miR-421 as a potent regulator of DPC4/Smad4, which may provide a novel therapeutic strategy for treatment of DPC4/Smad4-driven pancreatic cancer.« less

Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

PubMed

Sasseville, Maxime; Ritter, Lesley J; Nguyen, Thao M; Liu, Fang; Mottershead, David G; Russell, Darryl L; Gilchrist, Robert B

2010-09-15

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

Retraction: Myostatin Induces Degradation of Sarcomeric Proteins through a Smad3 Signaling Mechanism During Skeletal Muscle Wasting

PubMed Central

Lokireddy, Sudarsanareddy; McFarlane, Craig; Ge, Xiaojia; Zhang, Huoming; Sze, Siu Kwan; Sharma, Mridula

2011-01-01

Ubiquitination-mediated proteolysis is a hallmark of skeletal muscle wasting manifested in response to negative growth factors, including myostatin. Thus, the characterization of signaling mechanisms that induce the ubiquitination of intracellular and sarcomeric proteins during skeletal muscle wasting is of great importance. We have recently characterized myostatin as a potent negative regulator of myogenesis and further demonstrated that elevated levels of myostatin in circulation results in the up-regulation of the muscle-specific E3 ligases, Atrogin-1 and muscle ring finger protein 1 (MuRF1). However, the exact signaling mechanisms by which myostatin regulates the expression of Atrogin-1 and MuRF1, as well as the proteins targeted for degradation in response to excess myostatin, remain to be elucidated. In this report, we have demonstrated that myostatin signals through Smad3 (mothers against decapentaplegic homolog 3) to activate forkhead box O1 and Atrogin-1 expression, which further promotes the ubiquitination and subsequent proteasome-mediated degradation of critical sarcomeric proteins. Smad3 signaling was dispensable for myostatin-dependent overexpression of MuRF1. Although down-regulation of Atrogin-1 expression rescued approximately 80% of sarcomeric protein loss induced by myostatin, only about 20% rescue was seen when MuRF1 was silenced, implicating that Atrogin-1 is the predominant E3 ligase through which myostatin manifests skeletal muscle wasting. Furthermore, we have highlighted that Atrogin-1 not only associates with myosin heavy and light chain, but it also ubiquitinates these sarcomeric proteins. Based on presented data we propose a model whereby myostatin induces skeletal muscle wasting through targeting sarcomeric proteins via Smad3-mediated up-regulation of Atrogin-1 and forkhead box O1. PMID:21964591

Therapeutic potential of trichostatin A to control inflammatory and fibrogenic disorders of the ocular surface.

PubMed

Kitano, Ai; Okada, Yuka; Yamanka, Osamu; Shirai, Kumi; Mohan, Rajiv R; Saika, Shizuya

2010-12-31

To examine the effects of a histone deacetylase inhibitor, Trichostatin A (TSA), on the behavior of macrophages and subconjunctival fibroblasts in vitro and on ocular surface inflammation and scarring in vivo using an alkali burn wound healing model. Effects of TSA on expression of inflammation-related growth factors or collagen I were examined by real-time RT-PCR or immunoassay in mouse macrophages or human subconjunctival fibroblasts. Effects of TSA on trans forming growth factor β (TGFβ)/Smad signaling were evaluated with western blotting and/or immunocytochemistry. Alkali-burn injuries on the eyes of mice were performed with three µl of 0.5 N NaOH under general and topical anesthesia. TSA (600 µg/Kg daily) or vehicle was administered to animals via intraperitoneal (i.p.) injection. Histology and real-time RT-PCR investigations evaluated the effects of TSA on the healing process of the cornea. TSA inhibited TGFβ 1 and vascular endothelial growth factor (VEGF) expression in macrophages, and TGFβ1 and collagen I in ocular fibroblasts. It elevated the expression of 5'-TG-3'-interacting factor (TGIF) and Smad7 in fibroblasts and blocked nuclear translocation of phospho-Smad2. Real-time PCR and immunocytochemistry studies showed that systemic administration of TSA suppressed the inflammation and fibrotic response in the stroma and accelerated epithelial healing in the alkali-burned mouse cornea. Systemic administration of TSA reduces inflammatory and fibrotic responses in the alkali-burned mouse ocular surface in vivo. The mechanisms of action involve attenuation of Smad signal in mesenchymal cells and reduction in the activation and recruitment of macrophages. TSA has the potential to treat corneal scarring in vivo.

TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Ling, E-mail: fangling_1984@126.com; Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032; Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032

Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7more » protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 protein in human fibrotic livers • Upregulation of TRPM7 by TGF-β1 elicited Smad signaling in HSC-T6 cells • Blockade of TRPM7 decreased α-SMA and Col1α1 expressions in activated HSC-T6 cells • Silencing of TRPM7 led to collagen degradation in TGF-β1 stimulated HSC-T6 cells • TRPM7 upregulation contributes to the activation of TGF-β1/Smad pathway.« less

RGC32 induces epithelial-mesenchymal transition by activating the Smad/Sip1 signaling pathway in CRC.

PubMed

Wang, Xiao-Yan; Li, Sheng-Nan; Zhu, Hui-Fang; Hu, Zhi-Yan; Zhong, Yan; Gu, Chuan-Sha; Chen, Shi-You; Liu, Teng-Fei; Li, Zu-Guo

2017-05-04

Response gene to complement 32 (RGC32) is a transcription factor that regulates the expression of multiple genes involved in cell growth, viability and tissue-specific differentiation. However, the role of RGC32 in tumorigenesis and tumor progression in colorectal cancer (CRC) has not been fully elucidated. Here, we showed that the expression of RGC32 was significantly up-regulated in human CRC tissues versus adjacent normal tissues. RGC32 expression was significantly correlated with invasive and aggressive characteristics of tumor cells, as well as poor survival of CRC patients. We also demonstrated that RGC32 overexpression promoted proliferation, migration and tumorigenic growth of human CRC cells in vitro and in vivo. Functionally, RGC32 facilitated epithelial-mesenchymal transition (EMT) in CRC via the Smad/Sip1 signaling pathway, as shown by decreasing E-cadherin expression and increasing vimentin expression. In conclusion, our findings suggested that overexpression of RGC32 facilitates EMT of CRC cells by activating Smad/Sip1 signaling.

Smad3 mutant mice develop colon cancer with overexpression of COX-2

PubMed Central

Zhu, Yu-Ping; Liu, Zhuo; Fu, Zhi-Xuan; Li, De-Chuan

2017-01-01

Colon cancer is the second most common cause of cancer-associated mortality in human populations. The aim of the present study was to identify the role of cyclooxygenase-2 (COX-2) in Smad3 mutant mice, which are known to develop colon cancer. Homozygous Smad3 (−/−) mutant mice were generated from inbred and hybrid Smad3 mouse strains by intercrossing the appropriate heterozygotes. Immunohistochemistry with COX-2 antibody was performed throughout this experiment and the data was validated and cross-checked with reverse transcription-polymerase chain reaction (RT-PCR). Homozygous mutant Smad3 mice were generated and the overexpression pattern of COX-2 was identified by immunohistochemistry and validated with RT-PCR. The results of the present study demonstrated a link between the Smad3 mutant mice, colon cancer and COX-2. In addition, the overexpression pattern of COX-2 in Smad3 mutant mice that develop colon cancer was identified. PMID:28454287

Lipopolysaccharide inhibits transforming growth factor-beta1-stimulated Smad6 expression by inducing phosphorylation of the linker region of Smad3 through a TLR4-IRAK1-ERK1/2 pathway.

PubMed

Kim, Eun-Ye; Kim, Byung-Chul

2011-03-09

Smad6, one of the inhibitory Smads, plays an important role in transforming growth factor-beta1 (TGF-β1)-mediated negative regulation of pro-inflammatory signaling. In this study, we found that bacterial endotoxin lipopolysaccharide (LPS) inhibits TGF-β1-induced expression of Smad6 in RAW264.7 cells. This repression was accompanied by increased Smad3 linker phosphorylation at Thr-179 and Ser-208 and was dependent on ERK1/2 activity via the TLR4-IRAK1-linked signaling cascade. The expression of a mutant Smad3, that lacks the phosphorylation sites in the linker regions, significantly reversed the inhibitory effect of LPS on TGF-β1-induced Smad6 expression and its anti-inflammatory capacity. Collectively, our findings show how LPS pro-inflammatory signal antagonizes the anti-inflammatory activity of TGF-β1. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A critical role for transcription factor Smad4 in T cell function independent of transforming growth factor beta receptor signaling

PubMed Central

Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A.; Wan, Yisong Y.

2014-01-01

Summary Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. While Smad4 was dispensable for T cell generation, homeostasis and effector function, it was essential for T cell proliferation following activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity and anti-tumor immunity. PMID:25577439

A critical role for transcription factor Smad4 in T cell function that is independent of transforming growth factor β receptor signaling.

PubMed

Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A; Wan, Yisong Y

2015-01-20

Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. Although Smad4 was dispensable for T cell generation, homeostasis, and effector function, it was essential for T cell proliferation after activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity, and anti-tumor immunity. Copyright © 2015 Elsevier Inc. All rights reserved.

Podocyte-derived microparticles promote proximal tubule fibrotic signaling via p38 MAPK and CD36

PubMed Central

Munkonda, Mercedes N.; Akbari, Shareef; Landry, Chloe; Sun, Suzy; Xiao, Fengxia; Turner, Maddison; Holterman, Chet E.; Nasrallah, Rania; Hébert, Richard L.; Kennedy, Christopher R. J.; Burger, Dylan

2018-01-01

ABSTRACT Tubulointerstitial fibrosis is a hallmark of advanced diabetic kidney disease that is linked to a decline in renal function, however the pathogenic mechanisms are poorly understood. Microparticles (MPs) are 100–1000 nm vesicles shed from injured cells that are implicated in intercellular signalling. Our lab recently observed the formation of MPs from podocytes and their release into urine of animal models of type 1 and 2 diabetes and in humans with type 1 diabetes. The purpose of the present study was to examine the role of podocyte MPs in tubular epithelial cell fibrotic responses. MPs were isolated from the media of differentiated, untreated human podocytes (hPODs) and administered to cultured human proximal tubule epithelial cells (PTECs). Treatment with podocyte MPs increased p38 and Smad3 phosphorylation and expression of the extracellular matrix (ECM) proteins fibronectin and collagen type IV. MP-induced responses were attenuated by co-treatment with the p38 inhibitor SB202190. A transforming growth factor beta (TGF-β) receptor inhibitor (LY2109761) blocked MP-induced Smad3 phosphorylation and ECM protein expression but not p38 phosphorylation suggesting that these responses occurred downstream of p38. Finally, blockade of the class B scavenger receptor CD36 completely abrogated MP-mediated p38 phosphorylation, downstream Smad3 activation and fibronectin/collagen type IV induction. Taken together our results suggest that podocyte MPs interact with proximal tubule cells and induce pro-fibrotic responses. Such interactions may contribute to the development of tubular fibrosis in glomerular disease. PMID:29435202

Modulation of TGF-beta signaling during progression of chronic liver diseases.

