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Sample records for suppressor protein caveolin-1

  1. E-cadherin is required for caveolin-1-mediated down-regulation of the inhibitor of apoptosis protein survivin via reduced beta-catenin-Tcf/Lef-dependent transcription.

    PubMed

    Torres, Vicente A; Tapia, Julio C; Rodriguez, Diego A; Lladser, Alvaro; Arredondo, Cristian; Leyton, Lisette; Quest, Andrew F G

    2007-11-01

    Caveolin-1 reportedly acts as a tumor suppressor and promotes events associated with tumor progression, including metastasis. The molecular mechanisms underlying such radical differences in function are not understood. Recently, we showed that caveolin-1 inhibits expression of the inhibitor of apoptosis protein survivin via a transcriptional mechanism involving the beta-catenin-Tcf/Lef pathway. Surprisingly, while caveolin-1 expression decreased survivin mRNA and protein levels in HT29(ATCC) human colon cancer cells, this was not the case in metastatic HT29(US) cells. Survivin down-regulation was paralleled by coimmunoprecipitation and colocalization of caveolin-1 with beta-catenin in HT29(ATCC) but not HT29(US) cells. Unlike HT29(ATCC) cells, HT29(US) cells expressed small amounts of E-cadherin that accumulated in intracellular patches rather than at the cell surface. Re-expression of E-cadherin in HT29(US) cells restored the ability of caveolin-1 to down-regulate beta-catenin-Tcf/Lef-dependent transcription and survivin expression, as seen in HT29(ATCC) cells. In addition, coimmunoprecipitation and colocalization between caveolin-1 and beta-catenin increased upon E-cadherin expression in HT29(US) cells. In human embryonic kidney HEK293T and HT29(US) cells, caveolin-1 and E-cadherin cooperated in suppressing beta-catenin-Tcf/Lef-dependent transcription as well as survivin expression. Finally, mouse melanoma B16-F10 cells, another metastatic cell model with low endogenous caveolin-1 and E-cadherin levels, were characterized. In these cells, caveolin-1-mediated down-regulation of survivin in the presence of E-cadherin coincided with increased apoptosis. Thus, the absence of E-cadherin severely compromises the ability of caveolin-1 to develop activities potentially relevant to its role as a tumor suppressor.

  2. Caveolin-1 is an aggresome-inducing protein

    PubMed Central

    Tiwari, Ajit; Copeland, Courtney A.; Han, Bing; Hanson, Caroline A.; Raghunathan, Krishnan; Kenworthy, Anne K.

    2016-01-01

    Caveolin-1 (Cav1) drives the formation of flask-shaped membrane invaginations known as caveolae that participate in signaling, clathrin-independent endocytosis and mechanotransduction. Overexpression or mutations of Cav1 can lead to its mistrafficking, including its accumulation in a perinuclear compartment previously identified as the Golgi complex. Here, we show that in the case of overexpressed Cav1-GFP, this perinuclear compartment consists of cytoplasmic inclusion bodies generated in response to the accumulation of aggregates of misfolded proteins, known as aggresomes. Aggresomes containing Cav1-GFP are encased within vimentin cages, form in a microtubule-dependent manner, and are enriched in a number of key regulators of protein turnover, including ubiquitin, VCP/p97 and proteasomes. Interestingly, aggresome induction was cell-type dependent and was observed for many but not all Cav1 constructs tested. Furthermore, endogenous Cav1 accumulated in aggresomes formed in response to proteosomal inhibition. Our finding that Cav1 is both an aggresome-inducing and aggresome-localized protein provides new insights into how cells handle and respond to misfolded Cav1. They also raise the possibility that aggresome formation may contribute to some of reported phenotypes associated with overexpressed and/or mutant forms of Cav1. PMID:27929047

  3. Cholesterol and phytosterols differentially regulate the expression of caveolin 1 and a downstream prostate cell growth-suppressor gene

    PubMed Central

    Ifere, Godwin O.; Equan, Anita; Gordon, Kereen; Nagappan, Peri; Igietseme, Joseph U.; Ananaba, Godwin A.

    2010-01-01

    Background The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream regulated gene1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor. Methods PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72 h, followed by trypan blue dye exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members. Results Physiological doses (16 µM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol enrichment promoted cell growth (P<0.05), while phytosterols significantly induced growth-suppression (P<0.05) and apoptosis. Cell cycle analysis showed that contrary to cholesterol, phytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1(cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis. Conclusions Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor-suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1

  4. Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent.

    PubMed

    Waschke, Jens; Golenhofen, Nikola; Kurzchalia, Teymuras V; Drenckhahn, Detlev

    2006-07-01

    Protein kinase C (PKC) is activated in response to various inflammatory mediators and contributes significantly to the endothelial barrier breakdown. However, the mechanisms underlying PKC-mediated permeability regulation are not well understood. We prepared microvascular myocardial endothelial cells from both wild-type (WT) and caveolin-1-deficient mice. Activation of PKC by phorbol myristate acetate (PMA) (100 nM) for 30 min induced intercellular gap formation and fragmentation of VE-cadherin immunoreactivity in WT but not in caveolin-1-deficient monolayers. To test the effect of PKC activation on VE-cadherin-mediated adhesion, we allowed VE-cadherin-coated microbeads to bind to the endothelial cell surface and probed their adhesion by laser tweezers. PMA significantly reduced bead binding to 78+/-6% of controls in WT endothelial cells without any effect in caveolin-1-deficient cells. In WT cells, PMA caused an 86+/-18% increase in FITC-dextran permeability whereas no increase in permeability was observed in caveolin-1-deficient monolayers. Inhibition of PKC by staurosporine (50 nM, 30 min) did not affect barrier functions in both WT and caveolin-1-deficient MyEnd cells. Theses data indicate that PKC activation reduces endothelial barrier functions at least in part by the reduction of VE-cadherin-mediated adhesion and demonstrate that PKC-mediated permeability regulation depends on caveolin-1.

  5. Phospholipase D1 in caveolae: regulation by protein kinase Calpha and caveolin-1.

    PubMed

    Kim, J H; Han, J M; Lee, S; Kim, Y; Lee, T G; Park, J B; Lee, S D; Suh, P G; Ryu, S H

    1999-03-23

    Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.

  6. Absence of caveolin-1 alters heat shock protein expression in spontaneous mammary tumors driven by Her-2/neu expression.

    PubMed

    Ciocca, Daniel R; Cuello-Carrión, F Darío; Natoli, Anthony L; Restall, Christina; Anderson, Robin L

    2012-02-01

    In a previous study, we measured caveolin-1 protein levels, both in the normal breast and in breast cancer. The study revealed no association between caveolin-1 expression in the epithelial compartment and clinical disease outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor associated strongly with reduced metastasis and improved survival. Using an animal model, we found that the onset of mammary tumors driven by Her-2/neu expression was accelerated in mice lacking caveolin-1. We have analysed the heat shock protein (Hsp) response in the tumors of mice lacking caveolin-1. In all cases, the mammary tumors were estrogen and progesterone receptor negative, and the levels of Her-2/neu (evaluated by immunohistochemistry) were not different between the caveolin-1 +/+ (n = 8) and the caveolin-1 -/- (n = 7) tumors. However, a significant reduction in the extent of apoptosis was observed in mammary tumors from animals lacking caveolin-1. While Bcl-2, Bax, and survivin levels in the tumors were not different, the amount of HSPA (Hsp70) was almost double in the caveolin-1 -/- tumors. In contrast, HSPB1 (Hsp27/Hsp25) levels were significantly lower in the caveolin-1 -/- tumors. The mammary tumors from caveolin-1 null mice expressed more HSPC4 (gp96 or grp94), but HSPC1 (Hsp90), HSPA5 (grp78), HSPD1 (Hsp60), and CHOP were not altered. No significant changes in these proteins were found in the stroma surrounding these tumors. These results demonstrate that the disruption of the Cav-1 gene can cause alterations of specific Hsps as well as tumor development.

  7. Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment

    PubMed Central

    Trimmer, Casey; Sotgia, Federica; Whitaker-Menezes, Diana; Balliet, Renee M; Eaton, Gregory; Martinez-Outschoorn, Ubaldo E; Pavlides, Stephanos; Howell, Anthony; Iozzo, Renato V; Pestell, Richard G; Scherer, Philipp E

    2011-01-01

    We have recently proposed a new model for understanding tumor metabolism, termed: “The Autophagic Tumor Stroma Model of Cancer Metabolism”. In this new paradigm, catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. Mechanistically, tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. To further test this hypothesis, here we created human Cav-1 deficient immortalized fibroblasts using a targeted sh-RNA knock-down approach. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by ∼4-fold. Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was clearly independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified >40 protein biomarkers that were upregulated, most of which were associated with (i) myofibroblast differentiation or (ii) oxidative stress/hypoxia. In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant overexpression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress. In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in this process. In summary, here we provide new evidence directly supporting a key role for a loss of stromal Cav-1 expression and oxidative stress in cancerassociated fibroblasts, in promoting tumor growth, which is consistent with “The Autophagic Tumor Stroma Model of

  8. Calcium regulates caveolin-1 expression at the transcriptional level

    SciTech Connect

    Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming; Li, Yan; Liu, Dan; Zhang, Xue-Cheng; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. Black-Right-Pointing-Pointer An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. Black-Right-Pointing-Pointer Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. Black-Right-Pointing-Pointer Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca{sup 2+}/calcineurin/NFAT.

  9. The metastatic suppressor NDRG1 inhibits EMT, migration and invasion through interaction and promotion of caveolin-1 ubiquitylation in human colorectal cancer cells.

    PubMed

    Mi, L; Zhu, F; Yang, X; Lu, J; Zheng, Y; Zhao, Q; Wen, X; Lu, A; Wang, M; Zheng, M; Ji, J; Sun, J

    2017-03-27

    N-myc downstream-regulated gene 1 (NDRG1) has been reported to act as a key regulatory molecule in tumor progression-related signaling pathways, especially in tumor metastasis. However, the related mechanism has not been fully discovered yet. Herein we demonstrated that the novel molecule of cell migration and invasion, caveolin-1, has direct interaction with NDRG1 in human colorectal cancer (CRC) cells. Moreover, we discovered that NDRG1 reduces caveolin-1 protein expression through promoting its ubiquitylation and subsequent degradation via the proteasome in CRC cells. In addition, caveolin-1 mediates the suppressive function of NDRG1 in epithelial-mesenchymal transition, migration and invasion in vitro and metastasis in vivo. These results help to fulfill the potential mechanisms of NDRG1 in anti-metastatic treatment for human colorectal cancer.Oncogene advance online publication, 27 March 2017; doi:10.1038/onc.2017.74.

  10. Effect of caveolin-1 on the expression of tight junction-associated proteins in rat glioma-derived microvascular endothelial cells

    PubMed Central

    Li, Yao; Liu, Li-Bo; Ma, Teng; Wang, Ping; Xue, Yi-Xue

    2015-01-01

    Caveolin-1 affects the permeability of blood-tumor barrier (BTB) by regulating the expression of tight junction-associated proteins. However, the effect is still controversial. In the present work, we studied the regulative effect of caveolin-1 on the expression of tight junction-associated proteins and BTB via directly silencing and overexpressing of caveolin-1 by recombinant adenovirus transduction of glioma-derived microvascular endothelial cells in rat brain. The results show that the caveolin-1 downregulation resulted in decreased expression of tight junction-associated proteins, opening of tight junctions, and increasing the permeability of BTB, whereas the overexpression of caveolin-1 presented the opposite effects. Therefore, we conclude that caveolin-1 regulates the expression of tight junction-associated proteins in a positive manner, which further plays a role in the regulation of BTB permeability. This finding provides a novel therapeutic target for selectively opening of BTB. PMID:26722502

  11. The cytoplasmic domain of influenza M2 protein interacts with caveolin-1.

    PubMed

    Zou, Peng; Wu, Fan; Lu, Lu; Huang, Jing-He; Chen, Ying-Hua

    2009-06-15

    The cytoplasmic domain of influenza M2 protein (M2c) consists of 54 amino acid (aa) residues from aa44 to aa97. In this paper, M2c and its deletion mutant M2c(delta47-55) were expressed using prokaryotic expression system. First, glutaraldehyde crosslinking assay showed that M2c had multimerization potential mediated by aa47-55. Then, M2c, instead of M2c(delta47-55), directed eGFP from the whole cell localization to a predominately perinuclear region in CHO cells, which indicated that aa47-55 of M2c mediated the localization. Moreover, M2c colocalized with caveolin-1 (Cav) when CHO cells were cotransfected with Cav. A caveolin-1 binding motif phixxxxphixxphi (phi represents aromatic amino acid residues) in aa47-55 of M2c was found by sequence alignment and analysis. Further overlay ELISA result showed that M2c, but not M2c(delta47-55), bound to prokaryotically expressed cholesterol-free Cav(2-101), which illustrated the interaction could be cholesterol-independent. That was the first report of cellular protein bound to M2c.

  12. Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4{sup +}T cells

    SciTech Connect

    Ohnuma, Kei; Uchiyama, Masahiko; Hatano, Ryo; Takasawa, Wataru; Endo, Yuko; Dang, Nam H.; Morimoto, Chikao

    2009-08-21

    CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

  13. Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands

    PubMed Central

    Thompson, Christopher; Hielscher, Abigail

    2017-01-01

    The extracellular matrix (ECM) is abnormal in breast tumors and has been reported to contribute to breast tumor progression. One factor, which may drive ongoing matrix synthesis in breast tumors, is the loss of stromal caveolin-1 (cav-1), a scaffolding protein of caveolae, which has been linked to breast tumor aggressiveness. To determine whether loss of cav-1 results in the abnormal expression of matrix proteins, mammary glands from cav- 1-/- and cav- 1 +/+ mice were investigated for differences in expression of several ECM proteins. In addition, the presence of myofibroblasts, changes in the vessel density, and differences in duct number and size were assessed in the mammary glands of both animal models. Using immunohistochemistry, expression of fibronectin, tenascin-C, collagens and αSMA were significantly increased in the mammary glands of cav-1-/- mice. Second harmonic generation revealed more organized collagen fibers in cav-1 -/- glands and supported immunohistochemical analyses of increased collagen abundance in the glands of cav-1 -/- mice. Analysis of the ductal structure demonstrated a significant increase in the number of proliferating ducts in addition to significant increases in the duct circumference and area in cav-1 -/- glands compared to cav- 1 +/+ glands. Differences in microvessel density weren’t apparent between the animal models. In summary, we found that the loss of cav-1 resulted in increased ECM and α-SMA protein expression in murine mammary glands. Furthermore, we found that an abnormal ductal architecture accompanied the loss of cav-1. These data support a role for cav-1 in maintaining mammary gland structure. PMID:28187162

  14. Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands.

    PubMed

    Thompson, Christopher; Rahim, Sahar; Arnold, Jeremiah; Hielscher, Abigail

    2017-01-01

    The extracellular matrix (ECM) is abnormal in breast tumors and has been reported to contribute to breast tumor progression. One factor, which may drive ongoing matrix synthesis in breast tumors, is the loss of stromal caveolin-1 (cav-1), a scaffolding protein of caveolae, which has been linked to breast tumor aggressiveness. To determine whether loss of cav-1 results in the abnormal expression of matrix proteins, mammary glands from cav- 1-/- and cav- 1 +/+ mice were investigated for differences in expression of several ECM proteins. In addition, the presence of myofibroblasts, changes in the vessel density, and differences in duct number and size were assessed in the mammary glands of both animal models. Using immunohistochemistry, expression of fibronectin, tenascin-C, collagens and αSMA were significantly increased in the mammary glands of cav-1-/- mice. Second harmonic generation revealed more organized collagen fibers in cav-1 -/- glands and supported immunohistochemical analyses of increased collagen abundance in the glands of cav-1 -/- mice. Analysis of the ductal structure demonstrated a significant increase in the number of proliferating ducts in addition to significant increases in the duct circumference and area in cav-1 -/- glands compared to cav- 1 +/+ glands. Differences in microvessel density weren't apparent between the animal models. In summary, we found that the loss of cav-1 resulted in increased ECM and α-SMA protein expression in murine mammary glands. Furthermore, we found that an abnormal ductal architecture accompanied the loss of cav-1. These data support a role for cav-1 in maintaining mammary gland structure.

  15. Caveolin1/protein arginine methyltransferase1/sirtuin1 axis as a potential target against endothelial dysfunction.

    PubMed

    Charles, Soniya; Raj, Vijay; Arokiaraj, Jesu; Mala, Kanchana

    2017-01-23

    Endothelial dysfunction (ED), an established response to cardiovascular risk factors, is characterized by increased levels of soluble molecules secreted by endothelial cells (EC). Evidence suggest that ED is an independent predictor of cardiac events and that it is associated with a deficiency in production or bioavailability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors. ED can be reversed by treating cardiovascular risk factors, hence, beyond ambiguity, ED contributes to initiation and progression of atherosclerotic disease. Majority of cardiovascular risk factors act by a common pathway, oxidative stress (OS), characterized by an imbalance in bioavailability of NO and reactive oxygen species (ROS). Enhanced ROS, through several mechanisms, alters competence of EC that leads to ED, reducing its potential to maintain homeostasis and resulting in development of cardiovascular disease (CVD). Influential mechanisms that have been implicated in the development of ED include (i) presence of elevated levels of NOS inhibitor, asymmetric dimethylarginine (ADMA) due to augmented enzyme activity of protein arginine methyl transferase-1 (PRMT1); (ii) decrease in NO generation by endothelial nitric oxide synthase (eNOS) uncoupling, or by reaction of NO with free radicals and (iii) impaired post translational modification of protein (PTM) such as eNOS, caveolin-1 (cav1) and sirtuin-1 (SIRT1). However, the inter-related mechanisms that concur to developing ED is yet to be understood. The events that possibly overlay include OS-induced sequestration of SIRT1 to caveolae facilitating cav1-SIRT1 association; potential increase in lysine acetylation of enzymes such as eNOS and PRMT1 leading to enhanced ADMA formation; imbalance in acetylation-methylation ratio (AMR); diminished NO generation and ED. Here we review current literature from research showing interdependent association between cav1-PRMT1

  16. Caveolin-1 and prostate cancer progression.

    PubMed

    Freeman, Michael R; Yang, Wei; Di Vizio, Dolores

    2012-01-01

    Caveolin-1 was identified in the 1990s as a marker of aggressive prostate cancer. The caveolin-1 protein localizes to vesicular structures called caveolae and has been shown to bind and regulate many signaling proteins involved in oncogenesis. Caveolin-1 also has lipid binding properties and mediates aspects of cholesterol and fatty acid metabolism and can elicit biological responses in a paracrine manner when secreted. Caveolin-1 is also present in the serum of prostate cancer patients and circulating levels correlate with extent of disease. Current evidence indicates that increased expression of caveolin-1 in prostate adenocarcinoma cells and commensurate downregulation of the protein in prostate stroma, mediate progression to the castration-resistant phase of prostate cancer through diverse pathways. This chapter summarizes the current state of our understanding of the cellular and physiologic mechanisms in which caveolin-1 participates in the evolution of prostate cancer cell phenotypes.

  17. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    SciTech Connect

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing . E-mail: jnzhang@dlmedu.edu.cn

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  18. Up-regulation of endothelial monocyte chemoattractant protein-1 by coplanar PCB77 is caveolin-1-dependent

    SciTech Connect

    Majkova, Zuzana; Smart, Eric; Toborek, Michal; Hennig, Bernhard

    2009-05-15

    Atherosclerosis, the primary cause of heart disease and stroke is initiated in the vascular endothelium, and risk factors for its development include environmental exposure to persistent organic pollutants. Caveolae are membrane microdomains involved in regulation of many signaling pathways, and in particular in endothelial cells. We tested the hypothesis that intact caveolae are required for coplanar PCB77-induced up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of the atherosclerosis development. Atherosclerosis-prone LDL-R{sup -/-} mice (control) or caveolin-1{sup -/-}/LDL-R{sup -/-} mice were treated with PCB77. PCB77 induced aortic mRNA expression and plasma protein levels of MCP-1 in control, but not caveolin-1{sup -/-}/LDL-R{sup -/-} mice. To study the mechanism of this effect, primary endothelial cells were used. PCB77 increased MCP-1 levels in endothelial cells in a time- and concentration-dependent manner. This effect was abolished by caveolin-1 silencing using siRNA. Also, MCP-1 up-regulation by PCB77 was prevented by inhibiting p38 and c-Jun N-terminal kinase (JNK), but not ERK1/2, suggesting regulatory functions via p38 and JNK MAPK pathways. Finally, pre-treatment of endothelial cells with the aryl hydrocarbon receptor (AhR) inhibitor {alpha}-naphthoflavone ({alpha}-NF) partially blocked MCP-1 up-regulation. Thus, our data demonstrate that coplanar PCB77 can induce MCP-1 expression by endothelial cells and that this effect is mediated by AhR, as well as p 38 and JNK MAPK pathways. Intact caveolae are required for these processes both in vivo and in vitro. This further supports a key role for caveolae in vascular inflammation induced by persistent organic pollutants.

  19. Opposing effects of protein kinase Calpha and protein kinase Cepsilon on collagen expression by human lung fibroblasts are mediated via MEK/ERK and caveolin-1 signaling.

    PubMed

    Tourkina, Elena; Gooz, Pal; Pannu, Jaspreet; Bonner, Michael; Scholz, Dimitri; Hacker, Sharon; Silver, Richard M; Trojanowska, Maria; Hoffman, Stanley

    2005-04-08

    The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.

  20. Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1.

    PubMed

    Yu, Cuijuan; Alterman, Michail; Dobrowsky, Rick T

    2005-08-01

    Addition of exogenous ceramide causes a significant displacement of cholesterol in lipid raft model membranes. However, whether ceramide-induced cholesterol displacement is sufficient to alter the protein composition of caveolin-enriched lipid raft membranes is unknown. Therefore, we examined whether increasing endogenous ceramide levels with bacterial sphingomyelinase (bSMase) depleted cholesterol and changed the protein composition of caveolin-enriched membranes (CEMs) isolated from immortalized Schwann cells. bSMase increased ceramide levels severalfold and decreased the cholesterol content of detergent-insoluble CEMs by 25-50% within 2 h. To examine the effect of ceramide on the protein composition of the CEMs, we performed a quantitative proteomic analysis using stable isotope labeling of cells in culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although ceramide rapidly depleted lipid raft cholesterol, the levels of the cholesterol binding protein caveolin-1 (Cav-1) decreased by 25% only after 8 h. Importantly, replenishing the cells with cholesterol rapidly reversed the loss of Cav-1 from the CEMs. Ceramide-induced cholesterol depletion increased the association of 5'-nucleotidase and ATP synthase beta-subunit with the CEMs but had a minimal effect on changing the abundance of other lipid raft proteins, such as flotillin-1 and G-proteins. These results suggest that the ceramide-induced loss of cholesterol from CEMs may contribute to altering the lipid raft proteome.

  1. Expression and potential correlation among Forkhead box protein M1, Caveolin-1 and E-cadherin in colorectal cancer

    PubMed Central

    Zhang, Jing; Zhang, Kundong; Zhou, Lisheng; Wu, Weidong; Jiang, Tao; Cao, Jun; Huang, Kejian; Qiu, Zhengjun; Huang, Chen

    2016-01-01

    The aim of the present study was to investigate the expression and functions of Forkhead box protein M1 (FoxM1), Caveolin-1 (Cav-1) and E-cadherin in colorectal cancer (CRC), and to determine the correlations among these proteins in CRC development and progression. The protein expression of FoxM1, Cav-1 and E-cadherin was identified using a human CRC and normal tissue microarray. A standard immunohistochemistry assay was performed employing anti-FoxM1, anti-Cav-1 and anti-E-cadherin antibodies. The clinicopathological significance of FoxM1, Cav-1 and E-cadherin in CRC was determined, and correlations were investigated between FoxM1 and Cav-1, FoxM1 and E-cadherin, Cav-1 and E-cadherin, respectively. The level of FoxM1, Cav-1 and E-Cadherin protein expression in CRC was found to be associated with pathological grade, tumor clinical stages and the presence of metastasis, respectively. Elevated expression of FoxM1 and Cav-1 was observed in the CRC tissues, and a significant correlation was found between the two proteins in CRC. However, it was also observed that FoxM1 was overexpressed while E-cadherin expression was low, indicating that there was a negative correlation between FoxM1 expression and E-cadherin expression. Moreover, there was also a negative correlation between Cav-1 and E-cadherin expression. Overall, the elevated expression of FoxM1 and Cav-1 in a human CRC microarray provided novel clinical evidence to elucidate the fact that they may play a critical role in the development and progression of CRC by negatively regulating E-cadherin expression. Furthermore, the positive correlation between FoxM1 and Cav-1 suggested that the proteins may constitute a novel signaling pathway in human CRC. PMID:27698803

  2. Caveolin-1 - A Novel Interacting Partner of Organic Cation/Carnitine Transporter (Octn2): Effect of Protein Kinase C on This Interaction in Rat Astrocytes

    PubMed Central

    Czeredys, Magdalena; Samluk, Łukasz; Michalec, Katarzyna; Tułodziecka, Karolina; Skowronek, Krzysztof; Nałęcz, Katarzyna A.

    2013-01-01

    OCTN2 - the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine - a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes - cells in which β-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14–22 and 447–454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency. PMID:24349196

  3. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality might be due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet characterized non-estrogenic pathway. We report here that SPI-fed rat serum inhibited osteoblastic c...

  4. Caveolin-1-deficient Mice Show Accelerated Mammary Gland Development During Pregnancy, Premature Lactation, and Hyperactivation of the Jak-2/STAT5a Signaling Cascade

    PubMed Central

    Park, David S.; Lee, Hyangkyu; Frank, Philippe G.; Razani, Babak; Nguyen, Andrew V.; Parlow, Albert F.; Russell, Robert G.; Hulit, James; Pestell, Richard G.; Lisanti, Michael P.

    2002-01-01

    It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (−/−) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (−/−) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling. PMID:12388746

  5. Bile acids down-regulate caveolin-1 in esophageal epithelial cells through sterol responsive element-binding protein.

    PubMed

    Prade, Elke; Tobiasch, Moritz; Hitkova, Ivana; Schäffer, Isabell; Lian, Fan; Xing, Xiangbin; Tänzer, Marc; Rauser, Sandra; Walch, Axel; Feith, Marcus; Post, Stefan; Röcken, Christoph; Schmid, Roland M; Ebert, Matthias P A; Burgermeister, Elke

    2012-05-01

    Bile acids are synthesized from cholesterol and are major risk factors for Barrett adenocarcinoma (BAC) of the esophagus. Caveolin-1 (Cav1), a scaffold protein of membrane caveolae, is transcriptionally regulated by cholesterol via sterol-responsive element-binding protein-1 (SREBP1). Cav1 protects squamous epithelia by controlling cell growth and stabilizing cell junctions and matrix adhesion. Cav1 is frequently down-regulated in human cancers; however, the molecular mechanisms that lead to this event are unknown. We show that the basal layer of the nonneoplastic human esophageal squamous epithelium expressed Cav1 mainly at intercellular junctions. In contrast, Cav1 was lost in 95% of tissue specimens from BAC patients (n = 100). A strong cytoplasmic expression of Cav1 correlated with poor survival in a small subgroup (n = 5) of BAC patients, and stable expression of an oncogenic Cav1 variant (Cav1-P132L) in the human BAC cell line OE19 promoted proliferation. Cav1 was also detectable in immortalized human squamous epithelial, Barrett esophagus (CPC), and squamous cell carcinoma cells (OE21), but was low in BAC cell lines (OE19, OE33). Mechanistically, bile acids down-regulated Cav1 expression by inhibition of the proteolytic cleavage of 125-kDa pre-SREBP1 from the endoplasmic reticulum/Golgi apparatus and nuclear translocation of active 68-kDa SREBP1. This block in SREBP1's posttranslational processing impaired transcriptional activation of SREBP1 response elements in the proximal human Cav1 promoter. Cav1 was also down-regulated in esophagi from C57BL/6 mice on a diet enriched with 1% (wt/wt) chenodeoxycholic acid. Mice deficient for Cav1 or the nuclear bile acid receptor farnesoid X receptor showed hyperplasia and hyperkeratosis of the basal cell layer of esophageal epithelia, respectively. These data indicate that bile acid-mediated down-regulation of Cav1 marks early changes in the squamous epithelium, which may contribute to onset of Barrett esophagus

  6. ∆Np73beta induces caveolin-1 in human non-small cell lung cancer cell line H1299.

    PubMed

    Caiola, Elisa; Marrazzo, Eleonora; Alesci, Simona; Broggini, Massimo; Marabese, Mirko

    2016-02-01

    Caveolins have recently attracted attention for their possible involvement in signal transduction. Their role in cancer is debated, being reported both a suppressive and oncogenic role in different experimental conditions. Caveolin-1 is regulated by the tumor suppressor p53 which is able to bind its promoter and activate transcription. We had previous evidences indicating that a specific p73 isoform, namely ∆Np73β, when overexpressed in NCI-H1299 induced growth arrest and cell death. By gene expression analysis in cell transiently overexpressed with ∆Np73β, a strong induction of caveolin-1 was found. Caveolin was induced both at mRNA and protein level, and we characterised the promoter sequence of the gene encoding for caveolin-1 and found that the promoter region containing the putative p53 (and hence p73) binding sequence was responsive to ∆Np73β, but not to ∆Np73α and ∆Np73γ which do not induce growth arrest as ∆Np73β does. A reduction in cell adhesion was observed in ∆Np73β overexpressing cells, again supporting a possible role of caveolins in determining these effects. By using specific siRNA directed against human caveolin-1, we could not however antagonize the effects induced by ∆Np73β. Although caveolin-1 represents one of the genes whose expression is strongly activated by ∆Np73β, we could not define a role of caveolin-1 as a mediator of ∆Np73β associated growth arrest. It could well be that the expression of caveolin-1 is able to mediate other activities of ∆Np73β, and studies are in progress to determine whether its expression is mainly associated to metastatic spread.

  7. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1*

    PubMed Central

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-01-01

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1. PMID:26797118

  8. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1.

    PubMed

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-03-18

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1.

  9. DNA methylation profiling identifies PTRF/Cavin-1 as a novel tumor suppressor in Ewing sarcoma when co-expressed with caveolin-1.

    PubMed

    Huertas-Martínez, Juan; Court, Franck; Rello-Varona, Santiago; Herrero-Martín, David; Almacellas-Rabaiget, Olga; Sáinz-Jaspeado, Miguel; Garcia-Monclús, Silvia; Lagares-Tena, Laura; Buj, Raquel; Hontecillas-Prieto, Lourdes; Sastre, Ana; Azorin, Daniel; Sanjuan, Xavier; López-Alemany, Roser; Moran, Sebastian; Roma, Josep; Gallego, Soledad; Mora, Jaume; García Del Muro, Xavier; Giangrande, Paloma H; Peinado, Miquel A; Alonso, Javier; de Alava, Enrique; Monk, Dave; Esteller, Manel; Tirado, Oscar M

    2017-02-01

    Epigenetic modifications have been shown to be important in developmental tumors as Ewing sarcoma. We profiled the DNA methylation status of 15 primary tumors, 7 cell lines, 10 healthy tissues and 4 human mesenchymal stem cells lines samples using the Infinium Human Methylation 450K. Differential methylation analysis between Ewing sarcoma and reference samples revealed 1166 hypermethylated and 864 hypomethylated CpG sites (Bonferroni p < 0.05, δ-β-value with absolute difference of >0.20) corresponding to 392 and 470 genes respectively. Gene Ontology analysis of genes differentially methylated in Ewing sarcoma samples showed a significant enrichment of developmental genes. Membrane and cell signal genes were also enriched, among those, 11 were related to caveola formation. We identified differential hypermethylation of CpGs located in the body and S-Shore of the PTRF gene in Ewing sarcoma that correlated with its repressed transcriptional state. Reintroduction of PTRF/Cavin-1 in Ewing sarcoma cells revealed a role of this protein as a tumor suppressor. Restoration of caveolae in the membrane of Ewing sarcoma cells, by exogenously reintroducing PTRF, disrupts the MDM2/p53 complex, which consequently results in the activation of p53 and the induction of apoptosis.

  10. Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible

    NASA Technical Reports Server (NTRS)

    Parat, M. O.; Fox, P. L.

    2001-01-01

    Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents

  11. Identification of Filamin as a Novel Ligand for Caveolin-1: Evidence for the Organization of Caveolin-1–associated Membrane Domains by the Actin Cytoskeleton

    PubMed Central

    Stahlhut, Martin; van Deurs, Bo

    2000-01-01

    Reports on the ultrastructure of cells as well as biochemical data have, for several years, been indicating a connection between caveolae and the actin cytoskeleton. Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-associated protein caveolin-1. Binding of caveolin-1 to filamin involved the N-terminal region of caveolin-1 and the C terminus of filamin close to the filamin-dimerization domain. In in vitro binding assays, recombinant caveolin-1 bound to both nonmuscle and muscle filamin, indicating that the interaction might not be cell type specific. With the use of confocal microscopy, colocalization of caveolin-1 and filamin was observed in elongated patches at the plasma membrane. Remarkably, when stress fiber formation was induced with Rho-stimulating Escherichia coli cytotoxic necrotizing factor 1, the caveolin-1–positive structures became coaligned with stress fibers, indicating that there was a physical link connecting them. Immunogold double-labeling electron microscopy confirmed that caveolin-1–labeled racemose caveolae clusters were positive for filamin. The actin network, therefore, seems to be directly involved in the spatial organization of caveolin-1–associated membrane domains. PMID:10637311

  12. Regulation of Phagolysosomal Digestion by Caveolin-1 of the Retinal Pigment Epithelium Is Essential for Vision*

    PubMed Central

    Sethna, Saumil; Chamakkala, Tess; Gu, Xiaowu; Thompson, Timothy C.; Cao, Guangwen; Elliott, Michael H.; Finnemann, Silvia C.

    2016-01-01

    Caveolin-1 associates with the endo/lysosomal machinery of cells in culture, suggesting that it functions at these organelles independently of its contribution to cell surface caveolae. Here we explored mice lacking caveolin-1 specifically in the retinal pigment epithelium (RPE). The RPE supports neighboring photoreceptors via diurnal phagocytosis of spent photoreceptor outer segment fragments. Like mice lacking caveolin-1 globally, RPECAV1−/− mice developed a normal RPE and neural retina but showed reduced rod photoreceptor light responses, indicating that lack of caveolin-1 affects photoreceptor function in a non-cell-autonomous manner. RPECAV1−/− RPE in situ showed normal particle engulfment but delayed phagosome clearance and reversed diurnal profiles of levels and activities of lysosomal enzymes. Therefore, eliminating caveolin-1 specifically impairs phagolysosomal degradation by the RPE in vivo. Endogenous caveolin-1 was recruited to maturing phagolysosomes in RPE cells in culture. Consistent with these in vivo data, a moderate increase (to ∼2.5-fold) or decrease (by half) of caveolin-1 protein levels in RPE cells in culture was sufficient to accelerate or impair phagolysosomal digestion, respectively. A mutant form of caveolin-1 that fails to reach the cell surface augmented degradation like wild-type caveolin-1. Acidic lysosomal pH and increased protease activity are essential for digestion. We show that halving caveolin-1 protein levels significantly alkalinized lysosomal pH and decreased lysosomal enzyme activities. Taken together, our results reveal a novel role for intracellular caveolin-1 in modulating phagolysosomal function. Moreover, they show, for the first time, that organellar caveolin-1 significantly affects tissue functionality in vivo. PMID:26814131

  13. Caveolin-1 signaling in lung fibrosis.

    PubMed

    Tourkina, Elena; Hoffman, Stanley

    2012-01-01

    Caveolin-1 is a master regulator of several signaling cascades because it is able to bind to and thereby inhibit members of a variety of kinase families. While associated with caveolae and involved in their generation, caveolin-1 is also present at other sites. A variety of studies have suggested that caveolin-1 may be a useful therapeutic target in fibrotic diseases of the lung and other tissues because in these diseases a low level of caveolin-1 expression is associated with a high level of collagen expression and fibrosis. Reduced caveolin-1 expression is observed not only in the fibroblasts that secrete collagen, but also in epithelial cells and monocytes. This is intriguing because both epithelial cells and monocytes have been suggested to be precursors of fibroblasts. Likely downstream effects of loss of caveolin-1 in fibrosis include activation of TGF-β signaling and upregulation of CXCR4 in monocytes resulting in their enhanced migration into damaged tissue where its ligand CXCL12 is produced. Finally, it may be possible to target caveolin-1 in fibrotic diseases without the use of gene therapy. A caveolin-1 peptide (caveolin-1 scaffolding domain) has been identified that retains the function of the full-length molecule to inhibit kinases and that can be modified by addition of the Antennapedia internalization sequence to allow it to enter cells both in vitro and in vivo.

  14. Spatiotemporal expression of caveolin-1 and EMMPRIN during mouse tooth development.

    PubMed

    Shi, Lu; Li, Lingyun; Wang, Ding; Li, Shu; Chen, Zhi; An, Zhengwen

    2016-06-01

    Caveolin-1 is a scaffolding protein involved in the formation of cholesterol-rich caveolae lipid rafts within the plasma membrane and is capable of collecting signaling molecules into the caveolae and regulating their activity, including extracellular matrix metalloproteinase inducer (EMMPRIN). However, detailed expression patterns of caveolin-1 and EMMPRIN in the developing dental germ are largely unknown. The present study investigated the expression patterns of caveolin-1 and EMMPRIN in the developing mouse tooth germ by immunohistochemistry and real-time polymerase chain reaction. At the bud stage, caveolin-1 expression was initiated in the epithelium bud and mesenchymal cells, while EMMPRIN was weakly expressed at this stage. At the cap stage, caveolin-1 protein was located in the lingual part of the tooth germ; however, EMMPRIN protein was located in the labial part. From the bell stage to 2 days postnatal, caveolin-1 expression was detected in the ameloblasts and cervical loop area; with EMMPRIN expression in the ameloblasts and odontoblasts. Real-time polymerase chain reaction results showed that both caveolin-1 and EMMPRIN mRNA levels increased gradually with progression of developmental stages, and peaked at day two postnatal. The current finding suggests that both caveolin-1 and EMMPRIN take part in mouse tooth development, especially in the differentiation and organization of odontogenic tissues.

  15. A role for caveolin-1 in post-injury reactive neuronal plasticity.

    PubMed

    Gaudreault, Sophie B; Blain, Jean-François; Gratton, Jean-Philippe; Poirier, Judes

    2005-02-01

    Remodeling and plasticity in the adult brain require cholesterol redistribution and synthesis for the formation of new membrane components. Caveolin-1 is a cholesterol-binding membrane protein involved in cellular cholesterol transport and homeostasis. Evidence presented here demonstrates an up-regulation of caveolin-1 in the hippocampus, which was temporally correlated with an increase in synaptophysin during the reinnervation phase in a mouse model of hippocampal deafferentation. Using an in vitro model of neuronal reactive plasticity, we examined the effect of virally mediated overexpression of caveolin-1 on injured differentiated PC12 cells undergoing terminal remodeling. Three days post lesion, caveolin-1-overexpressing cells revealed increases in synaptophysin and GAP-43, two markers of neurite sprouting and synaptogenesis. Morphologically, caveolin-1-overexpressing cells showed a decrease in primary neurite outgrowth and branching as well as an increase in neurite density. Caveolin-1-overexpressing cells also revealed the presence of terminal swelling and beading along processes, consistent with a possible alteration of microtubules stability. Moreover, a focal enrichment of caveolin-1 immunofluorescence was observed at the bases of axonal and dendritic terminals of mouse primary hippocampal neurons. Altogether, these results indicate that caveolin-1 plays an active role in the regulation of injury-induced synaptic and terminal remodeling in the adult CNS.

  16. Caveolin-1 regulates contractility in differentiated vascular smooth muscle.

    PubMed

    Je, Hyun-Dong; Gallant, Cynthia; Leavis, Paul C; Morgan, Kathleen G

    2004-01-01

    Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.

  17. Growth suppression of MCF-7 cancer cell-derived xenografts in nude mice by caveolin-1

    SciTech Connect

    Wu Ping; Wang Xiaohui; Li Fei; Qi Baoju; Zhu Hua; Liu Shuang; Cui Yeqing; Chen Jianwen

    2008-11-07

    Caveolin-1 is an essential structural constituent of caveolae membrane domains that has been implicated in mitogenic signaling and oncogenesis. However, the exact functional role of caveolin-1 still remains controversial. In this report, utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/cav-1 cells), we demonstrate that caveolin-1 expression dramatically inhibits invasion and migration of these cells. Importantly, in vivo experiments employing xenograft tumor models demonstrated that expression of caveolin-1 results in significant growth inhibition of breast tumors. Moreover, a dramatic delay in tumor progression was observed in MCF-7/cav-1 cells as compared with MCF-7 cells. Histological analysis of tumor sections demonstrated a marked decrease in the percentage of proliferating tumor cells (Ki-67 assay) along with an increase in apoptotic tumor cells (TUNEL assay) in MCF-7/cav-1-treated animals. Our current findings provide for the first time in vivo evidence that caveolin-1 can indeed function as a tumor suppressor in human breast adenocarcinoma derived from MCF-7 cells rather than as a tumor promoter.

  18. Caveolin-1 controls mitochondrial function through regulation of m-AAA mitochondrial protease

    PubMed Central

    Volonte, Daniela; Liu, Zhongmin; Shiva, Sruti; Galbiati, Ferruccio

    2016-01-01

    Mitochondrial proteases ensure mitochondrial integrity and function after oxidative stress by providing mitochondrial protein quality control. However, the molecular mechanisms that regulate this basic biological function in eukaryotic cells remain largely unknown. Caveolin-1 is a scaffolding protein involved in signal transduction. We find that AFG3L2, a m-AAA type of mitochondrial protease, is a novel caveolin-1-interacting protein in vitro. We show that oxidative stress promotes the translocation of both caveolin-1 and AFG3L2 to mitochondria, enhances the interaction of caveolin-1 with AFG3L2 in mitochondria and stimulates mitochondrial protease activity in wild-type fibroblasts. Localization of AFG3L2 to mitochondria after oxidative stress is inhibited in fibroblasts lacking caveolin-1, which results in impaired mitochondrial protein quality control, an oxidative phosphorylation to aerobic glycolysis switch and reduced ATP production. Mechanistically, we demonstrate that a lack of caveolin-1 does not alter either mitochondrial number or morphology but leads to the cytoplasmic and proteasome-dependent degradation of complexes I, III, IV and V upon oxidant stimulation. Restoration of mitochondrial respiratory chain complexes in caveolin-1 null fibroblasts reverts the enhanced glycolysis observed in these cells. Expression of a mutant form of AFG3L2, which has reduced affinity for caveolin-1, fails to localize to mitochondria and promotes degradation of complex IV after oxidative stress. Thus, caveolin-1 maintains mitochondrial integrity and function when cells are challenged with free radicals by promoting the mitochondrial localization of m-AAA protease and its quality control functions. PMID:27705926

  19. Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

    NASA Technical Reports Server (NTRS)

    Parat, Marie-Odile; Stachowicz, Rafal Z.; Fox, Paul L.

    2002-01-01

    During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

  20. Downregulation of caveolin-1 contributes to the synaptic plasticity deficit in the hippocampus of aged rats

    PubMed Central

    Liu, Yang; Liang, Zhanhua; Liu, Jing; Zou, Wei; Li, Xiaoyan; Wang, Yachen; An, Lijia

    2013-01-01

    Caveolin-1 is involved in the regulation of synaptic plasticity, but the relationship between its pression and cognitive function during aging remains controversial. To explore the relationship be-tween synaptic plasticity in the aging process and changes in learning and memory, we examined caveolin-1 expression in the hippocampus, cortex and cerebellum of rats at different ages. We also examined the relationship between the expression of caveolin-1 and synaptophysin, a marker of synaptic plasticity. Hippocampal caveolin-1 and synaptophysin expression in aged (22–24 month old) rats was significantly lower than that in young (1 month old) and adult (4 months old) rats. pression levels of both proteins were significantly greater in the cortex of aged rats than in that of young or adult rats, and levels were similar between the three age groups in the cerebellum. Linear regression analysis revealed that hippocampal expression of synaptophysin was associated with memory and learning abilities. Moreover, synaptophysin expression correlated positively with caveolin-1 expression in the hippocampus, cortex and cerebellum. These results confirm that caveolin-1 has a regulatory effect on synaptic plasticity, and suggest that the downregulation of hippocampal caveolin-1 expression causes a decrease in synaptic plasticity during physiological aging. PMID:25206583

  1. The less-often-traveled surface of stem cells: caveolin-1 and caveolae in stem cells, tissue repair and regeneration

    PubMed Central

    2013-01-01

    Stem cells are an important resource for tissue repair and regeneration. While a great deal of attention has focused on derivation and molecular regulation of stem cells, relatively little research has focused on how the subcellular structure and composition of the cell membrane influences stem cell activities such as proliferation, differentiation and homing. Caveolae are specialized membrane lipid rafts coated with caveolin scaffolding proteins, which can regulate cholesterol transport and the activity of cell signaling receptors and their downstream effectors. Caveolin-1 is involved in the regulation of many cellular processes, including growth, control of mitochondrial antioxidant levels, migration and senescence. These activities are of relevance to stem cell biology, and in this review evidence for caveolin-1 involvement in stem cell biology is summarized. Altered stem and progenitor cell populations in caveolin-1 null mice suggest that caveolin-1 can regulate stem cell proliferation, and in vitro studies with isolated stem cells suggest that caveolin-1 regulates stem cell differentiation. The available evidence leads us to hypothesize that caveolin-1 expression may stabilize the differentiated and undifferentiated stem cell phenotype, and transient downregulation of caveolin-1 expression may be required for transition between the two. Such regulation would probably be critical in regenerative applications of adult stem cells and during tissue regeneration. We also review here the temporal changes in caveolin-1 expression reported during tissue repair. Delayed muscle regeneration in transgenic mice overexpressing caveolin-1 as well as compromised cardiac, brain and liver tissue repair and delayed wound healing in caveolin-1 null mice suggest that caveolin-1 plays an important role in tissue repair, but that this role may be negative or positive depending on the tissue type and the nature of the repair process. Finally, we also discuss how caveolin-1

  2. Versatile Functions of Caveolin-1 in Aging-related Diseases

    PubMed Central

    Nguyen, Kim Cuc Thi

    2017-01-01

    Caveolin-1 (Cav-1) is a trans-membrane protein that is a major component of the caveolae structure on the plasma membrane. Cav-1 is involved in the regulation of various cellular processes, including cell growth, differentiation, endocytosis, and in particular it has been implied in cellular senescence. Here we review current knowledge about Cav-1 in cellular signaling and discuss the role of Cav-1 in aging-related diseases. PMID:28184336

  3. Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance.

    PubMed

    Thomas, Nicholas B P; Hutcheson, Iain R; Campbell, Lee; Gee, Julia; Taylor, Kathryn M; Nicholson, Robert I; Gumbleton, Mark

    2010-02-01

    Caveolin-1 displays both tumour-suppressor and tumour-promoter properties in breast cancer. Using characterised preclinical cell models for the transition of oestrogen-sensitive (WT-MCF-7 cells) to a tamoxifen-resistant (TAM-R cells) phenotype we examined the role caveolin-1 in the development of hormone-resistant breast cancer. The WT-MCF-7 cells showed abundant expression of caveolin-1 which potentiated oestrogen-receptor (ERalpha) signalling and promoted cell growth despite caveolin-1 mediating inhibition of ERK signalling. In TAM-R cells caveolin-1 expression was negligible, repressed by EGF-R/ERK signalling. Pharmacological inhibition of EGFR/ERK in TAM-R cells restored caveolin-1 and also resulted in the emergence of pools of phosphorylated caveolin-1. WT-MCF-7 cells exposed to tamoxifen for upto 12 weeks displayed increased caveolin-1 (peaking by week 2) followed (after week 8) by a marked decrease as the cells progress to develop a stable tamoxifen-resistant phenotype. The targeted down-regulation (siRNA) of caveolin-1 in WT-MCF-7 cells reduced growth but did not affect their sensitivity to tamoxifen, suggesting loss of caveolin-1 alone is not sufficient to confer tamoxifen-resistance. Hyperactivation of EGFR/ERK is a feature of tamoxifen-resistant breast cancer cells, a principal driver of cell growth. Recombinant expression of caveolin-1 in TAM-R cells did not affect EGFR/ERK activity, potentially due to mislocalisation of caveolin-1 through hyperactivation of the mTOR pathway or altered caveolin-1 phosphorylation. This work defines a novel role for caveolin-1 with implications for the clinical course of breast cancer and identifies caveolin-1 as a potential drug target for the treatment of early oestrogen-dependent breast cancers. Further, the loss of caveolin-1 may have benefit as a molecular signature for tamoxifen resistance.

  4. Altered Arachidonate Distribution in Macrophages from Caveolin-1 Null Mice Leading to Reduced Eicosanoid Synthesis*

    PubMed Central

    Astudillo, Alma M.; Pérez-Chacón, Gema; Meana, Clara; Balgoma, David; Pol, Albert; del Pozo, Miguel A.; Balboa, María A.; Balsinde, Jesús

    2011-01-01

    In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E2 and LTB4 production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response. PMID:21852231

  5. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

    NASA Technical Reports Server (NTRS)

    Park, H.; Go, Y. M.; Darji, R.; Choi, J. W.; Lisanti, M. P.; Maland, M. C.; Jo, H.

    2000-01-01

    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

  6. Expression of caveolin-1 in the early phase of beta-TCP implanted in dog mandible.

    PubMed

    Chou, Cherng-Tzeh; Bhawal, Ujjal K; Watanabe, Nobuyuki; Kuboyama, Noboru; Chang, Wei-Jen; Lee, Sheng-Yang; Abiko, Yoshimitsu

    2013-07-01

    Caveolin is an essential and signature protein of caveolae. Caveolin-1 participates in signal transduction processes by acting as a scaffolding protein that concentrates, organizes and functional regulates signalling molecules within caveolar membranes. Beta-tricalcium phosphate (β-TCP) has been widely used for scaffold in tissue engineering due to its high biodegradability, osteoconductivity, easy manipulation, and lack of histotoxicity. To better understand the role of caveolin-1 in bone homeostasis and response to β-TCP scaffold, β-TCP was implanted into the dog mandible defects in beagle dogs, and gene expression profiles were examined focused on the molecular components involved in caveolin-1 regulation. Here we showed the quantitative imageology analysis characterized using in vivo micro-computed tomography (CT) images at 4 and 7 days after β-TCP implanted in dog mandibles. The bone reformation by using the β-TCP scaffolds began within 4 days of surgery, and was healing well at 7 days after surgery. Higher mRNA level of caveolin-1 was observed in β-TCP-implanted Beagle dog mandibles compared with controls at day 4 and day 7 post-surgery. The enhancement of caveolin-1 by β-TCP was further confirmed by immunohistochemistry and immunofluorescence analysis. We further revealed increased Smad7 and Phospho Stat3 expression in β-TCP-implanted specimens. Taken together, these results suggest that the enhancement of caveolin-1 play an important role in accelerating bone formation by β-TCP.

  7. Effect of alteration of caveolin-1 expression on doxorubicin-induced apoptosis in H9c2 cardiac cells.

    PubMed

    Takaguri, Akira; Kamato, Maiko; Satoh, Yoshiaki; Ohtsuki, Kazuaki; Satoh, Kumi

    2015-09-01

    Doxorubicin is an anthracycline antibiotic widely used in cancer treatment. Although its antitumor efficacy appears to be dose dependent, its clinical use is greatly restricted by the development of cardiotoxicity associated with apoptosis. Although caveolin-1, the major structural protein in caveolae, can positively or negatively regulate apoptosis depending on the stimulus or cell types, the contribution of caveolin-1 to doxorubicin-induced apoptosis remains unknown. This study was performed to identify the regulatory role of caveolin-1 on doxorubicin-induced apoptosis in H9c2 cardiac cells using a genetic approach. Caveolin-1 knockdown with a short hairpin (sh) RNA adenovirus, but not overexpression of wild-type caveolin-1, resulted in a marked inhibition of doxorubicin-induced caspase-3 cleavage. However, caveolin-1 knockdown tended to protect against doxorubicin-induced decrease in cell viability, but it did not significantly reverse cell death induced by doxorubicin. Doxorubicin stimulated the phosphorylation of p38 and extracellular signal regulated kinase (ERK). Doxorubicin-induced caspase-3 cleavage was inhibited by U0126, a MEK inhibitor or SB203580, a p38 inhibitor. Caveolin-1 knockdown markedly inhibited doxorubicin-induced p-38 phosphorylation but not ERK-mediated p-53 phosphorylation in H9c2 cardiac cells. Our results suggest that reduced caveolin-1 expression plays an anti-apoptotic role in doxorubicin-induced apoptosis but that it is insufficient to prevent such an apoptosis in H9c2 cardiac cells.

  8. Status of caveolin-1 in various membrane domains of the bovine lens.

    PubMed

    Cenedella, Richard J; Sexton, Patricia S; Brako, Lawrence; Lo, Woo-Kuen; Jacob, Robert F

    2007-10-01

    Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25-30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells.

  9. Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells

    SciTech Connect

    Shatz, Maria; Lustig, Gila; Reich, Reuven; Liscovitch, Mordechai

    2010-06-10

    Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells. Caveolin-1 knockdown inhibited cell proliferation and reduced focal adhesion kinase (Fak) phosphorylation. Matrix invasion and cell migration as well as expression and activity of matrix metalloproteases were attenuated following caveolin-1 RNAi-mediated knockdown or overexpression of Y14F and P132L mutants, demonstrating dominant-negative activity of these mutants. Time-lapse fluorescence microscopy revealed that caveolin-1 and its mutants P132L and Y14F are localized to the trailing edge of migrating cells during both random and directed cell movement, implying an active role of caveolin-1 in the migration process. Suppression of caveolin-1 function greatly elevated the percentage of H1299 cells exhibiting focal adhesions. In addition, cell aggregation was increased by wild type caveolin-1 and attenuated by both P132L and Y14F mutants. Overexpression of wild type caveolin-1 increased caveolae density, however, P132L and Y14F mutants did not affect caveolae formation, suggesting that in this respect that the mutants do not act in a dominant negative manner, and that effects of caveolin-1 on caveolae and cell invasion, migration, focal adhesion and aggregation, are separable. Our data provide novel mechanistic insights into the role of caveolin-1 in cell motility, invasiveness and aggregation, therefore, expanding our understanding of the tumor-promoting activities of caveolin-1 in advanced-stage cancer.

  10. Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells.

    PubMed

    Shatz, Maria; Lustig, Gila; Reich, Reuven; Liscovitch, Mordechai

    2010-06-10

    Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells. Caveolin-1 knockdown inhibited cell proliferation and reduced focal adhesion kinase (Fak) phosphorylation. Matrix invasion and cell migration as well as expression and activity of matrix metalloproteases were attenuated following caveolin-1 RNAi-mediated knockdown or overexpression of Y14F and P132L mutants, demonstrating dominant-negative activity of these mutants. Time-lapse fluorescence microscopy revealed that caveolin-1 and its mutants P132L and Y14F are localized to the trailing edge of migrating cells during both random and directed cell movement, implying an active role of caveolin-1 in the migration process. Suppression of caveolin-1 function greatly elevated the percentage of H1299 cells exhibiting focal adhesions. In addition, cell aggregation was increased by wild type caveolin-1 and attenuated by both P132L and Y14F mutants. Overexpression of wild type caveolin-1 increased caveolae density, however, P132L and Y14F mutants did not affect caveolae formation, suggesting that in this respect that the mutants do not act in a dominant negative manner, and that effects of caveolin-1 on caveolae and cell invasion, migration, focal adhesion and aggregation, are separable. Our data provide novel mechanistic insights into the role of caveolin-1 in cell motility, invasiveness and aggregation, therefore, expanding our understanding of the tumor-promoting activities of caveolin-1 in advanced-stage cancer.