PubMed

Matsuzaki, Koichi

2009-01-01

A large body of work has established roles for epithelial cells as important mediators of progressive fibrosis and carcinogenesis. Transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines are important inducers of fibro-carcinogenesis. TGF-beta signaling involves phosphorylation of Smad3 at middle linker and/or C-terminal regions. Reversible shifting of Smad3-dependent signaling between tumor-suppression and oncogenesis in hyperactive Ras-expressing epithelial cells indicates that Smad3 phosphorylated at the C-terminal region (pSmad3C) transmits a tumor-suppressive TGF-beta signal, while oncogenic activities such as cell proliferation and invasion are promoted by Smad3 phosphorylated at the linker region (pSmad3L). Notably, pSmad3L-mediated signaling promotes extracellular matrix deposition by activated mesenchymal cells. During progression of chronic liver diseases, hepatic epithelial hepatocytes undergo transition from the tumor-suppressive pSmad3C pathway to the fibrogenic/oncogenic pSmad3L pathway, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma. c-Jun N-terminal kinase activated by pro-inflammatory cytokines is mediating this perturbed hepatocytic TGF-beta signaling. Thus, TGF-beta signaling of hepatocytes affected by chronic inflammation offers a general framework for understanding the molecular mechanisms of human fibro-carcinogenesis during progression of chronic liver diseases.

Hepatitis B virus X promotes hepatocellular carcinoma development via nuclear protein 1 pathway.

PubMed

Bak, Yesol; Shin, Hye-jun; Bak, In seon; Yoon, Do-young; Yu, Dae-Yeul

2015-10-30

Hepatocellular carcinoma (HCC) is one of the most common malignancies and chronic hepatitis B virus (HBV) infection is a major risk factor for HCC. Hepatitis B virus X (HBx) protein relates to trigger oncogenesis. HBx has oncogenic properties with a hyperproliferative response to HCC. Nuclear protein 1 (NUPR1) is a stress-response protein, frequently upregulated in several cancers. Recent data revealed that NUPR1 is involved in tumor progression, but its function in HCC is not revealed yet. Here we report HBx can induce NUPR1 in patients, mice, and HCC cell lines. In an HBx transgenic mouse model, we found that HBx overexpression upregulates NUPR1 expression consistently with tumor progression. Further, in cultured HBV positive cells, HBx knockdown induces downregulation of NUPR1. Smad4 is a representative transcription factor, regulated by HBx, and we showed that HBx upregulates NUPR1 by Smad4 dependent way. We found that NUPR1 can inhibit cell death and induce vasculogenic mimicry in HCC cell lines. Moreover, NUPR1 silencing in HepG2-HBx showed reduced cell motility. These results suggest that HBx can modulate NUPR1 expression through the Smad4 pathway and NUPR1 has a role in hepatocellular carcinoma progression. Copyright © 2015 Elsevier Inc. All rights reserved.

The Impact of SMAD4 Loss on Outcome in Patients with Advanced Pancreatic Cancer Treated with Systemic Chemotherapy

PubMed Central

Ormanns, Steffen; Haas, Michael; Remold, Anna; Kruger, Stephan; Holdenrieder, Stefan; Kirchner, Thomas; Heinemann, Volker; Boeck, Stefan

2017-01-01

The role of the tumor suppressor mothers against decapentaplegic homolog 4 (SMAD4) has not yet been defined in patients (pts) with advanced pancreatic cancer (aPC). This translational research study was designed to evaluate the impact of tumoral SMAD4 loss on clinicopathological parameters and outcome in PC patients receiving palliative chemotherapy. Using immunohistochemistry, we examined SMAD4 expression in tumor tissue of 143 aPC pts treated within completed prospective clinical and biomarker trials. In uni- and multivariate analyses, SMAD4 expression status was correlated to clinicopathological patient characteristics and outcome. At chemotherapy initiation, 128 pts had metastatic PC; most pts (n = 99) received a gemcitabine-based regimen. SMAD4 loss was detected in 92 pts (64%); patient characteristics such as gender, age, tumor grading, disease stage or number of metastatic sites had no significant impact on tumoral SMAD4 status. In univariate analyses, SMAD4 loss had no impact on overall survival (hazard ratio (HR) 1.008, p = 0.656); however, we observed a prolonged progression-free survival (HR 1.565, p = 0.038) in pts with tumoral SMAD4 loss. This finding was confirmed in multivariate analyses (HR 1.790, p = 0.040), but only for gemcitabine-treated pts. In contrast to previous studies in resectable PC, loss of SMAD4 expression was not associated with a negative outcome in patients with advanced PC receiving systemic chemotherapy. PMID:28534865

Triptolide inhibits TGF-β1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ming; Lv, Zhiqiang; Huang, Linjie

Background: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-β1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. Methods: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-β1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. Results: Triptolidemore » significantly inhibited TGF-β1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-β1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. Conclusions: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway. - Highlights: • In this study, rat airway smooth muscle cells were cultured and made synchronized. • Triptolide inhibited TGF-β1-induced airway smooth muscle cells proliferation. • Triptolide inhibited ASMCs proliferation via negative regulation of Smad signaling pathway.« less

The Impact of SMAD4 Loss on Outcome in Patients with Advanced Pancreatic Cancer Treated with Systemic Chemotherapy.

PubMed

Ormanns, Steffen; Haas, Michael; Remold, Anna; Kruger, Stephan; Holdenrieder, Stefan; Kirchner, Thomas; Heinemann, Volker; Boeck, Stefan

2017-05-19

The role of the tumor suppressor mothers against decapentaplegic homolog 4 (SMAD4) has not yet been defined in patients (pts) with advanced pancreatic cancer (aPC). This translational research study was designed to evaluate the impact of tumoral SMAD4 loss on clinicopathological parameters and outcome in PC patients receiving palliative chemotherapy. Using immunohistochemistry, we examined SMAD4 expression in tumor tissue of 143 aPC pts treated within completed prospective clinical and biomarker trials. In uni- and multivariate analyses, SMAD4 expression status was correlated to clinicopathological patient characteristics and outcome. At chemotherapy initiation, 128 pts had metastatic PC; most pts ( n = 99) received a gemcitabine-based regimen. SMAD4 loss was detected in 92 pts (64%); patient characteristics such as gender, age, tumor grading, disease stage or number of metastatic sites had no significant impact on tumoral SMAD4 status. In univariate analyses, SMAD4 loss had no impact on overall survival (hazard ratio (HR) 1.008, p = 0.656); however, we observed a prolonged progression-free survival (HR 1.565, p = 0.038) in pts with tumoral SMAD4 loss. This finding was confirmed in multivariate analyses (HR 1.790, p = 0.040), but only for gemcitabine-treated pts. In contrast to previous studies in resectable PC, loss of SMAD4 expression was not associated with a negative outcome in patients with advanced PC receiving systemic chemotherapy.

CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

PubMed

Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

2009-02-15

Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

Role of Rho/ROCK and p38 MAP kinase pathways in transforming growth factor-beta-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo.

PubMed

Kamaraju, Anil K; Roberts, Anita B

2005-01-14

TGF-beta is a multifunctional cytokine known to exert its biological effects through a variety of signaling pathways of which Smad signaling is considered to be the main mediator. At present, the Smad-independent pathways, their interactions with each other, and their roles in TGF-beta-mediated growth inhibitory effects are not well understood. To address these questions, we have utilized a human breast cancer cell line MCF10CA1h and demonstrate that p38 MAP kinase and Rho/ROCK pathways together with Smad2 and Smad3 are necessary for TGF-beta-mediated growth inhibition of this cell line. We show that Smad2/3 are indispensable for TGF-beta-mediated growth inhibition, and that both p38 and Rho/ROCK pathways affect the linker region phosphorylation of Smad2/3. Further, by using Smad3 mutated at the putative phosphorylation sites in the linker region, we demonstrate that phosphorylation at Ser203 and Ser207 residues is required for the full transactivation potential of Smad3, and that these residues are targets of the p38 and Rho/ROCK pathways. We demonstrate that activation of the p38 MAP kinase pathway is necessary for the full transcriptional activation potential of Smad2/Smad3 by TGF-beta, whereas activity of Rho/ROCK is necessary for both down-regulation of c-Myc protein and up-regulation of p21waf1 protein, directly interfering with p21waf1 transcription. Our results not only implicate Rho/ROCK and p38 MAPK pathways as necessary for TGF-beta-mediated growth inhibition, but also demonstrate their individual contributions and the basis for their cooperation with each other.

Reversible phospho-Smad3 signalling between tumour suppression and fibrocarcinogenesis in chronic hepatitis B infection.

PubMed

Deng, Y-R; Yoshida, K; Jin, Q L; Murata, M; Yamaguchi, T; Tsuneyama, K; Moritoki, Y; Niu, J Q; Matsuzaki, K; Lian, Z-X

2014-04-01

Transforming growth factor (TGF)-β, type I receptor (TβRI) and c-Jun N-terminal kinases (JNK) phosphorylate Smad3 differentially to create 2 isoforms phosphorylated (p) at the COOH-terminus (C) or at the linker region (L) and regulate hepatocytic fibrocarcinogenesis. This study aimed to compare the differences between how hepatitis B virus (HBV) infection affected hepatocytic Smad3 phosphorylated isoforms before and after anti-viral therapy. To clarify the relationship between Smad3 phosphorylation and liver disease progression, we studied 10 random patients in each stage of HBV-related fibrotic liver disease (F1-4) and also 10 patients with HBV-associated HCC. To examine changes in phosphorylated Smad3 signalling before and after anti-HBV therapies, we chose 27 patients with chronic hepatitis B who underwent baseline and follow-up biopsies at 52 weeks from the start of nucleoside analogue treatments (Lamivudine 100 mg daily or Telbivudine 600 mg daily). Fibrosis stage, inflammatory activity and phosphorylated Smad3 positivity in the paired biopsy samples were compared. Hepatocytic pSmad3C signalling shifted to fibrocarcinogenic pSmad3L signalling as the livers progressed from chronic hepatitis B infection to HCC. After nucleoside analogue treatment, serum alanine aminotransferase (ALT) and HBV-DNA levels in 27 patients with HBV-related chronic liver diseases were decreased dramatically. Decrease in HBV-DNA restored pSmad3C signalling in hepatocytes, while eliminating prior fibrocarcinogenic pSmad3L signalling. Oral nucleoside analogue therapies can suppress fibrosis and reduce HCC incidence by successfully reversing phosphorylated Smad3 signalling; even liver disease progressed to cirrhosis in chronic hepatitis B patients. © 2013 British Society for Immunology.

Smad3 Deficiency in Mice Protects Against Insulin Resistance and Obesity Induced by a High-Fat Diet

PubMed Central

Tan, Chek Kun; Leuenberger, Nicolas; Tan, Ming Jie; Yan, Yew Wai; Chen, Yinghui; Kambadur, Ravi; Wahli, Walter; Tan, Nguan Soon

2011-01-01

OBJECTIVE Obesity and associated pathologies are major global health problems. Transforming growth factor-β/Smad3 signaling has been implicated in various metabolic processes, including adipogenesis, insulin expression, and pancreatic β-cell function. However, the systemic effects of Smad3 deficiency on adiposity and insulin resistance in vivo remain elusive. This study investigated the effects of Smad3 deficiency on whole-body glucose and lipid homeostasis and its contribution to the development of obesity and type 2 diabetes. RESEARCH DESIGN AND METHODS We compared various metabolic profiles of Smad3-knockout and wild-type mice. We also determined the mechanism by which Smad3 deficiency affects the expression of genes involved in adipogenesis and metabolism. Mice were then challenged with a high-fat diet to study the impact of Smad3 deficiency on the development of obesity and insulin resistance. RESULTS Smad3-knockout mice exhibited diminished adiposity with improved glucose tolerance and insulin sensitivity. Chromatin immunoprecipitation assay revealed that Smad3 deficiency increased CCAAT/enhancer-binding protein β-C/EBP homologous protein 10 interaction and exerted a differential regulation on proliferator-activated receptor β/δ and proliferator-activated receptor γ expression in adipocytes. Focused gene expression profiling revealed an altered expression of genes involved in adipogenesis, lipid accumulation, and fatty acid β-oxidation, indicative of altered adipose physiology. Despite reduced physical activity with no modification in food intake, these mutant mice were resistant to obesity and insulin resistance induced by a high-fat diet. CONCLUSIONS Smad3 is a multifaceted regulator in adipose physiology and the pathogenesis of obesity and type 2 diabetes, suggesting that Smad3 may be a potential target for the treatment of obesity and its associated disorders. PMID:21270259

Prognostic value of loss of heterozygosity and sub-cellular localization of SMAD4 varies with tumor stage in colorectal cancer.

PubMed

Jia, Xu; Shanmugam, Chandrakumar; Paluri, Ravi K; Jhala, Nirag C; Behring, Michael P; Katkoori, Venkat R; Sugandha, Shajan P; Bae, Sejong; Samuel, Temesgen; Manne, Upender

2017-03-21

Although loss of heterozygosity (LOH) at chromosome location 18q21 and decreased expression of SMAD4 in invasive colorectal cancers (CRCs) correlate with poor patient survival, the prognostic value of LOH at 18q21 and sub-cellular localization of SMAD4 have not been evaluated in relation to tumor stage. Genomic DNA samples from 209 formalin-fixed, paraffin-embedded sporadic CRC tissues and their matching controls were analyzed for 18q21 LOH, and corresponding tissue sections were evaluated by immunohistochemistry for expression of SMAD4 and assessed for its sub-cellular localization (nuclear vs. cytoplasmic). In addition, 53 frozen CRCs and their matching control tissues were analyzed for their mutational status and mRNA expression of SMAD4. The phenotypic expression pattern and LOH status were evaluated for correlation with patient survival by the use of Kaplan-Meier and Cox regression models. LOH of 18q21 was detected in 61% of the informative cases. In 8% of the cases, missense point mutations were detected in Smad4. In CRCs, relative to controls, there was increased SMAD4 staining in the cytoplasm (74%) and decreased staining in the nuclei (37%). LOH of 18q21 and high cytoplasmic localization of SMAD4 were associated with shortened overall survival of Stage II patients, whereas low nuclear expression of SMAD4 was associated with worse survival, but only for patients with Stage III CRCs. LOH of 18q21 and high cytoplasmic localization of SMAD4 in Stage II CRCs and low nuclear SMAD4 in Stage III CRCs are predictors of shortened patient survival.

Smad4/Fascin index is highly prognostic in patients with diffuse type EBV-associated gastric cancer.

PubMed

Son, Byoung Kwan; Kim, Dong-Hoon; Min, Kyueng-Whan; Kim, Eun-Kyung; Kwon, Mi Jung

2018-04-01

Gastric cancer is a heterogeneous disorder for which predicting clinical outcomes is challenging, although various biomarkers have been suggested. The Smad4 and Fascin proteins are known prognostic indicators of different types of malignancy. Smad4 primarily functions as a key regulator of tumor suppression, whereas Fascin exhibits oncogenic function by enhancing tumor infiltration. A combined marker based on these opposing roles may improve prognostic accuracy in gastric cancer. Smad4 and Fascin expression was assessed in tissue microarrays obtained from 285 primary gastric adenocarcinoma, 201 normal tissue, and 51 metastatic adenocarcinoma samples. A Smad4/Fascin index based on the relative expression of each protein was divided into low- and high-expression groups using receiver operating characteristic curves. We compared normal tissue, primary adenocarcinoma, and metastatic adenocarcinoma in Smad4 and Fascin expression and the differences in clinicopathological findings between low Smad4/Fascin and high Smad4/Fascin expression in gastric adenocarcinoma. High Smad4/Fascin expression was significantly associated with worse outcomes, such as old age, advanced T and N category, large tumor size, high histological grade, lymphatic and vascular invasion, and presence of Epstein-Barr virus (EBV) (all p < 0.05). Univariate and multivariate analyses revealed a significant relationship between disease-free or overall survival and Smad4/Fascin index in diffuse-type or EBV-associated gastric cancer (all p < 0.05). A dual marker system using Smad4 and Fascin may be a reliable indicator for predicting clinical outcomes in patients with diffuse-type or EBV-associated gastric cancer. Copyright © 2018 Elsevier GmbH. All rights reserved.

Anterior segment dysgenesis correlation with epithelial-mesenchymal transition in Smad4 knockout mice.

PubMed

Li, Jing; Qin, Yu; Zhao, Fang-Kun; Wu, Di; He, Xue-Fei; Liu, Jia; Zhao, Jiang-Yue; Zhang, Jin-Song

2016-01-01

To explore the molecular mechanisms in lens development and the pathogenesis of Peters anomaly in Smad4 defective mice. Le-Cre transgenic mouse line was employed to inactivate Smad4 in the surface ectoderm selectively. Pathological techniques were used to reveal the morphological changes of the anterior segment in Smad4 defective eye. Immunohistochemical staining was employed to observe the expression of E-cadherin, N-cadherin and α-SMA in anterior segment of Smad4 defective mice and control mice at embryonic (E) day 16.5. Real-time quantitative polymerase chain reaction (qPCR) was performed to detect the expression of Snail, Zeb1, Zeb2 and Twist2 in lens of Smad4 defective mice and control mice at E16.5. Statistical evaluations were performed using the unpaired Student's t-test (two-tailed) by SPSS 11.0 software. Conditional deletion of Smad4 on eye surface ectoderm resulted in corneal dysplasia, iridocorneal angle closure, corneolenticular adhesions and cataract resembling Peters anomaly. Loss of Smad4 function inhibited E-cadherin expression in the lens epithelium cells and corneal epithelium cells in Smad4 defective eye. Expression of N-cadherin was up-regulated in corneal epithelium and corneal stroma. Both E-cadherin and N-cadherin were down-regulated at the future trabecular meshwork region in mutant eye. The qPCR results showed that the expression of Twist2 was increased significantly in the mutant lens (P<0.01). Smad4 is essential to eye development and likely a candidate pathogenic gene to Peters anomaly by regulating epithelial-mesenchymal transition. Twist2 can be regulated by Smad4 and plays an essential role in lens development.

Prognostic value of loss of heterozygosity and sub-cellular localization of SMAD4 varies with tumor stage in colorectal cancer

PubMed Central

Jia, Xu; Shanmugam, Chandrakumar; Paluri, Ravi K.; Jhala, Nirag C.; Behring, Michael P.; Katkoori, Venkat R.; Sugandha, Shajan P.; Bae, Sejong; Samuel, Temesgen; Manne, Upender

2017-01-01

Background Although loss of heterozygosity (LOH) at chromosome location 18q21 and decreased expression of SMAD4 in invasive colorectal cancers (CRCs) correlate with poor patient survival, the prognostic value of LOH at 18q21 and sub-cellular localization of SMAD4 have not been evaluated in relation to tumor stage. Methods Genomic DNA samples from 209 formalin-fixed, paraffin-embedded sporadic CRC tissues and their matching controls were analyzed for 18q21 LOH, and corresponding tissue sections were evaluated by immunohistochemistry for expression of SMAD4 and assessed for its sub-cellular localization (nuclear vs. cytoplasmic). In addition, 53 frozen CRCs and their matching control tissues were analyzed for their mutational status and mRNA expression of SMAD4. The phenotypic expression pattern and LOH status were evaluated for correlation with patient survival by the use of Kaplan-Meier and Cox regression models. Results LOH of 18q21 was detected in 61% of the informative cases. In 8% of the cases, missense point mutations were detected in Smad4. In CRCs, relative to controls, there was increased SMAD4 staining in the cytoplasm (74%) and decreased staining in the nuclei (37%). LOH of 18q21 and high cytoplasmic localization of SMAD4 were associated with shortened overall survival of Stage II patients, whereas low nuclear expression of SMAD4 was associated with worse survival, but only for patients with Stage III CRCs. Conclusions LOH of 18q21 and high cytoplasmic localization of SMAD4 in Stage II CRCs and low nuclear SMAD4 in Stage III CRCs are predictors of shortened patient survival. PMID:28423626

SUMO-Specific Cysteine Protease 1 Promotes Epithelial Mesenchymal Transition of Prostate Cancer Cells via Regulating SMAD4 deSUMOylation

PubMed Central

Zhang, Xiaoyan; Wang, Hao; Wang, Hua; Xiao, Fengjun; Seth, Prem; Xu, Weidong; Jia, Qinghua; Wu, Chutse; Yang, Yuefeng; Wang, Lisheng