  11. Oxidative stress-induced inhibition of Sirt1 by caveolin-1 promotes p53-dependent premature senescence and stimulates the secretion of interleukin 6 (IL-6).

    PubMed

    Volonte, Daniela; Zou, Huafei; Bartholomew, Janine N; Liu, Zhongmin; Morel, Penelope A; Galbiati, Ferruccio

    2015-02-13

    Oxidative stress can induce premature cellular senescence. Senescent cells secrete various growth factors and cytokines, such as IL-6, that can signal to the tumor microenvironment and promote cancer cell growth. Sirtuin 1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including senescence. We found that caveolin-1, a structural protein component of caveolar membranes, is a direct binding partner of Sirt1, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain of Sirt1 (amino acids 310-317). Our data show that oxidative stress promotes the sequestration of Sirt1 into caveolar membranes and the interaction of Sirt1 with caveolin-1, which lead to inhibition of Sirt1 activity. Reactive oxygen species stimulation promotes acetylation of p53 and premature senescence in wild-type but not caveolin-1 null mouse embryonic fibroblasts (MEFs). Either down-regulation of Sirt1 expression or re-expression of caveolin-1 in caveolin-1 null MEFs restores reactive oxygen species-induced acetylation of p53 and premature senescence. In addition, overexpression of caveolin-1 induces stress induced premature senescence in p53 wild-type but not p53 knockout MEFs. Phosphorylation of caveolin-1 on tyrosine 14 promotes the sequestration of Sirt1 into caveolar membranes and activates p53/senescence signaling. We also identified IL-6 as a caveolin-1-specific cytokine that is secreted by senescent fibroblasts following the caveolin-1-mediated inhibition of Sirt1. The caveolin-1-mediated secretion of IL-6 by senescent fibroblasts stimulates the growth of cancer cells. Therefore, by inhibiting Sirt1, caveolin-1 links free radicals to the activation of the p53/senescence pathway and the protumorigenic properties of IL-6.

  12. Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa

    PubMed Central

    Bajmoczi, Milan; Gadjeva, Mihaela; Alper, Seth L.; Pier, Gerald B.; Golan, David E.

    2009-01-01

    Patients with cystic fibrosis (CF) exhibit defective innate immunity and are susceptible to chronic lung infection with Pseudomonas aeruginosa. To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (ΔF508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)-CFTR with an NH2-terminal green fluorescent protein (GFP) tag. GFP-CFTR exhibited posttranslational modification, subcellular localization, and anion transport function typical of WT-CFTR. P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. P. aeruginosa internalization also required free lateral diffusion of GFP-CFTR, allowing for bacterial coclustering with GFP-CFTR and caveolin-1 at the plasma membrane. Thus efficient initiation of innate immunity to P. aeruginosa requires formation of an epithelial “internalization platform” involving both caveolin-1 and functional, laterally mobile CFTR. PMID:19386787

  13. Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa.

    PubMed

    Bajmoczi, Milan; Gadjeva, Mihaela; Alper, Seth L; Pier, Gerald B; Golan, David E

    2009-08-01

    Patients with cystic fibrosis (CF) exhibit defective innate immunity and are susceptible to chronic lung infection with Pseudomonas aeruginosa. To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (DeltaF508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)-CFTR with an NH(2)-terminal green fluorescent protein (GFP) tag. GFP-CFTR exhibited posttranslational modification, subcellular localization, and anion transport function typical of WT-CFTR. P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. P. aeruginosa internalization also required free lateral diffusion of GFP-CFTR, allowing for bacterial coclustering with GFP-CFTR and caveolin-1 at the plasma membrane. Thus efficient initiation of innate immunity to P. aeruginosa requires formation of an epithelial "internalization platform" involving both caveolin-1 and functional, laterally mobile CFTR.

  14. Rosiglitazone ameliorates diffuse axonal injury by reducing loss of tau and up-regulating caveolin-1 expression

    PubMed Central

    Zhao, Yong-lin; Song, Jin-ning; Ma, Xu-dong; Zhang, Bin-fei; Li, Dan-dong; Pang, Hong-gang

    2016-01-01

    Rosiglitazone up-regulates caveolin-1 levels and has neuroprotective effects in both chronic and acute brain injury. Therefore, we postulated that rosiglitazone may ameliorate diffuse axonal injury via its ability to up-regulate caveolin-1, inhibit expression of amyloid-beta precursor protein, and reduce the loss and abnormal phosphorylation of tau. In the present study, intraperitoneal injection of rosiglitazone significantly reduced the levels of amyloid-beta precursor protein and hyperphosphorylated tau (phosphorylated at Ser404(p-tau (S404)), and it increased the expression of total tau and caveolin-1 in the rat cortex. Our results show that rosiglitazone inhibits the expression of amyloid-beta precursor protein and lowers p-tau (S404) levels, and it reduces the loss of total tau, possibly by up-regulating caveolin-1. These actions of rosiglitazone may underlie its neuroprotective effects in the treatment of diffuse axonal injury. PMID:27482223

  15. Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1.

    PubMed

    Bhattacharya, Sayak; Mahavadi, Sunila; Al-Shboul, Othman; Rajagopal, Senthilkumar; Grider, John R; Murthy, Karnam S

    2013-08-01

    Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

  16. The different functions and clinical significances of caveolin-1 in human adenocarcinoma and squamous cell carcinoma

    PubMed Central

    Fu, Pin; Chen, Fuchun; Pan, Qi; Zhao, Xianda; Zhao, Chen; Cho, William Chi-Shing; Chen, Honglei

    2017-01-01

    Caveolin-1 (Cav-1), a major structural protein of caveolae, is an integral membrane protein which plays an important role in the progression of carcinoma. However, whether Cav-1 acts as a tumor promoter or a tumor suppressor still remains controversial. For example, the tumor-promoting function of Cav-1 has been found in renal cancer, prostate cancer, tongue squamous cell carcinoma (SCC), lung SCC and bladder SCC. In contrast, Cav-1 also plays an inhibitory role in esophagus adenocarcinoma, lung adenocarcinoma and cutaneous SCC. The role of Cav-1 is still controversial in thyroid cancer, hepatocellular carcinoma, gastric adenocarcinoma, colon adenocarcinoma, breast cancer, pancreas cancer, oral SCC, laryngeal SCC, head and neck SCC, esophageal SCC and cervical SCC. Besides, it has been reported that the loss of stromal Cav-1 might predict poor prognosis in breast cancer, gastric cancer, pancreas cancer, prostate cancer, oral SCC and esophageal SCC. However, the accumulation of stromal Cav-1 has been found to be promoted by the progression of tongue SCC. Taken together, Cav-1 seems playing a different role in different cancer subtypes even of the same organ, as well as acting differently in the same cancer subtype of different organs. Thus, we hereby explore the functions of Cav-1 in human adenocarcinoma and SCC from the perspective of clinical significances and pathogenesis. We envision that novel targets may come with the further investigation of Cav-1 in carcinogenesis. PMID:28243118

  17. Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

    PubMed

    Abousaab, Abeer; Warsi, Jamshed; Elvira, Bernat; Lang, Florian

    2016-06-01

    Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5 µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.

  18. Peroxisome Proliferator–Activated Receptor γ–Mediated Inhibition on Hypoxia-Triggered Store-Operated Calcium Entry. A Caveolin-1–Dependent Mechanism

    PubMed Central

    Yang, Kai; Lu, Wenju; Jiang, Qian; Yun, Xin; Zhao, Mingming; Jiang, Haiyang

    2015-01-01

    Our previous publication demonstrated that peroxisome proliferator–activated receptor γ (PPARγ) inhibits the pathogenesis of chronic hypoxia (CH)–induced pulmonary hypertension by targeting store-operated calcium entry (SOCE) in rat distal pulmonary arterial smooth muscle cells (PASMCs). In this study, we aim to determine the role of a membrane scaffolding protein, caveolin-1, during the suppressive process of PPARγ on SOCE. Adult (6–8 weeks) male Wistar rats (200–250 g) were exposed to CH (10% O2) for 21 days to establish CH-induced pulmonary hypertension. Primary cultured rat distal PASMCs were applied for the molecular biological experiments. First, hypoxic exposure led to 2.5-fold and 1-fold increases of caveolin-1 protein expression in the distal pulmonary arteries and PASMCs, respectively. Second, effective knockdown of caveolin-1 significantly reduced hypoxia-induced SOCE for 58.2% and 41.5%, measured by Mn2+ quenching and extracellular Ca2+ restoration experiments, respectively. These results suggested that caveolin-1 acts as a crucial regulator of SOCE, and hypoxia–up-regulated caveolin-1 largely accounts for hypoxia-elevated SOCE in PASMCs. Then, by using a high-potency PPARγ agonist, GW1929, we detected that PPARγ activation inhibited SOCE and caveolin-1 protein for 62.5% and 59.8% under hypoxia, respectively, suggesting that caveolin-1 also acts as a key target during the suppressive process of PPARγ on SOCE in PASMCs. Moreover, by using effective small interfering RNAs against PPARγ and caveolin-1, and PPARγ antagonist, T0070907, we observed that PPARγ plays an inhibitory role on caveolin-1 protein by promoting its lysosomal degradation, without affecting the messenger RNA level. PPARγ inhibits SOCE, at least partially, by suppressing cellular caveolin-1 protein in PASMCs. PMID:26020612

  19. Peroxisome Proliferator-Activated Receptor γ-Mediated Inhibition on Hypoxia-Triggered Store-Operated Calcium Entry. A Caveolin-1-Dependent Mechanism.

    PubMed

    Yang, Kai; Lu, Wenju; Jiang, Qian; Yun, Xin; Zhao, Mingming; Jiang, Haiyang; Wang, Jian

    2015-12-01

    Our previous publication demonstrated that peroxisome proliferator-activated receptor γ (PPARγ) inhibits the pathogenesis of chronic hypoxia (CH)-induced pulmonary hypertension by targeting store-operated calcium entry (SOCE) in rat distal pulmonary arterial smooth muscle cells (PASMCs). In this study, we aim to determine the role of a membrane scaffolding protein, caveolin-1, during the suppressive process of PPARγ on SOCE. Adult (6-8 weeks) male Wistar rats (200-250 g) were exposed to CH (10% O2) for 21 days to establish CH-induced pulmonary hypertension. Primary cultured rat distal PASMCs were applied for the molecular biological experiments. First, hypoxic exposure led to 2.5-fold and 1-fold increases of caveolin-1 protein expression in the distal pulmonary arteries and PASMCs, respectively. Second, effective knockdown of caveolin-1 significantly reduced hypoxia-induced SOCE for 58.2% and 41.5%, measured by Mn(2+) quenching and extracellular Ca(2+) restoration experiments, respectively. These results suggested that caveolin-1 acts as a crucial regulator of SOCE, and hypoxia-up-regulated caveolin-1 largely accounts for hypoxia-elevated SOCE in PASMCs. Then, by using a high-potency PPARγ agonist, GW1929, we detected that PPARγ activation inhibited SOCE and caveolin-1 protein for 62.5% and 59.8% under hypoxia, respectively, suggesting that caveolin-1 also acts as a key target during the suppressive process of PPARγ on SOCE in PASMCs. Moreover, by using effective small interfering RNAs against PPARγ and caveolin-1, and PPARγ antagonist, T0070907, we observed that PPARγ plays an inhibitory role on caveolin-1 protein by promoting its lysosomal degradation, without affecting the messenger RNA level. PPARγ inhibits SOCE, at least partially, by suppressing cellular caveolin-1 protein in PASMCs.

  20. Pulmonary hypertension and metabolic syndrome: Possible connection, PPARγ and Caveolin-1.

    PubMed

    Mathew, Rajamma

    2014-08-26

    A number of disparate diseases can lead to pulmonary hypertension (PH), a serious disorder with a high morbidity and mortality rate. Recent studies suggest that the associated metabolic dysregulation may be an important factor adversely impacting the prognosis of PH. Furthermore, metabolic syndrome is associated with vascular diseases including PH. Inflammation plays a significant role both in PH and metabolic syndrome. Adipose tissue modulates lipid and glucose metabolism, and also produces pro- and anti-inflammatory adipokines that modulate vascular function and angiogenesis, suggesting a close functional relationship between the adipose tissue and the vasculature. Both caveolin-1, a cell membrane scaffolding protein and peroxisome proliferator-activated receptor (PPAR) γ, a ligand-activated transcription factor are abundantly expressed in the endothelial cells and adipocytes. Both caveolin-1 and PPARγ modulate proliferative and anti-apoptotic pathways, cell migration, inflammation, vascular homeostasis, and participate in lipid transport, triacylglyceride synthesis and glucose metabolism. Caveolin-1 and PPARγ regulate the production of adipokines and in turn are modulated by them. This review article summarizes the roles and inter-relationships of caveolin-1, PPARγ and adipokines in PH and metabolic syndrome.

  1. Nitroglycerin Tolerance in Caveolin-1 Deficient Mice

    PubMed Central

    Mao, Mao; Varadarajan, Sudhahar; Fukai, Tohru; Bakhshi, Farnaz R.; Chernaya, Olga; Dudley, Samuel C.; Minshall, Richard D.; Bonini, Marcelo G.

    2014-01-01

    Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48–72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation. PMID:25158065

  2. Transcriptional repression of Caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.

    PubMed

    Boopathi, Ettickan; Gomes, Cristiano Mendes; Goldfarb, Robert; John, Mary; Srinivasan, Vittala Gopal; Alanzi, Jaber; Malkowicz, S Bruce; Kathuria, Hasmeena; Zderic, Stephen A; Wein, Alan J; Chacko, Samuel

    2011-05-01

    Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits down-regulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Silencing of GATA6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in bladder smooth muscle cells. Together, these data suggest that GATA-6 acts as a transcriptional repressor of CAV1 gene expression in PBOO-induced BSM hypertrophy in men and mice. GATA-6-induced transcriptional repression represents a new regulatory mechanism of CAV1 gene expression in pathologic BSM, and may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men.

  3. Polychlorinated biphenyl-induced VCAM-1 expression is attenuated in aortic endothelial cells isolated from caveolin-1 deficient mice

    SciTech Connect

    Han, Sung Gu; Eum, Sung Yong; Toborek, Michal; Smart, Eric; Hennig, Bernhard

    2010-07-15

    Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of cardiovascular diseases such as atherosclerosis. Vascular cell adhesion molecule-1 (VCAM-1) is a critical mediator for adhesion and uptake of monocytes across the endothelium in the early stages of atherosclerosis development. The upregulation of VCAM-1 by PCBs may be dependent on functional membrane domains called caveolae. Caveolae are particularly abundant in endothelial cell membranes and involved in trafficking and signal transduction. The objective of this study was to investigate the role of caveolae in PCB-induced endothelial cell dysfunction. Primary mouse aortic endothelial cells (MAECs) isolated from caveolin-1-deficient mice and background C57BL/6 mice were treated with coplanar PCBs, such as PCB77 and PCB126. In addition, siRNA gene silencing technique was used to knockdown caveolin-1 in porcine vascular endothelial cells. In MAECs with functional caveolae, VCAM-1 protein levels were increased after exposure to both coplanar PCBs, whereas expression levels of VCAM-1 were not significantly altered in cells deficient of caveolin-1. Furthermore, PCB-induced monocyte adhesion was attenuated in caveolin-1-deficient MAECs. Similarly, siRNA silencing of caveolin-1 in porcine endothelial cells confirmed the caveolin-1-dependent VCAM-1 expression. Treatment of cells with PCB77 and PCB126 resulted in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), and pharmacological inhibition of ERK1/2 diminished the observed PCB-induced increase in monocyte adhesion. These findings suggest that coplanar PCBs induce adhesion molecule expression, such as VCAM-1, in endothelial cells, and that this response is regulated by caveolin-1 and functional caveolae. Our data demonstrate a critical role of functional caveolae in the activation and dysfunction of endothelial cells by coplanar PCBs.

  4. Caveolin-1 Expression and Membrane Cholesterol Content Modulate N-Type Calcium Channel Activity in NG108-15 Cells

    PubMed Central

    Toselli, M.; Biella, G.; Taglietti, V.; Cazzaniga, E.; Parenti, M.

    2005-01-01

    Caveolins are the main structural proteins of glycolipid/cholesterol-rich plasmalemmal invaginations, termed caveolae. In addition, caveolin-1 isoform takes part in membrane remodelling as it binds and transports newly synthesized cholesterol from endoplasmic reticulum to the plasma membrane. Caveolin-1 is expressed in many cell types, including hippocampal neurons, where an abundant SNAP25-caveolin-1 complex is detected after induction of persistent synaptic potentiation. To ascertain whether caveolin-1 influences neuronal voltage-gated Ca2+ channel basal activity, we stably expressed caveolin-1 into transfected neuroblastoma × glioma NG108-15 hybrid cells [cav1(+) clone] that lack endogenous caveolins but express N-type Ca2+ channels upon cAMP-induced neuronal differentiation. Whole-cell patch-clamp recordings of cav1(+) cells demonstrated that N-type current density was reduced in size by ∼70% without any significant change in the time course of activation and inactivation and voltage dependence. Moreover, the cav1(+) clone exhibited a significantly increased proportion of membrane cholesterol compared to wild-type NG108-15 cells. To gain insight into the mechanism underlying caveolin-1 lowering of N-current density, and more precisely to test whether this was indirectly caused by caveolin-1-induced enhancement of membrane cholesterol, we compared single N-type channel activities in cav1(+) clone and wild-type NG108-15 cells enriched with cholesterol after exposure to a methyl-β-cyclodextrin-cholesterol complex. A lower Ca2+ channel activity was recorded from cell-attached patches of both cell types, thus supporting the view that the increased proportion of membrane cholesterol is ultimately responsible for the effect. This is due to a reduction in the probability of channel opening caused by a significant decrease of channel mean open time and by an increase of the frequency of null sweeps. PMID:16040758

  5. Increased caveolin-1 expression in Alzheimer's disease brain.

    PubMed

    Gaudreault, Sophie B; Dea, Doris; Poirier, Judes

    2004-07-01

    Increasing evidence suggests that cholesterol plays a central role in the pathophysiology of Alzheimer's disease (AD). Caveolin is a cholesterol-binding membrane protein involved in cellular cholesterol transport. We investigated the changes in the protein amount of hippocampal caveolin of autopsy-confirmed AD and aged-matched control subjects. Our results demonstrate that caveolin protein levels in the hippocampus and caveolin mRNA in the frontal cortex are up-regulated in AD by approximately two-fold, compared to control brains. These results suggest a relationship between caveolin-1 expression levels and a dysregulation of cholesterol homeostasis at the plasma membrane of brain cells. In support of this hypothesis, a significant increase in caveolin protein levels has also been observed in hippocampal tissue from ApoE-deficient (knockout) and aged wild-type mice; two situations associated with modifications of transbilayer distribution of cholesterol in brain synaptic plasma membranes. These results indicate that caveolin over-expression is linked to alterations of cholesterol distribution in the plasma membrane of brain cells and are consistent with the notion of a deterioration of cholesterol homeostasis in AD.

  6. Expression of Stromal Caveolin- 1 May Be a Predictor for Aggressive Behaviour of Breast Cancer.

    PubMed

    Eliyatkin, Nuket; Aktas, Safiye; Diniz, Gulden; Ozgur, Halil Hakan; Ekin, Zubeyde Yildirim; Kupelioglu, Ali

    2017-02-24

    Caveolin-1 (Cav-1) is well known as a principal scaffolding protein of caveolae which are specialized plasma membrane structures. The role of Cav-1 in tumorigenesis of breast cancers is relatively less studied. The aim of the present study is to describe the biological roles of Cav-1 in breast cancers considering its contrasting dual functions as an oncogene and as a tumor suppressor. This study included 71 females with breast cancer who had been histopathologically diagnosed in Private Gunes Pathology Laboratory between the years 2007, and 2012. The mean age is 52.48 ± 12.8 years. Patients were followed up for a mean period of 47.97 ± 20.48 months. We didn't determine Cav-1 positive tumor cells. In 36 cases (50.7%), there were stromal expressions of Cav-1. In the statistical analysis, there was a statistically significant correlation between Cav-1 expression and ER (p = 0.033), metastasis (p = 0.005), lymphatic invasion (p = 0.000), nodal metastasis (p = 0,003), perinodal invasion (p = 0.003), metastasis (p = 0.005) and survival (p = 0.009). We found that Cav-1 expression is associated with tumor size, histological grade, lymph node involvement. Accordingly, we have suggested that Cav-1 may be a predictive biomarker for breast cancer.

  7. Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

    PubMed

    Ortiz, Rina; Díaz, Jorge; Díaz, Natalia; Lobos-Gonzalez, Lorena; Cárdenas, Areli; Contreras, Pamela; Díaz, María Inés; Otte, Ellen; Cooper-White, Justin; Torres, Vicente; Leyton, Lisette; Quest, Andrew F G

    2016-06-28

    Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

  8. Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis

    PubMed Central

    Ortiz, Rina; Díaz, Jorge; Díaz, Natalia; Lobos-Gonzalez, Lorena; Cárdenas, Areli; Contreras, Pamela; Díaz, María Inés; Otte, Ellen; Cooper-White, Justin; Torres, Vicente; Leyton, Lisette; Quest, Andrew F.G.

    2016-01-01

    Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1. PMID:27259249

  9. Alterations of caveolin-1 expression in a mouse model of delayed cerebral vasospasm following subarachnoid hemorrhage

    PubMed Central

    Xiong, Ye; Wang, Xue-Min; Zhong, Ming; Li, Ze-Qun; Wang, Zhi; Tian, Zuo-Fu; Zheng, Kuang; Tan, Xian-Xi

    2016-01-01

    The aim of the present study was to evaluate the expression levels of caveolin-1 in the basilar artery following delayed cerebral vasospasm (DCVS) in a rat model of subarachnoid hemorrhage (SAH), in order to investigate the association between caveolin-1 and DCVS, and its potential as a treatment for DCVS of SAH. A total of 150 Sprague Dawley rats were randomly allocated into blank, saline and SAH groups. The SAH and saline groups were subdivided into days 3, 5, 7 and 14 following the establishment of the model. The murine model of SAH was established by double injection of autologous arterial blood into the cisterna magana and DCVS was detected using Bederson neurological severity scores. Hematoxylin and eosin (HE) staining was used to observe the inner perimeter of the basilar artery pipe and variations in the thickness of the basilar artery wall. Alterations in the levels of caveolin-1 protein in the basilar artery were measured using immunofluorescence and western blot analysis; whereas alterations in the mRNA expression levels of caveolin-1 were detected by reverse transcription-quantitative polymerase chain reaction. In the present study, 15 mice succumbed to SAH-induced DCVS in the day 3 (n=3), 5 (n=5) and 7 (n=2) groups. No mortality was observed in the blank control and saline groups during the process of observation in the SAH group, All mice in the SAH groups exhibited Bederson neurological severity scores ≥1; whereas no neurological impairment was detected in the blank and normal saline groups, demonstrating the success of the model. HE staining was used to assess vasospasm and the results demonstrated that the inner perimeter of the basal artery pipe decreased at day 3 in the SAH group; whereas values peaked in the day 7 group. The thickness of the basal artery wall significantly increased (P<0.05), as compared with the blank and saline groups, in which no significant alterations in the wall thickness and the inner perimeter of the basal artery pipe

  10. Association of caveolin-1 genotypes with gastric cancer in Taiwan.

    PubMed

    Lin, Chih-Hsueh; Lin, Cheng-Chieh; Tsai, Chia-Wen; Chang, Wen-Shin; Yang, Chuan-Wei; Bau, Da-Tian

    2014-05-01

    Gastric cancer is one of the leading causes of tumor-related death worldwide, for which the prevalence and mortality rates are very high in developed countries. Caveolin-1 (Cav-1) is the main protein in the caveolin family and plays a role in tumorigenesis signaling. The contribution of CAV1 genetic variants to gastric cancer is still largely unknown. In the present study, we aimed to investigate the role of CAV1 genotypes in gastric cancer risk. We recruited 358 gastric patients and 358 cancer-free controls for CAV1 genotyping analysis. Six single-nucleotide polymorphisms (SNPs) of CAV1, C521A (rs1997623), G14713A (rs3807987), G21985A (12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992), were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. There was a significant difference between the gastric cancer and control groups in the genotypic frequency distribution of the CAV1 G14713A genotypes (p=1.24*10(-5)), with those carrying the A allele having a higher risk for gastric cancer compared to those with the GG genotype (p=0.0001). Our findings suggested that CAV1 genotype may determine the individual susceptibility to gastric cancer, and that the CAV1 G14713A genotype may serve as a novel biomarker for early detection and prediction of gastric cancer.

  11. Caveolin-1: a critical regulator of lung injury

    PubMed Central

    Lee, Seon-Jin; Minshall, Richard D.; Choi, Augustine M. K.

    2011-01-01

    Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. Cav-1 regulates critical cell functions including proliferation, apoptosis, cell differentiation, and transcytosis via diverse signaling pathways. Abundant in almost every cell type in the lung, including type I epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, macrophages, and neutrophils, cav-1 plays a crucial role in the pathogenesis of acute lung injury (ALI). ALI and its severe form, acute respiratory distress syndrome (ARDS), are responsible for significant morbidity and mortality in intensive care units, despite improvement in ventilation strategies. The pathogenesis of ARDS is still poorly understood, and therapeutic options remain limited. In this article, we summarize recent data regarding the regulation and function of cav-1 in lung biology and pathology, in particular as it relates to ALI. We further discuss the potential molecular and cellular mechanisms by which cav-1 expression contributes to ALI. Investigating the cellular functions of cav-1 may provide new insights for understanding the pathogenesis of ALI and provide novel targets for therapeutic interventions in the future. PMID:21097526

  12. Flagellin-dependent TLR5/caveolin-1 as a promising immune activator in immunosenescence

    PubMed Central

    Lim, Jae Sung; Nguyen, Kim Cuc Thi; Nguyen, Chung Truong; Jang, Ik-Soon; Han, Jung Min; Fabian, Claire; Lee, Shee Eun; Rhee, Joon Haeng; Cho, Kyung A

    2015-01-01

    The age-associated decline of immune responses causes high susceptibility to infections and reduced vaccine efficacy in the elderly. However, the mechanisms underlying age-related deficits are unclear. Here, we found that the expression and signaling of flagellin (FlaB)-dependent Toll-like receptor 5 (TLR5), unlike the other TLRs, were well maintained in old macrophages, similar to young macrophages. The expression and activation of TLR5/MyD88, but not TLR4, were sensitively regulated by the upregulation of caveolin-1 in old macrophages through direct interaction. This interaction was also confirmed using macrophages from caveolin-1 or MyD88 knockout mice. Because TLR5 and caveolin-1 were well expressed in major old tissues including lung, skin, intestine, and spleen, we analyzed in vivo immune responses via a vaccine platform with FlaB as a mucosal adjuvant for the pneumococcal surface protein A (PspA) against Streptococcus pneumoniae infection in young and aged mice. The FlaB-PspA fusion protein induced a significantly higher level of PspA-specific IgG and IgA responses and demonstrated a high protective efficacy against a lethal challenge with live S. pneumoniae in aged mice. These results suggest that caveolin-1/TLR5 signaling plays a key role in age-associated innate immune responses and that FlaB-PspA stimulation of TLR5 may be a new strategy for a mucosal vaccine adjuvant against pneumococcal infection in the elderly. PMID:26223660

  13. The role of caveolin-1 and syndecan-4 in the internalization of PEGylated PAMAM dendrimer polyplexes into myoblast and hepatic cells.

    PubMed

    Shen, Wenwen; van Dongen, Mallory A; Han, Yingchun; Yu, Maomao; Li, Yanzhi; Liu, George; Banaszak Holl, Mark M; Qi, Rong

    2014-11-01

    To improve gene delivery efficiency of PEGylated poly(amidoamine) dendrimers in livers and muscles, the roles of syndecan-4 receptor and caveolin-1 protein in the endocytosis of PEGylated generation 5 (G5-PEG) or 7 (G7-PEG) dendrimers and plasmid DNA polyplexes were explored in C2C12 and HepG2 cells. Expression levels of syndecan-4 for both cell lines were downregulated by transfection of the cells with syndecan-4 specific siRNA. Caveolin-1 was upregulated by infecting the cells with adenovirus vector expressed caveolin-1 (Ad-CAV-1). The impact of syndecan-4 and caveolin-1 on endocytosis of G5-PEG/DNA or G7-PEG/DNA polyplexes was then measured by flow cytometry. Our results demonstrate that downregulation of syndecan-4 and upregulation of caveolin-1 significantly improved internalization of PEG-PAMAM dendrimer polyplexes in HepG2 cells; however, in C2C12 cells, downregulation of syndecan-4 decreased the internalization of the polyplexes while upregulation of caveolin-1 had no effect on internalization. Gene expression results for G5-PEG/pGFP on the two cell lines exhibited the same trends for syndecan-4 and caveolin-1 as was observed for endocytosis of the polyplexes. This study gives a clue how to take strategies by up- or down-regulation of the expressions of syndecan-4 and caveolin-1 to improve in vivo gene delivery efficiency of the PEG-PAMAM dendrimers in clinical transgenic therapy.

  14. The Role of Caveolin-1 and Syndecan-4 in the Internalization of PEGylated PAMAM Dendrimer Polyplexes into Myoblast and Hepatic Cells

    PubMed Central

    Shen, Wenwen; van Dongen, Mallory A.; Han, Yingchun; Yu, Maomao; Li, Yanzhi; Liu, George; BanaszakHoll, Mark M.; Qi, Rong

    2014-01-01

    To improve gene delivery efficiency of PEGylated poly(amidoamine) dendrimers in livers and muscles, the roles of syndecan-4 receptor and caveolin-1 protein in the endocytosis of PEGylated generation 5 (G5-PEG) or 7 (G7-PEG) dendrimers and plasmid DNA polyplexes were explored in C2C12 and HepG2 cells. Expression levels of syndecan-4 for both cell lines were downregulated by transfection of the cells with syndecan-4 specific siRNA. Caveolin-1 was upregulated by infecting the cells with adenovirus vector expressed caveolin-1 (Ad-CAV-1). The impact of syndecan-4 and caveolin-1 on endocytosis of G5-PEG/DNA or G7-PEG/DNA polyplexes was then measured by flow cytometry. Our results demonstrate that downregulation of syndecan-4 and upregulation of caveolin-1 significantly improved internalization of PEG-PAMAM dendrimer polyplexes in HepG2 cells; however, in C2C12 cells, downregulation of syndecan-4 decreased the internalization of the polyplexes while upregulation of caveolin-1 had no effect on internalization. Gene expression results for G5-PEG/pGFP on the two cell lines exhibited the same trends for syndecan-4 and caveolin-1 as was observed for endocytosis of the polyplexes. This study gives a clue how to take strategies by up- or down-regulation of the expressions of Syndecan-4 and Caveolin-1 to improve in vivo gene delivery efficiency of the PEG-PAMAM dendrimers in clinical transgenic therapy. PMID:25083608

  15. Expression of caveolin-1 and podocalyxin in rat lungs challenged with 2-kDa macrophage-activating lipopeptide and Flt3L.

    PubMed

    Tschernig, T; Pabst, R; Kasper, M; El-Hadi, Mustafa; Singh, B

    2014-04-01

    Caveolin-1 is one of the important regulators of vascular permeability in inflamed lungs. Podocalyxin is a CD34 protein expressed on vascular endothelium and has a role in podocyte development in the kidney. Few data are available on the expression of caveolin-1 and podocalyxin in lungs challenged with Toll-like receptor 2 (TLR2) agonists such as mycoplasma-derived macrophage activating lipopeptide or with immune modulators such as Fms-like tyrosine kinase receptor-3 ligand (Flt3L), which expands dendritic cell populations in the lung. Because of the significance of pathogen-derived molecules that act through TLR2 and of the role of immune modulators in lung physiology, we examine the immunohistochemical expression of caveolin-1 and podocalyxin in lungs from rats challenged with a 2-kDa macrophage-activating lipopeptide (MALP-2) and Flt3L. Normal rat lungs expressed caveolin-1 in alveolar septa, vascular endothelium and airway epithelium, especially along the lateral borders of epithelial cells but not in alveolar macrophages. MALP-2 and Flt3L decreased and increased, respectively, the expression of caveolin-1. Caveolin-1 expression seemed to increase in microvessels in bronchiole-associated lymphoid tissue (BALT) in Flt3L-challenged lungs but not in normal or MALP-2-treated lungs. Podocalyxin was absent in the epithelium and alveolar macrophages but was present in the vasculature of control, Flt3L- and MALP-2-treated rats. Compared with control and MALP-2-treated rats, Flt3L-treated lungs showed greater expression of podocalyxin in BALT vasculature and at the interface of monocytes and the endothelium. These immunohistochemical data describing the altered expression of caveolin-1 and podocalyxin in lungs treated with MALP-2 or Flt3L encourage further mechanistic studies on the role of podocalyxin and caveolin-1 in lung inflammation.

  16. Potential role of caveolin-1 in acetaminophen-induced hepatotoxicity

    SciTech Connect

    Gardner, Carol R.; Gray, Joshua P.; Joseph, Laurie B.; Cervelli, Jessica; Bremer, Nicole; Kim, Yunjung; Mishin, Vladimir; Laskin, Jeffrey D.; Laskin, Debra L.

    2010-05-15

    Caveolin-1 (Cav-1) is a membrane scaffolding protein, which functions to regulate intracellular compartmentalization of various signaling molecules. In the present studies, transgenic mice with a targeted disruption of the Cav-1 gene (Cav-1{sup -/-}) were used to assess the role of Cav-1 in acetaminophen-induced hepatotoxicity. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was correlated with decreased expression of Cav-1 in the liver. Acetaminophen-induced hepatotoxicity was significantly attenuated in Cav-1{sup -/-} mice, an effect that was independent of acetaminophen metabolism. Acetaminophen administration resulted in increased hepatic expression of the oxidative stress marker, lipocalin 24p3, as well as hemeoxygenase-1, but decreased glutathione and superoxide dismutase-1; no differences were noted between the genotypes suggesting that reduced toxicity in Cav-1{sup -/-} mice is not due to alterations in antioxidant defense. In wild-type mice, acetaminophen increased mRNA expression of the pro-inflammatory cytokines, interleukin-1beta, and monocyte chemoattractant protein-1 (MCP-1), as well as cyclooxygenase-2, while 15-lipoxygenase (15-LOX), which generates anti-inflammatory lipoxins, decreased. Acetaminophen-induced changes in MCP-1 and 15-LOX expression were greater in Cav-1{sup -/-} mice. Although expression of tumor necrosis factor-alpha, a potent hepatocyte mitogen, was up-regulated in the liver of Cav-1{sup -/-} mice after acetaminophen, expression of proliferating cell nuclear antigen and survivin, markers of cellular proliferation, were delayed, which may reflect the reduced need for tissue repair. Taken together, these data demonstrate that Cav-1 plays a role in promoting inflammation and toxicity during the pathogenesis of acetaminophen-induced injury.

  17. ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

    SciTech Connect

    Colonna, Cecilia . E-mail: ccolonna@fmed.uba.ar; Podesta, Ernesto J.

    2005-04-01

    Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.

  18. ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells.

    PubMed

    Colonna, Cecilia; Podestá, Ernesto J

    2005-04-01

    Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.

  19. Caveolin-1 Protects B6129 Mice against Helicobacter pylori Gastritis

    PubMed Central

    Hitkova, Ivana; Yuan, Gang; Anderl, Florian; Gerhard, Markus; Kirchner, Thomas; Reu, Simone; Röcken, Christoph; Schäfer, Claus; Schmid, Roland M.; Vogelmann, Roger; Ebert, Matthias P. A.; Burgermeister, Elke

    2013-01-01

    Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies (“humming bird”) compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells. PMID:23592983

  20. [Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

    PubMed

    Beniaminov, A D; Krasnov, G S; Dmitriev, A A; Puzanov, G A; Snopok, B A; Senchenko, V N; Kashuba, V I

    2016-01-01

    Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation.

  1. Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

    PubMed

    Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

    2015-03-01

    Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation.

  2. Caveolin-1, caveolin-3 and VEGF expression in the masticatory muscles of mdx mice.

    PubMed

    Kunert-Keil, Christiane; Gredes, Tomasz; Lucke, Silke; Morgenstern, Sven; Mielczarek, Agnieszka; Sporniak-Tutak, Katarzyna; Gedrange, Tomasz; Spassov, Alexander

    2011-01-01

    Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx), its murine model, are characterized by muscle damage and muscle weakness associated with inflammation and new vessel formation. Caveolins, dystrophin-associated proteins, are involved in the pathogenesis of DMD, because increased numbers of caveolae are found in DMD and mdx hindlimb muscles. Caveolae influence angiogenesis due to their content of vascular endothelial growth factor (VEGF) receptors. Orofacial muscles in mdx mice undergo muscle necrosis followed by muscle regeneration. To ascertain the role of caveolins and VEGF in the pathogenesis of dystrophic masticatory muscles, we examined the expression of caveolin-1 (cav-1), caveolin-3 (cav-3) and VEGF in control and mdx mice. In mdx masticatory muscles, no changes in transcript and protein levels of VEGF were found, whereas cav-1 and cav-3 expression was increased. Using immunohistochemistry, a strong sarcolemmal staining of caveolin-3 in regenerated muscle fibers was found. Furthermore, immunohistochemistry with the caveolin-1 antibody showed an increase in the amount of blood vessels in areas with regenerating muscle fibers. Dystrophic masticatory muscles showed changes comparable to those of hindlimb muscles in the expression of cav-1 and cav-3. The angiogenesis seems to be unaffected in the jaw muscles of mdx mice. We speculate that the increased caveolin expression could cause extensive and efficient muscle regeneration.

  3. Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells.

    PubMed

    Stuart, Elizabeth S; Webley, Wilmore C; Norkin, Leonard C

    2003-07-01

    Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se.

  4. Caveolin-1 is down-regulated in alveolar rhabdomyosarcomas and negatively regulates tumor growth

    PubMed Central

    Huertas-Martínez, Juan; Rello-Varona, Santiago; Herrero-Martín, David; Barrau, Ignasi; García-Monclús, Silvia; Sáinz-Jaspeado, Miguel; Lagares-Tena, Laura; Núñez-Álvarez, Yaiza; Mateo-Lozano, Silvia; Mora, Jaume; Roma, Josep; Toran, Nuria; Moran, Sebastian; López-Alemany, Roser; Gallego, Soledad; Esteller, Manel; Peinado, Miguel A.; Xavier García del, Muro; Tirado, Oscar M.

    2014-01-01

    Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with histological variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of less than 30%. Caveolin-1 (CAV1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signaling. In the present study we report that compared to other forms of rhabdomyosarcoma (RMS) CAV1 expression is either undetectable or very low in ARMS cell lines and tumor samples. DNA methylation analysis of the promoter region and azacytidine-induced re-expression suggest the involvement of epigenetic mechanisms in the silencing of CAV1. Reintroduction of CAV1 in three of these cell lines impairs their clonogenic capacity and promotes features of muscular differentiation. In vitro, CAV1-expressing cells show high expression of Caveolin-3 (CAV3), a muscular differentiation marker. Blockade of MAPK signaling is also observed. In vivo, CAV1-expressing xenografts show growth delay, features of muscular differentiation and increased cell death. In summary, our results suggest that CAV1 could function as a potent tumor suppressor in ARMS tumors. Inhibition of CAV1 function therefore, could contribute to aberrant cell proliferation, leading to ARMS development. PMID:25313138

  5. Down-regulation of caveolin-1 in glioma vasculature: modulation by radiotherapy.

    PubMed

    Régina, Anthony; Jodoin, Julie; Khoueir, Paul; Rolland, Yannève; Berthelet, France; Moumdjian, Robert; Fenart, Laurence; Cecchelli, Romeo; Demeule, Michel; Béliveau, Richard

    2004-01-15

    Primary brain tumors, particularly glioblastomas (GB), remain a challenge for oncology. An element of the malignant brain tumors' aggressive behavior is the fact that GB are among the most densely vascularized tumors. To determine some of the molecular regulations occuring at the brain tumor endothelium level during tumoral progression would be an asset in understanding brain tumor biology. Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling, oncogenesis, and angiogenesis. In this work we investigated regulation of caveolin-1 expression in brain endothelial cells (ECs) under angiogenic conditions. In vitro, brain EC caveolin-1 is down-regulated by angiogenic factors treament and by hypoxia. Coculture of brain ECs with tumoral cells induced a similar down-regulation. In addition, activation of the p42/44 MAP kinase is demonstrated. By using an in vivo brain tumor model, we purified ECs from gliomas as well as from normal brain to investigate possible regulation of caveolin-1 expression in tumoral brain vasculature. We show that caveolin-1 expression is strikingly down-regulated in glioma ECs, whereas an increase of phosphorylated caveolin-1 is observed. Whole-brain radiation treatment, a classical way in which GB is currently being treated, resulted in increased caveolin-1 expression in tumor isolated ECs. The level of tumor cells spreading around newly formed blood vessels was also elevated. The regulation of caveolin-1 expression in tumoral ECs may reflect the tumoral vasculature state and correlates with angiogenesis kinetics.

  6. Caveolae and Caveolin-1 Integrate Reverse Cholesterol Transport and Inflammation in Atherosclerosis.

    PubMed

    Qin, Li; Zhu, Neng; Ao, Bao-Xue; Liu, Chan; Shi, Ya-Ning; Du, Ke; Chen, Jian-Xiong; Zheng, Xi-Long; Liao, Duan-Fang

    2016-03-22

    Lipid disorder and inflammation play critical roles in the development of atherosclerosis. Reverse cholesterol transport is a key event in lipid metabolism. Caveolae and caveolin-1 are in the center stage of cholesterol transportation and inflammation in macrophages. Here, we propose that reverse cholesterol transport and inflammation in atherosclerosis can be integrated by caveolae and caveolin-1.

  7. Caveolae and Caveolin-1 Integrate Reverse Cholesterol Transport and Inflammation in Atherosclerosis

    PubMed Central

    Qin, Li; Zhu, Neng; Ao, Bao-Xue; Liu, Chan; Shi, Ya-Ning; Du, Ke; Chen, Jian-Xiong; Zheng, Xi-Long; Liao, Duan-Fang

    2016-01-01

    Lipid disorder and inflammation play critical roles in the development of atherosclerosis. Reverse cholesterol transport is a key event in lipid metabolism. Caveolae and caveolin-1 are in the center stage of cholesterol transportation and inflammation in macrophages. Here, we propose that reverse cholesterol transport and inflammation in atherosclerosis can be integrated by caveolae and caveolin-1. PMID:27011179

  8. Stromal cell expression of caveolin-1 predicts outcome in breast cancer.

    PubMed

    Sloan, Erica K; Ciocca, Daniel R; Pouliot, Normand; Natoli, Anthony; Restall, Christina; Henderson, Michael A; Fanelli, Mariel A; Cuello-Carrión, Fernando D; Gago, Francisco E; Anderson, Robin L

    2009-06-01

    Caveolin-1 has been linked to tumor progression and clinical outcome in breast cancer, but a clear resolution of its role as a prognostic marker is lacking. We assessed caveolin-1 levels in normal breast tissue and two breast cancer cohorts for which outcome data were available. We found that caveolin-1 was not expressed in normal breast luminal epithelium but was present in the epithelial compartment of some tumors. We found no association between caveolin-1 expression in the epithelial compartment and clinical outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor, rather than within tumor cells, associated strongly with reduced metastasis and improved survival (P < 0.0001). The onset of mammary tumors driven by Her2/neu overexpression was accelerated in mice lacking caveolin-1, thereby supporting the observation that the presence of caveolin-1 in the tumor microenvironment modulates tumor development. These studies suggest that stromal caveolin-1 expression may be a potential therapeutic target and a valuable prognostic indicator of breast cancer progression.

  9. Stromal Cell Expression of Caveolin-1 Predicts Outcome in Breast Cancer

    PubMed Central

    Sloan, Erica K.; Ciocca, Daniel R.; Pouliot, Normand; Natoli, Anthony; Restall, Christina; Henderson, Michael A.; Fanelli, Mariel A.; Cuello-Carrión, Fernando D.; Gago, Francisco E.; Anderson, Robin L.

    2009-01-01

    Caveolin-1 has been linked to tumor progression and clinical outcome in breast cancer, but a clear resolution of its role as a prognostic marker is lacking. We assessed caveolin-1 levels in normal breast tissue and two breast cancer cohorts for which outcome data were available. We found that caveolin-1 was not expressed in normal breast luminal epithelium but was present in the epithelial compartment of some tumors. We found no association between caveolin-1 expression in the epithelial compartment and clinical outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor, rather than within tumor cells, associated strongly with reduced metastasis and improved survival (P < 0.0001). The onset of mammary tumors driven by Her2/neu overexpression was accelerated in mice lacking caveolin-1, thereby supporting the observation that the presence of caveolin-1 in the tumor microenvironment modulates tumor development. These studies suggest that stromal caveolin-1 expression may be a potential therapeutic target and a valuable prognostic indicator of breast cancer progression. PMID:19411449

  10. Nanoscale imaging of caveolin-1 membrane domains in vivo.

    PubMed

    Gabor, Kristin A; Kim, Dahan; Kim, Carol H; Hess, Samuel T

    2015-01-01

    Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200-250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.

  11. Caveolin-1 and cancer metabolism in the tumor microenvironment: markers, models, and mechanisms.

    PubMed

    Sotgia, Federica; Martinez-Outschoorn, Ubaldo E; Howell, Anthony; Pestell, Richard G; Pavlides, Stephanos; Lisanti, Michael P

    2012-01-01

    Caveolins are a family of membrane-bound scaffolding proteins that compartmentalize and negatively regulate signal transduction. Recent studies have implicated a loss of caveolin-1 (Cav-1) expression in the pathogenesis of human cancers. Loss of Cav-1 expression in cancer-associated fibroblasts results in an activated tumor microenvironment, thereby driving early tumor recurrence, metastasis, and poor clinical outcome in breast and prostate cancers. We describe various paracrine signaling mechanism(s) by which the loss of stromal Cav-1 promotes tumor progression, including fibrosis, extracellular matrix remodeling, and the metabolic/catabolic reprogramming of cancer-associated fibroblast, to fuel the growth of adjacent tumor cells. It appears that oxidative stress is the root cause of initiation of the loss of stromal Cav-1 via autophagy, which provides further impetus for the use of antioxidants in anticancer therapy. Finally, we discuss the functional role of Cav-1 in epithelial cancer cells.

  12. Differential proteomic analysis of caveolin-1 KO cells reveals Sh2b3 and Clec12b as novel interaction partners of caveolin-1 and capns1 as potential mediator of caveolin-1-induced apoptosis

    PubMed Central

    Kulkarni, Yogesh M; Liu, Changxing; Qi, Qi; Zhu, Yanmei; Klinke, David J; Liu, Jun

    2014-01-01

    Caveolin-1 (Cav1) is a small scaffolding protein implicated in a variety of cellular functions, including cell signaling, lipid transport and membrane traffic. The objective of this study was to use comparative proteomics to identify differentially expressed proteins in Cav1 knockout (KO) mouse embryonic fibroblasts. These deregulated proteins were then analyzed using systems biology tools to gain insight into the local network properties and to identify the interaction partners of Cav1. We identified five proteins that were up-regulated and ten proteins that were down-regulated in Cav1 KO cells, suggesting that the local network behaves as a complex system. Protein interaction network analysis revealed two proteins, Sh2b3 and Clec12b, as novel interaction partners of Cav1. Functional annotation showed apoptosis signaling as the most significant pathway. To validate this functional annotation, Cav1 KO cells showed more than 1.5-fold increase in caspase-3 activity over wild type cells upon apoptotic stimulation. We also found that calpain small subunit 1 is up-regulated in Cav1 KO cells and directly influences cell response to apoptotic stimuli. Moreover, Capns1 was reduced in Cav1 KO cells following re-expression of Cav1 and suppression of Capns1 activity in Cav1 KO cells significantly inhibited the sensitivity to apoptotic stimuli, as measured by caspase 3 activity. In conclusion, our results suggest that Sh2b3 and Clec12b functionally interact with Cav1 and that calpain small subunit 1 may mediate Cav1-induced apoptosis. PMID:24091439

  13. Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

    SciTech Connect

    Luo, Di-xian; Xia, Cheng-lai; Li, Jun-mu; Xiong, Yan; Yuan, Hao-yu; TANG, Zhen-Wang; Zeng, Yixin; Liao, Duan-fang

    2010-12-03

    Research highlights: {yields} Vertical static pressure accelerates ox-LDL-induced cholesterol accumulation in cultured vascular smooth muscle cells. {yields} Static pressure induces SREBP-1 activation. {yields} Static pressure downregulates the expressions of caveolin-1 by activating SREBP-1. {yields} Static pressure also downregulates the transcription of ABCA1 by activating SREBP-1. {yields} Static pressure increases ox-LDL-induced cholesterol accumulation by SREBP-1-mediated caveolin-1 downregulation in vascular smooth muscle cells cultured in vitro. -- Abstract: Objective: To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism. Methods: Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different static pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure. Results: Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 {+-} 2.8 mg/g, 31.8 {+-} 0.7 mg/g, 92.3 {+-} 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 {+-} 9.4 mg/g, 235.9 {+-} 3.0 mg/g, 386.7 {+-} 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels were

  14. Caveolin-1 is necessary for hepatic oxidative lipid metabolism: evidence for crosstalk between caveolin-1 and bile acid signaling.

    PubMed

    Fernández-Rojo, Manuel A; Gongora, Milena; Fitzsimmons, Rebecca L; Martel, Nick; Martin, Sheree D; Nixon, Susan J; Brooks, Andrew J; Ikonomopoulou, Maria P; Martin, Sally; Lo, Harriet P; Myers, Stephen A; Restall, Christina; Ferguson, Charles; Pilch, Paul F; McGee, Sean L; Anderson, Robin L; Waters, Michael J; Hancock, John F; Grimmond, Sean M; Muscat, George E O; Parton, Robert G

    2013-07-25

    Caveolae and caveolin-1 (CAV1) have been linked to several cellular functions. However, a model explaining their roles in mammalian tissues in vivo is lacking. Unbiased expression profiling in several tissues and cell types identified lipid metabolism as the main target affected by CAV1 deficiency. CAV1-/- mice exhibited impaired hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent oxidative fatty acid metabolism and ketogenesis. Similar results were recapitulated in CAV1-deficient AML12 hepatocytes, suggesting at least a partial cell-autonomous role of hepatocyte CAV1 in metabolic adaptation to fasting. Finally, our experiments suggest that the hepatic phenotypes observed in CAV1-/- mice involve impaired PPARα ligand signaling and attenuated bile acid and FXRα signaling. These results demonstrate the significance of CAV1 in (1) hepatic lipid homeostasis and (2) nuclear hormone receptor (PPARα, FXRα, and SHP) and bile acid signaling.

  15. Fyn is required for oxidative- and hyperosmotic-stress-induced tyrosine phosphorylation of caveolin-1.

    PubMed Central

    Sanguinetti, Amy R; Cao, Haiming; Corley Mastick, Cynthia

    2003-01-01

    Caveolin-1 is phosphorylated on Tyr(14) in response to both oxidative and hyperosmotic stress. In the present paper, we show that this phosphorylation requires activation of the Src family kinase Fyn. Stress-induced caveolin phosphorylation was abolished by three Src kinase inhibitors, SU6656, PP2 and PD180970, and was not observed in fibroblasts derived from a Src, Yes and Fyn triple-knockout mouse (SYF-/-). Using cell lines derived from single-kinase-knockout mice (Src-/-, Yes-/- and Fyn-/-), we show that expression of Fyn, but not Src or Yes, is required for stress-induced caveolin phosphorylation. Heterologous expression of Fyn in the SYF-/- and Fyn-/- cells was sufficient to reconstitute stress-induced caveolin phosphorylation, and overexpression of Fyn in wild-type cells induced hyperphosphorylation of caveolin. Fyn was autophosphorylated following oxidative stress, verifying activation of this kinase. Interestingly, there was a concomitant increase in the phosphorylation of Fyn on its Csk (C-terminal Src kinase) site, indicating feedback inhibition. Csk binds to phosphocaveolin [Cao, Courchesne and Mastick (2002) J. Biol. Chem. 277, 8771-8774] and should phosphorylate any co-localized Src-family kinases. Oxidative-stress-induced phosphorylation of caveolin-1 also requires expression of Abl [Sanguinetti and Mastick (2003) Cell Signal. 15, 289-298]. Using inhibitors and cells derived from knockout mice, we verified a requirement for both Abl and Fyn in stress-induced caveolin phosphorylation in a single cell type. Our data suggest a novel mechanism for attenuation of Src-kinase activity by Abl: stable tyrosine phosphorylation of a scaffolding protein, caveolin, and recruitment of Csk. Paxillin, a substrate of both Abl and Src, organizes a similar regulatory complex. PMID:12921535

  16. The tumor suppressor protein Fhit. A novel interaction with tubulin.

    PubMed

    Chaudhuri, A R; Khan, I A; Prasad, V; Robinson, A K; Ludueña, R F; Barnes, L D

    1999-08-20

    FHIT (fragile histidine triad) is a candidate human tumor suppressor gene located at chromosome 3p14.2, a location that encompasses the FRA3B chromosomal fragile site. Aberrant transcripts have been detected in a variety of primary tumors, and homozygous deletions in the FHIT locus have been detected in different tumor cell lines. The gene product Fhit in vitro possesses the ability to hydrolyze diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A). The mechanism of action of Fhit as a tumor suppressor is unknown. Because the tubulin-microtubule system plays an important role in cell division and cell proliferation, we investigated the interaction between wild-type Fhit or mutant Fhit (H96N) and tubulin in vitro. The mutant form of Fhit (H96N) lacks Ap(3)A hydrolase activity but retains tumor suppressor activity. We found that both wild-type and mutated forms of Fhit bind to tubulin strongly and specifically with K(d) values of 1.4 and 2.1 microM, respectively. Neither wild-type nor mutant Fhit cause nucleation or formation of microtubules, but in the presence of microtubule-associated proteins, both wild-type and mutant Fhit promote assembly to a greater extent than do microtubule-associated proteins alone, and the microtubules formed appear normal by electron microscopy. Our results suggest the possibility that Fhit may exert its tumor suppressor activity by interacting with microtubules and also indicate that the interaction between Fhit and tubulin is not related to the Ap(3)A hydrolase activity of Fhit.

  17. Clathrin- and caveolin-1–independent endocytosis

    PubMed Central

    Damm, Eva-Maria; Pelkmans, Lucas; Kartenbeck, Jürgen; Mezzacasa, Anna; Kurzchalia, Teymuras; Helenius, Ari

    2005-01-01

    Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1. PMID:15668298

  18. Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells.

    PubMed

    Matsui, Takanori; Higashimoto, Yuichiro; Taira, Junichi; Yamagishi, Sho-ichi

    2013-11-15

    Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36×10(-7) M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.