2017-01-01

In advanced prostate cancer, small ubiquitin-like modifier (SUMO)-specific cysteine protease 1 (SENP1) is up-regulated. However, the role of SENP1 in regulating deSUMOylation of TGF-β/SMADs signaling is unknown. In this study, we developed a lentiviral vector, PLKO.1-shSENP1, to silence SENP1 in prostate cancer cells with high metastatic characteristics (PC3M). Likewise, we also created an adenovirus vector, Ad5/F11p-SENP1 to over-express SENP1 in prostate cancer cells with low metastatic potential (LNCaP). We showed that silencing of SENP1 promoted cellular apoptosis, and inhibited proliferation and migration of PC3M cells. Moreover, SENP1 silencing increased the SMAD4 expression at protein level, up-regulated E-cadherin and down-regulated Vimentin expression, indicating the inhibition of epithelial mesenchymal transition (EMT). Furthermore, SMAD4 interference abolished SENP1-mediated up-regulation of E-cadherin, suggesting that SENP1 regulated E-cadherin expression via SMAD4. SENP1 over-expression in LNCaP cells reduced SMAD4 protein, and promoted EMT via decreasing E-cadherin and increasing Vimentin. Moreover, down-regulation of SMAD4 and E-cadherin were blocked, after transfection with two SUMOylation sites mutated SMAD4, suggesting that SENP1 might reduce SMAD4 levels to regulate E-cadherin expression via deSUMOylation of SMAD4. In conclusion, SENP1 deSUMOylated SMAD4 to promote EMT via up-regulating E-cadherin in prostate cancer cells. Therefore, SENP1 is a potential target for treatment of advanced prostate cancer. PMID:28417919

Angiotensin II Receptor Antagonism Reduces Transforming Growth Factor Beta and Smad Signaling in Thoracic Aortic Aneurysm

PubMed Central

Nataatmadja, Maria; West, Jennifer; Prabowo, Sulistiana; West, Malcolm

2013-01-01

ABSTRACT Background The expression of transforming growth factor beta (TGF-β) and Smad3 regulates extracellular matrix homeostasis and inflammation in aortic aneurysms. The expression of Smad3 depends on signaling by angiotensin II (AngII) receptor pathways through TGF-β receptor–dependent and –independent pathways. Methods To determine the expression of AngII type 1 (AT1R) and type 2 receptors (AT2R), TGF-β, and Smad3 in thoracic aortic aneurysms, we performed immunohistochemistry testing on tissue and cultured cells derived from subjects with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) malformation and from normal aortas of subjects who were organ donors. Results MFS and BAV aneurysm tissue showed enhanced accumulation of TGF-β and Smad3 in vascular smooth muscle cells (VSMCs) and in inflammatory cells in the subintimal layer and tunica media. The normal aortic wall exhibited minimal TGF-β and Smad3 staining. Cultured VSMCs from MFS and BAV samples showed nuclear Smad3 and strong cytoplasmic TGF-β expression in the cytoplasmic vesicles. In control cells, Smad3 was located mainly in the cytoplasm, and weak cytoplasmic TGF-β was distributed with a pattern similar to that of the aneurysm-derived cells. Compared to normal aorta cells, AT1R and AT2R expression was increased in both aneurysm types. Treatment of cultured VSMCs with the AT1R antagonist losartan caused both reduced TGF-β vesicle localization and nuclear expression of Smad3. Conclusions Increased TGF-β and Smad3 expression in aneurysm tissue and cultured VSMCs is consistent with aberrant TGF-β expression and the activation of Smad3 signaling. Losartan-mediated reduction in TGF-β expression and the cytoplasmic localization of Smad3 support a role for AT1R antagonism in the inhibition of aneurysm progression. PMID:23532685

SMAD4 Loss Is Associated with Cetuximab Resistance and Induction of MAPK/JNK Activation in Head and Neck Cancer Cells.

PubMed

Ozawa, Hiroyuki; Ranaweera, Ruchira S; Izumchenko, Evgeny; Makarev, Eugene; Zhavoronkov, Alex; Fertig, Elana J; Howard, Jason D; Markovic, Ana; Bedi, Atul; Ravi, Rajani; Perez, Jimena; Le, Quynh-Thu; Kong, Christina S; Jordan, Richard C; Wang, Hao; Kang, Hyunseok; Quon, Harry; Sidransky, David; Chung, Christine H

2017-09-01

Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC. Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4 -low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss. Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162-75. ©2017 AACR . ©2017 American Association for Cancer Research.

Smad1 and WIF1 genes are downregulated during saccular stage of lung development in the nitrofen rat model.

PubMed

Fujiwara, Naho; Doi, Takashi; Gosemann, Jan-Hendrik; Kutasy, Balazs; Friedmacher, Florian; Puri, Prem

2012-02-01

The exact pathogenesis of pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) still remains unclear. Smad1, one of the bone morphogenesis protein (BMP) receptor downstream signaling proteins, plays a key role in organogenesis including lung development and maturation. Smad1 knockout mice display reduced sacculation, an important feature of pulmonary hypoplasia. Wnt inhibitor factor 1 (Wif1) is a target gene of Smad1 in the developing lung epithelial cells (LECs). Smad1 directly regulates Wif1 gene expression and blockade of Smad1 function in fetal LECs is reported to downregulate Wif1 gene expression. We designed this study to test the hypothesis that pulmonary Smad1 and Wif1 gene expression is downregulated during saccular stage of lung development in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18, and D21. Fetal lungs were dissected and divided into 2 groups: control and nitrofen (n = 9 at each time point, respectively). Pulmonary gene expression of Smad1 and Wif1 were analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution of Smad1 and Wif1. The relative mRNA expression levels of Smad1 and Wif1 were significantly downregulated in the nitrofen group compared to controls on D18 and D21 (*p < 0.01, **p < 0.05). Immunoreactivity of Smad1 and Wif1 was also markedly decreased in nitrofen lungs compared to controls on D18 and D21. We provide evidence, for the first time, that the pulmonary gene expression of Smad1 and Wif1 is downregulated on D18 and D21 (saccular stage of lung development) in the nitrofen-induced hypoplastic lung. These findings suggest that the downregulation of Smad1/Wif1 gene expression may contribute to pulmonary hypoplasia in the nitrofen CDH model by retardation of lung development during saccular stage.

Single Chain Antibodies as Tools to Study transforming growth factor-β-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.

PubMed

Blokzijl, Andries; Zieba, Agata; Hust, Michael; Schirrmann, Thomas; Helmsing, Saskia; Grannas, Karin; Hertz, Ellen; Moren, Anita; Chen, Lei; Söderberg, Ola; Moustakas, Aristidis; Dübel, Stefan; Landegren, Ulf

2016-06-01

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-β signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-β signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-β signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

PubMed Central

Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R.

2015-01-01

Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling. PMID:25602473

AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription.

PubMed

Takai, Hideki; Araki, Shouta; Mezawa, Masaru; Kim, Dong-Soon; Li, Xinyue; Yang, Li; Li, Zhengyang; Wang, Zhitao; Nakayama, Youhei; Ogata, Yorimasa

2008-02-29

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.

Studying the Roles of GRK2-Mediated Smad2/3 Phosphorylation as a Negative Feedback Mechanism of TGF-Beta Signaling and a Target of Breast Cancer Therapeutics

DTIC Science & Technology

2014-01-01

as blocking this pathway could slow down metastasis in animal models. Since Smad2 and Smad3 are transcription factors, they are not ideal drug...through different mechanisms. Whereas GRK2 phosphorylates a defined serine/threonine residue on the linker region of the Smad, BCAR3 recruits another...500) and a rabbit anti-phospho- Smad3 antibody (#9520, Cell Signaling, 1:500), or Alexa568-labled Phalloidin (Life Technologies). Cells were then

MiR-34a-3p alters proliferation and apoptosis of meningioma cells in vitro and is directly targeting SMAD4, FRAT1 and BCL2

PubMed Central

Werner, Tamara V.; Hart, Martin; Nickels, Ruth; Kim, Yoo-Jin; Menger, Michael D.; Bohle, Rainer M.; Keller, Andreas; Ludwig, Nicole; Meese, Eckart

2017-01-01

Micro (mi)RNAs are short, noncoding RNAs and deregulation of miRNAs and their targets are implicated in tumor generation and progression in many cancers. Meningiomas are mostly benign, slow growing tumors of the central nervous system with a small percentage showing a malignant phenotype. Following in silico prediction of potential targets of miR-34a-3p, SMAD4, FRAT1, and BCL2 have been confirmed as targets by dual luciferase assays with co-expression of miR-34a-3p and reporter gene constructs containing the respective 3'UTRs. Disruption of the miR-34a-3p binding sites in the 3'UTRs resulted in loss of responsiveness to miR-34a-3p overexpression. In meningioma cells, overexpression of miR-34a-3p resulted in decreased protein levels of SMAD4, FRAT1 and BCL2, while inhibition of miR-34a-3p led to increased levels of these proteins as confirmed by Western blotting. Furthermore, deregulation of miR-34a-3p altered cell proliferation and apoptosis of meningioma cells in vitro. We show that SMAD4, FRAT1 and BCL2 are direct targets of miR-34a-3p and that deregulation of miR-34a-3p alters proliferation and apoptosis of meningioma cells in vitro. As part of their respective signaling pathways, which are known to play a role in meningioma genesis and progression, deregulation of SMAD4, FRAT1 and BCL2 might contribute to the aberrant activation of these signaling pathways leading to increased proliferation and inhibition of apoptosis in meningiomas. PMID:28340489

The inhibition of macrophage foam cell formation by tetrahydroxystilbene glucoside is driven by suppressing vimentin cytoskeleton.

PubMed

Yao, Wenjuan; Huang, Lei; Sun, Qinju; Yang, Lifeng; Tang, Lian; Meng, Guoliang; Xu, Xiaole; Zhang, Wei

2016-10-01

Macrophage foam cell formation triggered by oxLDL is an important event that occurs during the development of atherosclerosis. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside (TSG) exhibits significant anti-atherosclerotic activity. Herein we used U937 cells induced by PMA and oxLDL in vitro to investigate the inhibitory effects of TSG on U937 differentiation and macrophage foam cell formation. TSG pretreatment markedly inhibited cell differentiation induced by PMA, macrophage apoptosis and foam cell formation induced by oxLDL. The inhibition of vimentin expression and cleavage was involved in these inhibitory effects of TSG. The suppression of vimentin by siRNA in U937 significantly inhibited cell differentiation, apoptosis and foam cell formation. Using inhibitors for TGFβR1 and PI3K, we found that vimentin production in U937 cells is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG pretreatment inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by PMA and oxLDL. Furthermore, TSG attenuated the induced caspase-3 activation and adhesion molecules levels by PMA and oxLDL. PMA and oxLDL increased the co-localization of vimentin with ICAM-1, which was attenuated by pretreatment with TSG. These results suggest that TSG inhibits macrophage foam cell formation through suppressing vimentin expression and cleavage, adhesion molecules expression and vimentin-ICAM-1 co-localization. The interruption of TGFβ/Smad pathway and caspase-3 activation is responsible for the downregulation of TSG on vimentin expression and degradation, respectively. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

MiR-34a-3p alters proliferation and apoptosis of meningioma cells in vitro and is directly targeting SMAD4, FRAT1 and BCL2.