  19. Prognostic and predictive values of SPP1, PAI and caveolin-1 in patients with oral squamous cell carcinoma.

    PubMed

    Huang, Cong-Fa; Yu, Guang-Tao; Wang, Wei-Ming; Liu, Bing; Sun, Zhi-Jun

    2014-01-01

    SPP1, PAI and caveolin-1 are known to be closely associated with tumor progression in several kinds of human tumors. This study aimed to investigate the expression of SPP1, PAI and caveolin-1 in oral squamous cell carcinoma (OSCC), and to evaluate their association with the prognosis in oral carcinoma. Immunohistochemical staining was used to examine the expression of SPP1, PAI and caveolin-1 in 17 normal oral mucosa, 6 oral epithelial dysplasia and 43 OSCC specimens by tissue microarrays. High expression of SPP1, PAI and caveolin-1 was found in OSCC patients, and SPP1 and PAI expression were significantly higher in OSCC than in normal oral mucosa. No significant correlations were found between SPP1, PAI and caveolin-1 expression and clinicopathological factors. Expression of SPP1, PAI and caveolin-1 was also not associated with overall survival. Moreover, SPP1 was closely correlated with PAI, caveolin-1 and Keap1, and PAI had significant correlations with caveolin-1, Keap1 and Nrf2, and caveolin-1 was associated with Keap1 by using the Pearson correlation coefficient test. Our findings suggest that overexpressed SPP1, PAI and caveolin-1 were linked to carcinogenesis and progression, and thus they may serve as potential prognostic factors in OSCC.

  20. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  1. Chloride channel ClC- 2 enhances intestinal epithelial tight junction barrier function via regulation of caveolin-1 and caveolar trafficking of occludin.

    PubMed

    Nighot, Prashant K; Leung, Lana; Ma, Thomas Y

    2017-03-01

    Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2(CLCN2)) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2(CLCN2)) significantly enhanced TJ barrier (increased TER by ≥2 times and reduced inulin flux by 50%) compared to control Caco-2(pEZ) cells. ClC-2 over-expression (Caco-2(CLCN2)) increased occludin protein level compared to control Caco-2(pEZ) cells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2(CLCN2) cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2(CLCN2) cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.

  2. C-terminus of human BKca channel alpha subunit enhances the permeability of the brain endothelial cells by interacting with caveolin-1 and triggering caveolin-1 intracellular trafficking.

    PubMed

    Song, Yang; Wang, Ping; Ma, Jun; Xue, Yixue

    2014-06-01

    The blood-tumor barrier (BTB) significantly limits the delivery of chemotherapeutic drugs to brain tumors. In this study, we found a significant increase in the permeability of BTB by mediating the association of the C-terminus of alpha subunit of human large-conductance calcium-activated potassium channels (hSlo1c) with caveolin-1 (Cav-1). We present evidence for the first time that hSlo1c associates with Cav-1 in human brain microvascular endothelial cells (HBMECs). A 57-amino acid (966-1022) fragment in hSlo1c was identified to be critical for hSlo1c/Cav-1 interaction. Activation of HBMECs transfected with fusion plasmids of pCMV-hSlo1c containing aa966-1022 by NS1619 selectively enhanced BTB permeability in a BTB model from the co-culture of HBMECs and U87 MG cells but not if the fusion plasmid lacks this fragment. This effect was attenuated by filipin, an agent disrupting caveolae or deletion of the potential interaction fragment, suggesting hSlo1c/Cav-1 association is crucial for regulating the permeability of BTB. Furthermore, we found that hSlo1c/Cav-1 association boosted Cav-1 transferring from the cell membrane to the cytoplasm of HBMECs. Our study indicates that cytoplasmic hSlo1c not only associates with Cav-1 but also has functional consequences on the permeability of BTB by triggering the intracellular trafficking of its interacting protein partner, Cav-1.

  3. Caveolin-1 regulates TCR signal strength and regulatory T-cell differentiation into alloreactive T cells.

    PubMed

    Schönle, Anne; Hartl, Frederike A; Mentzel, Jan; Nöltner, Theresa; Rauch, Katharina S; Prestipino, Alessandro; Wohlfeil, Sebastian A; Apostolova, Petya; Hechinger, Anne-Kathrin; Melchinger, Wolfgang; Fehrenbach, Kerstin; Guadamillas, Marta C; Follo, Marie; Prinz, Gabriele; Ruess, Ann-Katrin; Pfeifer, Dietmar; del Pozo, Miguel Angel; Schmitt-Graeff, Annette; Duyster, Justus; Hippen, Keli I; Blazar, Bruce R; Schachtrup, Kristina; Minguet, Susana; Zeiser, Robert

    2016-04-14

    Caveolin-1 (Cav-1) is a key organizer of membrane specializations and a scaffold protein that regulates signaling in multiple cell types. We found increased Cav-1 expression in human and murine T cells after allogeneic hematopoietic cell transplantation. Indeed, Cav-1(-/-)donor T cells caused less severe acute graft-versus-host disease (GVHD) and yielded higher numbers of regulatory T cells (Tregs) compared with controls. Depletion of Tregs from the graft abrogated this protective effect. Correspondingly, Treg frequencies increased when Cav-1(-/-)T cells were exposed to transforming growth factor-β/T-cell receptor (TCR)/CD28 activation or alloantigen stimulation in vitro compared with wild-type T cells. Mechanistically, we found that the phosphorylation of Cav-1 is dispensable for the control of T-cell fate by using a nonphosphorylatable Cav-1 (Y14F/Y14F) point-mutation variant. Moreover, the close proximity of lymphocyte-specific protein tyrosine kinase (Lck) to the TCR induced by TCR-activation was reduced in Cav-1(-/-)T cells. Therefore, less TCR/Lck clustering results in suboptimal activation of the downstream signaling events, which correlates with the preferential development into a Treg phenotype. Overall, we report a novel role for Cav-1 in TCR/Lck spatial distribution upon TCR triggering, which controls T-cell fate toward a regulatory phenotype. This alteration translated into a significant increase in the frequency of Tregs and reduced GVHD in vivo.

  4. Caveolin-1 modulates intraocular pressure: implications for caveolae mechanoprotection in glaucoma

    PubMed Central

    Elliott, Michael H.; Ashpole, Nicole E.; Gu, Xiaowu; Herrnberger, Leonie; McClellan, Mark E.; Griffith, Gina L.; Reagan, Alaina M.; Boyce, Timothy M.; Tanito, Masaki; Tamm, Ernst R.; Stamer, W. Daniel

    2016-01-01

    Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1−/−) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm’s canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1−/− outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1−/− eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology. PMID:27841369

  5. Akt phosphorylates and regulates Pdcd4 tumor suppressor protein.

    PubMed

    Palamarchuk, Alexey; Efanov, Alexey; Maximov, Vadim; Aqeilan, Rami I; Croce, Carlo M; Pekarsky, Yuri

    2005-12-15

    Programmed cell death 4 (Pdcd4) is a tumor suppressor protein that interacts with eukaryotic initiation factor 4A and inhibits protein synthesis. Pdcd4 also suppresses the transactivation of activator protein-1 (AP-1)-responsive promoters by c-Jun. The Akt (protein kinase B) serine/threonine kinase is a key mediator of phosphoinositide 3-kinase pathway involved in the regulation of cell proliferation, survival, and growth. Because Pdcd4 has two putative Akt phosphorylation sites at Ser(67) and Ser(457), we investigated whether Akt phosphorylates and regulates Pdcd4. Our results show that Akt specifically phosphorylates Ser(67) and Ser(457) residues of Pdcd4 in vitro and in vivo. We further show that phosphorylation of Pdcd4 by Akt causes nuclear translocation of Pdcd4. Using luciferase assay, we show that phosphorylation of Pdcd4 by Akt also causes a significant decrease of the ability of Pdcd4 to interfere with the transactivation of AP-1-responsive promoter by c-Jun.

  6. Reciprocal Activating Crosstalk between c-Met and Caveolin 1 Promotes Invasive Phenotype in Hepatocellular Carcinoma

    PubMed Central

    Korhan, Peyda; Erdal, Esra; Kandemiş, Emine; Çokaklı, Murat; Nart, Deniz; Yılmaz, Funda; Can, Alp; Atabey, Neşe

    2014-01-01

    c-Met, the receptor for Hepatocyte Growth Factor (HGF), overexpressed and deregulated in Hepatocellular Carcinoma (HCC). Caveolin 1 (CAV1), a plasma membrane protein that modulates signal transduction molecules, is also overexpressed in HCC. The aim of this study was to investigate biological and clinical significance of co-expression and activation of c-Met and CAV1 in HCC. We showed that c-Met and CAV1 were co-localized in HCC cells and HGF treatment increased this association. HGF-triggered c-Met activation caused a concurrent rise in both phosphorylation and expression of CAV1. Ectopic expression of CAV1 accelerated c-Met signaling, resulted in enhanced migration, invasion, and branching-morphogenesis. Silencing of CAV1 downregulated c-Met signaling, and decreased migratory/invasive capability of cells and attenuated branching morphogenesis. In addition, activation and co-localization of c-Met and CAV1 were elevated during hepatocarcinogenesis. In conclusion reciprocal activating crosstalk between c-Met and CAV1 promoted oncogenic signaling of c-Met contributed to the initiation and progression of HCC. PMID:25148256

  7. Caveolin-1 Promotes the Imbalance of Th17/Treg in Patients with Chronic Obstructive Pulmonary Disease.

    PubMed

    Sun, Nina; Wei, Xiaofang; Wang, Jingluan; Cheng, Zhaozhong; Sun, Weihong

    2016-12-01

    The imbalance of Th17/Treg cells plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Caveolin-1 (Cav-1) has been regarded as a potential critical regulatory protein in pathological mechanisms of chronic inflammatory respiratory diseases. Therefore, we investigated whether the loss of Cav-1 is involved in the homeostasis of Th17/Treg cells in COPD. We examined the expressions of plasma Cav-1 and circulating Th17, Treg cells, and the related cytokines in patients with COPD. Enzyme-linked immunosorbent assay (ELISA) analyses showed a significant reduction of plasma Cav-1 levels in patients with stable COPD (SCOPD) and acutely exacerbated COPD (AECOPD) compared to smokers without COPD. This loss was associated with an increase in frequency of Treg and decreased in frequency of Th17 cells. To further identify the role of Cav-1, we studied the effects of Cav-1 overexpression or downregulation on frequencies of Treg and Th17 cells in peripheral blood mononuclear cells (PBMCs) from subjects. Interestingly, small interfering RNA (siRNA) downregulation of Cav-1 was accompanied by an augmentation of Treg and reduction of Th17 expression. Together, our study demonstrated that the loss of Cav-1 contributed to the imbalance of Th17/Treg cells in patients with COPD.

  8. Caveolin-1 regulates genomic action of the glucocorticoid receptor in neural stem cells.

    PubMed

    Peffer, Melanie E; Chandran, Uma R; Luthra, Soumya; Volonte, Daniela; Galbiati, Ferruccio; Garabedian, Michael J; Monaghan, A Paula; DeFranco, Donald B

    2014-07-01

    While glucocorticoids (GCs) are used clinically to treat many conditions, their neonatal and prenatal usage is increasingly controversial due to reports of delayed adverse outcomes, especially their effects on brain development. Such alterations may reflect the impact of GCs on neural progenitor/stem cell (NPSC) function. We previously demonstrated that the lipid raft protein caveolin-1 (Cav-1) was required for rapid GC signaling in embryonic mouse NPSCs operating through plasma membrane-bound glucocorticoid receptors (GRs). We show here that genomic GR signaling in NPSCs requires Cav-1. Loss of Cav-1 impacts the transcriptional response of many GR target genes (e.g., the serum- and glucocorticoid-regulated kinase 1 gene) that are likely to mediate the antiproliferative effects of GCs. Microarray analysis of wild-type C57 or Cav-1-deficient NPSCs identified approximately 100 genes that are differentially regulated by GC treatment. These changes in hormone responsiveness in Cav-1 knockout NPSCs are associated with the loss of GC-regulated phosphorylation of GR at serine 211 but not at serine 226. Chromatin recruitment of total GR to regulatory regions of target genes such as Fkbp-5, RhoJ, and Sgk-1, as well as p211-GR recruitment to Sgk-1, are compromised in Cav-1 knockout NPSCs. Cav-1 is therefore a multifunctional regulator of GR in NPSCs influencing both rapid and genomic action of the receptor to impact cell proliferation.

  9. Caveolin-1 and ATP binding cassette transporter A1 and G1-mediated cholesterol efflux.

    PubMed

    Wang, Faqi; Gu, Hong-mei; Zhang, Da-wei

    2014-01-01

    Atherosclerosis is one major cause of cardiovascular diseases, the leading cause of death in industrialized countries. Reverse cholesterol transport (RCT) is thought to be one primary pathway to protect against atherosclerosis. The first and rate-limiting step of RCT is ATP-binding cassette transport A1 (ABCA1) and ABCG1-mediated cholesterol efflux from the cells. Recently, caveolin-1 (CAV1), a scaffolding protein that organizes and concentrates certain caveolin-interacting signaling molecules and receptors within caveolae membranes, has been shown to regulate ABCA1 and ABCG1-mediated cholesterol efflux probably via interacting with them. In the present review, we summarize the current knowledge and views on the regulatory role of CAV1 on the cholesterol homeostasis with emphasis on the association of CAV1 with ABCA1 and ABCG1. We conclude that the dominance of the positive regulation by CAV1 on the ABCA1 and ABCG1-mediated cholesterol efflux is depending on the species, cell types, as well as the levels of CAV1 expression.

  10. Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration

    PubMed Central

    Cao, Jingli; Navis, Adam; Cox, Ben D.; Dickson, Amy L.; Gemberling, Matthew; Karra, Ravi; Bagnat, Michel; Poss, Kenneth D.

    2016-01-01

    In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration. PMID:26657776

  11. Pro-metastatic NEDD9 regulates individual cell migration via caveolin-1-dependent trafficking of integrins

    PubMed Central

    Kozyulina, Polina Y.; Loskutov, Yuriy V.; Kozyreva, Varvara K.; Rajulapati, Anuradha; Ice, Ryan J.; Jones, Brandon. C.; Pugacheva, Elena N.

    2014-01-01

    The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of pro-metastatic protein, NEDD9, in breast cancer (BC) cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and co-localizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand/integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Re-expression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9 depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. PMID:25319010

  12. Expression of Caveolin 1 is enhanced by DNA demethylation during adipocyte differentiation. status of insulin signaling.

    PubMed

    Palacios-Ortega, Sara; Varela-Guruceaga, Maider; Milagro, Fermín Ignacio; Martínez, José Alfredo; de Miguel, Carlos

    2014-01-01

    Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.

  13. Expression of Caveolin 1 Is Enhanced by DNA Demethylation during Adipocyte Differentiation. Status of Insulin Signaling

    PubMed Central

    Palacios-Ortega, Sara; Varela-Guruceaga, Maider; Milagro, Fermín Ignacio; Martínez, José Alfredo; de Miguel, Carlos

    2014-01-01

    Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised. PMID:24751908

  14. Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts

    PubMed Central

    Wang, Shuai; Kaartinen, Mari T.

    2015-01-01

    Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9—which can alter TG enzyme conformation—localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts. PMID:26231113

  15. PML tumor suppressor protein is required for HCV production

    SciTech Connect

    Kuroki, Misao; Ariumi, Yasuo; Hijikata, Makoto; Ikeda, Masanori; Dansako, Hiromichi; Wakita, Takaji; Shimotohno, Kunitada; Kato, Nobuyuki

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer PML tumor suppressor protein is required for HCV production. Black-Right-Pointing-Pointer PML is dispensable for HCV RNA replication. Black-Right-Pointing-Pointer HCV could not alter formation of PML-NBs. Black-Right-Pointing-Pointer INI1 and DDX5, PML-related proteins, are involved in HCV life cycle. -- Abstract: PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.

  16. Identification, expression pattern, cellular location and potential role of the caveolin-1 gene from Artemia sinica.

    PubMed

    Li, Xuejie; Yao, Feng; Zhang, Wei; Cheng, Cheng; Chu, Bing; Liu, Yan; Mei, Yanli; Wu, Yang; Zou, Xiangyang; Hou, Lin

    2014-05-01

    Caveolins are integral membrane proteins that serve as scaffolds to recruit numerous signaling molecules. Caveolins play an important role in membrane trafficking, signal transduction, substrate transport and endocytosis in differentiated cells. In this study, a caveolin-1 gene from Artemia sinica (As-cav-1) was successfully cloned for the first time. The full-length cDNA of As-cav-1 comprises 974 bp, with a 675 bp open reading frame (ORF) that encodes a polypeptide of 224 amino acids with a caveolin scaffolding domain (CSD) and two transmembrane domains. Multiple sequence alignment revealed that the putative As-CAV-1 protein sequence was relatively conserved across species, especially in the CSD domain. Real-time PCR revealed high levels of the As-cav-1 transcript at 0h of embryo development. Furthermore, As-cav-1 transcripts were highly upregulated under high salinity (200‰) and low temperature stresses (15°C). To further characterize As-cav-1, recombinant pET30a-cav-1 protein was expressed using a prokaryotic expression system. The recombinant protein comprised 290 amino acids with a theoretical molecular weight of 32kDa, and a predicted isoelectric point of 5.6. Western blotting of the expression levels of As-CAV-1 during different embryo development stages revealed that As-CAV-1 levels decreased gradually during development stages from 0 h to 40 h, and increased at 3d. Furthermore, western blotting showed that As-CAV-1 was upregulated to its highest expression level by low temperature stress (15°C) and high salinity. Confocal laser microscopy analysis, using antibodies generated against the recombinant As-CAV-1 protein, showed that As-CAV-1 was mostly located in the cell membrane. Our results suggested that As-cav-1 plays a vital role in protecting embryos from high salt damage and low temperature stress, especially during post-diapause embryonic development.

  17. Hepatic caveolin-1 is enhanced in Cyp27a1/ApoE double knockout mice.

    PubMed

    Zurkinden, Line; Mansour, Yosef T; Rohrbach, Beatrice; Vogt, Bruno; Mistry, Hiten D; Escher, Geneviève

    2016-10-01

    Sterol 27-hydroxylase (CYP27A1) is involved in bile acid synthesis and cholesterol homoeostasis. Cyp27a1((-/-))/Apolipoprotein E((-/-)) double knockout mice (DKO) fed a western diet failed to develop atherosclerosis. Caveolin-1 (CAV-1), the main component of caveolae, is associated with lipid homoeostasis and has regulatory roles in vascular diseases. We hypothesized that liver CAV-1 would contribute to the athero-protective mechanism in DKO mice. Cyp27a1((+/+))/ApoE((-/-)) (ApoE KO), Cyp27a1((+/-))/ApoE((-/-)) (het), and DKO mice were fed a western diet for 2 months. Atherosclerotic plaque and CAV-1 protein were quantified in aortas. Hepatic Cav-1 mRNA was assessed using qPCR, CAV-1 protein by immunohistochemistry and western blotting. Total hepatic and plasma cholesterol was measured using chemiluminescence. Cholesterol efflux was performed in RAW264.7 cells, using mice plasma as acceptor. CAV-1 protein expression in aortas was increased in endothelial cells of DKO mice and negatively correlated with plaque surface (P < 0.05). In the liver, both CAV-1 protein and mRNA expression doubled in DKO, compared to ApoE KO and het mice (P < 0.001 for both) and was negatively correlated with total hepatic cholesterol (P < 0.05). Plasma from DKO, ApoE KO and het mice had the same efflux capacity. In the absence of CYP27A1, CAV-1 overexpression might have an additional athero-protective role by partly overcoming the defect in CYP27A1-mediated cholesterol efflux.

  18. Caveolin-1 promotes Ewing sarcoma metastasis regulating MMP-9 expression through MAPK/ERK pathway

    PubMed Central

    Lagares-Tena, Laura; García-Monclús, Silvia; López-Alemany, Roser; Almacellas-Rabaiget, Olga; Huertas-Martínez, Juan; Sáinz-Jaspeado, Miguel; Mateo-Lozano, Silvia; Rodríguez-Galindo, Carlos; Rello-Varona, Santiago; Herrero-Martín, David; Tirado, Oscar M.

    2016-01-01

    Ewing sarcoma (ES) is a bone and soft tissue sarcoma affecting mostly children and young adults. Caveolin-1 (CAV1) is a well-known target of EWS/FLI1, the main driver of ES, with an oncogenic role in ES. We have previously described how CAV1 is able to induce metastasis in ES via matrix metalloproteinase-9 (MMP-9). In the present study we showed how CAV1 silencing in ES reduced MEK1/2 and ERK1/2 phosphorylation. Accordingly, chemical inhibition of MEK1/2 resulted in reduction in MMP-9 expression and activity that correlated with reduced migration and invasion. IQ Motif Containing GTPase Activating Protein 1 (IQGAP1) silencing reduced MEK1/2 and ERK1/2 phosphorylation and MMP-9 expression. Furthermore, IQGAP1 silenced cells showed a marked decrease in their migratory and invasive capacity. We demonstrated that CAV1 and IQGAP1 localize in close proximity at the cellular edge, thus IQGAP1 could be the connecting node between CAV1 and MEK/ERK in ES metastatic phenotype. Analysis of the phosphorylation profile of CAV1-silenced cells showed a decrease of p-ribosomal protein S6 (RPS6). RPS6 can be phosphorylated by p90 ribosomal S6 kinases (RSK) proteins. CAV1-silenced cells showed reduced levels of p-RSK1 and treatment with U0126 provoked the same effect. Despite not affecting ERK1/2 and RPS6 phosphorylation status neither MMP-9 expression nor activity, RSK1 silencing resulted in a reduced migratory and invasive capacity in vitro and reduced incidence of metastases in vivo in a novel orthotopic model. The present work provides new insights into CAV1-driven metastatic process in ES unveiling novel key nodes. PMID:27487136

  19. Caveolin-1 contributes to realgar nanoparticle therapy in human chronic myelogenous leukemia K562 cells

    PubMed Central

    Shi, Dan; Liu, Yan; Xi, Ronggang; Zou, Wei; Wu, Lijun; Zhang, Zhiran; Liu, Zhongyang; Qu, Chao; Xu, Baoli; Wang, Xiaobo

    2016-01-01

    Chronic myelogenous leukemia (CML) is characterized by the t(9;22) (q34;q11)-associated Bcr-Abl fusion gene, which is an essential element of clinical diagnosis. As a traditional Chinese medicine, realgar has been widely used for the treatment of various diseases for >1,500 years. Inspired by nano-drug, realgar nanoparticles (NPs) have been prepared with an average particle size of <100 nm in a previous work. Compared with coarse realgar, the realgar NPs have higher bioavailability. As a principal constituent protein of caveolae, caveolin-1 (Cav-1) participates in regulating various cellular physiological and pathological processes including tumorigenesis and tumor development. In previous studies, it was found that realgar NPs can inhibit several types of tumor cell proliferation. However, the therapeutic effect of realgar NPs on CML has not been fully elucidated. In the present paper, it was demonstrated that realgar NPs can inhibit the proliferation of K562 cells and degrade Bcr-Abl fusion protein effectively. Both apoptosis and autophagy were activated in a dose-dependent manner in realgar NPs treated cells, and the induction of autophagy was associated with class I phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway. Morphological analysis indicated that realgar NPs induced differentiation effectively in CML cells. Furthermore, it was identified that Cav-1 might play a crucial role in realgar NP therapy. In order to study the effects of Cav-1 on K562 cells during realgar NP treatment, a Cav-1 overexpression cell model was established by using transient transfection. The results indicated that Cav-1 overexpression inhibited K562 cell proliferation, promoted endogenic autophagy, and increased the sensitivity of K562 cells to realgar NPs. Therefore, the results demonstrated that realgar NPs degraded Bcr-Abl oncoprotein, while the underlying mechanism might be related to apoptosis and autophagy, and Cav-1 might be considered as a

  20. Caveolin-1 regulates P2X7 receptor signaling in osteoblasts.

    PubMed

    Gangadharan, Vimal; Nohe, Anja; Caplan, Jeffrey; Czymmek, Kirk; Duncan, Randall L

    2015-01-01

    The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.

  1. Regulation of pancreatic cancer cell migration and invasion by RhoC GTPase and Caveolin-1

    PubMed Central

    Lin, Min; DiVito, Melinda M; Merajver, Sofia D; Boyanapalli, Madanamohan; van Golen, Kenneth L

    2005-01-01

    Background In the current study we investigated the role of caveolin-1 (cav-1) in pancreatic adenocarcinoma (PC) cell migration and invasion; initial steps in metastasis. Cav-1 is the major structural protein in caveolae; small Ω-shaped invaginations within the plasma membrane. Caveolae are involved in signal transduction, wherein cav-1 acts as a scaffolding protein to organize multiple molecular complexes regulating a variety of cellular events. Recent evidence suggests a role for cav-1 in promoting cancer cell migration, invasion and metastasis; however, the molecular mechanisms have not been described. The small monomeric GTPases are among several molecules which associate with cav-1. Classically, the Rho GTPases control actin cytoskeletal reorganization during cell migration and invasion. RhoC GTPase is overexpressed in aggressive cancers that metastasize and is the predominant GTPase in PC. Like several GTPases, RhoC contains a putative cav-1 binding motif. Results Analysis of 10 PC cell lines revealed high levels of cav-1 expression in lines derived from primary tumors and low expression in those derived from metastases. Comparison of the BxPC-3 (derived from a primary tumor) and HPAF-II (derived from a metastasis) demonstrates a reciprocal relationship between cav-1 expression and p42/p44 Erk activation with PC cell migration, invasion, RhoC GTPase and p38 MAPK activation. Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression. Conclusion Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion. In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells. PMID:15969750

  2. Electroacupuncture Exerts Neuroprotection through Caveolin-1 Mediated Molecular Pathway in Intracerebral Hemorrhage of Rats

    PubMed Central

    Li, Hui-Qin; Li, Yan; Chen, Zi-Xian; Zhang, Xiao-Guang; Zheng, Xia-wei; Yang, Wen-ting; Chen, Shuang

    2016-01-01

    Spontaneous intracerebral hemorrhage (ICH) is one of the most devastating types of stroke. Here, we aim to demonstrate that electroacupuncture on Baihui (GV20) exerts neuroprotection for acute ICH possibly via the caveolin-1/matrix metalloproteinase/blood-brain barrier permeability pathway. The model of ICH was established by using collagenase VII. Rats were randomly divided into three groups: Sham-operation group, Sham electroacupuncture group, and electroacupuncture group. Each group was further divided into 4 subgroups according to the time points of 6 h, 1 d, 3 d, and 7 d after ICH. The methods were used including examination of neurological deficit scores according to Longa's scale, measurement of blood-brain barrier permeability through Evans Blue content, in situ immunofluorescent detection of caveolin-1 in brains, western blot analysis of caveolin-1 in brains, and in situ zymography for measuring matrix metalloproteinase-2/9 activity in brains. Compared with Sham electroacupuncture group, electroacupuncture group has resulted in a significant improvement in neurological deficit scores and in a reduction in Evans Blue content, expression of caveolin-1, and activity of matrix metalloproteinase-2/9 at 6 h, 1 d, 3 d, and 7 d after ICH (P < 0.05). In conclusion, the present results suggested that electroacupuncture on GV20 can improve neurological deficit scores and reduce blood-brain barrier permeability after ICH, and the mechanism possibly targets caveolin-1/matrix metalloproteinase/blood-brain barrier permeability pathway. PMID:27725888

  3. Associated inflammation or increased flow-mediated shear stress, but not pressure alone, disrupts endothelial caveolin-1 in infants with pulmonary hypertension

    PubMed Central

    Dereddy, Narendra; Huang, Jing; Erb, Markus; Guzel, Sibel; Wolk, John H; Sett, Suvro S; Gewitz, Michael H; Mathew, Rajamma

    2012-01-01

    Endothelial caveolin-1 loss is an important feature of pulmonary hypertension (PH); the rescue of caveolin-1 abrogates experimental PH. Recent studies in human PH suggest that the endothelial caveolin-1 loss is followed by an enhanced expression of caveolin-1 in smooth muscle cells (SMC) with subsequent neointima formation. In order to evaluate caveolin-1 expression in infants with PH, we examined the available clinical histories, hemodynamic data, and the expression of caveolin-1, PECAM-1, vWF, and smooth muscle α-actin in the lung biopsy/autopsy specimens obtained from infants with congenital heart disease (CHD, n = 8) and lung disease (n = 9). In CHD group, PH associated with increased pulmonary blood flow exhibited loss of endothelial caveolin-1 and PECAM-1 in pulmonary arteries; additional vWF loss was associated with enhanced expression of caveolin-1 in SMC. In the absence of PH, increased or decreased pulmonary blood flow did not disrupt endothelial caveolin-1, PECAM-1, or vWF; nor was there any enhanced expression of caveolin-1 in SMC. In Lung Disease + PH group, caveolin-1, PECAM-1, and vWF were well preserved in seven infants, and importantly, SMC in these arteries did not exhibit enhanced caveolin-1 expression. Two infants with associated inflammatory disease exhibited loss of endothelial caveolin-1 and PECAM-1; additional loss of vWF was accompanied by enhanced expression of caveolin-1 in SMC. Thus, associated flow-induced shear stress or inflammation, but not elevated pulmonary artery pressure alone, disrupts endothelial caveolin-1. Subsequent vWF loss, indicative of extensive endothelial damage is associated with enhanced expression of caveolin-1 in SMC, which may worsen the disease. PMID:23372934

  4. Impairment of Transforming Growth Factor β Signaling in Caveolin-1-deficient Hepatocytes

    PubMed Central

    Mayoral, Rafael; Valverde, Ángela M.; Llorente Izquierdo, Cristina; González-Rodríguez, Águeda; Boscá, Lisardo; Martín-Sanz, Paloma

    2010-01-01

    Caveolin-1 (Cav-1) is the main structural protein of caveolae and plays an important role in various cellular processes such as vesicular transport, cholesterol homeostasis, and signal transduction pathways. The expression and functional role of Cav-1 have been reported in liver and in hepatocyte cell lines, in human cirrhotic liver, and in hepatocellular carcinomas. Previous studies demonstrated that Cav-1 was dispensable for liver regeneration, because Cav-1−/− animals survived and fully regenerated liver function and size after partial hepatectomy. In this study, we have investigated the mechanisms by which the lack of Cav-1 accelerates liver regeneration after partial hepatectomy. The data show that transforming growth factor β (TGF-β) signaling is impaired in regenerating liver of Cav-1−/− mice and in hepatocytes derived from these animals. TGF-β receptors I and II do not colocalize in the same membrane fraction in the hepatocytes derived from Cav-1−/− mice, as Smad2/3 signaling decreased in the absence of Cav-1 at the time that the transcriptional corepressor SnoN accumulates. Accordingly, the expression of TGF-β target genes, such as plasminogen activator inhibitor-1, is decreased due to the presence of the high levels of SnoN. Moreover, hepatocyte growth factor inhibited TGF-β signaling in the absence of Cav-1 by increasing SnoN expression. Taken together, these data might help to unravel why Cav-1-deficient mice exhibit an accelerated liver regeneration after partial hepatectomy and add new insights on the molecular mechanisms controlling the initial commitment to hepatocyte proliferation. PMID:19966340

  5. Ciprofloxacin mediates cancer stem cell phenotypes in lung cancer cells through caveolin-1-dependent mechanism.

    PubMed

    Phiboonchaiyanan, Preeyaporn Plaimee; Kiratipaiboon, Chayanin; Chanvorachote, Pithi

    2016-04-25

    Cancer stem cells (CSCs), a subpopulation of cancer cells with high aggressive behaviors, have been identified in many types of cancer including lung cancer as one of the key mediators driving cancer progression and metastasis. Here, we have reported for the first time that ciprofloxacin (CIP), a widely used anti-microbial drug, has a potentiating effect on CSC-like features in human non-small cell lung cancer (NSCLC) cells. CIP treatment promoted CSC-like phenotypes, including enhanced anchorage-independent growth and spheroid formation. The known lung CSC markers: CD133, CD44, ABCG2 and ALDH1A1 were found to be significantly increased, while the factors involving in epithelial to mesenchymal transition (EMT): Slug and Snail, were depleted. Also, self-renewal transcription factors Oct-4 and Nanog were found to be up-regulated in CIP-treated cells. The treatment of CIP on CSC-rich populations obtained from secondary spheroids resulted in the further increase of CSC markers. In addition, we have proven that the mechanistic insight of the CIP induced stemness is through Caveolin-1 (Cav-1)-dependent mechanism. The specific suppression of Cav-1 by stably transfected Cav-1 shRNA plasmid dramatically reduced the effect of CIP on CSC markers as well as the CIP-induced spheroid formation ability. Cav-1 was shown to activate protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways in CSC-rich population; however, such an effect was rarely found in the main lung cancer cells population. These findings reveal a novel effect of CIP in positively regulating CSCs in lung cancer cells via the activation of Cav-1, Akt and ERK, and may provoke the awareness of appropriate therapeutic strategy in cancer patients.

  6. Intestinal epithelial cell caveolin 1 regulates fatty acid and lipoprotein cholesterol plasma levels

    PubMed Central

    Shen, Meng-Chieh; Quinlivan, Vanessa; Anderson, Jennifer L.; Farber, Steven A.

    2017-01-01

    ABSTRACT Caveolae and their structural protein caveolin 1 (CAV1) have roles in cellular lipid processing and systemic lipid metabolism. Global deletion of CAV1 in mice results in insulin resistance and increases in atherogenic plasma lipids and cholesterol, but protects from diet-induced obesity and atherosclerosis. Despite the fundamental role of the intestinal epithelia in the regulation of dietary lipid processing and metabolism, the contributions of CAV1 to lipid metabolism in this tissue have never been directly investigated. In this study the cellular dynamics of intestinal Cav1 were visualized in zebrafish and the metabolic contributions of CAV1 were determined with mice lacking CAV1 in intestinal epithelial cells (CAV1IEC-KO). Live imaging of Cav1–GFP and fluorescently labeled caveolae cargos shows localization to the basolateral and lateral enterocyte plasma membrane (PM), suggesting Cav1 mediates transport between enterocytes and the submucosa. CAV1IEC-KO mice are protected from the elevation in circulating fasted low-density lipoprotein (LDL) cholesterol associated with a high-fat diet (HFD), but have increased postprandial LDL cholesterol, total free fatty acids (FFAs), palmitoleic acid, and palmitic acid. The increase in circulating FAs in HFD CAV1IEC-KO mice is mirrored by decreased hepatic FAs, suggesting a non-cell-autonomous role for intestinal epithelial cell CAV1 in promoting hepatic FA storage. In conclusion, CAV1 regulates circulating LDL cholesterol and several FA species via the basolateral PM of enterocytes. These results point to intestinal epithelial cell CAV1 as a potential therapeutic target to lower circulating FFAs and LDL cholesterol, as high levels are associated with development of type II diabetes and cardiovascular disease. PMID:28130355

  7. The Importance of Caveolin-1 as Key-Regulator of Three-Dimensional Growth in Thyroid Cancer Cells Cultured under Real and Simulated Microgravity Conditions

    PubMed Central

    Riwaldt, Stefan; Bauer, Johann; Pietsch, Jessica; Braun, Markus; Segerer, Jürgen; Schwarzwälder, Achim; Corydon, Thomas J.; Infanger, Manfred; Grimm, Daniela

    2015-01-01

    We recently demonstrated that the CAV1 gene was down-regulated, when poorly differentiated thyroid FTC-133 cancer cells formed spheroids under simulated microgravity conditions. Here, we present evidence that the caveolin-1 protein is involved in the inhibition of spheroid formation, when confluent monolayers are exposed to microgravity. The evidence is based on proteins detected in cells and their supernatants of the recent spaceflight experiment: “NanoRacks-CellBox-Thyroid Cancer”. The culture supernatant had been collected in a special container adjacent to the flight hardware incubation chamber and stored at low temperature until it was analyzed by Multi-Analyte Profiling (MAP) technology, while the cells remaining in the incubation chamber were fixed by RNAlater and examined by mass spectrometry. The soluble proteins identified by MAP were investigated in regard to their mutual interactions and their influence on proteins, which were associated with the cells secreting the soluble proteins and had been identified in a preceding study. A Pathway Studio v.11 analysis of the soluble and cell-associated proteins together with protein kinase C alpha (PRKCA) suggests that caveolin-1 is involved, when plasminogen enriched in the extracellular space is not activated and the vascular cellular adhesion molecule (VCAM-1) mediated cell–cell adhesion is simultaneously strengthened and activated PRKCA is recruited in caveolae, while the thyroid cancer cells do not form spheroids. PMID:26633361

  8. The Importance of Caveolin-1 as Key-Regulator of Three-Dimensional Growth in Thyroid Cancer Cells Cultured under Real and Simulated Microgravity Conditions.

    PubMed

    Riwaldt, Stefan; Bauer, Johann; Pietsch, Jessica; Braun, Markus; Segerer, Jürgen; Schwarzwälder, Achim; Corydon, Thomas J; Infanger, Manfred; Grimm, Daniela

    2015-11-30

    We recently demonstrated that the CAV1 gene was down-regulated, when poorly differentiated thyroid FTC-133 cancer cells formed spheroids under simulated microgravity conditions. Here, we present evidence that the caveolin-1 protein is involved in the inhibition of spheroid formation, when confluent monolayers are exposed to microgravity. The evidence is based on proteins detected in cells and their supernatants of the recent spaceflight experiment: "NanoRacks-CellBox-Thyroid Cancer". The culture supernatant had been collected in a special container adjacent to the flight hardware incubation chamber and stored at low temperature until it was analyzed by Multi-Analyte Profiling (MAP) technology, while the cells remaining in the incubation chamber were fixed by RNAlater and examined by mass spectrometry. The soluble proteins identified by MAP were investigated in regard to their mutual interactions and their influence on proteins, which were associated with the cells secreting the soluble proteins and had been identified in a preceding study. A Pathway Studio v.11 analysis of the soluble and cell-associated proteins together with protein kinase C alpha (PRKCA) suggests that caveolin-1 is involved, when plasminogen enriched in the extracellular space is not activated and the vascular cellular adhesion molecule (VCAM-1) mediated cell-cell adhesion is simultaneously strengthened and activated PRKCA is recruited in caveolae, while the thyroid cancer cells do not form spheroids.

  9. Caveolin-1-Enhanced Motility and Focal Adhesion Turnover Require Tyrosine-14 but Not Accumulation to the Rear in Metastatic Cancer Cells

    PubMed Central

    Ortiz, Rina J.; Lobos, Lorena; Díaz, María I.; Díaz, Natalia; Härtel, Steffen; Leyton, Lisette; Quest, Andrew F. G.

    2012-01-01

    Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells. PMID:22505999

  10. Modified Panax ginseng Extract Inhibits uPAR-Mediated α[Formula: see text]β1-Integrin Signaling by Modulating Caveolin-1 to Induce Early Apoptosis in Lung Cancer Cells.

    PubMed

    Hwang, In-Hu; Kwon, Yong-Kyun; Cho, Chong-Kwan; Lee, Yeon-Weol; Sung, Jung-Suk; Joo, Jong-Cheon; Lee, Kyung-Bok; Yoo, Hwa-Seung; Jang, Ik-Soon

    2016-01-01

    Urokinase receptor (uPAR) is enhanced in many human cancer cells and is frequently an indicator of poor prognosis. Activation of [Formula: see text]1-integrin requires caveolin-1 and is regulated by uPAR. However, the underlying molecular mechanism responsible for the interaction between uPAR and [Formula: see text]1-integrin remains obscure. We found that modified regular Panax ginseng extract (MRGX) had a negative modulating effect on the uPAR/[Formula: see text]1-integrin interaction, disrupted the uPAR/integrin interaction by modulating caveoline-1, and caused early apoptosis in cancer cells. Additionally, we found that siRNA-mediated caveoline-1 downregulation inhibited uPAR-mediated [Formula: see text]1-integrin signaling, whereas caveoline-1 up-regulation stimulated the signaling, which suppressed p53 expression, thereby indicating negative crosstalk exists between the integrin [Formula: see text]1 and the p53 pathways. Thus, these findings identify a novel mechanism whereby the inhibition of [Formula: see text]1 integrin and the activation of p53 modulate the expression of the anti-apoptotic proteins that are crucially involved in inducing apoptosis in A549 lung cancer cells. Furthermore, MRGX causes changes in the expressions of members of the Bcl-2 family (Bax and Bcl-2) in a pro-apoptotic manner. In addition, MGRX-mediated inhibition of [Formula: see text]1 integrin attenuates ERK phosphorylation (p-ERK), which up-regulates caspase-8 and Bax. Therefore, ERK may affect mitochondria through a negative regulation of caspase-8 and Bax. Taken together, these findings reveal that MRGX is involved in uPAR-[Formula: see text]1-integrin signaling by modulating caveolin-1 signaling to induce early apoptosis in A549 lung-cancer cells and strongly indicate that MRGX might be useful as a herbal medicine and may lead to the development of new herbal medicine that would suppress the growth of lung-cancer cells.

  11. Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1

    PubMed Central

    Matsumura, Shigeru; Kojidani, Tomoko; Kamioka, Yuji; Uchida, Seiichi; Haraguchi, Tokuko; Kimura, Akatsuki; Toyoshima, Fumiko

    2016-01-01

    Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1–LGN–NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry. PMID:27292265

  12. Inhibition of macrophage-derived foam cell formation by ezetimibe via the caveolin-1/MAPK pathway.

    PubMed

    Qin, Li; Yang, Yun-Bo; Yang, Yi-Xin; Zhu, Neng; Liu, Zheng; Ni, Ya-Guang; Li, Shun-Xiang; Zheng, Xi-Long; Liao, Duan-Fang

    2016-02-01

    Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, effectively reduces plasma cholesterol, but its effect on atherosclerosis is unclear. Foam cell formation has been implicated as a key mediator during the development of atherosclerosis. The purpose of this study was to investigate the effects of ezetimibe on foam cell formation and explore the underlying mechanism. The results presented here show that ezetimibe reduces atherosclerotic lesions in apolipoprotein E deficient (apoE-/-) mice by lowering cholesterol levels. Treatment of macrophages with Chol:MβCD resulted in foam cell formation, which was concentration-dependently inhibited by the presence of ezetimibe. Mechanically, ezetimibe treatment downregulated the expression of CD36 and scavenger receptor class B1 (SR-B1), but upregulated the expression of apoE and caveolin-1 in macrophage-derived foam cells, which kept consistent with our microarray results. Moreover, treatment with ezetimibe abrogated the increase of phospho-extracellular signal regulated kinase (ERK) 1/2 and their nuclear accumulation in foam cells. Inhibition of the MAPK pathway by the MEK inhibitor PD98059 attenuated the inhibitory effect of ezetimibe on the expression of p-ERK1/2 and caveolin-1. Taken together, these results showed that ezetimibe suppressed foam cell formation via the caveolin-1/MAPK signalling pathway, suggesting that inhibition of foam cell formation might be a novel mechanism underlying the anti-atherosclerotic effect of ezetimibe.

  13. High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

    PubMed

    Parker, Katherine H; Horn, Lucas A; Ostrand-Rosenberg, Suzanne

    2016-09-01

    Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by

  14. CAVEOLIN-1 expression in brain metastasis from lung cancer predicts worse outcome and radioresistance, irrespective of tumor histotype.

    PubMed

    Duregon, Eleonora; Senetta, Rebecca; Pittaro, Alessandra; Verdun di Cantogno, Ludovica; Stella, Giulia; De Blasi, Pierpaolo; Zorzetto, Michele; Mantovani, Cristina; Papotti, Mauro; Cassoni, Paola

    2015-10-06

    Brain metastases develop in one-third of patients with non-small-cell lung cancer and are associated with a dismal prognosis, irrespective of surgery or chemo-radiotherapy. Pathological markers for predicting outcomes after surgical resection and radiotherapy responsiveness are still lacking. Caveolin 1 has been associated with chemo- and radioresistance in various tumors, including non-small-cell lung cancer. Here, caveolin 1 expression was assessed in a series of 69 brain metastases from non-small-cell lung cancer and matched primary tumors to determine its role in predicting survival and radiotherapy responsiveness. Only caveolin 1 expression in brain metastasis was associated with poor prognosis and an increased risk of death (log rank test, p = 0.015). Moreover, in the younger patients (median age of <54 years), caveolin 1 expression neutralized the favorable effect of young age on survival compared with the older patients. Among the radiotherapy-treated patients, an increased risk of death was detected in the group with caveolin 1-positive brain metastasis (14 out of 22 patients, HR=6.839, 95% CI 1.849 to 25.301, Wald test p = 0.004). Overall, caveolin 1 expression in brain metastasis from non-small-cell lung cancer is independently predictive of worse outcome and radioresistance and could become an additional tool for personalized therapy in the critical subset of brain-metastatic non-small-cell lung cancer patients.

  15. Caveolin-1 mediates inflammatory breast cancer cell invasion via the Akt1 pathway and RhoC GTPase.

    PubMed

    Joglekar, Madhura; Elbazanti, Weam O; Weitzman, Matthew D; Lehman, Heather L; van Golen, Kenneth L

    2015-06-01

    With a propensity to invade the dermal lymphatic vessels of the skin overlying the breast and readily metastasize, inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer. We previously reported that caveolin-1 is overexpressed in IBC and that RhoC GTPase is a metastatic switch responsible for the invasive phenotype. RhoC-driven invasion requires phosphorylation by Akt1. Using a reliable IBC cell line we set out to determine if caveolin-1 expression affects RhoC-mediated IBC invasion. Caveolin-1 was down regulated by introduction of siRNA or a caveolin scaffolding domain. The ability of the cells to invade was tested and the status of Akt1 and RhoC GTPase examined. IBC cell invasion is significantly decreased when caveolin-1 is down regulated. Activation of Akt1 is decreased when caveolin-1 is down regulated, leading to decreased phosphorylation of RhoC GTPase. Thus, we report here that caveolin-1 overexpression mediates IBC cell invasion through activation Akt1, which phosphorylates RhoC GTPase.

  16. Dissociation of Hyperglycemia from Altered Vascular Contraction and Relaxation Mechanisms in Caveolin-1 Null Mice

    PubMed Central

    Pojoga, Luminita H.; Yao, Tham M.; Opsasnick, Lauren A.; Garza, Amanda E.; Reslan, Ossama M.; Adler, Gail K.; Williams, Gordon H.

    2014-01-01

    Hyperglycemia and endothelial dysfunction are associated with hypertension, but the specific causality and genetic underpinning are unclear. Caveolin-1 (cav-1) is a plasmalemmal anchoring protein and modulator of vascular function and glucose homeostasis. Cav-1 gene variants are associated with reduced insulin sensitivity in hypertensive individuals, and cav-1−/− mice show endothelial dysfunction, hyperglycemia, and increased blood pressure (BP). On the other hand, insulin-sensitizing therapy with metformin may inadequately control hyperglycemia while affecting the vascular outcome in certain patients with diabetes. To test whether the pressor and vascular changes in cav-1 deficiency states are related to hyperglycemia and to assess the vascular mechanisms of metformin under these conditions, wild-type (WT) and cav-1−/− mice were treated with either placebo or metformin (400 mg/kg daily for 21 days). BP and fasting blood glucose were in cav-1−/− > WT and did not change with metformin. Phenylephrine (Phe)- and KCl-induced aortic contraction was in cav-1−/− < WT; endothelium removal, the nitric-oxide synthase (NOS) blocker l-NAME (Nω-nitro-l-arginine methyl ester), or soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced Phe contraction, and metformin blunted this effect. Acetylcholine-induced relaxation was in cav-1−/− > WT, abolished by endothelium removal, l-NAME or ODQ, and reduced with metformin. Nitric oxide donor sodium nitroprusside was more potent in inducing relaxation in cav-1−/− than in WT, and metformin reversed this effect. Aortic eNOS, AMPK, and sGC were in cav-1−/− > WT, and metformin decreased total and phosphorylated eNOS and AMPK in cav-1−/−. Thus, metformin inhibits both vascular contraction and NO-cGMP-dependent relaxation but does not affect BP or blood glucose in cav-1−/− mice, suggesting dissociation of hyperglycemia from altered vascular function in cav-1

  17. Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells

    PubMed Central

    Felley-Bosco, Emanuela; Bender, Florent C.; Courjault-Gautier, Françoise; Bron, Claude; Quest, Andrew F. G.

    2000-01-01

    To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway. PMID:11114180

  18. Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

    PubMed

    Felley-Bosco, E; Bender, F C; Courjault-Gautier, F; Bron, C; Quest, A F

    2000-12-19

    To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

  19. Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesis

    SciTech Connect

    Robb, Victoria A.; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H.

    2003-06-11

    Meningiomas are common tumors of the central nervous system, however, the mechanisms under lying their pathogenesis are largely undefined. Two members of the Protein 4.1 super family, the neuro fibromatosis 2 (NF2) gene product (merlin/schwannomin) and Protein 4.1B have been implicated as meningioma tumor suppressors. In this report, we demonstrate that another Protein 4.1 family member, Protein 4.1R, also functions as a meningioma tumor suppressor. Based on the assignment of the Protein 4.1R gene to chromosome 1p32-36, a common region of deletion observed in meningiomas, we analyzed Protein 4.1R expression in meningioma cell lines and surgical tumor specimens. We observed loss of Protein 4.1R protein expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningioma as by immuno histo chemistry (IHC). Analysis of a subset of these sporadic meningiomas by fluorescent in situ hybridization (FISH) with a Protein 4.1R specific probe demonstrated 100 percent concordance with the IHC results. In support of a meningioma tumor suppressor function, over expression of Protein 4.1R resulted in suppression of IOMM-Lee and CH157MN cell proliferation. Similar to the Protein 4.1B and merlin meningioma tumor suppressors, Protein 4.1R localization in the membrane fraction increased significantly under conditions of growth arrest in vitro. Lastly, Protein 4.1R interacted with some known merlin/Protein 4.1B interactors such as CD44 and bII-spectrin, but did not associate with the Protein 4.1B interactors 14-3-3 and PRMT3 or the merlin binding proteins SCHIP-1 and HRS. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor important in the molecular pathogenesis of meningioma.

  20. Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations.

    PubMed

    Ritz, Danilo; Vuk, Maja; Kirchner, Philipp; Bug, Monika; Schütz, Sabina; Hayer, Arnold; Bremer, Sebastian; Lusk, Caleb; Baloh, Robert H; Lee, Houkeun; Glatter, Timo; Gstaiger, Matthias; Aebersold, Ruedi; Weihl, Conrad C; Meyer, Hemmo

    2011-08-07

    The AAA-ATPase VCP (also known as p97) cooperates with distinct cofactors to process ubiquitylated proteins in different cellular pathways. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds to the plasma membrane protein caveolin-1 (CAV1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitylated CAV1 in SDS-resistant high-molecular-weight complexes on endosomes, which are en route to degradation in endolysosomes. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific caveolin-3 accumulates in sarcoplasmic pools and specifically delocalizes from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations.

  1. Triptolide inhibits the migration and invasion of human prostate cancer cells via Caveolin-1/CD147/MMPs pathway.

    PubMed

    Yuan, Shiqi; Wang, Liping; Chen, Xixi; Fan, Bo; Yuan, Qingmin; Zhang, Han; Yang, Deyong; Wang, Shujing

    2016-12-01

    Prostate cancer (PCa) is the second most common type of carcinoma and the 5th leading cause of cancer-related death in males. Triptolide, is a main and effective component of Tripterygium wilfordii Hook F, which exerts an broad-spectrum anti-malignant tumor function. However, the effect of triptolide on migration and invasion of human prostate cancer cells is still poorly understood. In this study, we demonstrated that triptolide significantly inhibited the proliferation, migration and invasion of prostate cancer cells in a time- and dose-dependent manner. Caveolin-1 (Cav-1) is regarded as a major structural protein of caveolae and participated in lipid transport, signal transduction and tumor progression. Triptolide treatment inhibited the expression of tumor promoter Cav-1 and reduced CD147 and MMPs activities at both mRNA and protein levels. Meanwhile, triptolide treatment combined with Cav-1 knockdown in PCa cells enhanced the effects of anti-migration and anti-invasion, and those effects were restored following Cav-1-rescued. Together, our research indicates that triptolide represses the migration and invasion through Cav-1/CD147/MMPs pathway in PCa cells, which gives a better understanding of triptolide in clinical aggressive prostate cancer therapy.

  2. Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes.

    PubMed

    He, Kangmin; Yan, Xiaohua; Li, Nan; Dang, Song; Xu, Li; Zhao, Bing; Li, Zijian; Lv, Zhizhen; Fang, Xiaohong; Zhang, Youyi; Chen, Ye-Guang

    2015-06-01

    Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.

  3. Downregulation of caveolin-1 increases the sensitivity of drug-resistant colorectal cancer HCT116 cells to 5-fluorouracil

    PubMed Central

    Li, Zhaoyang; Wang, Ning; Huang, Changxin; Bao, Yanhong; Jiang, Yiqian; Zhu, Guiting

    2017-01-01

    Colorectal cancer is the third most common type of cancer in men and women. Chemotherapy is an important treatment strategy for patients with terminal stage cancer. However, the development of drug resistance hampers the effectiveness of chemotherapy. Therefore, an effective therapeutic approach to target chemoresistance-associated cellular molecules is required. In the present study, drug-resistant human colorectal cancer HCT116 cells were developed by treating HCT116 cells with increasing concentrations of 5-fluorouracil (5-FU). The present study indicated that the drug-resistance cells (DRC) were resistant to 5-FU compared with parental HCT116 cells by detecting cell survival using an MTT assay. Additionally, the expression of the chemoresistance-associated protein caveolin-1 (Cav-1) was assessed by reverse transcription-quantitative polymerase chain reaction and western blotting. The results revealed that the Cav-1 expression level was significantly higher in DRC compared with that in the parental HCT116 cells. Next, Cav-1 was silenced by small interfering RNA (siRNA) or was inhibited with its specific inhibitor methyl β-cyclodextrin (MCD). MTT assay demonstrated that Cav-1 siRNA and MCD resensitized DRC to 5-FU. These data reveal that Cav-1 was involved in the development of resistance, suggesting that Cav-1 is a potential target for the treatment of colorectal cancer chemoresistance. In addition, 5-FU combined with Cav-1 siRNA or its specific inhibitor may increase the effectiveness of the treatment strategy. PMID:28123586

  4. Effects of high glucose on caveolin-1 and insulin signaling in 3T3-L1 adipocytes

    PubMed Central

    Palacios-Ortega, Sara; Varela-Guruceaga, Maider; Martínez, J. Alfredo; de Miguel, Carlos; Milagro, Fermín I.

    2016-01-01

    ABSTRACT Adipocytes exposed to high glucose concentrations exhibit impaired metabolic function, including an increase of oxidative and proinflammatory factors that might favor the development of insulin resistance. Caveolin-1 (Cav-1) is a key mediator of the insulin transduction pathway whose expression is significantly enhanced during adipocyte differentiation. In this work, we studied the effects of high glucose concentration on the regulation of Cav-1 expression and activation and its relation to the insulin signaling pathway during the adipogenic process and in long-term differentiated adipocytes. Both, long-term high glucose exposure during adipogenesis and short-term glucose incubation of mature adipocytes, promoted triglyceride accumulation in 3T3-L1 cells. The short-term exposure of mature adipocytes to high glucose significantly reduced the sensitivity to insulin of Cav-1, insulin receptor (IR) and potein kinase B (AKT-2) phosphorylation, as well as insulin-induced deoxyglucose uptake. Adipocytes differentiated in the presence of high glucose lost Cav-1 and IR response to insulin-stimulated phosphorylation, but maintained the insulin sensitivity of AKT-2 phosphorylation and deoxyglucose uptake. Although long-term high glucose exposure increased DNA methylation in Cav-1 promoter, Cav-1 expression was not affected. Moreover, these cells showed an increase of Cav-1, IR and AKT-2 protein content, pointing to an adaptive response induced by the long-term high glucose exposure. PMID:27144098

  5. Effects of high glucose on caveolin-1 and insulin signaling in 3T3-L1 adipocytes.

    PubMed

    Palacios-Ortega, Sara; Varela-Guruceaga, Maider; Martínez, J Alfredo; de Miguel, Carlos; Milagro, Fermín I

    2016-01-01

    Adipocytes exposed to high glucose concentrations exhibit impaired metabolic function, including an increase of oxidative and proinflammatory factors that might favor the development of insulin resistance. Caveolin-1 (Cav-1) is a key mediator of the insulin transduction pathway whose expression is significantly enhanced during adipocyte differentiation. In this work, we studied the effects of high glucose concentration on the regulation of Cav-1 expression and activation and its relation to the insulin signaling pathway during the adipogenic process and in long-term differentiated adipocytes. Both, long-term high glucose exposure during adipogenesis and short-term glucose incubation of mature adipocytes, promoted triglyceride accumulation in 3T3-L1 cells. The short-term exposure of mature adipocytes to high glucose significantly reduced the sensitivity to insulin of Cav-1, insulin receptor (IR) and potein kinase B (AKT-2) phosphorylation, as well as insulin-induced deoxyglucose uptake. Adipocytes differentiated in the presence of high glucose lost Cav-1 and IR response to insulin-stimulated phosphorylation, but maintained the insulin sensitivity of AKT-2 phosphorylation and deoxyglucose uptake. Although long-term high glucose exposure increased DNA methylation in Cav-1 promoter, Cav-1 expression was not affected. Moreover, these cells showed an increase of Cav-1, IR and AKT-2 protein content, pointing to an adaptive response induced by the long-term high glucose exposure.