PubMed

Werner, Tamara V; Hart, Martin; Nickels, Ruth; Kim, Yoo-Jin; Menger, Michael D; Bohle, Rainer M; Keller, Andreas; Ludwig, Nicole; Meese, Eckart

2017-03-23

Micro (mi)RNAs are short, noncoding RNAs and deregulation of miRNAs and their targets are implicated in tumor generation and progression in many cancers. Meningiomas are mostly benign, slow growing tumors of the central nervous system with a small percentage showing a malignant phenotype.Following in silico prediction of potential targets of miR-34a-3p, SMAD4 , FRAT1 , and BCL2 have been confirmed as targets by dual luciferase assays with co-expression of miR-34a-3p and reporter gene constructs containing the respective 3'UTRs. Disruption of the miR-34a-3p binding sites in the 3'UTRs resulted in loss of responsiveness to miR-34a-3p overexpression. In meningioma cells, overexpression of miR-34a-3p resulted in decreased protein levels of SMAD4, FRAT1 and BCL2, while inhibition of miR-34a-3p led to increased levels of these proteins as confirmed by Western blotting. Furthermore, deregulation of miR-34a-3p altered cell proliferation and apoptosis of meningioma cells in vitro .We show that SMAD4 , FRAT1 and BCL2 are direct targets of miR-34a-3p and that deregulation of miR-34a-3p alters proliferation and apoptosis of meningioma cells in vitro . As part of their respective signaling pathways, which are known to play a role in meningioma genesis and progression, deregulation of SMAD4 , FRAT1 and BCL2 might contribute to the aberrant activation of these signaling pathways leading to increased proliferation and inhibition of apoptosis in meningiomas.

Gain-of-function mutations in SMAD4 cause a distinctive repertoire of cardiovascular phenotypes in patients with Myhre syndrome.

PubMed

Lin, Angela E; Michot, Caroline; Cormier-Daire, Valerie; L'Ecuyer, Thomas J; Matherne, G Paul; Barnes, Barrett H; Humberson, Jennifer B; Edmondson, Andrew C; Zackai, Elaine; O'Connor, Matthew J; Kaplan, Julie D; Ebeid, Makram R; Krier, Joel; Krieg, Elizabeth; Ghoshhajra, Brian; Lindsay, Mark E

2016-10-01

Myhre syndrome is a rare, distinctive syndrome due to specific gain-of-function mutations in SMAD4. The characteristic phenotype includes short stature, dysmorphic facial features, hearing loss, laryngotracheal anomalies, arthropathy, radiographic defects, intellectual disability, and a more recently appreciated spectrum of cardiovascular defects with a striking fibroproliferative response to surgical intervention. We report four newly described patients with typical features of Myhre syndrome who had (i) a mildly narrow descending aorta and restrictive cardiomyopathy; (ii) recurrent pericardial and pleural effusions; (iii) a large persistent ductus arteriosus with juxtaductal aortic coarctation; and (iv) restrictive pericardial disease requiring pericardiectomy. Additional information is provided about a fifth previously reported patient with fatal pericardial disease. A literature review of the cardiovascular features of Myhre syndrome was performed on 54 total patients, all with a SMAD4 mutation. Seventy percent had a cardiovascular abnormality including congenital heart defects (63%), pericardial disease (17%), restrictive cardiomyopathy (9%), and systemic hypertension (15%). Pericarditis and restrictive cardiomyopathy are associated with high mortality (three patients each among 10 deaths); one patient with restrictive cardiomyopathy also had epicarditis. Cardiomyopathy and pericardial abnormalities distinguish Myhre syndrome from other disorders caused by mutations in the TGF-β signaling cascade (Marfan, Loeys-Dietz, or Shprintzen-Goldberg syndromes). We hypothesize that the expanded spectrum of cardiovascular abnormalities relates to the ability of the SMAD4 protein to integrate diverse signaling pathways, including canonical TGF-β, BMP, and Activin signaling. The co-occurrence of congenital and acquired phenotypes demonstrates that the gene product of SMAD4 is required for both developmental and postnatal cardiovascular homeostasis. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Heat shock protein 22 (HSPB8) limits TGF-β-stimulated migration of osteoblasts.

PubMed

Yamamoto, Naohiro; Tokuda, Haruhiko; Kuroyanagi, Gen; Kainuma, Shingo; Matsushima-Nishiwaki, Rie; Fujita, Kazuhiko; Kozawa, Osamu; Otsuka, Takanobu

2016-11-15

Heat shock proteins (HSPs) are induced in response to various physiological and environmental conditions such as chemical and heat stress, and recognized to function as molecular chaperones. HSP22 (HSPB8), a low-molecular weight HSP, is ubiquitously expressed in many cell types. However, the precise role of HSP22 in bone metabolism remains to be clarified. In the present study, we investigated whether HSP22 is implicated in the transforming growth factor-β (TGF-β)-stimulated migration of osteoblast-like MC3T3-E1 cells. Although protein levels of HSP22 were clearly detected in unstimulated MC3T3-E1 cells, TGF-β failed to induce the protein levels. The TGF-β-stimulated migration was significantly up-regulated by knockdown of HSP22 expression. The cell migration stimulated by platelet-derived growth factor-BB was also enhanced by HSP22 knockdown. SB203580, an inhibitor of p38 mitogen-activated protein kinase, PD98059, an inhibitor of MEK1/2, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase had no effects on the TGF-β-induced migration. SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the migration with or without TGF-β stimulation. Smad2, Smad3, Smad4 or Smad7 was not coimmunoprecipitated with HSP22. On the other hand, the TGF-β-induced Smad2 phosphorylation was enhanced by HSP22 down-regulation. The protein levels of TGF-β type II receptor (TGF-β RII) but not TGF-β type I receptor (TGF-β RI) was significantly up-regulated in HSP22 knockdown cells compared with those in the control cells. However, the levels of TGF-β RII mRNA in HSP22 knockdown cells were little different from those of the control cells. Neither TGF-β RI nor TGF-β RII was coimmunoprecipitated with HSP22. SIS3 reduced the amplification by HSP22 knockdown of the TGF-β-stimulated cell migration almost to the basal level. Our results strongly suggest that HSP22 functions as a negative regulator in the TGF-β-stimulated migration of osteoblasts via suppression of the Smad-dependent pathway, resulting from modulating the protein levels of TGF-β RII. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Activin A amplifies dysregulated BMP signaling and induces chondro-osseous differentiation of primary connective tissue progenitor cells in patients with fibrodysplasia ossificans progressiva (FOP).

PubMed

Wang, Haitao; Shore, Eileen M; Pignolo, Robert J; Kaplan, Frederick S

2018-04-01

Fibrodysplasia ossificans progressiva (FOP), is caused by mutations in the type I BMP receptor ACVR1 that lead to increased activation of the BMP-pSmad1/5/8 signaling pathway. Recent findings suggest that Activin A (Act A) promiscuously stimulates the bone morphogenetic protein (BMP) signaling pathway in vitro and mediates heterotopic ossification (HO) in mouse models of FOP, but primary data from FOP patient cells are lacking. To examine BMP-pSmad1/5/8 pathway signaling and chondro-osseous differentiation in response to endogenous and exogenous Act A in primary connective tissue progenitor cells [CTPCs; also known as stem cells from human exfoliated deciduous teeth (SHED) cells] from patients with FOP. These cells express the common FOP mutation, ACVR1 (R206H). We found that Act A amplifies dysregulated BMP pathway signaling in human FOP primary CTPCs cells through the Smad1/5/8 pathway and induces chondro-osseous differentiation. Amplification of BMP-pSmad1/5/8 signaling was inhibited by Follistatin and by a neutralizing antibody to Activin A. The increased basal pSmad1/5/8 activity, as well as the hypoxia-induced stimulation of FOP CTPCs cells, were BMP4 and Act A independent. Importantly, either BMP4 or Act A stimulated pSmad1/5/8 pathway signaling but BMP4 signaling was not dependent on Activin A and vice versa. Circulating plasma levels of Act A or BMP4 are similar in controls compared to FOP patients, and suggest the potential for an autocrine or paracrine route for pathological signaling. The mutated FOP receptor [ACVR1 (R206H)] is hypersensitive to BMP4 and uniquely sensitive (compared to the wild type receptor) to Act A. Both canonical and non-canonical ligands have a synergistic effect on BMP-pSmad1/5/8 signaling in FOP CTPCs and may cooperate to alter the threshold for HO in FOP. Our findings in primary human FOP CTPCs have important implications for the design of clinical trials to inhibit dysregulated BMP pathway signaling in humans who have FOP. Copyright © 2017 Elsevier Inc. All rights reserved.

Low-Dose Paclitaxel Ameliorates Pulmonary Fibrosis by Suppressing TGF-β1/Smad3 Pathway via miR-140 Upregulation

PubMed Central

Wang, Congjie; Song, Xiaodong; Li, Youjie; Han, Fang; Gao, Shuyan; Wang, Xiaozhi; Xie, Shuyang; Lv, Changjun

2013-01-01

Abnormal TGF-β1/Smad3 activation plays an important role in the pathogenesis of pulmonary fibrosis, which can be prevented by paclitaxel (PTX). This study aimed to investigate an antifibrotic effect of the low-dose PTX (10 to 50 nM in vitro, and 0.6 mg/kg in vivo). PTX treatment resulted in phenotype reversion of epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs) with increase of miR-140. PTX resulted in an amelioration of bleomycin (BLM)-induced pulmonary fibrosis in rats with reduction of the wet lung weight to body weight ratios and the collagen deposition. Our results further demonstrated that PTX inhibited the effect of TGF-β1 on regulating the expression of Smad3 and phosphorylated Smad3 (p-Smad3), and restored the levels of E-cadherin, vimentin and α-SMA. Moreover, lower miR-140 levels were found in idiopathic pulmonary fibrosis (IPF) patients, TGF-β1-treated AECs and BLM-instilled rat lungs. Through decreasing Smad3/p-Smad3 expression and upregulating miR-140, PTX treatment could significantly reverse the EMT of AECs and prevent pulmonary fibrosis of rats. The action of PTX to ameliorate TGF-β1-induced EMT was promoted by miR-140, which increased E-cadherin levels and reduced the expression of vimentin, Smad3 and p-Smad3. Collectively, our results demonstrate that low-dose PTX prevents pulmonary fibrosis by suppressing the TGF-β1/Smad3 pathway via upregulating miR-140. PMID:23967091

Intracellular mediators of transforming growth factor beta superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo.

PubMed

Rajagopal, Ramya; Ishii, Shunsuke; Beebe, David C

2007-06-25

Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Proteins that are downstream of the transforming growth factor-beta superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFbeta superfamily for their normal development. Phosphorylated Smad1 (pSmad1), pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA) and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-beta superfamily to endosomes is important for the regulation of growth factor signaling.

Effects of SARA on oxygen-glucose deprivation in PC12 cell line.

PubMed

Wang, Jiao-Qi; He, Jin-Ting; Du, Zhen-Wu; Li, Zong-Shu; Liu, Yong-Feng; Mang, Jing; Xu, Zhong-Xin

2013-05-01

Ischemic stroke is a major composition of cerebrovascular disease, seriously threatening to human health in the world. Activin A (ActA), belonging to transforming growth factor-beta (TGF-β) super family, plays an important role in the hypoxic-ischemic brain injury through ActA/Smads pathway. While as an essential phosphorylation assistor in TGF-β signaling, the functions and mechanisms of smad anchor for receptor activation (SARA) in ischemic brain injury remain poorly understood. To solve this problem and explore the pathological processes of ischemic stroke, we used an Oxygen-Glucose deprivation (OGD) model in nerve growth factor-induced differentiated rattus PC12 pheochromocytoma cells and down regulated the expressions of SARA by RNA interference technology. Our results showed that the repression of SARA before OGD exposure reduced the expressions of Smad2, 3, 4 mRNA and the phosphorylation rate of Smad2 protein, but it did not affect the mRNA expressions of Smad7. After OGD treatment, ActA/Smads pathway was activated and the expression of SARA in the SARA pre-repression group was significantly up-regulated. The pre-repression of SARA increased the sensitivities of nerve-like cells to OGD damage. Moreover, the mRNA expression of Smad7 which was supposed to participate in the negative feedback of ActA/Smads pathway was also elevated due to OGD injury. Taken together, these results suggest a positive role of SARA in assisting the phosphorylation of Smad2 and maintaining the neuron protective effect of ActA/Smads pathway.

Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Koichi, E-mail: shimada-ki@dent.nihon-u.ac.jp; Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo; Ikeda, Kyoko

2009-12-18

Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1.more » To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-{alpha} stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-{alpha}-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-{alpha} and BMP signaling pathways.« less

Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis.

PubMed

Dufton, Neil P; Peghaire, Claire R; Osuna-Almagro, Lourdes; Raimondi, Claudio; Kalna, Viktoria; Chuahan, Abhishek; Webb, Gwilym; Yang, Youwen; Birdsey, Graeme M; Lalor, Patricia; Mason, Justin C; Adams, David H; Randi, Anna M

2017-10-12

The role of the endothelium in protecting from chronic liver disease and TGFβ-mediated fibrosis remains unclear. Here we describe how the endothelial transcription factor ETS-related gene (ERG) promotes liver homoeostasis by controlling canonical TGFβ-SMAD signalling, driving the SMAD1 pathway while repressing SMAD3 activity. Molecular analysis shows that ERG binds to SMAD3, restricting its access to DNA. Ablation of ERG expression results in endothelial-to-mesenchymal transition (EndMT) and spontaneous liver fibrogenesis in EC-specific constitutive hemi-deficient (Erg cEC-Het ) and inducible homozygous deficient mice (Erg iEC-KO ), in a SMAD3-dependent manner. Acute administration of the TNF-α inhibitor etanercept inhibits carbon tetrachloride (CCL 4 )-induced fibrogenesis in an ERG-dependent manner in mice. Decreased ERG expression also correlates with EndMT in tissues from patients with end-stage liver fibrosis. These studies identify a pathogenic mechanism where loss of ERG causes endothelial-dependent liver fibrogenesis via regulation of SMAD2/3. Moreover, ERG represents a promising candidate biomarker for assessing EndMT in liver disease.The transcription factor ERG is key to endothelial lineage specification and vascular homeostasis. Here the authors show that ERG balances TGFβ signalling through the SMAD1 and SMAD3 pathways, protecting the endothelium from endothelial-to-mesenchymal transition and consequent liver fibrosis in mice via a SMAD3-dependent mechanism.

Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice.

PubMed

Petit, Fabrice G; Deng, Chuxia; Jamin, Soazik P

2016-01-01

Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism. Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct.

Smad4 Loss Correlates With Higher Rates of Local and Distant Failure in Pancreatic Adenocarcinoma Patients Receiving Adjuvant Chemoradiation.

PubMed

Herman, Joseph M; Jabbour, Salma K; Lin, Steven H; Deek, Matthew P; Hsu, Charles C; Fishman, Elliot K; Kim, Sinae; Cameron, John L; Chekmareva, Marina; Laheru, Daniel A; Narang, Amol K; Pawlik, Timothy M; Hruban, Ralph H; Wolfgang, Christopher L; Iacobuzio-Donahue, Christine A

2018-02-01

The tumor suppressor gene SMAD4 (DPC4) is genetically inactivated in approximately half of pancreatic ductal adenocarcinomas (PDAs). We examined whether Smad4 tumor status was associated with outcomes after adjuvant chemoradiation (CRT) for resected PDAs. Patients treated with adjuvant CRT were identified (N = 145). Smad4 status was determined by immunolabeling and graded as intact or lost. Kaplan-Meier method and multivariable competing risk analyses were performed. On multivariate competing risk analysis, Smad4 loss was associated with increased risk of local recurrence (LR) (hazard ratio, 2.37; 95% confidence interval, 1.10-5.11; P = 0.027), distant failure (DF) (hazard ratio, 1.71; 95% confidence interval, 1.03-2.83; P = 0.037), and synchronous LR and DF at first recurrence (14.9 % vs 5.3%, P = 0.07) compared with Smad4 intact cancers. Smad4 loss was not associated with median overall survival (22 vs 22 months; P = 0.63) or disease-free survival (lost [13.6 months] vs intact [13.5 months], P = 0.79). After PDA resection and adjuvant CRT, Smad4 loss correlated with higher risk of LR and DF, but not with survival. Smad4 loss may help predict which surgical patients are at higher risk for failure after definitive management and may benefit from intensified adjuvant therapy.

Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch, craniofacial and cardiac defects

PubMed Central

Nie, Xuguang; Deng, Chu-xia; Wang, Qin; Jiao, Kai

2008-01-01

TGFβ/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFβ/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2α, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFβ/BMP signals are essential for appropriate NCC development. PMID:18334251

Decreased SMAD4 expression is associated with induction of epithelial-to-mesenchymal transition and cetuximab resistance in head and neck squamous cell carcinoma

PubMed Central

Cheng, Haixia; Fertig, Elana J; Ozawa, Hiroyuki; Hatakeyama, Hiromitsu; Howard, Jason D; Perez, Jimena; Considine, Michael; Thakar, Manjusha; Ranaweera, Ruchira; Krigsfeld, Gabriel; Chung, Christine H

2015-01-01

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting this receptor, is widely used to treat these patients. In the following investigation, we examined the role of SMAD4 down-regulation in mediating epithelial-to-mesenchymal transition (EMT) and cetuximab resistance in HNSCC. We determined that SMAD4 downregulation was significantly associated with increased cell motility, increased expression of vimentin, and cetuximab resistance in HNSCC cell lines. In the HNSCC genomic dataset obtained from The Cancer Genome Atlas, SMAD4 was altered in 20/279 (7%) of HNSCC via homozygous deletion, and nonsense, missense, and silent mutations. When SMAD4 expression was compared with respect to human papillomavirus (HPV) status, HPV-positive tumors had higher expression compared to HPV-negative tumors. Furthermore, higher SMAD4 expression also correlated with higher CDKN2A (p16) expression. Our data suggest that SMAD4 down-regulation plays an important role in the induction of EMT and cetuximab resistance. Patients with higher SMAD4 expression may benefit from cetuximab use in the clinic. PMID:26046389

Mutation analysis of the Smad3 gene in human osteoarthritis.

PubMed

Yao, Jun-Yan; Wang, Yan; An, Jing; Mao, Chun-Ming; Hou, Ning; Lv, Ya-Xin; Wang, You-Liang; Cui, Fang; Huang, Min; Yang, Xiao

2003-09-01

Osteoarthritis (OA) is the most common joint disease worldwide. Recent studies have shown that targeted disruption of Smad3 in mouse results in OA. To reveal the possible association between the Smad3 gene mutation and human OA, we employed polymerase chain reaction-single strand conformation polymorphism and sequencing to screen mutations in all nine exons of the Smad3 gene in 32 patients with knee OA and 50 patients with only bone fracture. A missense mutation of the Smad3 gene was found in one patient. The single base mutation located in the linker region of the SMAD3 protein was A --> T change in the position 2 of codon 197 and resulted in an asparagine to isoleucine amino-acid substitution. The expressions of matrix metalloproteinase 2 (MMP-2) and MMP-9 in sera of the patient carrying the mutation were higher than other OA patients and controls. This is the first report showing that the Smad3 gene mutations could be associated with the pathogenesis of human OA.

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling.

PubMed

Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

2017-01-30

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Characterization of dSnoN and its relationship to Decapentaplegic signaling in Drosophila.

PubMed

Barrio, Rosa; López-Varea, Ana; Casado, Mar; de Celis, Jose F

2007-06-01

Vertebrate members of the ski/snoN family of proto-oncogenes antagonize TGFbeta and BMP signaling in a variety of experimental situations. This activity of Ski/SnoN proteins is related to their ability to interact with Smads, the proteins acting as key mediators of the transcriptional response to the TGFbeta superfamily members. However, despite extensive efforts to identify the physiological roles of the Ski/SnoN proteins, it is not yet clear whether they participate in regulating Activin and/or BMP signaling during normal development. It is therefore crucial to examine their roles in vivo mostly because of the large number of known Ski/SnoN-interacting proteins and the association between the up-regulation of these genes and cancer progression. Here we characterize the Drosophila homolog to vertebrate ski and snoN genes. The Drosophila dSnoN protein retains the ability of its vertebrate counterparts to antagonize BMP signaling in vivo and in cultured cells. dSnoN does not interfere with Mad phosphorylation but it interacts genetically with Mad, Medea and dSmad2. Mutations in either the Smad2-3 or Smad4 putative binding sites of dSnoN prevent the antagonism of dSnoN towards Dpp signaling, although homozygous flies for these mutations or for a genetic deficiency of the locus are viable and have wings of normal size and pattern.

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

NASA Astrophysics Data System (ADS)

Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

2017-01-01

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Modularized TGFbeta-Smad Signaling Pathway

NASA Technical Reports Server (NTRS)

Li, Yongfeng; Wang, M.; Carra, C.; Cucinotta, F. A.