  6. Caveolin-1 knockdown is associated with the metastasis and proliferation of human lung cancer cell line NCI-H460.

    PubMed

    Song, Yang; Xue, Liyan; Du, Sha; Sun, Mingzhong; Hu, Jun; Hao, Lihong; Gong, Linlin; Yeh, Dongmei; Xiong, Hai; Shao, Shujuan

    2012-09-01

    Caveolin-1 (CAV-1), one component of caveolae, involves in multiple cellular processes and signal transductions. We previously showed that the expression of CAV-1 gene in NCI-H446 cells inhibited cell proliferation and promoted cell metastasis. Here we explore the function of CAV-1 on tumor growth and metastasis by using NCI-H460 in vitro. First, we established NCI-H460 cell line, which CAV-1 was stably knockdown. Then we investigated the effects of CAV-1 on the morphology, proliferation, cell cycle and metastasis potential for NCI-H460 cell by crystal violet stains, CCK-8, colony formation, flow cytometry, scratch-wound assay and transwell assay. Western blot was used to examine the expression changes of cyclin D1, PCNA, E-cadherin and β-catenin. Our results showed stable knockdown of CAV-1 inhibited the proliferation of NCI-H460 cells. Cell cycle of the transfected cells was arrested in G1/S phase and the expressions of cyclin D1 and PCNA protein were downregulated. Downregulation of CAV-1 promoted the migration and invasion abilities of NCI-H460 cells in vitro. The expression of β-catenin increased and the level of E-cadherin decreased. In summary, our findings provide experimental evidence that CAV-1 may function as a proproliferative and antimetastatic gene in NCI-H460 cell line.

  7. Functional association between Wwox tumor suppressor protein and p73, a p53 homolog.

    PubMed

    Aqeilan, Rami I; Pekarsky, Yuri; Herrero, Juan J; Palamarchuk, Alexey; Letofsky, Jean; Druck, Teresa; Trapasso, Francesco; Han, Shuang-Yin; Melino, Gerry; Huebner, Kay; Croce, Carlo M

    2004-03-30

    The WWOX gene is a recently cloned tumor suppressor gene that spans the FRA16D fragile region. Wwox protein contains two WW domains that are generally known to mediate protein-protein interaction. Here we show that Wwox physically interacts via its first WW domain with the p53 homolog, p73. The tyrosine kinase, Src, phosphorylates Wwox at tyrosine 33 in the first WW domain and enhances its binding to p73. Our results further demonstrate that Wwox expression triggers redistribution of nuclear p73 to the cytoplasm and, hence, suppresses its transcriptional activity. In addition, we show that cytoplasmic p73 contributes to the proapoptotic activity of Wwox. Our findings reveal a functional cross-talk between p73 and Wwox tumor suppressor protein.

  8. Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein*

    PubMed Central

    Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R. B.; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C.; Mackey, John; Baksh, Shairaz

    2015-01-01

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis. PMID:26269600

  9. Involvement of regucalcin as a suppressor protein in human carcinogenesis: insight into the gene therapy.

    PubMed

    Yamaguchi, Masayoshi

    2015-08-01

    Regucalcin, which its gene is located on the X chromosome, plays a multifunctional role as a suppressor protein in cell signal transduction in various types of cells and tissues. The suppression of regucalcin gene expression has been shown to involve in carcinogenesis. Regucalcin gene expression was uniquely downregulated in carcinogenesis of rat liver in vivo, although the expression of other many genes was upregulated, indicating that endogenous regucalcin plays a suppressive role in the development of hepatocarcinogenesis. Overexpression of endogenous regucalcin was found to suppress proliferation of rat cloned hepatoma cells in vitro. Moreover, the regucalcin gene and its protein levels were demonstrated specifically to downregulate in human hepatocellular carcinoma by analysis with multiple gene expression profiles and proteomics. Regucalcin gene expression was also found to suppress in human tumor tissues including kidney, lung, brain, breast and prostate, suggesting that repressed regucalcin gene expression leads to the development of carcinogenesis in various tissues. Regucalcin may play a role as a suppressor protein in carcinogenesis. Overexpression of endogenous regucalcin is suggested to reveal preventive and therapeutic effects on carcinogenesis. Delivery of the regucalcin gene may be a novel useful tool in the gene therapy of carcinogenesis. This review will discuss regarding to an involvement of regucalcin as a suppressor protein in human carcinogenesis in insight into the gene therapy.

  10. Interplay between hepatic mitochondria-associated membranes, lipid metabolism and caveolin-1 in mice.

    PubMed

    Sala-Vila, Aleix; Navarro-Lérida, Inmaculada; Sánchez-Alvarez, Miguel; Bosch, Marta; Calvo, Carlos; López, Juan Antonio; Calvo, Enrique; Ferguson, Charles; Giacomello, Marta; Serafini, Annalisa; Scorrano, Luca; Enriquez, José Antonio; Balsinde, Jesús; Parton, Robert G; Vázquez, Jesús; Pol, Albert; Del Pozo, Miguel A

    2016-06-06

    The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed.

  11. Interplay between hepatic mitochondria-associated membranes, lipid metabolism and caveolin-1 in mice

    PubMed Central

    Sala-Vila, Aleix; Navarro-Lérida, Inmaculada; Sánchez-Alvarez, Miguel; Bosch, Marta; Calvo, Carlos; López, Juan Antonio; Calvo, Enrique; Ferguson, Charles; Giacomello, Marta; Serafini, Annalisa; Scorrano, Luca; Enriquez, José Antonio; Balsinde, Jesús; Parton, Robert G.; Vázquez, Jesús; Pol, Albert; Del Pozo, Miguel A.

    2016-01-01

    The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed. PMID:27272971

  12. Regulation of protein phosphatase 2A (PP2A) tumor suppressor function by PME-1.

    PubMed

    Kaur, Amanpreet; Westermarck, Jukka

    2016-12-15

    Protein phosphatase 2A (PP2A) plays a major role in maintaining cellular signaling homeostasis by dephosphorylation of a variety of signaling proteins and acts as a tumor suppressor. Protein phosphatase methylesterase-1 (PME-1) negatively regulates PP2A activity by highly complex mechanisms that are reviewed here. Importantly, recent studies have shown that PME-1 promotes oncogenic MAPK/ERK and AKT pathway activities in various cancer types. In human glioma, high PME-1 expression correlates with tumor progression and kinase inhibitor resistance. We discuss the emerging cancer-associated function of PME-1 and its potential clinical relevance.

  13. Caveolin-1 plays a key role in the oleanolic acid-induced apoptosis of HL-60 cells.

    PubMed

    Ma, Wei; Wang, Di-Di; Li, Li; Feng, Yu-Kuan; Gu, Hong-Mei; Zhu, Gui-Ming; Piao, Jin-Hua; Yang, Yu; Gao, Xu; Zhang, Peng-Xia

    2014-07-01

    Our previous study found that caveolin-1 (CAV-1) protein expression is upregulated during oleanolic acid (OA)-induced inhibition of proliferation and promotion of apoptosis in HL-60 cells. CAV-1 is the main structural protein component of caveolae, playing important roles in tumorigenesis and tumor development. It has been shown that cav-1 expression is lower in leukemia cancer cell lines SUP-B15, HL-60, THP-1 and K562 and in chronic lymphocytic leukemia primary (CLP) cells when compared with normal white blood cells, with the lowest cav-1 expression level found in HL-60 cells. To study the effects of cav-1 in HL-60 cells and the effects of cav-1 overexpression on OA drug efficacy, cav-1 was overexpressed in HL-60 cells using lentiviral-mediated transfection combined with OA treatment. The results showed that cav-1 overexpression inhibited HL-60 cell proliferation, promoted apoptosis, arrested the cell cycle in the G1 phase and inhibited activation of the PI3K/AKT/mTOR signaling pathway. Overexpression of CAV-1 also increased HL-60 cell sensitivity to OA. To further verify whether OA affects HL-60 cells via the activation of downstream signaling pathways by CAV-1, cav-1 gene expression was silenced using RNAi, and the cells were treated with OA to examine its efficacy. The results showed that after cav-1 silencing, OA had little effect on cell activity, apoptosis, the cell cycle and phosphorylation of HL-60 cells. This study is the first to show that CAV-1 plays a crucial role in the effects of OA on HL-60 cells.

  14. Inflammatory cytokines induce phosphorylation and ubiquitination of prostate suppressor protein NKX3.1.

    PubMed

    Markowski, Mark C; Bowen, Cai; Gelmann, Edward P

    2008-09-01

    Inflammation of the prostate is a risk factor for the development of prostate cancer. In the aging prostate, regions of inflammatory atrophy are foci for prostate epithelial cell transformation. Expression of the suppressor protein NKX3.1 is reduced in regions of inflammatory atrophy and in preinvasive prostate cancer. Inflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1beta accelerate NKX3.1 protein loss by inducing rapid ubiquitination and proteasomal degradation. The effect of TNF-alpha is mediated via the COOH-terminal domain of NKX3.1 where phosphorylation of serine 196 is critical for cytokine-induced degradation. Mutation of serine 196 to alanine abrogates phosphorylation at that site and the effect of TNF-alpha on NKX3.1 ubiquitination and protein loss. This is in contrast to control of steady-state NKX3.1 turnover, which is mediated by serine 185. Mutation of serine 185 to alanine increases NKX3.1 protein stability by inhibiting ubiquitination and doubling the protein half-life. A third COOH-terminal serine at position 195 has a modulating effect on both steady-state protein turnover and on ubiquitination induced by TNF-alpha. Thus, cellular levels of the NKX3.1 tumor suppressor are affected by inflammatory cytokines that target COOH-terminal serine residues to activate ubiquitination and protein degradation. Our data suggest that strategies to inhibit inflammation or to inhibit effector kinases may be useful approaches to prostate cancer prevention.

  15. Increased PDE5 activity and decreased Rho kinase and PKC activities in colonic muscle from caveolin-1-/- mice impair the peristaltic reflex and propulsion.

    PubMed

    Mahavadi, Sunila; Bhattacharya, Sayak; Kumar, Divya P; Clay, Chereena; Ross, Gracious; Akbarali, Hamid I; Grider, John R; Murthy, Karnam S

    2013-12-01

    Caveolae are specialized regions of the plasma membrane that concentrate receptors and associated signaling molecules critical in regulation of cellular response to transmitters and hormones. We have determined the effects of caveolin-1 (Cav-1) deletion, caveolin-1 siRNA, and caveolar disruption in mice on the signaling pathways that mediate contraction and relaxation in colonic smooth muscle and on the components of the peristaltic reflex in isolated tissue and propulsion in intact colonic segments. In Cav-1-/- mice, both relaxation and contraction were decreased in smooth muscle cells and muscle strips, as well as during both phases of the peristaltic reflex and colonic propulsion. The decrease in relaxation in response to the nitric oxide (NO) donor was accompanied by a decrease in cGMP levels and an increase in phosphodiesterase 5 (PDE5) activity. Relaxation by a PDE5-resistant cGMP analog was not affected in smooth muscle of Cav-1-/- mice, suggesting that inhibition of relaxation was due to augmentation of PDE5 activity. Similar effects on relaxation, PDE5 and cGMP were obtained in muscle cells upon disruption of caveolae by methyl-β-cyclodextrin or suppression of Cav-1. Sustained contraction mediated via inhibition of myosin light chain phosphatase (MLCP) activity is regulated by Rho kinase and PKC via phosphorylation of two endogenous inhibitors of MLCP: myosin phosphatase-targeting subunit (MYPT1) and 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), respectively. The activity of both enzymes and phosphorylation of MYPT1 and CPI-17 were decreased in smooth muscle from Cav-1-/- mice. We conclude that the integrity of caveolae is essential for contractile and relaxant activity in colonic smooth muscle and the maintenance of neuromuscular function at organ level.

  16. Reduced caveolin-1 promotes hyper-inflammation due to abnormal heme oxygenase-1 localizationin LPS challenged macrophages with dysfunctional CFTR

    PubMed Central

    Zhang, Ping-Xia; Murray, Thomas S.; Villella, Valeria Rachela; Ferrari, Eleonora; Esposito, Speranza; D'Souza, Anthony; Raia, Valeria; Maiuri, Luigi; Krause, Diane S.; Egan, Marie E.; Bruscia, Emanuela M.

    2013-01-01

    We have previously reported that TLR4 signaling is increased in lipopolysaccharide (LPS) -stimulated Cystic Fibrosis (CF) macrophages (MΦs), contributing to the robust production of pro-inflammatory cytokines. The heme oxygenase (HO-1)/carbon monoxide (CO) pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. Here, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules (CORM2)enhancesCAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations reestablished HO-1 and CAV-1 cell surface localization in CF MΦ's. Consistent with restoration of HO-1/CAV-1 negative regulation of TLR4 signaling, genetic or pharmacological (CORM2)-induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counter-regulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ's response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease. PMID:23606537

  17. p53 Protein Is a Suppressor of Papillomavirus DNA Amplificational Replication

    PubMed Central

    Lepik, Dina; Ilves, Ivar; Kristjuhan, Arnold; Maimets, Toivo; Ustav, Mart

    1998-01-01

    p53 protein was able to block human and bovine papillomavirus DNA amplificational replication while not interfering with Epstein-Barr virus oriP once-per-cell cycle replication. Oligomerization, intact DNA-binding, replication protein A-binding, and proline-rich domains of the p53 protein were essential for efficient inhibition, while the N-terminal transcriptional activation and C-terminal regulatory domains were dispensable for the suppressor activity of the p53 protein. The inhibition of replication was caused neither by the downregulation of expression of the E1 and E2 proteins nor by cell cycle block or apoptosis. Our data suggest that the intrinsic activity of p53 to suppress amplificational replication of the papillomavirus origin may have an important role in the virus life cycle and in virus-cell interactions. PMID:9658131

  18. Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells

    PubMed Central

    Liu, Jie; Weaver, John; Jin, Xinchun; Zhang, Yuan; Xu, Ji; Liu, Ke J.; Li, Weiping; Liu, Wenlan

    2017-01-01

    Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation. PMID:26515186

  19. Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells.

    PubMed

    Liu, Jie; Weaver, John; Jin, Xinchun; Zhang, Yuan; Xu, Ji; Liu, Ke J; Li, Weiping; Liu, Wenlan

    2016-11-01

    Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation.

  20. Hydroxylation-Dependent Interaction of Substrates to the Von Hippel-Lindau Tumor Suppressor Protein (VHL).

    PubMed

    Heir, Pardeep; Ohh, Michael

    2016-01-01

    Oxygen-dependent hydroxylation of critical proline residues, catalyzed by prolyl hydroxylase (PHD1-3) enzymes, is a crucial posttranslational modification (PTM) within the canonical hypoxia-inducible factor (HIF)-centric cellular oxygen-sensing pathway. Alteration of substrates in this way often leads to proteasomal degradation mediated by the von Hippel-Lindau Tumor Suppressor protein (VHL) containing E3-ubiquitin ligase complex known as ECV (Elongins B/C, CUL2, VHL). Here, we outline in vitro protocols to demonstrate the ability of VHL to bind to a prolyl-hydroxylated substrate.

  1. [HINT1--a novel tumor suppressor protein of the HIT superfamily].

    PubMed

    Ozga, Magdalena

    2010-01-01

    The histidine triad nucleotide binding protein1 (Hint1) belongs to the first branch of the HIT superfamily. Hint1 catalyses the process of hydrolysis of the P-N bond in AMP-lysine, AMP-alanine, AMP-NH2. The physiological role of this enzyme is still unclear. There is accumulating evidence that HINT1 is a novel tumor suppressor protein, albeit the mechanism of action of HINT1 in respect to tumor suppression is not fully understood. Recent findings have shown that Hint1 inhibits the activity of the transcription factors AP1, MITF and USF2, as well as influences the transcription process of some genes of Wnt/beta-catenin pathway. Thereby, it seems that Hint1 exerts its major cellular function as gene transcription regulator, and thus, this function provides its potential role as a tumor suppressor protein. The clinical relevance of impairments in the Hint1 expression with the respect to specific human cancers is still a matter of extensive studies.

  2. Mobility of the von Hippel-Lindau tumour suppressor protein is regulated by kinesin-2.

    PubMed

    Mans, Dorus A; Lolkema, Martijn P; van Beest, Moniek; Daenen, Laura G; Voest, Emile E; Giles, Rachel H

    2008-04-01

    The von Hippel-Lindau tumour suppressor protein (pVHL) participates in many cellular processes including oxygen sensing, microtubule stability and primary cilia regulation. Recently, we identified ATP-dependent motor complex kinesin-2 to endogenously bind the full-length variant of VHL (pVHL30) in primary kidney cells, and mediate its association to microtubules. Here we show that pVHL also endogenously binds the neuronal kinesin-2 complex, which slightly differs from renal kinesin-2. To investigate the role of kinesin-2 in pVHL mobility, we performed fluorescence recovery after photobleaching (FRAP) experiments in neuroblastoma cells. We observe that pVHL30 is a highly mobile cytoplasmic protein, which becomes an immobile centrosomal protein after ATP-depletion in living cells. This response to ATP-depletion is independent of GSK3beta-dependent phosphorylation of pVHL30. Furthermore, VHL variant alleles with reduced binding to kinesin-2 fail to respond to ATP-depletion. Accordingly, interfering with pVHL30-KIF3A interaction by either overexpressing a dominant negative construct or by reducing endogenous cellular levels of KIF3A by RNAi abolishes pVHL's response to ATP-depletion. From these data we suggest that mobility of a subcellular pool of pVHL is regulated by the ATP-dependent kinesin-2 motor. Kinesin-2 driven mobility of cytoplasmic pVHL might enable pVHL to function as a tumour suppressor.

  3. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells.

    PubMed

    Lobos-González, Lorena; Aguilar, Lorena; Diaz, Jorge; Diaz, Natalia; Urra, Hery; Torres, Vicente A; Silva, Veronica; Fitzpatrick, Christopher; Lladser, Alvaro; Hoek, Keith S; Leyton, Lisette; Quest, Andrew F G

    2013-07-01

    The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells.

  4. Significance of caveolin-1 and matrix metalloproteinase 14 gene expression in canine mammary tumours.

    PubMed

    Ebisawa, M; Iwano, H; Nishikawa, M; Tochigi, Y; Komatsu, T; Endou, Y; Hirayama, K; Taniyama, H; Kadosawa, T; Yokota, H

    2015-11-01

    Canine mammary tumours (CMTs) are the most common neoplasms affecting female dogs. There is an urgent need for molecular biomarkers that can detect early stages of the disease in order to improve accuracy of CMT diagnosis. The aim of this study was to examine whether caveolin-1 (Cav-1) and matrix metalloproteinase 14 (MMP14) are associated with CMT histological malignancy and invasion. Sixty-five benign and malignant CMT samples and six normal canine mammary glands were analysed using quantitative reverse transcription-polymerase chain reaction. Cav-1 and MMP14 genes were highly expressed in CMT tissues compared to normal tissues. Cav-1 especially was overexpressed in malignant and invasive CMT tissues. When a CMT cell line was cultured on fluorescent gelatin-coated coverslips, localisation of Cav-1 was observed at invadopodia-mediated degradation sites of the gelatin matrix. These findings suggest that Cav-1 may be involved in CMT invasion and that the markers may be useful for estimating CMT malignancy.

  5. Caveolin-1 Expression Level in Cancer Associated Fibroblasts Predicts Outcome in Gastric Cancer

    PubMed Central

    Gao, Jun; Fan, Lifang; Li, Zonghuan; Yang, Guifang; Chen, Honglei

    2013-01-01

    Aims Altered expression of epithelial or stromal caveolin-1 (Cav-1) is observed in various types of human cancers. However, the clinical significance of Cav-1 expression in gastric cancer (GC) remains largely unknown. The present study aims to explore the clinicopathological significance and prognostic value of both tumor cells and cancer associated fibroblasts (CAFs) Cav-1 in GC. Methods and Results Quantum dots immunofluorescence histochemistry was performed to examine the expression of Cav-1 in 20 cases of gastritis without intestinal metaplasia (IM), 20 cases of gastritis with IM and 286 cases of GC. Positive rates of epithelial Cav-1 in gastritis without IM, gastritis with IM and GC showed a decreasing trend (P = 0.012). Low expression of Cav-1 in CAFs but not in tumor cells was an independent predictor of poor prognosis in GC patients (P = 0.034 and 0.005 respectively in disease free survival and overall survival). Cav-1 level in tumor cells and CAFs showed no significant correlation with classic clinicopathological features. Conclusions Loss of epithelial Cav-1 may promote malignant progression and low CAFs Cav-1 level herald worse outcome of GC patient, suggesting CAFs Cav-1 may be a candidate therapeutic target and a useful prognostic marker of GC. PMID:23527097

  6. Paired hormone response elements predict caveolin-1 as a glucocorticoid target gene.

    PubMed

    van Batenburg, Marinus F; Li, Hualing; Polman, J Annelies; Lachize, Servane; Datson, Nicole A; Bussemaker, Harmen J; Meijer, Onno C

    2010-01-21

    Glucocorticoids act in part via glucocorticoid receptor binding to hormone response elements (HREs), but their direct target genes in vivo are still largely unknown. We developed the criterion that genomic occurrence of paired HREs at an inter-HRE distance less than 200 bp predicts hormone responsiveness, based on synergy of multiple HREs, and HRE information from known target genes. This criterion predicts a substantial number of novel responsive genes, when applied to genomic regions 10 kb upstream of genes. Multiple-tissue in situ hybridization showed that mRNA expression of 6 out of 10 selected genes was induced in a tissue-specific manner in mice treated with a single dose of corticosterone, with the spleen being the most responsive organ. Caveolin-1 was strongly responsive in several organs, and the HRE pair in its upstream region showed increased occupancy by glucocorticoid receptor in response to corticosterone. Our approach allowed for discovery of novel tissue specific glucocorticoid target genes, which may exemplify responses underlying the permissive actions of glucocorticoids.

  7. Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase

    PubMed Central

    Cai, Ting; Wang, Haojie; Chen, Yiliang; Liu, Lijun; Gunning, William T; Quintas, Luis Eduardo M.; Xie, Zi-Jian

    2008-01-01

    Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool. PMID:18794328

  8. Loss of Caveolin-1 Impairs Retinal Function Due to Disturbance of Subretinal Microenvironment*

    PubMed Central

    Li, Xiaoman; McClellan, Mark E.; Tanito, Masaki; Garteiser, Philippe; Towner, Rheal; Bissig, David; Berkowitz, Bruce A.; Fliesler, Steven J.; Woodruff, Michael L.; Fain, Gordon L.; Birch, David G.; Khan, M. Suhaib; Ash, John D.; Elliott, Michael H.

    2012-01-01

    Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K+ affinity of the RPE-expressed α1-Na+/K+-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis. PMID:22451674

  9. Growth suppression by ursodeoxycholic acid involves caveolin-1 enhanced degradation of EGFR

    PubMed Central

    Feldman, Rebecca; Martinez, Jesse D.

    2009-01-01

    Summary Ursodeoxycholic acid (UDCA) has been shown to prevent colon tumorigenesis in animal models and in humans. In vitro work indicates that this bile acid can suppress cell growth and mitogenic signaling suggesting that UDCA may be an anti-proliferative agent. However, the mechanism by which UDCA functions is unclear. Previously we showed that bile acids may alter cellular signaling by acting at the plasma membrane. Here we utilized EGFR as a model membrane receptor and examined the effects that UDCA has on its functioning. We found that UDCA promoted an interaction between EGFR and caveolin-1 and this interaction enhanced UDCA-mediated suppression of MAP kinase activity and cell growth . Importantly, UDCA treatment led to recruitment of the ubiquitin ligase, c-Cbl, to the membrane, ubiquitination of EGFR, and increased receptor degradation. Moreover, suppression of c-Cbl activity abrogated UDCA's growth suppression activities suggesting that receptor ubiquitination plays an important role in UDCA's biological activities. Taken together these results suggest that UDCA may act to suppress cell growth by inhibiting the mitogenic activity of receptor tyrosine kinases such as EGFR through increased receptor degradation. PMID:19446582

  10. Caveolin-1–dependent apoptosis induced by fibrin degradation products

    PubMed Central

    Guo, Yi-He; Hernandez, Irene; Isermann, Berend; Kang, Tae-bong; Medved, Leonid; Sood, Rashmi; Kerschen, Edward J.; Holyst, Trudy; Mosesson, Michael W.

    2009-01-01

    In mice lacking the blood coagulation regulator thrombomodulin, fibrinolytic degradation products (FDP) of fibrin induce apoptotic cell death of a specialized cell type in the placenta, polyploid trophoblast giant cells. Here, we document that this bioactivity of FDP is conserved in human FDP, is not limited to trophoblast cells, and is associated with an Aα-chain segment of fibrin fragment E (FnE). The majority of proapoptotic activity is arginine-glycine-aspartic acid (RGD)-independent and requires caveolin-1–dependent cellular internalization of FnE. Internalization through caveoli is mediated by an epitope contained within Aα52-81 that is necessary and sufficient for cellular uptake of FnE. Aα52-81 does not cause apoptosis itself, and competitively inhibits FnE internalization and apoptosis induction. Apoptotic activity per se resides within Aα17-37 and requires the N-terminal neoepitope generated by release of fibrinopeptide A. Cellular internalization of FnE elicits depression of mitochondrial function and consequent apoptosis that is strictly dependent on the activity of caspases 9 and 3. These findings describe the molecular details of a novel mechanism linking fibrin degradation to cell death in the placenta, which may also contribute to pathologic alterations in nonplacental vascular beds that are associated with fibrinolysis. PMID:19074731

  11. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells

    PubMed Central

    Lobos-González, L; Aguilar, L; Diaz, J; Diaz, N; Urra, H; Torres, V; Silva, V; Fitzpatrick, C; Lladser, A; Hoek, K.S.; Leyton, L; Quest, AFG

    2013-01-01

    SUMMARY The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis, and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10(cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10(E-cad) cells reduces subcutaneous tumor formation, and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10(cav1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity and cell migration observed with B16F10(cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells. PMID:23470013

  12. Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide.

    PubMed

    Pleasant-Jenkins, Dorea; Reese, Charles; Chinnakkannu, Panneerselvem; Kasiganesan, Harinath; Tourkina, Elena; Hoffman, Stanley; Kuppuswamy, Dhandapani

    2017-04-01

    Chronic ventricular pressure overload (PO) results in congestive heart failure (CHF) in which myocardial fibrosis develops in concert with ventricular dysfunction. Caveolin-1 is important in fibrosis in various tissues due to its decreased expression in fibroblasts and monocytes. The profibrotic effects of low caveolin-1 can be blocked with the caveolin-1 scaffolding domain peptide (CSD, a caveolin-1 surrogate) using both mouse models and human cells. We have studied the beneficial effects of CSD on mice in which PO was induced by trans-aortic constriction (TAC). Beneficial effects observed in TAC mice receiving CSD injections daily included: improved ventricular function (increased ejection fraction, stroke volume, and cardiac output; reduced wall thickness); decreased collagen I, collagen chaperone HSP47, fibronectin, and CTGF levels; decreased activation of non-receptor tyrosine kinases Pyk2 and Src; and decreased activation of eNOS. To determine the source of cells that contribute to fibrosis in CHF, flow cytometric studies were performed that suggested that myofibroblasts in the heart are in large part bone marrow-derived. Two CD45+ cell populations were observed. One (Zone 1) contained CD45+/HSP47-/macrophage marker+ cells (macrophages). The second (Zone 2) contained CD45(moderate)/HSP47+/macrophage marker- cells often defined as fibrocytes. TAC increased the number of cells in Zones 1 and 2 and the level of HSP47 in Zone 2. These studies are a first step in elucidating the mechanism of action of CSD in heart fibrosis and promoting the development of CSD as a novel treatment to reduce fibrosis and improve ventricular function in CHF patients.

  13. Modification of an apparatus for tumor-suppressor protein crystal growth in the International Space Station

    NASA Astrophysics Data System (ADS)

    de Morais Mendonca Teles, Antonio

    Some human diseases as tumors are being studied continuously for the development of vaccines against them. And a way of doing that is by means of proteins research. There are some kinds of proteins, like the p53 and p73 proteins, which are tumor suppressors. There are other diseases such as A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases which are protein-related. The determination of how proteins geometrically order themselves, during its biological functions is very necessary to understand how a protein's structure affects its function, to design vaccines that intercede in tumor-protein activities and in other proteins related to those other diseases. The protein crystal growth in microgravity environment produces purer crystallization than on the ground, and it is a powerful tool to produce better vaccines. Several data have already been acquired using ground-based research and in spaceflight experiments aboard the Spacelab and Space Shuttle missions, and in the MIR and in the International Space Station (ISS). Here in this paper, I propose to be performed in the ISS Biological Research Facility (which is being developed), multiple crystal growth of proteins related to cancer (as tumors suppressors and oncoproteins), A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases, for the future obtaining of possible vaccines against them. I also propose a simple and practical equipment, a modification of the crystallization plates (which use a vapor diffusion technique) inside each cylinder of the Protein Crystallization Apparatus in Microgravity (PCAM), with multiple chambers with different sizes. Instead of using some chambers with the same size it is better to use several chambers with different sizes. Why is that? The answer is: the energy associated with the surface tension of the liquid in the chamber is directly related to the circle area of it. So, to minimize the total energy of the surface tension of a proteins liquid -making it more stable

  14. The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1.

    PubMed

    Zhou, Mina I; Wang, Hongmei; Ross, Jonathan J; Kuzmin, Igor; Xu, Chengen; Cohen, Herbert T

    2002-10-18

    The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.

  15. Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway

    SciTech Connect

    Luo, Di-xian; Cheng, Jiming; Xiong, Yan; Li, Junmo; Xia, Chenglai; Xu, Canxin; Wang, Chun; Zhu, Bingyang; Hu, Zhuowei; Liao, Duan-fang

    2010-01-22

    Intimal hyperplasia plays an important role in various types of vascular remodeling. Mechanical forces derived from blood flow are associated with the proliferation of vascular smooth muscle cells (VSMC). This contributes to many vascular disorders such as hypertension, atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTA). In this study, we show that static pressure induces the proliferation of VSMC and activates its related signal pathway. VSMC from a rat aorta were treated with different pressures (0, 60, 90, 120, 150 and 180 mm Hg) in a custom-made pressure incubator for 24 h. The most active proliferation of VSMC was detected at a pressure of 120 mm Hg. VSMC was also incubated under a static pressure of 120 mm Hg for different time intervals (0, 2, 4, 8, 12 and 24 h). We found that static pressure significantly stimulates VSMC proliferation. Extracellular signal-regulated kinases 1/2 (ERK1/2) activation showed a peak at the pressure of 120 mm Hg at 4-h time point. Moreover, caveolin-1 expression was significantly inhibited by rising static pressure. Downregulation of VSMC proliferation could be found after PD98059 (ERK1/2 phosphorylation inhibitor) treatment. Our data also showed that a siRNA-mediated caveolin-1 knock down increased ERK1/2 phosphorylation and VSMC proliferation. These results demonstrate that static pressure promotes VSMC proliferation via the Caveolin-1/ERK1/2 pathway.

  16. The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

    SciTech Connect

    Fusaro, Adriana F.; Correa, Regis L.; Nakasugi, Kenlee; Jackson, Craig; Kawchuk, Lawrence; Vaslin, Maite F.S.; Waterhouse, Peter M.

    2012-05-10

    The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0{sup PE}, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0{sup PE} has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0{sup PE} destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

  17. Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein

    PubMed Central

    Minervini, Giovanni; Mazzotta, Gabriella M.; Masiero, Alessandro; Sartori, Elena; Corrà, Samantha; Potenza, Emilio; Costa, Rodolfo; Tosatto, Silvio C. E.

    2015-01-01

    Deregulation of the von Hippel-Lindau tumor suppressor protein (pVHL) is considered one of the main causes for malignant renal clear-cell carcinoma (ccRCC) insurgence. In human, pVHL exists in two isoforms, pVHL19 and pVHL30 respectively, displaying comparable tumor suppressor abilities. Mutations of the p53 tumor suppressor gene have been also correlated with ccRCC insurgence and ineffectiveness of treatment. A recent proteomic analysis linked full length pVHL30 with p53 pathway regulation through complex formation with the p14ARF oncosuppressor. The alternatively spliced pVHL19, missing the first 53 residues, lacks this interaction and suggests an asymmetric function of the two pVHL isoforms. Here, we present an integrative bioinformatics and experimental characterization of the pVHL oncosuppressor isoforms. Predictions of the pVHL30 N-terminus three-dimensional structure suggest that it may exist as an ensemble of structured and disordered forms. The results were used to guide Yeast two hybrid experiments to highlight isoform-specific binding properties. We observed that the physical pVHL/p14ARF interaction is specifically mediated by the 53 residue long pVHL30 N-terminal region, suggesting that this N-terminus acts as a further pVHL interaction interface. Of note, we also observed that the shorter pVHL19 isoform shows an unexpected high tendency to form homodimers, suggesting an additional isoform-specific binding specialization. PMID:26211615

  18. The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein.

    PubMed

    Miyakawa, Kei; Nishi, Mayuko; Matsunaga, Satoko; Okayama, Akiko; Anraku, Masaki; Kudoh, Ayumi; Hirano, Hisashi; Kimura, Hirokazu; Morikawa, Yuko; Yamamoto, Naoki; Ono, Akira; Ryo, Akihide

    2017-01-30

    Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1.

  19. Gastrokines: stomach-specific proteins with putative homeostatic and tumor suppressor roles.

    PubMed

    Menheniott, Trevelyan R; Kurklu, Bayzar; Giraud, Andrew S

    2013-01-15

    During the past decade, a new family of stomach-specific proteins has been recognized. Known as "gastrokines" (GKNs), these secreted proteins are products of gastric mucus-producing cell lineages. GKNs are highly conserved in physical structure, and emerging data point to convergent functions in the modulation of gastric mucosal homeostasis and inflammation. While GKNs are highly prevalent in the normal stomach, frequent loss of GKN expression in gastric cancers, coupled with established antiproliferative activity, suggests putative tumor suppressor roles. Conversely, ectopic expression of GKNs in reparative lesions of Crohn's disease alludes to additional activity in epithelial wound healing and/or repair. Modes of action remain unsolved, but the recent demonstration of a GKN2-trefoil factor 1 heterodimer implicates functional interplay with trefoil factors. This review aims to provide a historical account of GKN biology and encapsulate the rapidly accumulating evidence supporting roles in gastric epithelial homeostasis and tumor suppression.

  20. The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein

    PubMed Central

    Miyakawa, Kei; Nishi, Mayuko; Matsunaga, Satoko; Okayama, Akiko; Anraku, Masaki; Kudoh, Ayumi; Hirano, Hisashi; Kimura, Hirokazu; Morikawa, Yuko; Yamamoto, Naoki; Ono, Akira; Ryo, Akihide

    2017-01-01

    Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1. PMID:28134256

  1. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity.

    PubMed

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K; Sunil, Sujatha

    2016-11-30

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models.

  2. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity

    PubMed Central

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K.; Sunil, Sujatha

    2016-01-01

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models. PMID:27901124

  3. Genetic variation in caveolin-1 correlates with long-term pancreas transplant function.

    PubMed

    Hamilton, A; Mittal, S; Barnardo, M C N M; Fuggle, S V; Friend, P; Gough, S C L; Simmonds, M J

    2015-05-01

    Pancreas transplantation is a successful treatment for a selected group of people with type 1 diabetes. Continued insulin production can decrease over time and identifying predictors of long-term graft function is key to improving survival. The aim of this study was to screen subjects for variation in the Caveolin-1 gene (Cav1), previously shown to correlate with long-term kidney transplant function. We genotyped 435 pancreas transplant donors and 431 recipients who had undergone pancreas transplantation at the Oxford Transplant Centre, UK, for all known common variation in Cav1. Death-censored cumulative events were analyzed using Kaplan-Meier and Cox regression. Unlike kidney transplantation, the rs4730751 variant in our pancreas donors or transplant recipients did not correlate with long-term graft function (p = 0.331-0.905). Presence of rs3801995 TT genotype (p = 0.009) and rs9920 CC/CT genotype (p = 0.010) in our donors did however correlate with reduced long-term graft survival. Multivariate Cox regression (adjusted for donor and recipient transplant factors) confirmed the association of rs3801995 (p = 0.009, HR = 1.83;[95% CI = 1.16-2.89]) and rs9920 (p = 0.037, HR = 1.63; [95% CI = 1.03-2.73]) with long-term graft function. This is the first study to provide evidence that donor Cav1 genotype correlates with long-term pancreas graft function. Screening Cav1 in other datasets is required to confirm these pilot results.

  4. Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

    PubMed Central

    Zimnicka, Adriana M.; Husain, Yawer S.; Shajahan, Ayesha N.; Sverdlov, Maria; Chaga, Oleg; Chen, Zhenlong; Toth, Peter T.; Klomp, Jennifer; Karginov, Andrei V.; Tiruppathi, Chinnaswamy; Malik, Asrar B.; Minshall, Richard D.

    2016-01-01

    Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or “spreading” of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane. PMID:27170175

  5. Cell-Specific Modulation of Papovavirus Replication by Tumor Suppressor Protein p53

    PubMed Central

    Lepik, Dina; Ustav, Mart

    2000-01-01

    Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication. PMID:10775606

  6. Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy.

    PubMed

    Shi, Yin; Tan, Shi-Hao; Ng, Shukie; Zhou, Jing; Yang, Na-Di; Koo, Gi-Bang; McMahon, Kerrie-Ann; Parton, Robert G; Hill, Michelle M; Del Pozo, Miguel A; Kim, You-Sun; Shen, Han-Ming

    2015-01-01

    CAV1 (caveolin 1, caveolae protein, 22kDa) is well known as a principal scaffolding protein of caveolae, a specialized plasma membrane structure. Relatively, the caveolae-independent function of CAV1 is less studied. Autophagy is a process known to involve various membrane structures, including autophagosomes, lysosomes, and autolysosomes for degradation of intracellular proteins and organelles. Currently, the function of CAV1 in autophagy remains largely elusive. In this study, we demonstrate for the first time that CAV1 deficiency promotes both basal and inducible autophagy. Interestingly, the promoting effect was found mainly in the late stage of autophagy via enhancing lysosomal function and autophagosome-lysosome fusion. Notably, the regulatory function of CAV1 in lysosome and autophagy was found to be caveolae-independent, and acts through lipid rafts. Furthermore, the elevated autophagy level induced by CAV1 deficiency serves as a cell survival mechanism under starvation. Importantly, downregulation of CAV1 and enhanced autophagy level were observed in human breast cancer cells and tissues. Taken together, our data reveal a novel function of CAV1 and lipid rafts in breast cancer development via modulation of lysosomal function and autophagy.

  7. Endolysosomal sorting of ubiquitinated caveolin-1 is regulated by VCP/p97 and UBXD1 and impaired by VCP disease mutations

    PubMed Central

    Ritz, Danilo; Vuk, Maja; Kirchner, Philipp; Bug, Monika; Schütz, Sabina; Hayer, Arnold; Bremer, Sebastian; Lusk, Caleb; Baloh, Robert H.; Lee, Houkeun; Glatter, Timo; Gstaiger, Matthias; Aebersold, Ruedi; Weihl, Conrad C.; Meyer, Hemmo

    2011-01-01

    The AAA-ATPase VCP/p97 cooperates with distinct cofactors to process ubiquitinated proteins in different cellular pathways 1–3. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear 4, 5. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds the plasma membrane protein caveolin-1 (Cav1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitinated Cav1 in SDS-resistant high molecular weight complexes on endosomes, which are en route to degradation in endolysosomes 6. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of Cav1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific Caveolin-3 (Cav3) accumulates in sarcoplasmic pools and specifically delocalises from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitinated cargo in the endocytic pathway and suggest that impaired trafficking of caveolin may contribute to the pathogenesis in individuals with VCP mutations. PMID:21822278

  8. The metabolic/pH sensor soluble adenylyl cyclase is a tumor suppressor protein

    PubMed Central

    Ramos-Espiritu, Lavoisier; Diaz, Ana; Nardin, Charlee; Saviola, Anthony J.; Shaw, Fiona; Plitt, Tamar; Yang, Xia; Wolchok, Jedd; Pirog, Edyta C.; Desman, Garrett; Sboner, Andrea; Zhang, Tuo; Xiang, Jenny; Merghoub, Taha; Levin, Lonny R.; Buck, Jochen; Zippin, Jonathan H.

    2016-01-01

    cAMP signaling pathways can both stimulate and inhibit the development of cancer; however, the sources of cAMP important for tumorigenesis remain poorly understood. Soluble adenylyl cyclase (sAC) is a non-canonical, evolutionarily conserved, nutrient- and pH-sensing source of cAMP. sAC has been implicated in the metastatic potential of certain cancers, and it is differentially localized in human cancers as compared to benign tissues. We now show that sAC expression is reduced in many human cancers. Loss of sAC increases cellular transformation in vitro and malignant progression in vivo. These data identify the metabolic/pH sensor soluble adenylyl cyclase as a previously unappreciated tumor suppressor protein. PMID:27323809

  9. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

    PubMed

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

    2015-09-01

    RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression

  10. The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity.

    PubMed

    Lisztwan, J; Imbert, G; Wirbelauer, C; Gstaiger, M; Krek, W

    1999-07-15

    pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin-protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.

  11. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    PubMed

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  12. Simultaneous detection of the tumor suppressor FHIT gene and protein using the multi-functional biochip.

    PubMed

    Askari, Minoo D F; Miller, Gordon H; Vo-Dinh, Tuan

    2002-01-01

    The tumor suppressor gene, fragile histidine triad (FHIT), encompasses the most common human chromosomal fragile site, at 3pl4.2. Detection of FHIT gene is important in cancer diagnostics since its alterations have been associated with several human cancers. A unique multi-functional biochip for simultaneous detection of FHIT DNA and FHIT protein on the same platform was applied. The design of the biochip is based on miniaturization of photodiodes, where functioning of multiple optical sensing elements, amplifiers, discriminators, and logic circuitry are integrated on a single IC board. Performance of biochip is based on biomolecular recognition processes using both DNA and protein bioreceptors, Cy5-labeled probes and laser excitation. Application of biochip for concurrent detection of various immobilized target DNA and protein molecules and multiplex of DNA and protein on the same microarray was accomplished. Linearity of biochip for quantitative measurements was demonstrated. Results demonstrated utility of this multi-functional biochip as a useful detection technology with applications in biological and clinical laboratories.

  13. Interferon-Inducible Protein 16: Insight into the Interaction with Tumor Suppressor p53

    SciTech Connect

    Liao, Jack C.C.; Lam, Robert; Brazda, Vaclav; Duan, Shili; Ravichandran, Mani; Ma, Justin; Xiao, Ting; Tempel, Wolfram; Zuo, Xiaobing; Wang, Yun-Xing; Chirgadze, Nickolay Y.; Arrowsmith, Cheryl H.

    2011-08-24

    IFI16 is a member of the interferon-inducible HIN-200 family of nuclear proteins. It has been implicated in transcriptional regulation by modulating protein-protein interactions with p53 tumor suppressor protein and other transcription factors. However, the mechanisms of interaction remain unknown. Here, we report the crystal structures of both HIN-A and HIN-B domains of IFI16 determined at 2.0 and 2.35 {angstrom} resolution, respectively. Each HIN domain comprises a pair of tightly packed OB-fold subdomains that appear to act as a single unit. We show that both HIN domains of IFI16 are capable of enhancing p53-DNA complex formation and transcriptional activation via distinctive means. HIN-A domain binds to the basic C terminus of p53, whereas the HIN-B domain binds to the core DNA-binding region of p53. Both interactions are compatible with the DNA-bound state of p53 and together contribute to the effect of full-length IFI16 on p53-DNA complex formation and transcriptional activation.

  14. Impaired Cd14 and Cd36 expression, bacterial clearance, and Toll-like receptor 4-Myd88 signaling in caveolin-1-deleted macrophages and mice.

    PubMed

    Tsai, Tsung-Huang; Chen, Shu-Fen; Huang, Tai-Yu; Tzeng, Chun-Fu; Chiang, Ann-Shyn; Kou, Yu Ru; Lee, Tzong-Shyuan; Shyue, Song-Kun

    2011-01-01

    An overwhelming immune response, particularly from macrophages, with gram-negative bacteria-induced sepsis plays a critical role in survival of and organ damage in infected patients. Caveolin-1 (Cav-1), a major structure protein of caveolae, regulates many cellular functions. We examined the vital role of Cav-1 in the response of macrophages and mice to bacteria or LPS exposure. Deletion of Cav-1 decreased the expression of CD14 and CD36 during macrophage differentiation and suppressed their phagocytotic ability. As well, the ability to kill bacteria was inhibited in Cav-1 macrophages and mice peritoneal cavity, tissue, and plasma, which was partly attributed to hindered expression of iNOS induced by bacteria or LPS. Furthermore, deletion of Cav-1 attenuated the expression of Toll-like receptor 4 and myeloid differentiation factor 88 and the activation of nuclear factor κB, all of which impeded the production of inflammatory cytokines in response to bacterial exposure in Cav-1 macrophages and mice. Thus, Cav-1 participates in the regulation of CD14, CD36, Toll-like receptor 4 and myeloid differentiation factor 88 protein expression and is crucial for the immune response of macrophages to bacterial infection. Cav-1 may be a therapeutic target in the treatment of sepsis.

  15. Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma

    PubMed Central

    Marko, Tracy A.; Shamsan, Ghaidan A.; Edwards, Elizabeth N.; Hazelton, Paige E.; Rathe, Susan K.; Cornax, Ingrid; Overn, Paula R.; Varshney, Jyotika; Diessner, Brandon J.; Moriarity, Branden S.; O’Sullivan, M. Gerard; Odde, David J.; Largaespada, David A.

    2016-01-01

    Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype. PMID:27966608

  16. Nuclear γ-tubulin associates with nucleoli and interacts with tumor suppressor protein C53.

    PubMed

    Hořejší, Barbora; Vinopal, Stanislav; Sládková, Vladimíra; Dráberová, Eduarda; Sulimenko, Vadym; Sulimenko, Tetyana; Vosecká, Věra; Philimonenko, Anatoly; Hozák, Pavel; Katsetos, Christos D; Dráber, Pavel

    2012-01-01

    γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).

  17. How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein.

    PubMed

    Goodrich, David W

    2003-11-01

    The retinoblastoma tumor suppressor gene (RB1) is currently the only known gene whose mutation is necessary and sufficient for the development of a human cancer. Mutation or deregulation of RB1 is observed so frequently in other tumor types that compromising RB1 function may be a prerequisite for malignant transformation. Identifying the molecular mechanisms that provide the basis for RB1-mediated tumor suppression has become an important goal in the quest to understand and treat cancer. The lion's share of research on these mechanisms has focused on the carboxy-terminal half of the RB1 encoded protein (pRB). This focus is with good reason since this part of the protein, now called the "large pocket," is required for most of its known activities identified in vitro and in vivo. Large pocket mediated mechanisms alone, however, cannot account for all observed properties of pRB. The thesis presented here is that the relatively uncharacterized amino-terminal half of the protein makes important contributions to pRB-mediated tumor suppression. The goals of this review are to summarize evidence indicating that an amino-terminal structural domain is important for pRB function and to suggest a general hypothesis as to how this domain can be integrated with current models of pRB function.

  18. Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma.

    PubMed

    Marko, Tracy A; Shamsan, Ghaidan A; Edwards, Elizabeth N; Hazelton, Paige E; Rathe, Susan K; Cornax, Ingrid; Overn, Paula R; Varshney, Jyotika; Diessner, Brandon J; Moriarity, Branden S; O'Sullivan, M Gerard; Odde, David J; Largaespada, David A

    2016-12-14

    Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype.

  19. The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis

    PubMed Central

    Baxley, Ryan M.; Soshnev, Alexey A.; Koryakov, Dmitry E.; Zhimulev, Igor F.; Geyer, Pamela K.

    2011-01-01

    The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190 kD (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions. PMID:21651900

  20. Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    PubMed Central

    Panic, Andrej; Ketteler, Julia; Reis, Henning; Sak, Ali; Herskind, Carsten; Maier, Patrick; Rübben, Herbert; Jendrossek, Verena; Klein, Diana

    2017-01-01

    Despite good treatment results in localized prostate tumors, advanced disease stages usually have a pronounced resistance to chemotherapy and radiotherapy. The membrane protein caveolin-1 (Cav1) functions here as an important oncogene. Therefore we examined the impact of stromal Cav1 expression for tumor growth and sensitivity to ionizing radiation (IR). Silencing of Cav1 expression in PC3 cells resulted in increased tumor growth and a reduced growth delay after IR when compared to tumors generated by Cav1-expressing PC3 cells. The increased radiation resistance was associated with increasing amounts of reactive tumor stroma and a Cav1 re-expression in the malignant epithelial cells. Mimicking the human situation these results were confirmed using co-implantation of Cav1-silenced PC3 cells with Cav1-silenced or Cav1-expressing fibroblasts. Immunohistochemically analysis of irradiated tumors as well as human prostate tissue specimen confirmed that alterations in stromal-epithelial Cav1 expressions were accompanied by a more reactive Cav1-reduced tumor stroma after radiation and within advanced prostate cancer tissues which potentially mediates the resistance to radiation treatment. Conclusively, the radiation response of human prostate tumors is critically regulated by Cav1 expression in stromal fibroblasts. Loss of stromal Cav1 expression in advanced tumor stages may thus contribute to resistance of these tumors to radiotherapy. PMID:28112237

  1. Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways.

    PubMed

    Xiong, Niya; Li, Shun; Tang, Kai; Bai, Hongxia; Peng, Yueting; Yang, Hong; Wu, Chunhui; Liu, Yiyao

    2017-01-01

    Tumor cells translocating to distant sites are subjected to hemodynamic shear forces during their passage in the blood vessels. Low shear stress (LSS) plays a critical role in the regulation of various aspects of tumor cells functions, including motility and adhesion. Beyond its structural role, caveolin-1 (Cav-1), the important component of caveolae, represents a modulator of several cancer-associated functions as tumor progression and metastasis. However, the role of Cav-1 in regulating tumor cells response to shear stress remains poorly explored. Here, we characterized the role of LSS and Cav-1 in mediating cell motility and adhesion on human breast carcinoma MDA-MB-231 cells. We first showed that LSS exposure promoted cell polarity and focal adhesion (FA) dynamics, thus indicating elevated cell migration. Silencing of Cav-1 leaded to a significantly lower formation of stress fibers. However, LSS exposure was able to rescue it via the alteration of actin-associated proteins expression, including ROCK, p-MLC, cofilin and filamin A. Time-lapse migration assay indicated that Cav-1 expression fostered MDA-MB-231 cells motility and LSS triggered cells to rapidly generate new lamellipodia. Furthermore, Cav-1 and LSS significantly influenced cell adhesion. Taken together, our findings provide insights into mechanisms underlying LSS triggered events mediated by downstream Cav-1, including FAK/Src and ROCK/p-MLC pathways, involved in the reorganization of the cytoskeleton, cell motility, FA dynamics and breast cancer cell adhesion.

  2. Determinants of the tumor suppressor INPP4B protein and lipid phosphatase activities.

    PubMed

    Lopez, Sandra M; Hodgson, Myles C; Packianathan, Charles; Bingol-Ozakpinar, Ozlem; Uras, Fikriye; Rosen, Barry P; Agoulnik, Irina U

    2013-10-18

    The tumor suppressor INPP4B is an important regulator of phosphatidyl-inositol signaling in the cell. Reduced INPP4B expression is associated with poor outcomes for breast, prostate, and ovarian cancer patients. INPP4B contains a CX5R catalytic motif characteristic of dual-specificity phosphatases, such as PTEN. Lipid phosphatase activity of INPP4B has previously been described. In this report we show that INPP4B can dephosphorylate para-nitrophenyl phosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl (DiFMUP), synthetic phosphotyrosine analogs, suggesting that INPP4B has protein tyrosine phosphatase (PTP) activity. Using mutagenesis, we examined the functional role of specific amino acids within the INPP4B C842KSAKDR catalytic site. The K843M mutant displayed increased pNPP hydrolysis, the K846M mutant lost lipid phosphatase activity with no effect on PTP activity, and the D847E substitution ablated PTP activity and significantly reduced lipid phosphatase activity. Further, we show that INPP4B but not PTEN is able to reduce tyrosine phosphorylation of Akt1 and both the lipid and PTP activity of INPP4B likely contribute to the reduction of Akt1 phosphorylation. Taken together our data identified key residues in the INPP4B catalytic domain associated with lipid and protein phosphatase activities and found a robust downstream target regulated by INPP4B but not PTEN.

  3. The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein.

    PubMed

    Chen, Muhan; Nowak, Dawid G; Narula, Navneet; Robinson, Brian; Watrud, Kaitlin; Ambrico, Alexandra; Herzka, Tali M; Zeeman, Martha E; Minderer, Matthias; Zheng, Wu; Ebbesen, Saya H; Plafker, Kendra S; Stahlhut, Carlos; Wang, Victoria M Y; Wills, Lorna; Nasar, Abu; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Wilkinson, John E; Powers, Scott; Sordella, Raffaella; Altorki, Nasser K; Mittal, Vivek; Stiles, Brendon M; Plafker, Scott M; Trotman, Lloyd C

    2017-03-06

    Phosphatase and tensin homologue (PTEN) protein levels are critical for tumor suppression. However, the search for a recurrent cancer-associated gene alteration that causes PTEN degradation has remained futile. In this study, we show that Importin-11 (Ipo11) is a transport receptor for PTEN that is required to physically separate PTEN from elements of the PTEN degradation machinery. Mechanistically, we find that the E2 ubiquitin-conjugating enzyme and IPO11 cargo, UBE2E1, is a limiting factor for PTEN degradation. Using in vitro and in vivo gene-targeting methods, we show that Ipo11 loss results in degradation of Pten, lung adenocarcinoma, and neoplasia in mouse prostate with aberrantly high levels of Ube2e1 in the cytoplasm. These findings explain the correlation between loss of IPO11 and PTEN protein in human lung tumors. Furthermore, we find that IPO11 status predicts disease recurrence and progression to metastasis in patients choosing radical prostatectomy. Thus, our data introduce the IPO11 gene as a tumor-suppressor locus, which is of special importance in cancers that still retain at least one intact PTEN allele.