2011-01-01

The Transforming Growth Factor beta (TGFbeta) signaling pathway is a prominent regulatory signaling pathway controlling various important cellular processes. It can be induced by several factors, including ionizing radiation. It is regulated by Smads in a negative feedback loop through promoting increases in the regulatory Smads in the cell nucleus, and subsequent expression of inhibitory Smad, Smad7 to form a ubiquitin ligase with Smurf targeting active TGF receptors for degradation. In this work, we proposed a mathematical model to study the radiation-induced Smad-regulated TGF signaling pathway. By modularization, we are able to analyze each module (subsystem) and recover the nonlinear dynamics of the entire network system. Meanwhile the excitability, a common feature observed in the biological systems, along the TGF signaling pathway is discussed by mathematical analysis and numerical simulation.

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

PubMed

Takahashi, Y; Fukui, T; Kishimoto, M; Suzuki, R; Mitsuyama, T; Sumimoto, K; Okazaki, T; Sakao, M; Sakaguchi, Y; Yoshida, K; Uchida, K; Nishio, A; Matsuzaki, K; Okazaki, K

2016-01-01

The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle. © 2016 International Society for Diseases of the Esophagus.

Identification of a novel intergenic miRNA located between the human DDC and COBL genes with a potential function in cell cycle arrest.

PubMed

Hoballa, Mohamad Hussein; Soltani, Bahram M; Mowla, Seyed Javad; Sheikhpour, Mojgan; Kay, Maryam

2018-07-01

Frequent abnormalities in 7p12 locus in different tumors like lung cancer candidate this region for novel regulatory elements. MiRNAs as novel regulatory elements encoded within the human genome are potentially oncomiRs or miR suppressors. Here, we have used bioinformatics tools to search for the novel miRNAs embedded within human chromosome 7p12. A bona fide stem loop (named mirZa precursor) had the features of producing a real miRNA (named miRZa) which was detected through RT-qPCR following the overexpression of its precursor. Then, endogenous miRZa was detected in human cell lines and tissues and sequenced. Consistent to the bioinformatics prediction, RT-qPCR as well as dual luciferase assay indicated that SMAD3 and IGF1R genes were targeted by miRZa. MiRZa-3p and miRZa-5p were downregulated in lung tumor tissue samples detected by RT-qPCR, and mirZa precursor overexpression in SW480 cells resulted in increased sub-G1 cell population. Overall, here we introduced a novel miRNA which is capable of targeting SMAD3 and IGF1R regulatory genes and increases the cell population in sub-G1 stage.

The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis.

PubMed

Fukui, Toshiro; Kishimoto, Masanobu; Nakajima, Atsushi; Yamashina, Masao; Nakayama, Shinji; Kusuda, Takeo; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2011-04-01

The gastric corpus and antrum are believed to contain epithelial stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin. We explored whether Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), could serve as a marker for stem cells. Stomachs, small intestines, and colons from Helicobacter felis-infected and noninfected C57BL/6 mice were examined. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, or doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) was performed, and pSmad2/3L-Thr immunostaining-positive cells were counted. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. In infected mice, pSmad2/3L-Thr immunostaining-positive cells were significantly increased in the corpus and antrum compared with those of noninfected mice (p < 0.0001). The number of Ki67 immunostaining-positive cells in the corpus and antrum of infected mice was also much greater than in the noninfected mice. Although pSmad2/3L-Thr immunostaining-positive cells were detected among the Ki67 cells, immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells showed immunohistochemical co-localization with cytokeratin 8, but some of them showed co-localization or adjacent localization with DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features and were confirmed in the isthmus. In small intestines and colons, pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around crypt bases, where the respective putative stem cells are thought to exist. We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells of the murine stomach and have suggested these cells to be epithelial stem cells.

TGF-β signalling pathway and breast cancer susceptibility

PubMed Central

Scollen, Serena; Luccarini, Craig; Baynes, Caroline; Driver, Kristy; Humphreys, Manjeet K.; Garcia-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Pharoah, Paul D.; Easton, Douglas F.; Hesketh, Robin; Metcalfe, James C; Dunning, Alison M

2011-01-01

Background TGF-β acts as a suppressor of primary tumour initiation but has been implicated as a promoter of the later malignant stages. Here associations with risk of invasive breast cancer are assessed for SNPs tagging seventeen genes in the canonical TGF-β ALK5/SMADs 2&3 and ALK1/SMADs 1&5 signalling pathways: LTBP1, LTBP2, LTBP4, TGFB1, TGFB2, TGFB3, TGFBR1(ALK5), ALK1, TGFBR2, Endoglin, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6 and SMAD72. Methods 354 tag SNPs (minor allele frequency>0.05) were selected for genotyping in a staged study design using 6,703 cases and 6,840 controls from the SEARCH study. Significant associations were meta-analysed with data from the NCI Polish Breast Cancer Study (PBCS) (1,966 cases and 2,347 controls) and published data from the Breast Cancer Association Consortium (BCAC). Results Associations of three SNPs, tagging TGFB1 (rs1982073), TGFBR1 (rs10512263) and TGFBR2 (rs4522809) were detected in SEARCH; however associations became weaker in meta-analyses including data from PBCS and BCAC. Tumour sub-type analyses indicated that the TGFB1 rs1982073 association may be confined to increased risk of developing progesterone receptor negative (PR−) tumours (1.18 (95% CI 1.09-1.28), 4.1×10−5 (P value for heterogeneity of ORs by PR status = 2.3 × 10−4)). There was no evidence for breast cancer risk associations with SNPs in the endothelial-specific pathway utilising ALK1/SMADs 1&5 that promotes angiogenesis. Conclusion Common variation in the TGF-β ALK5/SMADs 2&3 signalling pathway, which initiates signalling at the cell surface to inhibit cell proliferation, might be related to risk of specific tumour sub-types. Impact The subtype specific associations require very large studies to be confirmed. PMID:21527583

Glucocorticoids Recruit Tgfbr3 and Smad1 to Shift Transforming Growth Factor-β Signaling from the Tgfbr1/Smad2/3 Axis to the Acvrl1/Smad1 Axis in Lung Fibroblasts*

PubMed Central

Schwartze, Julian T.; Becker, Simone; Sakkas, Elpidoforos; Wujak, Łukasz A.; Niess, Gero; Usemann, Jakob; Reichenberger, Frank; Herold, Susanne; Vadász, István; Mayer, Konstantin; Seeger, Werner; Morty, Rory E.

2014-01-01

Glucocorticoids represent the mainstay therapy for many lung diseases, providing outstanding management of asthma but performing surprisingly poorly in patients with acute respiratory distress syndrome, chronic obstructive pulmonary disease, lung fibrosis, and blunted lung development associated with bronchopulmonary dysplasia in preterm infants. TGF-β is a pathogenic mediator of all four of these diseases, prompting us to explore glucocorticoid/TGF-β signaling cross-talk. Glucocorticoids, including dexamethasone, methylprednisolone, budesonide, and fluticasone, potentiated TGF-β signaling by the Acvrl1/Smad1/5/8 signaling axis and blunted signaling by the Tgfbr1/Smad2/3 axis in NIH/3T3 cells, as well as primary lung fibroblasts, smooth muscle cells, and endothelial cells. Dexamethasone drove expression of the accessory type III TGF-β receptor Tgfbr3, also called betaglycan. Tgfbr3 was demonstrated to be a “switch” that blunted Tgfbr1/Smad2/3 and potentiated Acvrl1/Smad1 signaling in lung fibroblasts. The Acvrl1/Smad1 axis, which was stimulated by dexamethasone, was active in lung fibroblasts and antagonized Tgfbr1/Smad2/3 signaling. Dexamethasone acted synergistically with TGF-β to drive differentiation of primary lung fibroblasts to myofibroblasts, revealed by acquisition of smooth muscle actin and smooth muscle myosin, which are exclusively Smad1-dependent processes in fibroblasts. Administration of dexamethasone to live mice recapitulated these observations and revealed a lung-specific impact of dexamethasone on lung Tgfbr3 expression and phospho-Smad1 levels in vivo. These data point to an interesting and hitherto unknown impact of glucocorticoids on TGF-β signaling in lung fibroblasts and other constituent cell types of the lung that may be relevant to lung physiology, as well as lung pathophysiology, in terms of drug/disease interactions. PMID:24347165

Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle.

PubMed

Rezaei, Hossein B; Kamato, Danielle; Ansari, Ghazaleh; Osman, Narin; Little, Peter J

2012-08-01

The transforming growth factor (TGF)-β superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-β induces its biological effects through Type I and Type II transmembrane receptors that have serine/threonine kinase activities and weak tyrosine kinase activity. In vascular smooth muscle, TGF-β binds to the TGF-β Type II receptor (TβRII) at the cell surface, recruiting the Type I receptor (TβRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TβRI, the transcription factors receptor activated (R-) Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Overall, Smad2/3 and co-Smad4 have similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. Phosphorylation of the Smad linker region appears to have an important role in the regulation of Smad activity and function. The mitogen-activated protein kinase (MAPK) family, CDK2, CDK4 and calcium-calmodulin dependent kinase are the main kinases that phosphorylate sites in the linker region. The role of the linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAPK and other kinases. In some instances, linker region phosphorylation regulates the inhibition of the nuclear translocation of Smads. In the present review, we describe TGF-β signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-β superfamily ligands in the context of vascular smooth muscle. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-inducedmore » Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells. - Highlights: • Hsc70 siRNA treatment suppressed the expression of Hsc70 but induced the expression of Hsp70 in NRK-49F cells. • Hsc70 siRNA treatment suppressed the activation of Smad2/3 in the cells treated with TGF-β. • Hsc70 interacted with Smad2/3 on stimulation with TGF-β in the cells. • Hsp70 did not influence the TGF-β-induced activation of Smad2/3 in the cells overexpressing Hsp70.« less

Poricoic acid ZA, a novel RAS inhibitor, attenuates tubulo-interstitial fibrosis and podocyte injury by inhibiting TGF-β/Smad signaling pathway.