  4. Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression

    PubMed Central

    Chou, Ting-Fang; Chuang, Ya-Ting; Hsieh, Wan-Chen; Chang, Pei-Yun; Liu, Hsin-Yu; Mo, Shu-Ting; Hsu, Tzu-Sheng; Miaw, Shi-Chuen; Chen, Ruey-Hwa; Kimchi, Adi; Lai, Ming-Zong

    2016-01-01

    Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK–HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of Dapk−/− Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases. PMID:27312851

  5. 4-cholesten-3-one suppresses lung adenocarcinoma metastasis by regulating translocation of HMGB1, HIF1α and Caveolin-1

    PubMed Central

    Ma, Jinben; Fu, Guobin; Wu, Jing; Han, Shaoxian; Zhang, Lishan; Yang, Ming; Yu, Yong; Zhang, Mengyuan; Lin, Yanliang; Wang, Yibing

    2016-01-01

    Metastasis is a great challenge in lung adenocarcinoma (ADC) therapy. Cholesterol has been implicated in ADC metastasis. 4-cholesten-3-one, as cholesterol metabolite and analog, can substitute membrane cholesterol and increase membrane fluidity. In this study, we explored the possibility that 4-cholesten-3-one inhibited ADC metastasis. Low-dose 4-cholesten-3-one significantly restrained ADC cells migration and invasion with little effects on cells viabilities. Further investigation showed that 4-cholesten-3-one promoted ROS generation, which transiently activated AMPKα1, increased HIF1α expression, reduced Bcl-2 expression and caused autophagy. AMPKα1 knockdown partly suppressed 4-cholesten-3-one-induced autophagy but, neither prevented 4-cholesten-3-one-induced upregulation of HIF1α or downregulation of Bcl-2. 4-cholesten-3-one-induced autophagy facilitated the release of HMGB1 from nuclei to cytoplasm, blocking nuclear translocation of HIF1α and activation of MMP2 and MMP9. Also, 4-cholesten-3-one induced time-dependent phosphorylation of caveolin-1, Akt and NF-κB. With increasing treatment time, 4-cholesten-3-one accelerated caveolin-1 internalization, but reduced the phosphorylation of Akt and NF-κB, and inhibited the expression of snail and twist. These data suggested that 4-cholesten-3-one could be a potential candidate for anti-metastasis of lung adenocarcinoma. PMID:27899819

  6. Cleavage of metastasis suppressor gene product KiSS-1 protein/metastin by matrix metalloproteinases.

    PubMed

    Takino, Takahisa; Koshikawa, Naohiko; Miyamori, Hisashi; Tanaka, Motohiro; Sasaki, Takuma; Okada, Yasunori; Seiki, Motoharu; Sato, Hiroshi

    2003-07-24

    A human placenta cDNA library was screened by the expression cloning method for gene products that interact with matrix metalloproteinases (MMPs), and we isolated a cDNA whose product formed a stable complex with pro-MMP-2 and pro-MMP-9. The cDNA encoded the metastasis suppressor gene KiSS-1. KiSS-1 protein was shown to form a complex with pro-MMP. KiSS-1 protein is known to be processed to peptide ligand of a G-protein-coupled receptor (hOT7T175) named metastin, and suppresses metastasis of tumors expressing the receptor. Active MMP-2, MMP-9, MT1-MMP, MT3-MMP and MT5-MMP cleaved the Gly118-Leu119 peptide bond of not only full-length KiSS-1 protein but also metastin decapeptide. Metastin decapeptide induced formation of focal adhesion and actin stress fibers in cells expressing the receptor, and digestion of metastin decapeptide by MMP abolished its ligand activity. Migration of HT1080 cells expressing hOT7T175 that harbor a high-level MMP activity was only slightly suppressed by either metastin decapeptide or MMP inhibitor BB-94 alone, but the combination of metastin decapeptide and BB-94 showed a synergistic effect in blocking cell migration. We propose that metastin could be used as an antimetastatic agent in combination with MMP inhibitor, or MMP-resistant forms of metastin could be developed and may also be efficacious.

  7. Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells.

    PubMed

    Sinha, Pratima; Okoro, Chinonyerem; Foell, Dirk; Freeze, Hudson H; Ostrand-Rosenberg, Suzanne; Srikrishna, Geetha

    2008-10-01

    Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1beta, IL-6, and PGE(2) induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1(high)CD11b(high)F4/80(-)CD80(+)IL4Ralpha(+/-)Arginase(+) MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-kappaB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-kappaB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.

  8. The tumor suppressor protein menin inhibits AKT activation by regulating its cellular localization

    PubMed Central

    Wang, Yan; Ozawa, Atsushi; Zaman, Shadia; Prasad, Nijaguna B.; Chandrasekharappa, Settara C.; Agarwal, Sunita K.; Marx, Stephen J.

    2010-01-01

    Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder associated mainly with tumors of multiple endocrine organs. Mutations in the MEN1 gene that encodes for the menin protein are the predominant cause for hereditary MEN1 syndrome. Though menin is a tumor suppressor, its molecular mechanism of action has not been defined. Here we report that menin interacts with AKT1 in vitro and in vivo. Menin downregulates the level of active AKT and its kinase activity. Through interaction with AKT1, menin suppresses both AKT1 induced proliferation and anti-apoptosis in non-endocrine and endocrine cells. Confocal microscopy analysis revealed that menin regulates AKT1 in part by reducing the translocation of AKT1 from the cytoplasm to the plasma membrane during growth factor stimulation. Our findings may be generalizable to other cancers, insofar as we found that loss of menin expression was also associated with AKT activation in a mouse model of pancreatic islet adenoma. Together, our results suggest menin as an important novel negative regulator of AKT kinase activity. PMID:21127195

  9. The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells

    PubMed Central

    Parasido, Erika; Tricoli, Lucas; Sivakumar, Angiela; Mikhaiel, John P.; Yenugonda, Venkata; Rodriguez, Olga C.; Karam, Sana D.; Rood, Brian R.; Avantaggiati, Maria Laura; Albanese, Chris

    2015-01-01

    Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB. PMID:26540407

  10. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

    PubMed

    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival.

  11. Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1)

    SciTech Connect

    Yates, Luke A. Durrant, Benjamin P.; Barber, Michael; Harlos, Karl; Fleurdépine, Sophie; Norbury, Chris J.; Gilbert, Robert J. C.

    2015-02-21

    The use of truncation and RNA-binding mutations of caffeine induced death suppressor protein 1 (Cid1) as a means to enhance crystallogenesis leading to an improvement of X-ray diffraction resolution by 1.5 Å is reported. The post-transcriptional addition of uridines to the 3′-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3′-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction. In metazoans, the Cid1 orthologues ZCCHC6 and ZCCHC11 uridylate histone mRNAs, targeting them for degradation, but also uridylate microRNAs, altering their maturation. Cid1 has been studied as a model TUT that has provided insights into the larger and more complex metazoan enzyme system. In this paper, two strategies are described that led to improvements both in the crystallogenesis of Cid1 and in the resolution of diffraction by ∼1.5 Å. These advances have allowed high-resolution crystallo@@graphic studies of this TUT system to be initiated.

  12. Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells.

    PubMed

    Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae

    2012-04-01

    This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.

  13. Green tea polyphenols down-regulate caveolin-1 expression via ERK1/2 and p38MAPK in endothelial cells.

    PubMed

    Li, Yanrong; Ying, Chenjiang; Zuo, Xuezhi; Yi, Haiwei; Yi, Weijie; Meng, Yi; Ikeda, Katsumi; Ye, Xiaolei; Yamori, Yukio; Sun, Xiufa

    2009-12-01

    Caveolin-1 (Cav-1), a negative regulator of endothelial nitric oxide synthase (eNOS), influences various aspects of the cardiovascular functions. We had reported that a high-fat diet up-regulated aortic Cav-1 expressions in rats. In this study, we investigated the effects of green tea polyphenols (GTPs) on endothelial Cav-1 expression and phosphorylation in vitro. Bovine aortic endothelial cells (BAECs) were treated with 4 microg/ml GTPs for 0, 4, 8, 12, 16 and 24 h, and with 0, 0.04, 0.4, 4 and 40 microg/ml GTPs for 16 h, respectively. Cav-1 protein and mRNA were detected using Western blot and reverse transcriptase polymerase chain reaction. Cav-1 protein expression was down-regulated after treatment of BAECs with 4 microg/ml GTPs for 12, 16 and 24 h. And decrease in the level of Cav-1 mRNA was observed after GTP treatment for 4 and 8 h. GTPs (0.04-4 microg/ml) down-regulate Cav-1 protein expressions and mRNA levels dose dependently. PD98059, an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2), up-regulated Cav-1 expression in BAECs alone and abolished the down-regulation effects of GTPs in BAECs while pretreatment with it. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) with SB203580, which down-regulates Cav-1 expression in BAECs alone, deteriorated the Cav-1 down-regulating effects by GTPs. In addition to the effects on expression of Cav-1, GTP treatment inhibited phosphorylation of Cav-1 [tyrosine 14 (Tyr14)]. These data indicate that GTPs down-regulate gene expression of Cav-1 time- and dose- dependently via activating ERK1/2 and inhibiting p38MAPK signaling.

  14. Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

    PubMed Central

    Mendoza-Rodriguez, Mónica; Arreola, Hugo; Valdivia, Alejandra; Peralta, Raúl; Serna, Humberto; Villegas, Vanessa; Romero, Pablo; Alvarado-Hernández, Beatriz; Paniagua, Lucero; Marrero-Rodríguez, Daniel; Meraz, Marco A; Salcedo, Mauricio

    2013-01-01

    Aims: Cervical Cancer (CC) is one of the most important health problems in women. It frequently presents genetic changes at chromosome region 3q21. This region contains the Cellular Retinol Binding Protein 1 gene (CRBP1) which has been implicated as an important element in the development of other types of cancer. The main goal of the present work was to determine the molecular alterations of CRBP1 and its relationship to CC. Methods: To determine the molecular alterations of CRBP1 gene in CC; twenty-six CC and twenty-six healthy cervix samples were evaluated for: 1) Copy number gain by real-time PCR analysis, 2) expression levels by an immunohistochemistry assay on tissue microarray, and 3) the methylation status of the CRBP1 promoter region. Results: The increase in CRBP1 copy number was observed in 10 out of the 26 CC samples analyzed, while healthy cervices samples showed no changes in the copy number. In addition, there was a lack of expression of the CRBP1 gene in an important number of the CC samples (17/26), and the CRBP1 gene promoter was methylated in 15/26 of the CC samples. Interestingly, there was a significant association between the lack of expression of the CRBP1 gene and its methylation status. Conclusions: The data indicates that, both activating and inactivating changes in the CRBP1 gene could be significant events in the development and progression of CC, and the lack of expression of the CRBP1 protein could be related with to the development of CC. We believe that there is enough evidence to consider to CRBP1 gene as a tumor suppressor gene for CC. PMID:24040446

  15. The regulation of tumor suppressor protein, p53, and estrogen receptor (ERα) by resveratrol in breast cancer cells

    PubMed Central

    Saluzzo, Julieta; Hallman, Kelly M.; Aleck, Katie; Dwyer, Brigitte; Quigley, Meghan; Mladenovik, Viktoria; Siebert, Amy E.; Dinda, Sumi

    2016-01-01

    Resveratrol (RES) is a natural antioxidant found abundantly in grapes, peanuts, and berries, and is known to possess anti-tumorigenic properties. However, there is a noticeable lack of studies on the mechanistic effects of Resveratrol on tumor suppressors. Previous studies from our laboratory have shown the tumor suppressor protein p53 and estrogen receptor-alpha (ERα) to be possible molecular targets for RES. In this study, the anti-estrogenic effects of RES were analyzed on the expression of ERα and p53. The breast cancer cells grown in stripped serum were treated with 60 μM RES, as the optimum concentration based on data obtained from a concentration study using 1-100 μM RES. Our studies indicate that RES caused a decrease in the levels of protein expression of p53 and ERα as compared to the control. Increasing concentrations of RES caused a four-fold decrease in cell number in comparison to estradiol. RES, in conjunction with ICI 182,780 (ICI), caused a down-regulation of both p53 and ERα as compared to the control. These observed effects on cell proliferation and regulation of both p53 and ERα by RES may lead to further understanding of the relationship between tumor suppressor proteins and steroid receptors in breast cancer cells. PMID:28191286

  16. Metronomic Ceramide Analogs Inhibit Angiogenesis in Pancreatic Cancer through Up-regulation of Caveolin-1 and Thrombospondin-1 and Down-regulation of Cyclin D112

    PubMed Central

    Bocci, Guido; Fioravanti, Anna; Orlandi, Paola; Di Desidero, Teresa; Natale, Gianfranco; Fanelli, Giovanni; Viacava, Paolo; Naccarato, Antonio Giuseppe; Francia, Giulio; Danesi, Romano

    2012-01-01

    Aims To evaluate the antitumor and antiangiogenic activity of metronomic ceramide analogs and their relevant molecular mechanisms. Methods Human endothelial cells [human dermal microvascular endothelial cells and human umbilical vascular endothelial cell (HUVEC)] and pancreatic cancer cells (Capan-1 and MIA PaCa-2) were treated with the ceramide analogs (C2, AL6, C6, and C8), at low concentrations for 144 hours to evaluate any antiproliferative and proapoptotic effects and inhibition of migration and to measure the expression of caveolin-1 (CAV-1) and thrombospondin-1 (TSP-1) mRNAs by real-time reverse transcription-polymerase chain reaction. Assessment of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt phosphorylation and of CAV-1 and cyclin D1 protein expression was performed by ELISA. Maximum tolerated dose (MTD) gemcitabine was compared against metronomic doses of the ceramide analogs by evaluating the inhibition of MIA PaCa-2 subcutaneous tumor growth in nude mice. Results Metronomic ceramide analogs preferentially inhibited cell proliferation and enhanced apoptosis in endothelial cells. Low concentrations of AL6 and C2 caused a significant inhibition of HUVEC migration. ERK1/2 and Akt phosphorylation were significantly decreased after metronomic ceramide analog treatment. Such treatment caused the overexpression of CAV-1 and TSP-1 mRNAs and proteins in endothelial cells, whereas cyclin D1 protein levels were reduced. The antiangiogenic and antitumor impact in vivo of metronomic C2 and AL6 regimens was similar to that caused by MTD gemcitabine. Conclusions Metronomic C2 and AL6 analogs have antitumor and antiangiogenic activity, determining the up-regulation of CAV-1 and TSP-1 and the suppression of cyclin D1. PMID:23019415

  17. Potential RNA Binding Proteins in Saccharomyces Cerevisiae Identified as Suppressors of Temperature-Sensitive Mutations in Npl3

    PubMed Central

    Henry, M.; Borland, C. Z.; Bossie, M.; Silver, P. A.

    1996-01-01

    The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a protein with similarity to heterogeneous nuclear ribonucleoproteins (hnRNPs). Npl3p has been implicated in many nuclear-related events including RNA export, protein import, and rRNA processing. Several temperature-sensitive alleles of NPL3 have been isolated. We now report the sequence of these alleles. For one allele, npl3-1, four complementation groups of suppressors have been isolated. The cognate genes for the two recessive mutants were cloned. One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family. The other suppressor corresponds to a newly defined gene we term HRP1, which also encodes a protein with similarity to the hnRNP A/B proteins of vertebrates. Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele. We show that HRP1 is essential for cell growth and that the corresponding protein is located in the nucleus. The discovery of two hnRNP homologues that can partially suppress the function of Np13p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism. PMID:8770588

  18. Physical and functional interactions between the Wwox tumor suppressor protein and the AP-2gamma transcription factor.

    PubMed

    Aqeilan, Rami I; Palamarchuk, Alexey; Weigel, Ronald J; Herrero, Juan J; Pekarsky, Yuri; Croce, Carlo M

    2004-11-15

    The WWOX gene encodes a tumor suppressor WW domain-containing protein, Wwox. Alterations of WWOX have been demonstrated in multiple types of cancer, and introduction of Wwox into Wwox-negative tumor cells has resulted in tumor suppression and apoptosis. The Wwox protein contains two WW domains that typically bind proline-rich motifs and mediate protein-protein interactions. Recently, we have described functional cross-talk between the Wwox protein and the p53 homologue, p73. To further explore the biological function of Wwox, we investigated other interacting candidates. In this report, we demonstrate a physical and functional association between AP-2gamma transcription factor and the Wwox protein. AP-2gamma at 20q13.2 encodes a transcription factor and is frequently amplified in breast carcinoma. We show that Wwox binds to the PPPY motif of AP-2gamma via its first WW domain. Alterations of tyrosine 33 in the first WW domain of Wwox or the proline-rich motif in AP-2gamma dramatically reduce this interaction. In addition, our results demonstrate that Wwox expression triggers redistribution of nuclear AP-2gamma to the cytoplasm, hence suppressing its transactivating function. Our results suggest that Wwox tumor suppressor protein inhibits AP-2gamma oncogenic activity by sequestering it in the cytoplasm.

  19. Down-regulation of Connexin43 expression reveals the involvement of caveolin-1 containing lipid rafts in human U251 glioblastoma cell invasion.

    PubMed

    Strale, Pierre-Olivier; Clarhaut, Jonathan; Lamiche, Coralie; Cronier, Laurent; Mesnil, Marc; Defamie, Norah

    2012-11-01

    Glioblastoma cells are characterized by high proliferation and invasive capacities. Tumor development has been associated with a decrease of gap-junctional intercellular communication, but the concrete involvement of gap junction proteins, connexins, remains elusive since they are also suspected to promote cell invasion. In order to better understand how connexins control the glioma cell phenotype, we studied the consequences of inhibiting the intrinsic expression of the major astrocytic connexin, Connexin43, in human U251 glioblastoma cells by the shRNA strategy. The induced down-regulation of Cx43 expression has various effects on the U251 cells such as increased clonogenicity, angiogenesis and decreased adhesion on specific extracellular matrix proteins. We demonstrate that the invasion capacity measured in vitro and ex vivo correlates with Cx43 expression level. For the first time in a cancer cell context, our work demonstrates that Cx43 cofractionates, colocalizes and coimmunoprecipitates with a lipid raft marker, caveolin-1 and that this interaction is inversely correlated to the level of Cx43. This localization of Cx43 in these lipid raft microdomains regulates both homo- and heterocellular gap junctional communications (respectively between U251 cells, or between U251 cells and astrocytes). Moreover, the adhesive and invasive capacities are not dependent, in our model, on Cav-1 expression level. Our results tend to show that heterocellular gap junctional communication between cancer and stroma cells may affect the behavior of the tumor cells. Altogether, our data demonstrate that Cx43 controls the tumor phenotype of glioblastoma U251 cells and in particular, invasion capacity, through its localization in lipid rafts containing Cav-1.

  20. Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischemic stroke stage.

    PubMed

    Liu, Jie; Jin, Xinchun; Liu, Ke J; Liu, Wenlan

    2012-02-29

    Blood-brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here, we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran and promoted the secretion of metalloproteinase-2 and -9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT (2-[[(4-phenoxyphenyl)sulfonyl]methyl]-thiirane) or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity, and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2 h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis.

  1. Restoration of caveolin-1 expression suppresses growth, membrane-type-4 metalloproteinase expression and metastasis-associated activities in colon cancer cells.

    PubMed

    Nimri, Lili; Barak, Hossei; Graeve, Lutz; Schwartz, Betty

    2013-11-01

    Caveolin-1 (cav-1) and flotillin-1 are two major structural proteins associated with lipid rafts in mammalian cells. The membrane-type matrix metalloproteinases (MT-MMPs) are expressed at the cell surface, hydrolyze extracellular matrix, and play an important role in cancer cell migration and metastasis. Expression of cav-1, flotillin-1, and MT4-MMP in lysates and lipid rafts of LS174T and HM-7 colon cancer cells was determined. The impact of restoration of cav-1 expression on proliferation, adhesion, motility in vitro, and growth of implanted tumors in vivo was characterized. Cav-1 is not expressed in lipid rafts of the highly metastatic colon cancer cell line (HM-7), but expressed in cytosolic fractions of the parental lower metastatic cell line (LS174T). In contrast, MT4-MMP was expressed in lipid rafts of HM-7 cells but not in LS174T cells. Overexpression of cav-1 in HM-7 cells down-regulate proliferation, viability, wound closure, adhesion to laminin, invasion, and development of filopodial and lamellipodial structures in a dose-dependent manner. Cav-1 positive HM-7 clones ceased to express MT4-MMP in their lipid rafts. Comparative proteomic analyses of lipid rafts from cav-1 positive and cav-1 negative cells demonstrated de novo expression of flotillin-1 only on the cells expressing cav-1. Xenografting control cells devoid of cav-1 in nude mice induced development of bigger tumors expressing higher levels of proliferating cell nuclear antigen as compared to mice injected with cells expressing the highest cav-1 levels. We conclude that cav-1 orchestrates and reorganize several proteins in lipid rafts, activities directly associated with reduced tumorigenic and metastatic ability of colon cancer cells.

  2. Protective Effect of Ginsenoside Rg1 on Bleomycin-Induced Pulmonary Fibrosis in Rats: Involvement of Caveolin-1 and TGF-β1 Signal Pathway.

    PubMed

    Zhan, Heqin; Huang, Feng; Ma, Wenzhuo; Zhao, Zhenghang; Zhang, Haifang; Zhang, Chong

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and high mortality rate. Panax Notoginseng Saponins (PNS), extracted from Panax Notoginseng as a traditional Asian medicine, displayed a significant anti-fibrosis effect in liver and lung. However, whether Ginsenoside Rg1 (Rg1), an important and active ingredient of PNS, exerts anti-fibrotic activity on IPF still remain unclear. In this study, we investigated the effect of Rg1 on bleomycin-induced pulmonary fibrosis in rats. Bleomycin (5 mg/kg body weight) was intratracheally administrated to male rats. Rg1 (18, 36 and 72 mg/kg) was orally administered on the next day after bleomycin. Lungs were harvested at day 7 and 28 for the further experiments. Histological analysis revealed that bleomycin successfully induced pulmonary fibrosis, and that Rg1 restored the histological alteration of bleomycin-induced pulmonary fibrosis (PF), significantly decreased lung coefficient, scores of alveolitis, scores of PF as well as contents of alpha smooth muscle actin (α-SMA) and hydroxyproline (Hyp) in a dose-dependent manner in PF rats. Moreover, Rg1 increased the expression levels of Caveolin-1 (Cav-1) mRNA and protein, lowered the expression of transforming growth factor-β1 (TGF-β1) mRNA and protein in the lung tissues of PF rats. These data suggest that Rg1 exhibits protective effect against bleomycin-induced PF in rats, which is potentially associated with the down-regulation of TGF-β1 and up-regulation of Cav-1.

  3. Identification of a myeloid-derived suppressor cell cystatin-like protein that inhibits metastasis.

    PubMed

    Boutté, Angela M; Friedman, David B; Bogyo, Matthew; Min, Yongfen; Yang, Li; Lin, P Charles

    2011-08-01

    Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by ∼ 40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.

  4. Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer

    PubMed Central

    2014-01-01

    Background Thyroid cancer is the most common endocrine malignancy, and many patients with metastatic differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) fail to respond to conventional therapies, resulting in morbidity and mortality. Additional therapeutic targets and treatment options are needed for these patients. We recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ATC and confers an aggressive phenotype when overexpressed in DTC cells. Methods Microarray analysis was used to identify downstream targets of PPARγ in ATC cells. Western blot analysis and immunohistochemistry (IHC) were used to assess thioredoxin interacting protein (TXNIP) expression in thyroid cancer cell lines and primary tumor specimens. Retroviral transduction was used to generate ATC cell lines that overexpress TXNIP, and assays that assess glucose uptake, viable cell proliferation, and invasion were used to characterize the in vitro properties of these cells. An orthotopic thyroid cancer mouse model was used to assess the effect of TXNIP overexpression in ATC cell lines in vivo. Results Using microarray analysis, we show that TXNIP is highly upregulated when PPARγ is depleted from ATC cells. Using Western blot analysis and IHC, we show that DTC and ATC cells exhibit differential TXNIP expression patterns. DTC cell lines and patient tumors have high TXNIP expression in contrast to low or absent expression in ATC cell lines and tumors. Overexpression of TXNIP decreases the growth of HTh74 cells compared to vector controls and inhibits glucose uptake in the ATC cell lines HTh74 and T238. Importantly, TXNIP overexpression in T238 cells results in attenuated tumor growth and decreased metastasis in an orthotopic thyroid cancer mouse model. Conclusions Our findings indicate that TXNIP functions as a tumor suppressor in thyroid cells, and its downregulation is likely important in

  5. Regulator of G protein signaling 6 is a novel suppressor of breast tumor initiation and progression

    PubMed Central

    Fisher, Rory A.

    2013-01-01

    Breast cancer is a large global health burden and the most frequently diagnosed malignancy in women worldwide. Here, we utilize RGS6− /− mice to interrogate the role of regulator of G protein signaling 6 (RGS6), localized to the ductal epithelium in mouse and human breast, as a novel tumor suppressor in vivo. RGS6− /− mice exhibit accelerated 7,12-dimethylbenza[α]anthracene (DMBA)-induced tumor initiation and progression, as well as decreased overall survival. Analysis of carcinogenic aberrations in the mammary glands of DMBA-treated mice revealed a failure of the DNA damage response concurrent with augmented oncogenesis in RGS6−/− animals. Furthermore, RGS6 suppressed cell growth induced by either human epidermal growth factor receptor 2 or estrogen receptor activation in both MCF-7 breast cancer cells and mammary epithelial cells (MECs). MECs isolated from RGS6−/− mice also showed a deficit in DMBA-induced ATM/p53 activation, reactive oxygen species generation and apoptosis confirming that RGS6 is required for effective activation of the DNA damage response in these cells, a critical countermeasure against carcinogen-mediated genotoxic stress. The ability of RGS6 to simultaneously enhance DNA-damage-induced apoptotic signaling and suppress oncogenic cell growth likely underlie the accelerated tumorigenesis and cellular transformation observed in DMBA-treated RGS6−/− mice and isolated MECs, respectively. Unsurprisingly, spontaneous tumor formation was also seen in old female RGS6−/− but not in wild-type mice. Our finding that RGS6 is downregulated in all human breast cancer subtypes independent of their molecular classification indicates that obtaining a means to restore the growth suppressive and pro-apoptotic actions of RGS6 in breast might be a viable means to treat a large spectrum of breast tumors. PMID:23598467

  6. Regulator of G protein signaling 6 is a novel suppressor of breast tumor initiation and progression.

    PubMed

    Maity, Biswanath; Stewart, Adele; O'Malley, Yunxia; Askeland, Ryan W; Sugg, Sonia L; Fisher, Rory A

    2013-08-01

    Breast cancer is a large global health burden and the most frequently diagnosed malignancy in women worldwide. Here, we utilize RGS6(-/-) mice to interrogate the role of regulator of G protein signaling 6 (RGS6), localized to the ductal epithelium in mouse and human breast, as a novel tumor suppressor in vivo. RGS6(-/-) mice exhibit accelerated 7,12-dimethylbenza[α]anthracene (DMBA)-induced tumor initiation and progression, as well as decreased overall survival. Analysis of carcinogenic aberrations in the mammary glands of DMBA-treated mice revealed a failure of the DNA damage response concurrent with augmented oncogenesis in RGS6(-/-) animals. Furthermore, RGS6 suppressed cell growth induced by either human epidermal growth factor receptor 2 or estrogen receptor activation in both MCF-7 breast cancer cells and mammary epithelial cells (MECs). MECs isolated from RGS6(-/-) mice also showed a deficit in DMBA-induced ATM/p53 activation, reactive oxygen species generation and apoptosis confirming that RGS6 is required for effective activation of the DNA damage response in these cells, a critical countermeasure against carcinogen-mediated genotoxic stress. The ability of RGS6 to simultaneously enhance DNA-damage-induced apoptotic signaling and suppress oncogenic cell growth likely underlie the accelerated tumorigenesis and cellular transformation observed in DMBA-treated RGS6(-/-) mice and isolated MECs, respectively. Unsurprisingly, spontaneous tumor formation was also seen in old female RGS6(-/-) but not in wild-type mice. Our finding that RGS6 is downregulated in all human breast cancer subtypes independent of their molecular classification indicates that obtaining a means to restore the growth suppressive and pro-apoptotic actions of RGS6 in breast might be a viable means to treat a large spectrum of breast tumors.

  7. The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein.

    PubMed

    Vincent, A; Liebman, S W

    1992-10-01

    The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.

  8. HMG-CoA reductase inhibitor improves endothelial dysfunction in spontaneous hypertensive rats via down-regulation of caveolin-1 and activation of endothelial nitric oxide synthase.

    PubMed

    Suh, Jung-Won; Choi, Dong-Ju; Chang, Hyuk-Jae; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Kim, Kwang-Il; Kim, Cheol-Ho; Kim, Hyo-Soo; Oh, Buyng-Hee; Park, Young-Bae

    2010-01-01

    Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10(-9)-10(-4) M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status.

  9. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.

    PubMed Central

    Munemitsu, S; Albert, I; Souza, B; Rubinfeld, B; Polakis, P

    1995-01-01

    The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. We examined the effects of exogenous APC expression on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC caused a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction was due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lacked this activity. Mutational analysis revealed that the central region of the APC protein, which is typically deleted or severely truncated in tumors, was responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7708772

  10. Quantum Dots-Based Immunofluorescent Imaging of Stromal Fibroblasts Caveolin-1 and Light Chain 3B Expression and Identification of Their Clinical Significance in Human Gastric Cancer

    PubMed Central

    He, Yuyu; Zhao, Xianda; Gao, Jun; Fan, Lifang; Yang, Guifang; Cho, William Chi-shing; Chen, Honglei

    2012-01-01

    Caveolin-1 (Cav-1) expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts) are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC) and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV)-associated GC (EBVaGC) is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs)-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029) that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032) and was positively associated with Cav-1 expression (r = 0.432, p < 0.001). EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis. PMID:23203033

  11. Caveolae and caveolin-1 are implicated in 1alpha,25(OH)2-vitamin D3-dependent modulation of Src, MAPK cascades and VDR localization in skeletal muscle cells.

    PubMed

    Buitrago, Claudia; Boland, Ricardo

    2010-07-01

    We previously reported that 1alpha,25(OH)2D3 induces non-transcriptional rapid responses through activation of MAPKs in C2C12 skeletal muscle cells. However, there is little information on the molecular mechanism underlying the initiation of 1alpha,25(OH)2D3 signaling through this pathway. Plasma membrane components have been involved in some non-genomic effects. In this work, we investigated the role of caveolae and caveolin-1 (cav-1) in 1alpha,25(OH)2D3-stimulation of c-Src and MAPKs. When proliferating cells were pretreated with methyl beta cyclodextrin (MbetaCD), a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1alpha,25(OH)2D3-dependent activation of ERK1/2, p38 MAPK and c-Src was suppressed. Similar results were obtained by siRNA technology whereby silencing of cav-1 expression abolished activation of c-Src and MAPKs induced by the hormone. By confocal immunocytochemistry it was observed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions. Hormone treatment disrupted the colocalization of these proteins and redistributed them into cytoplasm and nucleus. Co-immunoprecipitation assays corroborated these observations. Changes in VDR localization after 1alpha,25(OH)2D3 exposure were also investigated. Confocal microscopy images showed that the hormone induces VDR translocation to the plasma membrane, and this effect is abolished by MbetaCD. Altogether, these data suggest that caveolae is involved upstream in c-Src-MAPKs activation by 1alpha,25(OH)2D3 and that VDR and cav-1 participate in the rapid signaling elicited by the hormone.

  12. Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter, Niemann-Pick type C1.

    PubMed

    Mundy, Dorothy I; Lopez, Adam M; Posey, Kenneth S; Chuang, Jen-Chieh; Ramirez, Charina M; Scherer, Philipp E; Turley, Stephen D

    2014-07-01

    Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1(-/-)), and subsequently in Cav-1(-/-) mice that also lacked the lysosomal cholesterol transporter Niemann-Pick C1 (Npc1) (Cav-1(-/-):Npc1(-/-)). In 50-day-old Cav-1(-/-) mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1(+/+) controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1(-/-):Npc1(-/-) mice (0.356±0.022) markedly exceeded that in their Cav-1(+/+):Npc1(+/+) controls (0.137±0.009), as well as in their Cav-1(-/-):Npc1(+/+) (0.191±0.013) and Cav-1(+/+):Npc1(-/-) (0.213±0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74±0.17, 0.71±0.05, 0.96±0.05 and 3.12±0.43, respectively, with the extra cholesterol in the Cav-1(-/-):Npc1(-/-) and Cav-1(+/+):Npc1(-/-) mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1(-/-):Npc1(-/-) mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.

  13. Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells.

    PubMed

    Tirado, Oscar M; Mateo-Lozano, Silvia; Villar, Joaquín; Dettin, Luis E; Llort, Anna; Gallego, Soledad; Ban, Jozef; Kovar, Heinrich; Notario, Vicente

    2006-10-15

    Tumors of the Ewing's sarcoma family (ESFT), such as Ewing's sarcoma (EWS) and primitive neuroectodermal tumors (PNET), are highly aggressive malignancies predominantly affecting children and young adults. ESFT express chimeric transcription factors encoded by hybrid genes fusing the EWS gene with several ETS genes, most commonly FLI-1. EWS/FLI-1 proteins are responsible for the malignant phenotype of ESFT, but only few of their transcriptional targets are known. Using antisense and short hairpin RNA-mediated gene expression knockdown, array analyses, chromatin immunoprecipitation methods, and reexpression studies, we show that caveolin-1 (CAV1) is a new direct target of EWS/FLI-1 that is overexpressed in ESFT cell lines and tumor specimens and is necessary for ESFT tumorigenesis. CAV1 knockdown led to up-regulation of Snail and the concomitant loss of E-cadherin expression. Consistently, loss of CAV1 expression inhibited the anchorage-independent growth of EWS cells and markedly reduced the growth of EWS cell-derived tumors in nude mice xenografts, indicating that CAV1 promotes the malignant phenotype in EWS carcinogenesis. Reexpression of CAV1 or E-cadherin in CAV1 knockdown EWS cells rescued the oncogenic phenotype of the original EWS cells, showing that the CAV1/Snail/E-cadherin pathway plays a central role in the expression of the oncogenic transformation functions of EWS/FLI-1. Overall, these data identify CAV1 as a key determinant of the tumorigenicity of ESFT and imply that targeting CAV1 may allow the development of new molecular therapeutic strategies for ESFT patients.

  14. A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

    PubMed

    Koonin, E V; Bork, P; Sander, C

    1994-06-11

    Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.

  15. Valproic acid inhibits the invasion of PC3 prostate cancer cells by upregulating the metastasis suppressor protein NDRG1.

    PubMed

    Lee, Jae Eun; Kim, Jung Hwa

    2015-12-01

    Valproic acid (VPA) is a clinically available histone deacetylase inhibitor with promising anticancer attributes. Recent studies have demonstrated the anticancer effects of VPA on prostate cancer cells. However, little is known about the differential effects of VPA between metastatic and non-metastatic prostate cancer cells and the relationship between the expression of metastasis suppressor proteins and VPA. In the present study, we demonstrate that inhibition of cell viability and invasion by VPA was more effective in the metastatic prostate cancer cell line PC3 than in the tumorigenic but non-metastatic prostate cell line, RWPE2. Further, we identified that the metastasis suppressor NDRG1 is upregulated in PC3 by VPA treatment. In contrast, NDRG1 was not increased in RWPE2 cells. Also, the suppressed invasion of PC3 cells by VPA treatment was relieved by NDRG1 knockdown. Taken together, we suggest that the anticancer effect of VPA on prostate cancer cells is, in part, mediated through upregulation of NDRG1. We also conclude that VPA has differential effects on the metastasis suppressor gene and invasion ability between non-metastatic and metastatic prostate cancer cells.

  16. Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins

    PubMed Central

    Köhrer, Caroline; Xie, Liang; Kellerer, Susanne; Varshney, Umesh; RajBhandary, Uttam L.

    2001-01-01

    A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoacylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only requirement is that the suppressor tRNA must not be a substrate for any of the cellular aminoacyl–tRNA synthetases. Here, we describe conditions for the import of amber and ochre suppressor tRNAs derived from Escherichia coli initiator tRNA into mammalian COS1 cells, and we present evidence for their activity in the specific suppression of amber (UAG) and ochre (UAA) codons, respectively. We show that an aminoacylated amber suppressor tRNA (supF) derived from the E. coli tyrosine tRNA can be imported into COS1 cells and acts as a suppressor of amber codons, whereas the same suppressor tRNA imported without prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl–tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid analogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility further of importing a mixture of amber and ochre suppressor tRNAs for the insertion of two different amino acid analogues into a protein and the potential use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations. PMID:11717406

  17. Age-Related Modulations of AQP4 and Caveolin-1 in the Hippocampus Predispose the Toxic Effect of Phoneutria nigriventer Spider Venom

    PubMed Central

    Soares, Edilene S.; Stávale, Leila M.; Mendonça, Monique C. P.; Coope, Andressa; da Cruz-Höfling, Maria Alice

    2016-01-01

    We have previously demonstrated that Phoneutria nigriventer venom (PNV) causes blood–brain barrier (BBB) breakdown, swelling of astrocytes end-feet and fluid permeation into brain interstitium in rats. Caveolae and water channels respond to BBB alterations by co-participation in shear stress response and edema formation/resolution. Herein, we showed post-natal developmental-related changes of two BBB-associated transporter proteins: the endothelial caveolin-1 (Cav-1), the major scaffolding protein from caveolae frame, and the astroglial aquaporin-4 (AQP4), the main water channel protein expressed in astrocytic peri-vascular end-feet processes, in the hippocampus of rats intraperitoneally-administered PNV. Western blotting protein levels; immunohistochemistry (IHC) protein distribution in CA1, CA2, and CA3 subfields; and gene expression by Real Time-Polymerase Chain Reaction (qPCR) were assessed in post-natal Day 14 (P14) and 8–10-week-old rats over critical periods of envenomation. The intensity and duration of the toxic manifestations indicate P14 neonate rats more vulnerable to PNV than adults. Histologically, the capillaries of P14 and 8–10-week-old rats treated with PNV showed perivascular edema, while controls did not. The intensity of the toxic manifestations in P14 decreases temporally (2 > 5 > 24 h), while inversely the expression of AQP4 and Cav-1 peaked at 24 h when clinically PNV-treated animals do not differ from saline controls. IHC of AQP4 revealed that hippocampal CA1 showed the least expression at 2 h when toxic manifestation was maximal. Subfield IHC quantification revealed that in P14 rats Cav-1 peaked at 24 h when toxic manifestations were absent, whereas in 8–10-week-old rats Cav-1 peaked at 2 h when toxic signs were highest, and progressively attenuated such increases until 24 h, remaining though significantly above baseline. Considering astrocyte-endothelial physical and functional interactions, we hypothesize that age

  18. Ubiquitin-specific protease 11 (USP11) functions as a tumor suppressor through deubiquitinating and stabilizing VGLL4 protein

    PubMed Central

    Zhang, Encheng; Shen, Bing; Mu, Xingyu; Qin, Yan; Zhang, Fang; Liu, Yong; Xiao, Jiantao; Zhang, Pingzhao; Wang, Chenji; Tan, Mingyue; Fan, Yu

    2016-01-01

    VGLL4 is a transcriptional repressor that interacts with transcription factors TEADs and inhibits YAP-induced overgrowth and tumorigenesis. VGLL4 protein was dramatically reduced in various types of human cancers. But how VGLL4 protein is post-transcriptional regulated is poorly understood. In this study, we identify deubiquitinating enzyme USP11 as a novel VGLL4 interactor. We reveal that the USP domain of USP11 and the N-terminal region of VGLL4 are required for mutual binding. USP11 controls VGLL4 protein stability by promoting its deubiquitination. Furthermore, our results show that knockdown of USP11 promotes cell growth, migration, and invasion in a YAP-dependent manner. Together, our results suggest that USP11 may exert its tumor suppressor role by modulating VGLL4/YAP-TEADs regulatory loop. PMID:28042509

  19. Human melanoma cells express FGFR/Src/Rho signaling that entails an adhesion-independent caveolin-1 membrane association.

    PubMed

    Fecchi, Katia; Travaglione, Sara; Spadaro, Francesca; Quattrini, Adriano; Parolini, Isabella; Piccaro, Giovanni; Raggi, Carla; Fabbri, Alessia; Felicetti, Federica; Carè, Alessandra; Fiorentini, Carla; Sargiacomo, Massimo

    2012-03-15

    Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin-1 (Cav-1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolae's asset, through bFGF/FGFR/c-Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav-1 enriched detergent-resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav-1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav-1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav-1 disassembly, together with persisting cell signal activity, being Src and Cav-1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.

  20. Evidence for Dsg3 in regulating Src signaling by competing with it for binding to caveolin-1

    PubMed Central

    Wan, Hong; Lin, Kuang; Tsang, Siu Man; Uttagomol, Jutamas

    2015-01-01

    This data article contains extended, complementary analysis related to the research articles entitled “Desmoglein 3, via an interaction with E-cadherin, is associated with activation of Src” (Tsang et al., 2010) [1] and figures related to the review article entitled “Desmoglein 3: a help or a hindrance in cancer progression?” (Brown et al., 2014) [2]. We show here that both Src and caveolin-1 (Cav-1) associate with Dsg3 in a non-ionic detergent soluble pool and that modulation of Dsg3 levels inversely alters the expression of Src in the Cav-1 complex. Furthermore, immunofluorescence analysis revealed a reduced colocalization of Cav-1/total Src in cells with overexpression of Dsg3 compared to control cells. In support, the sequence analysis has identified a region within the carboxyl-terminus of human Dsg3 for a likelihood of binding to the scaffolding domain of Cav-1, the known Src binding site in Cav-1, and this region is highly conserved across most of 18 species as well as within desmoglein family members. Based on these findings, we propose a working model that Dsg3 activates Src through competing with its inactive form for binding to Cav-1, thus leading to release of Src followed by its auto-activation. PMID:26858977

  1. Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains.

    PubMed

    Rimoldi, Valeria; Reversi, Alessandra; Taverna, Elena; Rosa, Patrizia; Francolini, Maura; Cassoni, Paola; Parenti, Marco; Chini, Bice

    2003-09-04

    We have recently shown that oxytocin inhibits cell proliferation when the vast majority of oxytocin receptors are excluded from caveolin-1-enriched microdomains, and that, on the contrary, it has a mitogenic effect when the receptors are targeted to these plasma membrane domains. In this study, we investigated whether the receptors located inside and outside caveolar microdomains initiate different signalling pathways and how this may lead to opposite effects on cell proliferation. Our data indicate that, depending on their localization, oxytocin receptors transactivate EGFR and activate ERK1/2 using different signalling intermediates. The final outcome is a different temporal pattern of EGFR and ERK1/2 phosphorylation, which is more persistent when the receptors are located outside caveolar microdomains and inhibit cell growth, and very transient when they are located in caveolar microdomains and stimulate cell growth. Finally, only the activation of receptors located outside caveolar microdomains correlates with the activation of the cell cycle inhibitor p21(WAF1/CIP1), thus suggesting that the antiproliferative OTR effects may, in this case, be achieved by a sustained activation of EGFR and MAPK leading to the induction of this cell cycle regulator.

  2. Secreted frizzled-related protein-5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer.

    PubMed

    Kawakami, Kazumori; Yamamura, Soichiro; Hirata, Hiroshi; Ueno, Koji; Saini, Sharanjot; Majid, Shahana; Tanaka, Yuichiro; Kawamoto, Ken; Enokida, Hideki; Nakagawa, Masayuki; Dahiya, Rajvir

    2011-02-01

    Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.

  3. The G-protein Alpha Subunit Gsα Is A Tumor Suppressor In Sonic Hedgehog-driven Medulloblastoma

    PubMed Central

    He, Xuelian; Zhang, Liguo; Chen, Ying; Remke, Marc; Shih, David; Lu, Fanghui; Wang, Haibo; Deng, Yaqi; Yu, Yang; Xia, Yong; Wu, Xiaochong; Ramaswamy, Vijay; Hu, Tom; Wang, Fan; Zhou, Wenhao; Burns, Dennis K.; Kim, Se Hoon; Kool, Marcel; Pfister, Stefan M.; Weinstein, Lee S.; Pomeroy, Scott L.; Gilbertson, Richard J.; Rubin, Joshua B.; Hou, Yiping; Wechsler-Reya, Robert; Taylor, Michael D.; Lu, Q. Richard

    2014-01-01

    Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G-protein Gsα, as a potent tumor suppressor gene that defines a subset of aggressive Sonic Hedgehog (Shh)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically-distinct progenitors is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gsα is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh-signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation of a Gsα effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas mutants. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gsα that acts as a molecular link across Shh-group medulloblastomas of disparate cellular and anatomical origins, illuminating G-protein modulation as a potential therapeutic avenue. PMID:25150496

  4. Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA

    PubMed Central

    Samuel, Glady Hazitha; Wiley, Michael R.; Badawi, Atif; Adelman, Zach N.; Myles, Kevin M.

    2016-01-01

    Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen’s ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito’s RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular “arms race” between vector and pathogen underlies the continued existence of flaviviruses in nature. PMID:27849599

  5. Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster

    SciTech Connect

    Holdridge, C.; Dorsett, D. )

    1991-04-01

    The suppressor of hairy-wing [su(Hw)] locus of Drosophila melanogaster encodes a zinc finger protein that binds a repeated motif in the gypsy retroposon. Mutations of su(Hw) suppress the phenotypes associated with mutations caused by gypsy insertions. To examine the mechanisms by which su(Hw) alters gene expression, a fragment of gypsy containing multiple su(Hw) protein-binding sites was inserted into various locations in the well-characterized Drosophila hsp70 heat shock gene promoter. The authors found no evidence for activation of basal hsp70 transcription by su(Hw) protein in cultured Drosophila cells but observed that it can repress heat shock-induced transcription. Repression occurred only when su(Hw) protein-binding sites were positioned between binding sites for proteins required for heat shock transcription. They propose that su(Hw) protein interferes nonspecifically with protein-protein interactions required for heat shock transcription, perhaps sterically, or by altering the ability of DNA to bend or twist.

  6. Overexpression of caveolin-1 attenuates brain edema by inhibiting tight junction degradation

    PubMed Central

    Choi, Kang-Ho; Lee, Eun-Bin; Lee, Jung-Kil; Kim, Joon-Tae; Kim, Ja-Hae; Lee, Min-Cheol; Lee, Hong-Joon; Cho, Ki-Hyun

    2016-01-01

    Cerebral edema from the disruption of the blood-brain barrier (BBB) after cerebral ischemia is a major cause of morbidity and mortality as well as a common event in patients with stroke. Caveolins (Cavs) are thought to regulate BBB functions. Here, we report for the first time that Cav-1 overexpression (OE) decreased brain edema from BBB disruption following ischemic insult. Edema volumes and Cav-1 expression levels were measured following photothrombosis and middle cerebral artery occlusion (MCAO). Endothelial cells that were transduced with a Cav-1 lentiviral expression vector were transplanted into rats. BBB permeability was quantified with Evans blue extravasation. Edema volume was determined from measures of the extravasation area, brain water content, and average fluorescence intensity after Cy5.5 injections. Tight junction (TJ) protein expression was measured with immunoblotting. Cav-1 expression levels and vasogenic brain edema correlated strongly after ischemic insult. Cav-1 expression and BBB disruption peaked 3 d after the MCAO. In addition, intravenous administration of endothelial cells expressing Cav-1 effectively increased the Cav-1 levels 3 d after the MCAO ischemic insult. Importantly, Cav-1 OE ameliorated the vasogenic edema by inhibiting the degradation of TJ protein expression in the acute phase of ischemic stroke. These results suggested that Cav-1 OE protected the integrity of the BBB mainly by preventing the degradation of TJ proteins in rats. These findings need to be confirmed in a clinical setting in human subjects. PMID:27708218

  7. NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

    PubMed Central

    Boguta, M; Dmochowska, A; Borsuk, P; Wrobel, K; Gargouri, A; Lazowska, J; Slonimski, P P; Szczesniak, B; Kruszewska, A

    1992-01-01

    We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation. Images PMID:1729612

  8. Caveolin-1 is critical for abdominal aortic aneurysm formation induced by angiotensin II and inhibition of lysyl oxidase

    PubMed Central

    Takayanagi, Takehiko; Crawford, Kevin J.; Kobayashi, Tomonori; Obama, Takashi; Tsuji, Toshiyuki; Elliott, Katherine J.; Hashimoto, Tomoki; Rizzo, Victor; Eguchi, Satoru

    2014-01-01

    Although angiotensin II (Ang II) and its receptor AT1 have been implicated in abdominal aortic aneurysm (AAA) formation, the proximal signaling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signaling platform to facilitate the temporal and spatial localization of signal transduction events including those stimulated by Ang II. Caveolin-1 (Cav1) enriched caveolae in vascular smooth muscle cells mediate ADAM17-dependent epidermal growth factor receptor (EGFR) transactivation, which is linked to vascular remodeling induced by Ang II. Here, we have tested our hypothesis that Cav1 plays a critical role for development of AAA at least in part via its specific alteration of Ang II signaling within caveolae. Cav1−/− mice and the control wild-type mice were co-infused with Ang II and β-aminopropionitrile to induce AAA. We found that Cav1−/− mice with the co-infusion did not develop AAA compared to control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1−/− aortae with the co-infusion. Furthermore, Cav1−/− mice aortae with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared to aortae from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented Ang II-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress presumably through the regulation of caveolae compartmentalized signals induced by Ang II. PMID:24329494

  9. Roles of Caveolin-1 in Angiotensin II-Induced Hypertrophy and Inward Remodeling of Cerebral Pial Arterioles.

    PubMed

    Umesalma, Shaikamjad; Houwen, Frederick Keith; Baumbach, Gary L; Chan, Siu-Lung

    2016-03-01

    Angiotensin II (Ang II) is a major determinant of inward remodeling and hypertrophy in pial arterioles that may have an important role in stroke during chronic hypertension. Previously, we found that epidermal growth factor receptor is critical in Ang II-mediated hypertrophy that may involve caveolin-1 (Cav-1). In this study, we examined the effects of Cav-1 and matrix metalloproteinase-9 (MMP9) on Ang II-mediated structural changes in pial arterioles. Cav-1-deficient (Cav-1(-/-)), MMP9-deficient (MMP9(-/-)), and wild-type mice were infused with either Ang II (1000 ng/kg per minute) or saline via osmotic minipumps for 28 days (n=6-8 per group). Systolic arterial pressure was measured by a tail-cuff method. Pressure and diameter of pial arterioles were measured through an open cranial window in anesthetized mice. Cross-sectional area of the wall was determined histologically in pressurized fixed pial arterioles. Expression of Cav-1, MMP9, phosphorylated epidermal growth factor receptor, and Akt was determined by Western blotting and immunohistochemistry. Deficiency of Cav-1 or MMP9 did not affect Ang II-induced hypertension. Ang II increased the expression of Cav-1, phosphorylated epidermal growth factor receptor, and Akt in wild-type mice, which was attenuated in Cav-1(-/-) mice. Ang II-induced hypertrophy, inward remodeling, and increased MMP9 expression in pial arterioles were prevented in Cav-1(-/-) mice. Ang II-mediated increases in MMP9 expression and inward remodeling, but not hypertrophy, were prevented in MMP9(-/-) mice. In conclusion, Cav-1 is essential in Ang II-mediated inward remodeling and hypertrophy in pial arterioles. Cav-1-induced MMP9 is exclusively involved in inward remodeling, not hypertrophy. Further studies are needed to determine the role of Akt in Ang II-mediated hypertrophy.

  10. Pyrogallol abates VSMC migration via modulation of Caveolin-1, matrix metalloproteinase and intima hyperplasia in carotid ligation mouse.

    PubMed

    Ma, Yu-Dong; Thiyagarajan, Varadharajan; Tsai, May-Jywan; Lue, Sheng-I; Chia, Yi-Chen; Shyue, Song-Kun; Weng, Ching-Feng

    2016-12-01

    Migration of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia and other vascular diseases. Caveolin-1 (Cav-1) has been recognized as a proliferative inhibitor of VSMCs and is likely to be an important regulator of VSMC migration. The underlying mechanism of pyrogallol on the VSMC migration is not fully understood. This study attempted to dissect the role of Cav-1 and matrix metalloproteinase (MMP) in VSMC migration and to investigate the effect of pyrogallol on VSMC mobility during carotid artery ligation mice. The mRNA expression of MMP-3 and MMP-13 was down-regulated in cultured VSMC prepared from Cav-1-deficient (Cav-1 KO) mice whereas MMP-14 expression was up-regulated. Pyrogallol effectively inhibited the migration of Cav-1 KO VSMC by promoting the expression of tissue inhibitors of metalloproteinase (TIMP)-2. Pyrogallol also inhibited the migration of Cav-1 wild type (WT) VSMC, however, by increasing TIMP-1 expression and repressing MMP-2 activity. In a parallel in vivo study, intra-peritoneal (ip) of pyrogallol to carotid artery ligated mice significantly suppressed intima formation in mice carotid artery. Furthermore, the proMMP-9 activity in pyrogallol-treated mice serum significantly increased from Day 0 to Day 2 and decreased from Day 2 to Day 7 in a time-dependent manner. In addition, WT mice treated with pyrogallol had significantly reduced neointima formation, whereas no differences were observed in Cav-1 knock out (KO) mice. These results suggest that pyrogallol not only inhibited VSMC migration but also effectively diminishes the severity of neointima hyperplasia, implying that pyrogallol possesses potential anti-atherogenic effects for the treatment of vascular diseases.

  11. Driver gene classification reveals a substantial overrepresentation of tumor suppressors among very large chromatin-regulating proteins

    PubMed Central

    Waks, Zeev; Weissbrod, Omer; Carmeli, Boaz; Norel, Raquel; Utro, Filippo; Goldschmidt, Yaara

    2016-01-01

    Compiling a comprehensive list of cancer driver genes is imperative for oncology diagnostics and drug development. While driver genes are typically discovered by analysis of tumor genomes, infrequently mutated driver genes often evade detection due to limited sample sizes. Here, we address sample size limitations by integrating tumor genomics data with a wide spectrum of gene-specific properties to search for rare drivers, functionally classify them, and detect features characteristic of driver genes. We show that our approach, CAnceR geNe similarity-based Annotator and Finder (CARNAF), enables detection of potentially novel drivers that eluded over a dozen pan-cancer/multi-tumor type studies. In particular, feature analysis reveals a highly concentrated pool of known and putative tumor suppressors among the <1% of genes that encode very large, chromatin-regulating proteins. Thus, our study highlights the need for deeper characterization of very large, epigenetic regulators in the context of cancer causality. PMID:28008934

  12. The effects of turmeric (curcumin) on tumor suppressor protein (p53) and estrogen receptor (ERα) in breast cancer cells

    PubMed Central

    Hallman, Kelly; Aleck, Katie; Dwyer, Brigitte; Lloyd, Victoria; Quigley, Meghan; Sitto, Nada; Siebert, Amy E; Dinda, Sumi

    2017-01-01

    Curcumin (CUR) is a compound that has antibacterial, antiviral, anti-inflammatory, and anticancer properties. In this study, we have analyzed the effects of CUR on the expression of ERα and p53 in the presence of hormones and anti-hormones in breast cancer cells. Cells were cultured in a medium containing charcoal-stripped fetal bovine serum to deplete any endogenous steroids and treated with CUR at varying concentrations or in combination with hormones and anti-hormones. Protein analysis revealed a relative decrease in the levels of p53 and ERα upon treatment with 5–60 µM CUR. In cell proliferation studies, CUR alone caused a 10-fold decrease compared with the treatment with estrogen, which suggests its antiproliferative effects. Delineating the role of CUR in the regulation of p53, ERα, and their mechanisms of action may be important in understanding the influence of CUR on tumor suppressors and hormone receptors in breast cancer. PMID:28331366

  13. Driver gene classification reveals a substantial overrepresentation of tumor suppressors among very large chromatin-regulating proteins.

    PubMed

    Waks, Zeev; Weissbrod, Omer; Carmeli, Boaz; Norel, Raquel; Utro, Filippo; Goldschmidt, Yaara

    2016-12-23

    Compiling a comprehensive list of cancer driver genes is imperative for oncology diagnostics and drug development. While driver genes are typically discovered by analysis of tumor genomes, infrequently mutated driver genes often evade detection due to limited sample sizes. Here, we address sample size limitations by integrating tumor genomics data with a wide spectrum of gene-specific properties to search for rare drivers, functionally classify them, and detect features characteristic of driver genes. We show that our approach, CAnceR geNe similarity-based Annotator and Finder (CARNAF), enables detection of potentially novel drivers that eluded over a dozen pan-cancer/multi-tumor type studies. In particular, feature analysis reveals a highly concentrated pool of known and putative tumor suppressors among the <1% of genes that encode very large, chromatin-regulating proteins. Thus, our study highlights the need for deeper characterization of very large, epigenetic regulators in the context of cancer causality.

  14. Suppressors of a Host Range Mutation in the Rabbitpox Virus Serpin SPI-1 Map to Proteins Essential for Viral DNA Replication

    PubMed Central

    Luttge, Benjamin G.; Moyer, Richard W.

    2005-01-01

    The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism. PMID:15994811

  15. Suppressors of a host range mutation in the rabbitpox virus serpin SPI-1 map to proteins essential for viral DNA replication.

    PubMed

    Luttge, Benjamin G; Moyer, Richard W

    2005-07-01

    The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.

  16. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    PubMed Central

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  17. Epigenetic regulation of protein-coding and microRNA genes by the Gfi1-interacting tumor suppressor PRDM5.

    PubMed

    Duan, Zhijun; Person, Richard E; Lee, Hu-Hui; Huang, Shi; Donadieu, Jean; Badolato, Raffaele; Grimes, H Leighton; Papayannopoulou, Thalia; Horwitz, Marshall S

    2007-10-01

    Gfi1 transcriptionally governs hematopoiesis, and its mutations produce neutropenia. In an effort to identify Gfi1-interacting proteins and also to generate new candidate genes causing neutropenia, we performed a yeast two-hybrid screen with Gfi1. Among other Gfi1-interacting proteins, we identified a previously uncharacterized member of the PR domain-containing family of tumor suppressors, PRDM5. PRDM5 has 16 zinc fingers, and we show that it acts as a sequence-specific, DNA binding transcription factor that targets hematopoiesis-associated protein-coding and microRNA genes, including many that are also targets of Gfi1. PRDM5 epigenetically regulates transcription similarly to Gfi1: it recruits the histone methyltransferase G9a and class I histone deacetylases to its target gene promoters and demonstrates repressor activity on synthetic reporters; on endogenous target genes, however, it functions as an activator, in addition to a repressor. Interestingly, genes that PRDM5 activates, as opposed to those it represses, are also targets of Gfi1, suggesting a competitive mechanism through which two repressors could cooperate in order to become transcriptional activators. In neutropenic patients, we identified PRDM5 protein sequence variants perturbing transcriptional function, suggesting a potentially important role in hematopoiesis.

  18. Cullin 1 functions as a centrosomal suppressor of centriole multiplication by regulating Polo-like kinase 4 protein levels

    PubMed Central

    Korzeniewski, Nina; Zheng, Leon; Cuevas, Rolando; Parry, Joshua; Chatterjee, Payel; Anderton, Brittany; Duensing, Anette; Münger, Karl; Duensing, Stefan

    2009-01-01

    Abnormal centrosome and centriole numbers are frequently detected in tumor cells where they can contribute to mitotic aberrations that cause chromosome missegregation and aneuploidy. The molecular mechanisms of centriole overduplication in malignant cells, however, are poorly characterized. Here, we show that the core SCF component CUL1 localizes to maternal centrioles and that CUL1 is critical for suppressing centriole overduplication through multiplication, a recently discovered mechanism whereby multiple daughter centrioles form concurrently at single maternal centrioles. We found that this activity of CUL1 involves the degradation of Polo-like kinase 4 (PLK4) at maternal centrioles. PLK4 is required for centriole duplication and strongly stimulates centriole multiplication when aberrantly expressed. We found that CUL1 is critical for the degradation of active PLK4 following deregulation of cyclin E/CDK2 activity, as is frequently observed in human cancer cells, as well as for baseline PLK4 protein stability. Collectively, our results suggest that CUL1 may function as a tumor suppressor by regulating PLK4 protein levels and thereby restraining excessive daughter centriole formation at maternal centrioles. PMID:19679553

  19. SUPPRESSOR OF FRIGIDA3 Encodes a Nuclear ACTIN-RELATED PROTEIN6 Required for Floral Repression in ArabidopsisW⃞

    PubMed Central

    Choi, Kyuha; Kim, Sanghee; Kim, Sang Yeol; Kim, Minsoo; Hyun, Youbong; Lee, Horim; Choe, Sunghwa; Kim, Sang-Gu; Michaels, Scott; Lee, Ilha

    2005-01-01

    Flowering traits in winter annual Arabidopsis thaliana are conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FLC acts as a flowering repressor and is regulated by multiple flowering pathways. We isolated an early-flowering mutant, suppressor of FRIGIDA3 (suf3), which also shows leaf serration, weak apical dominance, and infrequent conversion of the inflorescence shoot to a terminal flower. The suf3 mutation caused a decrease in the transcript level of FLC in both a FRI-containing line and autonomous pathway mutants. However, suf3 showed only a partial reduction of FLC transcript level, although it largely suppressed the late-flowering phenotype. In addition, the suf3 mutation caused acceleration of flowering in both 35S-FLC and a flc null mutant, indicating that SUF3 regulates additional factor(s) for the repression of flowering. SUF3 is highly expressed in the shoot apex, but the expression is not regulated by FRI, autonomous pathway genes, or vernalization. SUF3 encodes the nuclear ACTIN-RELATED PROTEIN6 (ARP6), the homolog of which in yeast is a component of an ATP-dependent chromatin-remodeling SWR1 complex. Our analyses showed that SUF3 regulates FLC expression independent of vernalization, FRI, and an autonomous pathway gene, all of which affect the histone modification of FLC chromatin. Subcellular localization using a green fluorescent protein fusion showed that Arabidopsis ARP6 is located at distinct regions of the nuclear periphery. PMID:16155178

  20. Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Scotcher, Jenna; Clarke, David J.; Weidt, Stefan K.; Mackay, C. Logan; Hupp, Ted R.; Sadler, Peter J.; Langridge-Smith, Pat R. R.

    2011-05-01

    The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.

  1. Overexpression of tumor suppressor protein OSCP1/NOR1 induces ER stress and apoptosis during development of Drosophila melanogaster

    PubMed Central

    Huu, Nguyen Tho; Yoshida, Hideki; Yamaguchi, Masamitsu

    2015-01-01

    OSCP1/NOR1 (organic solute carrier partner 1/oxidored-nitro domain-containing protein 1) is known as a transporter of various organic solutes into cells and also is reported to act as a tumor suppressor protein. Although overexpression of OSCP1 has been shown to play multiple roles in mammalian cell lines, its biological significance in living organisms is not fully understood. To explore the effects of OSCP1/NOR1 on development, we performed genetic studies in flies featuring overexpression of its Drosophila orthologue, dOSCP1. Overexpression of dOSCP1 in eye imaginal discs induced a rough eye phenotype in adult flies, likely resulting from a delay in S phase progression and induction of caspase-dependent apoptosis followed by compensatory proliferation. However, it did not appear to be involved in differentiation of R7 photoreceptor cells. We also found that overexpression of dOSCP1 caused endoplasmic reticulum stress in salivary gland cells. These results indicate that overexpression of dOSCP1 exerts effects on various biological processes during Drosophila development. PMID:26175940

  2. Silencing suppressor activity of a begomovirus DNA β encoded protein and its effect on heterologous helper virus replication.

    PubMed

    Eini, Omid; Dogra, Satish C; Dry, Ian B; Randles, John W

    2012-07-01

    DNA β satellites are circular single-stranded molecules associated with some monopartite begomoviruses in the family Geminiviridae. They co-infect with their helper viruses to induce severe disease in economically important crops. The βC1 protein encoded by DNA β is a pathogenicity determinant and has been reported to suppress post-transcriptional gene silencing (PTGS). The βC1 proteins from various DNA β molecules show low levels of amino acid sequence conservation. We show here that the βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV) is a suppressor of systemic PTGS. When this DNA β satellite co-inoculated with a heterologous helper virus, Tomato leaf curl virus (ToLCV), reduced the level of ToLCV siRNA and this was associated with a higher level of virus accumulation in infected tobacco plants. This may be a mechanism by which βC1 protects a heterologous virus from host gene silencing.

  3. The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila.

    PubMed

    Hsu, Shih-Jui; Plata, Maria P; Ernest, Ben; Asgarifar, Saghi; Labrador, Mariano

    2015-07-01

    Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

  4. The Drosophila suppressor of underreplication protein binds to late-replicating regions of polytene chromosomes.

    PubMed Central

    Makunin, I V; Volkova, E I; Belyaeva, E S; Nabirochkina, E N; Pirrotta, V; Zhimulev, I F

    2002-01-01

    In many late-replicating euchromatic regions of salivary gland polytene chromosomes, DNA is underrepresented. A mutation in the SuUR gene suppresses underreplication and leads to normal levels of DNA polytenization in these regions. We identified the SuUR gene and determined its structure. In the SuUR mutant stock a 6-kb insertion was found in the fourth exon of the gene. A single SuUR transcript is present at all stages of Drosophila development and is most abundant in adult females and embryos. The SuUR gene encodes a protein of 962 amino acids whose putative sequence is similar to the N-terminal part of SNF2/SWI2 proteins. Staining of salivary gland polytene chromosomes with antibodies directed against the SuUR protein shows that the protein is localized mainly in late-replicating regions and in regions of intercalary and pericentric heterochromatin. PMID:11901119

  5. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  6. Conserved Molecular Underpinnings and Characterization of a Role for Caveolin-1 in the Tumor Microenvironment of Mature T-Cell Lymphomas

    PubMed Central

    Herek, Tyler A.; Shew, Timothy D.; Spurgin, Heather N.; Cutucache, Christine E.

    2015-01-01

    Neoplasms of extra-thymic T-cell origin represent a rare and difficult population characterized by poor clinical outcome, aggressive presentation, and poorly defined molecular characteristics. Much work has been done to gain greater insights into distinguishing features among malignant subtypes, but there also exists a need to identify unifying characteristics to assist in rapid diagnosis and subsequent potential treatment. Herein, we investigated gene expression data of five different mature T-cell lymphoma subtypes (n = 187) and found 21 genes to be up- and down-regulated across all malignancies in comparison to healthy CD4+ and CD8+ T-cell controls (n = 52). From these results, we sought to characterize a role for caveolin-1 (CAV1), a gene with previous description in the progression of both solid and hematological tumors. Caveolin-1 was upregulated, albeit with a heterogeneous nature, across all mature T-cell lymphoma subtypes, a finding confirmed using immunohistochemical staining on an independent sampling of mature T-cell lymphoma biopsies (n = 65 cases). Further, stratifying malignant samples in accordance with high and low CAV1 expression revealed that higher expression of CAV1 in mature T-cell lymphomas is analogous with an enhanced inflammatory and invasive gene expression profile. Taken together, these results demonstrate a role for CAV1 in the tumor microenvironment of mature T-cell malignancies and point toward potential prognostic implications. PMID:26566034

  7. Per os administered refined olive oil and marine PUFA-rich oils reach the cornea: possible role on oxidative stress through caveolin-1 modulation

    PubMed Central

    2009-01-01

    Background Olive oil and fish oils are known to possess beneficial properties for human health. We investigated whether different oils and fatty acids alone were able to decrease oxidative stress induced on corneal cells. Methods In our in vivo study, rats were fed with marine oils rich in polyunsaturated fatty acids (PUFA) or refined olive oil during 28 days. At the end of the protocol, corneas were analysed for their fatty acids composition to study the incorporation of fatty acids in cell membranes. In our in vitro study, a human corneal cell line was incubated with marine oils or refined olive oil and subjected to oxidative stress (tBHP 50 μM, 1 hour). Effects on reactive oxygen species generation, mitochondria and caveolin-1 expression were studied using microcytofluorometry, flow cytometry and confocal microscopy. Results Our results indicate that dietary oils changed the fatty acids composition of corneal cell membranes. According to our results, PUFA-rich oils and refined olive oil (free of antioxidants) blocked reactive oxygen species production. Oleic acid, the major fatty acid of olive oil, also decreased oxidative stress. Moreover, oleic acid modified caveolin-1 expression. Antioxidant properties of oleic acid could be due to disruption of membrane microdomains such as caveolae. Conclusion Oleic acid, a potential potent modulator of oxidative stress, could be added to PUFA-rich oils to prevent oxidative stress-linked corneal pathology. PMID:19930652

  8. Palmitoylation of cysteine 415 of CB1 receptor affects ligand-stimulated internalization and selective interaction with membrane cholesterol and caveolin 1.

    PubMed

    Oddi, Sergio; Stepniewski, Tomasz Maciej; Totaro, Antonio; Selent, Jana; Scipioni, Lucia; Dufrusine, Beatrice; Fezza, Filomena; Dainese, Enrico; Maccarrone, Mauro

    2017-02-12

    We previously demonstrated that CB1 receptor is palmitoylated at cysteine 415, and that such a post-translational modification affects its biological activity. To assess the molecular mechanisms responsible for modulation of CB1 receptor function by S-palmitoylation, in this study biochemical and morphological approaches were paralleled with computational analyses. Molecular dynamics simulations suggested that this acyl chain stabilizes helix 8 as well as the interaction of CB1 receptor with membrane cholesterol. In keeping with these in silico data, experimental results showed that the non-palmitoylated CB1 receptor was unable to interact efficaciously with caveolin 1, independently of its activation state. Moreover, in contrast with the wild-type receptor, the lack of S-palmitoylation in the helix 8 made the mutant CB1 receptor completely irresponsive to agonist-induced effects in terms of both lipid raft partitioning and receptor internalization. Overall, our results support the notion that palmitoylation of cysteine 415 modulates the conformational state of helix 8 and influences the interactions of CB1 receptor with cholesterol and caveolin 1, suggesting that the palmitoyl chain may serve as a functional interface for CB1 receptor localization and function.

  9. Novel Molecular Interactions and Biological Functions of the Neurofibromatosis 2 Tumor Suppressor Protein, Merlin

    DTIC Science & Technology

    2008-08-01

    eluted fusion proteins for the PKA assay were dialyzed against 20 mM Tris–HCl, 10 mM MgCl2, pH 7.4 at þ48C o/n. Wild-type merlin was produced in Sf9 ...BaculoGOLD system (BD Biosciences). Sf9 cells were used to produce recombinant merlin according to user’s manual (Invitrogen). Full-length merlin was...42) and recombinant merlin was produced in Sf9 insect cells as described in the GST-fusion protein section. Nocodazole 20 mM, Taxol 20 mM (Sigma) and

  10. Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

    PubMed Central

    Clark, R F; Elgin, S C

    1992-01-01

    The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin. Images PMID:1461737

  11. Novel Molecular Interactions and Biological Functions of the Neurofibromatosis 2 Tumor Suppressor Protein, Merlin

    DTIC Science & Technology

    2008-08-01

    interactions, in particular during protein oligomerization ( Burkhard et al., 2001). Inter- estingly, although the sequence in ezrin is very similar (B90...586–599. Burkhard P, Stetefeld J, , Strelkov SV. (2001). Trends Cell Biol 11: 82–88. Chambers DN, Bretscher A. (2005). Biochemistry 44: 3926– 3932

  12. The NOR1/OSCP1 proteins in cancer: from epigenetic silencing to functional characterization of a novel tumor suppressor

    PubMed Central

    Yi, Mei; Yang, Jianbo; Li, Wenjuan; Li, Xiaoling; Xiong, Wei; McCarthy, James B.; Li, Guiyuan; Xiang, Bo

    2017-01-01

    NOR1 (Oxidored-nitro domain-containing protein 1), also known as OSCP1, was first identified in nasopharyngeal carcinoma (NPC) cells in 2003. NOR1 is evolutionarily conserved among species with its expression is restricted to brain, testis and respiratory epithelial cells. NOR1 was downregulated in NPC and the downregulation associates with poor prognosis. Previous study demonstrated that hypermethylation of NOR1 promoter was observed in NPC and hematological malignancies, which has been believed to be the main epigenetic cause for NOR1 silencing in these cancers. Recently, the NOR1 tumor suppressor status has been fully established. NOR1 inhibited cancer cell growth by disturbing tumor cell energe metabolism. NOR1 also promote tumor cells apoptosis in oxidative stress and hypoxia by inhibition of stress induced autophagy. Moreover, NOR1 suppressed cancer cell epithelial-mesenchymal transition, invasion and metastasis via activation of FOXA1/HDAC2-slug regulatory network. Deciphering the molecular mechanisms underlying NOR1 mediated tumor suppressive role would be helpful to a deeper understanding of carcinogenesis and, furthermore, to the development of new therapeutic approaches. Here we summarize the current knowledge on NOR1 focusing on its expression pattern, epigenetic and genetic association with human cancers and its biological functions. This review will also elucidate the potential application of NOR1/OSCP1 for some human malignancies. PMID:28367242

  13. Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2.

    PubMed

    Okumura, Fumihiko; Joo-Okumura, Akiko; Nakatsukasa, Kunio; Kamura, Takumi

    2017-01-01

    Ubiquitin ligase von Hippel-Lindau tumor suppressor (pVHL) negatively regulates protein levels of hypoxia-inducible factor-α (HIF-α). Loss of pVHL causes HIF-α accumulation, which contributes to the pathogenesis of von Hippel-Lindau (VHL) disease. In contrast, v-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2; B-Myb), a transcription factor, prevents VHL pathogenesis by regulating gene expression of HIF-independent pathways. Both HIF-α and B-Myb are targets of pVHL-mediated polyubiquitination and proteasomal degradation. Here, we show that knockdown of HIF-2α induces downregulation of B-Myb in 786-O cells, which are deficient in pVHL, and this downregulation is prevented by proteasome inhibition. In the presence of pVHL and under hypoxia-like conditions, B-Myb and HIF-2α are both upregulated, and the upregulation of B-Myb requires expression of HIF-2α. We also show that HIF-2α and B-Myb interact in the nucleus, and this interaction is mediated by the central region of HIF-2α and the C-terminal region of B-Myb. These data indicate that oncogenic HIF-2α stabilizes B-Myb to suppress VHL pathogenesis.

  14. Targeting A Tumor Suppressor To Suppress Tumor Growth: News and Views on Protein Phosphatase 2A (PP2A) as a Target for Anti-cancer Therapy

    PubMed Central

    Perrotti, Danilo; Neviani, Paolo

    2013-01-01

    Protein phosphatase 2A (PP2A), one of the major serine-threonine phosphatases in mammalian cells, maintains cell homeostasis by counteracting most of the kinase-driven intracellular signaling pathways. Unrestrained activation of oncogenic kinases together with inhibition of tumor suppressors is frequently required for the development of cancer. Because it has been found genetically altered or functionally inactivated in many solid cancers and leukemias, PP2A is indeed a bona fide tumor suppressor. For example, the phosphatase activity of PP2A is suppressed in chronic myelogenous leukemia and other malignancies characterized by the aberrant activity of oncogenic kinases. Notably, preclinical studies indicate that pharmacologic restoration of PP2A tumor suppressor activity by PP2A activating drugs (PADs, e.g. FTY720) effectively antagonizes cancer development and progression. Herein, we systematically discuss the importance of PP2A as a druggable tumor suppressor in light of the possible introduction of PADs into anti-cancer therapeutic protocols. PMID:23639323

  15. Cooperative Role of Mineralocorticoid Receptor and Caveolin-1 in Regulating the Vascular Response to Low Nitric Oxide-High Angiotensin II-Induced Cardiovascular Injury.

    PubMed

    Pojoga, Luminita H; Yao, Tham M; Opsasnick, Lauren A; Siddiqui, Waleed T; Reslan, Ossama M; Adler, Gail K; Williams, Gordon H; Khalil, Raouf A

    2015-10-01

    Aldosterone interacts with mineralocorticoid receptor (MR) to stimulate sodium reabsorption in renal tubules and may also affect the vasculature. Caveolin-1 (cav-1), an anchoring protein in plasmalemmal caveolae, binds steroid receptors and also endothelial nitric oxide synthase, thus limiting its translocation and activation. To test for potential MR/cav-1 interaction in the vasculature, we investigated if MR blockade in cav-1-replete or -deficient states would alter vascular function in a mouse model of low nitric oxide (NO)-high angiotensin II (AngII)-induced cardiovascular injury. Wild-type (WT) and cav-1 knockout mice (cav-1(-/-)) consuming a high salt diet (4% NaCl) received Nω-nitro-l-arginine methyl ester (L-NAME) (0.1-0.2 mg/ml in drinking water at days 1-11) plus AngII (0.7-2.8 mg/kg per day via an osmotic minipump at days 8-11) ± MR antagonist eplerenone (EPL) 100 mg/kg per day in food. In both genotypes, blood pressure increased with L-NAME + AngII. EPL minimally changed blood pressure, although its dose was sufficient to block MR and reverse cardiac expression of the injury markers cluster of differentiation 68 and plasminogen activator inhibitor-1 in L-NAME+AngII treated mice. In aortic rings, phenylephrine and KCl contraction was enhanced with EPL in L-NAME+AngII treated WT mice, but not cav-1(-/-) mice. AngII-induced contraction was not different, and angiotensin type 1 receptor expression was reduced in L-NAME + AngII treated WT and cav-1(-/-) mice. In WT mice, acetylcholine-induced relaxation was enhanced with L-NAME + AngII treatment and reversed with EPL. Acetylcholine relaxation in cav-1(-/-) mice was greater than in WT mice, not modified by L-NAME + AngII or EPL, and blocked by ex vivo L-NAME, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or endothelium removal, suggesting the role of NO-cGMP. Cardiac endothelial NO synthase was increased in cav-1(-/-) versus WT mice, further increased with L-NAME + AngII, and not affected by EPL

  16. Cooperative Role of Mineralocorticoid Receptor and Caveolin-1 in Regulating the Vascular Response to Low Nitric Oxide–High Angiotensin II–Induced Cardiovascular Injury

    PubMed Central

    Pojoga, Luminita H.; Yao, Tham M.; Opsasnick, Lauren A.; Siddiqui, Waleed T.; Reslan, Ossama M.; Adler, Gail K.; Williams, Gordon H.

    2015-01-01

    Aldosterone interacts with mineralocorticoid receptor (MR) to stimulate sodium reabsorption in renal tubules and may also affect the vasculature. Caveolin-1 (cav-1), an anchoring protein in plasmalemmal caveolae, binds steroid receptors and also endothelial nitric oxide synthase, thus limiting its translocation and activation. To test for potential MR/cav-1 interaction in the vasculature, we investigated if MR blockade in cav-1–replete or –deficient states would alter vascular function in a mouse model of low nitric oxide (NO)–high angiotensin II (AngII)–induced cardiovascular injury. Wild-type (WT) and cav-1 knockout mice (cav-1−/−) consuming a high salt diet (4% NaCl) received Nω-nitro-l-arginine methyl ester (L-NAME) (0.1–0.2 mg/ml in drinking water at days 1–11) plus AngII (0.7–2.8 mg/kg per day via an osmotic minipump at days 8–11) ± MR antagonist eplerenone (EPL) 100 mg/kg per day in food. In both genotypes, blood pressure increased with L-NAME + AngII. EPL minimally changed blood pressure, although its dose was sufficient to block MR and reverse cardiac expression of the injury markers cluster of differentiation 68 and plasminogen activator inhibitor-1 in L-NAME+AngII treated mice. In aortic rings, phenylephrine and KCl contraction was enhanced with EPL in L-NAME+AngII treated WT mice, but not cav-1−/− mice. AngII-induced contraction was not different, and angiotensin type 1 receptor expression was reduced in L-NAME + AngII treated WT and cav-1−/− mice. In WT mice, acetylcholine-induced relaxation was enhanced with L-NAME + AngII treatment and reversed with EPL. Acetylcholine relaxation in cav-1−/− mice was greater than in WT mice, not modified by L-NAME + AngII or EPL, and blocked by ex vivo L-NAME, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or endothelium removal, suggesting the role of NO-cGMP. Cardiac endothelial NO synthase was increased in cav-1−/− versus WT mice, further increased with L-NAME + AngII, and

  17. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  18. Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML)

    PubMed Central

    Milutinovic, Snezana; Heynen-Genel, Susanne; Chao, Elizabeth; Dewing, Antimone; Solano, Ricardo; Milan, Loribelle; Barron, Nikki; He, Min; Diaz, Paul W.; Matsuzawa, Shu-ichi; Reed, John C.; Hassig, Christian A.

    2016-01-01

    Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli. PMID:27031987

  19. The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery.

    PubMed

    Lolkema, Martijn P; Mehra, Niven; Jorna, Anita S; van Beest, Moniek; Giles, Rachel H; Voest, Emile E

    2004-12-10

    The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.

  20. Regulation of cell polarity determinants by the Retinoblastoma tumor suppressor protein

    PubMed Central

    Payankaulam, Sandhya; Yeung, Kelvin; McNeill, Helen; Henry, R. William; Arnosti, David N.

    2016-01-01

    In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical–basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis. PMID:26971715

  1. Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells

    PubMed Central

    Selga, Elisabet; Morales, Cristina; Noé, Véronique; Peinado, Miguel A; Ciudad, Carlos J

    2008-01-01

    Background Methotrexate is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the dhfr gene or as a result of a transcriptome screening using microarrays. Methods Gene expression levels were compared between triplicate samples from either HT29 sensitive cells and resistant to 10-5 M MTX by hybridization to the GeneChip® HG U133 PLUS 2.0 from Affymetrix. After normalization, a list of 3-fold differentially expressed genes with a p-value < 0.05 including multiple testing correction (Benjamini and Hochberg false discovery rate) was generated. RT-Real-time PCR was used to validate the expression levels of selected genes and copy-number was determined by qPCR. Functional validations were performed either by siRNAs or by transfection of an expression plasmid. Results Genes adjacent to the dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCα genes in resistant cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward

  2. Is PML a Tumor Suppressor?

    PubMed Central

    Mazza, Massimiliano; Pelicci, Pier Giuseppe

    2013-01-01

    The role of the promyelocytic leukemia (PML) protein has been widely tested in many different contexts, as attested by the hundreds of papers present in the literature. In most of these studies, PML is regarded as a tumor suppressor, a notion on the whole accepted by the scientific community. In this review, we examine how the concept of tumor-suppressor gene has evolved until now and then systematically assess whether this assumption for PML is supported by unambiguous experimental evidence. PMID:23847764

  3. Epidermal growth factor promotes protein degradation of epithelial protein lost in neoplasm (EPLIN), a putative metastasis suppressor, during epithelial-mesenchymal transition.

    PubMed

    Zhang, Shumin; Wang, Xu; Iqbal, Shareen; Wang, Yanru; Osunkoya, Adeboye O; Chen, Zhengjia; Chen, Zhuo; Shin, Dong M; Yuan, Hongwei; Wang, Yongqiang A; Zhau, Haiyen E; Chung, Leland W K; Ritenour, Chad; Kucuk, Omer; Wu, Daqing

    2013-01-18

    Aberrant expression of EGF receptors has been associated with hormone-refractory and metastatic prostate cancer (PCa). However, the molecular mechanism for EGF signaling in promoting PCa metastasis remains elusive. Using experimental models of PCa metastasis, we demonstrated that EGF could induce robust epithelial-mesenchymal transition (EMT) and increase invasiveness. Interestingly, EGF was found to be capable of promoting protein turnover of epithelial protein lost in neoplasm (EPLIN), a putative suppressor of EMT and tumor metastasis. Mechanistic study revealed that EGF could activate the phosphorylation, ubiquitination, and degradation of EPLIN through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signaling cascade. Pharmacological inhibition of the ERK1/2 pathway effectively antagonized EGF-induced EPLIN degradation. Two serine residues, i.e. serine 362 and serine 604, were identified as putative ERK1/2 phosphorylation sites in human EPLIN, whose point mutation rendered resistance to EGF-induced protein turnover. This study elucidated a novel molecular mechanism for EGF regulation of EMT and invasiveness in PCa cells, indicating that blockade of EGF signaling could be beneficial in preventing and retarding PCa metastasis at early stages.

  4. Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus-induced gene silencing.

    PubMed

    Qiu, Wenping; Scholthof, Karen-Beth G

    2004-03-01

    The capsid protein (CP) of satellite panicum mosaic virus (SPMV) has been implicated as a pathogenicity factor, inducing severe chlorosis on millet plants co-infected with SPMV and its helper virus, Panicum mosaic virus (PMV). In this study, we tested the effects of SPMV CP on Nicotiana benthamiana, a plant that does not support PMV+SPMV infections. SPMV CP expressed from a Potato virus X (PVX) gene vector elicited necrotic lesions on N. benthamiana. Pathogenicity factors often have the additional feature of acting as suppressors of gene silencing; therefore, several assays were developed to test if SPMV CP could act in such a capacity. The results showed that SPMV CP failed to act as a suppressor of posttranscriptional gene silencing when such tests were performed with transgenic N. benthamiana plants silenced for green fluorescent protein (GFP) expression by agroinfiltration or plant virus vectors. However SPMV CP expressed from the PVX gene vector did interfere with suppressor activity associated with PVX p25. This included a rebounded level of GFP silencing along the vascular tissues, including the veins on upper noninoculated leaves. Therefore, the roles of the SPMV CP now include encapsidation of the SPMV RNA, activity as a pathogenicity factor in both host and nonhost plants, and the enigmatic feature of interfering with suppression of gene silencing.

  5. Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway.

    PubMed

    Naim, Fatima; Shrestha, Pushkar; Singh, Surinder P; Waterhouse, Peter M; Wood, Craig C

    2016-06-01

    Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

  6. Targeting myeloid-derived suppressor cells using a novel adenosine monophosphate-activated protein kinase (AMPK) activator.

    PubMed

    Trikha, Prashant; Plews, Robert L; Stiff, Andrew; Gautam, Shalini; Hsu, Vincent; Abood, David; Wesolowski, Robert; Landi, Ian; Mo, Xiaokui; Phay, John; Chen, Ching-Shih; Byrd, John; Caligiuri, Michael; Tridandapani, Susheela; Carson, William

    2016-01-01

    Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that accumulate in the blood and tumors of patients with cancer. MDSC play a critical role during tumor evasion and promote immune suppression through variety of mechanisms, such as the generation of reactive oxygen and nitrogen species (ROS and RNS) and cytokines. AMPactivated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress. However, the role of AMPK in the regulation of MDSC function remains largely unexplored. This study was designed to investigate whether treatment of MDSC with OSU-53, a PPAR-inactive derivative that stimulates AMPK kinase, can modulate MDSC function. Our results demonstrate that OSU-53 treatment increases the phosphorylation of AMPK, significantly reduces nitric oxide production, inhibits MDSC migration, and reduces the levels of IL-6 in murine MDSC cell line (MSC2 cells). OSU53 treatment mitigated the immune suppressive functions of murine MDSC, promoting T-cell proliferation. Although OSU-53 had a modest effect on tumor growth in mice inoculated with EMT-6 cells, importantly, administration of OSU53 significantly (p < 0.05) reduced the levels of MDSC in the spleens and tumors. Furthermore, mouse MDSC from EMT-6 tumor-bearing mice and human MDSC isolated from melanoma patients treated with OSU-53 showed a significant reduction in the expression of immune suppressive genes iNOS and arginase. In summary, these results demonstrate a novel role of AMPK in the regulation of MDSC functions and provide a rationale of combining OSU-53 with immune checkpoint inhibitors to augment their response in cancer patients.

  7. Virus-Induced Necrosis Is a Consequence of Direct Protein-Protein Interaction between a Viral RNA-Silencing Suppressor and a Host Catalase[C][W

    PubMed Central

    Inaba, Jun-ichi; Kim, Bo Min; Shimura, Hanako; Masuta, Chikara

    2011-01-01

    Many plant host factors are known to interact with viral proteins during pathogenesis, but how a plant virus induces a specific disease symptom still needs further research. A lily strain of Cucumber mosaic virus (CMV-HL) can induce discrete necrotic spots on infected Arabidopsis (Arabidopsis thaliana) plants; other CMV strains can induce similar spots, but they are not as distinct as those induced by CMV-HL. The CMV 2b protein (2b), a known RNA-silencing suppressor, is involved in viral movement and symptom induction. Using in situ proximity ligation assay immunostaining and the protoplast assays, we report here that CMV 2b interacts directly with Catalase3 (CAT3) in infected tissues, a key enzyme in the breakdown of toxic hydrogen peroxide. Interestingly, CAT3, normally localized in the cytoplasm (glyoxysome), was recruited to the nucleus by an interaction between 2b and CAT3. Although overexpression of CAT3 in transgenic plants decreased the accumulation of CMV and delayed viral symptom development to some extent, 2b seems to neutralize the cellular catalase contributing to the host defense response, thus favoring viral infection. Our results thus provide evidence that, in addition to altering the type of symptom by disturbing microRNA pathways, 2b can directly bind to a host factor that is important in scavenging cellular hydrogen peroxide and thus interfere specifically with that host factor, leading to the induction of a specific necrosis. PMID:21622812

  8. An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4.

    PubMed

    Fehler, Olesja; Singh, Priyanka; Haas, Astrid; Ulrich, Diana; Müller, Jan P; Ohnheiser, Johanna; Klempnauer, Karl-Heinz

    2014-01-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4.

  9. Rice grassy stunt virus nonstructural protein p5 serves as a viral suppressor of RNA silencing and interacts with nonstructural protein p3.

    PubMed

    Zhang, Chao; Liu, Xiao-juan; Wu, Kang-cheng; Zheng, Lu-Ping; Ding, Zuo-mei; Li, Fei; Zou, Peng; Yang, Liang; Wu, Jian-guo; Wu, Zu-jian

    2015-11-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes serious rice disease in Southeast Asian countries. In this study, a green fluorescent protein (GFP)-based transient expression assay was conducted to show that p5, encoded on RNA5 in the viral sense, is a viral suppressor of RNA silencing (VSR). Protein-protein interactions (PPIs) between p5 and all RGSV proteins except pC1 and pC2 were investigated using Gal4-based yeast two-hybrid (Y2H) experiments. The results demonstrated that p5 interacts with itself and with p3 encoded on RNA3 in the viral sense. p5-p5 and p5-p3 interactions were detected by bimolecular fluorescence complementation (BiFC) assay, and the p5-p3 interaction was confirmed by subcellular co-localization and co-immunoprecipitation (Co-IP) assays. Using the Y2H system, we demonstrated that the p5-p3 interaction requires both the N-terminal (amino acid residues 1 to 99) and C-terminal (amino acid residues 94 to 191) domains of p5. In addition, either p5 or p3 could enhance the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana plants. A much more significant enhancement of PVX pathogenicity and accumulation was observed when p5 and p3 were expressed together. Our data also showed that RGSV p3 does not function as a VSR, and it had no effect on the VSR activity of p5 or the subcellular localization pattern of p5 in plant cells from Nicotiana benthamiana.

  10. FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53

    PubMed Central

    Dixit, Updesh; Pandey, Ashutosh K.; Liu, Zhihe; Kumar, Sushil; Neiditch, Matthew B.; Klein, Kenneth M.

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with the CHC background. Oncomine data analysis of normal versus HCC tumors with the CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53 and regulating BCCIP and TCTP, which are positive and negative regulators of p53, respectively. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that the activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1 but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1 knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), we found no significant difference in in vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, the DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53 and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with an HCV background, it may have a role in promoting persistent virus infection and tumorigenesis. IMPORTANCE It is our novel finding that FUSE binding protein 1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host cell factor required for HCV replication. Oncomine data analysis of a large number of samples has revealed that overexpression of

  11. P53 tumor suppressor gene and protein expression is altered in cell lines derived from spontaneous and alpha-radiation-induced canine lung tumors

    SciTech Connect

    Tierney, L.A.; Johnson, N.F.; Lechner, J.F.

    1994-11-01

    Mutations in the p53 tumor suppressor gene are the most frequently occurring gene alterations in malignant human cancers, including lung cancer. In lung cancer, common point mutations within conserved exons of the p53 gene result in a stabilized form of mutant protein which is detectable in most cases by immunohistochemistry. In addition to point mutations, allelic loss, rearrangements, and deletions of the p53 gene have also been detected in both human and rodent tumors. It has been suggested that for at least some epithelial neoplasms, the loss of expression of wild-type p53 protein may be more important for malignant transformation than the acquisition of activating mutations. Mechanisms responsible for the loss of expression of wild-type protein include gene deletion or rearrangement, nonsense or stop mutations, mutations within introns or upstream regulatory regions of the gene, and accelerated rates of degradation of the protein by DNA viral oncoproteins.

  12. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

    PubMed Central

    2009-01-01

    Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant

  13. An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification.

    PubMed

    Dittrich, Anna C Nelson; Devarenne, Timothy P

    2012-02-01

    Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the "gatekeeper" residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3(M385G) can also specifically utilize N(6)-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.

  14. Myrtenal ameliorates diethylnitrosamine-induced hepatocarcinogenesis through the activation of tumor suppressor protein p53 and regulation of lysosomal and mitochondrial enzymes.

    PubMed

    Lingaiah, Hari Babu; Natarajan, Nandakumar; Thamaraiselvan, Rengarajan; Srinivasan, Perumal; Periyasamy, Balasubramanian Maruthaiveeran

    2013-08-01

    Myrtenal is a novel class of compound belongs to monoterpenes found predominantly in mint, pepper, etc., and it was shown to have excellent pharmacological activities against many diseases among which cancer is imperative. Hepatocellular carcinoma is a primary malignancy of the hepatocytes, which rapidly leads to death in short periods. The aim of this study was to investigate the possible therapeutic efficiency of myrtenal against diethylnitrosamine-induced experimental hepatocarcinogenesis by analyzing the key enzymes of carbohydrate metabolism, lysosomal and mitochondrial TCA cycle enzymes, and also the possible role of tumor suppressor protein p53, and scanning electron microscopic studies. The results revealed that myrtenal significantly ameliorated the altered enzymes of carbohydrate metabolism, lysosomal and mitochondrial enzymes, and interestingly the tumor suppressor protein p53 was found to be significantly accumulated in myrtenal-treated animals, which inevitably confirms that myrtenal has a prominent role in preventing the liver cancer during treatment. Furthermore, the antineoplastic property was well evidenced by the mRNA expression of p53 protein by the reverse-transcriptase polymerase chain reaction and immunoblot analysis. The observed anticancer property of myrtenal may be due to the involvement and expression of p53 and influence in the mitochondrial and lysosomal membrane integrity and also interference in the gluconeogenesis process of cancer cells. Our results suggest that myrtenal is very efficient and useful compound in the treatment of liver cancer in future.

  15. The 1.35 A resolution structure of the phosphatase domain of the suppressor of T cell receptor signaling protein in complex with sulfate

    SciTech Connect

    Jakoncic, J.; Sondgeroth, B.; Carpino, N.; Nassar, N.

    2010-04-19

    The suppressor of T-cell signaling (Sts) proteins are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including the T-cell receptor (TCR) and the epidermal growth-factor receptor (EGFR). They contain at their C-terminus a 2H-phosphatase homology (PGM) domain that is responsible for their protein tyrosine phosphatase activity. Here, the crystal structure of the phosphatase domain of Sts-1, Sts-1PGM, was determined at pH 4.6. The asymmetric unit contains two independent molecules and each active site is occupied by a sulfate ion. Each sulfate is located at the phosphate-binding site and makes similar interactions with the catalytic residues. The structure suggests an explanation for the lower Michaelis-Menten constants at acidic pH.

  16. The 2b protein of Asparagus virus 2 functions as an RNA silencing suppressor against systemic silencing to prove functional synteny with related cucumoviruses.

    PubMed

    Shimura, Hanako; Masuta, Chikara; Yoshida, Naoto; Sueda, Kae; Suzuki, Masahiko

    2013-08-01

    Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1-3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing.

  17. Secreted frizzled-related protein-5 (sFRP-5) is epigenetically downregulated and functions as a tumor suppressor in kidney cancer

    PubMed Central

    Kawakami, Kazumori; Yamamura, Soichiro; Hirata, Hiroshi; Ueno, Koji; Saini, Sharanjot; Majid, Shahana; Tanaka, Yuichiro; Kawamoto, Ken; Enokida, Hideki; Nakagawa, Masayuki; Dahiya, Rajvir

    2014-01-01

    Secreted frizzled-related protein-5 (sFRP-5) has been identified as one of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells. PMID:20340127

  18. Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma.

    PubMed

    Jarmalavicius, Saulius; Trefzer, Uwe; Walden, Peter

    2010-03-01

    The aim of the study was to identify as potential therapeutic targets specific molecular alterations in tumor cells recognized by the immune system. To identify such targets, we analyzed the human leukocyte antigen (HLA) peptidomes of human melanoma cells by 2-dimensional nano-HPLC/mass spectrometry and tested the immunological significance of the peptides by ex vivo ELISpot assays with lymphocytes from melanoma patients. The peptide SQNPRFYHK was identified as derived from the regulator of the nuclear corepressor complex (NCoR) G-protein pathway suppressor 2 (GPS-2) and to be differentially unmethylated, monomethylated or asymmetrically dimethylated at the arginine. The methylation state was specifically recognized by the immune system in that only the monomethylated variant induced T-cell responses and significantly stronger responses in patients than in healthy controls. The methylations were confirmed with synthetic analogues and in vitro radiolabeling assays with recombinant GPS-2 and synthetic peptides. The immunity of the 3 variants of GPS-2 was tested in T-cell assays with T lymphocytes of melanoma patients compared with healthy donors. The results show for the first time that GPS-2 is differentially methylated at a site that lacks known methylation motifs and that the methylation state is detected by the immune system.-Jarmalavicius, S., Trefzer, U., Walden, P. Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma.

  19. WWOX: A fragile tumor suppressor

    PubMed Central

    Schrock, Morgan S

    2015-01-01

    WWOX, the WW domain-containing oxidoreductase gene at chromosome region 16q23.3–q24.1, spanning chromosomal fragile site FRA16D, encodes the 46 kDa Wwox protein, a tumor suppressor that is lost or reduced in expression in a wide variety of cancers, including breast, prostate, ovarian, and lung. The function of Wwox as a tumor suppressor implies that it serves a function in the prevention of carcinogenesis. Indeed, in vitro studies show that Wwox protein interacts with many binding partners to regulate cellular apoptosis, proliferation, and/or maturation. It has been reported that newborn Wwox knockout mice exhibit nascent osteosarcomas while Wwox+/− mice exhibit increased incidence of spontaneous and induced tumors. Furthermore, absence or reduction of Wwox expression in mouse xenograft models results in increased tumorigenesis, which can be rescued by Wwox re-expression, though there is not universal agreement among investigators regarding the role of Wwox loss in these experimental models. Despite this proposed tumor suppressor function, the overlap of the human WWOX locus with FRA16D sensitizes the gene to protein-inactivating deletions caused by replication stress. The high frequency of deletions within the WWOX locus in cancers of various types, without the hallmark protein inactivation-associated mutations of “classical” tumor suppressors, has led to the proposal that WWOX deletions in cancers are passenger events that occur in early cancer progenitor cells due to fragility of the genetic locus, rather than driver events which provide the cancer cell a selective advantage. Recently, a proposed epigenetic cause of chromosomal fragility has suggested a novel mechanism for early fragile site instability and has implications regarding the involvement of tumor suppressor genes at chromosomal fragile sites in cancer. In this review, we provide an overview of the evidence for WWOX as a tumor suppressor gene and put this into the context of fragility

  20. Biochemical Characterization of Arabidopsis Complexes Containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA Proteins in Light Control of Plant Development[W

    PubMed Central

    Zhu, Danmeng; Maier, Alexander; Lee, Jae-Hoon; Laubinger, Sascha; Saijo, Yusuke; Wang, Haiyang; Qu, Li-Jia; Hoecker, Ute; Deng, Xing Wang

    2008-01-01

    COP1 (for CONSTITUTIVELY PHOTOMORPHOGENIC1) and the four partially redundant SPA (for SUPPRESSOR OF PHYA) proteins work in concert to repress photomorphogenesis in Arabidopsis thaliana by targeting key transcription factors and phytochrome A for degradation via the 26S proteasome. Here, we report a detailed biochemical characterization of the SPA-COP1 complexes. The four endogenous SPA proteins can form stable complexes with COP1 in vivo regardless of light conditions but exhibit distinct expression profiles in different tissues and light conditions. The SPA proteins can self-associate or interact with each other, forming a heterogeneous group of SPA-COP1 complexes in which the exact SPA protein compositions vary depending on the abundance of individual SPA proteins. The four SPA proteins could be divided into two functional groups depending on their interaction affinities, their regulation of ELONGATED HYPOCOTYL5 degradation, and their opposite effects on COP1 protein accumulation. Loss-of-function mutations in a predominant SPA protein may cause a significant reduction in the overall SPA-COP1 E3 ligase activity, resulting in a partial constitutive photomorphogenic phenotype. This study thus provides an in-depth biochemical view of the SPA-COP1 E3 ligase complexes and offers new insights into the molecular basis for their distinct roles in the light control of plant development. PMID:18812498

  1. Fast-suppressor screening for new components in protein trafficking, organelle biogenesis and silencing pathway in Arabidopsis thaliana using DEX-inducible FREE1-RNAi plants.

    PubMed

    Zhao, Qiong; Gao, Caiji; Lee, PoShing; Liu, Lin; Li, Shaofang; Hu, Tangjin; Shen, Jinbo; Pan, Shuying; Ye, Hao; Chen, Yunru; Cao, Wenhan; Cui, Yong; Zeng, Peng; Yu, Sheng; Gao, Yangbin; Chen, Liang; Mo, Beixin; Liu, Xin; Xiao, Shi; Zhao, Yunde; Zhong, Silin; Chen, Xuemei; Jiang, Liwen

    2015-06-20

    Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F2 mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

  2. Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53

    PubMed Central

    Yue, Hong; Wang, Liming; Jin, Jessica; Ghosh, Santosh K.; Kawsar, Hameem I.; Zender, Chad; Androphy, Elliot J.; Weinberg, Aaron; McCormick, Thomas S.; Jin, Ge

    2016-01-01

    Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer. PMID:27034006

  3. The Parkinson disease-related protein DJ-1 counteracts mitochondrial impairment induced by the tumour suppressor protein p53 by enhancing endoplasmic reticulum-mitochondria tethering.

    PubMed

    Ottolini, Denis; Calì, Tito; Negro, Alessandro; Brini, Marisa

    2013-06-01

    DJ-1 was first identified as an oncogene. More recently, mutations in its gene have been found causative for autosomal recessive familial Parkinson disease. Numerous studies support the DJ-1 role in the protection against oxidative stress and maintenance of mitochondria structure; however, the mechanism of its protective function remains largely unknown. We investigated whether mitochondrial Ca(2+) homeostasis, a key parameter in cell physiology, could be a target for DJ-1 action. Here, we show that DJ-1 modulates mitochondrial Ca(2+) transients induced upon cell stimulation with an 1,4,5-inositol-tris-phosphate agonist by favouring the endoplasmic reticulum (ER)-mitochondria tethering. A reduction of DJ-1 levels results in mitochondria fragmentation and decreased mitochondrial Ca(2+) uptake in stimulated cells. To functionally couple these effects with the well-recognized cytoprotective role of DJ-1, we investigated its action in respect to the tumour suppressor p53. p53 overexpression in HeLa cells impairs their ability to accumulate Ca(2+) in the mitochondrial matrix, causes alteration of the mitochondrial morphology and reduces ER-mitochondria contact sites. Mitochondrial impairments are independent from Drp1 activation, since the co-expression of the dominant negative mutant of Drp1 failed to abolish them. DJ-1 overexpression prevents these alterations by re-establishing the ER-mitochondria tethering. Similarly, the co-expression of the pro-fusion protein Mitofusin 2 blocks the effects induced by p53 on mitochondria, confirming that the modulation of the ER-mitochondria contact sites is critical to mitochondria integrity. Thus, the impairment of ER-mitochondria communication, as a consequence of DJ-1 loss-of-function, may be detrimental for mitochondria-related processes and be at the basis of mitochondrial dysfunction observed in Parkinson disease.

  4. Sirtuin1 protects endothelial Caveolin-1 expression and preserves endothelial function via suppressing miR-204 and endoplasmic reticulum stress

    PubMed Central

    Kassan, M.; Vikram, A.; Kim, Y. R.; Li, Q.; Kassan, A.; Patel, H. H.; Kumar, S.; Gabani, M.; Liu, J.; Jacobs, J. S.; Irani, K.

    2017-01-01

    Sirtuin1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including endothelial function. Caveolin1 (Cav1) is also an important determinant of endothelial function. We asked if Sirt1 governs endothelial Cav1 and endothelial function by regulating miR-204 expression and endoplasmic reticulum (ER) stress. Knockdown of Sirt1 in endothelial cells, and in vivo deletion of endothelial Sirt1, induced endothelial ER stress and miR-204 expression, reduced Cav1, and impaired endothelium-dependent vasorelaxation. All of these effects were reversed by a miR-204 inhibitor (miR-204 I) or with overexpression of Cav1. A miR-204 mimic (miR-204 M) decreased Cav1 in endothelial cells. In addition, high-fat diet (HFD) feeding induced vascular miR-204 and reduced endothelial Cav1. MiR-204-I protected against HFD-induced downregulation of endothelial Cav1. Moreover, pharmacologic induction of ER stress with tunicamycin downregulated endothelial Cav1 and impaired endothelium-dependent vasorelaxation that was rescued by overexpressing Cav1. In conclusion, Sirt1 preserves Cav1-dependent endothelial function by mitigating miR-204-mediated vascular ER stress. PMID:28181559

  5. Caveolin-1 Mediates Low-Intensity Ultrasound-Induced Apoptosis via Downregulation of Signal Transducer and Activator of Transcription 3 Phosphorylation in Laryngeal Carcinoma Cells.

    PubMed

    Ye, Qingsheng; Meng, Cuida; Shen, Yannan; Ji, Jianjun; Wang, Xiaochun; Zhou, Sheng; Jia, Lili; Wang, Yanqun

    2016-09-01

    Low-intensity ultrasound therapy has been found to be a potential tool in the management of malignant tumors in recent years. However, the molecular mechanism underlying low-intensity ultrasound-induced apoptosis is still not clear. In this study, we investigated the effects of low-intensity ultrasound-induced apoptosis in HEp-2 cells. We found that low-intensity ultrasound significantly induced apoptosis, and the expression level of caveolin-1 (Cav-1) was dramatically increased after ultrasound treatment of HEp-2 cells. After inhibiting the expression level of Cav-1 using siRNA transfection, we found that the cellular apoptosis induced by low-intensity ultrasound was significantly suppressed. In addition, inhibition of Cav-1 expression promoted phosphorylation of signal transducer and activator of transcription 3 (STAT3), suggesting that the STAT3 signaling pathway was involved in low-intensity ultrasound-induced apoptosis via Cav-1 regulation. Our results indicate that Cav-1/STAT3 signaling pathway may mediate low-intensity ultrasound-induced apoptosis, and this technology could potentially be used clinically for the treatment of cancers.

  6. spr-2, a suppressor of the egg-laying defect caused by loss of sel-12 presenilin in Caenorhabditis elegans, is a member of the SET protein subfamily

    PubMed Central

    Wen, Chenhui; Levitan, Diane; Li, Xiajun; Greenwald, Iva

    2000-01-01

    Presenilin plays critical roles in the genesis of Alzheimer's disease and in LIN-12/Notch signaling during development. Here, we describe a screen for genes that influence presenilin level or activity in Caenorhabditis elegans. We identified four spr (suppressor of presenilin) genes by reverting the egg-laying defective phenotype caused by a null allele of the sel-12 presenilin gene. We analyzed the spr-2 gene in some detail. We show that loss of spr-2 activity suppresses the egg-laying defective phenotype of different sel-12 alleles and requires activity of the hop-1 presenilin gene, suggesting that suppression is accomplished by elevating presenilin activity rather than by bypassing the need for presenilin activity. We also show that SPR-2 is a nuclear protein and is a member of a protein subfamily that includes human SET, which has been identified in numerous different biochemical assays and at translocation breakpoints associated with a subtype of acute myeloid leukemia. PMID:11114162

  7. The p53 Tumor Suppressor-Like Protein nvp63 Mediates Selective Germ Cell Death in the Sea Anemone Nematostella vectensis

    PubMed Central

    Pankow, Sandra; Bamberger, Casimir

    2007-01-01

    Here we report the identification and molecular function of the p53 tumor suppressor-like protein nvp63 in a non-bilaterian animal, the starlet sea anemone Nematostella vectensis. So far, p53-like proteins had been found in bilaterians only. The evolutionary origin of p53-like proteins is highly disputed and primordial p53-like proteins are variably thought to protect somatic cells from genotoxic stress. Here we show that ultraviolet (UV) irradiation at low levels selectively induces programmed cell death in early gametes but not somatic cells of adult N. vectensis polyps. We demonstrate with RNA interference that nvp63 mediates this cell death in vivo. Nvp63 is the most archaic member of three p53-like proteins found in N. vectensis and in congruence with all known p53-like proteins, nvp63 binds to the vertebrate p53 DNA recognition sequence and activates target gene transcription in vitro. A transactivation inhibitory domain at its C-terminus with high homology to the vertebrate p63 may regulate nvp63 on a molecular level. The genotoxic stress induced and nvp63 mediated apoptosis in N. vectensis gametes reveals an evolutionary ancient germ cell protective pathway which relies on p63-like proteins and is conserved from cnidarians to vertebrates. PMID:17848985

  8. Evidence that noncoding RNA dutA is a multicopy suppressor of Dictyostelium discoideum STAT protein Dd-STATa.

    PubMed

    Shimada, Nao; Kawata, Takefumi

    2007-06-01

    Dd-STATa, a Dictyostelium discoideum homologue of metazoan STAT transcription factors, is necessary for culmination. We created a mutant strain with partial Dd-STATa activity and used it to screen for unlinked suppressor genes. We screened approximately 450,000 clones from a slug-stage cDNA library for their ability to rescue the culmination defect when overexpressed. There were 12 multicopy suppressors of Dd-STATa, of which 4 encoded segments of a known noncoding RNA, dutA. Expression of dutA is specific to the pstA zone, the region where Dd-STATa is activated. In suppressed strains the expression patterns of several putative Dd-STATa target genes become similar to the wild-type strain. In addition, the amount of the tyrosine-phosphorylated form of Dd-STATa is significantly increased in the suppressed strain. These results indicate that partial copies of dutA may act upstream of Dd-STATa to regulate tyrosine phosphorylation by an unknown mechanism.

  9. In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor[C][W][OPEN

    PubMed Central

    Okano, Yukari; Senshu, Hiroko; Hashimoto, Masayoshi; Neriya, Yutaro; Netsu, Osamu; Minato, Nami; Yoshida, Tetsuya; Maejima, Kensaku; Oshima, Kenro; Komatsu, Ken; Yamaji, Yasuyuki; Namba, Shigetou

    2014-01-01

    RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs. PMID:24879427

  10. Yeast frameshift suppressor mutations in the genes coding for transcription factor Mbf1p and ribosomal protein S3: evidence for autoregulation of S3 synthesis.

    PubMed Central

    Hendrick, J L; Wilson, P G; Edelman, I I; Sandbaken, M G; Ursic, D; Culbertson, M R

    2001-01-01

    The SUF13 and SUF14 genes were identified among extragenic suppressors of +1 frameshift mutations. SUF13 is synonymous with MBF1, a single-copy nonessential gene coding for a POLII transcription factor. The suf13-1 mutation is a two-nucleotide deletion in the SUF13/MBF1 coding region. A suf13::TRP1 null mutant suppresses +1 frameshift mutations, indicating that suppression is caused by loss of SUF13 function. The suf13-1 suppressor alters sensitivity to aminoglycoside antibiotics and reduces the accumulation of his4-713 mRNA, suggesting that suppression is mediated at the translational level. The SUF14 gene is synonymous with RPS3, a single-copy essential gene that codes for the ribosomal protein S3. The suf14-1 mutation is a missense substitution in the coding region. Increased expression of S3 limits the accumulation of SUF14 mRNA, suggesting that expression is autoregulated. A frameshift mutation in SUF14 that prevents full-length translation eliminated regulation, indicating that S3 is required for regulation. Using CUP1-SUF14 and SUF14-lacZ fusions, run-on transcription assays, and estimates of mRNA half-life, our results show that transcription plays a minor role if any in regulation and that the 5'-UTR is necessary but not sufficient for regulation. A change in mRNA decay rate may be the primary mechanism for regulation. PMID:11238400

  11. Caveolin-1 in Lipid Rafts Interacts with Dengue Virus NS3 during Polyprotein Processing and Replication in HMEC-1 Cells

    PubMed Central

    García Cordero, Julio; León Juárez, Moisés; González-Y-Merchand, Jorge A.; Cedillo Barrón, Leticia; Gutiérrez Castañeda, Benito

    2014-01-01

    Lipid rafts are ordered microdomains within cellular membranes that are rich in cholesterol and sphingolipids. Caveolin (Cav-1) and flotillin (Flt-1) are markers of lipid rafts, which serve as an organizing center for biological phenomena and cellular signaling. Lipid rafts involvement in dengue virus (DENV) processing, replication, and assembly remains poorly characterized. Here, we investigated the role of lipid rafts after DENV endocytosis in human microvascular endothelial cells (HMEC-1). The non-structural viral proteins NS3 and NS2B, but not NS5, were associated with detergent-resistant membranes. In sucrose gradients, both NS3 and NS2B proteins appeared in Cav-1 and Flt-1 rich fractions. Additionally, double immunofluorescence staining of DENV-infected HMEC-1 cells showed that NS3 and NS2B, but not NS5, colocalized with Cav-1 and Flt-1. Furthermore, in HMEC-1cells transfected with NS3 protease, shown a strong overlap between NS3 and Cav-1, similar to that in DENV-infected cells. In contrast, double-stranded viral RNA (dsRNA) overlapped weakly with Cav-1 and Flt-1. Given these results, we investigated whether Cav-1 directly interacted with NS3. Cav-1 and NS3 co-immunoprecipitated, indicating that they resided within the same complex. Furthermore, when cellular cholesterol was depleted by methyl-beta cyclodextrin treatment after DENV entrance, lipid rafts were disrupted, NS3 protein level was reduced, besides Cav-1 and NS3 were displaced to fractions 9 and 10 in sucrose gradient analysis, and we observed a dramatically reduction of DENV particles release. These data demonstrate the essential role of caveolar cholesterol-rich lipid raft microdomains in DENV polyprotein processing and replication during the late stages of the DENV life cycle. PMID:24643062

  12. Suppressor mutations in the Glutamine Dumper1 protein dissociate disturbance in amino acid transport from other characteristics of the Gdu1D phenotype.

    PubMed

    Yu, Shi; Pratelli, Réjane; Denbow, Cynthia; Pilot, Guillaume

    2015-01-01

    Intracellular amino acid transport across plant membranes is critical for metabolic pathways which are often split between different organelles. In addition, transport of amino acids across the plasma membrane enables the distribution of organic nitrogen through the saps between leaves and developing organs. Amino acid importers have been studied for more than two decades, and their role in this process is well-documented. While equally important, amino acid exporters are not well-characterized. The over-expression of GDU1, encoding a small membrane protein with one transmembrane domain, leads to enhancement of amino acid export by Arabidopsis cells, glutamine secretion at the leaf margin, early senescence and size reduction of the plant, possibly caused by the stimulation of amino acid exporter(s). Previous work reported the identification of suppressor mutations of the GDU1 over-expression phenotype, which affected the GDU1 and LOG2 genes, the latter encoding a membrane-bound ubiquitin ligase interacting with GDU1. The present study focuses on the characterization of three additional suppressor mutations affecting GDU1. Size, phenotype, glutamine transport and amino acid tolerance were recorded for recapitulation plants and over-expressors of mutagenized GDU1 proteins. Unexpectedly, the over-expression of most mutated GDU1 led to plants with enhanced amino acid export, but failing to display secretion of glutamine and size reduction. The results show that the various effects triggered by GDU1 over-expression can be dissociated from one another by mutagenizing specific residues. The fact that these residues are not necessarily conserved suggests that the diverse biochemical properties of the GDU1 protein are not only born by the characterized transmembrane and VIMAG domains. These data provide a better understanding of the structure/function relationships of GDU1 and may enable modifying amino acid export in plants without detrimental effects on plant fitness.

  13. The overgrown hematopoietic organs-31 tumor suppressor gene of Drosophila encodes an Importin-like protein accumulating in the nucleus at the onset of mitosis

    PubMed Central

    1995-01-01

    The tumor suppressor gene overgrown hematopoietic organs-31 (oho31) of Drosophila encodes a protein with extensive homology to the Importin protein of Xenopus (50% identity), the related yeast SRP1 protein, and the mammalian hSRP1 and RCH1 proteins. A strong reduction in the expression of oho31 by a P element inserted in the 5' untranslated region of the oho31 transcript or a complete inactivation of oho31 by imprecise P element excision leads to malignant development of the hematopoietic organs and the genital disc, as shown by their growth autonomy in transplantation assays. We have cloned the oho31 gene of Drosophila melanogaster and determined its nucleotide sequence. The gene encodes a phosphoprotein of 522 amino acids made of three domains: a central hydrophobic domain of eight repeats of 42-44 amino acids each, displaying similarity to the arm motif found in junctional and nucleopore complex proteins, and flanked by two hydrophilic NH2- and COOH-terminal domains. Immunostaining revealed that the OHO31 protein is supplied maternally and rapidly degraded during the first 13 nuclear divisions. Thereafter, the OHO31 protein is predominantly expressed, albeit at reduced levels, in proliferating tissues. During the interphase of early embryonic cell cycles, the OHO31 protein is present in the cytoplasm and massively accumulates in the nucleus at the onset of mitosis in late interphase and prophase. The nuclear import of OHO31 is, however, less pronounced during later developmental stages. These results suggest that, similar to Importin, OHO31 may act as a cytosolic factor in nuclear transport. Moreover, the cell cycle-dependent accumulation of OHO31 in the nucleus indicates that this protein may be required for critical nuclear reactions occurring at the onset of mitosis. PMID:7790349

  14. The milk protein α-casein functions as a tumor suppressor via activation of STAT1 signaling, effectively preventing breast cancer tumor growth and metastasis.

    PubMed

    Bonuccelli, Gloria; Castello-Cros, Remedios; Capozza, Franco; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Tsirigos, Aristotelis; Xuanmao, Jiao; Whitaker-Menezes, Diana; Howell, Anthony; Lisanti, Michael P; Sotgia, Federica

    2012-11-01

    Here, we identified the milk protein α-casein as a novel suppressor of tumor growth and metastasis. Briefly, Met-1 mammary tumor cells expressing α-casein showed a ~5-fold reduction in tumor growth and a near 10-fold decrease in experimental metastasis. To identify the molecular mechanism(s), we performed genome-wide transcriptional profiling. Interestingly, our results show that α-casein upregulates gene transcripts associated with interferon/STAT1 signaling and downregulates genes associated with "stemness." These findings were validated by immunoblot and FACS analysis, which showed the upregulation and hyperactivation of STAT1 and a decrease in the number of CD44(+) "cancer stem cells." These gene signatures were also able to predict clinical outcome in human breast cancer patients. Thus, we conclude that a lactation-based therapeutic strategy using recombinant α-casein would provide a more natural and non-toxic approach to the development of novel anticancer therapies.

  15. Caveolin-1-dependent activation of the metalloprotease TACE/ADAM17 by TGF-β in hepatocytes requires activation of Src and the NADPH oxidase NOX1.

    PubMed

    Moreno-Càceres, Joaquim; Mainez, Jèssica; Mayoral, Rafael; Martín-Sanz, Paloma; Egea, Gustavo; Fabregat, Isabel

    2016-04-01

    Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, the balance between which decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β. We have previously shown that caveolin-1 (CAV1) is required for activation of the metalloprotease tumour necrosis factor (TNF)-α-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17), and hence transactivation of the EGFR pathway. The specific mechanism by which TACE/ADAM17 is activated has not yet been determined. Here we show that TGF-β induces phosphorylation of sarcoma kinase (Src) in hepatocytes, a process that is impaired in Cav1(-/-) hepatocytes, coincident with a decrease in phosphorylated Src in detergent-resistant membrane fractions. TGF-β-induced activation of TACE/ADAM17 and EGFR phosphorylation were blocked using the Src inhibitor PP2. Cav1(+/+) hepatocytes showed early production of reactive oxygen species (ROS) induced by TGF-β, which was not seen in Cav1(-/-) cells. Production of ROS was inhibited by both the NADPH oxidase 1 (NOX1) inhibitor STK301831 and NOX1 knock-down, which also impaired TACE/ADAM17 activation and thus EGFR phosphorylation. Finally, neither STK301831 nor NOX1 silencing impaired Src phosphorylation, but PP2 blocked early ROS production, showing that Src is involved in NOX1 activation. As expected, inhibition of Src or NOX1 increased TGF-β-induced cell death in Cav1(+/+) cells. In conclusion, CAV1 is required for TGF-β-mediated activation of TACE/ADAM17 through a mechanism that involves phosphorylation of Src and NOX1-mediated ROS production.

  16. Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression.

    PubMed

    Ayala, Gustavo; Morello, Matteo; Frolov, Anna; You, Sungyong; Li, Rile; Rosati, Fabiana; Bartolucci, Gianluca; Danza, Giovanna; Adam, Rosalyn M; Thompson, Timothy C; Lisanti, Michael P; Freeman, Michael R; Di Vizio, Dolores

    2013-09-01

    Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-β1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-β1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.

  17. Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression

    PubMed Central

    Frolov, Anna; You, Sungyong; Li, Rile; Rosati, Fabiana; Bartolucci, Gianluca; Danza, Giovanna; Adam, Rosalyn M; Thompson, Timothy C; Lisanti, Michael P; Freeman, Michael R; Vizio, Dolores Di

    2014-01-01

    Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-β1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-β1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma. PMID:23729330

  18. Caveolin-1 expression is variably displayed in astroglial-derived tumors and absent in oligodendrogliomas: concrete premises for a new reliable diagnostic marker in gliomas.

    PubMed

    Cassoni, Paola; Senetta, Rebecca; Castellano, Isabella; Ortolan, Erika; Bosco, Martino; Magnani, Ivana; Ducati, Alessandro

    2007-05-01

    Caveolins are basic constituents of flask-shaped cell membrane microdomains (caveolae), which are involved in many cell functions, including signalling, trafficking, and cellular growth control. The distribution of caveolae within the normal brain and in brain tumors is controversial. In the present study, we describe the expression of caveolin-1 (cav-1) in 64 brain tumors of different grade, of either astroglial or oligodendroglial origin. All studied astrocitomas of any grade (from II to IV) were cav-1 positive, displaying staining patterns and intensity specifically associated to the different tumor grades. In all glioblastomas and gliosarcomas, cav-1 staining was extremely intense, typically localized at the cell membrane and recognized a variable percentage of cells, including the majority of spindle cells and palisade-oriented perinecrotic cells. In anaplastic astrocytomas, a less intense membrane staining or a cytoplasmic dotlike immunoreactivity were present, the latter being almost the exclusive pattern observed in diffuse astrocitomas grade II. In contrast to astroglial tumors, the striking totality of grade II oligodendrogliomas and the large majority of grade III were lacking cav-1 expression. Interestingly, a cav-1 distribution overlapping the pattern described in tissues was observed also in primary cell cultures of human glioblastomas and astrocytomas, and also in one established glioblastoma cell line (U251 MG), analyzed by means of confocal microscopy and flow cytometry. In conclusion, among astroglial tumors cav-1 expression varies in distribution, pattern, and intensity specifically according to tumor types and grades. The association between tumor progression and a more structured membranous pattern of cav-1 expression could suggest the hypothesis of a neoplastic shift towards a mesenchymal phenotype, whose behavioral and biologic significance worth further studies. Finally, the lack of cav-1 immunoreactivity in oligodendrogliomas suggests its

  19. Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells

    PubMed Central

    Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

    2016-01-01

    Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke. PMID:26881424

  20. Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells.

    PubMed

    Song, Haoming; Cheng, Youjun; Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

    2016-01-01

    Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.

  1. The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFβ-induced Autophagy.

    PubMed

    Korah, Juliana; Canaff, Lucie; Lebrun, Jean-Jacques

    2016-01-29

    TGFβ is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.

  2. FACT Proteins, SUPT16H and SSRP1, Are Transcriptional Suppressors of HIV-1 and HTLV-1 That Facilitate Viral Latency.

    PubMed

    Huang, Huachao; Santoso, Netty; Power, Derek; Simpson, Sydney; Dieringer, Michael; Miao, Hongyu; Gurova, Katerina; Giam, Chou-Zen; Elledge, Stephen J; Zhu, Jian

    2015-11-06

    Our functional genomic RNAi screens have identified the protein components of the FACT (facilitates chromatin transcription) complex, SUPT16H and SSRP1, as top host factors that negatively regulate HIV-1 replication. FACT interacts specifically with histones H2A/H2B to affect assembly and disassembly of nucleosomes, as well as transcription elongation. We further investigated the suppressive role of FACT proteins in HIV-1 transcription. First, depletion of SUPT16H or SSRP1 protein enhances Tat-mediated HIV-1 LTR (long terminal repeat) promoter activity. Second, HIV-1 Tat interacts with SUPT16H but not SSRP1 protein. However, both SUPT16H and SSRP1 are recruited to LTR promoter. Third, the presence of SUPT16H interferes with the association of Cyclin T1 (CCNT1), a subunit of P-TEFb, with the Tat-LTR axis. Removing inhibitory mechanisms to permit HIV-1 transcription is an initial and key regulatory step to reverse post-integrated latent HIV-1 proviruses for purging of reservoir cells. We therefore evaluated the role of FACT proteins in HIV-1 latency and reactivation. Depletion of SUPT16H or SSRP1 protein affects both HIV-1 transcriptional initiation and elongation and spontaneously reverses latent HIV-1 in U1/HIV and J-LAT cells. Similar effects were observed with a primary CD4+ T cell model of HIV-1 latency. FACT proteins also interfere with HTLV-1 Tax-LTR-mediated transcription and viral latency, indicating that they may act as general transcriptional suppressors for retroviruses. We conclude that FACT proteins SUPT16H and SSRP1 play a key role in suppressing HIV-1 transcription and promoting viral latency, which may serve as promising gene targets for developing novel HIV-1 latency-reversing agents.

  3. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.

    PubMed

    Němcová, Kateřina; Sebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-09-01

    In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT20 tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.

  4. Tumor suppressors: enhancers or suppressors of regeneration?

    PubMed Central

    Pomerantz, Jason H.; Blau, Helen M.

    2013-01-01

    Tumor suppressors are so named because cancers occur in their absence, but these genes also have important functions in development, metabolism and tissue homeostasis. Here, we discuss known and potential functions of tumor suppressor genes during tissue regeneration, focusing on the evolutionarily conserved tumor suppressors pRb1, p53, Pten and Hippo. We propose that their activity is essential for tissue regeneration. This is in contrast to suggestions that tumor suppression is a trade-off for regenerative capacity. We also hypothesize that certain aspects of tumor suppressor pathways inhibit regenerative processes in mammals, and that transient targeted modification of these pathways could be fruitfully exploited to enhance processes that are important to regenerative medicine. PMID:23715544

  5. The Anti-Aging and Tumor Suppressor Protein Klotho Enhances Differentiation of a Human Oligodendrocytic Hybrid Cell Line

    PubMed Central

    Chen, Ci-Di; Liang, Jennifer; Hixson, Kathryn; Zeldich, Ella; Abraham, Carmela R.

    2016-01-01

    Klotho functions as an aging suppressor, which, in mice, extends lifespan when overexpressed and accelerates development of aging-like phenotypes when disrupted. Klotho is mainly expressed in brain and kidney and is secreted into the serum and CSF. We have previously shown that Klotho is reduced in brains of old monkeys, rats, and mice. We further reported the ability of Klotho to enhance oligodendrocyte differentiation and myelination. Here, we examined the signaling pathways induced by Klotho in MO3.13, a human oligodendrocytic hybrid cell line. We show that exogenous Klotho affects the ERK and Akt signaling pathways, decreases the proliferative abilities and enhances differentiation of MO3.13 cells. Furthermore, microarray analysis of Klotho-treated MO3.13 cells reveals a massive change in gene expression with 80 % of the differentially expressed genes being downregulated. Using gene set enrichment analysis, we predicted potential transcription factors involved in regulating Klotho-treated MO3.13 cells and found that these cells are highly enriched in the gene sets, that are similarly observed in cancer, cardiovascular disease, stress, aging, and hormone-related chemical and genetic perturbations. Since Klotho is downregulated in all brain tumors tested to date, enhancing Klotho has therapeutic potential for treating brain and other malignancies. PMID:24907942

  6. The metastasis suppressor KISS1 is an intrinsically disordered protein slightly more extended than a random coil

    PubMed Central

    Ibáñez de Opakua, Alain; Merino, Nekane; Villate, Maider; Cordeiro, Tiago N.; Ormaza, Georgina; Sánchez-Carbayo, Marta; Diercks, Tammo; Bernadó, Pau

    2017-01-01

    The metastasis suppressor KISS1 is reported to be involved in the progression of several solid neoplasias, making it a promising molecular target for controlling their metastasis. The KISS1 sequence contains an N-terminal secretion signal and several dibasic sequences that are proposed to be the proteolytic cleavage sites. We present the first structural characterization of KISS1 by circular dichroism, multi-angle light scattering, small angle X-Ray scattering and NMR spectroscopy. An analysis of the KISS1 backbone NMR chemical shifts does not reveal any preferential conformation and deviation from a random coil ensemble. The backbone 15N transverse relaxation times indicate a mildly reduced mobility for two regions that are rich in bulky residues. The small angle X-ray scattering curve of KISS1 is likewise consistent with a predominantly random coil ensemble, although an ensemble optimization analysis indicates some preference for more extended conformations possibly due to positive charge repulsion between the abundant basic residues. Our results support the hypothesis that KISS1 mostly samples a random coil conformational space, which is consistent with its high susceptibility to proteolysis and the generation of Kisspeptin fragments. PMID:28207895

  7. Dickkopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer.

    PubMed

    D'Amico, Lucia; Mahajan, Sahil; Capietto, Aude-Hélène; Yang, Zhengfeng; Zamani, Ali; Ricci, Biancamaria; Bumpass, David B; Meyer, Melissa; Su, Xinming; Wang-Gillam, Andrea; Weilbaecher, Katherine; Stewart, Sheila A; DeNardo, David G; Faccio, Roberta

    2016-05-02

    Tumor-stroma interactions contribute to tumorigenesis. Tumor cells can educate the stroma at primary and distant sites to facilitate the recruitment of heterogeneous populations of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs suppress T cell responses and promote tumor proliferation. One outstanding question is how the local and distant stroma modulate MDSCs during tumor progression. Down-regulation of β-catenin is critical for MDSC accumulation and immune suppressive functions in mice and humans. Here, we demonstrate that stroma-derived Dickkopf-1 (Dkk1) targets β-catenin in MDSCs, thus exerting immune suppressive effects during tumor progression. Mice bearing extraskeletal tumors show significantly elevated levels of Dkk1 in bone microenvironment relative to tumor site. Strikingly, Dkk1 neutralization decreases tumor growth and MDSC numbers by rescuing β-catenin in these cells and restores T cell recruitment at the tumor site. Recombinant Dkk1 suppresses β-catenin target genes in MDSCs from mice and humans and anti-Dkk1 loses its antitumor effects in mice lacking β-catenin in myeloid cells or after depletion of MDSCs, demonstrating that Dkk1 directly targets MDSCs. Furthermore, we find a correlation between CD15(+) myeloid cells and Dkk1 in pancreatic cancer patients. We establish a novel immunomodulatory role for Dkk1 in regulating tumor-induced immune suppression via targeting β-catenin in MDSCs.

  8. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

    PubMed

    Peuchant, Evelyne; Bats, Marie-Lise; Moranvillier, Isabelle; Lepoivre, Michel; Guitton, Jérôme; Wendum, Dominique; Lacombe, Marie-Lise; Moreau-Gaudry, François; Boissan, Mathieu; Dabernat, Sandrine

    2017-04-01

    NME1 (nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. NME1-knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of NME1 reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to H2O2 and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. NME1-deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

  9. A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

    NASA Technical Reports Server (NTRS)

    Ratner, N.; Bloom, G. S.; Brady, S. T.

    1998-01-01

    Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

  10. Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor.

    PubMed

    Semba, Shuho; Han, Shuang-Yin; Qin, Haiyan R; McCorkell, Kelly A; Iliopoulos, Dimitrios; Pekarsky, Yuri; Druck, Teresa; Trapasso, Francesco; Croce, Carlo M; Huebner, Kay

    2006-09-22

    The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies.

  11. Rational optimization of amber suppressor tRNAs toward efficient incorporation of a non-natural amino acid into protein in a eukaryotic wheat germ extract.

    PubMed

    Ogawa, Atsushi; Namba, Yuki; Gakumasawa, Mai

    2016-03-07

    Amber suppression is a useful method of genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA). A wheat germ extract (WGE) is suitable for this method by virtue of its high productivity and versatility in addition to its advantages as a cell-free translation system. However, in spite of this high potential, a genetic NAA incorporation system in WGE has not been sufficiently optimized in terms of sup-tRNAs, in contrast to that in E. coli and its cell extracts. We herein rationally optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein in WGE, via flexizyme-based aminoacylation. The optimized sup-tRNA (named tLys-opt) that was pre-charged with AcPhe exclusively yielded up to 220 μg mL(-1) of AcPhe-incorporated protein (yellow fluorescent protein, YPet) under the optimal conditions. This high productivity is comparable to the best reported yield of a similar NAA-incorporated protein synthesized with an engineered aminoacyl-tRNA synthetase/sup-tRNA pair in WGE, despite the fact that tLys-opt that has released AcPhe was not reused at all in this study. The results clearly show both the necessity of optimizing sup-tRNAs for efficient NAA incorporation and the validity of our strategy for their optimization. Because the optimization strategy described here is expected to be applicable not only to amber sup-tRNAs for other NAAs but also to ones used in other acylation methods, it would facilitate the synthesis of large amounts of various types of NAA-incorporated proteins in WGE.

  12. Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer

    PubMed Central

    2011-01-01

    Background MAP2K4 is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer. Methods We screened for mutations in MAP2K4 using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in MAP2K4 using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing MAP2K4 expression in cell lines. Results In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of MAP2K4 homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of MAP2K4 was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between MAP2K4 expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with MAP2K4 siRNA showed some reduction in proliferation. Conclusions MAP2K4 is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort. PMID:21575258

  13. A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

    PubMed Central

    Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

    2016-01-01

    Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene

  14. Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells

    SciTech Connect

    Yuan Baozhu Jefferson, Amy M.; Millecchia, Lyndell; Popescu, Nicholas C.; Reynolds, Steven H.

    2007-11-01

    We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.

  15. Adenomatous Polyposis Coli Tumor Suppressor Protein Has Signaling Activity in Xenopus laevis Embryos Resulting in the Induction of an Ectopic Dorsoanterior Axis

    PubMed Central

    Vleminckx, Kris; Wong, Ellen; Guger, Kathy; Rubinfeld, Bonnee; Polakis, Paul; Gumbiner, Barry M.

    1997-01-01

    Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are linked to both familial and sporadic human colon cancer. So far, a clear biological function for the APC gene product has not been determined. We assayed the activity of APC in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein β-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induced a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic β-catenin expression. In fact, axis induction by APC required the availability of cytosolic β-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of β-catenin, indicating that it has direct positive signaling activity in addition to its role in β-catenin turnover. From these results we propose a model in which APC acts as part of the Wnt/β-catenin signaling pathway, either upstream of, or in conjunction with, β-catenin. PMID:9015311

  16. Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

    PubMed Central

    Liwak, Urszula; Thakor, Nehal; Jordan, Lindsay E.; Roy, Rajat; Lewis, Stephen M.; Pardo, Olivier E.; Seckl, Michael

    2012-01-01

    Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-xL. We now show that the FGF-2–S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-xL messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-xL translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-xL) that mediate FGF-2–S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression. PMID:22431522

  17. RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4.

    PubMed

    Poria, D K; Guha, A; Nandi, I; Ray, P S

    2016-03-31

    Translation control of proinflammatory genes has a crucial role in regulating the inflammatory response and preventing chronic inflammation, including a transition to cancer. The proinflammatory tumor suppressor protein programmed cell death 4 (PDCD4) is important for maintaining the balance between inflammation and tumorigenesis. PDCD4 messenger RNA translation is inhibited by the oncogenic microRNA, miR-21. AU-rich element-binding protein HuR was found to interact with the PDCD4 3'-untranslated region (UTR) and prevent miR-21-mediated repression of PDCD4 translation. Cells stably expressing miR-21 showed higher proliferation and reduced apoptosis, which was reversed by HuR expression. Inflammatory stimulus caused nuclear-cytoplasmic relocalization of HuR, reversing the translation repression of PDCD4. Unprecedentedly, HuR was also found to bind to miR-21 directly, preventing its interaction with the PDCD4 3'-UTR, thereby preventing the translation repression of PDCD4. This suggests that HuR might act as a 'miRNA sponge' to regulate miRNA-mediated translation regulation under conditions of stress-induced nuclear-cytoplasmic translocation of HuR, which would allow fine-tuned gene expression in complex regulatory environments.

  18. Proteins related to St. John's Wort p27SJ, a suppressor of HIV-1 expression, are ubiquitous in plants.

    PubMed

    Perera, Tekla; Berna, Anne; Scott, Ken; Lemaitre-Guillier, Christelle; Bernier, François

    2008-02-01

    Proteins belonging to the family of DING proteins are ubiquitous in animals and several of them are associated with various diseases. Their presence in a few plant species has previously been reported and the St John's Wort DING protein was recently described as an inhibitor of HIV replication and transcription. However, data about DING protein occurrence in plants and their biochemical properties remain almost nonexistent. We describe methods for the purification of DING proteins from plants that may have general applicability since they are not dependent upon specific affinity ligands, contrary to previously described protocols. Cibacron Blue chromatography, sometimes preceded by an ion-exchange chromatographic step, is suitable for most plant extracts. DING proteins were purified from various species and cell types and their identity was confirmed immunologically and, in some cases, by N-terminal sequence analysis, indicating that they are ubiquitous in the plant kingdom. They are associated with the cell wall and sometimes secreted in the medium for in vitro grown cells. High-molecular-weight DING precursors were often observed. Internal peptides were also sequenced, as a prelude to gene cloning experiments.

  19. High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

    NASA Astrophysics Data System (ADS)

    Lösche, Matthias

    2012-02-01

    The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration < 5 å) but binds the membrane tightly with its two major domains, the C2 and

  20. The Arabidopsis transcription factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 is a direct substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and regulates immunity.

    PubMed

    Kang, Sining; Yang, Fan; Li, Lin; Chen, Huamin; Chen, She; Zhang, Jie

    2015-03-01

    Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Mitogen-activated protein kinases (MAPKs), as well as other cytoplasmic kinases, integrate upstream immune signals and, in turn, dissect PTI signaling via different substrates to regulate defense responses. However, only a few direct substrates of these signaling kinases have been identified. Here, we show that PAMP perception enhances phosphorylation of BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1), a transcription factor involved in brassinosteroid (BR) signaling pathway, through pathogen-induced MAPKs in Arabidopsis (Arabidopsis thaliana). BES1 interacts with MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and is phosphorylated by MPK6. bes1 loss-of-function mutants display compromised resistance to bacterial pathogen Pseudomonas syringae pv tomato DC3000. BES1 S286A/S137A double mutation (BES1(SSAA)) impairs PAMP-induced phosphorylation and fails to restore bacterial resistance in bes1 mutant, indicating a positive role of BES1 phosphorylation in plant immunity. BES1 is phosphorylated by glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2), a negative regulator of BR signaling. BR perception inhibits BIN2 activity, allowing dephosphorylation of BES1 to regulate plant development. However, BES1(SSAA) does not affect BR-mediated plant growth, suggesting differential residue requirements for the modulation of BES1 phosphorylation in PTI and BR signaling. Our study identifies BES1 as a unique direct substrate of MPK6 in PTI signaling. This finding reveals MAPK-mediated BES1 phosphorylation as another BES1 modulation mechanism in plant cell signaling, in addition to GSK3-like kinase-mediated BES1 phosphorylation and F box protein-mediated BES1 degradation.

  1. Cutaneous HPV8 and MmuPV1 E6 Proteins Target the NOTCH and TGF-β Tumor Suppressors to Inhibit Differentiation and Sustain Keratinocyte Proliferation

    PubMed Central

    Meyers, Jordan M.; Grace, Miranda; Munger, Karl

    2017-01-01

    Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV) or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV) 5 and HPV8 inhibit NOTCH and TGF-β signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis. PMID:28107544

  2. Immunohistochemical detection of p53 tumor-suppressor protein is a poor indicator of prognosis for canine cutaneous mast cell tumors.

    PubMed

    Ginn, P E; Fox, L E; Brower, J C; Gaskin, A; Kurzman, I D; Kubilis, P S

    2000-01-01

    Eighty-three canine cutaneous mast cell tumors were graded histologically and evaluated immunohistochemically for p53 tumor-suppressor protein expression. An avidin-biotin immunohistochemical protocol incorporated a rabbit polyclonal antibody (CM-1) directed against normal and mutant p53 protein. Positive staining was observed in 44.6% (37/83) of tumors and included 50% (12/24) of grade I (well differentiated) tumors, 46.9% (23/49) of grade II (intermediate differentiation) tumors, and 20% (2/10) of grade III (poorly differentiated) tumors. A statistically significantly higher proportion (P < 0.019) of tumors from the head and neck (83.3%, 10/12), stained positive for p53 than tumors from the thorax, back, abdomen, and axilla (39.4%, 13/33), legs (35.7%, 10/28), or prepuce, scrotal, or inguinal areas (44.4%, 4/9). No statistically significant difference between p53 labeling and histologic grade, breed, or tumor size was present. Survival data were available for 53/83 (63.9%) of dogs. Positive reactivity for p53 was observed in 47% (25/53) of tumors within this group, with 57.9% (11/19) of grade I, 43.3% (13/30) of grade II, and 25% (1/4) of grade III tumors labeled. Mean survival time for the 53 dogs was 12.1 months. The median survival time for dogs with grade III tumors or tumors >5 cm was statistically significantly shorter (P < 0.0001) than for dogs with grades I and II or smaller tumors. Although p53 protein abnormalities may play a role in tumor development or behavior in some canine cutaneous mast cell tumors, immunoreactivity was not associated with lack of tumor differentiation, tumor locations previously shown to demonstrate aggressive biological behavior, breed predisposition, or survival times.

  3. The Tumor Suppressor Mst1 Promotes Changes in the Cellular Redox State by Phosphorylation and Inactivation of Peroxiredoxin-1 Protein*

    PubMed Central

    Rawat, Sonali Jalan; Creasy, Caretha L.; Peterson, Jeffrey R.; Chernoff, Jonathan

    2013-01-01

    The serine/threonine protein kinases Mst1 and Mst2 can be activated by cellular stressors including hydrogen peroxide. Using two independent protein interaction screens, we show that these kinases associate, in an oxidation-dependent manner, with Prdx1, an enzyme that regulates the cellular redox state by reducing hydrogen peroxide to water and oxygen. Mst1 inactivates Prdx1 by phosphorylating it at Thr-90 and Thr-183, leading to accumulation of hydrogen peroxide in cells. These results suggest that hydrogen peroxide-stimulated Mst1 activates a positive feedback loop to sustain an oxidizing cellular state. PMID:23386615

  4. Interleukin-27-Mediated Suppression of Human Th17 Cells Is Associated with Activation of STAT1 and Suppressor of Cytokine Signaling Protein 1

    PubMed Central

    Liu, Hong

    2011-01-01

    Accumulating evidence indicates that interleukin (IL)-27, a member of the IL-12 family of cytokines, antagonizes pathological Th17 effector cell responses. Relatively little is known about the cytokines that regulate human Th17 cells. In this study, we investigated the effect of IL-27 on the differentiation of human Th17 cells and on committed memory Th17 cells. We demonstrate that IL-27 suppresses the development of human Th17 cells by downregulating retinoid orphan nuclear receptor C expression and that this inhibition is associated with the induction of the intracellular signaling factors STAT1 and induction of the suppressor of cytokine signaling protein 1. The IL-27-mediated inhibition of IL-17 is independent of IL-10. We show that IL-27 inhibits differentiation of naïve T cells into IL-17+ T cells under different Th17 polarizing conditions. IL-27 suppresses other Th17 subset cytokines such as IL-22 and IL-21 but not tumor necrosis factor-α. Moreover, we also show that IL-27 inhibits IL-17 production by committed Th17 memory cells, which is independent of IL-10. These studies show that IL-27 negatively regulates both the developing and committed human Th17 responses and therefore may be a promising therapeutic approach in the treatment of Th17-mediated diseases. PMID:21235411

  5. Conversion of the LIMA1 tumour suppressor into an oncogenic LMO-like protein by API2-MALT1 in MALT lymphoma.

    PubMed

    Nie, Zilin; Du, Ming-Qing; McAllister-Lucas, Linda M; Lucas, Peter C; Bailey, Nathanael G; Hogaboam, Cory M; Lim, Megan S; Elenitoba-Johnson, Kojo S J

    2015-01-08

    MALT1 is the only known paracaspase and is a critical mediator of B- and T-cell receptor signalling. The function of the MALT1 gene is subverted by oncogenic chimeric fusions arising from the recurrent t(11;18)(q21;q21) aberration, which is the most frequent translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. API2-MALT1-positive MALT lymphomas manifest antibiotic resistance and aggressive clinical behaviour with poor clinical outcome. However, the mechanisms underlying API2-MALT1-induced MALT lymphomagenesis are not fully understood. Here we show that API2-MALT1 induces paracaspase-mediated cleavage of the tumour suppressor protein LIMA1. LIMA1 binding by API2-MALT1 is API2 dependent and proteolytic cleavage is dependent on MALT1 paracaspase activity. Intriguingly, API2-MALT1-mediated proteolysis generates a LIM domain-only (LMO)-containing fragment with oncogenic properties in vitro and in vivo. Importantly, primary MALT lymphomas harbouring the API2-MALT1 fusion uniquely demonstrate LIMA1 cleavage fragments. Our studies reveal a novel paracaspase-mediated oncogenic gain-of-function mechanism in the pathogenesis of MALT lymphoma.

  6. The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency*

    PubMed Central

    Sengupta, Satyaki; Lingnurkar, Raj; Carey, Timothy S.; Pomaville, Monica; Kar, Parimal; Feig, Michael; Wilson, Catherine A.; Knott, Jason G.; Arnosti, David N.; Henry, R. William

    2015-01-01

    The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system. PMID:25903125

  7. Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi.

    PubMed

    Zhang, Weiguo; Fraiture, Malou; Kolb, Dagmar; Löffelhardt, Birgit; Desaki, Yoshitake; Boutrot, Freddy F G; Tör, Mahmut; Zipfel, Cyril; Gust, Andrea A; Brunner, Frédéric

    2013-10-01

    Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called sclerotinia culture filtrate elicitor1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to receptor-like protein30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases brassinosteroid insensitive1-associated receptor kinase1 (BAK1) and Suppressor of BIR1-1/evershed (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.

  8. Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs

    PubMed Central

    2014-01-01

    Background The genome is pervasively transcribed but most transcripts do not code for proteins, constituting non-protein-coding RNAs. Despite increasing numbers of functional reports of individual long non-coding RNAs (lncRNAs), assessing the extent of functionality among the non-coding transcriptional output of mammalian cells remains intricate. In the protein-coding world, transcripts differentially expressed in the context of processes essential for the survival of multicellular organisms have been instrumental in the discovery of functionally relevant proteins and their deregulation is frequently associated with diseases. We therefore systematically identified lncRNAs expressed differentially in response to oncologically relevant processes and cell-cycle, p53 and STAT3 pathways, using tiling arrays. Results We found that up to 80% of the pathway-triggered transcriptional responses are non-coding. Among these we identified very large macroRNAs with pathway-specific expression patterns and demonstrated that these are likely continuous transcripts. MacroRNAs contain elements conserved in mammals and sauropsids, which in part exhibit conserved RNA secondary structure. Comparing evolutionary rates of a macroRNA to adjacent protein-coding genes suggests a local action of the transcript. Finally, in different grades of astrocytoma, a tumor disease unrelated to the initially used cell lines, macroRNAs are differentially expressed. Conclusions It has been shown previously that the majority of expressed non-ribosomal transcripts are non-coding. We now conclude that differential expression triggered by signaling pathways gives rise to a similar abundance of non-coding content. It is thus unlikely that the prevalence of non-coding transcripts in the cell is a trivial consequence of leaky or random transcription events. PMID:24594072

  9. Omnipotent Suppressors Effective in psi Strains of SACCHAROMYCES CEREVISIAE: Recessiveness and Dominance.

    PubMed

    Ono, B; Moriga, N; Ishihara, K; Ishiguro, J; Ishino, Y; Shinoda, S

    1984-06-01

    We have characterized recessive and dominant omnipotent suppressor mutations obtained by conversion of the leu2-1 UAA mutation and the met8-UAG mutation in a psi(+) strain of Saccharomyces cerevisiae. The suppressors that act recessively upon these markers fell into two complementation groups; the sup47 and sup36 suppressors show linkage to the tyr1 locus and the aro1 locus, respectively. Of the suppressors acting dominantly upon both markers, those linked to the tyr1 locus are alleles of the SUP46 ribosomal mutation. The sup47 suppressors differ from the SUP46 suppressors not only in their suppressor activities in heterozygous diploids but also in their map positions relative to the tyr1 locus and their effects on the S11 ribosomal protein. The remaining dominant suppressors are not alleles of sup36 as judged by linkage analysis. The recessive suppressors and the dominant suppressors also differ in their effects on cell growth.

  10. Tumor Suppressor Bromodomain-containing Protein 7 Cooperates with Smads to Promote Transforming Growth Factor-β Responses

    PubMed Central

    Liu, Jinquan; He, Zhou; Zhang, Ye; You, Han; Huang, Jun; Lin, Xia; Feng, Xin-Hua

    2016-01-01

    Smad proteins are central mediators in the canonical transforming growth factor-β (TGF-β) signaling pathway in mammalian cells. We report here that bromodomain-containing protein 7 (BRD7) functions as a novel transcription coactivator for Smads in TGF-β signaling. BRD7 forms a TGF-β inducible complex with Smad3/4 through its N-terminal Smad-binding domain. BRD7 simultaneously binds to acetylated histones to promote Smad-chromatin association, and associates with histone acetyltransferase p300 to enhance Smad transcriptional activity. Ectopic expression of BRD7, but not its mutants defective in Smad binding, enhances TGF-β transcriptional, tumor suppressing and epithelial-mesenchymal transition (EMT) responses. Conversely, depletion of BRD7 inhibits TGF-β responses. Thus, our study provides compelling evidence for a new function of BRD7 in fine-tuning TGF-β physiological responses. PMID:27270427

  11. Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs▿

    PubMed Central

    Kuan, Yung-Shu; Brewer-Jensen, Paul; Bai, Wen-Li; Hunter, Cedric; Wilson, Carrie B.; Bass, Sarah; Abernethy, John; Wing, James S.; Searles, Lillie L.

    2009-01-01

    RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated αβ/αγ elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the αβ RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an αβ element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome. PMID:19687295

  12. Interaction of UAG suppressors and omnipotent suppressors in Saccharomyces cerevisiae.

    PubMed

    Song, J M; Liebman, S W

    1985-02-01

    Haploids bearing the dominant UAG suppressor, SUP7-a, and various alleles of the omnipotent suppressor sup35 were examined. The presence of the UAG suppressor reduced the efficiency of some alleles of sup35, and caused other sup35 alleles to be lethal. A nonclassical interaction of the dominant suppressor tRNA and the ribosome is proposed to explain these observations.

  13. Expression of ankyrin repeat and suppressor of cytokine signaling box protein 4 (Asb-4) in proopiomelanocortin neurons of the arcuate nucleus of mice produces a hyperphagic, lean phenotype.

    PubMed

    Li, Ji-Yao; Chai, Biao-Xin; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W

    2010-01-01

    Ankyrin repeat and suppressor of cytokine signaling box-containing protein 4 (Asb-4) is specifically expressed in the energy homeostasis-related brain areas and colocalizes with proopiomelanocortin (POMC) neurons of the arcuate nucleus (ARC). Injection of insulin into the third ventricle of the rat brain increased Asb-4 mRNA expression in the paraventricular nucleus but not in the ARC of the hypothalamus, whereas injection of leptin (ip) increased Asb-4 expression in both mouse paraventricular nucleus and ARC. A transgenic mouse in which Myc-tagged Asb-4 is specifically expressed in POMC neurons of the ARC was made and used to study the effects of Asb-4 on ingestive behavior and metabolic rate. Animals with overexpression of Asb-4 in POMC neurons demonstrated an increase in food intake. However, POMC-Asb-4 transgenic animals gained significantly less weight from 6-30 wk of age. The POMC-Asb-4 mice had reduced fat mass and increased lean mass and lower levels of blood leptin. The transgenic animals were resistant to high-fat diet-induced obesity. Transgenic mice had significantly higher rates of oxygen consumption and carbon dioxide production than wild-type mice during both light and dark periods. The locomotive activity of transgenic mice was increased. The overexpression of Asb-4 in POMC neurons increased POMC mRNA expression in the ARC. The transgenic animals had no observed effect on peripheral glucose metabolism and the activity of the autonomic nervous system. These results indicate that Asb-4 is a key regulatory protein in the central nervous system, involved in the control of feeding behavior and metabolic rate.

  14. Protein phosphatase 2A is essential to maintain active Wnt signaling and its Aβ tumor suppressor subunit is not expressed in colon cancer cells.

    PubMed

    Carmen Figueroa-Aldariz, M; Castañeda-Patlán, M Cristina; Santoyo-Ramos, Paula; Zentella, Alejandro; Robles-Flores, Martha

    2015-11-01

    Canonical Wnt signaling is altered in most cases of colorectal cancer. Experimental evidence indicates that protein phosphatase 2A (PP2A) may play either positive or negative roles in Wnt signaling but its precise in vivo functions remain elusive. In this work, using colon cultured cell lines we showed that basal PP2A activity is markedly reduced in malignant cells compared to non-malignant counterparts. We found that whereas normal or cancer cells displaying not altered Wnt signaling express mRNAs coding for PP2A-A scaffold α and β isoforms, cancer cells which have altered Wnt signaling do not express the Aβ isoform mRNA. Remarkably, we found that the Aβ protein levels are lost in all colon cancer cells, and in patients' tumor biopsies. In addition, all cancer cells exhibit higher levels of RalA activity, compared to non-malignant cells. Rescue experiments to restore Aβ expression in malignant RKO cells, diminished the RalGTPase activation and cell proliferation, indicating that the Aβ isoform acts as tumor suppressor in colon cancer cells. Reciprocal co-immunoprecipitation and immunofluorescence studies showed that the PP2A-C and -Aα subunits, expressed in all colon cells, interact in vivo with β-catenin only in malignant cells. Selective inhibition of PP2A did not significantly affect cellular apoptosis but induced dose-dependent negative effects in β-catenin-mediated transcriptional activity and in cell proliferation of malignant cells, indicating that the residual PP2A activity found in malignant cells, mediated by -C and Aα core subunits, is essential to maintain active Wnt signaling and cell proliferation in colon cancer cells.

  15. Identification of a genetic interaction between the tumor suppressor EAF2 and the retinoblastoma protein (Rb) signaling pathway in C. elegans and prostate cancer cells

    SciTech Connect

    Cai, Liquan; Wang, Dan; Fisher, Alfred L.; Wang, Zhou

    2014-05-02

    Highlights: • RNAi screen identified genetic enhancers for the C. elegans homolog of EAF2. • EAF2 and RBBP4 proteins physically bind to each other and alter transcription. • Overexpression of EAF2 and RBBP4 induces the cell death in prostate cancer cells. - Abstract: The tumor suppressor EAF2 is regulated by androgen signaling and associated with prostate cancer. While EAF2 and its partner ELL have been shown to be members of protein complexes involved in RNA polymerase II transcriptional elongation, the biologic roles for EAF2 especially with regards to the development of cancer remains poorly understood. We have previously identified the eaf-1 gene in Caenorhabditiselegans as the ortholog of EAF2, and shown that eaf-1 interacts with the ELL ortholog ell-1 to control development and fertility in worms. To identify genetic pathways that interact with eaf-1, we screened RNAi libraries consisting of transcription factors, phosphatases, and chromatin-modifying factors to identify genes which enhance the effects of eaf-1(tm3976) on fertility. From this screen, we identified lin-53, hmg-1.2, pha-4, ruvb-2 and set-6 as hits. LIN-53 is the C. elegans ortholog of human retinoblastoma binding protein 4/7 (RBBP 4/7), which binds to the retinoblastoma protein and inhibits the Ras signaling pathway. We find that lin-53 showed a synthetic interaction with eaf-1(tm3976) where knockdown of lin-53 in an eaf-1(tm3976) mutant resulted in sterile worms. This phenotype may be due to cell death as the treated worms contain degenerated embryos with increased expression of the ced-1:GFP cell death marker. Further we find that the interaction between eaf-1 and lin-53/RBBP4/7 also exists in vertebrates, which is reflected by the formation of a protein complex between EAF2 and RBBP4/7. Finally, overexpression of either human EAF2 or RBBP4 in LNCaP cells induced the cell death while knockdown of EAF2 in LNCaP enhanced cell proliferation, indicating an important role of EAF2 in

  16. Genetic Modeling of PIM Proteins in Cancer: Proviral Tagging and Cooperation with Oncogenes, Tumor Suppressor Genes, and Carcinogens

    PubMed Central

    Aguirre, Enara; Renner, Oliver; Narlik-Grassow, Maja; Blanco-Aparicio, Carmen

    2014-01-01

    The PIM proteins, which were initially discovered as proviral insertion sites in Moloney-murine leukemia virus infection, are a family of highly homologous serine/threonine kinases that have been reported to be overexpressed in hematological malignancies and solid tumors. The PIM proteins have also been associated with metastasis and overall treatment responses and implicated in the regulation of apoptosis, metabolism, the cell cycle, and homing and migration, which makes these proteins interesting targets for anti-cancer drug discovery. The use of retroviral insertional mutagenesis and refined approaches such as complementation tagging has allowed the identification of myc, pim, and a third group of genes (including bmi1 and gfi1) as complementing genes in lymphomagenesis. Moreover, mouse modeling of human cancer has provided an understanding of the molecular pathways that are involved in tumor initiation and progression at the physiological level. In particular, genetically modified mice have allowed researchers to further elucidate the role of each of the Pim isoforms in various tumor types. PIM kinases have been identified as weak oncogenes because experimental overexpression in lymphoid tissue, prostate, and liver induces tumors at a relatively low incidence and with a long latency. However, very strong synergistic tumorigenicity between Pim1/2 and c-Myc and other oncogenes has been observed in lymphoid tissues. Mouse models have also been used to study whether the inhibition of specific PIM isoforms is required to prevent carcinogen-induced sarcomas, indicating that the absence of Pim2 and Pim3 greatly reduces sarcoma growth and bone invasion; the extent of this effect is similar to that observed in the absence of all three isoforms. This review will summarize some of the animal models that have been used to understand the isoform-specific contribution of PIM kinases to tumorigenesis. PMID:24860787

  17. Foxa2, a novel protein partner of the tumour suppressor menin, is deregulated in mouse and human MEN1 glucagonomas.

    PubMed

    Bonnavion, Rémy; Teinturier, Romain; Gherardi, Samuele; Leteurtre, Emmanuelle; Yu, Run; Cordier-Bussat, Martine; Du, Rui; Pattou, François; Vantyghem, Marie-Christine; Bertolino, Philippe; Lu, Jieli; Zhang, Chang Xian

    2017-02-11

    Foxa2, known as one of the pioneer factors, plays a crucial role in islet development and endocrine functions. Its expression and biological functions are regulated by various factors, including, in particular, insulin and glucagon. However, its expression and biological role in adult pancreatic α-cells remain elusive. In the current study, we showed that Foxa2 was overexpressed in islets from α-cell-specific Men1 mutant mice, at both the transcriptional level and the protein level. More importantly, immunostaining analyses showed its prominent nuclear accumulation, specifically in α-cells, at a very early stage after Men1 disruption. Similar nuclear FOXA2 expression was also detected in a substantial proportion (12/19) of human multiple endocrine neoplasia type 1 (MEN1) glucagonomas. Interestingly, our data revealed an interaction between Foxa2 and menin encoded by the Men1 gene. Furthermore, using several approaches, we demonstrated the relevance of this interaction in the regulation of two tested Foxa2 target genes, including the autoregulation of the Foxa2 promoter by Foxa2 itself. The current study establishes menin, a novel protein partner of Foxa2, as a regulator of Foxa2, the biological functions of which extend beyond the pancreatic endocrine cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  18. An antisuppressor that acts on omnipotent suppressors in yeast.

    PubMed

    Liebman, S W; Cavenagh, M

    1980-05-01

    Six partially dominant antisuppressors were obtained that reduce the efficiency of two omnipotent yeast suppressors, sup45 and sup35, thought to be ribosomal ambiguity mutations. Each of these six antisuppressors was shown to fall within a single Mendelian locus, named asu9. The asu9 mutations are specific for omnipotent suppressors; they have no effect on several dominant tRNA-like suppressors. In the absence of suppressors, asu9 causes sensitivity to the aminoglycoside antibiotic, paromomycin. The properties of asu9 are consistent with the hypothesis that asu9 alters yeast ribosomal proteins.

  19. Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

    SciTech Connect

    Gestl, Erin E.; Anne Boettger, S.

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated with p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53

  20. Suppressor of cytokine signaling 3 inhibits LPS-induced IL-6 expression in osteoblasts by suppressing CCAAT/enhancer-binding protein ß activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Suppressors of cytokine signaling 3 (SOCS3) is an important intracellular regulator of TLR4 signaling and has been implicated in several inflammatory diseases. Although SOCS3 seems to contribute to the balance between the pro-inflammatory effects of IL-6 and antiinflammatory signaling of IL-10 by ne...

  1. Dispersion suppressors with bending

    SciTech Connect

    Garren, A.

    1985-10-01

    Dispersion suppressors of two main types are usually used. In one the cell quadrupole focussing structure is the same as in normal cells but some of the dipoles are replaced by drifts. In the other, the quadrupole strengths and/or spacings are different from those of the normal cells, but the bending is about the same as it is in the cells. In SSC designs to date, dispersion suppressors of the former type have been used, consisting of two cells with bending equivalent to one. In this note a suppressor design with normal bending and altered focussing is presented. The advantage of this scheme is that circumference is reduced. The disadvantages are that additional special quadrupoles must be provided (however, they need not be adjustable), and the maximum beta values within them are about 30% higher than the cell maxima.

  2. Tumor suppressor identified as inhibitor of inflammation

    Cancer.gov

    Scientists at NCI have found that a protein, FBXW7, which acts as a tumor suppressor, is also important for the reduction in strength of inflammatory pathways. It has long been recognized that a complex interaction exists between cancer causing mechanisms

  3. Regulation of Hypoxia-Inducible mRNAs by the von Hippel-Lindau Tumor Suppressor Protein Requires Binding to Complexes Containing Elongins B/C and Cul2

    PubMed Central

    Lonergan, Kim M.; Iliopoulos, Othon; Ohh, Michael; Kamura, Takumi; Conaway, Ronald C.; Conaway, Joan Weliky; Kaelin, William G.

    1998-01-01

    The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis. PMID:9447969

  4. Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein.

    PubMed Central

    Frattini, M G; Hurst, S D; Lim, H B; Swaminathan, S; Laimins, L A

    1997-01-01

    Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5-fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post-transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53. PMID:9029152

  5. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    PubMed

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  6. Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt.

    PubMed

    McDaneld, T G; Spurlock, D M

    2008-11-01

    Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K

  7. Syndecan-2 Exerts Antifibrotic Effects by Promoting Caveolin-1–mediated Transforming Growth Factor-β Receptor I Internalization and Inhibiting Transforming Growth Factor-β1 Signaling

    PubMed Central

    Shi, Yuanyuan; Gochuico, Bernadette R.; Yu, Guoying; Tang, Xiaomeng; Osorio, Juan C.; Fernandez, Isis E.; Risquez, Cristobal F.; Patel, Avignat S.; Shi, Ying; Wathelet, Marc G.; Goodwin, Andrew J.; Haspel, Jeffrey A.; Ryter, Stefan W.; Billings, Eric M.; Kaminski, Naftali; Morse, Danielle

    2013-01-01

    Rationale: Alveolar transforming growth factor (TGF)-β1 signaling and expression of TGF-β1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-β receptor TβRI inhibits TGF-β signaling and could attenuate development of experimental lung fibrosis. Objectives: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-β1 signaling in alveolar epithelial cells. Methods: Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2–dependent changes in epithelial cell TGF-β1 signaling, TGF-β1, and TβRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. Measurements and Main Results: We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-β1 signaling and downstream expression of TGF-β1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1–dependent internalization of TGF-β1 and TβRI in alveolar epithelial cells, which inhibited TGF-β1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. Conclusions: Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1–dependent TGF-β1 and TβRI internalization and inhibiting TGF-β1 signaling in alveolar epithelial

  8. In MMTV-Her-2/neu transgenic mammary tumors the absence of caveolin-1-/- alters PTEN and NHERF1 but not β-catenin expression.

    PubMed

    Cuello-Carrión, F Darío; Cayado-Gutiérrez, Niubys; Natoli, Anthony L; Restall, Christina; Anderson, Robin L; Nadin, Silvina; Alvarez-Olmedo, Daiana; Castro, Gisela N; Gago, Francisco E; Fanelli, Mariel A; Ciocca, Daniel R

    2013-09-01

    In a recent study, we have shown that in mammary tumors from mice lacking the Cav-1 gene, there are alterations in specific heat shock proteins as well as in tumor development. With this in mind, we have now investigated other proteins in the same mammary mouse tumor model (Her-2/neu expressing mammary tumors from Cav-1 wild type and Cav-1 null mice), to further comprehend the complex tumor-stroma mechanisms involved in regulating stress responses during tumor development. In this tumor model the cancer cells always lacked of Cav-1, so the KO influenced the Cav-1 in the stroma. By immunohistochemistry, we have found a striking co-expression of β-catenin and Her-2/neu in the tumor cells. The absence of Cav-1 in the tumor stroma had no effect on expression or localization of β-catenin and Her-2/neu. Both proteins appeared co-localized at the cell surface during tumor development and progression. Since Her-2/neu activation induces MTA1, we next evaluated MTA1 in the mouse tumors. Although this protein was found in numerous nuclei, the absence of Cav-1 did not alter its expression level. In contrast, significantly more PTEN protein was noted in the tumors lacking Cav-1 in the stroma, with the protein localized mainly in the nuclei. P-Akt levels were relatively low in tumors from both Cav-1 WT and Cav-1 KO mice. There was also an increase in nuclear NHERF1 expression levels in the tumors arising from Cav-1 KO mice. The data obtained in the MMTV-neu model are consistent with a role for Cav-1 in adjacent breast cancer stromal cells in modulating the expression and localization of important proteins implicated in tumor cell behavior.

  9. Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma.

    PubMed

    Codenotti, Silvia; Vezzoli, Marika; Poliani, Pietro Luigi; Cominelli, Manuela; Bono, Federica; Kabbout, Hadi; Faggi, Fiorella; Chiarelli, Nicola; Colombi, Marina; Zanella, Isabella; Biasiotto, Giorgio; Montanelli, Alessandro; Caimi, Luigi; Monti, Eugenio; Fanzani, Alessandro

    2016-08-01

    Caveolins (Cav-1, -2 and -3) and Cavins (Cavin-1, -2, -3 and -4) are two protein families controlling the biogenesis and function of caveolae, plasma membrane omega-like invaginations representing the primary site of important cellular processes like endocytosis, cholesterol homeostasis and signal transduction. Caveolae are especially abundant in fat tissue, playing a consistent role in a number of processes, such as the insulin-dependent glucose uptake and transmembrane transport of lipids underlying differentiation, maintenance and adaptive hypertrophy of adipocytes. Based on this premise, in this work we have investigated the expression of caveolar protein components in liposarcoma (LPS), an adipocytic soft tissue sarcoma affecting adults categorized in well-differentiated, dedifferentiated, myxoid and pleomorphic histotypes. By performing an extensive microarray data analysis followed by immunohistochemistry on human LPS tumors, we demonstrated that Cav-1, Cav-2 and Cavin-1 always cluster in all the histotypes, reaching the highest expression in well-differentiated LPS, the least aggressive of the malignant forms composed by tumor cells with a morphology resembling mature adipocytes. In vitro experiments carried out using two human LPS cell lines showed that the expression levels of Cav-1, Cav-2 and Cavin-1 proteins were faintly detectable during cell growth, becoming consistently increased during the accumulation of intracellular lipid droplets characterizing the adipogenic differentiation. Moreover, in differentiated LPS cells the three proteins were also found to co-localize and form molecular aggregates at the plasma membrane, as shown via immunofluorescence and immunoprecipitation analysis. Overall, these data indicate that Cav-1, Cav-2 and Cavin-1 may be considered as reliable markers for identification of LPS tumors characterized by consistent adipogenic differentiation.

  10. Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

    PubMed

    Eustice, D C; Wakem, L P; Wilhelm, J M; Sherman, F

    1986-03-20

    The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

  11. Transcriptional Repression of Tumor Suppressor CDC73, Encoding an RNA Polymerase II Interactor, by Wilms Tumor 1 Protein (WT1) Promotes Cell Proliferation

    PubMed Central

    Rather, Mohammad Iqbal; Swamy, Shivananda; Gopinath, Kodaganur S.; Kumar, Arun

    2014-01-01

    The Wilms tumor 1 gene (WT1) can either repress or induce the expression of genes. Inconsistent with its tumor suppressor role, elevated WT1 levels have been observed in leukemia and solid tumors. WT1 has also been suggested to act as an oncogene by inducing the expression of MYC and BCL-2. However, these are only the correlational studies, and no functional study has been performed to date. Consistent with its tumor suppressor role, CDC73 binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex and causes transcriptional repression of oncogenes MYC and CCND1. It also represses β-catenin-mediated transcription. Based on the reduced level of CDC73 in oral squamous cell carcinoma (OSCC) samples in the absence of loss-of-heterozygosity, promoter methylation, and mutations, we speculated that an inhibitory transcription factor is regulating its expression. The bioinformatics analysis predicted WT1 as an inhibitory transcription factor to regulate the CDC73 level. Our results showed that overexpression of WT1 decreased CDC73 levels and promoted proliferation of OSCC cells. ChIP and EMSA results demonstrated binding of WT1 to the CDC73 promoter. The 5-azacytidine treatment of OSCC cells led to an up-regulation of WT1 with a concomitant down-regulation of CDC73, further suggesting regulation of CDC73 by WT1. Exogenous CDC73 attenuated the protumorigenic activity of WT1 by apoptosis induction. An inverse correlation between expression levels of CDC73 and WT1 was observed in OSCC samples. These observations indicated that WT1 functions as an oncogene by repressing the expression of CDC73 in OSCC. We suggest that targeting WT1 could be a therapeutic strategy for cancer, including OSCC. PMID:24257751

  12. Synergistic antineoplastic effect of DLC1 tumor suppressor protein and histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), on prostate and liver cancer cells: perspectives for therapeutics.

    PubMed

    Zhou, Xiaoling; Yang, Xu-Yu; Popescu, Nicholas C

    2010-04-01

    Inactivation of tumor suppressor genes is a major contributing alteration in the initiation or progression of cancer. The human tumor suppressor gene DLC1 (deleted in liver cancer 1) is frequently downregulated or silenced in multiple cancers, predominantly by epigenetic mechanisms. With the current considerable interest and progress in epigenetic therapy, a number of promising antineoplastic agents, particularly histone deacetylase (HDAC) inhibitors, have been developed and used successfully in clinical trials. Both DLC1 and HDAC inhibitors exert antineoplastic functions, and their combined action could be exploited for a more effective cancer therapy. To evaluate the potential benefits of this approach, we examined the antineoplastic effects of adenoviral (Ad)-DLC1-mediated transduction and exposure to suberoylanilide hydroxamic acid (SAHA), a powerful HDAC inhibitor, in two human cancer cell lines that lack intrinsic DLC1 expression, 22Rv1 prostate cancer cells and 7703K human hepatocellular carcinoma cells. Consistent with the oncosuppressive function of DLC1 in several cancers, including prostate and liver cancer, transduction of 22Rv1 and 7703K cells with an Ad-DLC1 expression vector resulted in alterations of cell morphology, induction of apoptosis, and inhibition of cell proliferation, migration, and anchorage-independent growth. A low concentration of SAHA (5 microM) efficiently restored the expression of DLC1 in 22Rv1 cells that lack DLC1 expression due to histone deacetylation but had a minimal effect in 7703K cells in which silencing of the DLC1 gene is due mainly to promoter hypermethylation. Regardless of the epigenetic mechanism of DLC1 inactivation, SAHA treatment of DLC1-transduced cells had a synergistic inhibitory effect on tumor cell proliferation and tumorigenesis in both cell lines. In 22Rv1 cells, this combination regimen nearly abolished the formation of colonies in semisolid media as a measure of tumorigenicity in vitro. Current in vitro

  13. Dysregulation of sterol regulatory element binding protein-1c in livers of morbidly obese women is associated with altered suppressor of cytokine signaling-3 and signal transducer and activator of transcription-1 signaling.

    PubMed

    Elam, Marshall B; Yellaturu, Chandrahasa; Howell, George E; Deng, Xiong; Cowan, George S; Kumar, Poonam; Park, Edwards A; Hiler, M Lloyd; Wilcox, Henry G; Hughes, Thomas A; Cook, George A; Raghow, Rajendra

    2010-04-01

    We compared hepatic expression of genes that regulate lipid biosynthesis and metabolic signaling in liver biopsy specimens from women who were undergoing gastric bypass surgery (GBP) for morbid obesity with that in women undergoing ventral hernia repair who had experienced massive weight loss (MWL) after prior GBP. Comprehensive metabolic profiles of morbidly obese (MO) (22 subjects) and MWL (9 subjects) were also compared. Analyses of gene expression in liver biopsies from MO and MWL were accomplished by Affymetrix microarray, real-time polymerase chain reaction, and Western blotting techniques. After GBP, MWL subjects had lost on average 102 lb as compared with MO subjects. This was accompanied by effective reversal of the dyslipidemia and insulin resistance that were present in MO. As compared with MWL, livers of MO subjects exhibited increased expression of sterol regulatory element binding protein (SREBP)-1c and its downstream lipogenic targets, fatty acid synthase and acetyl-coenzyme A-carboxylase-1. Livers of MO subjects also exhibited enhanced expression of suppressor of cytokine signaling-3 protein and attenuated Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling. Consistent with these findings, we found that the human SREBP-1c promoter was positively regulated by insulin and negatively regulated by STAT3. These data support the hypothesis that suppressor of cytokine signaling-3-mediated attenuation of the STAT signaling pathway and resulting enhanced expression of SREBP-1c, a key regulator of de novo lipid biosynthesis, are mechanistically related to the development of hepatic insulin resistance and dyslipidemia in MO women.

  14. RBP1 Recruits the mSIN3-Histone Deacetylase Complex to the Pocket of Retinoblastoma Tumor Suppressor Family Proteins Found in Limited Discrete Regions of the Nucleus at Growth Arrest

    PubMed Central

    Lai, Albert; Kennedy, Brian K.; Barbie, David A.; Bertos, Nicholas R.; Yang, Xiang Jiao; Theberge, Marie-Christine; Tsai, Shih-Chang; Seto, Edward; Zhang, Yi; Kuzmichev, Andrei; Lane, William S.; Reinberg, Danny; Harlow, Ed; Branton, Philip E.

    2001-01-01

    Retinoblastoma (RB) tumor suppressor family pocket proteins induce cell cycle arrest by repressing transcription of E2F-regulated genes through both histone deacetylase (HDAC)-dependent and -independent mechanisms. In this study we have identified a stable complex that accounts for the recruitment of both repression activities to the pocket. One component of this complex is RBP1, a known pocket-binding protein that exhibits both HDAC-dependent and -independent repression functions. RB family proteins were shown to associate via the pocket with previously identified mSIN3-SAP30-HDAC complexes containing exclusively class I HDACs. Such enzymes do not interact directly with RB family proteins but rather utilize RBP1 to target the pocket. This mechanism was shown to account for the majority of RB-associated HDAC activity. We also show that in quiescent normal human cells this entire RBP1-mSIN3-SAP30-HDAC complex colocalizes with both RB family members and E2F4 in a limited number of discrete regions of the nucleus that in other studies have been shown to represent the initial origins of DNA replication following growth stimulation. These results suggest that RB family members, at least in part, drive exit from the cell cycle by recruitment of this HDAC complex via RBP1 to repress transcription from E2F-dependent promoters and possibly to alter chromatin structure at DNA origins. PMID:11283269

  15. Targeting tumor suppressor genes for cancer therapy.

    PubMed

    Liu, Yunhua; Hu, Xiaoxiao; Han, Cecil; Wang, Liana; Zhang, Xinna; He, Xiaoming; Lu, Xiongbin

    2015-12-01

    Cancer drugs are broadly classified into two categories: cytotoxic chemotherapies and targeted therapies that specifically modulate the activity of one or more proteins involved in cancer. Major advances have been achieved in targeted cancer therapies in the past few decades, which is ascribed to the increasing understanding of molecular mechanisms for cancer initiation and progression. Consequently, monoclonal antibodies and small molecules have been developed to interfere with a specific molecular oncogenic target. Targeting gain-of-function mutations, in general, has been productive. However, it has been a major challenge to use standard pharmacologic approaches to target loss-of-function mutations of tumor suppressor genes. Novel approaches, including synthetic lethality and collateral vulnerability screens, are now being developed to target gene defects in p53, PTEN, and BRCA1/2. Here, we review and summarize the recent findings in cancer genomics, drug development, and molecular cancer biology, which show promise in targeting tumor suppressors in cancer therapeutics.

  16. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

    PubMed Central

    Yu, Lan; Shang, Zeng-Fu; Abdisalaam, Salim; Lee, Kyung-Jong; Gupta, Arun; Hsieh, Jer-Tsong; Asaithamby, Aroumougame; Chen, Benjamin P.C.; Saha, Debabrata

    2016-01-01

    Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability. PMID:27568005

  17. The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD-box RNA helicases are nucleolar- and chromocenter-localized proteins that undergo stress-mediated relocalization and are involved in epigenetic gene silencing.

    PubMed

    Khan, Asif; Garbelli, Anna; Grossi, Serena; Florentin, Assa; Batelli, Giorgia; Acuna, Tania; Zolla, Gaston; Kaye, Yuval; Paul, Laju K; Zhu, Jian-Kang; Maga, Giovanni; Grafi, Gideon; Barak, Simon

    2014-07-01

    DEAD-box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD-box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up-regulated stress-responsive gene expression. Here, we show that Arabidopsis STRS-overexpressing lines displayed a less tolerant phenotype and reduced expression of stress-induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP-STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis-localization in specific gene-silencing mutants and exhibited RNA-dependent ATPase and RNA-unwinding activities. In particular, STRS2 showed mis-localization in three out of four mutants of the RNA-directed DNA methylation (RdDM) pathway while STRS1 was mis-localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.

  18. Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells

    PubMed Central

    Tomchuck, Suzanne L.; Henkle, Sarah L.; Coffelt, Seth B.; Betancourt, Aline M.

    2012-01-01

    Background The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to “danger” signals – by-products of damaged, infected or inflamed tissues – via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC. Methodology/Principal Findings We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization. Conclusion/Significance These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals. PMID:22745793

  19. Increased myeloid-derived suppressor cells in gastric cancer correlate with cancer stage and plasma S100A8/A9 proinflammatory proteins.

    PubMed

    Wang, Linda; Chang, Esther W Y; Wong, Siew Cheng; Ong, Siew-Min; Chong, Debra Q Y; Ling, Khoon Lin

    2013-01-15

    Immune dysfunction may contribute to tumor progression in gastric cancer (GC) patients. One mechanism of immune dysfunction is the suppression of T cell activation and impairment of the efficacy of cancer immunotherapy by myeloid-derived suppressor cells (MDSCs). We assessed the phenotype and immunosuppressive function of MDSCs in GC patients. We further investigated the role of S100A8/A9 in GC and the relationship between S100A8/A9 and MDSC function. Lastly, the effect of MDSCs on survival rates and its potential as a prognostic factor in GC patients were investigated. MDSCs from PBMCs of GC patients were identified by comparing the expression of specific surface markers with PBMCs from healthy individuals. The ability of MDSCs to suppress T lymphocyte response and the effect of S100A8/A9 and RAGE blocking were tested in vitro by (autologous) MLR. GC patients had significantly more MDSCs than healthy individuals. These MDSCs suppressed both T lymphocyte proliferation and IFN-γ production and had high arginase-I expression. Levels of S100A8/A9 in plasma were higher in GC patients compared with healthy individuals, and they correlated with MDSC levels in the blood. Blocking of S100A8/A9 itself and the S100A8/A9 receptor RAGE on MDSCs from GC patients abrogated T cell effector function. We found that high levels of MDSCs correlated with more advanced cancer stage and with reduced survival (p = 0.006). S100A8/A9 has been identified as a potential target to modulate antitumor immunity by reversing MDSC-mediated immunosuppression.

  20. Tumor suppressor molecules and methods of use

    DOEpatents

    Welch, Peter J.; Barber, Jack R.

    2004-09-07

    The invention provides substantially pure tumor suppressor nucleic acid molecules and tumor suppressor polypeptides. The invention also provides hairpin ribozymes and antibodies selective for these tumor suppressor molecules. Also provided are methods of detecting a neoplastic cell in a sample using detectable agents specific for the tumor suppressor nucleic acids and polypeptides.

  1. The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF*

    PubMed Central

    Baljuls, Angela; Beck, Matthias; Oenel, Ayla; Robubi, Armin; Kroschewski, Ruth; Hekman, Mirko; Rudel, Thomas; Rapp, Ulf R.

    2012-01-01

    The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity. PMID:22605333

  2. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae.

    PubMed

    Van Rossom, Sofie; Op de Beeck, Ken; Franssens, Vanessa; Swinnen, Erwin; Schepers, Anne; Ghillebert, Ruben; Caldara, Marina; Van Camp, Guy; Winderickx, Joris

    2012-01-01

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2-6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  3. Piperlongumine exerts cytotoxic effects against cancer cells with mutant p53 proteins at least in part by restoring the biological functions of the tumor suppressor.

    PubMed

    Basak, Debasish; Punganuru, Surendra R; Srivenugopal, Kalkunte S

    2016-04-01

    Piperlongumine (PL), a small molecule alkaloid present in black pepper (Piper longum), has been reported to kill tumor cells irrespective of their p53 gene status, however, the mechanisms involved are unknown. Since p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by PL may affect the structure and/or function of the mutant p53 protein and promote cell death. We used two human colon cancer cell lines, the HT29 and SW620 which harbor the R273H DNA contact abrogatory mutation in p53. PL treatment induced significant ROS production and protein glutathionylation with a concomitant increase in Nrf-2 expression in both cell lines. Surprisingly, immunoprecipitation with wt-p53 specific antibodies (PAb1620) or direct western blotting showed a progressive generation of wild-type-like p53 protein along with a loss of its mutant counterpart in PL-treated HT29 and SW620 cells. Moreover, the EMSA and DNA-affinity blotting revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes, MDM2 and Bax. PL, while cytotoxic by itself, also increased the cell killing by many anticancer drugs. In nude mice bearing the HT29 tumors, PL alone (7.5 mg/kg daily) produced a 40% decrease in tumor volume, which was accompanied by diminished intratumoral mutant p53 protein levels. The antitumor efficacy of BCNU or doxorubicin in HT29 xenografts was highly potentiated by PL, followed by expression of apoptotic proteins. These clinically-relevant findings suggest that PL-induced oxidative milieu facilitates a weak functional restoration of mutant p53 through protein glutathionylation and contributes to the increased drug sensitivity.

  4. The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects.

    PubMed Central

    Craven, R J; Petes, T D

    2001-01-01

    Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family. Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing. We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p. We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis. Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells. Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p. PMID:11333225

  5. Suppressor of hairy‐wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi

    PubMed Central

    Carballar‐Lejarazú, R.; Brennock, P.

    2016-01-01

    Abstract DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy‐like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric‐a‐brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood‐fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes. PMID:27110891

  6. The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

    SciTech Connect

    Dougherty, Gerard W.; Chopp, Treasa; Qi Shengmei; Cutler, Mary Lou . E-mail: mcutler@usuhs.mil

    2005-05-15

    Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.

  7. The fold recognition of CP2 transcription factors gives new insights into the function and evolution of tumor suppressor protein p53.

    PubMed

    Kokoszynska, Katarzyna; Ostrowski, Jerzy; Rychlewski, Leszek; Wyrwicz, Lucjan S

    2008-09-15

    The CP2 transcription factor (TFCP2) is a critical regulator of erythroid gene expression. Apart from the involvement in the transcriptional switch of globin gene promoters it activates an array of cellular and viral gene promoters. A number of homologous proteins was identified in genomes of Metazoa, with additional five homologues encoded by the human genome (TFCP2L1, UBP1, GRHL1, GRHL2, GRHL3). Although several experimental studies have already been published, the knowledge on the molecular mechanism of activity of this transcription factors remains very limited. Here we present the application of fold recognition and protein structure prediction in drafting the structure-to-function relationship of the CP2 family. The employed procedure clearly shows that the family adopts a DNA binding immunoglobulin fold homologous to the p53 (TP53) core domain, and a novel type of ubiquitin-like domain and a sterile alpha motif (SAM) form oligomerization modules. With a traceable evolution of CP2 family throughout the Metazoa group this protein family is highly likely to represent an ancestor of the critical cell cycle regulator p53. Based on this observation several functional hypotheses on structure-to-function relationship of p53 were drawn. The DNA motif recognized by p53 is a result of further specialization of the CP2 DNA-binding module. The analysis also shows the critical role of protein oligomerization for the function of this protein superfamily. Finally, the identification of distant homologs of TP53 allowed performing a phylogenetic footprinting analysis explaining the role of the specific amino acids important for both - the protein folding and the binding of DNA.

  8. Novel tumor-suppressor gene epidermal growth factor-containing fibulin-like extracellular matrix protein 1 is epigenetically silenced and associated with invasion and metastasis in human gastric cancer.

    PubMed

    Zhu, Xin-Jiang; Liu, Jian; Xu, Xiao-Yang; Zhang, Chun-Dong; Dai, Dong-Qiu

    2014-06-01

    The present study aimed to investigate the role of histone modification and DNA methylation in epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) silencing in gastric cancer (GC). In the present study, four GC cell lines, and 45 paired normal and GC tissue samples were used to assess EFEMP1 expression using quantitative polymerase chain reaction (PCR), and EFEMP1 gene methylation status was evaluated by methylation-specific PCR. The involvement of histone modification in GC cell lines was examined by a chromatin immunoprecipitation (ChIP) assay. The results demonstrated that EFEMP1 mRNA level and methylation status in the EFEMP1 promoter region was associated with tumor differentiation, depth of tumor invasion and lymph node metastasis. DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) rapidly reduced DNA methylation and histone H3-K9 trimethylation at the silenced loci and reactivated EFEMP1 expression. By contrast, the histone deacetylase inhibitor trichostatin A markedly increased histone H3-K9 acetylation. However, it had no effect on DNA methylation, histone H3-K9 trimethylation or gene expression. In conclusion, the results suggested that EFEMP1 may function as a tumor suppressor in GC. Aberrant DNA methylation and histone H3-K9 trimethylation of EFEMP1 may be responsible for its downregulation in GC, and thus have an important role in tumor invasion and metastasis.

  9. Tumor suppressor von Hippel-Lindau (VHL) stabilization of Jade-1 protein occurs through plant homeodomains and is VHL mutation dependent.

    PubMed

    Zhou, Mina I; Wang, Hongmei; Foy, Rebecca L; Ross, Jonathan J; Cohen, Herbert T

    2004-02-15

    The von Hippel-Lindau (VHL) gene is the major renal cancer gene in adults. The mechanism of renal tumor suppression by VHL protein is only partly elucidated. VHL loss increases expression of the hypoxia-inducible factor alpha transcription factors. However, clinical and biochemical data indicate that the hypoxia-inducible factors are necessary but not sufficient for renal tumorigenesis, which suggests other VHL effector pathways are involved. Jade-1 protein interacts strongly with VHL and is most highly expressed in renal proximal tubules, precursor cells of renal cancer. Short-lived Jade-1 protein contains plant homeodomain (PHD) and candidate PEST degradation motifs and is substantially stabilized by VHL. The effect of VHL on Jade-1 protein abundance and relative protein stability was further examined in immunoblots and metabolic labeling experiments using two time points. VHL-Jade-1 binding was tested in coimmunoprecipitations. In cotransfection studies with wild-type VHL, the Jade-1 PHD-extended PHD module, not the candidate PEST domain, was required for full VHL-mediated stabilization. This module is also found in leukemia transcription factors AF10 and AF17, as well as closely related Jade-like proteins, which suggests all might be VHL regulated. Intriguingly, naturally occurring truncations and mutations of VHL affected wild-type Jade-1 binding and stabilization. Although the VHL beta domain was sufficient for Jade-1 binding, both the alpha and beta domains were required for Jade-1 stabilization. Thus, truncating VHL mutations, which are severe and associated with renal cancer development, prevented Jade-1 stabilization. Moreover, well-controlled cotransfection and metabolic labeling experiments revealed that VHL missense mutations that cause VHL disease without renal cancer, such as Tyr98His and Tyr112His, stabilized Jade-1 fully. In contrast, like the VHL truncations, VHL missense mutations commonly associated with renal cancer, such as Leu118Pro or Arg167

  10. "Ring-fencing" BRCA1 tumor suppressor activity.

    PubMed

    Patel, Ketan J; Crossan, Gerry P; Hodskinson, Michael R G

    2011-12-13

    BRCA1 is a crucial human breast and ovarian cancer tumor suppressor gene. The article by Drost et al. in this issue of Cancer Cell together with a recent paper in Science now provide a clearer picture of how this large and complex protein suppresses tumorigenesis.

  11. Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways

    PubMed Central

    Huang, Wei-Ru; Chiu, Hung-Chuan; Liao, Tsai-Ling; Chuang, Kuo-Pin; Shih, Wing-Ling; Liu, Hung-Jen

    2015-01-01

    Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication. PMID:26244501

  12. [The relationship of cytoplasmic and mitochondrial protein synthesis in yeasts: the mapping of mitochondrial mutations neutralizing the exhibition of nuclear omnipotent suppressors].

    PubMed

    Mironova, L N; Zelenaia, O A; Sudarikov, A B; Ter-Avanesian, M D

    1988-06-01

    The genetic and physical mapping of mitochondrial mutations [CRD] neutralizing respiratory deficiency in sup1 and sup2 mutants was performed. The genetical methods demonstrated improbability of location of these mutations in genes coding for the enzymes of respiratory chain and for 21S rRNA. Southern-blot analysis has shown these mutations to be localized in the Hinc10 fragment of mitochondrial genome. This fact was interpreted as indication that var1 gene is affected by [CRD] mutations. Our results are in agreement with the hypothesis on the participation of sup1(2) proteins in mitochondrial translation.

  13. Tumor suppressor p53 induces miR-15a processing to inhibit neuronal apoptosis inhibitory protein (NAIP) in the apoptotic response DNA damage in breast cancer cell

    PubMed Central

    Yang, Li; Zhao, Wei; Wei, Ping; Zuo, Wenshu; Zhu, Shouhui

    2017-01-01

    This study was aimed to investigate the functional role of miR-15a in breast cancer cells in response to DNA damage and to illustrate the possible potential underlying molecular mechanism(s). Human breast cancer cell lines MCF-7 cells and/or MDA-MB-231 cells were pre-treated with or without bleomycin. Cells were transfected with corresponding vectors. qRT-PCR was used to detect the expression of mRNA or miRNA, and immunoprecipitation and immunoblot analysis were performed to explore the status of protein association. Cell apoptosis was analyzed with flow cytometry. The results showed that neuronal apoptosis inhibitory protein (NAIP) was negatively regulated by p53 in MCF-7 cells, and NAIP expression was still high in bleomycin-treated MCF-7 cells. In addition, we observed that miR-15a expression was regulated by p53, and the effects of miR-15a on DNA damage was also mediated by p53. Furthermore, the results revealed that the cell apoptosis was mediated by miR-15a. Taken together, this study reveals that p53 negatively regulates NAIP expression by targeting miR-15a processing from primary into precursor miRNA in breast cancer. PMID:28337296

  14. Shear-stress-mediated refolding of proteins from aggregates and inclusion bodies.

    PubMed

    Yuan, Tom Z; Ormonde, Callum F G; Kudlacek, Stephan T; Kunche, Sameeran; Smith, Joshua N; Brown, William A; Pugliese, Kaitlin M; Olsen, Tivoli J; Iftikhar, Mariam; Raston, Colin L; Weiss, Gregory A

    2015-02-09

    Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.

  15. Oomycete pathogens encode RNA silencing suppressors.

    PubMed

    Qiao, Yongli; Liu, Lin; Xiong, Qin; Flores, Cristina; Wong, James; Shi, Jinxia; Wang, Xianbing; Liu, Xigang; Xiang, Qijun; Jiang, Shushu; Zhang, Fuchun; Wang, Yuanchao; Judelson, Howard S; Chen, Xuemei; Ma, Wenbo

    2013-03-01

    Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

  16. Characterization of DNA binding property of the HIV-1 host factor and tumor suppressor protein Integrase Interactor 1 (INI1/hSNF5).

    PubMed

    Das, Supratik; Banerjee, Baisakhi; Hossain, Maidul; Thangamuniyandi, Muruganandan; Dasgupta, Saumya; Chongdar, Nipa; Kumar, Gopinatha Suresh; Basu, Gautam

    2013-01-01

    Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105-183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and Kd  = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K'd1 = 222 µM and K'd2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = -29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH'1 = 115.7 KJ/mole, TΔS'1 = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH'2 = -106.3 KJ/mole, TΔS'2 = -75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1.

  17. pH-sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

    PubMed Central

    Law, Sean M; Zhang, Bin W; Brooks, Charles L

    2013-01-01

    Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short-interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH-dependent residues that affect CIRV p19–siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH-dependent binding profile. PMID:23450521

  18. pH-sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability.

    PubMed

    Law, Sean M; Zhang, Bin W; Brooks, Charles L

    2013-05-01

    Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short-interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH-dependent residues that affect CIRV p19-siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH-dependent binding profile.

  19. Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells.

    PubMed

    Lee, Yean-Jang; Kuo, Hsing-Chun; Chu, Chia-Yih; Wang, Chau-Jong; Lin, Wan-Chyi; Tseng, Tsui-Hwa

    2003-12-15

    Caffeic acid phenethyl ester (CAPE), an active component of propolis, has many biological and pharmacological activities including antioxidant, anti-inflammation, antiviral action, and anticancer effect. Our previous studies showed that CAPE exhibited significant cytotoxicity in oral cancer cells. Herein we further investigated the cytotoxicity potential of CAPE and the mechanism of its action in C6 glioma cells. The data exhibited that C6 glioma cells underwent internucleosomal DNA fragmentation 24 hr after the treatment of CAPE (50 microM). The proportion of C6 glioma cells with hypodiploid nuclei was increased to 24% at 36 hr after the exposure. Further results showed that CAPE induced the release of cytochrome c from mitochondria into cytosol, and the activation of CPP32. CAPE application also enhanced the expression of p53, Bax, and Bak. Finally, the potential signaling components underlying CAPE induction of apoptosis were elucidated. We found that CAPE activated extracellular signal-regulated kinase (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in C6 glioma cells. More importantly, p38 kinase formed a complex with p53 after the treatment of CAPE for 0.5 hr. The expression of p53, phospho-serine 15 of p53, and Bax, and inactivate form of CPP32 was suppressed by a pretreatment of a specific p38 MAPK inhibitor, SB203580. The resultant data suggest that p38 MAPK mediated the CAPE-induced p53-dependent apoptosis in C6 glioma cells.

  20. Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition.

    PubMed

    Lentine, Brandon; Antonucci, Lisa; Hunce, Ray; Edwards, Justina; Marallano, Valerie; Krucher, Nancy A

    2012-09-01

    The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G 1- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be that dephosphorylation of specific amino acids signals apoptosis vs. cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants of Rb to alter serine/threonine to glutamic acid to mimic the phosphorylated state. By calcium phosphate transfection, mutant plasmids were introduced into C33A Rb-null cells, and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. Our results show that only mutation of threonine-821 to glutamic acid (T821E) blocked apoptosis by 50%, whereas other sites tested had little effect. In Rb-null Saos-2 and SKUT-1 cells, the T821E mutation also blocked apoptosis induced by the cdk inhibitor, Roscovitine, by 50%. In addition, we show that endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Thus, our data indicates that dephosphorylation of threonine-821 of Rb is required for cells to undergo apoptosis.

  1. The Establishment of a Hyperactive Structure Allows the Tumour Suppressor Protein p53 to Function through P-TEFb during Limited CDK9 Kinase Inhibition

    PubMed Central

    Albert, Thomas K.; Antrecht, Claudia; Kremmer, Elisabeth; Meisterernst, Michael

    2016-01-01

    CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed—with significant delay—by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription. PMID:26745862

  2. miRNA-411 acts as a potential tumor suppressor miRNA via the downregulation of specificity protein 1 in breast cancer.

    PubMed

    Guo, Liangfeng; Yuan, Jianhui; Xie, Ni; Wu, Huisheng; Chen, Weicai; Song, Shufen; Wang, Xianming

    2016-10-01

    The expression and functions of microRNA (miR)-411 have been investigated in several types of cancer. However, until now, miR-411 in human breast cancer has not been examined. The present study investigated the expression, biological functions and molecular mechanisms of miR‑411 in human breast cancer, discussing whether it offers potential as a therapeutic biomarker for breast cancer in the future. The expression levels of miR‑411 in human breast cancer tissues and cells were measured using reverse transcription‑quantitative polymerase chain reaction analysis. Following transfection with miR‑411 mimics, an MTT assay, cell migration and invasion assay, western blot analysis and luciferase assay were performed in human breast cancer cell lines. According to the results, it was found that miR‑411 was significantly downregulated in breast cancer, and associated with lymph node metastasis and histological grade. Additionally, it was observed that miR‑411 suppressed cell growth, migration and invasion in the breast cancer cells. The present study also provided the first evidence, to the best of our knowledge, that miR‑411 was likely to directly target specificity protein 1 in breast cancer. These findings indicated that miR‑411 may be used a therapeutic biomarker for the treatment of breast cancer in the future.

  3. miRNA-411 acts as a potential tumor suppressor miRNA via the downregulation of specificity protein 1 in breast cancer

    PubMed Central

    Guo, Liangfeng; Yuan, Jianhui; Xie, Ni; Wu, Huisheng; Chen, Weicai; Song, Shufen; Wang, Xianming

    2016-01-01

    The expression and functions of microRNA (miR)-411 have been investigated in several types of cancer. However, until now, miR-411 in human breast cancer has not been examined. The present study investigated the expression, biological functions and molecular mechanisms of miR-411 in human breast cancer, discussing whether it offers potential as a therapeutic biomarker for breast cancer in the future. The expression levels of miR-411 in human breast cancer tissues and cells were measured using reverse transcription-quantitative polymerase chain reaction analysis. Following transfection with miR-411 mimics, an MTT assay, cell migration and invasion assay, western blot analysis and luciferase assay were performed in human breast cancer cell lines. According to the results, it was found that miR-411 was significantly downregulated in breast cancer, and associated with lymph node metastasis and histological grade. Additionally, it was observed that miR-411 suppressed cell growth, migration and invasion in the breast cancer cells. The present study also provided the first evidence, to the best of our knowledge, that miR-411 was likely to directly target specificity protein 1 in breast cancer. These findings indicated that miR-411 may be used a therapeutic biomarker for the treatment of breast cancer in the future. PMID:27572271

  4. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA)

    PubMed Central

    Nicol, Alcina F; Pires, Andréa Rodrigues Cordovil; de Souza, Simone R; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; e Silva, José R Lapa; Pirmez, Claude

    2008-01-01

    Background The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. Methods We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. Results HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Conclusion Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development. PMID:18840277

  5. Arcuate nucleus transcriptome profiling identifies ankyrin repeat and suppressor of cytokine signalling box-containing protein 4 as a gene regulated by fasting in central nervous system feeding circuits.

    PubMed

    Li, J-Y; Kuick, R; Thompson, R C; Misek, D E; Lai, Y-M; Liu, Y-Q; Chai, B-X; Hanash, S M; Gantz, I

    2005-06-01

    The arcuate nucleus of the hypothalamus is a primary site for sensing blood borne nutrients and hormonal messengers that reflect caloric status. To identify novel energy homeostatic genes, we examined RNA extracts from the microdissected arcuate nucleus of fed and 48-h fasted rats using oligonucleotide microarrays. The relative abundance of 118 mRNA transcripts was increased and 203 mRNA transcripts was decreased during fasting. One of the down-regulated mRNAs was ankyrin-repeat and suppressor of cytokine signalling box-containing protein 4 (Asb-4). The predicted structure of Asb-4 protein suggested that it might encode an intracellular regulatory protein, and therefore its mRNA expression was investigated further. Reverse transcription quantitative polymerase chain reaction was used to validate down-regulation of Asb-4 mRNA in the arcuate nucleus of the fasted Sprague-Dawley rat (relative expression of Asb-4 mRNA: fed = 4.66 +/- 0.26; fasted = 3.96 +/- 0.23; n = 4, P < 0.01). Down-regulation was also demonstrated in the obese fa/fa Zucker rat, another model of energy disequilibrium (relative expression of Asb-4 mRNA: lean Zucker = 3.91 +/- 0.32; fa/fa = 2.93 +/- 0.26; n = 5, P < 0.001). In situ hybridisation shows that Asb-4 mRNA is expressed in brain areas linked to energy homeostasis, including the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, lateral hypothalamus and posterodorsal medial amygdaloid area. Double in situ hybridisation revealed that Asb-4 mRNA colocalises with key energy homeostatic neurones. In the fed state, Asb-4 mRNA is expressed by 95.6% of pro-opiomelanocortin (POMC) neurones and 46.4% of neuropeptide Y (NPY) neurones. By contrast, in the fasted state, the percentage of POMC neurones expressing Asb-4 mRNA drops to 73.2% (P < 0.001). Moreover, the density of Asb-4 mRNA per fasted POMC neurone is markedly decreased. Conversely, expression of Asb-4 mRNA by NPY neurones in the fasted state is modestly increased to 52.7% (P < 0

  6. Therapeutic targeting of tumor suppressor genes.

    PubMed

    Morris, Luc G T; Chan, Timothy A

    2015-05-01

    Carcinogenesis is a multistep process attributable to both gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes. Currently, most molecular targeted therapies are inhibitors of oncogenes, because inactivated tumor suppressor genes have proven harder to "drug." Nevertheless, in cancers, tumor suppressor genes undergo alteration more frequently than do oncogenes. In recent years, several promising strategies directed at tumor suppressor genes, or the pathways controlled by these genes, have emerged. Here, we describe advances in a number of different methodologies aimed at therapeutically targeting tumors driven by inactivated tumor suppressor genes.

  7. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    SciTech Connect

    Kumari, Gita; Mahalingam, S.

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  8. Control of autophagy by oncogenes and tumor suppressor genes.

    PubMed

    Maiuri, M C; Tasdemir, E; Criollo, A; Morselli, E; Vicencio, J M; Carnuccio, R; Kroemer, G

    2009-01-01

    Multiple oncogenes (in particular phosphatidylinositol 3-kinase, PI3K; activated Akt1; antiapoptotic proteins from the Bcl-2 family) inhibit autophagy. Similarly, several tumor suppressor proteins (such as BH3-only proteins; death-associated protein kinase-1, DAPK1; the phosphatase that antagonizes PI3K, PTEN; tuberous sclerosic complex 1 and 2, TSC1 and TSC2; as well as LKB1/STK11) induce autophagy, meaning that their loss reduces autophagy. Beclin-1, which is required for autophagy induction acts as a haploinsufficient tumor suppressor protein, and other essential autophagy mediators (such as Atg4c, UVRAG and Bif-1) are bona fide oncosuppressors. One of the central tumor suppressor proteins, p53 exerts an ambiguous function in the regulation of autophagy. Within the nucleus, p53 can act as an autophagy-inducing transcription factor. Within the cytoplasm, p53 exerts a tonic autophagy-inhibitory function, and its degradation is actually required for the induction of autophagy. The role of autophagy in oncogenesis and anticancer therapy is contradictory. Chronic suppression of autophagy may stimulate oncogenesis. However, once a tumor is formed, autophagy inhibition may be a therapeutic goal for radiosensitization and chemosensitization. Altogether, the current state-of-the art suggests a complex relationship between cancer and deregulated autophagy that must be disentangled by further in-depth investigation.

  9. Electrodialytic membrane suppressors for ion chromatography make programmable buffer generators.

    PubMed

    Chen, Yongjing; Srinivasan, Kannan; Dasgupta, Purnendu K

    2012-01-03

    The use of buffer solutions is immensely important in a great variety of disciplines. The generation of continuous pH gradients in flow systems plays an important role in the chromatographic separation of proteins, high-throughput pK(a) determinations, etc. We demonstrate here that electrodialytic membrane suppressors used in ion chromatography can be used to generate buffers. The generated pH, computed from first principles, agrees well with measured values. We demonstrate the generation of phosphate and citrate buffers using a cation-exchange membrane (CEM) -based anion suppressor and Tris and ethylenediamine buffers using an anion-exchange membrane (AEM) -based cation suppressor. Using a mixture of phosphate, citrate, and borate as the buffering ions and using a CEM suppressor, we demonstrate the generation of a highly reproducible (avg RSD 0.20%, n = 3), temporally linear (pH 3.0-11.9, r(2) > 0.9996), electrically controlled pH gradient. With butylamine and a large concentration (0.5 M) of added NaCl, we demonstrate a similar linear pH gradient of large range with a near-constant ionic strength. We believe that this approach will be of value for the generation of eluents in the separation of proteins and other biomolecules and in online process titrations.

  10. viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence.

    PubMed

    Csorba, Tibor; Kontra, Levente; Burgyán, József

    2015-05-01

    RNA silencing is a homology-dependent gene inactivation mechanism that regulates a wide range of biological processes including antiviral defense. To deal with host antiviral responses viruses evolved mechanisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Besides working as silencing suppressors, these proteins may also fulfill other functions during infection. In many cases the interplay between the suppressor function and other "unrelated" functions remains elusive. We will present host factors implicated in antiviral pathways and summarize the current status of knowledge about the diverse viral suppressors' strategies acting at various steps of antiviral silencing in plants. Besides, we will consider the multi-functionality of these versatile proteins and related biochemical processes in which they may be involved in fine-tuning the plant-virus interaction. Finally, we will present the current applications and discuss perspectives of the use of these proteins in molecular biology and biotechnology.

  11. Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon.

    PubMed

    Pekow, Joel; Meckel, Katherine; Dougherty, Urszula; Butun, Fatma; Mustafi, Reba; Lim, John; Crofton, Charis; Chen, Xindi; Joseph, Loren; Bissonnette, Marc

    2015-02-01

    The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.

  12. Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae.

    PubMed

    Wakem, L P; Sherman, F

    1990-03-01

    Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness. Genetic analysis of 12 representative suppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP35 (chromosome IV), SUP45 (chromosome II) and SUP46 (chromosome II), as well as three new loci SUP42 (chromosome IV), SUP43 (chromosome XV) and SUP44 (chromosome VII). Suppressors belonging to the same locus had a wide range of different phenotypes. Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressors encode proteins that effect different functions and that altered forms of each of the proteins can effect the same function.

  13. A genome wide dosage suppressor network reveals genomic robustness

    PubMed Central

    Patra, Biranchi; Kon, Yoshiko; Yadav, Gitanjali; Sevold, Anthony W.; Frumkin, Jesse P.; Vallabhajosyula, Ravishankar R.; Hintze, Arend; Østman, Bjørn; Schossau, Jory; Bhan, Ashish; Marzolf, Bruz; Tamashiro, Jenna K.; Kaur, Amardeep; Baliga, Nitin S.; Grayhack, Elizabeth J.; Adami, Christoph; Galas, David J.; Raval, Alpan; Phizicky, Eric M.; Ray, Animesh

    2017-01-01

    Genomic robustness is the extent to which an organism has evolved to withstand the effects of deleterious mutations. We explored the extent of genomic robustness in budding yeast by genome wide dosage suppressor analysis of 53 conditional lethal mutations in cell division cycle and RNA synthesis related genes, revealing 660 suppressor interactions of which 642 are novel. This collection has several distinctive features, including high co-occurrence of mutant-suppressor pairs within protein modules, highly correlated functions between the pairs and higher diversity of functions among the co-suppressors than previously observed. Dosage suppression of essential genes encoding RNA polymerase subunits and chromosome cohesion complex suggests a surprising degree of functional plasticity of macromolecular complexes, and the existence of numerous degenerate pathways for circumventing the effects of potentially lethal mutations. These results imply that organisms and cancer are likely able to exploit the genomic robustness properties, due the persistence of cryptic gene and pathway functions, to generate variation and adapt to selective pressures. PMID:27899637

  14. Interaction between Nm23 and the tumor suppressor VHL.

    PubMed

    Lin, Chih-Hung; Dammai, Vincent; Adryan, Boris; Hsu, Tien

    2015-02-01

    Among the anti-tumor genes (tumor suppressors and metastasis suppressors), the von-Hippel Lindau gene and the Nm23 family of genes are among the more intriguing ones. Both are small (long and short forms of VHL are 30 and 19 kD, respectively, and Nm23 is ~17 kD), and both possess diverse molecular and cellular functions. Despite extensive studies, the entire spectra of functions and the molecular function-phenotype correlation of these two proteins have not been completely elucidated. In this report, we present data showing these two proteins interact physically. We also summarize and confirm the previous studies that demonstrated the endocytic function of these two genes and further show that the endocytic function of VHL is mediated through the activity of Nm23. These functional and molecular interactions are evolutionarily conserved from Drosophila to human.

  15. Noise suppressor for turbo fan jet engines

    NASA Technical Reports Server (NTRS)

    Cheng, D. Y. (Inventor)

    1983-01-01

    A noise suppressor is disclosed for installation on the discharge or aft end of a turbo fan engine. Within the suppressor are fixed annular airfoils which are positioned to reduce the relative velocity between the high temperature fast moving jet exhaust and the low temperature slow moving air surrounding it. Within the suppressor nacelle is an exhaust jet nozzle which constrains the shape of the jet exhaust to a substantially uniform elongate shape irrespective of the power setting of the engine. Fixed ring airfoils within the suppressor nacelle therefore have the same salutary effects irrespective of the power setting at which the engine is operated.

  16. Genetic and Molecular Analysis of Suppressors of Ras Mutations

    DTIC Science & Technology

    1999-10-01

    mediated signal transduction pathway during C. elegans vulval development. Mutations in sur-8 were identified as suppressors of an activated let-60...positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function...Protein Phosphatase 2A (PP2A) and also positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction

  17. Suppressors made from intermetallic materials

    SciTech Connect

    Klett, James W; Muth, Thomas R; Cler, Dan L

    2014-11-04

    Disclosed are several examples of apparatuses for suppressing the blast and flash produced as a projectile is expelled by gases from a firearm. In some examples, gases are diverted away from the central chamber to an expansion chamber by baffles. The gases are absorbed by the expansion chamber and desorbed slowly, thus decreasing pressure and increasing residence time of the gases. In other examples, the gases impinge against a plurality of rods before expanding through passages between the rods to decrease the pressure and increase the residence time of the gases. These and other exemplary suppressors are made from an intermetallic material composition for enhanced strength and oxidation resistance at high operational temperatures.

  18. Myeloid-derived suppressor cells

    PubMed Central

    Chandra, Dinesh; Gravekamp, Claudia

    2013-01-01

    While conventional anticancer therapies, including surgical resection, radiotherapy, and/or chemotherapy, are relatively efficient at eliminating primary tumors, these treatment modalities are largely ineffective against metastases. At least in part, this reflects the rather inefficient delivery of conventional anticancer agents to metastatic lesions. We have recently demonstrated that myeloid-derived suppressor cells (MDSCs) can be used as cellular missiles to selectively deliver a radioisotope-coupled attenuated variant of Listeria monocytogenes to both primary and metastatic neoplastic lesions in mice with pancreatic cancer. This novel immunotherapeutic intervention robustly inhibited tumor growth while promoting a dramatic decrease in the number of metastases. PMID:24427545

  19. Nuclear omnipotent suppressors of premature termination codons in mitochondrial genes affect the 37S mitoribosomal subunit.

    PubMed

    Boguta, M; Mieszczak, M; Zagórski, W

    1988-02-01

    nam3 and R705, yeast nuclear omnipotent suppressors of mitochondrial mit- mutations, reverse the superimposed spectrum of trans-recessive splicing defects by affecting the protein composition of the small mitoribosomal subunit. Analysis of the suppressor's interaction suggests that suppression results from mutations in the mitoribosomal polypeptides. These data indicate an obligatory connection between mitoribosome function and splicing of introns bI2, bI4 and aI1 in yeast mitochondria.

  20. Reversions to respiratory competence of omnipotent sup45 suppressor mutants may be caused by secondary sup45 mutations.

    PubMed

    Mironova, L N; Samsonova, M G; Zhouravleva, G A; Kulikov, V N; Soom, M J

    1995-02-01

    The molecular nature of the sup45 respiratory deficient omnipotent suppressor, and of three reversions to respiratory competence which removed the suppressor effect of the initial mutation, was examined. All reversions were caused by secondary sup45 mutations which indicates a direct connection between sup45 "respiratory" and "translational" functions. Computer analysis showed the local changes of Sup45 protein characteristics in the suppressor strain and revertants in comparison to the wild-type protein. The distribution of mutant sites in relation to evolutionary conserved, and tentatively functional, regions in the Sup45 protein is discussed.

  1. Regulation of herpes simplex virus-specific cell-mediated immunity by a specific suppressor factor.

    PubMed Central

    Horohov, D W; Wyckoff, J H; Moore, R N; Rouse, B T

    1986-01-01

    Our study was designed to investigate the nature of an antigen-specific suppressor factor generated by antigen-stimulated herpes simplex virus (HSV)-immune splenocytes. Factor SF-200, a 90,000- to 100,000-dalton fraction obtained after Sephacryl gel filtration, suppressed the generation of HSV-specific cytotoxic T-lymphocyte and lymphoproliferative responses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of SF-200 indicated that it contained an I-J+, anti-idiotypic protein. It was possible to adsorb the suppressor activity of SF-200 to an anti-I-J immunoaffinity column. The suppressor activity could be eluted from the immunoaffinity column with a low-pH buffer. The acid-eluted material was determined to be both I-J+ and reactive with anti-HSV antiserum by Western blot analysis. Both SF-200 and the I-J+ suppressor activity suppressed only HSV-specific cell-mediated immunity responses. However, it was possible to generate nonspecific suppressor activity by incubating the I-J+ suppressor factor with Lyt 1+ splenocytes from HSV-immune mice. The implication of these results with respect to the model for a suppressor cell circuit regulating HSV-specific cell-mediated immunity responses is discussed. Images PMID:3009850

  2. Identification of phosphatidylcholine transfer protein-like in the parasite Entamoeba histolytica.

    PubMed

    Piña-Vázquez, Carolina; Reyes-López, Magda; Mendoza-Hernández, Guillermo; Bermúdez-Cruz, Rosa María; de la Garza, Mireya

    2014-12-01

    Caveolin is the protein marker of caveola-mediated endocytosis. Previously, we demonstrated by immunoblotting and immunofluorescence that an anti-chick embryo caveolin-1 monoclonal antibody (mAb) recognizes a protein in amoeba extracts. Nevertheless, the caveolin-1 gene is absent in the Entamoeba histolytica genome database. In this work, the goal was to isolate, identify and characterize the protein that cross-reacts with chick embryo caveolin-1. We identified the protein using a proteomic approach, and the complete gene was cloned and sequenced. The identified protein, E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L), is a member of the StAR-related lipid transfer (START) protein superfamily. The human homolog binds and transfers phosphatidylcholine (PC) and phosphatidylethanolamine (PE) between model membranes in vitro; however, the physiological role of PCTP-L remains elusive. Studies in silico showed that EhPCTP-L has a central START domain and also contains a C-terminal intrinsically disordered region. The anti-rEhPCTP-L antibody demonstrated that EhPCTP-L is found in the plasma membrane and cytosol, which is in agreement with previous reports on the human counterpart. This result points to the plasma membrane as one possible target membrane for EhPCTP-L. Furthermore, assays using filipin and nystatin showed down regulation of EhPCTP-L, in an apparently cholesterol-independent way. Interestingly, EhPCTP-L binds primarily to anionic phospholipids phosphatidylserine (PS) and phosphatidic acid (PA), while its mammalian counterpart HsPCTP-L binds neutral phospholipids PC and PE. The present study provides information that helps reveal the possible function and regulation of PCTP-L expression in the primitive eukaryotic parasite E. histolytica.

  3. Suppressor Effects of Coping Strategies on Resilience

    ERIC Educational Resources Information Center

    Yoon, Jae ho; Lee, Ji hae; Lee, Chae Yeon; Cho, Minhee; Lee, Sang Min

    2014-01-01

    The purpose of the current study is to demonstrate a significant suppressor effect among coping strategies on resilience. Two different samples were used to replicate the suppressor effect. Participants in the first example were 391 adolescents (middle school students) in Korea, and participants in the second example were 282 young adults…

  4. Discovery of Tumor Suppressor Gene Function.

    ERIC Educational Resources Information Center

    Oppenheimer, Steven B.

    1995-01-01

    This is an update of a 1991 review on tumor suppressor genes written at a time when understanding of how the genes work was limited. A recent major breakthrough in the understanding of the function of tumor suppressor genes is discussed. (LZ)

  5. Two Replicable Suppressor Situations in Personality Research

    ERIC Educational Resources Information Center

    Paulhus, Delroy L.; Robins, Richard W.; Trzesniewski, Kali H.; Tracy, Jessica L.

    2004-01-01

    Suppressor situations occur when the simultaneous inclusion of two predictors improves one or both validities. A common allegation is that suppressor effects rarely replicate and have little substantive import. We present substantive examples from two established research domains to counter this skepticism. In the first domain, we show how…

  6. Properties of the SIRS suppressor pathway.

    PubMed

    Aune, T M; Pierce, C W

    1983-01-01

    The SIRS suppressor pathway is initiated by activation of Ly 2+ T lymphocytes by either con A or IFN beta. SIRS is a protein which has been purified and exists as two species with mol. wts. of 14,000 and 21,500. The target of SIRS is the macrophage and macrophages appear to oxidize or activate SIRS in a peroxide dependent process. Catalase blocks SIRS or IFN beta action by consuming H2O2 and levamisole blocks SIRS or IFN beta by preventing activation or oxidation of SIRS by H2O2. Other agents which block SIRS or IFN beta action include electron donors which can inactivate SIRSox. SIRSox is a potent inhibitor of immune responses and proliferation of normal and neoplastic cells. The mechanism of SIRSox-mediated inhibition of proliferation appears to involve oxidation or modification of protein sulfhydryls. Although the applicability of this pathway to the regulation of immune responses and cellular proliferation remains to be determined, both IFN beta and levamisole have been found to affect a wide variety of cellular processes. The involvement of both IFN beta and levamisole in the SIRS pathway suggests that this pathway may be an important host mechanism for regulating both immune responses and cellular proliferation in general.

  7. Targeted deletion of Wwox reveals a tumor suppressor function.

    PubMed

    Aqeilan, Rami I; Trapasso, Francesco; Hussain, Sadiq; Costinean, Stefan; Marshall, Dean; Pekarsky, Yuri; Hagan, John P; Zanesi, Nicola; Kaou, Mohamed; Stein, Gary S; Lian, Jane B; Croce, Carlo M

    2007-03-06

    The WW domain-containing oxidoreductase (WWOX) spans the second most common fragile site of the human genome, FRA16D, located at 16q23, and its expression is altered in several types of human cancer. We have previously shown that restoration of WWOX expression in cancer cells suppresses tumorigenicity. To investigate WWOX tumor suppressor function in vivo, we generated mice carrying a targeted deletion of the Wwox gene and monitored incidence of tumor formation. Osteosarcomas in juvenile Wwox(-/-) and lung papillary carcinoma in adult Wwox(+/-) mice occurred spontaneously. In addition, Wwox(+/-) mice develop significantly more ethyl nitrosourea-induced lung tumors and lymphomas in comparison to wild-type littermate mice. Intriguingly, these tumors still express Wwox protein, suggesting haploinsuffiency of WWOX itself is cancer predisposing. These results indicate that WWOX is a bona fide tumor suppressor.

  8. The hematopoietic tumor suppressor interferon regulatory factor 8 (IRF8) is upregulated by the antimetabolite cytarabine in leukemic cells involving the zinc finger protein ZNF224, acting as a cofactor of the Wilms' tumor gene 1 (WT1) protein.

    PubMed

    Montano, Giorgia; Ullmark, Tove; Jernmark-Nilsson, Helena; Sodaro, Gaetano; Drott, Kristina; Costanzo, Paola; Vidovic, Karina; Gullberg, Urban

    2016-01-01

    The transcription factor interferon regulatory factor-8 (IRF8) is highly expressed in myeloid progenitors, while most myeloid leukemias show low or absent expression. Loss of IRF8 in mice leads to a myeloproliferative disorder, indicating a tumor-suppressive role of IRF8. The Wilms tumor gene 1 (WT1) protein represses the IRF8-promoter. The zinc finger protein ZNF224 can a