PubMed

Wang, Ming; Chen, Dan-Qian; Wang, Min-Chang; Chen, Hua; Chen, Lin; Liu, Dan; Zhao, Hui; Zhao, Ying-Yong

2017-12-01

The pathogenesis of tubulo-interstitial fibrosis and glomerulosclerosisis was characterized by cellular hypertrophy, extracellular matrix accumulation and podocyte detachment. Poricoic acid ZA (PZA) is a tetracyclic triterpenoid compound extracted from the surface layer of Poria cocos (LPC), which have been used extensively for diuretic and renoprotective effects. The anti-fibrotic effect of PZA is investigated in HK-2 cells and podocytes induced by TGF-β1 and angiotensin II (ANGII). qRT-PCR, siRNA, immunofluorescence staining, co-immunoprecipitation and Western blot analyses are used to evaluate the expression of RAS signaling, TGF-β/Smad pathway, epithelial-to-mesenchymal transition (EMT) and podocyte markers. PZA restores the mRNA and protein expression of EMT in HK-2 cells. Specific TGF-β1-siRNA efficiently blocks ANGII-induced protein expression of TGF-β1 and further inhibits activated Smad signaling. PZA significantly attenuates up-regulation of angiotensinogen, renin, ACE and AT1. Further, PZA reverses up-regulation of TGFβRII and suppresses Smad proteins. Simultaneously, PZA inhibits the protein interaction of TGF-β receptor and Smads and PZA also inhibits activated RAS and TGF-β/Smad signaling cascade and up-regulates protein expression of podocyte markers and mitigates podocyte injury. This study demonstrated the beneficial role of PZA in renal fibrosis and podocyte injury. Our study highlighted that PZA inhibits RAS and further suppresses TGF-β/Smad pathway through inhibiting Smad2/3 phosphorylation via blocking Smad2/3-TGFβRI protein interaction. PZA is implicated in activation of RAS/TGF-β/Smad axis in HK-2 cells and podocytes. PZA could be considered as a novel RAS inhibitor for treating CKD. Copyright © 2017 Elsevier GmbH. All rights reserved.

Mechanisms of Action of Acetaldehyde in the Up-Regulation of the Human α2(I) Collagen Gene in Hepatic Stellate Cells

PubMed Central

Reyes-Gordillo, Karina; Shah, Ruchi; Arellanes-Robledo, Jaime; Hernández-Nazara, Zamira; Rincón-Sánchez, Ana Rosa; Inagaki, Yutaka; Rojkind, Marcos; Lakshman, M. Raj

2015-01-01

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4–containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties. PMID:24641900

Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation.

PubMed

Ji, Hong; Tang, Haiying; Lin, Hongli; Mao, Jingwei; Gao, Lili; Liu, Jia; Wu, Taihua

2014-11-01

The differentiation of fibroblasts, which are promoted by transforming growth factor-β (TGF-β)/Smad, is involved in the process of pulmonary fibrosis. The Rho/Rho-associated coiled-coil-forming protein kinase (Rock) pathway may regulate the fibroblast differentiation and myofibroblast expression of α-smooth muscle actin (α-SMA), however, the mechanism is not clear. The aim of the present study was to evaluate the role of Rho/Rock and TGF-β/Smad in TGF-β1-induced lung fibroblasts differentiation. Human embryonic lung fibroblasts were stimulated by TGF-β1, Y-27632 (inhibitor of Rho/Rock signaling) and staurosporine (inhibitor of TGF-β/Smad signaling). The α-SMA expression, cell cycle progression, content of the extracellular matrix (ECM) in cell culture supernatants and the expression of RhoA, RhoC, Rock1 and Smad2 were detected. The results demonstrated that α-SMA-positive cells significantly increased following TGF-β1 stimulation. Rho/Rock and TGF-β/Smad inhibitors suppressed TGF-β1-induced lung fibroblast differentiation. The inhibitors increased G 0 /G 1 and decreased S and G 2 /M percentages. The concentrations of the ECM proteins in the supernatant were significantly increased by TGF-β1 stimulation, whereas they were decreased by inhibitor stimulation. RhoA, RhoC, Rock1, Smad2 and tissue inhibitor of metalloproteinase-1 were upregulated by TGF-β1 stimulation. The Rho/Rock inhibitor downregulated Smad2 expression and the TGF-β/Smad inhibitor downregulated RhoA, RhoC and Rock1 expression. Therefore, the Rho/Rock pathway and Smad signaling were involved in the process of lung fibroblasts transformation, induced by TGF-β1, to myofibroblasts. The two pathways may undergo cross-talk in the lung fibroblasts differentiation in vitro .

Predictive value of the combination of SMAD4 expression and lymphocyte infiltration in malignant transformation of oral leukoplakia.

PubMed

Sakata, Junki; Yoshida, Ryoji; Matsuoka, Yuichiro; Nagata, Masashi; Hirosue, Akiyuki; Kawahara, Kenta; Nakamura, Takuya; Nakamoto, Masafumi; Hirayama, Masatoshi; Takahashi, Nozomu; Nakashima, Hikaru; Arita, Hidetaka; Ogi, Hidenao; Hiraki, Akimitsu; Shinohara, Masanori; Nakayama, Hideki

2017-04-01

Oral leukoplakia (OL) is a common, potentially malignant disorder of the oral cavity. SMAD4 was initially identified as a tumor suppressor and central mediator of transforming growth factor (TGF)-β signaling. In this study, we aimed to determine the expression patterns of SMAD4 in OL, its relationship with the degree of inflammation, and its clinical implications as a biomarker for OL malignant transformation. A total of 150 patients with OL were enrolled in this study. Paraffin-embedded sections obtained from biopsy or resection specimens were subjected to immunohistochemical analysis. Associations among the status of epithelial SMAD4 expression, stromal lymphocyte infiltration, and malignant transformation of OL were examined. Malignant transformation was significantly associated with the status of SMAD4 expression (P = 0.0017) and lymphocyte infiltration status (P = 0.0054). Cox regression analysis, based on the event-free survival (EFS), revealed that a low SMAD4 expression was a significant prognostic factor in OL patients (hazard ratio, 2.632; P = 0.043). In addition, a low SMAD4 expression was closely correlated with high lymphocyte infiltration (P = 0.00035), resulting in a significant correlation between the combination of low SMAD4 expression and high lymphocyte infiltration with malignant transformation of OL (P = 0.00027). The combination of the status of epithelial SMAD4 expression and stromal lymphocyte infiltration may be a useful biomarker for predicting malignant transformation in OL patients. These results suggest that not only epithelial SMAD4 loss, but also stromal features, may regulate the risk of malignant transformation of OL. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Reverse correlation of Jab1 and Smad4 in PANC-1 cells involved in the pathogenesis of pancreatic cancer.

PubMed

Li, Jun; Gu, Zhuoyu; Li, Siyuan; Xiao, Zhiwei; Sun, Kan

2015-01-01

Steps in the genetic basis of pancreatic cancer (PC) have been recently identified, however, Studies focusing on the relationship between Jab1 and Smad4 in PC are rarely reported. This study was performed to examine the expression patterns and association of Jab1 and Smad4 in PC cells for gaining a further understanding of PC pathogenesis. Human pancreatic cancer cell line PANC-1 cells were infected with retrovirus vector containing GFP, HA-Jab1, siGFP, and siJab1 respectively. The expression of Jab1 and Smad4 in PANC-1 cells was analyzed by Western blot and immunocytochemistry. Subsequently, the effect of overexpression of Jab1 on cell proliferation inhibition mediated by TGF-β was examined with MTT colorimetry. The expression of Smad4 in PANC-1 cells was inhibited after the overexpression of Jab1. Inversely, the expression of Smad4 was increased after the down-regulation of Jab1 silenced by SiRNA. Smad4 expression in PANC-1 cells was negatively correlated with Jab1 expression. In addition, the cell proliferation inhibitory effect induced by TGF-β in PANC-1 cells was attenuated after the overexpression of Jab1. The reverse correlation of Jab1 and Smad4 in PANC-1 cells may be involved in the Pathogenesis of PC. Jab1 can cause degradation of Smad4 via TGF-β signal pathway, consequently contributing to the proliferation of PC cells.

HIV-1 stimulates nuclear entry of amyloid beta via dynamin dependent EEA1 and TGF-β/Smad signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

András, Ibolya E., E-mail: iandras@med.miami; Toborek, Michal, E-mail: mtoborek@med.miami.edu

Clinical evidence indicates increased amyloid deposition in HIV-1-infected brains, which contributes to neurocognitive dysfunction in infected patients. Here we show that HIV-1 exposure stimulates amyloid beta (Aβ) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood–brain barrier (BBB). Treatment with HIV-1 and/or Aβ resulted in concurrent increase in early endosomal antigen-1 (EEA1), Smad, and phosphorylated Smad (pSmad) in nuclear fraction of HBMEC. A series of inhibition and silencing studies indicated that Smad and EEA1 closely interact by influencing their own nuclear entry; the effect that was attenuated by dynasore, a blocker of GTP-ase activity ofmore » dynamin. Importantly, inhibition of dynamin, EEA1, or TGF-β/Smad effectively attenuated HIV-1-induced Aβ accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aβ involves the dynamin-dependent EEA1 and TGF-β/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level. - Highlights: • HIV-1 induces nuclear accumulation of amyloid beta (Aβ) in brain endothelial cells. • EEA-1 and TGF-Β/Smad act in concert to regulate nuclear entry of Aβ. • Dynamin appropriates the EEA-1 and TGF-Β/Smad signaling. • Dynamin serves as a master regulator of HIV-1-induced nuclear accumulation of Aβ.« less

Antifibrotic effects of Smad4 small interfering RNAs in injured skeletal muscle after acute contusion.

PubMed

Li, H; Chen, J; Chen, S; Zhang, Q; Chen, S

2011-10-01

Muscle injuries are common musculoskeletal problems encountered in sports medicine clinics. In this study, we examined the effect of lentivirus-mediated small interfering RNA (siRNA) targeting Smad4 on the suppression of the fibrosis in injured skeletal muscles. We found that Smad4-siRNA could efficiently knock down the expression of Smad4 in the C2C12 myoblast cells and in the contunded mice gastrocnemius muscle. The expression of mRNA level of Smad4 decreased to 11% and 49% compared to the control group, respectively, and the expression of protein level decreased to 13% and 57% respectively. Moreover, the lentivirus-mediated siRNA was stably transfected only into the skeletal muscle and not into the liver of the animals. In contunded mice gastrocnemius, the collagenous and vimentin-positive area in the Smad4 siRNA group reduced to 36% and 37% compared to the control group, respectively. Furthermore, compared to the scrambled Smad4 siRNA-injected mice and PBS control-injected mice, the muscle function of the mice injected with lentivirus-mediated Smad4 siRNA improved in terms of both fast-twitch and tetanic strength (P<0.05). The results suggest that the gene therapy of inhibiting Smad4 by lentivirus-mediated siRNA could be a useful approach to prevent scar tissue formation and improve the function of injured skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo.

PubMed

Zeng, Yuanyuan; Zhu, Jianjie; Shen, Dan; Qin, Hualong; Lei, Zhe; Li, Wei; Liu, Zeyi; Huang, Jian-An

2017-05-09

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3'-untranslated region (3'-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3'-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment.