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Sample records for binds transforming growth

  1. The latent transforming growth factor beta binding protein (LTBP) family.

    PubMed Central

    Oklü, R; Hesketh, R

    2000-01-01

    The transforming growth factor beta (TGFbeta) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFbetas is regulated by their association with latency-associated proteins and latent TGFbeta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFbeta activity, is associated with a variety of cancers. PMID:11104663

  2. Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity.

    PubMed

    Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Kaku, Masaru; Fong, Keith S K; Csiszar, Katalin; Yamauchi, Mitsuo

    2008-12-01

    Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling. PMID:18835815

  3. E1A inhibits transforming growth factor-beta signaling through binding to Smad proteins.

    PubMed

    Nishihara, A; Hanai, J; Imamura, T; Miyazono, K; Kawabata, M

    1999-10-01

    Smads form a recently identified family of proteins that mediate intracellular signaling of the transforming growth factor (TGF)-beta superfamily. Smads bind to DNA and act as transcriptional regulators. Smads interact with a variety of transcription factors, and the interaction is likely to determine the target specificity of gene induction. Smads also associate with transcriptional coactivators such as p300 and CBP. E1A, an adenoviral oncoprotein, inhibits TGF-beta-induced transactivation, and the ability of E1A to bind p300/CBP is required for the inhibition. Here we determined the Smad interaction domain (SID) in p300 and found that two adjacent regions are required for the interaction. One of the regions is the C/H3 domain conserved between p300 and CBP, and the other is a nonconserved region. p300 mutants containing SID inhibit transactivation by TGF-beta in a dose-dependent manner. E1A inhibits the interaction of Smad3 with a p300 mutant that contains SID but lacks the E1A binding domain. We found that E1A interacts specifically with receptor-regulated Smads, suggesting a novel mechanism whereby E1A antagonizes TGF-beta signaling. PMID:10497242

  4. Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

    PubMed Central

    Gishizky, M L; Cortez, D; Pendergast, A M

    1995-01-01

    Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479904

  5. Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

    PubMed Central

    Allen-Hoffmann, B L; Crankshaw, C L; Mosher, D F

    1988-01-01

    Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis. Images PMID:3054513

  6. Specific binding of endocrine transforming growth factor-beta 1 to vascular endothelium.

    PubMed Central

    Dickson, K; Philip, A; Warshawsky, H; O'Connor-McCourt, M; Bergeron, J J

    1995-01-01

    The presentation of recombinant biologically active 125I-TGF-beta 1 via the bloodstream to potential target cells in mice and rats was evaluated by quantitative light and electron microscope radioautography. Specificity was evaluated by in vivo competition with excess unlabeled TGF-beta 1, and integrity of the ligand at the binding site was demonstrated by trichloroacetic acid precipitation after extraction from tissues. The distribution of radiolabel at 2.5, 15, 30, 45, and 60 min after 125I-TGF-beta 1 injection revealed radiolabel principally over microvasculature endothelium but at times > 2.5 min over endothelial endocytic components indicative of internalization. Nonspecific binding of 125I-TGF-beta 1 to the apex of the proximal convoluted tubule of the kidney indicated it as the likely site of rapid clearance of TGF-beta 1 from the circulation, while a comparison of the binding of 125I-TGF-beta 1 (endothelial) to that of 125I-TGF-beta 1 complexed with alpha 2-macroglobulin-methylamine (liver parenchyma) indicated that clearance of TGF-beta 1 complexed alpha 2-macroglobulin was likely via the hepatic alpha 2-macroglobulin receptor. The endothelial TGF-beta receptors uncovered here are likely involved in the local regulatory mechanism of leukocyte and monocyte adhesion and tissue infiltration regulated by endocrine TGF-beta 1. Images PMID:7539454

  7. Latent transforming growth factor β-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor

    PubMed Central

    Brooke, Joanna S; Cha, Jeong-Heon; Eidels, Leon

    2002-01-01

    Background The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF) contribute to many cellular developmental processes. The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence. Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor β-binding protein, TGF-β, and fibulin-1 in some of these processes. To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library. cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated. Results From ≈ 3 × 106 screened monkey cDNA clones, cDNA clones were recovered that contained nucleotide sequences encoding domains of the monkey latent transforming growth factor-β binding protein-3 (MkLTBP-3) and fibulin-1C protein. The amino acid sequence derived from the MkLTBP-3 gene shared 98.6% identity with human LTBP-3 and 86.7% identity with mouse LTBP-3 amino acid sequences. The amino acid sequence derived from the monkey fibulin-1C gene shared 97.2% identity with human fibulin-1C. Yeast two-hybrid screens indicate that LTBP-3 and fibulin-1C interact with proHB-EGF through their calcium-binding EGF-like modules. Conclusions The interactions of the extracellular domain of proHB-EGF with LTBP-3 and fibulin-1C suggest novel functions for HB-EGF between cell and tissue surfaces. PMID:11846885

  8. Insulin-like growth factor binding protein-2 mediates the inhibition of DNA synthesis by transforming growth factor-beta in mink lung epithelial cells.

    PubMed

    Dong, Feng; Wu, Hai-Bin; Hong, Jiang; Rechler, Matthew M

    2002-01-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) has been proposed to mediate the growth inhibitory effects of transforming growth factor (TGF)-beta in breast and prostate cancer cells. Both TGF-beta and exogenous IGFBP-3 inhibit DNA synthesis in Mv1 mink lung epithelial cells (CCL64). The present study asks whether IGFBPs synthesized by CCL64 cells mediate growth inhibition by TGF-beta. CCL64 cells synthesize and secrete a single 34-kDa IGFBP that was identified as IGFBP-2 by immunoprecipitation and immunodepletion. Recombinant bovine IGFBP-2 inhibited CCL64 DNA synthesis in serum-free media in an IGF-independent manner. Coincubation with Leu(60)-IGF-I, an IGF-I analog that binds to IGFBPs with higher affinity than to IGF-I receptors, decreased the inhibition by bIGFBP-2. Leu(60)-IGF-I also decreased the inhibition of CCL64 DNA synthesis by TGF-beta by up to 70%, whereas Long-R3-IGF-I, an IGF-I analog with higher affinity for IGF-I receptors than for IGFBPs, did not decrease inhibition, suggesting that the effect of Leu(60)-IGF-I resulted from its forming complexes with endogenous IGFBPs. Leu(60)-IGF-I did not decrease TGF-beta stimulation of a Smad3-dependent reporter gene. Following incubation of intact CCL64 cells with bIGFBP-2 at 0 degrees C, bIGFBP-2 was recovered in membrane fractions; membrane association was abolished by coincubation with Leu(60)-IGF-I. If exogenous and secreted IGFBP-2 must bind to CCL64 cells to inhibit DNA synthesis, Leu(60)-IGF-I might reduce the inhibition of DNA synthesis by bIGFBP-2 or TGF-beta by inhibiting the association of IGFBP-2 in the media with CCL64 cells. Since TGF-beta does not increase IGFBP-2 abundance, we propose that TGF-beta sensitizes CCL64 cells to the latent growth inhibitory activity of endogenous IGFBP-2 by potentiating an intracellular IGFBP-2 signaling pathway or by promoting the association of secreted IGFBP-2 with the plasma membrane. PMID:11807812

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

    PubMed

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

    2006-09-01

    Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter. PMID:16849317

  10. Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity.

    PubMed Central

    Bonita, D P; Miyake, S; Lupher, M L; Langdon, W Y; Band, H

    1997-01-01

    Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce

  11. The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor beta1 gene through an Egr-1 binding site.

    PubMed Central

    Yoo, Y D; Chiou, C J; Choi, K S; Yi, Y; Michelson, S; Kim, S; Hayward, G S; Kim, S J

    1996-01-01

    Increases in transforming growth factor beta1 (TGF-beta1) mRNA and biological activity in the early phase of human cytomegalovirus (CMV) infection in fibroblasts are paralleled by increased TGF-beta1-chloramphenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-beta1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV on 5'-deleted TGF-beta1 promoter-CAT reporter genes in transient DNA transfection assays. Two upstream TGF-beta1 promoter regions each containing an Egr-1 consensus site were shown to be important for IE2-induced transactivation in a cell type that displayed greatly reduced nonspecific activity. Furthermore, transfer of an Egr-l site from between positions -125 and -98, but not point mutant versions of this site, to a heterologous promoter also conveyed IE2 responsiveness. Addition of an IE2 expression vector or use of the U373 A45 astrocytoma cell line expressing IE2 also produced synergistic stimulation of GAL4-Egr-l-mediated activation of a target promoter containing GAL4 binding sites. The 80-kDa IE2 protein present in A45 cells proved to selectively bind to glutathione S-transferase (GST)-Egr-1 beads. The results of in vitro protein binding assays also revealed that an intact in vitro-translated IE2 protein bound directly to the GST-Egr-1 fusion protein through the zinc finger domain of the Egr-1 protein and that this binding activity was abolished by deletion of parts of the zinc finger DNA-binding domain. Similarly, the Egr-1 protein was found to associate preferentially with a small region within the C-terminal half of the IE2 protein adjacent to the DNA-binding and dimerization domains that are important for both transactivation and downregulation. We conclude from these observations that IE2 may regulate transcription of the TGF-beta1 gene as well as other potential cellular targets by virtue of its ability to interact with the Egr-1 DNA-binding

  12. Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1.

    PubMed Central

    Saharinen, J; Taipale, J; Keski-Oja, J

    1996-01-01

    Transforming growth factor-betas (TGF-betas) are produced by most cells in large latent complexes of TGF-beta and its propeptide (LAP) associated with a binding protein. The latent TGF-beta binding proteins (LTBPs-1, -2 and -3) mediate the secretion and, subsequently, the association of latent TGF-beta complexes with the extracellular matrix (ECM). The association of beta1-LAP with LTBP-1 was characterized at the molecular level with an expression system in mammalian cells, where TGF-beta1 and various fragments of LTBP-1 were co-expressed and secreted with the aid of a signal peptide synthesized to the LTBP-1 constructs. Immunoblotting of the fusion protein complexes indicated that the third 8-Cys repeat of LTBP-1 bound covalently to the LAP region of TGF-beta1. The cysteine required for the association between LTBP-1 and beta1-LAP was mapped to Cys33 of beta1-LAP. The N-terminal region of LTBP-1 consisting of the first 400 amino acids was found to associate covalently with the ECM. The data indicate that an 8-Cys repeat of LTBP is capable of covalent and specific protein-protein interactions. These interactions are mediated by exchanging cysteine disulfide bonds between the core 8-Cys repeat and an optionally associated protein during the secretion. This is, to our knowledge, the first demonstration of an extracellular protein module that is able to exchange cysteine disulfide bonds with heterologous ligand proteins. Images PMID:8617200

  13. Binding assay for the solubilized receptors of type beta transforming growth factor: adsorption and removal of free ligand by dextran-coated charcoal

    SciTech Connect

    Fanger, B.O.; Sporn, M.B.

    1986-08-01

    A binding assay was developed for the measurement of solubilized receptors for transforming growth factor type beta (TGF-beta). Solubilized receptors were incubated with /sup 125/I-TGF-beta, then the unbound ligand was removed by adsorption to dextran-coated charcoal. The binding of TGF-beta to solubilized receptors was saturable and specific, and increased in a linear manner with respect to the amount of membrane protein present. Crosslinking of radioactive complexes after adsorptive removal of unbound TGF-beta yielded complexes similar to affinity-labeled TGF-beta receptors from whole cells. Treatment of a 20% charcoal suspension with 0.2-0.4% dextran was optimal for the protection of receptors from adsorption to charcoal while allowing free TGF-beta to be removed; Mr approximately 250,000 dextran was most effective. This method can assay receptors from purified membranes and crude extracts of cells and tissues, and was used to demonstrate that TGF-beta receptors are glycosylated and retain a high affinity (Kd approximately 530 pM) for ligand after solubilization.

  14. The bovine papillomavirus type 1 E5 transforming protein specifically binds and activates the beta-type receptor for the platelet-derived growth factor but not other related tyrosine kinase-containing receptors to induce cellular transformation.

    PubMed Central

    Goldstein, D J; Li, W; Wang, L M; Heidaran, M A; Aaronson, S; Shinn, R; Schlegel, R; Pierce, J H

    1994-01-01

    The 44-amino-acid E5 protein of bovine papillomavirus type 1 is a highly hydrophobic protein which appears to transform cells through the activation of growth factor receptors. To investigate the specificity of E5-growth factor receptor interactions required for mitogenic signaling, we utilized a nontumorigenic, murine myeloid cell line (32D) which is strictly dependent on interleukin-3 (IL-3) for sustained proliferation in culture. This IL-3 dependence can be functionally substituted by the expression of a variety of surrogate growth factor receptors and the addition of the corresponding ligand. Several receptor cDNAs for the alpha- and beta-type platelet-derived growth factor receptors [alpha PDGFR and beta PDGFR], the epidermal growth factor receptor, and the colony-stimulating factor 1 receptor) were transfected into 32D cells constitutively expressing the E5 protein to test for IL-3-independent growth. Only beta PDGFR was capable of abrogating the IL-3 dependence of 32D cells. The proliferative signal induced by the coexpression of beta PDGFR and E5 was accompanied by stable complex formation between these proteins, constitutive tyrosine phosphorylation of the receptor, and tumorigenicity in nude mice. The lack of cooperative interaction between E5 and the epidermal growth factor receptor, the colony-stimulating factor 1 receptor, and the highly related alpha PDGFR was paralleled by the inability of E5 to bind to these receptors and failure to increase receptor tyrosine phosphorylation. Thus, these data indicate that the ability of E5 to induce sustained proliferation and transformation of 32D cells is a direct consequence of specific interaction between the E5 protein and the beta PDGFR signaling complex and the subsequent stimulation of receptor tyrosine phosphorylation. Images PMID:8207816

  15. Downregulation of transforming growth factor β (TGFβ) and latent TGFβ binding protein (LTBP)-4 expression in late stage canine mammary tumours.

    PubMed

    Klopfleisch, R; Schütze, M; Gruber, A D

    2010-12-01

    Although canine mammary tumours (CMT) are the most common malignant tumours in bitches the pathogenesis is far from understood. To analyse the role of the transforming growth factor β (TGFβ) family in the carcinogenesis of CMT, relative mRNA and protein expression of TGFβ-1, -2 and -3, TGFβ receptor (TGFβR)-1 and -3, latent TGFβ-binding protein (LTBP)-1, -3 and -4, and oestrogen receptor α in CMT were quantified. mRNA expression concentrations were measured in laser-microdissected tissue samples of mammary adenomas, carcinomas and their lymph node metastases and normalised to the non-neoplastic mammary gland of the same dog. Carcinomas and metastases had significantly decreased expression levels of TGFβ-3, TGFβR-3 and LTBP-4, but increased LTBP-1 expression when compared to non-neoplastic glands or adenomas. Decreased TGFβ and LTBP-4 expression were confirmed immunohistochemically. The data suggested that loss of TGFβ-3 and LTBP-4 may have growth-stimulatory effects in late-stage tumours, and loss of their expression, together with reduced TGFβR-3 expression, may be associated with increased proliferative activity of CMT similar to findings in human breast cancer. PMID:19836277

  16. Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor. beta. 1 and by the retinoblastoma gene product

    SciTech Connect

    Pietenpol, J.A.; Stein, R.W.; Moses, H.L. ); Muenger, K.; Howley, P.M. )

    1991-11-15

    Previous studies have shown that transforming growth factor {beta}1 (TGF-{beta}1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter region was required for regulation by both TGF-{beta}1 and pRB. These sequences, termed the TGF-{beta} control element (TCE), lie between positions {minus}86 and {minus}63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-{beta}1 treatment of TGF-{beta}-sensitive but not TGF-{beta}-insensitive cells. These data indicate that pRB can suppress c-myc transcription and growth inhibition.

  17. Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

    PubMed Central

    Ge, Jingjing; Chang, Na; Zhao, Zhongxin; Tian, Lei; Duan, Xianghui; Yang, Lin; Li, Liying

    2016-01-01

    RNA-binding protein HuR mediates transforming growth factor (TGF)-β1-induced profibrogenic actions. Up-regulation of Sphingosine kinase 1 (SphK1) is involved in TGF-β1-induced activation of hepatic stellate cells (HSCs) in liver fibrogenesis. However, the molecular mechanism of TGF-β1 regulates SphK1 remains unclear. This study was designed to investigate the role of HuR in TGF-β1-induced SphK1 expression and identify a new molecular mechanism in liver fibrogenensis. In vivo, HuR expression was increased, translocated to cytoplasm, and bound to SphK1 mRNA in carbon tetrachloride- and bile duct ligation-induced mouse fibrotic liver. HuR mRNA expression had a positive correlation with mRNA expressions of SphK1 and fibrotic markers, α-smooth muscle actin (α-SMA) and Collagen α1(I), respectively. In vitro, up-regulation of SphK1 and activation of HSCs stimulated by TGF-β1 depended on HuR cytoplasmic accumulation. The effects of TGF-β1 were diminished when HuR was silenced or HuR cytoplasmic translocation was blocked. Meanwhile, overexpression of HuR mimicked the effects of TGF-β1. Furthermore, TGF-β1 prolonged half-life of SphK1 mRNA by promoting its binding to HuR. Pharmacological or siRNA-induced SphK1 inhibition abrogated HuR-mediated HSC activation. In conclusion, our data suggested that HuR bound to SphK1 mRNA and played a crucial role in TGF-β1-induced HSC activation. PMID:26912347

  18. Loss of function mutations in the gene encoding latent transforming growth factor beta binding protein 2, LTBP2, cause primary congenital glaucoma.

    PubMed

    Narooie-Nejad, Mehrnaz; Paylakhi, Seyed Hassan; Shojaee, Seyedmehdi; Fazlali, Zeinab; Rezaei Kanavi, Mozhgan; Nilforushan, Naveed; Yazdani, Shahin; Babrzadeh, Farbod; Suri, Fatemeh; Ronaghi, Mostafa; Elahi, Elahe; Paisán-Ruiz, Coro

    2009-10-15

    Glaucoma is a heterogeneous group of optic neuropathies that manifests by optic nerve head cupping or degeneration of the optic nerve, resulting in a specific pattern of visual field loss. Glaucoma leads to blindness if left untreated, and is considered the second leading cause of blindness worldwide. The subgroup primary congenital glaucoma (PCG) is characterized by an anatomical defect in the trabecular meshwork, and age at onset in the neonatal or infantile period. It is the most severe form of glaucoma. CYP1B1 was the first gene genetically linked to PCG, and CYP1B1 mutations are the cause of disease in 20-100% of patients in different populations. Here, we report that LTBP2 encoding latent transforming growth factor beta binding protein 2 is a PCG causing gene, confirming results recently reported. A disease-associated locus on chromosome 14 was identified by performing whole genome autozygosity mapping in Iranian PCG families using high density single nucleotide polymorphism chips, and two disease-segregating loss of function mutations in LTBP2, p.Ser472fsX3 and p.Tyr1793fsX55, were observed in two families while sequencing candidate genes in the locus. The p.Tyr1793fsX55 mutation affects an amino acid close to the C-terminal of the encoded protein. Subsequently, LTBP2 expression was shown in human eyes, including the trabecular meshwork and ciliary processes that are thought to be relevant to the etiology of PCG. PMID:19656777

  19. Transforming growth factor-{beta} inhibits CCAAT/enhancer-binding protein expression and PPAR{gamma} activity in unloaded bone marrow stromal cells

    SciTech Connect

    Ahdjoudj, S.; Kaabeche, K.; Holy, X.; Fromigue, O.; Modrowski, D.; Zerath, E.; Marie, P.J. . E-mail: pierre.marie@larib.inserm.fr

    2005-02-01

    The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-{beta}2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP){alpha} and C/EBP{beta} {alpha} at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}2) transcripts at 7 days. TGF-{beta}2 administration in unloaded rats corrected the rise in C/EBP{alpha} and C/EBP{beta} transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPAR{gamma}2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBP{alpha} and C/EBP{beta} expression by TGF-{beta}2 was associated with increased PPAR{gamma} serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPAR{gamma} transactivating activity. The sequential inhibitory effect of TGF-{beta}2 on C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma}2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-{beta}2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma} expression and activity, which provides a sequential mechanism by which TGF-{beta}2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.

  20. Synthetic heparin-binding growth factor analogs

    DOEpatents

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  1. Nucleation and growth transformation kinetics

    NASA Astrophysics Data System (ADS)

    Erukhimovitch, V.; Baram, J.

    1995-03-01

    As a result of the reassessment of the Kolmogorov-Johnson-Mehl-Avrami (KJMA) theory for the kinetics of nucleation and growth transformations, an integral-equation formulation has been developed instead of the well-known and widely used Avrami equation. The presented formulation considers interfacial and diffusional growths, in one, two, and three dimensions, with both time-dependent and time-invariant nucleation and growth rates. The integral-equation model corrects reported inadequacies of the KJMA theory when applied in numerous experiments and various solid-state transformations. It is shown that in the example cases examined in this paper, crystallization from the amorphous state in melt-spun ribbons, isothermal aging of CuAlZn, pearlitic transition in an eutectoid steel, and crystallization in a PEKK polymer, the thermodynamic and kinetic interpretation and parameters extracted from best fits of the Avrami equations to the experimental data are erroneous. The KJMA formulation is a simplification of the real physical conditions. The main limitation of the new model is that almost all the integral equations representing the kinetics of solid-state transformations have no analytical solutions.

  2. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  3. Non-Smad transforming growth factor-β signaling regulated by focal adhesion kinase binding the p85 subunit of phosphatidylinositol 3-kinase.

    PubMed

    Hong, Min; Wilkes, Mark C; Penheiter, Sumedha G; Gupta, Shiv K; Edens, Maryanne; Leof, Edward B

    2011-05-20

    TGF-β modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-β. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-β treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-β-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-β and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases. PMID:21454615

  4. Characterization of transforming growth factor-beta (TGF-beta) receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein.

    PubMed Central

    Mitchell, E J; Lee, K; O'Connor-McCourt, M D

    1992-01-01

    Transforming growth factor-beta (TGF-beta) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-beta 1 and TGF-beta 2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGF-beta 1 and TGF-beta 2. The Kd values obtained from Scatchard analyses were approximately 65 pM for 125I-TGF-beta 1 and approximately 40 pM for 125I-TGF-beta 2, with 70,000 and 85,000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-beta 1 and TGF-beta 2 were equipotent (apparent half maximal inhibition [IC50] approximately 70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 125I-TGF-beta 1 and 125I-TGF-beta 2 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-beta binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-beta 1 than for -beta 2 yet exhibits a 5- to 10-fold higher affinity for TGF-beta 2 than for -beta 1. The 38-kDa TGF-beta binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-beta 2 than for -beta 1 that is strikingly similar to that of the type III/betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the

  5. Interactions Between β-Catenin and Transforming Growth Factor-β Signaling Pathways Mediate Epithelial-Mesenchymal Transition and Are Dependent on the Transcriptional Co-activator cAMP-response Element-binding Protein (CREB)-binding Protein (CBP)*

    PubMed Central

    Zhou, Beiyun; Liu, Yixin; Kahn, Michael; Ann, David K.; Han, Arum; Wang, Hongjun; Nguyen, Cu; Flodby, Per; Zhong, Qian; Krishnaveni, Manda S.; Liebler, Janice M.; Minoo, Parviz; Crandall, Edward D.; Borok, Zea

    2012-01-01

    Interactions between transforming growth factor-β (TGF-β) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated β-catenin-dependent and transforming growth factor-β1 (TGF-β1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the β-catenin/CBP (but not β-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-β1-mediated α-smooth muscle actin (α-SMA) and collagen induction in AEC. We now demonstrate that TGF-β1 induces LEF/TCF TOPFLASH reporter activation and nuclear β-catenin accumulation, while LiCl augments TGF-β-induced α-SMA expression, further confirming co-operation between β-catenin- and TGF-β-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of β-catenin and overexpression of ICAT abrogated effects of TGF-β1 on α-SMA transcription/expression, indicating a requirement for β-catenin in these Smad3-dependent effects. Following TGF-β treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and β-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of α-SMA via complex formation among Smad3, β-catenin, and CBP. ICG-001 inhibited α-SMA expression/transcription in response to TGF-β as well as α-SMA promoter occupancy by β-catenin and CBP, demonstrating a previously unknown requisite TGF-β1/β-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by β-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically

  6. NF-κB p65 Subunit Is Modulated by Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) in Nasopharyngeal Carcinoma HONE1 and HK1 Cells

    PubMed Central

    Lung, Hong Lok; Cheung, Arthur Kwok Leung; Dai, Wei; Kwong, Dora Lai-Wan; Ng, Wai Tong; Lee, Anne Wing Mui; Yau, Chun Chung; Ngan, Roger Kai Cheong; Tung, Stewart Yuk; Lung, Maria Li

    2015-01-01

    NF-κB is a well-characterized transcription factor, widely known as a key player in tumor-derived inflammation and cancer development. Herein, we present the functional and molecular relevance of the canonical NF-κB p65 subunit in nasopharyngeal carcinoma (NPC). Loss- and gain-of-function approaches were utilized to reveal the functional characteristics of p65 in propagating tumor growth, tumor-associated angiogenesis, and epithelial-to-mesenchymal transition in NPC cells. Extracellular inflammatory stimuli are critical factors that trigger the NF-κB p65 signaling; hence, we investigated the components of the tumor microenvironment that might potentially influence the p65 signaling pathway. This led to the identification of an extracellular matrix (ECM) protein that was previously reported as a candidate tumor suppressor in NPC. Our studies on the Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) protein provides substantial evidence that it can modulate the p65 transcriptional activity. Re-expression of LTBP2 elicits tumor suppressive effects that parallel the inactivation of p65 in NPC cells. LTBP2 was able to reduce phosphorylation of p65 at Serine 536, inhibit nuclear localization of active phosphorylated p65, and impair the p65 DNA-binding ability. This results in a consequential down-regulation of p65-related gene expression. Therefore, the data suggest that the overall up-regulation of p65 expression and the loss of this candidate ECM tumor suppressor are milestone events contributing to NPC development. PMID:25974126

  7. Grain nucleation and growth during phase transformations.

    PubMed

    Offerman, S E; van Dijk, N H; Sietsma, J; Grigull, S; Lauridsen, E M; Margulies, L; Poulsen, H F; Rekveldt, M Th; van der Zwaag, S

    2002-11-01

    The mechanical properties of polycrystalline materials are largely determined by the kinetics of the phase transformations during the production process. Progress in x-ray diffraction instrumentation at synchrotron sources has created an opportunity to study the transformation kinetics at the level of individual grains. Our measurements show that the activation energy for grain nucleation is at least two orders of magnitude smaller than that predicted by thermodynamic models. The observed growth curves of the newly formed grains confirm the parabolic growth model but also show three fundamentally different types of growth. Insight into the grain nucleation and growth mechanisms during phase transformations contributes to the development of materials with optimal mechanical properties. PMID:12411699

  8. Novel RNA-binding Protein P311 Binds Eukaryotic Translation Initiation Factor 3 Subunit b (eIF3b) to Promote Translation of Transforming Growth Factor β1-3 (TGF-β1-3)*

    PubMed Central

    Yue, Michael M.; Lv, Kaosheng; Meredith, Stephen C.; Martindale, Jennifer L.; Gorospe, Myriam; Schuger, Lucia

    2014-01-01

    P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-β1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a Kd of 1.26 μm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-β 5′UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-βs 5′UTRs and eIF3b, stimulates the translation of TGF-β1, 2, and 3. PMID:25336651

  9. Transforming growth factor-beta and transforming growth factor beta-receptor expression in human meningioma cells.

    PubMed Central

    Johnson, M. D.; Federspiel, C. F.; Gold, L. I.; Moses, H. L.

    1992-01-01

    The transforming growth factor-beta (TGF beta) family in mammals includes three closely related peptides that influence proliferation and numerous physiologic processes in most mesenchymal cells. In this study, Northern blots, immunohistochemistry, TGF beta radioreceptor assays, TGF beta receptor affinity labeling and [3H] thymidine incorporation were used to evaluate whether primary cell cultures of human meningiomas synthesize the three TGF beta isoforms, bear TGF beta receptors, and respond to TGF beta. Transcripts for TGF beta 1 and 2 were detected in the three cases analyzed. Transforming growth factor-beta 1 immunoreactivity was detected in three of six cases, and TGF beta 2 and 3 immunoreactivity were detected in each case analyzed. Media conditioned by cells cultured from six meningiomas also contained latent TGF beta-like activity. Transforming growth factor-beta receptor cross-linking studies identified TGF beta binding sites corresponding to the type 1, type 2, and type 3 receptors on meningioma cells. Treatment with active TGF beta 1 produced a statistically significant reduction in [3H] thymidine incorporation after stimulation with 10% fetal calf serum and epidermal growth factor in all six cases studied. Images Figure 1 Figure 2 Figure 4 PMID:1325741

  10. PDZ-binding kinase/T-LAK cell-originated protein kinase is a target of the fucoidan from brown alga Fucus evanescens in the prevention of EGF-induced neoplastic cell transformation and colon cancer growth

    PubMed Central

    Wang, Zhe; Ermakova, Svetlana P.; Xiao, JuanJuan; Lu, Tao; Xue, PeiPei; Zvyagintseva, Tatyana N.; Xiong, Hua; Shao, Chen; Yan, Wei; Duan, Qiuhong; Zhu, Feng

    2016-01-01

    The fucoidan with high anticancer activity was isolated from brown alga Fucus evanescens. The compound effectively prevented EGF-induced neoplastic cell transformation through inhibition of TOPK/ERK1/2/MSK 1 signaling axis. In vitro studies showed that the fucoidan attenuated mitogen-activated protein kinases downstream signaling in a colon cancer cells with different expression level of TOPK, resulting in growth inhibition. The fucoidan exerts its effects by directly interacting with TOPK kinase in vitro and ex vivo and inhibits its kinase activity. In xenograft animal model, oral administration of the fucoidan suppressed HCT 116 colon tumor growth. The phosphorylation of TOPK downstream signaling molecules in tumor tissues was also inhibited by the fucoidan. Taken together, our findings support the cancer preventive efficacy of the fucoidan through its targeting of TOPK for the prevention of neoplastic cell transformation and progression of colon carcinomas in vitro and ex vivo. PMID:26936995

  11. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua

    2012-04-24

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  12. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua

    2009-10-06

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  13. Epidermal growth factor signaling in transformed cells

    PubMed Central

    Lindsey, Stephan; Langhans, Sigrid A.

    2016-01-01

    Members of the epidermal growth factor receptor (EGFR/ErbB) family play a critical role in normal cell growth and development. However, many ErbB family members, especially EGFR, are aberrantly expressed or deregulated in tumors and are thought to play crucial roles in cancer development and metastatic progression. In this chapter, we provide an overview of key mechanisms contributing to aberrant EGFR/ErbB signaling in transformed cells which results in many phenotypic changes associated with the earliest stages of tumor formation, including several hallmarks of epithelial-to-mesenchymal transition (EMT). These changes often occur through interaction with other major signaling pathways important to tumor progression resulting in a multitude of transcriptional changes that ultimately impact cell morphology, proliferation and adhesion, all of which are crucial for tumor progression. The resulting mesh of signaling networks will need to be taken into account as new regimens are designed for targeting EGFR for therapeutic intervention. As new insights into the molecular mechanisms of the cross-talk of EGFR signaling with other signaling pathways and their role in therapeutic resistance to anti-EGFR therapies are gained a continual reassessment of clinical therapeutic regimes and strategies will be required. Understanding the consequences and complexity of EGF signaling and how it relates to tumor progression is critical for the development of clinical compounds and establishing clinical protocols for the treatment of cancer. PMID:25619714

  14. Cul7/p185/p193 binding to simian virus 40 large T antigen has a role in cellular transformation.

    PubMed

    Ali, Syed Hamid; Kasper, Jocelyn S; Arai, Takehiro; DeCaprio, James A

    2004-03-01

    Simian virus 40 large T antigen (TAg) is a viral oncoprotein that can promote cellular transformation. TAg's transforming activity results in part by binding and inactivating key tumor suppressors, including p53 and the retinoblastoma protein (pRb). We have identified a TAg-associated 185-kDa protein that has significant homology to the cullin family of E3 ubiquitin ligases. TAg binds to an SCF-like complex that contains p185/Cul7, Rbx1, and the F box protein Fbw6. This SCF-like complex binds to an N-terminal region of TAg. Several p185/Cul7-binding-deficient mutants of TAg were generated that retained binding to pRb and p53 and were capable of overcoming Rb-mediated repression of E2F transcription. Despite binding to pRb and p53, these p185/Cul7-binding-defective mutants of TAg were unable to transform primary mouse embryo fibroblasts. Cells expressing p185/Cul7-binding-defective mutants of TAg were unable to grow to high density or grow in an anchorage-independent manner as determined by growth in soft agar. Considering the significance of other TAg-interacting proteins in regulation of the cell cycle, p185/Cul7 may also regulate an important growth control pathway. PMID:14990695

  15. Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

    PubMed

    Jingushi, S; Scully, S P; Joyce, M E; Sugioka, Y; Bolander, M E

    1995-09-01

    Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7472755

  16. Transforming growth factor alpha and epidermal growth factor levels in bladder cancer and their relationship to epidermal growth factor receptor.

    PubMed Central

    Mellon, J. K.; Cook, S.; Chambers, P.; Neal, D. E.

    1996-01-01

    We have examined levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in neoplastic and non-neoplastic bladder tissue using a standard radioimmunoassay technique. Tumour samples had much higher TGF-alpha levels compared with EGF and TGF-alpha levels in malignant tissue were significantly higher than in benign bladder samples. There was, in addition, a difference in mean EGF levels from 'normal' bladder samples from non-tumour bearing areas of bladder in patients with bladder cancer compared with 'normal' bladder tissue obtained at the time of organ retrieval surgery. Levels of EGF and TGF-alpha did not correlate with levels of EGF receptor (EGFR) as determined by a radioligand binding method but levels of TGF-alpha > 10 ng gm-1 of tumour tissue did correlate with EGFR positivity defined using immunohistochemistry. These data suggest that TGF-alpha is the likely ligand for EGFR in bladder tumours. PMID:8605103

  17. Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery.

    PubMed

    Wing Moon Lam, Raymond; Abbah, Sunny Akogwu; Ming, Wang; Naidu, Mathanapriya; Ng, Felly; Tao, Hu; Goh Cho Hong, James; Ting, Kang; Hee Kit, Wong

    2016-01-01

    During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries. PMID:27585207

  18. Cellular Actions of Insulin-Like Growth Factor Binding Proteins

    PubMed Central

    Ferry, R. J.; Katz, L. E. L.; Grimberg, Adda; Cohen, P.; Weinzimer, S. A.

    2014-01-01

    The insulin-like growth factors (IGFs), insulin-like growth factor binding proteins (IGFBPs), and the IGFBP proteases are involved in the regulation of somatic growth and cellular proliferation both in vivo and in vitro. IGFs are potent mitogenic agents whose actions are determined by the availability of free IGFs to interact with the IGF receptors. IGFBPs comprise a family of proteins that bind IGFs with high affinity and specificity and thereby regulate IGF-dependent actions. IGFBPs have recently emerged as IGF-independent regulators of cell growth. Various IGFBP association proteins as well as cleavage of IGFBPs by specific proteases modulate levels of free IGFs and IGFBPs. The ubiquity and complexity of the IGF axis promise exciting discoveries and applications for the future. PMID:10226802

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

    PubMed

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

    2000-11-10

    In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells. PMID:11062049

  20. Cells transformed by murine herpesvirus 68 (MHV-68) release compounds with transforming and transformed phenotype suppressing activity resembling growth factors.

    PubMed

    Šupolíková, M; Staňová, A Vojs; Kúdelová, M; Marák, J; Zelník, V; Golais, F

    2015-12-01

    In this study, we investigated the medium of three cell lines transformed with murine herpesvirus 68 (MHV-68) in vitro and in vivo, 68/HDF, 68/NIH3T3, and S11E, for the presence of compounds resembling growth factors of some herpesviruses which have displayed transforming and transformed phenotype suppressing activity in normal and tumor cells. When any of spent medium was added to cell culture we observed the onset of transformed phenotype in baby hamster kidney cells (BHK-21) cells and transformed phenotype suppressing activity in tumor human epithelial cells (HeLa). In media tested, we have identified the presence of putative growth factor related to MHV-68 (MHGF-68). Its bivalent properties have been blocked entirely by antisera against MHV-68 and two monoclonal antibodies against glycoprotein B (gB) of MHV-68 suggesting viral origin of MHGF-68. The results of initial efforts to separate MHGF-68 on FPLC Sephadex G15 column in the absence of salts revealed the loss of its transforming activity but transformed phenotype suppressing activity retained. On the other hand, the use of methanol-water mobile phase on RP-HPLC C18 column allowed separation of MHGF-68 to two compounds. Both separated fractions, had only the transforming activity to normal cells. Further experiments exploring the nature and the structure of hitherto unknown MHGF-68 are now in the progress to characterize its molecular and biological properties. PMID:26666191

  1. Divergent effects of epidermal growth factor and transforming growth factors on a human endometrial carcinoma cell line.

    PubMed

    Korc, M; Haussler, C A; Trookman, N S

    1987-09-15

    Epidermal growth factor (EGF), at concentrations ranging from 0.83 to 4.98 nM, markedly inhibited the proliferation of RL95-2 cells that were seeded at low plating densities (4.7 X 10(3) cells/cm2). Under the same incubation conditions, 16.6 pM EGF enhanced cell proliferation. At high plating densities (2.5 X 10(4) cells/cm2) 0.83 nM EGF also stimulated cell proliferation. Both the inhibitory and stimulatory effects of EGF were mimicked by transforming growth factor-alpha (TGF-alpha). However, the inhibitory action of TGF-alpha was always greater that of EGF. Binding studies with 125I-labeled TGF-alpha indicated that maximal cell surface binding of TGF-alpha occurred at 15 min, whereas maximal internalization occurred at 45 min. Both cell surface and internalized radioactivity declined sharply thereafter. Analysis of radioactivity released into the incubation medium during pulse-chase experiments indicated that RL95-2 cells extensively degraded both TGF-alpha and EGF. The lysosomotropic compound methylamine arrested the generation of low-molecular-weight degradation products of EGF, but not of TGF-alpha. In contrast to EGF and TGF-alpha, transforming growth factor-beta (TGF-beta) inhibited the proliferation of RL95-2 cells that were seeded at either low or high plating densities. Further, transforming growth factor-beta induced the appearance of large cuboidal cells that were readily distinguished from cells treated with either EGF or TGF-alpha. These findings point to complex regulatory actions of growth factors on the proliferation of RL95-2 cells and suggest that the processing of TGF-alpha following EGF receptor activation is distinct from the processing of EGF. PMID:3497713

  2. The TATA-binding protein as a regulator of cellular transformation.

    PubMed

    Johnson, Sandra A S; Dubeau, Louis; White, Robert J; Johnson, Deborah L

    2003-01-01

    The TATA-binding protein, TBP, is used by all three RNA polymerases and is therefore central to the process of gene expression. TBP associates with several subsets of proteins, called TATA-binding protein-associated factors (TAFs). This results in the formation of at least three distinct complexes, SL1, TFIID, and TFIIIB, which dictates whether TBP functions in RNA polymerase (pol) I, pol II, or pol III transcription, respectively. The regulation of gene expression has focused largely on proteins that serve to modulate the efficiency by which the general transcription components, such as TBP, interact with promoters. The possibility of a basal transcription factor, itself, being regulated, and influencing cellular homeostasis, has not been extensively considered. However, recent studies have indicated that TBP is indeed regulated, and that modulation of its cellular concentration has a profound, and surprisingly selective, impact on gene expression that can mediate the normal proliferative responses of cells to growth stimuli as well as the transformation potential of cells. PMID:12963838

  3. Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

    PubMed Central

    Volz, Yvonne; Koschut, David; Matzke-Ogi, Alexandra; Dietz, Marina S.; Karathanasis, Christos; Richert, Ludovic; Wagner, Moritz G.; Mély, Yves; Heilemann, Mike; Niemann, Hartmut H.; Orian-Rousseau, Véronique

    2015-01-01

    CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs. PMID:26181364

  4. Source coding with a permutation-based reversible memory-binding transform for data compression in categorical data domains.

    PubMed

    Talbot, B G; Talbot, L M

    1998-01-01

    A general purpose reversible memory-binding transform (MBT) is developed, which uses a permutation transform technique to bind memory information to a transformed signal alphabet. The algorithm performs well in conjunction with a Huffman coder for both ordered sources, such as pixel intensities, and categorical sources, such as vector quantized codebook indices. PMID:18276336

  5. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    PubMed Central

    Lin, Yu-Feng; Chen, Jin-Yi

    2015-01-01

    We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified. PMID:26000290

  6. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  7. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    SciTech Connect

    Daughaday, W.H.; Trivedi, B.

    1987-07-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of /sup 125/I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound /sup 125/I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

  8. Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

    NASA Astrophysics Data System (ADS)

    Lee, L. S.

    1981-02-01

    The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

  9. Hydroxyapatite-binding peptides for bone growth and inhibition

    DOEpatents

    Bertozzi, Carolyn R.; Song, Jie; Lee, Seung-Wuk

    2011-09-20

    Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

  10. Metformin is a novel suppressor for transforming growth factor (TGF)-β1.

    PubMed

    Xiao, Han; Zhang, Jianshu; Xu, Zhonghe; Feng, Yenan; Zhang, Mingliang; Liu, Jianli; Chen, Ruifei; Shen, Jing; Wu, Jimin; Lu, Zhizhen; Fang, Xiaohong; Li, Jingyuan; Zhang, Youyi

    2016-01-01

    Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome, and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-β1 (TGF-β1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-β1 and found that metformin inhibits [(125)I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated. PMID:27349853

  11. Metformin is a novel suppressor for transforming growth factor (TGF)-β1

    NASA Astrophysics Data System (ADS)

    Xiao, Han; Zhang, Jianshu; Xu, Zhonghe; Feng, Yenan; Zhang, Mingliang; Liu, Jianli; Chen, Ruifei; Shen, Jing; Wu, Jimin; Lu, Zhizhen; Fang, Xiaohong; Li, Jingyuan; Zhang, Youyi

    2016-06-01

    Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome, and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-β1 (TGF-β1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-β1 and found that metformin inhibits [125I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated.

  12. Metformin is a novel suppressor for transforming growth factor (TGF)-β1

    PubMed Central

    Xiao, Han; Zhang, Jianshu; Xu, Zhonghe; Feng, Yenan; Zhang, Mingliang; Liu, Jianli; Chen, Ruifei; Shen, Jing; Wu, Jimin; Lu, Zhizhen; Fang, Xiaohong; Li, Jingyuan; Zhang, Youyi

    2016-01-01

    Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome, and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-β1 (TGF-β1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-β1 and found that metformin inhibits [125I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated. PMID:27349853

  13. Transforming growth factor-beta stimulates epithelial-mesenchymal transformation in the proepicardium.

    PubMed

    Olivey, Harold E; Mundell, Nathan A; Austin, Anita F; Barnett, Joey V

    2006-01-01

    The proepicardium (PE) migrates over the heart and forms the epicardium. A subset of these PE-derived cells undergoes epithelial-mesenchymal transformation (EMT) and gives rise to cardiac fibroblasts and components of the coronary vasculature. We report that transforming growth factor-beta (TGFbeta) 1 and TGFbeta2 increase EMT in PE explants as measured by invasion into a collagen gel, loss of cytokeratin expression, and redistribution of ZO1. The type I TGFbeta receptors ALK2 and ALK5 are both expressed in the PE. However, only constitutively active (ca) ALK2 stimulates PE-derived epithelial cell activation, the first step in transformation, whereas caALK5 stimulates neither activation nor transformation in PE explants. Overexpression of Smad6, an inhibitor of ALK2 signaling, inhibits epithelial cell activation, whereas BMP7, a known ligand for ALK2, has no effect. These data demonstrate that TGFbeta stimulates transformation in the PE and suggest that ALK2 partially mediates this effect. PMID:16245329

  14. Nanowire growth by an electron beam induced massive phase transformation

    SciTech Connect

    Sood, Shantanu; Kisslinger, Kim; Gouma, Perena

    2014-11-15

    Tungsten trioxide nanowires of a high aspect ratio have been synthesized in-situ in a TEM under an electron beam of current density 14A/cm² due to a massive polymorphic reaction. Sol-gel processed pseudocubic phase nanocrystals of tungsten trioxide were seen to rapidly transform to one dimensional monoclinic phase configurations, and this reaction was independent of the substrate on which the material was deposited. The mechanism of the self-catalyzed polymorphic transition and accompanying radical shape change is a typical characteristic of metastable to stable phase transformations in nanostructured polymorphic metal oxides. A heuristic model is used to confirm the metastable to stable growth mechanism. The findings are important to the control electron beam deposition of nanowires for functional applications starting from colloidal precursors.

  15. Nanowire growth by an electron beam induced massive phase transformation

    DOE PAGESBeta

    Sood, Shantanu; Kisslinger, Kim; Gouma, Perena

    2014-11-15

    Tungsten trioxide nanowires of a high aspect ratio have been synthesized in-situ in a TEM under an electron beam of current density 14A/cm² due to a massive polymorphic reaction. Sol-gel processed pseudocubic phase nanocrystals of tungsten trioxide were seen to rapidly transform to one dimensional monoclinic phase configurations, and this reaction was independent of the substrate on which the material was deposited. The mechanism of the self-catalyzed polymorphic transition and accompanying radical shape change is a typical characteristic of metastable to stable phase transformations in nanostructured polymorphic metal oxides. A heuristic model is used to confirm the metastable to stablemore » growth mechanism. The findings are important to the control electron beam deposition of nanowires for functional applications starting from colloidal precursors.« less

  16. Hepatocyte uptake and nuclear binding of epidermal growth factor (EGF)

    SciTech Connect

    Moriarity, D.M.; Underwood, T.

    1987-05-01

    The internalization of /sup 125/I-EGF and its cell-membrane receptor by target cells suggests a possible intracellular role for EGF and/or its receptor. They have examined the uptake of /sup 125/I-EGF by primary cultures of adult rat hepatocytes after 1, 24 and 48 hours of incubation in the presence of the growth factor. A significant increase in the association of radioactivity with various nuclear fractions was observed between 1 and 24 hours incubation. After 1 hour approximately 2% of the total specific binding was associated with both the nuclear sap proteins extractable with 0.14 M NaCl and with the residual nucleoplasm, while about 1% or less was associated with the nuclear membrane and the chromatin fractions. After 24 hours the percentage associated with the nuclear membrane and chromatin fractions increased 2-4 fold. Binding of /sup 125/I-EGF to isolated nuclei from intact livers of adult rats followed by fractionation of the nuclei after incubation with /sup 125/I-EGF indicated that after 60 min at 37/sup 0/C there was a substantial amount of specific binding associated with the nucleoplasm, nuclear membranes and chromatin fractions. These data indicate that specific interactions of EGF with nuclear components occur in both intact normal hepatocytes and in isolated nuclei from intact liver.

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

    PubMed

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

    2010-01-15

    Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells. PMID:19920136

  18. Transforming growth factor (TGF)-. alpha. in human milk

    SciTech Connect

    Okada, Masaki; Wakai, Kae; Shizume, Kazuo ); Iwashita, Mitsutoshi ); Ohmura, Eiji; Kamiya, Yoshinobu; Murakami, Hitomi; Onoda, Noritaka; Tsushima, Toshio

    1991-01-01

    Transforming growth factor (TGF)-{alpha} and epidermal growth factor (EGF) were measured in human milk by means of homologous radioimmunoassay. As previously reported, EGF concentration in the colostrum was approximately 200 ng/ml and decreased to 50 ng/ml by day 7 postpartum. The value of immunoreactive (IR)-TGF-{alpha} was 2.2-7.2 ng/ml, much lower than that of EGF. In contrast to EGF, the concentration of IR-TGF-{alpha} was fairly stable during the 7 postpartum days. There was no relationship between the concentrations of IR-TGF-{alpha} and IR-EGF, suggesting that the regulatory mechanism in the release of the two growth factors is different. On gel-chromatography using a Sephadex G-50 column, IR-EGF appeared in the fraction corresponding to that of authentic human EGF, while 70%-80% of the IR-TGF-{alpha} was eluted as a species with a molecular weight greater than that of authentic human TGF-{alpha}. Although the physiological role of TGF-{alpha} in milk is not known, it is possible that it is involved in the development of the mammary gland and/or the growth of newborn infants.

  19. Platelet-derived growth factor binds specifically to receptors on vascular smooth muscle cells and the binding becomes nondissociable.

    PubMed Central

    Williams, L T; Tremble, P; Antoniades, H N

    1982-01-01

    Radioiodinated platelet-derived growth factor (125I-PDGF) was used in studies of PDGF binding sites on vascular smooth muscle cells. There was an excellent correlation between the ability of 125I-PDGF to stimulate cell proliferation and to bind specifically to cultured vascular smooth muscle cells. The half-maximal concentration for both processes was 0.1 nM. There were 50,000 binding sites per cell. Reduced PDGF, prepared by treatment of PDGF with 20 mM dithiothreitol, had neither the ability to bind to smooth muscle cells nor to stimulate cellular proliferation. Epidermal growth factor, nerve growth factor, fibroblast growth factor, and histone B did not compete for the binding sites at a concentration of 10 nM. 125I-PDGF binding was slowly reversible at 4 degrees C and was rapidly and totally reversible after a 1-min incubation at 37 degrees C. After continued incubation at 37 degrees C, the binding became irreversible. The half-time for formation of the nondissociable state of 125I-PDGF binding was approximately equal to 5 min at 37 degrees C. The nondissociable state of binding was not formed at 4 degrees C even after 1 hr of incubation. These data suggest that the sites we labeled are the PDGF receptors that mediate PDGF's mitogenic action and that a nondissociable state of PDGF binding is formed at 37 degrees C. It is likely that nondissociable PDGF represents internalized ligand or binding to sites that are converted to a high-affinity state after the ligand binds. PMID:6310551

  20. Basic science for the clinician 57: transforming growth factor β.

    PubMed

    Sigal, Leonard H

    2012-08-01

    As is so often the case, a molecule gets named for its first identified activity or apparent role and then that initial name sticks, even as new and perhaps fundamentally different activities emerge from later studies. It is the special power of evolution that takes a certain activity and then uses it over and over again in pursuit of apparently disparate goals in a maturing or mature organism. In general terms, transforming growth factor β (TGF-β) is intimately involved in a variety of differentiation and growth inhibition processes, in apoptosis, and in deposition of the extracellular matrix. Initially identified in its role in oncogenesis, TGF-β is now implicated in a number of vascular and rheumatologic disorders, perhaps most notably the scleroderma. TGF-β has been identified as a powerful influence in angiogenesis, wound healing, joint inflammation, tumor growth and metastasis, and, of course, immunoregulation. So "what is in a name?" A rose by any other name would smell as sweet and would still be immunologically active, even if the name is "misleading." PMID:22832292

  1. Nerve growth factor binding domain of the nerve growth factor receptor

    SciTech Connect

    Welcher, A.A.; Bitler, C.M.; Radeke, M.J.; Shooter, E.M. )

    1991-01-01

    A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptors to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a K{sub d} comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cystein-rich sequence or the first and part of the second cystein-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.

  2. N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

    SciTech Connect

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang . E-mail: xudg@nic.bmi.ac.cn

    2007-06-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies.

  3. Role of growth factors in the growth of normal and transformed cells

    SciTech Connect

    Lokeshwar, V.B.

    1989-01-01

    Growth factors play an important role in the growth of normal cells. However, their untimely and/or excess production leads to neoplastic transformation. The role of growth factors in the growth of normal cells was studied by investigating the mechanism of transmodulation of the cell surface EGF receptor number by protamine. Protamine increased the EGF stimulated mitogenic response in Swiss mouse 3T3 cells and A431 cells by increasing the number of functionally active EGF receptors. Protamine also increased EGF receptor number in plasma membranes and solubilized membranes. This was evidenced by an increase in both {sup 125}I-EGF-EGF-receptor complex and EGF stimulated phosphorylation of the EGF receptor. The solubilized EGF receptor was retained on a protamine-agarose gel indicating that protamine might increase EGF receptor number by directly activating cryptic EGF receptors in the plasma membranes. The role of growth factors in neoplastic transformation was studied by investigating the role of the oncogene v-sis in the growth of Simian sarcoma virus (SSV) transformed cells. The product of the oncogene v-sis is 94% homologous to the B chain of PDGF. This study found that (i) v-sis gene product is synthesized as a 32 kDa unglycosylated monomer which is glycosylated, dimerized and proteolytically processed into p36, p72, p68, p58, p44 and p27 mol. wt. species respectively. (ii) p36, p72, p68 and p58 are very likely formed in the endoplasmic reticulum and/or Golgi complex. A fraction of newly synthesized p72, p68 and p58 is degraded intracellularly at a fast rate. (iii) p44 is a secretory product which remains tightly associated with the cell surface. p44 is recaptured by the cells through interaction with cell surface PDGF receptors and degraded into p27. (iv) During long term cultures p44 is extracellularly cleaved into a 27 kDa product.

  4. Heterogeneity of binding subunits of the human 150K insulin-like growth factor binding protein.

    PubMed

    Gelato, M C; Gaynes, L A; Greenstein, L A; Nissley, S P

    1990-04-01

    Models for the structure of the GH-dependent 150K insulin-like growth factor-binding protein (IGF-BP) complex include 1) a binding subunit of 40-60K mol wt associated with a larger nonbinding component, and 2) an oligomeric structure simply made up of six 25-28K monomeric IGF-BP complexes. To evaluate these alternative models we examined the IGF-binding characteristics and behavior on an SP-Sephadex ion exchange column of BP species identified by chemically cross-linking [125I]IGF-I and [125I]IGF-II. In addition, human serum was gel filtered on Sephadex G-200 in 0.05 M NH4HCO3, pH 8.0, and the 150K BP identified by binding of [125I]IGF-II to column fractions. When [125I]IGF-I or [125I]IGF-II was cross-linked to the 150K BP with disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10-15%) and autoradiography, four specifically labeled complexes of 20K, 24K, 33K, and 47K mol wt were identified. We examined the IGF-binding characteristics of these species by cross-linking [125I]IGF-I and [125I]IGF-II after incubation in the presence of increasing concentrations of unlabeled IGF-I or IGF-II. Formation of the 24K complex was inhibited more potently by IGF-II than IGF-I, whereas the relative potency of IGF-I vs. IGF-II for inhibition of the formation of the other complexes depended upon whether [125I]IGF-II or [125I]IGF-I was used. When the 150K BP complex generated from gel filtration on Sephadex G-200 was acid stripped, the only species seen with chemical cross-linking of either [125I]IGF-I or [125I]IGF-II was the 47K complex. By both conventional competitive binding studies and cross-linking [125I]IGF-I and [125I]IGF-II after incubation with increasing concentrations of unlabeled IGF-I or IGF-II, the formation of the 47K complex was usually more potently inhibited by IGF-I than IGF-II. When Cohn fraction IV extract was chromatographed on a SP-Sephadex column (pH 3) and cross-linking performed on the flow-through, the 47K

  5. Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

    NASA Technical Reports Server (NTRS)

    Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

    1999-01-01

    Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

  6. Growth hormone receptor/binding protein: Physiology and function

    SciTech Connect

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P.

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

  7. Nuclear translocation of type I transforming growth factor β receptor confers a novel function in RNA processing.

    PubMed

    Chandra, Manasa; Zang, Shengbing; Li, Haiqing; Zimmerman, Lisa J; Champer, Jackson; Tsuyada, Akihiro; Chow, Amy; Zhou, Weiying; Yu, Yang; Gao, Harry; Ren, Xiubao; Lin, Ren-Jang; Wang, Shizhen Emily

    2012-06-01

    Signaling of transforming growth factor β (TGF-β) is redirected in cancer to promote malignancy, but how TGF-β function is altered in a transformed cell is not fully understood. We investigated TGF-β signaling by profiling proteins that differentially bound to type I TGF-β receptor (TβRI) in nontransformed, HER2-transformed, and HER2-negative breast cancer cells using immunoprecipitation followed by protein identification. Interestingly, several nuclear proteins implicated in posttranscriptional RNA processing were uniquely identified in the TβRI coprecipitates from HER2-transformed cells. Ligand-inducible nuclear translocation of TβRI was observed only in transformed cells, and the translocation required importin β1, nucleolin, and Smad2/3. This trafficking was dependent on the high Ran GTPase activity resulting from oncogenic transformation. In the nucleus, TβRI associated with purine-rich RNA sequences in a synergistic manner with the RNA-binding factor hnRNP A1. We further found that nuclear translocation of TβRI specifically induced epidermal growth factor receptor (EGFR) transcript isoform c, which encodes a soluble EGFR protein, through alternative splicing or 3'-end processing. Our study confirms a cancer-specific nuclear translocation of TβRI and demonstrates its potential function in regulating nuclear RNA processing, as well as a novel gain-of-function mechanism of TGF-β signaling in cancer. PMID:22473997

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

    PubMed

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

    2009-04-10

    Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity. PMID:19218245

  9. Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cell-surface receptors.

    PubMed Central

    Raines, E W; Bowen-Pope, D F; Ross, R

    1984-01-01

    Evidence is presented that the binding of platelet-derived growth factor (PDGF) to plasma constituents inhibits the binding of PDGF to its cell-surface mitogen receptor. Approximately equivalent amounts of PDGF-binding activity were found in plasma from a number of different species known by radioreceptor assay to contain PDGF homologues in their clotted blood. Activation of the coagulation cascade did not significantly alter the PDGF-binding activity of the plasma components. Three molecular weight classes of plasma fractions that inhibit PDGF binding to its cell-surface receptor were defined by gel filtration: approximately equal to 40,000, 150,000, and greater than 500,000. Specific binding of 125I-labeled PDGF to the highest molecular weight plasma fraction could also be demonstrated by gel filtration. The binding of PDGF to these plasma components was reversible under conditions of low pH or with guanidine X HCl, and active PDGF could be recovered from the higher molecular weight fractions. Immunologic and functional evidence is presented that the highest molecular weight plasma fraction may be alpha 2-macroglobulin. A model is proposed in which the activity of PDGF released in vivo may be regulated by association with these plasma binding components and by high-affinity binding to cell-surface PDGF receptors. PMID:6203121

  10. Effects of transforming growth factor-beta on growth and differentiation of the continuous rat thyroid follicular cell line, FRTL-5

    SciTech Connect

    Morris, J.C. III; Ranganathan, G.; Hay, I.D.; Nelson, R.E.; Jiang, N.S.

    1988-09-01

    Transforming growth factor-beta (TGF beta) has been shown to influence the growth and differentiation of many widely varied cell types in vitro, including some that are endocrinologically active. We have investigated the previously unknown effects of this unique growth factor in the differentiated rat thyroid follicular cell line FRTL-5. The cells demonstrated specific, high affinity binding of TGF beta, and as with other epithelial cells, the growth of these thyroid follicular cells was potently inhibited by addition of TGF beta to the culture medium. TGF beta caused a significant reduction in TSH-sensitive adenylate cyclase activity in the cells. The addition of (Bu)2cAMP along with the growth factor to cultures partially reversed the characteristic morphological changes seen with TGF beta, but did not reverse the growth inhibition. To further investigate the possible mechanisms of the effects of TGF beta on the cells, we measured the influence of the growth factor on (125I)TSH binding. TGF beta did not compete for specific TSH-binding sites; however, exposure of the cells to TGF beta for 12 or more h resulted in a dose-dependent down-regulation of TSH receptors that was fully reversible. While cellular proliferation was potently inhibited by TGF beta, differentiated function, as manifest by iodine-trapping ability, was stimulated by the growth factor. This stimulation of iodine uptake was independent of, and additive to, the stimulatory effects of TSH. Finally, FRTL-5 cells in serum-free medium and in response to TSH were shown to secrete TGF beta-like activity that competed for (125I)TGF beta in a RRA. These studies suggest that TGF beta may represent an autocrine mechanism of controlling the growth response to TSH in thyroid follicular cells, while allowing the continuance of differentiated function.

  11. The Neuroprotective Functions of Transforming Growth Factor Beta Proteins

    PubMed Central

    Dobolyi, Arpád; Vincze, Csilla; Pál, Gabriella; Lovas, Gábor

    2012-01-01

    Transforming growth factor beta (TGF-β) proteins are multifunctional cytokines whose neural functions are increasingly recognized. The machinery of TGF-β signaling, including the serine kinase type transmembrane receptors, is present in the central nervous system. However, the 3 mammalian TGF-β subtypes have distinct distributions in the brain suggesting different neural functions. Evidence of their involvement in the development and plasticity of the nervous system as well as their functions in peripheral organs suggested that they also exhibit neuroprotective functions. Indeed, TGF-β expression is induced following a variety of types of brain tissue injury. The neuroprotective function of TGF-βs is most established following brain ischemia. Damage in experimental animal models of global and focal ischemia was shown to be attenuated by TGF-βs. In addition, support for their neuroprotective actions following trauma, sclerosis multiplex, neurodegenerative diseases, infections, and brain tumors is also accumulating. The review will also describe the potential mechanisms of neuroprotection exerted by TGF-βs including anti-inflammatory, -apoptotic, -excitotoxic actions as well as the promotion of scar formation, angiogenesis, and neuroregeneration. The participation of these mechanisms in the neuroprotective effects of TGF-βs during different brain lesions will also be discussed. PMID:22942700

  12. Transforming growth factor-betas and vascular disorders.

    PubMed

    Bobik, Alex

    2006-08-01

    Transforming growth factor-beta (TGF-beta) superfamily members, TGF-beta and bone morphogenetic proteins (BMPs), are potent regulatory cytokines with diverse functions on vascular cells. They signal through heteromeric type I and II receptor complexes activating Smad-dependent and Smad-independent signals, which regulate proliferation, differentiation, and survival. They are potent regulators of vascular development and vessel remodeling and play key roles in atherosclerosis and restenosis, regulating endothelial, smooth muscle cell, macrophage, T cell, and probably vascular calcifying cell responses. In atherosclerosis, TGF-beta regulates lesion phenotype by controlling T-cell responses and stimulating smooth muscle cells to produce collagen. It contributes to restenosis by augmenting neointimal cell proliferation and collagen accumulation. Defective TGF-beta signaling in endothelial cells attributable to mutations in endoglin or the type I receptor ALK-1 leads to hereditary hemorrhagic telangiectasia, whereas defective BMP signaling attributable to mutations in the BMP receptor II has been associated with development of primary pulmonary hypertension. The development of mouse models with either cell type-specific or general inactivation of TGF-beta/BMP signaling has started to reveal the importance of the regulatory network of TGF-beta/BMP pathways in vivo and their significance for atherosclerosis, hereditary hemorrhagic telangiectasia, and primary pulmonary hypertension. This review highlights recent findings that have advanced our understanding of the roles of TGF-beta superfamily members in regulating vascular cell responses and provides likely avenues for future research that may lead to novel pharmacological therapies for the treatment or prevention of vascular disorders. PMID:16675726

  13. Tissue localization of transforming growth factor-beta1 in pulmonary eosinophilic granuloma.

    PubMed

    Asakura, S; Colby, T V; Limper, A H

    1996-11-01

    Pulmonary eosinophilic granuloma is characterized by infiltration of the lungs with fibronodular lesions containing specialized Langerhans' cells. In some patients, progressive pulmonary fibrosis leads to significant respiratory impairment. Transforming growth factor-beta1 (TGF-beta1) promotes fibrosis by enhancing the synthesis of extracellular matrix components. The role of TGF-beta1 in promoting fibrosis in the setting of pulmonary eosinophilic granuloma is currently unknown. We used immunohistochemistry to evaluate the extent and distribution of TGF-beta1 and the extracellular matrix components type I collagen and decorin, a TGF-beta1-binding proteoglycan. Lung biopsies from 11 patients with pulmonary eosinophilic granuloma were evaluated. In biopsies with active inflammatory lesions containing Langerhans' cells, hyperplastic type 2 pneumocytes and alveolar macrophages within and surrounding the fibronodular lesions contained abundant TGF-beta1. Langerhans' cells were consistently devoid of immunoreactive TGF-beta1. Active inflammatory lesions also exhibited staining for decorin, in a loosely organized distribution. Advanced fibrotic lesions of eosinophilic granuloma, containing minimal inflammatory cells and few or no Langerhans' cells, exhibited weak or absent staining for TGF-beta1 within either hyperplastic type 2 pneumocytes or alveolar macrophages. The fibroconnective tissues of these advanced fibrotic lesions consistently revealed dense staining for decorin. Through their actions on extracellular matrix protein accumulation, TGF-beta1 and the TGF-beta1-binding proteoglycan decorin may modulate fibrotic repair accompanying pulmonary eosinophilic granuloma. PMID:8912775

  14. Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Hashiro, M.; Yoshimasa, H.; Yoshikawa, K. )

    1990-10-01

    The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of (3H)thymidine incorporation. The decrease of (3H)thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions.

  15. Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells

    PubMed Central

    Pi, Liya; Chung, Pei-Yu; Sriram, Sriniwas; Rahman, Masmudur M; Song, Wen-Yuan; Scott, Edward W; Petersen, Bryon E; Schultz, Gregory S

    2015-01-01

    AIM: To study the binding of connective tissue growth factor (CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor (PDGF)-B signaling. METHODS: The binding strengths of CTGF to cystine knot-containing growth factors including vascular endothelial growth factor (VEGF)-A, PDGF-B, bone morphogenetic protein (BMP)-4, and transforming growth factor (TGF)-β1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid β-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance (SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor β (PDGFRβ) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRβ and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis. RESULTS: In the LexA-based yeast two-hybrid system, cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein LexA. Yeast co-transformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine, tryptophan, and uracil. In liquid β-galactosidase assays, CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity, at 29.88 ± 0.91 fold above controls

  16. A tale of two paralogs: human Transformer2 proteins with differential RNA-binding affinities.

    PubMed

    Ghosh, Pritha; Grellscheid, Sushma Nagaraja; Sowdhamini, R

    2016-09-01

    The Transformer2 (Tra2) proteins in humans are homologues of the Drosophila Tra2 protein. One of the two RNA-binding paralogs, Tra2β, has been very well-studied over the past decade, but not much is known about Tra2α. It was very recently shown that the two proteins demonstrate the phenomenon of paralog compensation. Here, we provide a structural basis for this genetic backup circuit, using molecular modelling and dynamics studies. We show that the two proteins display similar binding specificities, but differential affinities to a short GAA-rich RNA stretch. Starting from the 6-nucleotide RNA in the solution structure, close to 4000 virtual mutations were modelled on RNA and the domain-RNA interactions were studied after energy minimisation to convergence. Separately, another known 13-nucleotide stretch was docked and the domain-RNA interactions were observed through a 100-ns dynamics trajectory. We have also demonstrated the 'compensatory' mechanism at the level of domains in one of the domain repeat-containing RNA-binding proteins. PMID:26414300

  17. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    SciTech Connect

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-04-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol /sup 125/I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function.

  18. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition.

    PubMed

    Green, M R; Couchman, J R

    1985-09-01

    Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map the distribution on frozen skin sections of an extracellular epitope on the EGF receptor. The [125I]EGF binding experiments showed accessible, unoccupied EGF receptors to be present on the epidermal basal cells (with reduced binding to spinous cells), the basal cells of the hair shaft and sebaceous gland, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing presumptive cortex cells, excluding the medulla, lying around and above the upper dermal papilla of anagen hair follicles, epithelial cells around the lower dermal papilla region, and in some tissue samples the cell margins of the viable differentiating layers of the epidermis. In a control study, to clarify whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar, indicating that, at least for SVK14 cells, EGF-R1 binding provides a reliable marker for EGF binding. Explanations for the discrepancies between these two methods for determining EGF receptor distribution in human skin are discussed, including the possibility that latent EGF receptors, unable to bind [125I]EGF, may be present in some differentiating epithelial compartments. PMID:2411822

  19. Transforming your professional self: encouraging lifelong personal and professional growth.

    PubMed

    Rodts, Mary F; Lamb, Karen V

    2008-01-01

    Transforming from student nurse to registered nurse is often discussed in a capstone class or a hospital orientation program. Changes in professional plans later in the career continuum often occur, but are not always planned. This article discusses the challenges of change, the need for career planning, stages of role acquisition, role socialization, and role transformation. In addition, it outlines the importance of creating a career plan to meet future career goals. PMID:18385597

  20. TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF)

    EPA Science Inventory

    TITLE:
    TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF). AUTHORS (ALL): Abbott, Barbara D.1; Best, Deborah S.1; Narotsky, Michael G.1. SPONSOR NAME: None INSTITUTIONS (ALL): 1. Repro Tox ...

  1. Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration.

    PubMed Central

    Kan, M; Huang, J S; Mansson, P E; Yasumitsu, H; Carr, B; McKeehan, W L

    1989-01-01

    Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation. Images PMID:2477840

  2. Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

    PubMed Central

    Riedel, H; Dull, T J; Honegger, A M; Schlessinger, J; Ullrich, A

    1989-01-01

    The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing. Images PMID:2583088

  3. RNA-binding motif protein 5 inhibits the proliferation of cigarette smoke-transformed BEAS-2B cells through cell cycle arrest and apoptosis.

    PubMed

    Lv, Xue-Jiao; Du, Yan-Wei; Hao, Yu-Qiu; Su, Zhen-Zhong; Zhang, Lin; Zhao, Li-Jing; Zhang, Jie

    2016-04-01

    Cigarette smoking has been shown to be the most significant risk factor for lung cancer. Recent studies have also indicated that RNA-binding motif protein 5 (RBM5) can modulate apoptosis and suppress tumor growth. The present study focused on the role of RBM5 in the regulation of cigarette smoke extract (CSE)-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. Herein, we exposed normal BEAS-2B cells for 8 days to varying concentrations of CSE or dimethylsulfoxide (DMSO), followed by a recovery period of 2 weeks. Next, the RBM5 protein was overexpressed in these transformed BEAS-2B cells though lentiviral infection. Later, the morphological changes, cell proliferation, cell cycle, apoptosis, invasion and migration were assessed. In addition, we analyzed the role of RBM5 in xenograft growth. The expression of RBM5 along with the genes related to cell cycle regulation, apoptosis and invasion were also examined. Finally, our results revealed that BEAS-2B cells exposed to 100 µg/ml CSE acquired phenotypic changes and formed tumors in nude mice, indicative of their cancerous transformation and had reduced RBM5 expression. Subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest, triggered apoptosis and inhibited their invasion and migration, including xenograft growth. Thus, we established an in vitro model of CSE-induced cancerous transformation and concluded that RBM5 overexpression inhibited the growth of these transformed cells through cell cycle arrest and induction of apoptosis. Therefore, our study suggests the importance of RBM5 in the pathogenesis of smoking-related cancer. PMID:26782095

  4. Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

    SciTech Connect

    Bikfalvi, A.; Dupuy, E.; Inyang, A.L.; Tobelem, G. ); Fayein, N.; Courtois, Y. ); Leseche, G. )

    1989-03-01

    The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells. The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely {sup 125}I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound {sup 125}I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At this temperature, degradation of the internalized ligand was followed after 1 hour by the appearance of three major bands of 15,000 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.

  5. Transforming Asian Pacific America: The Challenges of Growth and Diversity.

    ERIC Educational Resources Information Center

    Nakanishi, Don T.

    1994-01-01

    Examines the concept of Asian Pacific pan-ethnicity in light of the growing numbers and diversity of this population in the 1990s. While revised immigration laws, new immigrant groups, and continued hostility have brought new challenges, it is argued that Asian Pacific Americans can provide important leadership for the transformation of the United…

  6. Structural studies of neuropilin-2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket.

    PubMed

    Tsai, Yi-Chun Isabella; Fotinou, Constantina; Rana, Rohini; Yelland, Tamas; Frankel, Paul; Zachary, Ian; Djordjevic, Snezana

    2016-05-01

    Neuropilin-2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin-2 and its homologous protein neuropilin-1 have been implicated in cancers and infection. Molecular determinants of the ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterized a zinc ion binding site on human neuropilin-2. The neuropilin-2-specific zinc ion binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, remote from the neuropilin binding site for its physiological ligand, i.e. vascular endothelial growth factor. We also present an X-ray crystal structure of the neuropilin-2 b1 domain in a complex with the C-terminal sub-domain of VEGF-A. Zn(2+) binding to neuropilin-2 destabilizes the protein structure but this effect was counteracted by heparin, suggesting that modifications by glycans and zinc in the extracellular matrix may affect functional neuropilin-2 ligand binding and signalling activity. PMID:26991001

  7. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    SciTech Connect

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  8. Protection of transforming growth factor-beta 1 activity by heparin and fucoidan.

    PubMed

    McCaffrey, T A; Falcone, D J; Vicente, D; Du, B; Consigli, S; Borth, W

    1994-04-01

    The transforming growth factor-beta (TGF-beta) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-beta activity in vitro or in vivo. Our previous work indicated that 1) TGF-beta 1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and 2) heparin, and certain other polyanions, could block the binding of TGF-beta 1 to alpha 2-macroglobulin (alpha 2-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-beta 1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between 125I-TGF-beta 1 and activated alpha 2-M. Electrophoresis of 125I-TGF-beta 1 showed that fucoidan protects TGF-beta 1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-beta derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-beta, and purified TGF-beta 1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of 125I-TGF-beta 1 and doubled the amount of cell-associated 125I-TGF-beta 1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-beta activity. PMID:7511146

  9. Disruption of transforming growth factor beta signaling by a novel ligand-dependent mechanism.

    PubMed

    Fernandez, Tania; Amoroso, Stephanie; Sharpe, Shellyann; Jones, Gary M; Bliskovski, Valery; Kovalchuk, Alexander; Wakefield, Lalage M; Kim, Seong-Jin; Potter, Michael; Letterio, John J

    2002-05-20

    Transforming growth factor (TGF)-beta is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-beta receptors and one or more isoforms of TGF-beta, thus the synthesis and secretion of TGF-beta as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-beta results in TGF-beta ligand-receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-beta1 in the PCT binds to intracellular TGF-beta type II receptor (TbetaRII). Disruption of the expression of TGF-beta1 by antisense TGF-beta1 mRNA restores localization of TbetaRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TbetaRII (dnTbetaRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TbetaRII. Analysis of TGF-beta receptor-activated Smad2 suggests the intracellular ligand-receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-beta-receptor complex, and define a novel mechanism for modulating the TGF-beta signaling pathway. PMID:12021305

  10. COMPLEMENT C5 REGULATES THE EXPRESSION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS IN CHRONIC EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS

    PubMed Central

    Cudrici, Cornelia; Ito, Takahiro; Zafranskaia, Ekaterina; Weerth, Susanna; Rus, Violeta; Chen, Hegang; Niculescu, Florin; Soloviova, Katerina; Tegla, Cosmin; Gherman, Adrian; Raine, Cedric S.; Shin, Moon L.; Rus, Horea

    2008-01-01

    Complement activation plays a central role in autoimmune demyelination. To explore the possible effects of C5 on post-inflammatory tissue repair, we investigated the transcriptional profile induced by C5 in chronic experimental allergic encephalomyelitis (EAE) using oligonucleotide arrays. We used C5-deficient (C5-d) and C5-sufficient (C5-s) mice to compare the gene expression profile and we found that 390 genes were differentially regulated in C5-s mice as compared to C5-d mice during chronic EAE. Among them, a group of genes belonging to the family of insulin-like growth factor binding proteins (IGFBP) and transforming growth factor (TGF)-β3 were found most significantly differentially regulated by C5. The dysregulation of these genes suggests that these proteins might be responsible for the gliosis and lack of remyelination seen in C5-d mice with chronic EAE. PMID:18692252

  11. Osteogenic Responses in Fibroblasts Activated by Elastin Degradation Products and Transforming Growth Factor-β1

    PubMed Central

    Simionescu, Agneta; Simionescu, Dan T.; Vyavahare, Narendra R.

    2007-01-01

    Our objective was to establish the role of fibroblasts in medial vascular calcification, a pathological process known to be associated with elastin degradation and remodeling. Rat dermal fibroblasts were treated in vitro with elastin degradation products and transforming growth factor (TGF)-β1, factors usually present in deteriorated matrix environments. Cellular changes were monitored at the gene and protein level by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and von Kossa staining for calcium deposits. By 21 days, multicellular calcified nodules were formed in the presence of elastin degradation products and TGF-β1 separately and to a significantly greater extent when used together. Before mineralization, cells expressed α-smooth muscle actin and large amounts of collagen type I and matrix metalloproteinase-2, characteristic features of myofibroblasts, key elements in tissue remodeling and repair. Stimulated cells expressed increased levels of core-binding factor α1, osteocalcin, alkaline phosphatase, and osteoprotegerin, representative bone-regulating proteins. For most proteins analyzed, TGF-β1 synergistically amplified responses of fibroblasts to elastin degradation products. In conclusion, elastin degradation products and TGF-β1 promote myofibroblastic and osteogenic differentiation in fibroblasts. These results support the idea that elastin-related calcification involves dynamic remodeling events and suggest the possibility of a defective tissue repair process. PMID:17591959

  12. The type III transforming growth factor beta receptor regulates vascular and osteoblast development during palatogenesis

    PubMed Central

    Hill, Cynthia R.; Jacobs, Britni H.; Brown, Christopher B.; Barnett, Joey V.; Goudy, Steven L.

    2015-01-01

    Background Cleft palate occurs in up to 1:1000 live births and is associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palatal shelf elongation, elevation, and fusion. Transforming growth factor β (TGFβ) and bone morphogenetic protein 2 (BMP2) canonical signaling is required during each stage of palate development. The type III TGFβ receptor (TGFβR3) binds all three TGFβ ligands and BMP2, but its contribution to palatogenesis is unknown. Results The role of TGFβR3 during palate formation was found to be during palatal shelf elongation and elevation. Tgfbr3-/- embryos displayed reduced palatal shelf width and height, changes in proliferation and apoptosis, and reduced vascular and osteoblast differentiation. Abnormal vascular plexus organization as well as aberrant expression of arterial (Notch1, Alk1), venous (EphB4), and lymphatic (Lyve1) markers was also observed. Decreased osteoblast differentiation factors (Runx2, alk phos, osteocalcin, col1A1, and col1A2) demonstrated poor mesenchymal cell commitment to the osteoblast lineage within the maxilla and palatal shelves in Tgfbr3-/- embryos. Additionally, in vitro bone mineralization induced by osteogenic medium (OM+BMP2) was insufficient in Tgfbr3-/- palatal mesenchyme, but mineralization was rescued by overexpression of TGFβR3. Conclusions These data reveal a critical, previously unrecognized role for TGFβR3 in vascular and osteoblast development during palatogenesis. PMID:25382630

  13. Transforming growth factor receptor type II (ec-TβR II) behaves as a halophile.

    PubMed

    Saini, Komal; Khan, M Ashhar I; Chakrapani, Sumit; Deep, Shashank

    2015-01-01

    The members of transforming growth factor β family (TGF-β) are multifunctional proteins but their main role is to control cell proliferation and differentiation. Polypeptides of TGF-β family function by binding to two related, functionally distinct transmembrane receptor kinases, first to the type II (TβR II) followed by type I receptor (TβR I). The paper describes, in details, the stability of wt-ec-TβR II under different conditions. The stability of wt-ec-TβR II was observed at different pH and salt concentration using fluorescence spectroscopy. Stability of ec-TβR II decreases with decrease in pH. Interestingly, the addition of salt increases the stability of the TβRII at pH 5.0 as observed for halophiles. Computational analysis using DELPHI suggests that this is probably due to the decrease in repulsion between negatively charged residues at surface on the addition of salt. This is further confirmed by the change in the stability of receptor on mutation of some of the residues (D32A) at surface. PMID:25316422

  14. Characterization of insulin-like growth factor-binding proteins from sheep thyroid cells.

    PubMed

    Bachrach, L K; Liu, F R; Burrow, G N; Eggo, M C

    1989-12-01

    The insulin-like growth factors (IGFs) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when cross-linked to [125I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40-44 and 48-52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [125I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGF-stimulated binding proteins to 36-40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36-38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are non-glycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins

  15. The same site on the integrase-binding domain of lens epithelium–derived growth factor is a therapeutic target for MLL leukemia and HIV

    PubMed Central

    Murai, Marcelo J.; Pollock, Jonathan; He, Shihan; Miao, Hongzhi; Purohit, Trupta; Yokom, Adam; Hess, Jay L.; Muntean, Andrew G.; Grembecka, Jolanta

    2014-01-01

    Lens epithelium-derived growth factor (LEDGF) is a chromatin-associated protein implicated in leukemia and HIV type 1 infection. LEDGF associates with mixed-lineage leukemia (MLL) fusion proteins and menin and is required for leukemic transformation. To better understand the molecular mechanism underlying the LEDGF integrase-binding domain (IBD) interaction with MLL fusion proteins in leukemia, we determined the solution structure of the MLL-IBD complex. We found a novel MLL motif, integrase domain binding motif 2 (IBM2), which binds to a well-defined site on IBD. Point mutations within IBM2 abolished leukemogenic transformation by MLL-AF9, validating that this newly identified motif is essential for the oncogenic activity of MLL fusion proteins. Interestingly, the IBM2 binding site on IBD overlaps with the binding site for the HIV integrase (IN), and IN was capable of efficiently sequestering IBD from the menin-MLL complex. A short IBM2 peptide binds to IBD directly and inhibits both the IBD-MLL/menin and IBD-IN interactions. Our findings show that the same site on IBD is involved in binding to MLL and HIV-IN, revealing an attractive approach to simultaneously target LEDGF in leukemia and HIV. PMID:25305204

  16. DNA binding to crystalline silica characterized by Fourier-transform infrared spectroscopy.

    PubMed Central

    Mao, Y; Daniel, L N; Whittaker, N; Saffiotti, U

    1994-01-01

    The interaction of DNA with crystalline silica in buffered aqueous solutions at physiologic pH has been investigated by Fourier-transform infrared spectroscopy (FT-IR). In aqueous buffer, significant changes occur in the spectra of DNA and silica upon coincubation, suggesting that a DNA-silica complex forms as silica interacts with DNA. As compared to the spectrum of silica alone, the changes in the FT-IR spectrum of silica in the DNA-silica complex are consistent with an Si-O bond perturbation on the surface of the silica crystal. DNA remains in a B-form conformation in the DNA-silica complex. The most prominent changes in the DNA spectrum occur in the 1225 to 1000 cm-1 region. Upon binding, the PO2- asymmetric stretch at 1225 cm-1 is increased in intensity and slightly shifted to lower frequencies; the PO2- symmetric stretch at 1086 cm-1 is markedly increased in intensity and the band at 1053 cm-1, representing either the phosphodiester or the C-O stretch of DNA backbone, is significantly reduced in intensity. In D2O buffer, the DNA spectrum reveals a marked increase in intensity of the peak at 1086 cm-1 and a progressive decrease in intensity of the peak at 1053 cm-1 when DNA is exposed to increasing concentrations of silica. The carbonyl band at 1688 cm-1 diminishes and shifts to slightly lower frequencies with increasing concentrations of silica. The present study demonstrates that crystalline silica binds to the phosphate-sugar backbone of DNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7705292

  17. Stress-induced martensitic transformation in metastable austenitic stainless steels: Effect on fatigue crack growth rate

    NASA Astrophysics Data System (ADS)

    Khan, Z.; Ahmed, M.

    1996-04-01

    This paper addresses the influence of cyclic stress-induced martensitic transformation on fatigue crack growth rates in metastable austenitic stainless steels. At low applied stress and mean stress values in AISI type 301 stainless steel, fatigue crack growth rate is substantially retarded due to a cyclic stress-induced γ-α' and γ-ɛ martensitic transformation occurring at the crack-tip plastic zone. It is suggested that the transformation products produce a compressive residual stress at the tip of the fatigue crack, which essentially lowers the effective stress intensity and hence retards the fatigue crack growth rate. At high applied stress or mean stress values, fatigue crack growth rates in AISI type 301 steels become almost equal to those of stable AISI type 302 alloy. As the amount of transformed products increases (with an increase in applied or mean stress), the strain-hardening effect brought about by the transformed martensite phase appears to accelerate fatigue crack growth, offsetting the contribution from the compressive residual stress produced by the positive volume change of γ → α' or ɛ transformation.

  18. Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells.

    PubMed

    Gentilini, A; Feliers, D; Pinzani, M; Woodruff, K; Abboud, S

    1998-02-01

    Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-beta (TGF-beta) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-beta stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-beta is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal

  19. Regulation of type II transforming-growth-factor-beta receptors by protein kinase C iota.

    PubMed Central

    Chuang, Lea-Yea; Guh, Jinn-Yuh; Liu, Shu-Fen; Hung, Min-Yuan; Liao, Tung-Nan; Chiang, Tai-An; Huang, Jau-Shyang; Huang, Yu-Lun; Lin, Chi-Fong; Yang, Yu-Lin

    2003-01-01

    TGF-beta (transforming growth factor-beta) is implicated in the pathogenesis of diabetic nephropathy. We previously demonstrated that up-regulation of type II TGF-beta receptor (TbetaRII) induced by high glucose might contribute to distal tubular hypertrophy [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182-193]. We have elucidated the mechanism by using cultured Madin-Darby canine kidney cells. Enhancer assay and electrophoretic-mobility-shift assay were used to estimate the involvement of transcription factors. Western blotting and an in vitro kinase assay were used to evaluate the level and activity of protein kinase. We showed that glucose (100-900 mg/dl) induced an increase in mRNA level and promoter activity of TbetaRII (note: 'mg/dl' are the units commonly used in diabetes studies). The promoter region -209 to -177 appeared to contribute to positive transactivation of TbetaRII promoter by comparing five TbetaRII-promoter-CAT (chloramphenicol acetyl-transferase) plasmids. Moreover, the transcription factor AP-1 (activator protein 1) was significantly activated and specifically binds to TbetaRII promoter (-209 to -177). More importantly, we found that atypical PKC iota might be pivotal for high glucose-induced increase in both AP-1 binding and TbetaRII promoter activity. First, high glucose induced cytosolic translocation, activation and autophosphorylation of PKC iota. Secondly, antisense PKC iota expression plasmids attenuated high-glucose-induced increase in AP-1 binding and TbetaRII promoter activity; moreover, sense PKC iota expression plasmids enhanced these instead. Finally, we showed that antisense PKC iota expression plasmids might partly attenuate a high-glucose/TGF-beta1-induced increase in fibronectin. We conclude that PKC iota might mediate high-glucose-induced increase in TbetaRII promoter activity. In addition, antisense PKC iota expression plasmid effectively suppressed up-regulation of TbetaRII and

  20. Protein kinase A modulates transforming growth factor-β signaling through a direct interaction with Smad4 protein.

    PubMed

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

    2013-03-22

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

  1. The myelin oligodendrocyte glycoprotein directly binds nerve growth factor to modulate central axon circuitry

    PubMed Central

    Mei, Feng; Greenfield, Ariele; Jahn, Sarah; Shen, Yun-An A.; Reid, Hugh H.; McKemy, David D.

    2015-01-01

    Myelin oligodendrocyte glycoprotein (MOG) is a central nervous system myelin-specific molecule expressed on the outer lamellae of myelin. To date, the exact function of MOG has remained unknown, with MOG knockout mice displaying normal myelin ultrastructure and no apparent specific phenotype. In this paper, we identify nerve growth factor (NGF) as a binding partner for MOG and demonstrate that this interaction is capable of sequestering NGF from TrkA-expressing neurons to modulate axon growth and survival. Deletion of MOG results in aberrant sprouting of nociceptive neurons in the spinal cord. Binding of NGF to MOG may offer widespread implications into mechanisms that underlie pain pathways. PMID:26347141

  2. Mitogenic and metabolic actions of epidermal growth factor on rat articular chondrocytes: modulation by fetal calf serum, transforming growth factor-beta, and tyrphostin.

    PubMed

    Ribault, D; Khatib, A M; Panasyuk, A; Barbara, A; Bouizar, Z; Mitrovic, R D

    1997-01-15

    The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [3H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [35S]sulfate incorporation, and collagen synthesis by [3H]proline incorporation. The presence of specific receptors was confirmed by [125I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5-2.5 ng/ml) stimulated [3H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low (< or =1%) and was lost at high (> or =7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-beta and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites (Kd 0.18 nM). There were about 4.5 x 10(9) binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultured cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF-R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pathological conditions. PMID:9016808

  3. Substrate flexibility regulates growth and apoptosis of normal but not transformed cells

    NASA Technical Reports Server (NTRS)

    Wang, H. B.; Dembo, M.; Wang, Y. L.

    2000-01-01

    One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.

  4. Estimating binding free energy of a putative growth factors EGF-VEGF complex - a computational bioanalytical study.

    PubMed

    Lin, Meng-Han; Chang, C Allen; Fischer, Wolfgang B

    2016-08-01

    Epidermal growth factor (EGF) and homodimeric vascular endothelial growth factor (VEGF) bind to cell surface receptors. They are responsible for cell growth and angiogenesis, respectively. Docking of the individual proteins as monomeric units using ZDOCK 2.3.2 reveals a partial blocking of the receptor binding site of VEGF by EGF. The receptor binding site of EGF is not affected by VEGF. The calculated binding energy is found to be intermediate between the binding energies calculated for Alzheimer's Aß42 and the barnase/barstar complex. PMID:26338536

  5. Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

    PubMed

    2003-01-01

    Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is

  6. Non-instantaneous growth characteristics of martensitic transformation in high Cr ferritic creep-resistant steel

    NASA Astrophysics Data System (ADS)

    Liu, Chenxi; Shao, Yi; Chen, Jianguo; Liu, Yongchang

    2016-08-01

    Microstructural observation and high-resolution dilatometry were employed to investigate kinetics of martensitic transformation in high Cr ferritic creep-resistant steel upon different quenching/cooling rates. By incorporating the classical athermal nucleation and impingement correction, a non-instantaneous growth model for martensitic transformation has been developed. The developed model describes austenite/martensite interface mobility during martensite growth. The growth rate of martensite is found to be varied from 1 × 10-6 to 3 × 10-6 m/s. The low interface mobility suggests that it is not appropriate to presume the instantaneous growth behavior of martensite. Moreover, based on the proposed model, nucleation rate of martensite under different cooling rates is found to be nearly the same, while the growth rate of martensite is promoted by increasing the cooling rate.

  7. Insulin-like growth factor-binding protein-3 inhibition of prostate cancer growth involves suppression of angiogenesis.

    PubMed

    Liu, B; Lee, K-W; Anzo, M; Zhang, B; Zi, X; Tao, Y; Shiry, L; Pollak, M; Lin, S; Cohen, P

    2007-03-15

    Insulin-like growth factor-binding protein-3 (IGFBP-3) is a multifunctional protein that induces apoptosis utilizing both insulin-like growth factor receptor (IGF)-dependent and -independent mechanisms. We investigated the effects of IGFBP-3 on tumor growth and angiogenesis utilizing a human CaP xenograft model in severe-combined immunodeficiency mice. A 16-day course of IGFBP-3 injections reduced tumor size and increased apoptosis and also led to a reduction in the number of vessels stained with CD31. In vitro, IGFBP-3 inhibited both vascular endothelial growth factor- and IGF-stimulated human umbilical vein endothelial cells vascular network formation in a matrigel assay. This action is primarily IGF independent as shown by studies utilizing the non-IGFBP-binding IGF-1 analog Long-R3. Additionally, we used a fibroblast growth factor-enriched matrigel-plug assay and chick allantoic membrane assays to show that IGFBP-3 has potent antiangiogenic actions in vivo. Finally, overexpression of IGFBP-3 or the non-IGF-binding GGG-IGFBP-3 mutant in Zebrafish embryos confirmed that both IGFBP-3 and the non-IGF-binding mutant inhibited vessel formation in vivo, indicating that the antiangiogenic effect of IGFBP-3 is an IGF-independent phenomenon. Together, these studies provide the first evidence that IGFBP-3 has direct, IGF-independent inhibitory effects on angiogenesis providing an additional mechanism by which it exerts its tumor suppressive effects and further supporting its development for clinical use in the therapy of patients with prostate cancer. PMID:16983336

  8. Bereavement and transformation: a psycho-spiritual and post-traumatic growth perspective.

    PubMed

    Bray, Peter

    2013-09-01

    In the aftermath of loss, it is not uncommon for individuals to disclose psycho-spiritual experiences that lead to personal changes and psychological growth. This article argues for and outlines a broad conceptual framework that captures and acknowledges the significance of these subjective experiences. The model synthesises and integrates two approaches to healthy growth: Lawrence Calhoun and Richard Tedeschi's post-traumatic growth model and Stanislav and Christina Grof's model of psycho-spiritual transformation. A rationale for a broader model and underpinning theory is briefly discussed, and their shared understandings about the human potential for growth in the struggle through loss are explored. PMID:21948147

  9. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

    SciTech Connect

    Turley, E.A.; Moore, D.; Hayden, L.J.

    1987-06-02

    A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.

  10. Regulation of Transforming Growth Factor-β1–driven Lung Fibrosis by Galectin-3

    PubMed Central

    MacKinnon, Alison C.; Gibbons, Michael A.; Farnworth, Sarah L.; Leffler, Hakon; Nilsson, Ulf J.; Delaine, Tamara; Simpson, A. John; Forbes, Stuart J.; Hirani, Nik; Gauldie, Jack

    2012-01-01

    Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. Objectives: To examine the role of galectin-3 in pulmonary fibrosis. Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF. Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF. Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF. PMID:22095546

  11. A glycosylation-deficient endothelial cell mutant with modified responses to transforming growth factor-beta and other growth inhibitory cytokines: evidence for multiple growth inhibitory signal transduction pathways.

    PubMed Central

    Fafeur, V; O'Hara, B; Böhlen, P

    1993-01-01

    An endothelial cell line (M40) resistant to growth inhibition by transforming growth factor-beta type 1 (TGF beta 1) was isolated by chemical mutagenesis and growth in the presence of TGF beta 1. Like normal endothelial cells, this mutant is characterized by high expression of type II TGF beta receptor and low expression of type I TGF beta receptor. However, the mutant cells display a type II TGF beta receptor of reduced molecular weight as a result of a general defect in N-glycosylation of proteins. The alteration does not impair TGF beta 1 binding to cell surface receptors or the ability of TGF beta 1 to induce fibronectin or plasminogen activator inhibitor-type I production. M40 cells were also resistant to growth inhibition by tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) but were inhibited by interferon-gamma (IFN gamma) and heparin. These results imply that TGF beta 1, TNF alpha, and IL-1 alpha act through signal transducing pathways that are separate from pathways for IFN gamma and heparin. Basic fibroblast growth factor was still mitogenic for M40, further suggesting that TGF beta 1, TNF alpha, and IL-1 alpha act by direct inhibition of cell growth rather than by interfering with growth stimulatory pathways. Images PMID:8382975

  12. Heparin-Binding Epidermal Growth Factor-like Growth Factor/Diphtheria Toxin Receptor in Normal and Neoplastic Hematopoiesis

    PubMed Central

    Vinante, Fabrizio; Rigo, Antonella

    2013-01-01

    Heparin-binding EGF-like growth factor (HB-EGF) belongs to the EGF family of growth factors. It is biologically active either as a molecule anchored to the membrane or as a soluble form released by proteolytic cleavage of the extracellular domain. HB-EGF is involved in relevant physiological and pathological processes spanning from proliferation and apoptosis to morphogenesis. We outline here the main activities of HB-EGF in connection with normal or neoplastic differentiative or proliferative events taking place primitively in the hematopoietic microenvironment. PMID:23888518

  13. Effects of class I heparin binding growth factor and fibronectin on platelet adhesion and aggregation

    SciTech Connect

    Greisler, H.P.; Klosak, J.J.; Steinam, S.J.; Lam, T.M.; Burgess, W.H.; Kim, D.U. )

    1990-05-01

    Fibronectin and heparin binding growth factor-type 1 have been affixed to vascular graft surfaces to enhance the attachment and the proliferation of transplanted endothelial cells, respectively. The current study examines the effect of fibronectin and heparin binding growth factor-type 1 on platelet adhesion and activation in vivo and on platelet aggregation in vitro. Expanded polytetrafluoroethylene prostheses (5 cm x 4 mm internal diameter) were treated either with fibronectin (n = 9), fibronectin/heparin/heparin binding growth factor-type 1/heparin (n = 12), or neither (n = 13) and were interposed into canine aortoiliac systems bilaterally. Autogenous radiolabeled (Indium 111 oxine, 650 microCi) platelets were injected intravenously before reestablishment of circulation. Perfusion was maintained for 30 minutes, and prostheses were removed with segments of native aorta and distal iliac arteries bilaterally. Specimens were examined for thrombus-free surface area, by gamma well counting for adherent radiolabeled platelets, and by light microscopy and transmission and scanning electron microscopic techniques. Results showed that both the fibronectin and fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained significantly greater numbers of platelets and adherent radioactivity than did control graft segments when normalized to their ipsilateral iliac arteries. Fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained 27 +/- 16 times more radioactivity per square millimeter than ipsilateral iliac arteries, fibronectin pretreated prostheses had 13 +/- 8 times more radioactivity per square millimeter than ipsilateral iliac arteries, and untreated expanded polytetrafluoroethylene had 4 +/- 3 times more radioactivity per square millimeter than ipsilateral iliac arteries.

  14. TEM Study of the Growth Mechanism, Phase Transformation, and Core/shell Structure of Semiconductor Nanowires

    NASA Astrophysics Data System (ADS)

    Wong, Tai Lun

    In this thesis, the fabrication and characterization of one-dimensional nanostructures have been studied systematically to understand the growth mechanism and structure transformation of one-dimensional nanostructures. The growth behavior of the ultrathin ZnSe nanowires with diameter less than 60 nm was found to be different from classical vapor-liquid-solid (VLS) process. The growth rate increases when the diameter of nanowires decreases, in contrast to the classical VLS process in which the growth rate increases with the diameter. The nucleation, initial growth, growth rates, defects, interface structures and growth direction of the nanowires were investigated by high resolution transmission electron microscopy (HRTEM). We found the structure and growth direction of ultra-thin nanowires are highly sensitive to growth temperatures and diameters of nanowires. At a low growth temperature (380°C), the growth direction for most nanowires is along <111>. Planar defects were found throughout the nanowires. At a high growth temperature (530°C), uniform nanowires with diameters around 10nm were grown along <110> and <112> directions, and the nanowires with diameters larger than 20nm were mainly grown along <111> direction. The possible growth mechanism of ultrathin nanowires was proposed by combining the solid catalytic growth with the interface diffusion theory, in order to explain how the growth temperature and the size of the catalysts influent the morphology, growth direction and growth rate of ultrathin nanowires. Structural and phase transformation of a nickel coated Si nanowire to NiSi2/SiC core-shell nanowire heterostructures has been investigated by the in-situ Transmission Electron Microscope (TEM). The phase transformation is a single-site nucleation process and therefore a single crystalline NiSi2 core resulted in the core-shell nanowire heterostructures. The transformation of the Si nanowire to NiSi2/SiC core-shell nanowire heterostructures was extremely

  15. Heparin Binding Epidermal Growth Factor Like Growth Factor Heals Chronic Tympanic Membrane Perforations With Advantage Over Fibroblast Growth Factor 2 and Epidermal Growth Factor in an Animal Model

    PubMed Central

    Santa Maria, Peter Luke; Weierich, Kendall; Kim, Sungwoo; Yang, Yunzhi Peter

    2016-01-01

    Hypothesis That heparin binding epidermal growth factor like growth factor (HB-EGF) heals chronic tympanic membrane (TM) perforations at higher rates than fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) in an animal model. Background A non-surgical treatment for chronic TM perforation would benefit those unable to access surgery or those unable to have surgery, as well as reducing the cost of tympanoplasty. Growth factor (GF) treatments have been reported in the literature with variable success with the lack of a suitable animal providing a major obstacle. Methods The GFs were tested in a validated mouse model of chronic TM perforation. A bio absorbable hydrogel polymer was used to deliver the GF at a steady concentration as it dissolved over four weeks. A control (polymer only, n=18) was compared to polymer loaded with HB-EGF (5ug/ml, n=18), FGF2 (100ug/ml, n=19) and EGF (250ug/ml, n=19). Perforations were inspected at four weeks. Results The healing rates, as defined as one hundred percent perforation closure, were control (5/18, 27.8%), HB-EGF (15/18, 83.3%), FGF2 (6/19, 31.6%) and EGF (3/19, 15.8%). There were no differences between FGF2 (p=0.80) and EGF (p=0.31) with control healing rates. HB-EGF (p= 0.000001) showed a significant difference for healing. The HB-EGF healed TMs showed layers similar to a normal TM, whilst the other groups showed a lack of epithelial migration. Conclusion This study confirms the advantage of HB-EGF over two other commonly used growth factors and is a promising non-surgical treatment of chronic TM perforations. PMID:26075672

  16. Interaction of AIM with insulin-like growth factor-binding protein-4.

    PubMed

    You, Qiang; Wu, Yan; Yao, Nannan; Shen, Guannan; Zhang, Ying; Xu, Liangguo; Li, Guiying; Ju, Cynthia

    2015-09-01

    Apoptosis inhibitor of macrophages (AIM/cluster of differentiation 5 antigen-like/soluble protein α) has been shown to inhibit cellular apoptosis; however, the underlying molecular mechanism has not been elucidated. Using yeast two‑hybrid screening, the present study uncovered that AIM binds to insulin‑like growth factor binding protein‑4 (IGFBP‑4). AIM interaction with IGFBP‑4, as well as IGFBP‑2 and ‑3, but not with IGFBP‑1, ‑5 and ‑6, was further confirmed by co‑immunoprecipitation (co‑IP) using 293 cells. The binding activity and affinity between AIM and IGFBP‑4 in vitro were analyzed by co‑IP and biolayer interferometry. Serum depletion‑induced cellular apoptosis was attenuated by insulin‑like growth factor‑I (IGF‑I), and this effect was abrogated by IGFBP‑4. Of note, in the presence of AIM, the inhibitory effect of IGFBP‑4 on the anti‑apoptosis function of IGF‑I was attenuated, possibly through binding of AIM with IGFBP‑4. In conclusion, to the best of our knowledge, the present study provides the first evidence that AIM binds to IGFBP‑2, ‑3 and ‑4. The data suggest that this interaction may contribute to the mechanism of AIM-mediated anti-apoptosis function. PMID:26135353

  17. Integrin α7 Binds Tissue Inhibitor of Metalloproteinase 3 to Suppress Growth of Prostate Cancer Cells

    PubMed Central

    Tan, Lang-Zhu; Song, Yang; Nelson, Joel; Yu, Yan P.; Luo, Jian-Hua

    2014-01-01

    Integrin α7 (ITGA7) is a tumor-suppressor gene that is critical for suppressing the growth of malignant tumors; however, the mechanisms allowing ITGA7 to suppress the growth of cancer cells remain unclear. Herein, we show that ITGA7 binds to tissue inhibitor of metalloproteinase 3 (TIMP3) in prostate cancer cells. The ITGA7-TIMP3 binding led to a decreased protein level of tumor necrosis factor α, cytoplasmic translocation of NF-κB, and down-regulation of cyclin D1. These changes led to an accumulation of cells in G0/G1 and a dramatic suppression of cell growth. Knocking down TIMP3 or ITGA7/TIMP3 binding interference largely abrogated the signaling changes induced by ITGA7, whereas a mutant ITGA7 lacking TIMP3 binding activity had no tumor-suppressor activity. Interestingly, knocking down ITGA7 ligand laminin β1 enhanced ITGA7-TIMP3 signaling and the downstream tumor-suppressor activity, suggesting the existence of a counterbalancing role between extracellular matrix and integrin signaling. As a result, this report demonstrates a novel and critical signaling mechanism of ITGA7, through the TIMP3/NF-κB/cyclin D1 pathway. PMID:23830872

  18. Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis

    SciTech Connect

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-03-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that (/sup 125/I)EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

  19. Effects of transformed ferrite growth on the tensile fracture characteristics of a dual-phase steel

    NASA Astrophysics Data System (ADS)

    Jeong, W. C.; Kim, C. H.

    1988-02-01

    The effects of transformed ferrite growth on the tensile fracture characteristics of a dual-phase steel were investigated by observing crack initiation, propagation, and fracture behaviors. Crack initiation occurred by decohesion between martensite and ferrite. However, cracks propagated along the ferrite-martensite interface in a high temperature quenched specimen, whereas in specimens quenched from lower temperature cracks propagated into the martensite particle. Tensile fracture behaviors were not strongly influenced by the cooling rate. At both cooling rates of 5.6 and 0.1 °C/sec, specimens quenched from high temperature fractured by partially brittle fracture mode, but fracture mode changed to ductile mode as the quenching temperature decreased. The effect of transformed ferrite on the fracture mode was not substantially different from that of retained ferrite. However, the crack initiation and propagation was influenced by the variation in martensite distribution caused by different growth behavior of transformed ferrite.

  20. Stable Glass Transformation to Supercooled Liquid via Surface-Initiated Growth Front

    SciTech Connect

    Swallen, Stephen F.; Traynor, Katherine; McMahon, Robert J.; Ediger, M. D.; Mates, Thomas E.

    2009-02-13

    Highly stable glasses of tris-naphthylbenzene transform into a liquid when annealed above the glass transition temperature T{sub g}. In contrast to the predictions of standard models, the observed transformation is spatially inhomogeneous. Secondary ion mass spectrometry experiments on isotopically labeled multilayer films show that the liquid grows into the stable glass with sharp growth fronts initiated at the free surface and at the interface with the substrate. For the free surface, the growth velocity is constant in time and has the same temperature dependence as self-diffusion in the equilibrium supercooled liquid. These stable glasses are packed so efficiently that surfaces and interfaces are required to initiate the transformation to the liquid even well above T{sub g}.

  1. Interaction of transforming growth factor-beta-1 with alpha-2-macroglobulin from normal and inflamed equine joints.

    PubMed Central

    Coté, N; Trout, D R; Hayes, M A

    1998-01-01

    Binding between equine plasma alpha-2-macroglobulin (alpha 2M) and several cytokines known to participate in inflammatory reactions in other species was initially examined. Plasma was obtained from 5 horses with various abnormalities. Samples, both untreated and after reaction with methylamine, were incubated with exogenous, radiolabeled, porcine-derived transforming growth factor-beta-1 (125I-TGF-beta 1), recombinant human interleukin-1-beta (125I-IL-1 beta), and recombinant human tumor necrosis factor-alpha (125I-rhTNF-alpha). They were then subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE). Binding of the native (slow) and activated (fast) forms of alpha 2M to each cytokine was subjectively evaluated with autoradiography. Equine alpha 2M bound 125I-TGF-beta 1. However, poor or no binding was observed between alpha 2M and either of 125I-rhTNF-alpha or 125I-IL-1 beta. Synovial fluid was then obtained from 6 normal horses, 6 horses with septic arthritis, and 6 horses with degenerative joint disease. Untreated and methylamine-reacted samples were quantitatively examined for binding with 125I-TGF-beta 1, using the autoradiographic techniques described above and densitometry. Native and activated alpha 2M were also quantified by densitometry of PAGE gels. Native alpha 2M was significantly elevated in septic arthritis (6.4% to 29.5% of total protein detected) and degenerative joint disease (2.8% to 12.3%), compared to normal joints (0.9% to 4.2%). Activated alpha 2M, however, was not detected in untreated synovial fluid samples. In all plasma and joint fluid samples, whether untreated or reacted with methylamine, 125I-TGF-beta 1 bound predominantly to alpha 2M, and preferentially to the activated form of alpha 2M. In synovial fluid, the amount of 125I-TGF-beta 1 binding was proportional to the quantity of alpha 2M present. These results indicate that: 1) equine alpha 2M binds TGF-beta 1; 2) the native form of alpha 2M is present in both equine plasma

  2. Binding proteins for growth hormone and prolactin in rabbit kidney cytosol

    SciTech Connect

    Herington, A.C.; Stevenson, J.L.; Ymer, S.I. )

    1988-09-01

    Two soluble, receptor-like binding proteins with apparent somatotrophic (growth hormone (GH)) and lactogenic (prolactin (PRL)) specificities, respectively, and that are present in rabbit kidney cytosol have now been examined in more detail using specific GH receptor and PRL receptor monoclonal antibodies (MAb). Gel chromatography of {sup 125}I-labeled human GH ({sup 125}I-hGH) kidney cytosol complexes in the absence of these MAbs revealed two specifically bound regions of radioactivity at molecular weights (MW) of {approximately}120,000 and {approximately}60,000, which are similar in size to complexes formed by the native GH receptor of rabbit liver cytosol and the PRL receptor of mammary gland. Co-incubation with GH-receptor MAb inhibited {sup 125}I-hGH binding only to the higher MW (120,000) species, whereas the PRL-receptor MAb inhibited only the lower MW (60,000) species, thus establishing definitively the hormonal specificities of the two binding proteins. The presence of both GH- and PRL-specific binding subunits in cytosol was confirmed using covalent cross-linking techniques. No GH binding protein was detected in kidney membranes. The presence of naturally soluble, receptor-like binding proteins for GH and PRL in kidney cytosol preparations raises the possibility of their playing a role in the intracellular regulation of kidney function and/or metabolism.

  3. Binding of the growth factor glycyl-L-histidyl-L-lysine by heparin.

    PubMed

    Rabenstein, D L; Robert, J M; Hari, S

    1995-12-01

    Evidence is presented that the growth factor glycyl-histidyl-lysine (GHK) binds to heparin, and the interaction has been characterized by [1H]NMR spectroscopy. 1H chemical shifts indicate that GHK interacts with both the carboxylic acid and the carboxylate forms of heparin. The chemical shift data are consistent with a weak delocalized binding of the triprotonated (ImH+, GlyNH3+, LysNH3+) form of GHK by the carboxylic acid form of heparin. As the pD is increased and the carboxylic acid groups are titrated, chemical shift data indicate that ammonium groups of GHK are hydrogen bonded to heparin carboxylate groups, while the histidyl imidazolium ring occupies the imidazolium-binding site of heparin. Evidence for site-specific binding includes displacement of chemical shift titration curves for heparin to lower pD, increased shielding of specific heparin protons by the imidazolium ring current and displacement of chemical shift titration curves for GHK to higher pD. Specific binding constants were determined for binding of the (ImH+, GlyNH3+), LysNH3+) forms of GHK by the carboxylate form of heparin from chemical shift vs. pD titration data. PMID:7498545

  4. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    PubMed Central

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

  5. Novel Bioluminescent Binding Assays for Ligand-Receptor Interaction Studies of the Fibroblast Growth Factor Family.

    PubMed

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand-receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand-receptor interaction studies. PMID:27414797

  6. Atomic mechanisms of diffusional nucleation and growth and comparisons with their counterparts in shear transformations

    NASA Astrophysics Data System (ADS)

    Aaronson, Hubert I.

    1993-02-01

    An integrated overview is presented of a viewpoint on the present understanding of nucleation and growth mechanisms in both diffusional and shear (martensitic) transformations. Special emphasis is placed on the roles played by the anisotropy of interphase boundary structure and energy and also upon elastic shear strain energy in both types of transformation. Even though diffusional nucleation is based on random statistical fluctuations, use of the time reversal principle shows that interfacial energy anisotropy leads to accurately reproducible orientation relationships and hence to partially or fully coherent boundaries, even when nucleation at a grain boundary requires an irrational orientation relationship to obtain. Since the fully coherent boundary areas separating most linear misfit compensating defects are wholly immobile during diffusional growth because of the improbability of moving substitutional atoms even temporarily into interstitial sites under conditions normally encountered, partially and fully coherent interphase boundaries should be immovable without the intervention of growth ledges. These ledges, however, must be heavily kinked and usually irregular in both spacing and path if they, too, are not to be similarly trapped. On the other hand, the large shear strain energy usually associated with martensite requires that its formation be initiated through a process which avoids the activation barrier associated with nucleation, perhaps by the Olson-Cohen matrix dislocation rearrangement mechanism. During growth, certain ledges on martensite plates serve as transformation dislocations and perform the crystal structure change (Bain strain). However, the terraces between these ledges in martensite (unlike those present during diffusional growth) are also mobile during non-fcc/hcp transformations; glissile dislocations on these terraces perform the lattice invariant deformation. Growth ledges operative during both diffusional and shear growth probably

  7. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA

    PubMed Central

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-01-01

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein–protein interactions. In order to isolate the protein–DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1–VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1–VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. PMID:26044711

  8. The transforming growth factor beta type II receptor can replace the activin type II receptor in inducing mesoderm.

    PubMed Central

    Bhushan, A; Lin, H Y; Lodish, H F; Kintner, C R

    1994-01-01

    The type II receptors for the polypeptide growth factors transforming growth factor beta (TGF-beta) and activin belong to a new family of predicted serine/threonine protein kinases. In Xenopus embryos, the biological effects of activin and TGF-beta 1 are strikingly different; activin induces a full range of mesodermal cell types in the animal cap assay, while TGF-beta 1 has no effects, presumably because of the lack of functional TGF-beta receptors. In order to assess the biological activities of exogenously added TGF-beta 1, RNA encoding the TGF-beta type II receptor was introduced into Xenopus embryos. In animal caps from these embryos, TGF-beta 1 and activin show similar potencies for induction of mesoderm-specific mRNAs, and both elicit the same types of mesodermal tissues. In addition, the response of animal caps to TGF-beta 1, as well as to activin, is blocked by a dominant inhibitory ras mutant, p21(Asn-17)Ha-ras. These results indicate that the activin and TGF-beta type II receptors can couple to similar signalling pathways and that the biological specificities of these growth factors lie in their different ligand-binding domains and in different competences of the responding cells. Images PMID:8196664

  9. The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic Translation Initiation Factor 4A Binding Protein That Inhibits Translation

    PubMed Central

    Yang, Hsin-Sheng; Jansen, Aaron P.; Komar, Anton A.; Zheng, Xiaojing; Merrick, William C.; Costes, Sylvain; Lockett, Stephen J.; Sonenberg, Nahum; Colburn, Nancy H.

    2003-01-01

    Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5′ mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4D418A, a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4D418A bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1. PMID:12482958

  10. TRANSFORMATION

    SciTech Connect

    LACKS,S.A.

    2003-10-09

    Transformation, which alters the genetic makeup of an individual, is a concept that intrigues the human imagination. In Streptococcus pneumoniae such transformation was first demonstrated. Perhaps our fascination with genetics derived from our ancestors observing their own progeny, with its retention and assortment of parental traits, but such interest must have been accelerated after the dawn of agriculture. It was in pea plants that Gregor Mendel in the late 1800s examined inherited traits and found them to be determined by physical elements, or genes, passed from parents to progeny. In our day, the material basis of these genetic determinants was revealed to be DNA by the lowly bacteria, in particular, the pneumococcus. For this species, transformation by free DNA is a sexual process that enables cells to sport new combinations of genes and traits. Genetic transformation of the type found in S. pneumoniae occurs naturally in many species of bacteria (70), but, initially only a few other transformable species were found, namely, Haemophilus influenzae, Neisseria meningitides, Neisseria gonorrheae, and Bacillus subtilis (96). Natural transformation, which requires a set of genes evolved for the purpose, contrasts with artificial transformation, which is accomplished by shocking cells either electrically, as in electroporation, or by ionic and temperature shifts. Although such artificial treatments can introduce very small amounts of DNA into virtually any type of cell, the amounts introduced by natural transformation are a million-fold greater, and S. pneumoniae can take up as much as 10% of its cellular DNA content (40).

  11. Ethanol-induced loss of brain cyclic AMP binding proteins: correlation with growth suppression

    SciTech Connect

    Pennington, S.; Kalmus, G.

    1987-05-01

    Brain hypoplasia secondary to maternal ethanol consumption is a common fetal defect observed in all models of fetal alcohol syndrome. The molecular mechanism by which ethanol inhibits growth is unknown but has been hypothesized to involve ethanol-induced changes in the activity of cyclic-AMP stimulated protein kinase. Acute and chronic alcohol exposure elevate cyclic AMP level in many tissues, including brain. This increase in cyclic AMP should increase the phosphorylating activity of kinase by increasing the amount of dissociated (active) kinase catalytic subunit. In 7-day embryonic chick brains, ethanol-induced growth suppression was correlated with increased brain cyclic AMP content but neither basal nor cyclic AMP stimulated kinase catalytic activity was increased. However, the levels of cyclic AMP binding protein (kinase regulatory subunit) were significantly lowered by ethanol exposure. Measured as either /sup 3/H cyclic AMP binding or as 8-azido cyclic AM/sup 32/P labeling, ethanol-exposed brains had significantly less cyclic AMP binding activity (51 +/- 14 versus 29 +/- 10 units/..mu..g protein for 8-azido cyclic AMP binding). These findings suggest that ethanol's effect on kinase activity may involve more than ethanol-induced activation of adenylate cyclase.

  12. Handling Arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses.

    PubMed

    Rivero, Luz; Scholl, Randy; Holomuzki, Nicholas; Crist, Deborah; Grotewold, Erich; Brkljacic, Jelena

    2014-01-01

    Growing healthy plants is essential for the advancement of Arabidopsis thaliana (Arabidopsis) research. Over the last 20 years, the Arabidopsis Biological Resource Center (ABRC) has collected and developed a series of best-practice protocols, some of which are presented in this chapter. Arabidopsis can be grown in a variety of locations, growth media, and environmental conditions. Most laboratory accessions and their mutant or transgenic derivatives flower after 4-5 weeks and set seeds after 7-8 weeks, under standard growth conditions (soil, long day, 23 ºC). Some mutant genotypes, natural accessions, and Arabidopsis relatives require strict control of growth conditions best provided by growth rooms, chambers, or incubators. Other lines can be grown in less-controlled greenhouse settings. Although the majority of lines can be grown in soil, certain experimental purposes require utilization of sterile solid or liquid growth media. These include the selection of primary transformants, identification of homozygous lethal individuals in a segregating population, or bulking of a large amount of plant material. The importance of controlling, observing, and recording growth conditions is emphasized and appropriate equipment required to perform monitoring of these conditions is listed. Proper conditions for seed harvesting and preservation, as well as seed quality control, are also described. Plant transformation and genetic crosses, two of the methods that revolutionized Arabidopsis genetics, are introduced as well. PMID:24057358

  13. Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells.

    PubMed

    Slootweg, M C; Ohlsson, C; Salles, J P; de Vries, C P; Netelenbos, J C

    1995-10-01

    GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH

  14. Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells.

    PubMed Central

    Owada, M K; Hakura, A; Iida, K; Yahara, I; Sobue, K; Kakiuchi, S

    1984-01-01

    Caldesmon is a calmodulin-binding and F-actin-binding protein originally purified from chicken gizzard smooth muscle. This protein binds to F-actin filaments in a Ca2+- and calmodulin-dependent "flip-flop" fashion, thereby regulating the function of actin filaments. Here we report that various lines of cultured cells contain a Mr 77,000 protein that specifically reacts with the affinity-purified caldesmon antibody raised against chicken gizzard caldesmon . Among the fibroblast proteins that had been pulse-labeled with [35S]methionine, the Mr 77,000 protein was the only protein band detected on the NaDodSO4 gel that reacted with the anticaldesmon . The subcellular distribution of the Mr 77,000 protein was investigated by the indirect immunofluorescence technique using the anticaldesmon . In all fibroblast cell lines examined, the immunofluorescence localized along the cellular stress fibers and in leading edges of the cell. In Rous sarcoma virus-transformed cells (S7-1), however, the distribution of the fluorescence changed to a diffuse and blurred appearance. These staining patterns of anticaldesmon obtained with the normal and transformed cells coincided with those of antiactin in the corresponding states, strongly suggesting the functional linkage between the Mr 77,000 protein and actin filaments. We propose to refer to this Mr 77,000 protein as caldesmon 77. The cellular level of caldesmon 77 in transformed S7-1 cells decreased to about one-third of that in their normal counterparts (cell line no. 7). Essentially the same result was obtained with normal rat kidney cells infected with the temperature-sensitive transformation mutant Schmidt-Ruppin strain of Rous sarcoma virus (68 N2 clone). The cellular level of caldesmon 77 observed at a permissive temperature (35 degrees C) was about one-third of that at a nonpermissive temperature (38.5 degrees C). These changes of caldesmon 77 in transformed cells may correlate with the loss of Ca2+ regulation in the

  15. Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth

    PubMed Central

    PJ, Noy; P, Lodhia; K, Khan; X, Zhuang; DG, Ward; AR, Verissimo; A, Bacon; R, Bicknell

    2015-01-01

    We previously identified CLEC14A as a tumour endothelial marker. Here we show CLEC14A is a regulator of sprouting angiogenesis in vitro and in vivo. Using a HUVEC spheroid sprouting assay we found CLEC14A to be a regulator of sprout initiation. Analysis of endothelial sprouting in aortic ring and in vivo subcutaneous sponge assays from clec14a+/+ and clec14a−/− mice revealed defects in sprouting angiogenesis in CLEC14A deficient animals. Tumour growth was retarded and vascularity reduced in clec14a−/− mice. Pulldown and co-immunoprecipitation experiments confirmed MMRN2 binds to the extracellular region of CLEC14A. The CLEC14A-MMRN2 interaction was interrogated using mouse monoclonal antibodies. Monoclonal antibodies were screened for their ability to block this interaction. Clone C4 but not C2 blocked CLEC14A-MMRN2 binding. C4 antibody perturbed tube formation and endothelial sprouting in vitro and in vivo, with a similar phenotype to loss of CLEC14A. Significantly, tumour growth was impaired in C4 treated animals and vascular density was also reduced in the C4 treated group. We conclude that CLEC14A-MMRN2 binding has a role in inducing sprouting angiogenesis during tumour growth, that has the potential to be manipulated in future anti-angiogenic therapy design. PMID:25745997

  16. Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth.

    PubMed

    Noy, P J; Lodhia, P; Khan, K; Zhuang, X; Ward, D G; Verissimo, A R; Bacon, A; Bicknell, R

    2015-11-19

    We previously identified CLEC14A as a tumour endothelial marker. Here we show that CLEC14A is a regulator of sprouting angiogenesis in vitro and in vivo. Using a human umbilical vein endothelial cell spheroid-sprouting assay, we found CLEC14A to be a regulator of sprout initiation. Analysis of endothelial sprouting in aortic ring and in vivo subcutaneous sponge assays from clec14a(+/+) and clec14a(-/-) mice revealed defects in sprouting angiogenesis in CLEC14A-deficient animals. Tumour growth was retarded and vascularity reduced in clec14a(-/-) mice. Pull-down and co-immunoprecipitation experiments confirmed that MMRN2 binds to the extracellular region of CLEC14A. The CLEC14A-MMRN2 interaction was interrogated using mouse monoclonal antibodies. Monoclonal antibodies were screened for their ability to block this interaction. Clone C4, but not C2, blocked CLEC14A-MMRN2 binding. C4 antibody perturbed tube formation and endothelial sprouting in vitro and in vivo, with a similar phenotype to loss of CLEC14A. Significantly, tumour growth was impaired in C4-treated animals and vascular density was also reduced in the C4-treated group. We conclude that CLEC14A-MMRN2 binding has a role in inducing sprouting angiogenesis during tumour growth, which has the potential to be manipulated in future antiangiogenic therapy design. PMID:25745997

  17. Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane.

    PubMed

    te Welscher, Yvonne M; ten Napel, Hendrik H; Balagué, Miriam Masià; Souza, Cleiton M; Riezman, Howard; de Kruijff, Ben; Breukink, Eefjan

    2008-03-01

    Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol. PMID:18165687

  18. Transformer 2β and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

    PubMed

    Kuwano, Y; Nishida, K; Kajita, K; Satake, Y; Akaike, Y; Fujita, K; Kano, S; Masuda, K; Rokutan, K

    2015-05-01

    RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2β (Tra2β) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2β antibody and microarray analysis identified a subset of Tra2β-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2β knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2β knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2β mRNA having a short, alternatively spliced 3' UTR different from BCL2α 3' UTR. RIP assays with anti-Tra2β and anti-Argonaute 2 antibodies, respectively, showed that Tra2β bound to BCL2α 3' UTR, and that Tra2β knockdown facilitated association of miR-204 with BCL2α 3' UTR. The consensus sequence (GAA) for Tra2β-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2β to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2β-silenced or overexpressed cells revealed that Tra2β antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2β mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2β knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2β regulates apoptosis by

  19. Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

    PubMed Central

    Jiang, Chao; Wilkinson, Mark C.

    2016-01-01

    The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans. PMID:27030175

  20. Sustained activation of fibroblast transforming growth factor-beta/Smad signaling in a murine model of scleroderma.

    PubMed

    Takagawa, Shinsuke; Lakos, Gabriella; Mori, Yasuji; Yamamoto, Toshiyuki; Nishioka, Kiyoshi; Varga, John

    2003-07-01

    Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the

  1. Induction of Transforming Growth Factor Beta Receptors following Focal Ischemia in the Rat Brain

    PubMed Central

    Pál, Gabriella; Lovas, Gábor; Dobolyi, Arpád

    2014-01-01

    Transforming growth factor-βs (TGF-βs) regulate cellular proliferation, differentiation, and survival. TGF-βs bind to type I (TGF-βRI) and II receptors (TGF-βRII), which are transmembrane kinase receptors, and an accessory type III receptor (TGF-βRIII). TGF-β may utilize another type I receptor, activin-like kinase receptor (Alk1). TGF-β is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Recently, we reported the expression pattern of TGF-β1-3 after MCAO. To establish how TGF-βs exert their actions following MCAO, the present study describes the induction of TGF-βRI, RII, RIII and Alk1 at 24 h, 72 h and 1 mo after transient 1 h MCAO as well as following 24 h permanent MCAO using in situ hybridization histochemistry. In intact brain, only TGF-βRI had significant expression: neurons in cortical layer IV contained TGF-βRI. At 24 h after the occlusion, no TGF-β receptors showed induction. At 72 h following MCAO, all four types of TGF-β receptors were induced in the infarct area, while TGF-βRI and RII also appeared in the penumbra. Most cells with elevated TGF-βRI mRNA levels were microglia. TGF-βRII co-localized with both microglial and endothelial markers while TGF-βRIII and Alk1 were present predominantly in endothels. All four TGF-β receptors were induced within the lesion 1 mo after the occlusion. In particular, TGF-βRIII was further induced as compared to 72 h after MCAO. At this time point, TGF-βRIII signal was predominantly not associated with blood vessels suggesting its microglial location. These data suggest that TGF-β receptors are induced after MCAO in a timely and spatially regulated fashion. TGF-β receptor expression is preceded by increased TGF-β expression. TGF-βRI and RII are likely to be co-expressed in microglial cells while Alk1, TGF-βRII, and RIII in endothels within the infarct where TGF-β1 may be their ligand. At later time points, TGF-βRIII may also appear in glial cells to potentially

  2. Transforming growth factor-betas and their signaling receptors are coexpressed in Crohn's disease.

    PubMed Central

    di Mola, F F; Friess, H; Scheuren, A; Di Sebastiano, P; Graber, H; Egger, B; Zimmermann, A; Korc, M; Büchler, M W

    1999-01-01

    OBJECTIVE: To evaluate mechanisms that contribute to tissue repair and tissue remodeling in Crohn's disease (CD). SUMMARY BACKGROUND DATA: Transforming growth factor-betas (TGF-betas) are involved in different chronic inflammatory disorders. They function by binding to two receptors, type I (TbetaR-I) subtype ALK5 and type II (TbetaR-II), which are concomitantly required for signal transduction. METHODS: Tissues were obtained from 18 patients with CD (10 female patients, 8 male patients, median age 38.7 years [range 16 to 58 years]) undergoing surgery because of CD-related complications. Tissue samples of 18 healthy organ donors (10 female subjects, 8 male subjects, median age 50.3 years [range 15 to 65 years]) served as controls. The expression and localization of TGF-beta1, TGF-beta2, TGF-beta3, TbetaR-IALK5, TbetaR-II, and TbetaR-III were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: On Northern blot analysis, 94% of the CD samples exhibited enhanced TGF-beta1, TGF-beta3, and TbetaR-II mRNA expression compared with controls. TGF-beta2 was increased in 72%, TbetaR-IALK5 in 72%, and TbetaR-III in 82% of the patients with CD. On in situ hybridization and immunohistochemical analysis, TGF-beta1, TbetaR-IALK5, and TbetaR-II were seen to be colocalized in the lamina propria cells and in the lymphocytes closest to the luminal surface, but also in the remaining epithelial cells, and in fibroblasts of CD tissue samples. CONCLUSIONS: The concomitant overexpression of TGF-betas and their signaling receptors in CD points to a potential role of these regulatory molecules in the pathophysiology of CD. Activation of TGF-beta-mediated pathways might promote the repair of mucosal injury by enhancing the process of reepithelization, but might also contribute to extracellular matrix generation and subsequently to intramural fibrosis and intestinal obstruction. Images Figure 1. Figure 3. Figure 4. Figure 5. PMID:9923802

  3. TRANSFORMER

    DOEpatents

    Baker, W.R.

    1959-08-25

    Transformers of a type adapted for use with extreme high power vacuum tubes where current requirements may be of the order of 2,000 to 200,000 amperes are described. The transformer casing has the form of a re-entrant section being extended through an opening in one end of the cylinder to form a coaxial terminal arrangement. A toroidal multi-turn primary winding is disposed within the casing in coaxial relationship therein. In a second embodiment, means are provided for forming the casing as a multi-turn secondary. The transformer is characterized by minimized resistance heating, minimized external magnetic flux, and an economical construction.

  4. Crosstalk of carcinoembryonic antigen and transforming growth factor-β via their receptors: comparing human and canine cancer.

    PubMed

    Jensen-Jarolim, Erika; Fazekas, Judit; Singer, Josef; Hofstetter, Gerlinde; Oida, Kumiko; Matsuda, Hiroshi; Tanaka, Akane

    2015-05-01

    There is accumulating evidence that the transforming growth factor beta (TGF-β) and nuclear factor kappa-B (NFκB) pathways are tightly connected and play a key role in malignant transformation in cancer. Immune infiltration by regulatory T- and B-lymphocytes (Tregs, Bregs) has recently gained increased attention for being an important source of TGF-β. There is a plethora of studies examining the pro-tumorigenic functions of carcinoembryonic antigen (CEA), but its receptor CEAR is far less studied. So far, there is a single connecting report that TGF-β also may signal through CEAR. The crosstalk between cancer tissues is further complicated by the expression of CEAR and TGF-β receptors in stromal cells, and implications of TGF-β in epithelial-mesenchymal transition. Furthermore, tumor-infiltrating Tregs and Bregs may directly instruct cancer cells by secreting TGF-β binding to their CEAR. Therefore, both TGF-β and CEA may act synergistically in breast cancer and cause disease progression, and NFκB could be a common crossing point between their signaling. CEAR, TGF-β1-3, TGF-β-R types I-III and NFκB class I and II molecules have an outstanding human-canine sequence identity, and only a canine CEA homolog has not yet been identified. For these reasons, the dog may be a valid translational model patient for investigating the crosstalk of the interconnected CEA and TGF-β networks. PMID:25832000

  5. Amblyomma americanum tick saliva insulin-like growth factor binding protein-related protein 1 binds insulin but not insulin-like growth factors.

    PubMed

    Radulović, Ž M; Porter, L M; Kim, T K; Bakshi, M; Mulenga, A

    2015-10-01

    Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24-48 h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick-borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24-48 h of the tick starting to feed makes AamIGFBP-rP1 an attractive target for antitick vaccine development. PMID:26108887

  6. Analysis of growth hormone and lactogenic binding sites cross-linked to iodinated human growth hormone

    SciTech Connect

    Hughes, J.P.; Simpson, J.S.; Friesen, H.G.

    1983-06-01

    GH (GHR) and lactogenic receptors were analyzed after use of the cross-linking reagent ethylene glycol bis-(succinimidyl succinate) to attach covalently iodinated human GH (hGH) to binding proteins 1) on intact IM-9 lymphocytes, 2) in a partially purified GHR preparation from rabbit liver, and 3) in crude microsomal fractions from rabbit liver, rabbit mammary gland, and rat liver. The latter two microsomal preparations contain primarily lactogenic receptors, whereas in IM-9 lymphocytes and the rabbit liver preparations, GHR predominate. Cross-linked (125I)hGH-receptor complexes were solubilized, reduced, and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of proteins cross-linked to (125I)hGH in the microsomal fraction from rabbit liver showed a specifically labeled complex with an estimated molecular weight (mol wt) of 75K. A slightly lower mol wt (71K) was determined for the complex labeled in the purified GHR preparation. In contrast to the relatively low mol wt complexes in rabbit liver, a complex that migrated with an apparent mol wt of 130K was identified in IM-9 lymphocytes. Labeled complexes were identified at 66K from rat liver and 61K from rabbit mammary gland. If it is assumed that hGH contributes 21K to the mol wt of the radiolabeled complexes, then the approximate mol wts of hGH-binding sites are 50-54K from rabbit liver, 109K from IM-9 lymphocytes, 45K from rat liver, and 40K from rabbit mammary gland.

  7. Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants.

    PubMed Central

    Gershoni, J M

    1987-01-01

    Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots. Images PMID:3295881

  8. [THE ROLE OF TRANSFORMING GROWTH FACTOR-B IN IMMUNOPATHOGENESIS OF DISEASES OF CONNECTIVE TISSUE].

    PubMed

    Rudoi, A S; Moskalev, A V; Sboitchakov, V B

    2016-02-01

    The recent studies of molecular physiology of fibrillin and pathophysiology of inherent disorders of structure and function of connective tissue such as dissection and aneurysm of aorta, myxomatously altered cusps and prolapses of mitral valve, syndrome of hyper-mobility of joints, demonstrated that important role in development of these malformations play alterations of transfer of signals by growth factors and matrix cellular interaction. These conditions under manifesting Marfan's syndrome can be a consequence of anomalies of fibrillin-1 which deficiency unbrakes process of activation of transforming growth factor-β (TGFβ). The involvement of TGFβ in pathogenesis of Marfan's syndrome permits consider antagonists of angiotensin-transforming enzymes as potential pharmaceuticals in therapy of this disease. The article presents analysis of publications' data related to this problem. PMID:27455564

  9. Transformation of the Herbicide Sulcotrione into a Root Growth Enhancer Compound by Sequential Photolysis and Hydrolysis.

    PubMed

    Goujon, Eric; Maruel, Sandra; Richard, Claire; Goupil, Pascale; Ledoigt, Gérard

    2016-01-27

    Xanthene-1,9-dione-3,4-dihydro-6-methylsulfonyl (1), the main product of sulcotrione phototransformation on plant leaves, was slowly hydrolyzed into 2-hydroxy-4-methylsulfonylbenzoic acid (2) and 1,3-cyclohexanedione (3) in aqueous solution. Interestingly, the rate of hydrolysis was significantly enhanced in the presence of roots of monocotyledonous plants, while the same treatment showed adverse effects on broadleaf weeds. Root growth enhancement varied according to the plant species and concentrations of compound 2, as shown with Zea mays roots. Compound 2 is a derivative of salicylic acid that is known to be a plant signaling messenger. Compound 2 was, therefore, able to mimic some known effects of this phytohormone. This work showed that a pesticide like sulcotrione was transformed into a compound exhibiting a positive impact on plant growth. This study exemplified a rarely reported situation where chemical and biological chain reactions transformed a xenobiotic into a compound exhibiting potential beneficial effects. PMID:26654319

  10. Mediation of wound-related Rous sarcoma virus tumorigenesis by TFG (transforming growth factor)-. beta

    SciTech Connect

    Sieweke, M.H.; Bissell, M.J. ); Thompson, N.L.; Sporn, M.B. )

    1990-06-29

    In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-{beta} is present locally shortly after wounding, but not in unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor {beta}1 (TGF-{beta}1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-{beta} resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still led to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in would healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-{alpha} had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-{beta} release during the would-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system. 31 refs., 3 figs., 2 tabs.

  11. [Effects of nitrogen regulators on fertilizer nitrogen transformation in meadow cinnamon soil and on pakchoi growth].

    PubMed

    Sun, Zhi-Mei; Zhang, Kuo; Liu, Jian-Tao; Si, Huan-Sen; Wang, Yan-Qun

    2012-09-01

    Soil incubation test and pot experiment were conducted to investigate the effects of dicyandiamide (DCD) and its combination with nano-carbon on the transformation of fertilizers (urea and ammonium bicarbonate) nitrogen (N) in meadow cinnamon soil, a typical soil type in North China Plain, and on the growth of pakchoi (Brassica chinensis). In the first two weeks after applying urea and ammonium bicarbonate, the soil NH4+-N and NO3(-)-N contents varied greatly, but little variation was observed since then. The effects of the applied fertilizer N on the pakchoi growth and its N use efficiency differed significantly at early growth stages, but had little difference at harvesting stage. The DCD inhibited the transformation of the fertilizer N (especially ammonium bicarbonate N) into nitrate markedly, and this effect increased with increasing DCD dose. Under the conditions of our experiment, the optimal application rate of DCD was 1.0-1.5% of applied fertilize N, which could increase the pakchoi yield significantly, improve the leaf color, decrease the plant nitrate contents, and increase the fertilizer N use efficiency. The combination of DCD and nano-carbon exerted a synergistic effect on inhibiting soil ammonium oxidation, and also, promoted the pakchoi growth and N utilization at early growth stages significantly and decreased the plant nitrate level at harvesting stage. PMID:23286007

  12. Growth-inhibitory and metal-binding proteins in Chlorella vulgaris exposed to cadmium or zinc.

    PubMed

    Huang, Zhiyong; Li, Lianping; Huang, Gaoling; Yan, Qingpi; Shi, Bing; Xu, Xiaoqin

    2009-01-18

    Phytochelatins, with the general structure of (gamma-Glu-Cys)n-Gly (n=2-11), are usually recognized as being strongly induced by metals in microalgae and play an important role in the detoxification of heavy metals in environment. However, there have been few studies on metallothionein (MT) synthesis in Chlorella vulgaris (C. vulgaris) exposed to heavy metals. The present study describes the growth inhibition of C. vulgaris exposed to different concentrations of cadmium and zinc, and the induction of metal-binding MT-like proteins in the cells. The amounts of metal-binding proteins, induced in the alga exposed to different concentrations of Cd and Zn, were analyzed with a size-exclusion HPLC coupled to ICP-MS. After being purified with a gel filtration column (Sephadex G-75, 3.5cmx80cm) and a desalting column (G-25, 1.5cmx30cm), the isoforms and sub-isoforms of Zn-binding protein were characterized by a reverse phase-HPLC coupled to electrospray ionization and a triple quadrupole mass spectrometer (HPLC-ESI-MS/MS). In addition, the ultraviolet spectra of purified Zn-binding proteins were analyzed in media with different pH values. The results showed that the significant inhibitory effects (at p<0.05) on the cell growth were observed when excessive metals such as 80micromoll(-1) of Cd, and 60 and 80micromoll(-1) of Zn were added. The Cd/Zn-binding proteins induced in C. vulgaris exposed to Cd and Zn were referred to as Cd/Zn-MT-like proteins in which the mean molecular mass of the apo-MT-like was 6152Da. The induced Cd/Zn-MT-like proteins might be involved in the detoxification of heavy metals, such as cadmium and zinc, by the alga. PMID:19019465

  13. Estrogen receptor activation function 2 (AF-2) is essential for hormone-dependent transactivation and cell transformation induced by a v-Jun DNA binding domain-estrogen receptor chimera.

    PubMed

    Dunn, Catherine A; Clark, William; Black, Elizabeth J; Gillespie, David A F

    2003-08-25

    A chimeric protein consisting of the estrogen receptor alpha ligand binding domain (ER-alpha LBD) fused to the DNA binding domain (DBD) of the v-Jun oncoprotein, deltavJ-hER, was previously shown to elicit estradiol-dependent transcriptional activation and cell transformation. Remarkably, in the unliganded state deltavJ-hER is not inert, but rather inhibits cell proliferation. To understand the molecular basis for these opposite effects on cell growth, we investigated the effect of estradiol on deltavJ-hER function. We find that deltavJ-hER is localised to the cell nucleus and capable of binding TPA-response element (TRE) DNA recognition sites in the presence and absence of estradiol, indicating that these properties are unlikely to be the targets of hormonal regulation. In contrast, a mutant derivative of deltavJ-hER in which amino acid substitutions selectively disrupt activation function 2 (AF-2) function is unable to elicit estradiol-dependent transcription or cell transformation, even though DNA binding is not impaired. Taken together, these observations establish that estrogen receptor AF-2 activity is essential for cell transformation by deltavJ-hER. PMID:12932827

  14. Expression and subcellular targeting of human insulin-like growth factor binding protein-3 in transgenic tobacco plants.

    PubMed

    Cheung, Stanley C K; Sun, Samuel S M; Chan, Juliana C N; Tong, Peter C Y

    2009-12-01

    Human insulin-like growth factor binding protein-3 (hIGFBP-3) is a multifunctional protein which has high affinity for insulin-like growth factor-I (IGF-I). It combines with IGF-I to form a tertiary complex in circulation, thus regulating the activity of IGF-I. Furthermore, recombinant hIGFBP-3 (rhIGFBP-3) has been found to negatively regulate cell proliferation and induce apoptosis. In this study, we have established an efficient plant bioreactor platform for mass production of rhIGFBP-3. Different expression constructs, driven by the seed-specific phaseolin promoter, were designed and transformed into tobacco plant via Agrobacterium. To enhance protein expression level, the signal peptide (SP) and the C-terminal tetrapeptide AFVY of phaseolin were used to direct rhIGFBP-3 to protein storage vacuole (PSV) in tobacco seed for stable accumulation. Western blot analysis showed that rhIGFBP-3 was successfully synthesized in transgenic tobacco seeds, with the highest protein expression of 800 mug/g dry weight. The localization of rhIGFBP-3 in PSV was also evident by confocal immunofluorescence microscopy. Our results indicated that protein sorting sequences could benefit the expression level of rhIGFBP-3 and it is feasible to use plant as "bio-factory" to produce therapeutic recombinant proteins in large quantity. PMID:19504171

  15. Multifunctional roles of insulin-like growth factor binding protein 5 in breast cancer

    PubMed Central

    Akkiprik, Mustafa; Feng, Yumei; Wang, Huamin; Chen, Kexin; Hu, Limei; Sahin, Aysegul; Krishnamurthy, Savitri; Ozer, Ayse; Hao, Xishan; Zhang, Wei

    2008-01-01

    The insulin-like growth factor axis, which has been shown to protect cells from apoptosis, plays an essential role in normal cell physiology and in cancer development. The family of insulin-like growth factor binding proteins (IGFBPs) has been shown to have a diverse spectrum of functions in cell growth, death, motility, and tissue remodeling. Among the six IGFBP family members, IGFBP-5 has recently been shown to play an important role in the biology of breast cancer, especially in breast cancer metastasis; however, the exact mechanisms of action remain obscure and sometimes paradoxical. An in-depth understanding of IGFBP-5 would shed light on its potential role as a target for breast cancer therapeutics. PMID:18710598

  16. Severe inflammatory bowel disease associated with congenital alteration of transforming growth factor beta signaling.

    PubMed

    Naviglio, Samuele; Arrigo, Serena; Martelossi, Stefano; Villanacci, Vincenzo; Tommasini, Alberto; Loganes, Claudia; Fabretto, Antonella; Vignola, Silvia; Lonardi, Silvia; Ventura, Alessandro

    2014-08-01

    Transforming growth factor beta is a pleiotropic cytokine which plays a central role in the homeostasis of the immune system. A complex dysregulation of its signaling occurs in Loeys-Dietz syndrome, a monogenic disorder caused by mutations of transforming growth factor beta receptors type 1 or type 2, characterized by skeletal involvement, craniofacial abnormalities, and arterial tortuosity with a strong predisposition for aneurysm and dissection. In addition, several immunologic abnormalities have been described in these patients, including an increased risk of allergic disorders as well as eosinophilic gastrointestinal disorders. The occurrence of inflammatory bowel disorders has been also reported, but it is poorly documented. We describe two unrelated children with Loeys-Dietz syndrome affected by severe chronic inflammatory colitis appearing at an early age. The intestinal disease presented similar features in both patients, including a histopathological picture of non-eosinophilic chronic ulcerative colitis, striking elevation of inflammatory markers, and a distinctly severe clinical course leading to failure to thrive, with resistance to multiple immunosuppressive treatments. One of the patients also presented autoimmune thyroiditis. Our report confirms that chronic ulcerative colitis may be associated with Loeys-Dietz syndrome. This finding suggests that an alteration of transforming growth factor beta signaling may by itself predispose to inflammatory colitis in humans, and represent an invaluable model to understand inflammatory bowel diseases. PMID:24486179

  17. Deletion of ocular transforming growth factor β signaling mimics essential characteristics of diabetic retinopathy.

    PubMed

    Braunger, Barbara M; Leimbeck, Sarah V; Schlecht, Anja; Volz, Cornelia; Jägle, Herbert; Tamm, Ernst R

    2015-06-01

    Diabetic retinopathy, a major cause of blindness, is characterized by a distinct phenotype. The molecular causes of the phenotype are not sufficiently clear. Here, we report that deletion of transforming growth factor β signaling in the retinal microenvironment of newborn mice induces changes that largely mimic the phenotype of nonproliferative and proliferative diabetic retinopathy in humans. Lack of transforming growth factor β signaling leads to the formation of abundant microaneurysms, leaky capillaries, and retinal hemorrhages. Retinal capillaries are not covered by differentiated pericytes, but by a coat of vascular smooth muscle-like cells and a thickened basal lamina. Reactive microglia is found in close association with retinal capillaries. In older animals, loss of endothelial cells and the formation of ghost vessels are observed, findings that correlate with the induction of angiogenic molecules and the accumulation of retinal hypoxia-inducible factor 1α, indicating hypoxia. Consequently, retinal and vitreal neovascularization occurs, a scenario that leads to retinal detachment, vitreal hemorrhages, neuronal apoptosis, and impairment of sensory function. We conclude that transforming growth factor β signaling is required for the differentiation of retinal pericytes during vascular development of the retina. Lack of differentiated pericytes initiates a scenario of structural and functional changes in the retina that mimics those of diabetic retinopathy strongly indicating a common mechanism. PMID:25857227

  18. Binding and degradation of (/sup 125/I)human growth hormone in rat adipocytes

    SciTech Connect

    Gorin, E.; Grichting, G.; Goodman, H.M.

    1984-08-01

    Iodinated human growth hormone (( /sup 125/I)hGH) binds to both specific and nonspecific sites on the surface of adipocytes isolated from the epididymal fat of normal rats. When adipocytes were incubated at 37 C with 1 nM (/sup 125/I)hGH, specific binding increased for 30-60 min and thereafter remained approximately constant as long as the hormone was present in the medium. About 90% of the /sup 125/I released was soluble in 5% trichloroacetic acid and was in the form of iodotyrosine. The rate of /sup 125/I release from specific binding sites decreased by a factor of 4 when the temperature was lowered from 37 to 17 C. Replacement of some of the sodium chloride in the buffer with 25 mM ammonium chloride had little or no effect on the amount on /sup 125/I that bound to cells when (/sup 125/I)hGH was present in the medium, but completely blocked the release of /sup 125/I from cells transferred to hormone-free medium. Ammonium chloride also significantly reduced both the release of /sup 125/I from nonspecific binding sites and the amount of /sup 125/I recovered in trichloroacetic acid-soluble form. Cloroquine, leupeptin, or colchicine nearly doubled the specific binding of (/sup 125/I)hGH after 180 min and markedly slowed the release of /sup 125/I when cells were transferred to hormone-free medium. All of these agents also significantly reduced the rate of release of /sup 125/I from nonspecific binding sites. Incubation of adipose tissue from hypophysectomized rats with ammonium chloride, leupeptin, or colchicine failed to alter the ability of GH to increase glucose oxidation, induce refractoriness, or promote lipolysis in the presence of theophylline.

  19. Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

    PubMed

    Sanders, Jeffrey M; Wampole, Matthew E; Thakur, Mathew L; Wickstrom, Eric

    2013-01-01

    The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF

  20. Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

    PubMed Central

    McCauley, Micah J.; Rouzina, Ioulia; Manthei, Kelly A.; Gorelick, Robert J.; Musier-Forsyth, Karin; Williams, Mark C.

    2015-01-01

    Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA–DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA–DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription. PMID:26483503

  1. Magnetic field-responsive release of transforming growth factor beta 1 from heparin-modified alginate ferrogels.

    PubMed

    Kim, Hwi; Park, Honghyun; Lee, Jae Won; Lee, Kuen Yong

    2016-10-20

    Stimuli-responsive polymeric systems have been widely used for various drug delivery and tissue engineering applications. Magnetic stimulation can be also exploited to regulate the release of pharmaceutical drugs, growth factors, and cells from hydrogels in a controlled manner, on-demand. In the present study, alginate ferrogels containing iron oxide nanoparticles were fabricated via ionic cross-linking, and their various characteristics were investigated. The deformation of the ferrogels was dependent on the polymer concentration, calcium concentration, iron oxide concentration, and strength of magnetic field. To modulate the release of transforming growth factor beta 1 (TGF-β1) under magnetic stimulation, alginate was chemically modified with heparin, as TGF-β1 has a heparin-binding domain. Alginate was first modified with ethylenediamine, and heparin was then conjugated to the ethylenediamine-modified alginate via carbodiimide chemistry. Conjugation of heparin to alginate was confirmed by infrared spectroscopy and proton nuclear magnetic resonance spectroscopy. Sustained release of TGF-β1 from alginate-g-heparin ferrogels was achieved, and application of a magnetic field to the ferrogels regulated TGF-β1 release, resultantly enhancing chondrogenic differentiation of ATDC5 cells, which were used as a model chondrogenic cell line. Alginate-based ferrogels that release drugs in a controlled manner may therefore be useful in many biomedical applications. PMID:27474590

  2. Transforming growth factor-beta in the chicken fundal layers: an immunohistochemical study.

    PubMed

    Mathis, Ute; Schaeffel, Frank

    2010-06-01

    In the chicken model of myopia, it has first been shown that imposing defocus to the retina results in active remodelling of the sclera which, in turn, results in axial length changes of the eye. Transforming growth factor-beta (TGF-beta) is one of the scleral growth modulators but its cellular localization in the fundal layers, colocalization and function are not well known. The aim of the current study was to investigate the cellular distribution of the three isoforms TGF-beta1, 2 and 3 by immunohistochemical labelling. Furthermore, the effects of visual experience that induces refractive errors on TGF-beta2 labelling were examined. Transversal cryostat sections of the fundal layers were analyzed by indirect immunofluorescent labelling and cell counts. Visual experience was changed by having the chicks wear either diffusers, or positive or negative lenses of 7D power in front of the right eyes for various periods of time. Left eyes served as uncovered controls. All TGF-beta isoforms were localized in both scleral layers. In choroid, diffuse labelling of all isoforms was found. In retina, TGF-beta1 and 3 were detected in bipolar, amacrine and ganglion cells and TGF-beta2 in amacrine and ganglion cells. To further characterize these cells, double-labelling with known amacrine and bipolar cell markers was performed (calbindin, cellular retinoic acid binding protein (CRABP), Islet1, Lim3 and protein kinase C (PKC)). TGF-beta1, 2 and 3 could be colocalized with calbindin and CRABP in single amacrine cells. TGF-beta1-positive bipolar cells were immunoreactive to Lim3. TGF-beta1 and 3 were never colocalized with PKC in bipolar cells. Also, colocalization with peptides known to be involved in myopia development in chicks, such as glucagon, or vasointestinal polypeptide and the key enzyme for dopamine synthesis, tyrosine hydroxylase, was not observed. Lenses or diffusers, worn by the chicks for various periods of time, had no effect on TGF-beta2 immunoreactivity in

  3. Transforming growth factor beta mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation.

    PubMed Central

    Braun, L; Mead, J E; Panzica, M; Mikumo, R; Bell, G I; Fausto, N

    1988-01-01

    Transforming growth factor beta (TGF-beta) is a growth factor with multiple biological properties including stimulation and inhibition of cell proliferation. To determine whether TGF-beta is involved in hepatocyte growth responses in vivo, we measured the levels of TGF-beta mRNA in normal liver and during liver regeneration after partial hepatectomy in rats. TGF-beta mRNA increases in the regenerating liver and reaches a peak (about 8 times higher than basal levels) after the major wave of hepatocyte cell division and mitosis have taken place and after the peak expression of the ras protooncogenes. Although hepatocytes from normal and regenerating liver respond to TGF-beta, they do not synthesize TGF-beta mRNA. Instead, the message is present in liver nonparenchymal cells and is particularly abundant in cell fractions enriched for endothelial cells. TGF-beta inhibits epidermal growth factor-induced DNA synthesis in vitro in hepatocytes from normal or regenerating liver, although the dose-response curves vary according to the culture medium used. We conclude that TGF-beta may function as the effector of an inhibitory paracrine loop that is activated during liver regeneration, perhaps to prevent uncontrolled hepatocyte proliferation. Images PMID:3422749

  4. Tumor-host interactions in the gallbladder suppress distal angiogenesis and tumor growth: involvement of transforming growth factor beta1.

    PubMed

    Gohongi, T; Fukumura, D; Boucher, Y; Yun, C O; Soff, G A; Compton, C; Todoroki, T; Jain, R K

    1999-10-01

    Angiogenesis inhibitors produced by a primary tumor can create a systemic anti-angiogenic environment and maintain metastatic tumor cells in a state of dormancy. We show here that the gallbladder microenvironment modulates the production of transforming growth factor (TGF)-beta1, a multifunctional cytokine that functions as an endogenous anti-angiogenic and anti-tumor factor in a cranial window preparation. We found that a wide variety of human gallbladder tumors express TGF-beta1 irrespective of histologic type. We implanted a gel impregnated with basic fibroblast growth factor or Mz-ChA-2 tumor in the cranial windows of mice without tumors or mice with subcutaneous or gallbladder tumors to study angiogenesis and tumor growth at a secondary site. Angiogenesis, leukocyte-endothelial interaction in vessels and tumor growth in the cranial window were substantially inhibited in mice with gallbladder tumors. The concentration of TGF-beta1 in the plasma of mice with gallbladder tumors was 300% higher than that in the plasma of mice without tumors or with subcutaneous tumors. In contrast, there was no difference in the plasma levels of other anti- and pro-angiogenic factors. Treatment with neutralizing antibody against TGF-beta1 reversed both angiogenesis suppression and inhibition of leukocyte rolling induced by gallbladder tumors. TGF-beta1 also inhibited Mz-ChA-2 tumor cell proliferation. Our results indicate that the production of anti-angiogenesis/proliferation factors is regulated by tumor-host interactions. PMID:10502827

  5. Epidermal growth factor (EGF) antagonizes transforming growth factor (TGF)-beta1-induced collagen lattice contraction by human skin fibroblasts.

    PubMed

    Park, J S; Kim, J Y; Cho, J Y; Kang, J S; Yu, Y H

    2000-12-01

    Wound contraction plays an important role in healing, but in extreme conditions, it may lead to excessive scar formation and pathological wound contracture. To date, the key regulator of excessive contracture is known to be transforming growth factor-beta (TGF-beta1). In this study, we have evaluated epidermal growth factor (EGF) antagonism in fibroblast-populated collagen lattice (FPCL) gel contraction, which has been generally used as an in vitro model thought to mimic wound contraction in vivo. As expected, TGF-beta1 treatment enhanced normal fibroblast-induced collagen gel contraction in a dose-dependent manner. In contrast, EGF did not affect normal gel formation, but significantly antagonized TGF-beta1-induced gel formation (p<0.05 at 100 ng/ml), whereas the other growth factor, platelet-derived growth factor (PDGF), did not altered either normal or TGF-beta1-induced gel contractions. Similarly, EGF treatment, but not PDGF, also significantly suppressed TGF-beta1 release that was autologously elicited by TGF-beta1 treatment (p<0.01 at 100 ng/ml). Therefore, the results suggest that EGF may negatively regulate the role of TGF-beta1 through attenuating autologous release of TGF-beta1. PMID:11145189

  6. Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues.

    PubMed Central

    Van der Ven, L. T.; Roholl, P. J.; Gloudemans, T.; Van Buul-Offers, S. C.; Welters, M. J.; Bladergroen, B. A.; Faber, J. A.; Sussenbach, J. S.; Den Otter, W.

    1997-01-01

    To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues. Images Figure 1 Figure 2 Figure 4 PMID:9184179

  7. Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls

    NASA Technical Reports Server (NTRS)

    Basel, L. E.; Cleland, R. E.

    1992-01-01

    A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattern. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.

  8. The structure and regulation of expression of the mouse growth hormone receptor and binding protein

    SciTech Connect

    Talamantes, F.

    1994-12-31

    The mouse growth hormone receptor (mGHR) and the mouse growth hormone-binding protein (mGHBP) are products of a single gene which are generated alternative splicing. The factors that regulate the expression of mGHR and mGHBP mRNA and protein during pregnancy in the mouse are incompletely understood. During pregnancy in the mouse, there are parallel increases in circulating mouse growth hormone (mGH), liver mGHR, and serum mGHBP. The increase in both hepatic mGHR and serum mGHBP begins on Day 9 of gestation and by late gestation the hepatic mGHR content has increased 8-fold and serum mGHBP has increased 30-fold compared with values in nonpregnant controls. A parallel increase occurs in the steady state levels of liver GHR and GHBP encoding mRNAs. The increase in both messages begins on Day 9 of gestation; however, the GHR mRNA reaches maximum levels by Day 13, while the GHBP mRNA continues to increase until the end of pregnancy. The magnitude of the increase in the GHR-encoding message is 15- to 20-fold between nonpregnant and late pregnant mice, and the magnitude of the increase in the GHBP-encoding message is 30- to 50-fold. Both pituitary mGH and the number of conceptuses influence the receptors and binding protein for mGH during pregnancy. 22 refs.

  9. Elevated circulating insulin-like growth factor binding protein-1 is sufficient to cause fetal growth restriction.

    PubMed

    Watson, Carole S; Bialek, Peter; Anzo, Makoto; Khosravi, Javad; Yee, Siu-Pok; Han, Victor K M

    2006-03-01

    IGF binding protein-1 (IGFBP-1) inhibits the mitogenic actions of the IGFs. Circulating IGFBP-1 is elevated in newborns and experimental animals with fetal growth restriction (FGR). To establish a causal relationship between high circulating IGFBP-1 and FGR, we have generated transgenic mice using the mouse alpha-fetoprotein gene promoter to target overexpression of human IGFBP-1 (hIGFBP-1) in the fetal liver. These transgenic mice (AFP-BP1) expressed hIGFBP-1 mainly in the fetal hepatocytes, starting at embryonic d 14.5 (E14.5), with lower levels in the gut. The expression peaked at 1 wk postnatally (plasma concentration, 474 +/- 34 ng/ml). At birth, AFP-BP1 pups were 18% smaller [weighed 1.34 +/- 0.02 g compared with 1.62 +/- 0.04 g for wild type (WT); P < 0.05], and they did not demonstrate any postnatal catch-up growth. The placentas of the AFP-BP1 mice were larger than WT from E16.5 onwards (150 +/- 12 for AFP-BP1 vs. 100 +/- 5 mg for WT at E16.5; P < 0.05). Thus, this model of FGR is associated with a larger placenta, but without postnatal catch-up growth. Overall, these data clearly demonstrate that high concentrations of circulating IGFBP-1 are sufficient to cause FGR. PMID:16293667

  10. Delivering heparin-binding insulin-like growth factor 1 with self-assembling peptide hydrogels.

    PubMed

    Florine, Emily M; Miller, Rachel E; Liebesny, Paul H; Mroszczyk, Keri A; Lee, Richard T; Patwari, Parth; Grodzinsky, Alan J

    2015-02-01

    Heparin-binding insulin-like growth factor 1 (HB-IGF-1) is a fusion protein of IGF-1 with the HB domain of heparin-binding epidermal growth factor-like growth factor. A single dose of HB-IGF-1 has been shown to bind specifically to cartilage and to promote sustained upregulation of proteoglycan synthesis in cartilage explants. Achieving strong integration between native cartilage and tissue-engineered cartilage remains challenging. We hypothesize that if a growth factor delivered by the tissue engineering scaffold could stimulate enhanced matrix synthesis by both the cells within the scaffold and the adjacent native cartilage, integration could be enhanced. In this work, we investigated methods for adsorbing HB-IGF-1 to self-assembling peptide hydrogels to deliver the growth factor to encapsulated chondrocytes and cartilage explants cultured with growth factor-loaded hydrogels. We tested multiple methods for adsorbing HB-IGF-1 in self-assembling peptide hydrogels, including adsorption prior to peptide assembly, following peptide assembly, and with/without heparan sulfate (HS, a potential linker between peptide molecules and HB-IGF-1). We found that HB-IGF-1 and HS were retained in the peptide for all tested conditions. A subset of these conditions was then studied for their ability to stimulate increased matrix production by gel-encapsulated chondrocytes and by chondrocytes within adjacent native cartilage. Adsorbing HB-IGF-1 or IGF-1 prior to peptide assembly was found to stimulate increased sulfated glycosaminoglycan per DNA and hydroxyproline content of chondrocyte-seeded hydrogels compared with basal controls at day 10. Cartilage explants cultured adjacent to functionalized hydrogels had increased proteoglycan synthesis at day 10 when HB-IGF-1 was adsorbed, but not IGF-1. We conclude that delivery of HB-IGF-1 to focal defects in cartilage using self-assembling peptide hydrogels is a promising technique that could aid cartilage repair via enhanced matrix

  11. Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters.

    PubMed

    Thangasamy, Saminathan; Chen, Pei-Wei; Lai, Ming-Hsing; Chen, Jychian; Jauh, Guang-Yuh

    2012-07-01

    Tiller initiation and panicle development are important agronomical traits for grain production in Oryza sativa L. (rice), but their regulatory mechanisms are not yet fully understood. In this study, T-DNA mutant and RNAi transgenic approaches were used to functionally characterize a unique rice gene, LAGGING GROWTH AND DEVELOPMENT 1 (LGD1). The lgd1 mutant showed slow growth, reduced tiller number and plant height, altered panicle architecture and reduced grain yield. The fewer unelongated internodes and cells in lgd1 led to respective reductions in tiller number and to semi-dwarfism. Several independent LGD1-RNAi lines exhibited defective phenotypes similar to those observed in lgd1. Interestingly, LGD1 encodes multiple transcripts with different transcription start sites (TSSs), which were validated by RNA ligase-mediated rapid amplification of 5' and 3' cDNA ends (RLM-RACE). Additionally, GUS assays and a luciferase promoter assay confirmed the promoter activities of LGD1.1 and LGD1.5. LGD1 encoding a von Willebrand factor type A (vWA) domain containing protein is a single gene in rice that is seemingly specific to grasses. GFP-tagged LGD1 isoforms were predominantly detected in the nucleus, and weakly in the cytoplasm. In vitro northwestern analysis showed the RNA-binding activity of the recombinant C-terminal LGD1 protein. Our results demonstrated that LGD1 pleiotropically regulated rice vegetative growth and development through both the distinct spatiotemporal expression patterns of its multiple transcripts and RNA binding activity. Hence, the study of LGD1 will strengthen our understanding of the molecular basis of the multiple transcripts, and their corresponding polypeptides with RNA binding activity, that regulate pleiotropic effects in rice. PMID:22409537

  12. The CREB-binding protein inhibitor ICG-001 suppresses pancreatic cancer growth.

    PubMed

    Arensman, Michael D; Telesca, Donatello; Lay, Anna R; Kershaw, Kathleen M; Wu, Nanping; Donahue, Timothy R; Dawson, David W

    2014-10-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer due in part to a lack of highly robust cytotoxic or molecular-based therapies. Recent studies investigating ligand-mediated Wnt/β-catenin signaling have highlighted its importance in pancreatic cancer initiation and progression, as well as its potential as a therapeutic target in PDAC. The small-molecule ICG-001 binds cAMP-responsive element binding (CREB)-binding protein (CBP) to disrupt its interaction with β-catenin and inhibit CBP function as a coactivator of Wnt/β-catenin-mediated transcription. Given its ability to inhibit Wnt/β-catenin-mediated transcription in vitro and in vivo, as well as its efficacy in preclinical models of colorectal cancer and other Wnt-driven diseases, we examined ICG-001 and its potential role as a therapeutic in PDAC. ICG-001 alone significantly inhibited anchorage-dependent and -independent growth of multiple PDAC lines, and augmented in vitro growth inhibition when used in combination with gemcitabine. ICG-001 had only variable modest effects on PDAC apoptosis and instead mediated PDAC growth inhibition primarily through robust induction of G₁ cell-cycle arrest. These effects, however, seemed decoupled from its inhibition of Wnt/β-catenin-mediated transcription. DNA microarrays performed on PDAC cells in the context of ICG-001 treatment revealed ICG-001 altered the expression of several genes with well-established roles in DNA replication and cell-cycle progression, including direct actions on SKP2 and CDKN1A. ICG-001 also significantly prolonged survival in an in vivo orthotopic xenograft model of PDAC, indicating ICG-001 or derived compounds that disrupt CBP activity are potentially useful small-molecule therapeutics for pancreatic cancer. PMID:25082960

  13. Alginate Sulfates Mitigate Binding Kinetics of Proangiogenic Growth Factors with Receptors toward Revascularization.

    PubMed

    Schmidt, John; Lee, Min Kyung; Ko, Eunkyung; Jeong, Jae Hyun; DiPietro, Luisa A; Kong, Hyunjoon

    2016-07-01

    Ever since proangiogenic growth factors have been used as a vascular medicine to treat tissue ischemia, efforts have been increasingly made to develop a method to enhance efficacy of growth factors in recreating microvascular networks, especially at low dose. To this end, we hypothesized that polysaccharides substituted with sulfate groups would amplify growth factor receptor activation and stimulate phenotypic activities of endothelial cells involved in neovascularization. We examined this hypothesis by modifying alginate with a controlled number of sulfates and using it to derive a complex with vascular endothelial growth factor (VEGF), as confirmed with fluorescence resonance energy transfer (FRET) assay. Compared with the bare VEGF and with a mixture of VEGF and unmodified alginates, the VEGF complexed with alginate sulfates significantly reduced the dissociation rate with the VEGFR-2, elevated VEGFR-2 phosphorylation level, and increased the number of endothelial sprouts in vitro. Furthermore, the VEGF-alginate sulfate complex improved recovery of perfusion in an ischemic hindlimb of a mouse due to the increase of the capillary density. Overall, this study not only demonstrates an important cofactor of VEGF but also uncovers an underlying mechanism by which the cofactor mitigates the VEGF-induced signaling involved in the binding kinetics and activation of VEGFR. We therefore believe that the results of this study will be highly useful in improving the therapeutic efficacy of various growth factors and expediting their uses in clinical treatments of wounds and tissue defects. PMID:26881299

  14. CREB-binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway.

    PubMed

    Tang, Zhipeng; Yu, Wendan; Zhang, Changlin; Zhao, Shilei; Yu, Zhenlong; Xiao, Xiangsheng; Tang, Ranran; Xuan, Yang; Yang, Wenjing; Hao, Jiaojiao; Xu, Tingting; Zhang, Qianyi; Huang, Wenlin; Deng, Wuguo; Guo, Wei

    2016-02-01

    CBP (CREB-binding protein) is a transcriptional co-activator which possesses HAT (histone acetyltransferases) activity and participates in many biological processes, including embryonic development, growth control and homeostasis. However, its roles and the underlying mechanisms in the regulation of carcinogenesis and tumor development remain largely unknown. Here we investigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells. Elevated expression of CBP was detected in lung cancer cells and tumor tissues compared to the normal lung cells and tissues. Knockdown of CBP by siRNA or inhibition of its HAT activity using specific chemical inhibitor effectively suppressed cell proliferation, migration and colony formation and induced apoptosis in lung cancer cells by inhibiting MAPK and activating cytochrome C/caspase-dependent signaling pathways. Co-immunoprecipitation and immunofluorescence analyses revealed the co-localization and interaction between CBP and CPSF4 (cleavage and polyadenylation specific factor 4) proteins in lung cancer cells. Knockdown of CPSF4 inhibited hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating the synergetic effect of CBP and CPSF4 in the regulation of lung cancer cell growth and survival. Moreover, we found that high expression of both CBP and CPSF4 predicted a poor prognosis in the patients with lung adenocarcinomas. Collectively, our results indicate that CBP regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathways. PMID:26628108

  15. Heparin Binds Endothelial Cell Growth Factor, the Principal Endothelial Cell Mitogen in Bovine Brain

    NASA Astrophysics Data System (ADS)

    Maciag, Thomas; Mehlman, Tevie; Friesel, Robert; Schreiber, Alain B.

    1984-08-01

    Endothelial cell growth factor (ECGF), an anionic polypeptide mitogen, binds to immobilized heparin. The interaction between the acidic polypeptide and the anionic carbohydrate suggests a mechanism that is independent of ion exchange. Monoclonal antibodies to purified bovine ECGF inhibited the biological activity of ECGF in crude preparations of bovine brain. These data indicate that ECGF is the principal mitogen for endothelial cells from bovine brain, that heparin affinity chromatography may be used to purify and concentrate ECGF, and that the affinity of ECGF for heparin may have structural and perhaps biological significance.

  16. Transcriptional pathways associated with the slow growth phenotype of transformed Anaplasma marginale

    PubMed Central

    2013-01-01

    Background The ability to genetically manipulate bacteria has been fundamentally important for both basic biological discovery and translational research to develop new vaccines and antibiotics. Experimental alteration of the genetic content of prokaryotic pathogens has revealed both expected functional relationships and unexpected phenotypic consequences. Slow growth phenotypes have been reported for multiple transformed bacterial species, including extracellular and intracellular pathogens. Understanding the genes and pathways responsible for the slow growth phenotype provides the opportunity to develop attenuated vaccines as well as bacteriostatic antibiotics. Transformed Anaplasma marginale, a rickettsial pathogen, exhibits slow growth in vitro and in vivo as compared to the parent wild type strain, providing the opportunity to identify the underlying genes and pathways associated with this phenotype. Results Whole genome transcriptional profiling allowed for identification of specific genes and pathways altered in transformed A. marginale. Genes found immediately upstream and downstream of the insertion site, including a four gene operon encoding key outer membrane proteins, were not differentially transcribed between wild type and transformed A. marginale. This lack of significant difference in transcription of flanking genes and the large size of the insert relative to the genome were consistent with a trans rather than a cis effect. Transcriptional profiling across the complete genome identified the most differentially transcribed genes, including an iron transporter, an RNA cleaving enzyme and several genes involved in translation. In order to confirm the trend seen in translation-related genes, K-means clustering and Gene Set Enrichment Analysis (GSEA) were applied. These algorithms allowed evaluation of the behavior of genes as groups that share transcriptional status or biological function. Clustering and GSEA confirmed the initial observations and

  17. Shape and size transformation of gold nanorods (GNRs) via oxidation process: A reverse growth mechanism

    NASA Astrophysics Data System (ADS)

    Chandrasekar, Govindasamy; Mougin, Karine; Haidara, Hamidou; Vidal, Loïc; Gnecco, Enrico

    2011-02-01

    The anisotropic shape transformation of gold nanorods (GNRs) with H2O2 was observed in the presence of "cethyl trimethylammonium bromide" (CTAB). The adequate oxidative dissolution of GNR is provided by the following autocatalytic scheme with H2O2: Au0 → Au+, Au0 + Aun+ → 2Au3+, n = 1 and 3. The shape transformation of the GNRs was investigated by UV-vis spectroscopy and transmission electron microscopy (TEM). As-synthesised GNRs exhibit transverse plasmon band (TPB) at 523 nm and longitudinal plasmon band (LPB) at 731 nm. Upon H2O2 oxidation, the LPB showed a systematic hypsochromic (blue) shift, while TPB stays at ca. 523 nm. In addition, a new emerging peak observed at ca. 390 nm due to Au(III)-CTAB complex formation during the oxidation. TEM analysis of as-synthesised GNRs with H2O2 confirmed the shape transformation to spherical particles with 10 nm size in 2 h, whereas centrifuged nanorod solution showed no changes in the aspect ratio under the same condition. Au3+ ions produced from oxidation, complex with excess free CTAB and approach the nanorods preferentially at the end, leading to spatially directed oxidation. This work provides some information to the crystal stability and the growth mechanism of GNRs, as both growth and shortening reactions occur preferentially at the edge of single-crystalline GNRs, all directed by Br- ions.

  18. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    SciTech Connect

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun Nishina, Hiroshi

    2014-01-17

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.

  19. Growth hormone (GH) binding and effects of GH analogs in transgenic mice

    SciTech Connect

    Bartke, A.; Steger, R.W.; Turyn, D.

    1994-12-31

    Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb{sub 2} cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo. 35 refs.

  20. Isolation of bovine corneal keratan sulfate and its growth factor and morphogen binding.

    PubMed

    Weyers, Amanda; Yang, Bo; Solakyildirim, Kemal; Yee, Vienna; Li, Lingyun; Zhang, Fuming; Linhardt, Robert J

    2013-05-01

    Keratan sulfate (KS) is an important glycosaminoglycan that is found in cartilage, reproductive tissues, and neural tissues. Corneal KS glycosaminoglycan is found N-linked to lumican, keratocan and mimecan proteoglycans, and has been widely studied by investigators interested in corneal development and diseases. Recently, the availability of corneal KS has become severely limited, owing to restrictions on the shipment of bovine central nervous system byproducts across international borders in an effort to prevent additional cases of mad cow disease. We report a simple method for the purification of multi-milligram quantities of bovine corneal KS, and characterize its structural properties. We also examined its protein-binding properties, and discovered that corneal KS bound with high affinity to fibroblast growth factor-2 and sonic hedgehog, a growth factor and a morphogen involved in corneal development and healing. PMID:23402351

  1. Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth.

    PubMed Central

    Yingling, J M; Das, P; Savage, C; Zhang, M; Padgett, R W; Wang, X F

    1996-01-01

    The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8799132

  2. Expression of transforming growth factor alpha and epidermal growth factor receptor in rat lung neoplasms induced by plutonium-239

    SciTech Connect

    Stegelmeier, B.L.; Gillett, N.A.; Hahn, F.F.; Kelly, G.; Rebar, A.H.

    1994-11-01

    Ninety-two rat lung proliferative lesions and neoplasms induced by inhaled {sup 239}PuO{sub 2} were evaluated for aberrant expression of transforming growth factor alpha (TGF-{alpha}) and epidermal growth factor receptor (EGFR). Expression of TGF-{alpha} protein, measured by immunohistochemistry, was higher in 94% of the squamous cell carcinomas and 87% of the foci of alveolar epithelial squamous metaplasia than that exhibited by the normal-appearing, adjacent lung parenchyma. In contrast, only 20% of adenocarcinomas and foci of epithelial hyperplasia expressed elevated levels of TGF-{alpha}. Many neoplasms expressing TGF-{alpha} also expressed excessive levels of EGFR mRNA. Southern and DNA slot blot analyses showed that the elevated EGFR expression was not due to amplification of the EGFR gene. These data suggest that increased amounts of TGF-{alpha} were early alterations in the progression of plutonium-induced squamous cell carcinoma, and these increases may occur in parallel with overexpression of the receptor for this growth factor. Together, these alterations create a potential autocrine loop for sustaining clonal expansion of cells initiated by high-LET radiation. 44 refs., 4 figs., 1 tab.

  3. Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected

    PubMed Central

    Gómez-Cuadrado, A; Martín, M; Noël, M; Ruiz-Carrillo, A

    1995-01-01

    Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism. PMID:8524232

  4. The influence of adnectin binding on the extracellular domain of epidermal growth factor receptor

    PubMed Central

    Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

    2014-01-01

    The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the tenth type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography. PMID:25223306

  5. Increased insulin receptor binding in erythrocytes from growth hormone-deficient children.

    PubMed

    Dávila, N; Barceló, B; Carranza, M C; Calle, C

    1991-08-01

    Erythrocytes from growth hormone-deficient children (GHd-children) (n = 10) showed a statistically significant increase in insulin binding at low unlabeled insulin concentrations, together with a threefold decrease in apparent receptor affinity, as compared to control children (C) (n = 11). Scatchard analysis of the binding data using the two-site model revealed that both the receptor concentration R1 [GHd-children 0.10 +/- 0.01 ng/ml and C 0.03 +/- 0.002 ng/ml] and the dissociation constant KD1 [GHd-children (0.48 +/- 0.05) x 10(-9) M and C (0.19 +/- 0.01) x 10(-9) M] for high affinity-low capacity sites were significantly increased in erythrocytes from GHd-children, while neither receptor concentrations (R2) nor the dissociation constant (KD2) for low affinity-high capacity sites proved to be altered. These events were accompanied by a normal sensitivity to insulin as well as glucose tolerance in the GHd-group. The meaning of the increased insulin binding with normal insulin sensitivity in GH-deficiency is discussed. PMID:1760528

  6. Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

    SciTech Connect

    Chabot, J.G.; Walker, P.; Pelletier, G.

    1986-01-01

    The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of (/sup 125/I)EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of (/sup 125/I)EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of (/sup 125/I)EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.

  7. The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

    NASA Astrophysics Data System (ADS)

    Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

    2014-12-01

    The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

  8. The Marine-Derived Oligosaccharide Sulfate MS80, a Novel Transforming Growth Factor β1 Inhibitor, Reverses Epithelial Mesenchymal Transition Induced by Transforming Growth Factor-β1 and Suppresses Tumor Metastasis.

    PubMed

    Zhou, Ji; You, Wenjie; Sun, Guangqiang; Li, Yixuan; Chen, Bi; Ai, Jing; Jiang, Handong

    2016-10-01

    Metastasis accounts for the majority of cancer-related deaths. Transforming growth factor β (TGF-β) is believed to promote late-stage cancer progression and metastasis by inducing epithelial-mesenchymal transition (EMT). We previously reported that MS80, a novel oligosaccharide sulfate, inhibits TGF-β1-induced pulmonary fibrosis by binding TGF-β1. In our study MS80 effectively inhibited TGF-β/Smad signaling in lung cancer cells, breast cancer cells, and model cell lines. In addition, MS80 inhibited TGF-β1-induced EMT, motility, and invasion in vitro. Moreover, MS80 significantly inhibited lung metastasis in orthotopic 4T1 xenografts. Notably, the MS80 treatment significantly increased the infiltration of CD8(+) T cells and decreased the infiltration of regulatory T cells in primary tumors and spleens in mice bearing 4T1 xenografts. Therefore, MS80 is a novel and promising candidate for treating metastatic malignancies by targeting TGF-β1-induced EMT and mediating immunosuppression. PMID:27432893

  9. Emerging role of insulin-like growth factor-binding protein 7 in hepatocellular carcinoma.

    PubMed

    Akiel, Maaged; Rajasekaran, Devaraja; Gredler, Rachel; Siddiq, Ayesha; Srivastava, Jyoti; Robertson, Chadia; Jariwala, Nidhi Himanshu; Fisher, Paul B; Sarkar, Devanand

    2014-01-01

    Hepatocellular carcinoma (HCC) is a vicious and highly vascular cancer with a dismal prognosis. It is a life-threatening illness worldwide that ranks fifth in terms of cancer prevalence and third in cancer deaths. Most patients are diagnosed at an advanced stage by which time conventional therapies are no longer effective. Targeted molecular therapies, such as the multikinase inhibitor sorafenib, provide a modest increase in survival for advanced HCC patients and display significant toxicity. Thus, there is an immense need to identify novel regulators of HCC that might be targeted effectively. The insulin-like growth factor (IGF) axis is commonly abnormal in HCC. Upon activation, the IGF axis controls metabolism, tissue homeostasis, and survival. Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted protein of a family of low-affinity IGF-binding proteins termed "IGFBP-related proteins" that have been identified as a potential tumor suppressor in HCC. IGFBP7 has been implicated in regulating cellular proliferation, senescence, and angiogenesis. In this review, we provide a comprehensive discussion of the role of IGFBP7 in HCC and the potential use of IGFBP7 as a novel biomarker for drug resistance and as an effective therapeutic strategy. PMID:27508172

  10. Vascular endothelial growth factor from Trimeresurus jerdonii venom specifically binds to VEGFR-2.

    PubMed

    Zhong, Shurong; Wu, Jianbo; Cui, Yunpeng; Li, Rui; Zhu, Shaowen; Rong, Mingqiang; Lu, Qiumin; Lai, Ren

    2015-09-01

    Vascular endothelial growth factors (VEGFs) play important roles in angiogenesis. In this study, a vascular endothelial growth factor named TjsvVEGF was purified from the venom of Trimeresurus jerdonii by gel filtration, affinity, ion-exchange and high-performance liquid chromatography. TjsvVEGF was a homodimer with an apparent molecular mass of 29 kDa. The cDNA encoding TjsvVEGF was obtained by PCR. The open reading frame of the cloned TjsvVEGF was composed of 432 bp coding for a signal peptide of 24 amino acid residues and a mature protein of 119 amino acid residues. Compared with other snake venom VEGFs, the nucleotide and deduced protein sequences of the cloned TjsvVEGF were conserved. TjsvVEGF showed low heparin binding activity and strong capillary permeability increasing activity. The KD of TjsvVEGF to VEFGR-2 is 413 pM. However, the binding of TjsvVEGF to VEGFR-1 is too weak to detect. Though TjsvVEGF had high sequence identities (about 90%) with Crotalinae VEGFs, the receptor preference of TjsvVEGF was similar to Viperinae VEGFs which had lower sequence identities (about 60%) with it. TjsvVEGF might serve as a useful tool for the study of structure-function relationships of VEGFs and their receptors. PMID:26107411

  11. Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of acute stressor exposure on proximal (growth hormone; GH) and distal (insulin-like growth factor-I; IGF-I and IGF-binding proteins) components of the somatotropic axis are poorly understood in finfish. We exposed rainbow trout (Oncorhynchus mykiss) to a 5-minute handling disturbance to...

  12. Transforming growth factor-β in breast cancer: too much, too late

    PubMed Central

    Barcellos-Hoff, Mary Helen; Akhurst, Rosemary J

    2009-01-01

    The contribution of transforming growth factor (TGF)β to breast cancer has been studied from a myriad perspectives since seminal studies more than two decades ago. Although the action of TGFβ as a canonical tumor suppressor in breast is without a doubt, there is compelling evidence that TGFβ is frequently subverted in a malignant plexus that drives breast cancer. New knowledge that TGFβ regulates the DNA damage response, which underlies cancer therapy, reveals another facet of TGFβ biology that impedes cancer control. Too much TGFβ, too late in cancer progression is the fundamental motivation for pharmaceutical inhibition. PMID:19291273

  13. Influence of phase transformation on stress evolution during growth of metal thin films on silicon.

    PubMed

    Fillon, A; Abadias, G; Michel, A; Jaouen, C; Villechaise, P

    2010-03-01

    In situ stress measurements during two-dimensional growth of low mobility metal films on amorphous Si were used to demonstrate the impact of interface reactivity and phase transformation on stress evolution. Using Mo1-xSix films as examples, the results show that the tensile stress rise, which develops after the film has become crystalline, is correlated with an increase in lateral grain size. The origin of the tensile stress is attributed to the volume change resulting from the alloy crystallization, which occurs at a concentration-dependent critical thickness. PMID:20366996

  14. A novel transforming growth factor beta2 antisense transcript in mammalian lung.

    PubMed Central

    Coker, R K; Laurent, G J; Dabbagh, K; Dawson, J; McAnulty, R J

    1998-01-01

    Transforming growth factor (TGF) beta2 gene expression was examined in murine, rat and human lung by in situ hybridization with riboprobes. Hybridization signal was observed in a variety of cells with the sense probe, and Northern-blot analysis with this probe demonstrated the presence of a novel 3.5 kb transcript. This first report suggesting the existance of a natural TGFbeta2 antisense transcript raises the possibility that such a transcript may play a role in regulating TGFbeta2 production. PMID:9601055

  15. Phase transformations during the growth of paracetamol crystals from the vapor phase

    NASA Astrophysics Data System (ADS)

    Belyaev, A. P.; Rubets, V. P.; Antipov, V. V.; Bordei, N. S.

    2014-07-01

    Phase transformations during the growth of paracetamol crystals from the vapor phase are studied by differential scanning calorimetry. It is found that the vapor-crystal phase transition is actually a superposition of two phase transitions: a first-order phase transition with variable density and a second-order phase transition with variable ordering. The latter, being a diffuse phase transition, results in the formation of a new, "pretransition," phase irreversibly spent in the course of the transition, which ends in the appearance of orthorhombic crystals. X-ray diffraction data and micrograph are presented.

  16. Phase and structural transformations in annealed copper coatings in relation to oxide whisker growth

    NASA Astrophysics Data System (ADS)

    Dorogov, M. V.; Priezzheva, A. N.; Vlassov, S.; Kink, I.; Shulga, E.; Dorogin, L. M.; Lõhmus, R.; Tyurkov, M. N.; Vikarchuk, A. A.; Romanov, A. E.

    2015-08-01

    We describe structural and phase transformation in copper coatings made of microparticles during heating and annealing in air in the temperature range up to 400 °C. Such thermal treatment is accompanied by intensive CuO nanowhisker growth on the coating surface and the formation of the layered oxide phases (Cu2O and CuO) in the coating interior. X-ray diffraction and focused ion beam (FIB) are employed to characterize the multilayer structure of annealed copper coatings. Formation of volumetric defects such as voids and cracks in the coating is demonstrated.

  17. Time dependent impact of perinatal hypoxia on growth hormone, insulin-like growth factor 1 and insulin-like growth factor binding protein-3.

    PubMed

    Kartal, Ömer; Aydınöz, Seçil; Kartal, Ayşe Tuğba; Kelestemur, Taha; Caglayan, Ahmet Burak; Beker, Mustafa Caglar; Karademir, Ferhan; Süleymanoğlu, Selami; Kul, Mustafa; Yulug, Burak; Kilic, Ertugrul

    2016-08-01

    Hypoxic-ischemia (HI) is a widely used animal model to mimic the preterm or perinatal sublethal hypoxia, including hypoxic-ischemic encephalopathy. It causes diffuse neurodegeneration in the brain and results in mental retardation, hyperactivity, cerebral palsy, epilepsy and neuroendocrine disturbances. Herein, we examined acute and subacute correlations between neuronal degeneration and serum growth factor changes, including growth hormone (GH), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) after hypoxic-ischemia (HI) in neonatal rats. In the acute phase of hypoxia, brain volume was increased significantly as compared with control animals, which was associated with reduced GH and IGF-1 secretions. Reduced neuronal survival and increased DNA fragmentation were also noticed in these animals. However, in the subacute phase of hypoxia, neuronal survival and brain volume were significantly decreased, accompanied by increased apoptotic cell death in the hippocampus and cortex. Serum GH, IGF-1, and IGFBP-3 levels were significantly reduced in the subacute phase of HI. Significant retardation in the brain and body development were noted in the subacute phase of hypoxia. Here, we provide evidence that serum levels of growth-hormone and factors were decreased in the acute and subacute phase of hypoxia, which was associated with increased DNA fragmentation and decreased neuronal survival. PMID:26943480

  18. Transforming Growth Factor-β3 Therapy Delays Postoperative Reossification and Improves Craniofacial Growth in Craniosynostotic Rabbits.

    PubMed

    Gilbert, J; Karski, M; Smith, T D; Burrows, A M; Norbutt, C; Siegel, M I; Costello, B J; Cray, J J; Losee, J E; Moursi, A M; Cooper, G M; Mooney, M P

    2016-03-01

    Postoperative reossification is a common clinical correlate following surgery. It has been suggested that an underexpression of transforming growth factor-β3 (TGF-β3) may be related to craniosynostosis and postoperative reossification. Adding TGF-β3 may delay reossification and improve postoperative growth. The present study was designed to test this hypothesis. Thirty 10-day-old New Zealand white rabbits with hereditary coronal suture synostosis were divided into three groups: (1) suturectomy controls (n = 14), (2) suturectomy treated with bovine serum albumin (n = 8), and (3) suturectomy treated with TGF-β3 protein (n = 8). At 10 days of age, a 3-mm × 15-mm coronal suturectomy was performed, and serial three-dimensional (3D) computed tomography (CT) scans and cephalographs were taken at 10, 25, 42, and 84 days of age. Calvaria were harvested at 84 days of age for histomorphometric analysis. Mean differences were analyzed using a group by age analysis of variance. Analysis of the 3D CT scan data revealed that sites treated with TGF-β3 had significantly (P < .05) greater defect areas and significantly (P < .05) greater intracranial volumes through 84 days of age compared with controls. Histomorphometry showed that sites treated with TGF-β3 had patent suturectomy sites and significantly (P < .001) less new bone in the suturectomy site compared with controls. Serial radiograph data revealed significant (P < .05) differences in craniofacial growth from 25 to 84 days in TGF-β3-treated rabbits compared with controls. Data show that TGF-β3 administration delayed reossification and improved craniofacial growth in this rabbit model. These findings also suggest that this molecular-based therapy may have potential clinical use. PMID:26090789

  19. Harnessing high density lipoproteins to block transforming growth factor beta and to inhibit the growth of liver tumor metastases.

    PubMed

    Medina-Echeverz, José; Fioravanti, Jessica; Díaz-Valdés, Nancy; Frank, Kathrin; Aranda, Fernando; Gomar, Celia; Ardaiz, Nuria; Dotor, Javier; Umansky, Viktor; Prieto, Jesús; Berraondo, Pedro

    2014-01-01

    Transforming growth factor β (TGF-β) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFβ-inhibitory molecules. We constructed a plasmid encoding a potent TGF-β-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-β. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-β signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2-/-IL2rγ-/- immunodeficient mice. This effect was associated with downregulation of TGF-β target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8+ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-β-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms. PMID:24797128

  20. Harnessing High Density Lipoproteins to Block Transforming Growth Factor Beta and to Inhibit the Growth of Liver Tumor Metastases

    PubMed Central

    Medina-Echeverz, José; Fioravanti, Jessica; Díaz-Valdés, Nancy; Frank, Kathrin; Aranda, Fernando; Gomar, Celia; Ardaiz, Nuria; Dotor, Javier; Umansky, Viktor; Prieto, Jesús; Berraondo, Pedro

    2014-01-01

    Transforming growth factor β (TGF-β) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFβ-inhibitory molecules. We constructed a plasmid encoding a potent TGF-β-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-β. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-β signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2−/−IL2rγ−/− immunodeficient mice. This effect was associated with downregulation of TGF-β target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8+ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-β-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms. PMID:24797128

  1. Long [R3] insulin-like growth factor-I reduces growth, plasma growth hormone, IGF binding protein-3 and endogenous IGF-I concentrations in pigs.

    PubMed

    Dunaiski, V; Dunshea, F R; Walton, P E; Goddard, C

    1997-12-01

    Growth hormone (GH) improves growth performance in the pig. Analogues of insulin-like growth factor-I (IGF-I) that bind poorly to IGF binding proteins (IGFBP) stimulate growth in the rat but, in contrast, inhibit growth in the pig. This study was designed to determine the effect of IGF peptides alone or in combination with porcine GH (pGH) on growth characteristics and plasma hormone concentrations in finisher pigs. A four-day infusion of Long [R3] IGF-I (LR3IGF-I; 180 micrograms/kg/day) decreased the average daily gain, food intake, and plasma IGFBP-3, IGF-I and insulin concentrations. The mean plasma GH concentration was decreased by 23% and the area under the GH peaks was reduced by 60%. Co-administration of pGH (30 micrograms/kg/day) with LR3IGF-I had no interactive effect on growth performance, and plasma insulin, IGFBP-3 and IGF-I concentrations remained suppressed. The area under the GH peaks was not restored with this combination treatment although mean plasma GH concentrations were elevated in all animals receiving pGH. Infusion of IGF-I (180 micrograms/kg/day) decreased plasma insulin and mean GH concentrations but had no significant effect on IGFBP-3 concentrations. Average daily gain and feed intake were not changed by IGF-I treatment. A combination of IGF-I and pGH injection (30 micrograms/kg/day) increased plasma IGFBP-3 concentrations but plasma insulin levels remained suppressed. Plasma glucose levels were unaffected by any treatment. The study demonstrates that both IGF-I and LR3IGF-I suppress plasma GH concentrations in finisher pigs. This, in turn, may be responsible for the reduction in the plasma concentration of IGF-I, IGFBP-3 and insulin seen in LR3IGF-I-treated animals. The decrease in these parameters may contribute to the inhibitory effect of LR3IGF-I on growth performance in the pig. PMID:9488001

  2. MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor-{beta}.

    PubMed

    Louafi, Fethi; Martinez-Nunez, Rocio T; Sanchez-Elsner, Tilman

    2010-12-31

    Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with important effects on processes such as fibrosis, angiogenesis, and immunosupression. Using bioinformatics, we identified SMAD2, one of the mediators of TGF-β signaling, as a predicted target for a microRNA, microRNA-155 (miR-155). MicroRNAs are a class of small non-coding RNAs that have emerged as an important class of gene expression regulators. miR-155 has been found to be involved in the regulation of the immune response in myeloid cells. Here, we provide direct evidence of binding of miR-155 to a predicted binding site and the ability of miR-155 to repress SMAD2 protein expression. We employed a lentivirally transduced monocyte cell line (THP1-155) containing an inducible miR-155 transgene to show that endogenous levels of SMAD2 protein were decreased after sustained overexpression of miR-155. This decrease in SMAD2 led to a reduction in both TGF-β-induced SMAD-2 phosphorylation and SMAD-2-dependent activation of the expression of the CAGA(12)LUC reporter plasmid. Overexpression of miR-155 altered the cellular responses to TGF-β by changing the expression of a set of genes that is involved in inflammation, fibrosis, and angiogenesis. Our study provides firm evidence of a role for miR-155 in directly repressing SMAD2 expression, and our results demonstrate the relevance of one of the two predicted target sites in SMAD2 3'-UTR. Altogether, our data uncover an important role for miR-155 in modulating the cellular response to TGF-β with possible implications in several human diseases where homeostasis of TGF-β might be altered. PMID:21036908

  3. GSK3 inactivation is involved in mitochondrial complex IV defect in transforming growth factor (TGF) {beta}1-induced senescence

    SciTech Connect

    Byun, Hae-Ok; Jung, Hyun-Jung; Seo, Yong-Hak; Lee, Young-Kyoung; Hwang, Sung-Chul; Seong Hwang, Eun; Yoon, Gyesoon

    2012-09-10

    Transforming growth factor {beta}1 (TGF {beta}1) induces Mv1Lu cell senescence by persistently producing mitochondrial reactive oxygen species (ROS) through decreased complex IV activity. Here, we investigated the molecular mechanism underlying the effect of TGF {beta}1 on mitochondrial complex IV activity. TGF {beta}1 progressively phosphorylated the negative regulatory sites of both glycogen synthase kinase 3 (GSK3) {alpha} and {beta}, corresponding well to the intracellular ROS generation profile. Pre-treatment of N-acetyl cysteine, an antioxidant, did not alter this GSK3 phosphorylation (inactivation), whereas pharmacological inhibition of GSK3 by SB415286 significantly increased mitochondrial ROS, implying that GSK3 phosphorylation is an upstream event of the ROS generation. GSK3 inhibition by SB415286 decreased complex IV activity and cellular O{sub 2} consumption rate and eventually induced senescence of Mv1Lu cell. Similar results were obtained with siRNA-mediated knockdown of GSK3. Moreover, we found that GSK3 not only exists in cytosol but also in mitochondria of Mv1Lu cell and the mitochondrial GSK3 binds complex IV subunit 6b which has no electron carrier and is topologically located in the mitochondrial intermembrane space. Involvement of subunit 6b in controlling complex IV activity and overall respiration rate was proved with siRNA-mediated knockdown of subunit 6b. Finally, TGF {beta}1 treatment decreased the binding of the subunit 6b to GSK3 and subunit 6b phosphorylation. Taken together, our results suggest that GSK3 inactivation is importantly involved in TGF {beta}1-induced complex IV defects through decreasing phosphorylation of the subunit 6b, thereby contributing to senescence-associated mitochondrial ROS generation.

  4. [Transforming growth factor beta-1: structure, function, and regulation mechanisms in cancer].

    PubMed

    Peralta-Zaragoza, O; Lagunas-Martínez, A; Madrid-Marina, V

    2001-01-01

    Transforming growth factor beta-1 (TGF-beta 1) is produced by several cell lineages such as lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these and other cells. In general, TGF-beta 1 has pleiotropic properties on the immune response during the development of infection diseases and cancer; however, the mechanisms of action and regulation of gene expression of this cytokine are poorly understood, in this review, the biological properties and the molecular mechanisms that regulate TGF-beta 1 gene expression are described, to understand the role of this cytokine in growth and cell differentiation. The knowledge of molecular mechanisms of gene expression of TGF-beta 1 may serve to develop new cancer therapies. The English version of this paper is available at: http://www.insp.mx/salud/index.html PMID:11547595

  5. Insulin-like growth factor-binding protein-5 inhibits growth and induces differentiation of mouse osteosarcoma cells.

    PubMed

    Schneider, M R; Zhou, R; Hoeflich, A; Krebs, O; Schmidt, J; Mohan, S; Wolf, E; Lahm, H

    2001-10-26

    The precise role of insulin-like growth factor-binding protein-5 (IGFBP-5) in regulating the growth of tumor cells, especially of bone-derived malignant cells, is not well understood. We have investigated the biological activity of IGFBP-5 by transfecting OS/50-K8 mouse osteosarcoma cells with an expression vector containing the osteocalcin promoter and the complete mouse IGFBP-5 cDNA (OC-IGFBP-5). Overexpression of IGFBP-5 mRNA and secretion of increased amounts of bioactive protein in conditioned media were demonstrated in different clones. For the analysis of cell proliferation, three clones exhibiting high levels of IGFBP-5 expression were selected and compared to a mock clone and to nontransfected parental cells. IGFBP-5-secreting clones displayed reduced proliferation under both anchorage-dependent and -independent conditions (P < 0.05). The increase in proliferation observed in IGFBP-5-secreting clones after addition of exogenous IGF was significantly lower than that observed in mock-transfected or parental cells. A similar result was obtained with long[R3]IGF-I which has a low affinity for all IGFBPs, suggesting that the inhibitory effect of IGFBP-5 is only partially IGF-dependent. OC-IGFBP-5-transfected clones expressed significantly higher amounts of osteocalcin mRNA (P < 0.05) and secreted more osteocalcin protein than a mock clone or parental OS-50/K8 cells. Thus, part of the growth-inhibiting effect of IGFBP-5 may be due to an induction of differentiation in these cells. PMID:11606061

  6. Pharmacokinetics and distribution of heparin-binding growth factor I (endothelial cell growth factor) in the rat

    SciTech Connect

    Rosengart, T.K.; Kuperschmid, J.P.; Maciag, T.; Clark, R.E.

    1989-02-01

    Heparin-binding growth factor I (HBGF I), previously designated as endothelial cell growth factor, is a potent mitogen for endothelial cells in vitro, which may prove useful for promoting endothelial regeneration in vivo. Analysis of the pharmacokinetics and organ distribution of HBGF I is necessary before use of HBGF I as a pharmacological agent. Consequently, pharmacological studies were carried out with (125I)HBGF I in the rat. Intravenous injections of HBGF I were given with or without heparin (2.5 units/ng HBGF I). Blood concentrations of HBGF I decreased by one half 17 seconds after HBGF I bolus. This time was prolonged to 60 seconds when HBGF I was injected with heparin. The elimination half-life of HBGF I was 14 minutes in the presence of heparin. The highest concentrations of HBGF I following intravenous bolus were found in kidney, liver, and spleen, and the lowest in fat and brain. Heparin increased HBGF I concentrations in blood and all organs measured except kidney, which was significantly decreased (p less than 0.01). Intact HBGF I was recoverable from blood 5 minutes following intravenous administration. HBGF I underwent near-complete proteolytic digestion after more prolonged ex vivo incubation with rat plasma, but HBGF I was protected from proteolysis when incubations were conducted in the presence of heparin. Thus, it is feasible that HBGF I can be administered as a pharmacological agent in the presence of heparin. Further studies assessing acceleration of in vivo endothelial growth using HBGF I with heparin appear warranted.

  7. Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation.

    PubMed Central

    Pertovaara, L; Sistonen, L; Bos, T J; Vogt, P K; Keski-Oja, J; Alitalo, K

    1989-01-01

    Transforming growth factor beta (TGF beta) is a multifunctional polypeptide that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF beta signal transduction in cells is unknown. We report here that an early effect of TGF beta is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF beta, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF beta, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TGF beta. The increase in jun mRNA occurred with picomolar TGF beta concentrations within 1 h of TGF beta stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-line levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF beta, evidently in a protein synthesis-independent fashion. The junB response to TGF beta was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF beta may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF beta. Images PMID:2725496

  8. High glucose concentration induces the overexpression of transforming growth factor-beta through the activation of a platelet-derived growth factor loop in human mesangial cells.

    PubMed Central

    Di Paolo, S.; Gesualdo, L.; Ranieri, E.; Grandaliano, G.; Schena, F. P.

    1996-01-01

    High glucose concentration has been shown to induce the overexpression of transforming growth factor (TGF)-beta 1 mRNA and protein in different cell types, including murine mesangial cells, thus possibly accounting for the expansion of mesangial extracellular matrix observed in diabetic glomerulopathy. In the present study, we evaluated platelet-derived growth factor (PDGF) B-chain and PDGF-beta receptor gene expression in human mesangial cells (HMCs) exposed to different concentrations of glucose and then sought a possible relationship between a PDGF loop and the modulation of TGF-beta 1 expression. HMC [3H]thymidine incorporation was upregulated by 30 mmol/L glucose (HG) up to 24 hours, whereas it was significantly inhibited at later time points. Neutralizing antibodies to PDGF BB abolished the biphasic response to HG, whereas anti-TGF-beta antibodies reversed only the late inhibitory effect of hyperglycemic medium. HG induced an early and persistent increase of PDGF B-chain gene expression, as evaluated by reverse transcriptase polymerase chain reaction, whereas PDGF-beta receptor mRNA increased by twofold after 6 hours, thereafter declining at levels 70% lower than in controls after 24 hours. 125I-Labeled PDGF BB binding studies in HMCs exposed to HG for 24 hours confirmed the decrease of PDGF-beta receptor expression. TGF-beta 1-specific transcripts showed 43 and 78% increases after 24 and 48 hours of incubation in HG, respectively, which was markedly diminished by anti-PDGF BB neutralizing antibodies or suramin. We conclude that HG induces an early activation of a PDGF loop that, in turn, causes an increase of TGF-beta 1 gene expression, thus modulating both HMC proliferation and mesangial matrix production. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8952542

  9. Transforming Growth Factor-β-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells

    PubMed Central

    Kim, Yong-Eun; Kim, Jong Ok; Park, Ki-Sun; Won, Minho; Kim, Kyoon Eon; Kim, Kee K.

    2016-01-01

    The RNA-binding protein Rbfox3 is a well-known splicing regulator that is used as a marker for post-mitotic neurons in various vertebrate species. Although recent studies indicate a variable expression of Rbfox3 in non-neuronal tissues, including lung tissue, its cellular function in lung cancer remains largely unknown. Here, we report that the number of RBFOX3-positive cells in tumorous lung tissue is lower than that in normal lung tissue. As the transforming growth factor-β (TGF-β) signaling pathway is important in cancer progression, we investigated its role in RBFOX3 expression in A549 lung adenocarcinoma cells. TGF-β1 treatment inhibited RBFOX3 expression at the transcriptional level. Further, RBFOX3 depletion led to a change in the expression levels of a subset of proteins related to epithelial-mesenchymal transition (EMT), such as E-cadherin and Claudin-1, during TGF-β1-induced EMT. In immunofluorescence microscopic analysis, mesenchymal morphology was more prominent in RBFOX3-depleted cells than in control cells. These findings show that TGF-β-induced RBFOX3 inhibition plays an important role in EMT and propose a novel role for RBFOX3 in cancer progression. PMID:27432190

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

    PubMed

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

    2009-03-01

    Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation. PMID:19122240

  11. Estimating canopy leaf area index in the late stages of wheat growth using continuous wavelet transform

    NASA Astrophysics Data System (ADS)

    Huang, Yan; Tian, Qingjiu; Wang, Lei; Geng, Jun; Lyu, Chunguang

    2014-01-01

    The existing hyperspectral vegetation indices used for estimating the canopy leaf area index (LAI) of winter wheat (Triticum aestivum L.) performed well, but the use of such indices at late growth stages can lead to inaccurate results. To improve the performance of LAI models for wheat in late growth stages, the continuous wavelet transform (CWT) method was applied in this study and used to decompose the canopy reflectance and its first derivative into wavelet coefficients. The correlation scalograms of wavelet coefficients and the LAI were then constructed and used to extract the top 1% correlated region as the wavelet feature. The canopy LAI estimation model for late growth wheat was established at last and compared with models based on 12 different types of hyperspectral vegetation indices. The results showed that, compared with the estimation models using the hyperspectral vegetation indices (for which the R2 values were all less than 0.15 and the root-mean-square errors (RMSEs) were greater than 1), the CWT-based canopy LAI estimation model for late growth wheat had obvious improvements in accuracy (maximum R2 of 0.53 and minimum of RMSE of 0.78). Hence, this new method shows promise for use in agricultural and ecological applications.

  12. THE RNA-BINDING PROTEIN HUR PROMOTES GLIOMA GROWTH AND TREATMENT RESISTANCE

    PubMed Central

    Filippova, Natalia; Yang, Xiuhua; Wang, Yimin; Gillespie, G Yancey; Langford, Cathy; King, Peter H.; Wheeler, Crystal; Nabors, L. Burt

    2011-01-01

    Posttranscriptional regulation is a critical control point for the expression of genes that promote or retard tumor growth. We previously found that the mRNA binding protein, ELAV 1 (HuR), is upregulated in primary brain tumors and stabilizes growth factor mRNAs such as VEGF and IL-8. To better understand the role of HuR in brain tumor growth, we altered levels of HuR in glioma cells by shRNA or ectopic expression and measured tumor cell phenotype using in vitro and in vivo models. In HuR-silenced cells, we found a significant decrease in anchorage-independent growth and cell proliferation with a concomitant induction of apoptosis. Using an intracranial tumor model with primary glioblastoma cells, HuR silencing produced a significant decrease in tumor volume. In contrast, overexpression of HuR produced in vitro chemoresistance to standard glioma therapies. Since bcl-2 is abundantly expressed in glioma and associated with tumor growth and survival, we determined the impact of HuR on its regulation as a molecular validation to the cellular and animal studies. Using UV crosslinking and RNA immunoprecipitation, we show that HuR bound to the 3' untranslated region of all bcl-2 family members. Silencing of HuR led to transcript destabilization and reduced protein expression. Polysome profiling indicated loss of HuR from the translational apparatus. In summary, these findings reveal a HuR-dependent mechanism for cancer cell survival and sensitivity to chemotherapeutic drugs suggesting that HuR should be considered as a new therapeutic target. PMID:21498545

  13. Constitutive activation of epidermal growth factor receptor promotes tumorigenesis of Cr(VI)-transformed cells through decreased reactive oxygen species and apoptosis resistance development.

    PubMed

    Kim, Donghern; Dai, Jin; Fai, Leonard Yenwong; Yao, Hua; Son, Young-Ok; Wang, Lei; Pratheeshkumar, Poyil; Kondo, Kazuya; Shi, Xianglin; Zhang, Zhuo

    2015-01-23

    Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing

  14. Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors.

    PubMed Central

    Panganiban, G E; Rashka, K E; Neitzel, M D; Hoffmann, F M

    1990-01-01

    The decapentaplegic (dpp) gene of Drosophila melanogaster is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks. The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor beta (TGF-beta). We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells. Similar to other proteins in the TGF-beta family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells. The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish. The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-beta, was found extracellularly as a 30-kDa homodimer. Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish. The dpp protein could be released into solution by increased salt concentration and nonionic detergent. Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex. Images PMID:1692958

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

    PubMed

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

    2011-06-01

    Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition. PMID:21477078

  16. Growth Hormone Induces Transforming Growth Factor-Beta-Induced Protein in Podocytes: Implications for Podocyte Depletion and Proteinuria.

    PubMed

    Chitra, P Swathi; Swathi, T; Sahay, Rakesh; Reddy, G Bhanuprakash; Menon, Ram K; Kumar, P Anil

    2015-09-01

    The glomerular podocytes form a major size selective barrier for the filtration of serum proteins and reduced podocyte number is a critical event in the pathogenesis of proteinuria during diabetic nephropathy (DN). An elevated level of growth hormone (GH) is implicated as a causative factor in the development of nephropathy in patients with type 1 diabetes mellitus. We have previously shown that podocytes express GH receptor and are a target for GH action. To elucidate the molecular basis for the effects of GH on podocyte depletion, we conducted PCR-array analyses for extracellular matrix and adhesion molecules in podocytes. Our studies reveal that GH increases expression of a gene that encodes transforming growth factor-beta-induced protein (TGFBIp) expression. Similarly, microarray data retrieved from the Nephromine database revealed elevation of TGFBIp in patients with DN. Treatment with GH results in increased secretion of extracellular TGFBIp by podocytes. Both GH and TGFBIp induced apoptosis and epithelial mesenchymal transition (EMT) of podocytes. Exposure of podocytes to GH and TGFBIp resulted in increased migration of cells and altered podocyte permeability to albumin across podocyte monolayer. Administration of GH to rats induced EMT and apoptosis in the glomerular fraction of the kidney. Therefore, we conclude that the GH-dependent increase in TGFBIp in the podocyte is one of the mechanisms responsible for podocyte depletion in DN. PMID:25740786

  17. Dental pulp stem cells suppress the proliferation of lymphocytes via transforming growth factor-β1.

    PubMed

    Ding, Gang; Niu, Jianyi; Liu, Yi

    2015-04-01

    Dental pulp stem cells (DPSCs) possess self-renewal capability, multi-lineage differentiation potential, and can generate a dentin-pulp-like tissue in vivo, which is promising for tooth regeneration. To enlarge the cells resource of DPSCs and explore the feasibility of DPSCs-mediated immune therapy, it is prerequisite to investigate the immunological properties of DPSCs and the underlying mechanisms. Human DPSCs and peripheral blood mononuclear cells were isolated and cultured. Then we used lymphocytes proliferation assays, cytokines detection, Transwell cultures, neutralization experiments, and flow cytometry to examine the in vitro immune characteristics of DPSCs. We found that DPSCs failed to stimulate allogeneic T cells proliferation and suppressed T cells proliferation, B cells proliferation, and mixed lymphocyte reaction. In addition, DPSCs could up-regulate IL-10, down-regulate the production of IL-2, IL-17, and IFN-γ, and did not affect the production of IL-6. Monoclonal antibody against transforming growth factor-β1 restored the T cells proliferation inhibited by DPSCs. Moreover, the population of regulatory T cells increased significantly and T-helper 17 cells decreased significantly in peripheral blood mononuclear cells co-cultured with DPSCs. These data confirmed that DPSCs are low immunogenic, could inhibit the proliferation of lymphocytes, regulate the production of cytokines in vitro, and the secretion of transforming growth factor-β1 may be involved in this event. PMID:25605036

  18. Retinoic acid modulates rat Ito cell proliferation, collagen, and transforming growth factor beta production.

    PubMed Central

    Davis, B H; Kramer, R T; Davidson, N O

    1990-01-01

    Recent studies suggest that vitamin A plays an inhibitory role with respect to "activation" of the hepatic Ito cell, a likely effector of hepatic fibrogenesis. Ito cell "activation" during fibrogenesis is characterized by a decrease in intracellular vitamin A and an increase in cellular proliferation and collagen production. To explore the hypothesis that retinoids have the capacity to diminish Ito cell activation, cultured Ito cells were exposed to retinoic acid and its effects assessed on three key features: cell proliferation, collagen protein production and mRNA abundance, and transforming growth factor beta protein production. Retinoic acid was 100-1,000X more potent than retinol with respect to inhibition of Ito cell proliferation. Interstitial collagen and transforming growth factor beta production were also reduced by 10(-6) M retinoic acid. The relative abundance of type I collagen mRNA however, was not significantly altered. By contrast, retinoic acid administration to rats caused a marked reduction in the abundance of type I collagen mRNA in both total hepatic and purified Ito cell RNA. The relative abundance of rat hepatic fibronectin or apolipoprotein E mRNA was not significantly altered. These studies demonstrate that retinoic acid can differentially modulate several key features of hepatic fibrogenesis in vitro and in vivo. Images PMID:2254460

  19. Toll-Like Receptor 4 Signaling Augments Transforming Growth Factor-β Responses

    PubMed Central

    Bhattacharyya, Swati; Kelley, Kathleen; Melichian, Denisa S.; Tamaki, Zenshiro; Fang, Feng; Su, Yunyun; Feng, Gilbert; Pope, Richard M.; Budinger, G.R. Scott; Mutlu, Gökhan M.; Lafyatis, Robert; Radstake, Timothy; Feghali-Bostwick, Carol; Varga, John

    2014-01-01

    Because recent studies implicate Toll-like receptors (TLRs) in the pathogenesis of fibrosis, we sought to investigate the in vitro and in vivo role and mechanism of TLR4-mediated fibroblast responses in fibrogenesis. We found that TLR4 was constitutively expressed, and accumulation of endogenous TLR4 ligands significantly elevated, in lesional skin and lung tissues from patients with scleroderma. Activation of TLR4 signaling in explanted fibroblasts resulted in enhanced collagen synthesis and increased expression of multiple genes involved in tissue remodeling and extracellular matrix homeostasis. Moreover, TLR4 dramatically enhanced the sensitivity of fibroblasts to the stimulatory effect of transforming growth factor-β1. These profibrotic responses were abrogated by both genetic and pharmacological disruption of TLR4 signaling in vitro, and skin fibrosis induced by bleomycin in vivo was attenuated in mice harboring a mutated TLR4. Activation of TLR4 in fibroblasts augmented the intensity of canonical Smad signaling, and was accompanied by suppression of anti-fibrotic microRNA expression. Together, these results suggest a novel model to account for persistent fibrogenesis in scleroderma, in which activation of fibroblast TLR4 signaling, triggered by damage-associated endogenous TLR4 ligands, results in augmented transforming growth factor-β1 sensitivity with increased matrix production and progressive connective tissue remodeling. Under these conditions, fibroblast TLR4 serves as the switch for converting self-limited tissue repair into intractable fibrosis. PMID:23141927

  20. Strong magnetic field-assisted growth of carbon nanofibers and its microstructural transformation mechanism

    PubMed Central

    Luo, Chengzhi; Fu, Qiang; Pan, Chunxu

    2015-01-01

    It is well-known that electric and magnetic fields can control the growth direction, morphology and microstructure of one-dimensional carbon nanomaterials (1-DCNMs), which plays a key role for its potential applications in micro-nano-electrics and devices. In this paper, we introduce a novel process for controlling growth of carbon nanofibers (CNFs) with assistance of a strong magnetic field (up to 0.5 T in the center) in a chemical vapor deposition (CVD) system. The results reveal that: 1) The CNFs get bundled when grown in the presence of a strong magnetic field and slightly get aligned parallel to the direction of the magnetic field; 2) The CNFs diameter become narrowed and homogenized with increase of the magnetic field; 3) With the increase of the magnetic field, the microstructure of CNFs is gradually changed, i.e., the strong magnetic field makes the disordered “solid-cored” CNFs transform into a kind of bamboo-liked carbon nanotubes; 4) We propose a mechanism that the reason for these variations and transformation is due to diamagnetic property of carbon atoms, so that it has direction selectivity in the precipitation process. PMID:25761381

  1. Strong magnetic field-assisted growth of carbon nanofibers and its microstructural transformation mechanism.

    PubMed

    Luo, Chengzhi; Fu, Qiang; Pan, Chunxu

    2015-01-01

    It is well-known that electric and magnetic fields can control the growth direction, morphology and microstructure of one-dimensional carbon nanomaterials (1-DCNMs), which plays a key role for its potential applications in micro-nano-electrics and devices. In this paper, we introduce a novel process for controlling growth of carbon nanofibers (CNFs) with assistance of a strong magnetic field (up to 0.5 T in the center) in a chemical vapor deposition (CVD) system. The results reveal that: 1) The CNFs get bundled when grown in the presence of a strong magnetic field and slightly get aligned parallel to the direction of the magnetic field; 2) The CNFs diameter become narrowed and homogenized with increase of the magnetic field; 3) With the increase of the magnetic field, the microstructure of CNFs is gradually changed, i.e., the strong magnetic field makes the disordered "solid-cored" CNFs transform into a kind of bamboo-liked carbon nanotubes; 4) We propose a mechanism that the reason for these variations and transformation is due to diamagnetic property of carbon atoms, so that it has direction selectivity in the precipitation process. PMID:25761381

  2. Strong magnetic field-assisted growth of carbon nanofibers and its microstructural transformation mechanism

    NASA Astrophysics Data System (ADS)

    Luo, Chengzhi; Fu, Qiang; Pan, Chunxu

    2015-03-01

    It is well-known that electric and magnetic fields can control the growth direction, morphology and microstructure of one-dimensional carbon nanomaterials (1-DCNMs), which plays a key role for its potential applications in micro-nano-electrics and devices. In this paper, we introduce a novel process for controlling growth of carbon nanofibers (CNFs) with assistance of a strong magnetic field (up to 0.5 T in the center) in a chemical vapor deposition (CVD) system. The results reveal that: 1) The CNFs get bundled when grown in the presence of a strong magnetic field and slightly get aligned parallel to the direction of the magnetic field; 2) The CNFs diameter become narrowed and homogenized with increase of the magnetic field; 3) With the increase of the magnetic field, the microstructure of CNFs is gradually changed, i.e., the strong magnetic field makes the disordered ``solid-cored'' CNFs transform into a kind of bamboo-liked carbon nanotubes; 4) We propose a mechanism that the reason for these variations and transformation is due to diamagnetic property of carbon atoms, so that it has direction selectivity in the precipitation process.

  3. Alterations in cellular differentiation, mitogenesis, cytoskeleton and growth characteristics during Syrian hamster embryo cell multistep in vitro transformation.

    PubMed

    Isfort, R J; Cody, D B; Doersen, C J; Kerckaert, G A; Leboeuf, R A

    1994-10-01

    In vitro Syrian hamster embryo (SHE) cell transformation is a neoplastic process that proceeds through several identifiable consecutive stages including in vitro morphological transformation (mt), acquisition of immortality (I+), acquisition of tumorigenicity (T+) and tumor-derived cells (I'TD). Eight transformed lineages consisting of cells at the mt, I+, T+ and I'TD stages were assayed for alterations in general markers of cell differentiation, mitogenic signaling pathways, cytoskeleton and cellular growth in 3D matrix. Alterations in cellular differentiation markers included a decrease in H19 gene expression and placental alkaline phosphatase enzymatic activity at the mt stage in all lineages examined with a complete absence of H19 gene expression and placental alkaline phosphatase enzymatic activity by the I'TD stage in a majority of transformed lineages. Changes in mitogenic signaling pathways included the production of autocrine growth factors and alterations in growth factor-induced immediate early gene expression by the I'TD stage of transformation in the majority of transformed lineages investigated. By the I'TD stage of transformation in most lineages, changes in both the cytoskeleton (including a decrease in tropomyosin-I gene expression) and the Matrigel growth characteristics of SHE cells were observed. PMID:7927892

  4. Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.

    PubMed Central

    Panos, R J; Rubin, J S; Csaky, K G; Aaronson, S A; Mason, R J

    1993-01-01

    Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell

  5. Insulin-like growth factor binding protein-3 induces apoptosis in MCF7 breast cancer cells.

    PubMed

    Nickerson, T; Huynh, H; Pollak, M

    1997-08-28

    Insulin-like growth factors (IGFs) are known to have potent antiapoptotic activity. The antiestrogen ICI 182,780 (ICI) is a potent inhibitor of MCF7 human breast cancer cell growth and has recently been reported to act as an antiproliferative agent in part via upregulation of expression of insulin-like growth factor binding proteins (IGFBPs) -3 and -5, which attenuate the bioactivity of IGFs in many experimental systems. We show here that ICI and IGFBP-3 induce apoptosis in MCF7 cells. Treatment of MCF7 cells with 10 nM ICI or 36 nM recombinant human IGFBP. 3 for 72 hours increased apoptosis approximately 3.5-fold relative to control as quantitated by a cell death ELISA which measures DNA fragmentation. Long R3 IGF-I, an IGF-I analogue with greatly reduced affinity for IGFBPs yet similar affinity for IGF-I receptors, was a more potent inhibitor of IGFBP-3-induced and ICI-induced apoptosis than IGF-I. These results suggest that IGFBP-3 enhances apoptosis by reducing bioavailability of ligands for the IGF-I receptor and suggest that modulation of IGFBP-3 expression by ICI contributes to apoptosis induced by this compound. More generally, the data suggest that IGFBPs are regulators of apoptosis. PMID:9299428

  6. Emerging Roles of RNA-Binding Proteins in Plant Growth, Development, and Stress Responses.

    PubMed

    Lee, Kwanuk; Kang, Hunseung

    2016-03-01

    Posttranscriptional regulation of RNA metabolism, including RNA processing, intron splicing, editing, RNA export, and decay, is increasingly regarded as an essential step for fine-tuning the regulation of gene expression in eukaryotes. RNA-binding proteins (RBPs) are central regulatory factors controlling posttranscriptional RNA metabolism during plant growth, development, and stress responses. Although functional roles of diverse RBPs in living organisms have been determined during the last decades, our understanding of the functional roles of RBPs in plants is lagging far behind our understanding of those in other organisms, including animals, bacteria, and viruses. However, recent functional analysis of multiple RBP family members involved in plant RNA metabolism and elucidation of the mechanistic roles of RBPs shed light on the cellular roles of diverse RBPs in growth, development, and stress responses of plants. In this review, we will discuss recent studies demonstrating the emerging roles of multiple RBP family members that play essential roles in RNA metabolism during plant growth, development, and stress responses. PMID:26831454

  7. Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity.

    PubMed

    Bonnekoh, B; Wevers, A; Jugert, F; Merk, H; Mahrle, G

    1989-01-01

    The application of a simple, rapid, and inexpensive colorimetric growth assay was tested for human epidermal cells subcultured in uncoated plastic dishes. Cell layers were incubated with a crystal violet (CV) solution (0.2% with ethanol 2% in 0.5 M Tris-Cl buffer, pH 7.8) for 10 min at room temperature. After rinsing with 0.5 M Tris-Cl (pH 7.8) the cell layer was dried and decolorized with a sodium-dodecylsulfate solution (0.5% with ethanol 50% in 0.5 M Tris-Cl, pH 7.8) for 60 min at 37 degrees C. The extinction of the supernatant was read at the absorption maximum of 586 nm. The protein content of attached cells as classical parameter for quantifying cell growth was strongly related to CV extinction with a correlation coefficient of r = 0.98. Furthermore, the subcellular protein binding qualities of CV were analyzed. The water-soluble protein fraction of cultured epidermal cells was separated by sodium-dodecylsulfate polyacrylamide gel electrophoresis and stained with CV. We found a staining pattern which was qualitatively very similar to that of Coomassie blue, however less intense. Keratin electrophoresis revealed an affinity of CV to the 48, 50, and 56 kD cytokeratins. In conclusion, this CV assay is a reliable and simple method for the monitoring of epidermal cell growth in cultures. PMID:2482013

  8. ATP differentially upregulates fibroblast growth factor 2 and transforming growth factor α in neonatal and adult mice: effect on neuroproliferation.

    PubMed

    Jia, C; Cussen, A R; Hegg, C C

    2011-03-17

    Multiple neurotrophic factors play a role in proliferation, differentiation and survival in the olfactory epithelium (OE); however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic factors, fibroblast growth factor 2 (FGF2) and transforming growth factor alpha (TGFα), and that these neurotrophic factors have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased 20 h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse olfactory epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult olfactory epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal growth factor receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse olfactory epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development. PMID:21187124

  9. Antisense expression of an Arabidopsis ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress

    NASA Technical Reports Server (NTRS)

    Kim, S. H.; Arnold, D.; Lloyd, A.; Roux, S. J.

    2001-01-01

    We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.

  10. Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth

    PubMed Central

    2013-01-01

    Background Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear. Results We first investigated the effects of loss of SREBP function in non-transformed cells. Combined ablation of SREBP1 and SREBP2 by siRNA-mediated gene silencing or chemical inhibition of SREBP activation induced endoplasmic reticulum (ER)-stress and engaged the unfolded protein response (UPR) pathway, specifically under lipoprotein-deplete conditions in human retinal pigment epithelial cells. Induction of ER-stress led to inhibition of protein synthesis through increased phosphorylation of eIF2α. This demonstrates for the first time the importance of SREBP in the coordination of lipid and protein biosynthesis, two processes that are essential for cell growth and proliferation. SREBP ablation caused major changes in lipid composition characterized by a loss of mono- and poly-unsaturated lipids and induced accumulation of reactive oxygen species (ROS) and apoptosis. Alterations in lipid composition and increased ROS levels, rather than overall changes to lipid synthesis rate, were required for ER-stress induction. Next, we analyzed the effect of SREBP ablation in a panel of cancer cell lines. Importantly, induction of apoptosis following SREBP depletion was restricted to lipoprotein-deplete conditions. U87 glioblastoma cells were highly susceptible to silencing of either SREBP isoform, and apoptosis induced by SREBP1 depletion in these cells was rescued by antioxidants or by restoring the levels of mono-unsaturated fatty acids. Moreover, silencing of SREBP1

  11. Hammerhead Ribozyme-Mediated Knockdown of mRNA for Fibrotic Growth Factors: Transforming Growth Factor-Beta 1 and Connective Tissue Growth Factor

    PubMed Central

    Robinson, Paulette M.; Blalock, Timothy D.; Yuan, Rong; Lewin, Alfred S.; Schultz, Gregory S.

    2013-01-01

    Excessive scarring (fibrosis) is a major cause of pathologies in multiple tissues, including lung, liver, kidney, heart, cornea, and skin. The transforming growth factor- β (TGF- β) system has been shown to play a key role in regulating the formation of scar tissue throughout the body. Furthermore, connective tissue growth factor (CTGF) has been shown to mediate most of the fibrotic actions of TGF- β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. Currently, no approved drugs selectively and specifically regulate scar formation. Thus, there is a need for a drug that selectively targets the TGF- β cascade at the molecular level and has minimal off-target side effects. This chapter focuses on the design of hammerhead ribozymes, measurement of kinetic activity, and assessment of knockdown mRNAs of TGF- β and CTGF in cell cultures. PMID:22131029

  12. Reduced susceptibility of mice overexpressing transforming growth factor α to dextran sodium sulphate induced colitis

    PubMed Central

    Egger, B; Carey, H; Procaccino, F; Chai, N; Sandgren, E; Lakshmanan, J; Buslon, V; French, S; Buchler, M; Eysselein, V

    1998-01-01

    Background—Transforming growth factor α (TGF-α) knockout mice have increased susceptibility to dextran sodium sulphate (DSS) induced colitis. 
Aim—To substantiate the findings that TGF-α is a key mediator of colonic mucosal protection and/or repair mechanisms by evaluating the susceptibility of mice overexpressing TGF-α to DSS induced colitis. 
Methods—TGF-α overexpression was induced in transgenic mice by ZnSO4 administration in drinking water (TG+). Three groups were used as controls: one transgenic group without ZnSO4 administration (TG−), and two non-transgenic littermate groups receiving ZnSO4 (Non-TG+) or only water (Non-TG−). Acute colitis was induced in all groups by administration of DSS (5%, w/v) in drinking water for six days ad libitum. 
Results—About 35-39% of the entire colonic mucosa was destroyed in Non-TG−, Non-TG+, and TG− animals compared with 9% in TG+ mice. The crypt damage score was 18.7 (0.9), 18.2 (1.0), 18.9(0.8), and 6.8 (1.5) (means (SEM)) in Non-TG−, Non-TG+, TG−, and TG+ mice respectively. Mucin and bromodeoxyuridine staining were markedly enhanced in colons of TG+ mice compared with controls, indicating increased mucosal protection and regeneration. 
Conclusions—The significantly reduced susceptibility of mice overexpressing TGF-α to DSS further substantiates that endogenous TGF-α is a pivotal mediator of protection and/or healing mechanisms in the colon. 

 Keywords: transforming growth factor α; epidermal growth factor; dextran sodium sulphate; colitis; inflammatory bowel disease; transgenic mice PMID:9771407

  13. Transforming growth factor-beta as a differentiating factor for cultured smooth muscle cells.

    PubMed

    Gawaziuk, J P; X; Sheikh, F; Cheng, Z-Q; Cattini, P A; Stephens, N L

    2007-10-01

    The aim of the present study was to determine whether the development of supercontractile smooth muscle cells, contributing to the nonspecific hyperreactivity of airways in asthmatic patients, is due to transforming growth factor (TGF)-beta. In cultured smooth muscle cells starved by removal of 10% foetal bovine serum for 7 days, growth arrest was seen; 30% became elongated and demonstrated super contractility. Study of conditioned medium suggested that the differentiating factor was TGF-beta. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on conditioned medium from the arrested cells. Two protein bands were identified as matrix metalloproteinase (MMP)-2 and TGF-beta1. To determine second messenger signalling by SMAD2, Western blotting and confocal microscopy were employed. Conditioned medium from arrested cultures showed the presence of MMP-2 and TGF-beta1, as revealed by SDS-PAGE; 68- and 25-kDa bands were seen. Differentiation was confirmed by upregulation of marker proteins, smooth muscle type myosin heavy chain and myosin light chain kinase. Confirmation was obtained by downregulating these proteins with decorin treatment, which reduces the levels of active TGF-beta and an adenoviral dominant-negative vector coding for a mutated type II TGF-beta-receptor. Activation of second messenger signalling was demonstrated immunocytochemically by the presence of phosphorylated SMAD2 and SMAD4. Transforming growth factor-beta is likely to be the differentiating factor responsible for the development of these supercontractile smooth muscle cells. The development of such cells in vivo after cessation of an asthmatic attack could contribute to the nonspecific hyperreactivity of airways seen in patients. PMID:17596270

  14. RNA-binding proteins and translational regulation in axons and growth cones

    PubMed Central

    Hörnberg, Hanna; Holt, Christine

    2013-01-01

    RNA localization and regulation play an important role in the developing and adult nervous system. In navigating axons, extrinsic cues can elicit rapid local protein synthesis that mediates directional or morphological responses. The mRNA repertoire in axons is large and dynamically changing, yet studies suggest that only a subset of these mRNAs are translated after cue stimulation, suggesting the need for a high level of translational regulation. Here, we review the role of RNA-binding proteins (RBPs) as local regulators of translation in developing axons. We focus on their role in growth, guidance, and synapse formation, and discuss the mechanisms by which they regulate translation in axons. PMID:23734093

  15. The Anti-angiogenic Peptide, Loop 6, Binds Insulin-like Growth Factor-1 Receptor*

    PubMed Central

    Fernandez, Cecilia A.; Roy, Roopali; Lee, Sunyoung; Yang, Jiang; Panigrahy, Dipak; Van Vliet, Krystyn J.; Moses, Marsha A.

    2010-01-01

    Tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, have been shown to possess biological functions that are independent of their ability to inhibit matrix metalloproteinases. We have previously shown that the C-terminal domain of TIMP-2 and, in particular, Loop 6 inhibit capillary endothelial cell proliferation and angiogenesis both in vitro and in vivo. To elucidate the mechanism by which Loop 6 inhibits angiogenesis, we sought to determine whether its biological effects were the result of a known TIMP-2 protein-protein interaction or of a receptor-mediated event. In this study, we identify insulin-like growth factor-1 receptor as a binding partner of Loop 6/TIMP-2 and characterize this interaction on the endothelial cell surface and the consequences of this interaction on downstream receptor signaling. PMID:20940305

  16. Enhancing Peptide Ligand Binding to Vascular Endothelial Growth Factor by Covalent Bond Formation

    PubMed Central

    Marquez, Bernadette V.; Beck, Heather E.; Aweda, Tolulope A.; Phinney, Brett; Holsclaw, Cynthia; Jewell, William; Tran, Diana; Day, Jeffrey J.; Peiris, Malalage N.; Nwosu, Charles; Lebrilla, Carlito; Meares, Claude F.

    2012-01-01

    Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a target protein has many potential applications in biomedical science. For example, the properties of probes used as receptor-imaging ligands may be improved by increasing their residence time on the targeted receptor. Among the more interesting cases are peptide ligands, the strongest of which typically bind to receptors with micromolar dissociation constants, and which may depend on processes other than simple binding to provide images. The side chains of cysteine, histidine, or lysine are attractive for chemical attachment to improve binding to a receptor protein, and a system based on acryloyl probes attaching to engineered cysteine provides excellent positron emission tomographic images in animal models (Wei et al. (2008) J. Nucl. Med. 49, 1828-1835). In nature, lysine is a more common but less reactive residue than cysteine, making it an interesting challenge to modify. To seek practically useful cross-linking yields with naturally occurring lysine side chains, we have explored not only acryloyl but also other reactive linkers with different chemical properties. We employed a peptide-VEGF model system to discover that a 19mer peptide ligand, which carried a lysine-tagged dinitrofluorobenzene group, became attached stably and with good yield to a unique lysine residue on human vascular endothelial growth factor (VEGF), even in the presence of 70% fetal bovine serum. The same peptide carrying acryloyl and related Michael acceptors gave low yields of attachment to VEGF, as did the chloroacetyl peptide. PMID:22537066

  17. Valproic acid overcomes transforming growth factor-β-mediated sorafenib resistance in hepatocellular carcinoma

    PubMed Central

    Matsuda, Yasunobu; Wakai, Toshifumi; Kubota, Masayuki; Osawa, Mami; Hirose, Yuki; Sakata, Jun; Kobayashi, Takashi; Fujimaki, Shun; Takamura, Masaaki; Yamagiwa, Satoshi; Aoyagi, Yutaka

    2014-01-01

    Sorafenib is a multi-kinase inhibitor approved for hepatocellular carcinoma, but rarely causes tumor regression in patients with chronic liver diseases. To investigate whether growth factor-mediated signaling is involved in sorafenib resistance, HepG2 and PLC/PRF/5 hepatoma cells were exposed to epidermal growth factor (EGF), hepatocyte growth factor (HGF) or transforming growth factor-β (TGF-β) prior to treatment with sorafenib. Furthermore, to identify an effective combination treatment with sorafenib, growth factor-sensitized cells were treated with sorafenib alone or in combination with celecoxib, lovastatin or valproic acid (VPA). Trypan blue staining and Annexin V assays showed that the cytotoxic effect of sorafenib was inhibited by 15-54% in cells sensitized to TGF-β (P<0.05). Western blotting analysis showed that TGF-β significantly activated extracellular signal-regulated kinase (ERK)-mediated AKT signaling, and sorafenib failed to suppress both ERK and AKT in TGF-β-sensitized cells. The decreased anti-tumor effect of sorafenib was rescued by chemical inhibition of ERK and AKT. When TGF-β-sensitized cells were treated with sorafenib plus VPA, the levels of phosphorylated ERK and AKT were considerably suppressed and the numbers of dead cells were increased by 3.7-5.7-fold compared with those exposed to sorafenib alone (P<0.05). Moreover, low dose sorafenib-induced cell migration was effectively suppressed by combination treatment with sorafenib and VPA. Collectively, TGF-β/ERK/AKT signaling might play a critical role in sorafenib resistance in hepatoma cells, and combination treatment with VPA may be effective against this drug resistance. PMID:24817927

  18. Ternary Complex of Transforming Growth Factor-[beta]1 Reveals Isoform-specific Ligand Recognition and Receptor Recruitment in the Superfamily

    SciTech Connect

    Radaev, Sergei; Zou, Zhongcheng; Huang, Tao; Lafer, Eileen M.; Hinck, Andrew P.; Sun, Peter D.

    2010-11-03

    Transforming growth factor (TGF)-{beta}1, -{beta}2, and -{beta}3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-{angstrom} resolution crystal structure of the ternary complex between human TGF-{beta}1 and the extracellular domains of its type I and type II receptors, T{beta}RI and T{beta}RII. The TGF-{beta}1 ternary complex structure is similar to previously reported TGF-{beta}3 complex except with a 10{sup o} rotation in T{beta}RI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-{beta}. T{beta}RI showed significant binding to TGF-{beta}2 and TGF-{beta}3 but not TGF-{beta}1, and the binding to all three isoforms of TGF-{beta} was enhanced considerably in the presence of T{beta}RII. The preference of TGF-{beta}2 to T{beta}RI suggests a variation in its receptor recruitment in vivo. Although TGF-{beta}1 and TGF-{beta}3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-{beta} superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of T{beta}RII on TGF-{beta} is unique to TGF-{beta}s, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.

  19. Binding and internalization of nerve growth factor by PC12 cells

    SciTech Connect

    Kasaian, M.T.

    1987-01-01

    The interaction of nerve growth factor (NGF) with its cell surface receptors has been studied using both fluorescent- and radio-labelled NGF. The fluorescence studies were done by flow cytometry, and gave information about the concentration dependence and time course of NGF binding to rat pheochromocytoma cells (PC12) and human melanoma cells (A875). /sup 125/I-NGF was used to study the fate of NGF in PC12 cells following its association with cell surface receptors. Variations of the PC12 binding assay were used to distinguish ligand bound to fast and slowly dissociating receptors at the cell surface, internalized ligand, and cytoskeletally-associated NGF. Ligand uptake into each of these pools was followed in untreated cells, as well as in cells exposed to colchicine and/or cytochalasin B to disrupt the cytoskeleton. NGF degradation was also followed in these cells, and chloroquine was used to inhibit this process. In a separate project, NGF activity was assayed in samples of human amniotic fluid and cerebrospinal fluid (CSF). A range of activities was found in these samples, with the CSF samples containing somewhat more activity than the amniotic fluid samples.

  20. Identification of protein-protein binding sites by incorporating the physicochemical properties and stationary wavelet transforms into pseudo amino acid composition.

    PubMed

    Jia, Jianhua; Liu, Zi; Xiao, Xuan; Liu, Bingxiang; Chou, Kuo-Chen

    2016-09-01

    With the explosive growth of protein sequences entering into protein data banks in the post-genomic era, it is highly demanded to develop automated methods for rapidly and effectively identifying the protein-protein binding sites (PPBSs) based on the sequence information alone. To address this problem, we proposed a predictor called iPPBS-PseAAC, in which each amino acid residue site of the proteins concerned was treated as a 15-tuple peptide segment generated by sliding a window along the protein chains with its center aligned with the target residue. The working peptide segment is further formulated by a general form of pseudo amino acid composition via the following procedures: (1) it is converted into a numerical series via the physicochemical properties of amino acids; (2) the numerical series is subsequently converted into a 20-D feature vector by means of the stationary wavelet transform technique. Formed by many individual "Random Forest" classifiers, the operation engine to run prediction is a two-layer ensemble classifier, with the 1st-layer voting out the best training data-set from many bootstrap systems and the 2nd-layer voting out the most relevant one from seven physicochemical properties. Cross-validation tests indicate that the new predictor is very promising, meaning that many important key features, which are deeply hidden in complicated protein sequences, can be extracted via the wavelets transform approach, quite consistent with the facts that many important biological functions of proteins can be elucidated with their low-frequency internal motions. The web server of iPPBS-PseAAC is accessible at http://www.jci-bioinfo.cn/iPPBS-PseAAC , by which users can easily acquire their desired results without the need to follow the complicated mathematical equations involved. PMID:26375780

  1. Nuclear actions of insulin-like growth factor binding protein-3.

    PubMed

    Baxter, Robert C

    2015-09-10

    In addition to its actions outside the cell, cellular uptake and nuclear import of insulin-like growth factor binding protein-3 (IGFBP-3) has been recognized for almost two decades, but knowledge of its nuclear actions has been slow to emerge. IGFBP-3 has a functional nuclear localization signal and interacts with the nuclear transport protein importin-β. Within the nucleus IGFBP-3 appears to have a role in transcriptional regulation. It can bind to the nuclear receptor, retinoid X receptor-α and several of its dimerization partners, including retinoic acid receptor, vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-γ (PPARγ). These interactions modulate the functions of these receptors, for example inhibiting VDR-dependent transcription in osteoblasts and PPARγ-dependent transcription in adipocytes. Nuclear IGFBP-3 can be detected by immunohistochemistry in cancer and other tissues, and its presence in the nucleus has been shown in many cell culture studies to be necessary for its pro-apoptotic effect, which may also involve interaction with the nuclear receptor Nur77, and export from the nucleus. IGFBP-3 is p53-inducible and in response to DNA damage, forms a complex with the epidermal growth factor receptor (EGFR), translocating to the nucleus to interact with DNA-dependent protein kinase. Inhibition of EGFR kinase activity or downregulation of IGFBP-3 can inhibit DNA double strand-break repair by nonhomologous end joining. IGFBP-3 thus has the ability to influence many cell functions through its interactions with intranuclear pathways, but the importance of these interactions in vivo, and their potential to be targeted for therapeutic benefit, require further investigation. PMID:26074086

  2. Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

    PubMed

    Fresco, Victor M; Kern, Christine B; Mohammadi, Moosa; Twal, Waleed O

    2016-09-01

    Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development. PMID:27402846

  3. Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene

    SciTech Connect

    Miki, T.; Bottaro, D.P.; Fleming, T.P.; Smith, C.L.; Chan, A.M.L.; Aaronson, S.A. ); Burgess, W.H. )

    1992-01-01

    Expression cDNA cloning and structural analysis of the human keratinocyte growth factor receptor (KGFR) revealed identity with one of the fibroblast growth factor (FGF) receptors encoded by the bek gene (FGFR-2), except for a divergent stretch of 49 amino acids in their extracellular domains. Binding assays demonstrated that the KGFR was a high-affinity receptor for both KGF and acidic FGF, while FGFR-2 showed high affinity for basic and acidic FGF but no detectable binding by KGF. Genomic analysis of the bek gene revealed two alternative exons responsible for the region of divergence between the two receptors. The KGFR transcript was specific to epithelial cells, and it appeared to be differentially regulated with respect to the alternative FGFR-2 transcript. Thus, two growth factor receptors with different ligand-binding specificities and expression patterns are encoded by alternative transcripts of the same gene.

  4. Regulation of Transforming Growth Factor β1, Platelet-Derived Growth Factor, and Basic Fibroblast Growth Factor by Silicone Gel Sheeting in Early-Stage Scarring

    PubMed Central

    Choi, Jaehoon; Lee, Eun Hee; Park, Sang Woo

    2015-01-01

    Background Hypertrophic scars and keloids are associated with abnormal levels of growth factors. Silicone gel sheets are effective in treating and preventing hypertrophic scars and keloids. There has been no report on the change in growth factors in the scar tissue following the use of silicone gel sheeting for scar prevention. A prospective controlled trial was performed to evaluate whether growth factors are altered by the application of a silicone gel sheet on a fresh surgical scar. Methods Four of seven enrolled patients completed the study. Transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) were investigated immunohistochemically in biopsies taken from five scars at 4 months following surgery. Results In both the epidermis and the dermis, the expression of TGF-β1 (P=0.042 and P=0.042) and PDGF (P=0.043 and P=0.042) was significantly lower in the case of silicone gel sheet-treated scars than in the case of untreated scars. The expression of bFGF in the dermis was significantly higher in the case of silicone gel sheet-treated scars than in the case of untreated scars (P=0.042), but in the epidermis, the expression of bFGF showed no significant difference between the groups (P=0.655). Conclusions The levels of TGF-β1, PDGF, and bFGF are altered by the silicone gel sheet treatment, which might be one of the mechanisms of action in scar prevention. PMID:25606485

  5. Mammary tumor suppression by transforming growth factor beta 1 transgene expression.

    PubMed Central

    Pierce, D F; Gorska, A E; Chytil, A; Meise, K S; Page, D L; Coffey, R J; Moses, H L

    1995-01-01

    In cell culture, type alpha transforming growth factor (TGF-alpha) stimulates epithelial cell growth, whereas TGF-beta 1 overrides this stimulatory effect and is growth inhibitory. Transgenic mice that overexpress TGF-alpha under control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit mammary ductal hyperplasia and stochastic development of mammary carcinomas, a process that can be accelerated by administration of the chemical carcinogen 7,12-dimethylbenz[a]anthracene. MMTV-TGF-beta 1 transgenic mice display mammary ductal hypoplasia and do not develop mammary tumors. We report that in crossbreeding experiments involving the production of mice carrying both the MMTV-TGF-beta 1 and MMTV-TGF-alpha transgenes, there is marked suppression of mammary tumor formation and that MMTV-TGF-beta 1 transgenic mice are resistant to 7,12-dimethylbenz[a]anthracene-induced mammary tumor formation. These data demonstrate that overexpression of TGF-beta 1 in vivo can markedly suppress mammary tumor development. Images Fig. 1 Fig. 3 PMID:7753792

  6. Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia.

    PubMed Central

    Nabel, E G; Shum, L; Pompili, V J; Yang, Z Y; San, H; Shu, H B; Liptay, S; Gold, L; Gordon, D; Derynck, R

    1993-01-01

    The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8248168

  7. Capsaicin Inhibits Preferentially the NADH Oxidase and Growth of Transformed Cells in Culture

    NASA Astrophysics Data System (ADS)

    Morre, D. James; Chueh, Pin-Ju; Morre, Dorothy M.

    1995-03-01

    A hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells, all of human origin, by the naturally occurring quinone analog capsaicin (8-methyl-N-vanillyl-6-noneamide), compared with plasma membranes from human mammary epithelial, rat liver, normal rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. With cells in culture, capsaicin preferentially inhibited growth of HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells but was largely without effect on the mammary epithelial cells, rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. Inhibited cells became smaller and cell death was accompanied by a condensed and fragmented appearance of the nuclear DNA, as revealed by fluorescence microscopy with 4',6-diamidino-2-phenylindole, suggestive of apoptosis. The findings correlate capsaicin inhibition of cell surface NADH oxidase activity and inhibition of growth that correlate with capsaicin-induced apoptosis.

  8. Regulation of the ovarian reserve by members of the transforming growth factor beta family

    PubMed Central

    Pangas, Stephanie A.

    2012-01-01

    Genetic or environmental factors that affect the endowment of oocytes, their assembly nto primordial follicles, or their subsequent entry into the growing follicle pool can disrupt reproductive function and may underlie disorders such as primary ovarian insufficiency. Mouse models have been instrumental in identifying genes important in ovarian development, and a number of genes now associated with ovarian dysfunction in women were first identified as causing reproductive defects in knockout mice. The transforming growth factor beta (TGFB) family consists of developmentally important growth factors that include the TGFBs, anti-Müllerian hormone (AMH), activins, bone morphogenetic proteins (BMPs), and growth and differentiation factor 9 (GDF9). The ovarian primordial follicle pool is the source of oocytes in adults. Development of this pool can be grossly divided into three key processes: (1) establishment of oocytes during embryogenesis followed by (2) assembly and (3) activation of the primordial follicle. Disruptions in any of these processes may cause reproductive dysfunction. Most members of the TGFB family show pivotal roles in each of these areas. Understanding the phenotypes of various mouse models for this protein family will be directly relevant to understanding how disruptions in TGFB family signaling result in reproductive diseases in women and will present new areas for development of tailored diagnostics and interventions for infertility. PMID:22847922

  9. Microsatellite mutation of type II transforming growth factor-beta receptor is rare in atherosclerotic plaques.

    PubMed

    Clark, K J; Cary, N R; Grace, A A; Metcalfe, J C

    2001-04-01

    A somatic mutation within a microsatellite polyA tract in the coding region of the type II transforming growth factor (TGF)-beta receptor gene was reported to occur in human atherosclerotic and restenotic lesions. This mutation occurs frequently in colorectal cancer with the replication error repair phenotype and results in loss of sensitivity to the growth inhibitory effects of TGF-beta in cells from the tumors. The mutation was proposed to account for the clonal expansion of vascular smooth muscle cells observed in atherosclerotic plaques, through loss of the growth inhibitory effect of TGF-beta. The frequency of the mutation and the extent of clonal expansion of the mutated cells have major implications for the mechanism of atherogenesis and therapeutic strategies. We analyzed a set of 22 coronary arterial and 9 aortic samples containing early to advanced atherosclerotic lesions for the mutation in the type II TGF-beta receptor polyA tract. Only 1 coronary arterial sample from an advanced lesion showed detectable amounts of the mutation, present at a low level (8% of the DNA sample). The data imply that the mutation occurs only at low frequency and is not a major mechanistic contributor to the development of atherosclerosis. PMID:11304472

  10. Immunohistochemical localization of transforming growth factor beta isoforms in asbestos-related diseases.

    PubMed Central

    Jagirdar, J; Lee, T C; Reibman, J; Gold, L I; Aston, C; Bégin, R; Rom, W N

    1997-01-01

    Transforming growth factor beta (TGF-beta), a multifunctional cytokine and growth factor, plays a key role in scarring and fibrotic processes because of its ability to induce extracellular matrix proteins and modulate the growth and immune function of many cell types. These effects are important in inflammatory disorders with fibrosis and cancer. The asbestos-related diseases are characterized by fibrosis in the lower respiratory tract and pleura and increased occurrence of lung cancer and mesothelioma. We performed immunohistochemistry with isoform-specific antibodies to the three TGF-beta isoforms on 16 autopsy lungs from Quebec, Canada, asbestos miners and millers. There was increased immunolocalization of all three TGF-beta isoforms in the fibrotic lesions of asbestosis and pleural fibrosis. The hyperplastic type II pneumocytes contained all three isoforms. By contrast, there was differential spatial immunostaining for the TGF-beta isoforms in malignant mesothelioma, with TGF-beta 1 in the stroma but TGF-beta 2 in the tumor cells. These data are consistent with an important role for TGF-beta in accumulation of extracellular matrix and cell proliferation in asbestos-related diseases. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. PMID:9400723

  11. Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms.

    PubMed

    Pierce, G F; Mustoe, T A; Lingelbach, J; Masakowski, V R; Griffin, G L; Senior, R M; Deuel, T F

    1989-07-01

    Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In

  12. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

    PubMed Central

    Bilder, Patrick; Sun, Meihao; Lim, Jihyeon; Bielefeldt-Ohmann, Helle; Basaraba, Randall; So, Melvin; Zhu, Guofeng; Tufariello, JoAnn M.; Izzo, Angelo A.; Orme, Ian M.; Almo, Steve C.; Leyh, Thomas S.; Chan, John

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-Å-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection. PMID:19478878

  13. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    SciTech Connect

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  14. Dermatophyte-hormone relationships: characterization of progesterone-binding specificity and growth inhibition in the genera Trichophyton and Microsporum.

    PubMed Central

    Clemons, K V; Schär, G; Stover, E P; Feldman, D; Stevens, D A

    1988-01-01

    We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + and - mating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10(-7) to 2 X 10(-7) M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 X 10(-8) M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56 degrees C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding affinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings: DOC and DHT were effective competitors for [3H]progesterone binding. The growth of A. benhamiae + and -, M. canis, and T. rubrum were all inhibited by progesterone in a dose-responsive manner, with 50% inhibition achieved at concentrations of 9.8 x 10(-6), 1.2 x 10(-5), 1.5 x 10(-5), and 2.7 x 10(-6) M. respectively,. PMID:3182998

  15. Transforming growth factor-beta1-induced activation of the Raf-MEK-MAPK signaling pathway in rat lung fibroblasts via a PKC-dependent mechanism.

    PubMed

    Axmann, A; Seidel, D; Reimann, T; Hempel, U; Wenzel, K W

    1998-08-19

    In fibroblasts transforming growth factor-beta1 (TGF-beta1) regulates cell proliferation and turnover of macromolecular components of the extracellular matrix. Here, intracellular signaling events in growth-inhibited embryonic rat lung fibroblasts (RFL-6) upon stimulation with TGF-beta1 were investigated. TGF-beta1 rapidly induced the activation of c-Raf-1, MEK-1, and MAPK p42 and p44. The activation of this pathway by TGF-beta1 did not depend on autocrine platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). Inhibition of the binding of growth factors to their tyrosine kinase receptors did not affect MAPK activation by TGF-beta1. Ras activation by TGF-beta1 was significantly lower compared to the activation by PDGF or bFGF. The intracellular transduction of the TGF-beta1 signal was completely suppressed by depletion or inhibition of protein kinase C (PKC). It is shown that calcium-dependent isoforms of PKC are required for MAPK activation by TGF-beta1. PMID:9712718

  16. Expression of a Secreted Fibroblast Growth Factor Binding Protein-1 (FGFBP1) in Angioproliferative Kaposi Sarcoma

    PubMed Central

    Ray, Patricio E; Al-Attar, Ali; Liu, Xue-Hui; Das, Jharna R; Tassi, Elena; Wellstein, Anton

    2014-01-01

    Objective Kaposi’s sarcoma (KS) is an angioproliferative disease frequently seen in patients with the acquired immunodeficiency syndrome (AIDS). Previous studies suggest that the HIV-1 protein Tat and Fibroblast Growth Factor 2 (FGF-2) have synergistic angiogenic effects in AIDS-KS tumors. However, the mechanisms by which FGF-2 is released and activated in KS tumors are not clearly defined. We carried out this study to determine whether an FGF-binding protein (FGFBP1 or BP1) that enhances the angiogenic activity of FGF-2 is expressed in AIDS-KS tumors, and to define whether BP1, FGF-2, and HIV-Tat protein-protein interactions could play a potential clinically role in the pathogenesis of AIDS-KS. Methods BP1 was localized in AIDS-KS lesions by immunohistochemistry and in situ hybridization studies. The binding of radiolabeled FGF-2 to His-tagged BP1 or the FGF-receptor 1 was assessed in the presence and absence of HIV-Tat and other viral proteins. Mice carrying tetracycline-regulated BP1 transgene mice were used to determine whether activation of BP1 during wound healing induces KS-like lesions. Results BP1 expression was detected in AIDS-KS tumor keratinocytes, spindle cells, and infiltrating mononuclear cells. In addition, HIV-Tat competed for the binding of FGF-2 to immobilized BP1, but does not affect the interactions of FGF-2 with its high affinity receptor (FGFR-1). In contrast, two other HIV-proteins, Nef and gp120, did not affect the binding of FGF-2 to BP1 or to FGFR-1. Finally, up-regulation of BP1 expression in tetracycline-regulated –conditional BP1 transgenic mice subjected to skin wounds, induced KS-like skin lesions. Conclusion Taking into consideration the results of previous studies showing that both HIV-Tat and BP1 enhance the mitogenic and angiogenic activity of locally-stored FGF-2, both in vitro and in vivo, our findings suggest a novel mechanism by which the release and activity of FGFs can be modulated in AIDS-KS tumors by HIV-Tat as well

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

    SciTech Connect

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.

    2008-03-10

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

  18. Smad-Independent Transforming Growth Factor-β Regulation of Early Growth Response-1 and Sustained Expression in Fibrosis

    PubMed Central

    Bhattacharyya, Swati; Chen, Shu-Jen; Wu, Minghua; Warner-Blankenship, Matthew; Ning, Hongyan; Lakos, Gabriella; Mori, Yasuji; Chang, Eric; Nihijima, Chihiro; Takehara, Kazuhiro; Feghali-Bostwick, Carol; Varga, John

    2008-01-01

    Transforming growth factor-β (TGF-β) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-β and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-β causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-β to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-β, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-β enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-β signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis. PMID:18772333

  19. Role of Insulin-like Growth Factor Binding Protein-3 in Allergic Airway Remodeling

    PubMed Central

    Veraldi, Kristen L.; Gibson, Bethany T.; Yasuoka, Hidekata; Myerburg, Michael M.; Kelly, Elizabeth A.; Balzar, Silvana; Jarjour, Nizar N.; Pilewski, Joseph M.; Wenzel, Sally E.; Feghali-Bostwick, Carol A.

    2009-01-01

    Rationale: The hallmarks of allergic asthma are airway inflammation, obstruction, and remodeling. Airway remodeling may lead to irreversible airflow obstruction with increased morbidity and mortality. Despite advances in the treatment of asthma, the mechanisms underlying airway remodeling are still poorly understood. We reported that insulin-like growth factor (IGF) binding proteins (IGFBPs) contribute to extracellular matrix deposition in idiopathic pulmonary fibrosis; however, their contribution to airway remodeling in asthma has not been established. Objectives: We hypothesized that IGFBP-3 is overexpressed in asthma and contributes to airway remodeling. Methods: We evaluated levels of IGFBP-3 in tissues and bronchoalveolar lavage fluid from patients with asthma at baseline and 48 hours after allergen challenge, in reparative epithelium in an in vitro wounding assay, and in conditioned media from cytokine- and growth factor–stimulated primary epithelial cells. Measurements and Main Results: IGFBP-3 levels and distribution were evaluated by Western blot, ELISA, and immunofluorescence. IGFBP-3 is increased in vivo in the airway epithelium of patients with asthma compared with normal control subjects. The concentration of IGFBP-3 is increased in the bronchoalveolar lavage fluid of patients with asthma after allergen challenge, its levels are increased in reparative epithelium in an in vitro wounding assay and in the conditioned medium of primary airway epithelial cell cultures stimulated with IGF-I. Conclusions: Our results suggest that one mechanism of allergic airway remodeling is through the secretion of the profibrotic IGFBP-3 from IGF-I–stimulated airway epithelial cells during allergic inflammation. PMID:19608721

  20. Transcriptional and posttranslational regulation of insulin-like growth factor binding protein-3 by Akt3

    PubMed Central

    Jin, Quanri; Lee, Hyo-Jong; Min, Hye-Young; Smith, John Kendal; Hwang, Su Jung; Whang, Young Mi; Kim, Woo-Young; Kim, Yeul Hong; Lee, Ho-Young

    2014-01-01

    Insulin-like growth factor (IGF)-dependent and -independent antitumor activities of insulin-like growth factor binding protein-3 (IGFBP-3) have been proposed in human non-small cell lung cancer (NSCLC) cells. However, the mechanism underlying regulation of IGFBP-3 expression in NSCLC cells is not well understood. In this study, we show that activation of Akt, especially Akt3, plays a major role in the mRNA expression and protein stability of IGFBP-3 and thus antitumor activities of IGFBP-3 in NSCLC cells. When Akt was activated by genomic or pharmacologic approaches, IGFBP-3 transcription and protein stability were decreased. Conversely, suppression of Akt increased IGFBP-3 mRNA levels and protein stability in NSCLC cell lines. Characterization of the effects of constitutively active form of each Akt subtype (HA-Akt-DD) on IGFBP-3 expression in NSCLC cells and a xenograft model indicated that Akt3 plays a major role in the Akt-mediated regulation of IGFBP-3 expression and thus suppression of Akt effectively enhances the antitumor activities of IGFBP-3 in NSCLC cells with Akt3 overactivation. Collectively, these data suggest a novel function of Akt3 as a negative regulator of IGFBP-3, indicating the possible benefit of a combined inhibition of IGFBP-3 and Akt3 for the treatment of patients with NSCLC. PMID:24942865

  1. Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis.

    PubMed

    Chung, Y S; Breidt, F; Dubnau, D

    1998-08-01

    The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 pre-pilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo. ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results. PMID:9723928

  2. The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor

    PubMed Central

    Jin, Ming-hao; Sawamoto, Kazunobu; Ito, Mikiko; Okano, Hideyuki

    2000-01-01

    Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor α-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases. PMID:10688656

  3. Autocrine and exogenous transforming growth factor beta control cell cycle inhibition through pathways with different sensitivity.

    PubMed

    Wang, Jing; Sergina, Natalia; Ko, Tien C; Gong, Jiangeng; Brattain, Michael G

    2004-09-17

    Human colon carcinoma cells HCT116 that lack transforming growth factor beta (TGF-beta) type II receptor (RII) demonstrated restoration of autocrine TGF-beta activity upon reexpression of RII without restoring inhibitory responses to exogenous TGF-beta treatment. RII transfectants (designated RII Cl 37) had a longer lag phase relative to NEO-transfected control cells (designated NEO pool) before entering exponential growth in tissue culture. The prolonged growth arrest of RII Cl 37 cells was associated with markedly reduced cyclin-dependent kinase (CDK)2 activity. Our results demonstrate that p21 induction by autocrine TGF-beta is responsible for reduced CDK2 activity, which at least partially contributes to prolonged growth arrest and reduced cell proliferation in RII Cl 37 cells. In contrast to RII transfectants, HCT116 cells transfected with chromosome 3 (designated HCT116Ch3), which bears the RII gene, restored the response to exogenous TGF-beta as well as autocrine TGF-beta activity. Autocrine TGF-beta activity in HCT116Ch3 cells induced p21 expression as seen in RII Cl 37 cells; however, in addition to autocrine activity, HCT116Ch3 cells responded to exogenous TGF-beta as decreased CDK4 expression and reduced pRb phosphorylation mediated a TGF-beta inhibitory response in these cells. These results indicate that autocrine TGF-beta regulates the cell cycle through a pathway different from exogenous TGF-beta in the sense that p21 is a more sensitive effector of the TGF-beta signaling pathway, which can be induced and saturated by autocrine TGF-beta, whereas CDK4 inhibition is a less sensitive effector, which can only be activated by high levels of exogenous TGF-beta PMID:15271980

  4. Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta.

    PubMed Central

    Kooistra, A.; van den Eijnden-van Raaij, A. J.; Klaij, I. A.; Romijn, J. C.; Schröder, F. H.

    1995-01-01

    The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage-independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF-beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors. Images Figure 5 PMID:7543773

  5. Epithelial-to-mesenchymal transition in breast cancer: a role for insulin-like growth factor I and insulin-like growth factor–binding protein 3?

    PubMed Central

    Zielinska, Hanna A; Bahl, Amit; Holly, Jeff MP; Perks, Claire M

    2015-01-01

    Evidence indicates that for most human cancers the problem is not that gene mutations occur but is more dependent upon how the body deals with damaged cells. It has been estimated that only about 1% of human cancers can be accounted for by unmistakable hereditary cancer syndromes, only up to 5% can be accounted for due to high-penetrance, single-gene mutations, and in total only 5%–15% of all cancers may have a major genetic component. The predominant contribution to the causation of most sporadic cancers is considered to be environmental factors contributing between 58% and 82% toward different cancers. A nutritionally poor lifestyle is associated with increased risk of many cancers, including those of the breast. As nutrition, energy balance, macronutrient composition of the diet, and physical activity levels are major determinants of insulin-like growth factor (IGF-I) bioactivity, it has been proposed that, at least in part, these increases in cancer risk and progression may be mediated by alterations in the IGF axis, related to nutritional lifestyle. Localized breast cancer is a manageable disease, and death from breast cancer predominantly occurs due to the development of metastatic disease as treatment becomes more complicated with poorer outcomes. In recent years, epithelial-to-mesenchymal transition has emerged as an important contributor to breast cancer progression and malignant transformation resulting in tumor cells with increased potential for migration and invasion. Furthermore, accumulating evidence suggests a strong link between components of the IGF pathway, epithelial-to-mesenchymal transition, and breast cancer mortality. Here, we highlight some recent studies highlighting the relationship between IGFs, IGF-binding protein 3, and epithelial-to-mesenchymal transition. PMID:25632238

  6. Interferon-alpha inhibits murine macrophage transforming growth factor-beta mRNA expression.

    PubMed

    Dhanani, S; Huang, M; Wang, J; Dubinett, S M

    1994-06-01

    Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide is produced by a wide variety of cells and regulates a broad array of physiological and pathological functions. TGF-beta appears to play a central role in pulmonary fibrosis and may contribute to tumor-associated immunosuppression. Alveolar macrophages are a rich source of TGF-beta and are intimately involved in lung inflammation. We therefore chose to study TGF-beta regulation in murine alveolar macrophages as well as an immortalized peritoneal macrophage cell line (IC-21). Murine macrophages were incubated with cytokines to evaluate their role in regulating TGF-beta mRNA expression. We conclude that IFN-alpha downregulates TGF-beta mRNA expression in murine macrophages. PMID:8088926

  7. The pleiotropic roles of transforming growth factor beta inhomeostasis and carcinogenesis of endocrine organs.

    SciTech Connect

    Fleisch, Markus C.; Maxwell, Christopher A.; Barcellos-Hoff,Mary-Helen

    2006-01-13

    Transforming growth factor beta (TGF-beta) is a ubiquitous cytokine that plays a critical role in numerous pathways regulating cellular and tissue homeostasis. TGF-beta is regulated by hormones and is a primary mediator of hormone response in uterus, prostate and mammary gland. This review will address the role of TGF-beta in regulating hormone dependent proliferation and morphogenesis. The subversion of TGF-beta regulation during the processes of carcinogenesis, with particular emphasis on its effects on genetic stability and epithelial to mesenchymal transition (EMT), will also be examined. An understanding of the multiple and complex mechanisms of TGF-beta regulation of epithelial function, and the ultimate loss of TGF-beta function during carcinogenesis, will be critical in the design of novel therapeutic interventions for endocrine-related cancers.

  8. Role of transforming growth factor-beta (TGF) beta in the physiopathology of rheumatoid arthritis.

    PubMed

    Gonzalo-Gil, Elena; Galindo-Izquierdo, María

    2014-01-01

    Transforming growth factor-beta (TGF-β) is a cytokine with pleiotropic functions in hematopoiesis, angiogenesis, cell proliferation, differentiation, migration and apoptosis. Although its role in rheumatoid arthritis is not well defined, TGF-β activation leads to functional immunomodulatory effects according to environmental conditions. The function of TGF-β in the development of arthritis in murine models has been extensively studied with controversial results. Recent findings point to a non-relevant role for TGF-β in a mice model of collagen-induced arthritis. The study of TGF-β on T-cell responses has shown controversial results as an inhibitor or promoter of the inflammatory response. This paper presents a review of the role of TGF-β in animal models of arthritis. PMID:24685296

  9. Transforming growth factor-β: an important mediator in Helicobacter pylori-associated pathogenesis

    PubMed Central

    Li, Nianshuang; Xie, Chuan; Lu, Nong-Hua

    2015-01-01

    Helicobacter pylori (H.pylori) is a Gram-negative, microaerophilic, helical bacillus that specifically colonizes the gastric mucosa. The interaction of virulence factors, host genetic factors, and environmental factors contributes to the pathogenesis of H. pylori-associated conditions, such as atrophic gastritis and intestinal metaplasia. Infection with H. pylori has recently been recognized as the strongest risk factor for gastric cancer. As a pleiotropic cytokine, transforming growth factor (TGF)-β regulates various biological processes, including cell cycle, proliferation, apoptosis, and metastasis. Recent studies have shed new light on the involvement of TGF-β signaling in the pathogenesis of H. pylori infection. This review focuses on the potential etiological roles of TGF-β in H. pylori-mediated gastric pathogenesis. PMID:26583078

  10. Mechanistic basis and clinical relevance of the role of transforming growth factor-β in cancer

    PubMed Central

    Lin, Run-Long; Zhao, Lu-Jun

    2015-01-01

    Transforming growth factor-β (TGF-β) is a key factor in cancer development and progression. TGF-β can suppress tumorigenesis by inhibiting cell cycle progression and stimulating apoptosis in early stages of cancer progression. However, TGF-β can modulate cancer-related processes, such as cell invasion, distant metastasis, and microenvironment modification that may be used by cancer cells to their advantage in late stages. Corresponding mechanisms include angiogenesis promotion, anti-tumor immunity suppression, and epithelial-to-mesenchymal transition (EMT) induction. The correlation between TGF-β expression and cancer prognosis has also been extensively investigated. Results suggest that TGF-β pathway can be targeted to treat cancer; as such, the feasibility of this treatment is investigated in clinical trials. PMID:26779375

  11. Serum deprivation response inhibits breast cancer progression by blocking transforming growth factor-β signaling.

    PubMed

    Tian, Yao; Yu, Yue; Hou, Li-Kun; Chi, Jiang-Rui; Mao, Jie-Fei; Xia, Li; Wang, Xin; Wang, Ping; Cao, Xu-Chen

    2016-03-01

    Serum deprivation response (SDPR), a key substrate for protein kinase C, play a critical role in inducing membrane curvature and participate in the formation of caveolae. However, the function of SDPR in cancer development and progression is still not clear. Here, we found that SDPR is downregulated in human breast cancer. Overexpression of SDPR suppresses cell proliferation and invasion in MDA-MB-231 cells, while depletion of SDPR promotes cell proliferation and invasion in MCF10A cells. Subsequently, SDPR depletion induces epithelial-mesenchymal transition (EMT)-like phenotype. Finally, knockdown of SDPR activates transforming growth factor-β (TGF-β) signaling by upregulation of TGF-β1 expression. In conclusion, our results showed that SDPR inhibits breast cancer progression by blocking TGF-β signaling. Serum deprivation response suppresses cell proliferation and invasion in breast cancer cells. SDPR depletion induces epithelial-mesenchymal transition by activation of TGF-β signaling. PMID:26749136

  12. The Role of Transforming Growth Factor β1 in the Regulation of Blood Pressure

    PubMed Central

    Matsuki, Kota; Hathaway, Catherine K.; Lawrence, Marlon G.; Smithies, Oliver; Kakoki, Masao

    2016-01-01

    Although human association studies suggest a link between polymorphisms in the gene encoding transforming growth factor (TGF) β1 and differing blood pressure levels, a causative mechanism for this correlation remains elusive. Recently we have generated a series of mice with graded expression of TGFβ1, ranging from approximately 10% to 300% compared to normal. We have found that blood pressure and plasma volume are negatively regulated by TGFβ1. Of note, the 10% hypomorph exhibits primary aldosteronism and markedly impaired urinary excretion of water and electrolytes. We here review previous literature highlighting the importance of TGFβ signaling as a natriuretic system, which we postulate is a causative mechanism explaining how polymorphisms in TGFβ1 could influence blood pressure levels. PMID:25801626

  13. CYLD negatively regulates transforming growth factor-β-signalling via deubiquitinating Akt

    PubMed Central

    Lim, Jae Hyang; Jono, Hirofumi; Komatsu, Kensei; Woo, Chang-Hoon; Lee, Jiyun; Miyata, Masanori; Matsuno, Takashi; Xu, Xiangbin; Huang, Yuxian; Zhang, Wenhong; Park, Soo Hyun; Kim, Yu-Il; Choi, Yoo-Duk; Shen, Huahao; Heo, Kyung-Sun; Xu, Haodong; Bourne, Patricia; Koga, Tomoaki; Xu, Haidong; Yan, Chen; Wang, Binghe; Chen, Lin-Feng; Feng, Xin-Hua; Li, Jian-Dong

    2012-01-01

    Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis. PMID:22491319

  14. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  15. Enhanced jun gene expression is an early genomic response to transforming growth factor. beta. stimulation

    SciTech Connect

    Pertovaara, L.; Sistonen, L.; Keski-Oja, J.; Alitalo, K. ); Bos, T.J.; Vogt, P.K. . Dept. of Microbiology)

    1989-03-01

    Transforming growth factor {beta} (TGF{beta}) is a multifunctional polypeptide4 that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF{beta} signal transduction in cells is unknown. The authors report here that an early effect of TGF{beta} is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF{beta}, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF{beta}, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TFG{beta}. The increase in jun mRNA occurred with picomolar TGF{beta} concentrations within 1 h of TGF{beta} stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-fine levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF{beta}, evidently in a protein synthesis-independent fashion. The junB response to TGF{beta} was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF{beta} may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF{beta}.

  16. The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    PubMed Central

    Hospital, Véronique; Nishi, Eiichiro; Klagsbrun, Michael; Cohen, Paul; Seidah, Nabil G; Prat, Annik

    2002-01-01

    Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met(49). Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc-HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca(2+), via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase. PMID:12095415

  17. Transforming Growth Factor-Beta and Oxidative Stress Interplay: Implications in Tumorigenesis and Cancer Progression

    PubMed Central

    Krstić, Jelena; Trivanović, Drenka; Mojsilović, Slavko; Santibanez, Juan F.

    2015-01-01

    Transforming growth factor-beta (TGF-β) and oxidative stress/Reactive Oxygen Species (ROS) both have pivotal roles in health and disease. In this review we are analyzing the interplay between TGF-β and ROS in tumorigenesis and cancer progression. They have contradictory roles in cancer progression since both can have antitumor effects, through the induction of cell death, senescence and cell cycle arrest, and protumor effects by contributing to cancer cell spreading, proliferation, survival, and metastasis. TGF-β can control ROS production directly or by downregulating antioxidative systems. Meanwhile, ROS can influence TGF-β signaling and increase its expression as well as its activation from the latent complex. This way, both are building a strong interplay which can be taken as an advantage by cancer cells in order to increment their malignancy. In addition, both TGF-β and ROS are able to induce cell senescence, which in one way protects damaged cells from neoplastic transformation but also may collaborate in cancer progression. The mutual collaboration of TGF-β and ROS in tumorigenesis is highly complex, and, due to their differential roles in tumor progression, careful consideration should be taken when thinking of combinatorial targeting in cancer therapies. PMID:26078812

  18. Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells.

    PubMed Central

    Smith, G. H.; Sharp, R.; Kordon, E. C.; Jhappan, C.; Merlino, G.

    1995-01-01

    Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and

  19. Increased cAMP levels modulate transforming growth factor-beta/Smad-induced expression of extracellular matrix components and other key fibroblast effector functions.

    PubMed

    Schiller, Meinhard; Dennler, Sylviane; Anderegg, Ulf; Kokot, Agatha; Simon, Jan C; Luger, Thomas A; Mauviel, Alain; Böhm, Markus

    2010-01-01

    cAMP is a key messenger of many hormones and neuropeptides, some of which modulate the composition of extracellular matrix. Treatment of human dermal fibroblasts with dibutyryl cyclic AMP and forskolin antagonized the inductive effects of transforming growth factor-beta (TGF-beta) on the expression of collagen, connective tissue growth factor, tissue inhibitor of matrix metalloproteinase-1, and plasminogen activator inhibitor type I, four prototypical TGF-beta-responsive genes. Increased intracellular cAMP prevented TGF-beta-induced Smad-specific gene transactivation, although TGF-beta-mediated Smad phosphorylation and nuclear translocation remained unaffected. However, increased cAMP levels abolished TGF-beta-induced interaction of Smad3 with its transcriptional co-activator cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300. Overexpression of the transcriptional co-activator CBP/p300 rescued Smad-specific gene transcription in the presence of cAMP suggesting that sequestration of limited amounts of CBP/p300 by the activated cAMP/CREB pathway is the molecular basis of this inhibitory effect. These findings were extended by two functional assays. Increased intracellular cAMP levels suppressed the inductive activity of TGF-beta to contract mechanically unloaded collagen lattices and resulted in an attenuation of fibroblast migration of mechanically induced cell layer wounds. Of note, cAMP and TGF-beta synergistically induced hyaluronan synthase 2 (HAS2) expression and hyaluronan secretion, presumably via putative CREB-binding sites adjacent to Smad-binding sites within the HAS2 promoter. Our findings identify the cAMP pathway as a potent but differential and promoter-specific regulator of TGF-beta-mediated effects involved in extracellular matrix homeostasis. PMID:19858184

  20. Expression and localization of epidermal growth factor, transforming growth factor-α and epidermal growth factor receptor in the canine testis

    PubMed Central

    TAMADA, Hiromichi; TAKEMOTO, Kohei; TOMINAGA, Masato; KAWATE, Noritoshi; TAKAHASHI, Masahiro; HATOYA, Shingo; MATSUYAMA, Satoshi; INABA, Toshio; SAWADA, Tsutomu

    2015-01-01

    Gene expression of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and EGF receptor (EGF-R) and the localization of the corresponding proteins in the canine testis were studied. Levels of mRNA expressions were determined by semiquantitative reverse transcription polymerase chain reaction in the testes of the peripubertal (4–6 months), young adult (3–4 years), advanced adult (7–8 years) and senescent (11–16 years) groups. The EGF-R mRNA level in the testes of the peripubertal group was significantly higher than those in the other groups, whereas there was no difference in EGF and TGF-α mRNA levels among groups. Immunohistochemical stainings for EGF, TGF-α and EGF-R in the testis revealed that immunoreactivity in the seminiferous epithelium and Sertoli cell was weak and nonspecific for the stage of spermatogenesis, and distinct staining was found in Leydig cells. These results suggest that the EGF family of growth factors may be involved in testicular maturation and function in the dog. PMID:26498203

  1. Interrelationship of in situ growth stress evolution and phase transformations in Ti/W multilayered thin films

    NASA Astrophysics Data System (ADS)

    Wan, Li; Yu, Xiao-xiang; Zhou, Xuyang; Thompson, Gregory

    2016-06-01

    This paper addresses the in situ growth stress evolution and phase transformation of bcc to hcp Ti in Ti/W multilayered thin films. A series of equal layer thicknesses from 20 nm to 1 nm were deposited. As the bilayer thickness reduced, the overall film stress became less compressive until the Ti transformed from hcp (at the larger layer thicknesses) to bcc in the 1 nm/1 nm multilayer. The pseudomorphic bcc stabilization resulted in a recovery of the compressive stress to values near that for the bulk phase stabilized for the 5 nm/5 nm multilayer. A discernable change in stress slope was noted for the bcc to hcp Ti transition as a function of Ti layer thickness. The stress states for each film, during film growth, are rationalized by the lattice matching of the phase with the growth surface. These results are coupled to a molecular dynamics deposition simulation which revealed good agreement with the experimentally observed transformation thickness.

  2. G3139, an Anti-Bcl-2 Antisense Oligomer that Binds Heparin-Binding Growth Factors and Collagen I, Alters In Vitro Endothelial Cell Growth and Tubular Morphogenesis

    PubMed Central

    Stein, C.A.; Wu, SiJian; Voskresenskiy, Anatoliy M.; Zhou, Jin-Feng; Shin, Joongho; Miller, Paul; Souleimanian, Naira; Benimetskaya, Luba

    2009-01-01

    Purpose We examined the effects of G3139 on the interaction of heparin-binding proteins (e.g., FGF2 and collagen I) with endothelial cells. G3139 is an 18mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global Phase III trial in advanced melanoma (GM301) has evaluted G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood, as it is unlikely that Bcl-2 silencing is the sole mechanism for chemo-sensitization in melanoma cells. Experimental Design The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high affinity cell surface receptors, and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and HUVEC cells were determined. Results G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and from proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in 3D collagen gels, increases the mitogenesis of HUVEC cells similarly, and induces vessel sprouts in the rat aortic ring model. Conclusions G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with LDH observed clinically. PMID:19351753

  3. A Histologically Distinctive Interstitial Pneumonia Induced by Overexpression of the Interleukin 6, Transforming Growth Factor β1, or Platelet-Derived Growth Factor B Gene

    NASA Astrophysics Data System (ADS)

    Yoshida, Mitsuhiro; Sakuma, Junko; Hayashi, Seiji; Abe, Kin'ya; Saito, Izumu; Harada, Shizuko; Sakatani, Mitsunoir; Yamamoto, Satoru; Matsumoto, Norinao; Kaneda, Yasufumi; Kishmoto, Tadamitsu

    1995-10-01

    Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor β1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor β1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

  4. Membrane Morphology Is Actively Transformed by Covalent Binding of the Protein Atg8 to PE-Lipids

    PubMed Central

    Knorr, Roland L.; Nakatogawa, Hitoshi; Ohsumi, Yoshinori; Lipowsky, Reinhard; Baumgart, Tobias; Dimova, Rumiana

    2014-01-01

    Autophagy is a cellular degradation pathway involving the shape transformation of lipid bilayers. During the onset of autophagy, the water-soluble protein Atg8 binds covalently to phosphatdylethanolamines (PEs) in the membrane in an ubiquitin-like reaction coupled to ATP hydrolysis. We reconstituted the Atg8 conjugation system in giant and nm-sized vesicles with a minimal set of enzymes and observed that formation of Atg8-PE on giant vesicles can cause substantial tubulation of membranes even in the absence of Atg12-Atg5-Atg16. Our findings show that ubiquitin-like processes can actively change properties of lipid membranes and that membrane crowding by proteins can be dynamically regulated in cells. Furthermore we provide evidence for curvature sorting of Atg8-PE. Curvature generation and sorting are directly linked to organelle shapes and, thus, to biological function. Our results suggest that a positive feedback exists between the ubiquitin-like reaction and the membrane curvature, which is important for dynamic shape changes of cell membranes, such as those involved in the formation of autophagosomes. PMID:25522362

  5. Transforming growth factor beta 3 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint

    PubMed Central

    Li, Yingjie; El.Mozen, Loaye Abdelaziz; Cai, Hengxing; Fang, Wei; Meng, Qinggong; Li, Jian; Deng, Mohong; Long, Xing

    2015-01-01

    Synovial chondromatosis (SC) of temporomandibular joint is rare proliferative disorder featured by the formation of cartilaginous nodules in synovium and joint space. Transforming growth factor beta 3 (TGF-β3) is closely related to chondrogenic differentiation, and might participate in pathogenesis of SC. We discovered that increased quantity of synoviocytes and blood vessels were observed in SC synovium. The vessel wall and sublining fibroblasts were stained positively by the antibodies against TGF-β3, fibroblast growth factor 2 (FGF-2), and CD34. In loose bodies (LBs), TGF-β3 was mainly expressed in chondrocytes and FGF-2 was expressed in chondrocytes, fibroblasts, and vessel walls. Expressions of TGF-β1, TGF-β3, FGF-2, Sox9, Wnt-4, Foxc2, and VEGF-A mRNA were significantly higher in SC synovium. Stimulation of TGF-β3 on synoviocytes increased alkaline phosphatase (ALP) activity and expressions of chondrogenic genes (Sox9, Col2α1, Aggrecan, Wnt-4, and Wnt-11), osteogenic genes (Runx2, Foxc2, osteocalcin, and Col1α1), and VEGF-A, but failed to influence FGF-2 expression. However, the addition of FGF-2 increased TGF-β3 expression. In conclusion, TGF-β3 existed in synovium and LBs of SC, and was responsible for the pathogenesis of SC. PMID:25742744

  6. Transforming growth factor beta 3 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint.

    PubMed

    Li, Yingjie; El Mozen, Loaye Abdelaziz; Cai, Hengxing; Fang, Wei; Meng, Qinggong; Li, Jian; Deng, Mohong; Long, Xing

    2015-01-01

    Synovial chondromatosis (SC) of temporomandibular joint is rare proliferative disorder featured by the formation of cartilaginous nodules in synovium and joint space. Transforming growth factor beta 3 (TGF-β3) is closely related to chondrogenic differentiation, and might participate in pathogenesis of SC. We discovered that increased quantity of synoviocytes and blood vessels were observed in SC synovium. The vessel wall and sublining fibroblasts were stained positively by the antibodies against TGF-β3, fibroblast growth factor 2 (FGF-2), and CD34. In loose bodies (LBs), TGF-β3 was mainly expressed in chondrocytes and FGF-2 was expressed in chondrocytes, fibroblasts, and vessel walls. Expressions of TGF-β1, TGF-β3, FGF-2, Sox9, Wnt-4, Foxc2, and VEGF-A mRNA were significantly higher in SC synovium. Stimulation of TGF-β3 on synoviocytes increased alkaline phosphatase (ALP) activity and expressions of chondrogenic genes (Sox9, Col2α1, Aggrecan, Wnt-4, and Wnt-11), osteogenic genes (Runx2, Foxc2, osteocalcin, and Col1α1), and VEGF-A, but failed to influence FGF-2 expression. However, the addition of FGF-2 increased TGF-β3 expression. In conclusion, TGF-β3 existed in synovium and LBs of SC, and was responsible for the pathogenesis of SC. PMID:25742744

  7. Tumor-promoting functions of transforming growth factor-β in progression of cancer

    PubMed Central

    2012-01-01

    Transforming growth factor-β (TGF-β) elicits both tumor-suppressive and tumor-promoting functions during cancer progression. Here, we describe the tumor-promoting functions of TGF-β and how these functions play a role in cancer progression. Normal epithelial cells undergo epithelial-mesenchymal transition (EMT) through the action of TGF-β, while treatment with TGF-β and fibroblast growth factor (FGF)-2 results in transdifferentiation into activated fibroblastic cells that are highly migratory, thereby facilitating cancer invasion and metastasis. TGF-β also induces EMT in tumor cells, which can be regulated by oncogenic and anti-oncogenic signals. In addition to EMT promotion, invasion and metastasis of cancer are facilitated by TGF-β through other mechanisms, such as regulation of cell survival, angiogenesis, and vascular integrity, and interaction with the tumor microenvironment. TGF-β also plays a critical role in regulating the cancer-initiating properties of certain types of cells, including glioma-initiating cells. These findings thus may be useful for establishing treatment strategies for advanced cancer by inhibiting TGF-β signaling. PMID:22111550

  8. alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

    PubMed Central

    Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

    2002-01-01

    Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta. PMID:12234252

  9. alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

    PubMed

    Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

    2002-12-01

    Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta. PMID:12234252

  10. Demonstration of single crystal growth via solid-solid transformation of a glass

    DOE PAGESBeta

    Savytskii, Dmytro; Knorr, Brian; Dierolf, Volkmar; Jain, Himanshu

    2016-03-18

    Many advanced technologies have relied on the availability of single crystals of appropriate material such as silicon for microelectronics or superalloys for turbine blades. Similarly, many promising materials could unleash their full potential if they were available in a single crystal form. However, the current methods are unsuitable for growing single crystals of these oftentimes incongruently melting, unstable or metastable materials. Here we demonstrate a strategy to overcome this hurdle by avoiding the gaseous or liquid phase, and directly converting glass into a single crystal. Specifically, Sb2S3 single crystals are grown in Sb-S-I glasses as an example of this approach.more » In this first unambiguous demonstration of an all-solid-state glass → crystal transformation, extraneous nucleation is avoided relative to crystal growth via spatially localized laser heating and inclusion of a suitable glass former in the composition. Lastly, the ability to fabricate patterned single-crystal architecture on a glass surface is demonstrated, providing a new class of micro-structured substrate for low cost epitaxial growth, active planar devices, etc.« less

  11. Transforming growth factor-{beta}2 enhances differentiation of cardiac myocytes from embryonic stem cells

    SciTech Connect

    Kumar, Dinender . E-mail: Dinender.Kumar@uvm.edu; Sun, Baiming

    2005-06-24

    Stem cell therapy holds great promise for the treatment of injured myocardium, but is challenged by a limited supply of appropriate cells. Three different isoforms of transforming growth factor-{beta} (TGF-{beta}) -{beta}1, -{beta}2, and -{beta}3 exhibit distinct regulatory effects on cell growth, differentiation, and migration during embryonic development. We compared the effects of these three different isoforms on cardiomyocyte differentiation from embryonic stem (ES) cells. In contrast to TGF-{beta}1, or -{beta}3, treatment of mouse ES cells with TGF-{beta}2 isoform significantly increased embryoid body (EB) proliferation as well as the extent of the EB outgrowth that beat rhythmically. At 17 days, 49% of the EBs treated with TGF-{beta}2 exhibited spontaneous beating compared with 15% in controls. Cardiac myocyte specific protein markers sarcomeric myosin and {alpha}-actin were demonstrated in beating EBs and cells isolated from EBs. In conclusion, TGF-{beta}2 but not TGF-{beta}1, or -{beta}3 promotes cardiac myocyte differentiation from ES cells.

  12. Melanoma-associated expression of transforming growth factor-beta isoforms.

    PubMed Central

    Van Belle, P.; Rodeck, U.; Nuamah, I.; Halpern, A. C.; Elder, D. E.

    1996-01-01

    Melanocytic neoplasia is characterized by the aberrant overproduction of multiple cytokines in vitro. However, it is largely unknown how cytokine expression relates to melanoma progression in vivo. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine commonly produced by cultured melanoma cells. The in situ expression of all three TGF-beta isoforms (TGF-beta1, -2, and -3) was determined immunohistochemically in melanocytes and in 51 melanocytic lesions using isoform-specific antibodies. Significant linear trends of expression were observed from melanocytes through nevi and primary and metastatic melanomas for all three isoforms. TGF-beta1 was expressed by some melanocytes and almost uniformly by nevi and melanomas. TGF-beta2 and -3 were not expressed in normal melanocytes but were expressed in nevi and early and advanced primary (radial and vertical growth phase) and metastatic melanomas in a tumor-progression-related manner. TGF-beta2 was heterogeneously expressed in advanced primary and metastatic melanomas, whereas TGF-beta3 was uniformly and highly expressed in these lesions. Thus, expression of TGF-beta isoforms in melanocytes and melanocytic lesions is heterogeneous and related to tumor progression, and expression of TGF-beta2 and TGF-beta3 commences at critical junctures during progression of nevi to primary melanomas. Images Figure 2 Figure 3 PMID:8669474

  13. Changes in Maternal Serum Transforming Growth Factor Beta-1 during Pregnancy: A Cross-Sectional Study

    PubMed Central

    Orazulike, Ngozi C.; Ashmore, Jill; Konje, Justin C.

    2013-01-01

    Changes in circulating levels of maternal serum transforming growth factor beta-1 (TGF-β1), collected from 98 women (AGA) at different gestational ages (10–38 weeks) were measured and comparisons were made between levels in pregnant and nonpregnant controls and also between 10 women with small-for-gestational age (SGA) and 7 with appropriate-for-gestational age (AGA) fetuses. Maternal serum TGF-β1 levels at all stages of pregnancy were higher than those in normal healthy nonpregnant adults. The mean TGF-β1 levels in SGA pregnancies at 34-week gestation (32.5 + 3.2 ng/mL) were significantly less than those in AGA pregnancies (39.2 + 9.8 ng/mL) while at 38-week gestation, the levels were similar in the two groups (36.04 + 4.3 versus 36.7 + 7.0 ng/mL). This differential change in TGF-β1 levels is probably an important modulating factor in the aetiopathogenesis of abnormal intrauterine fetal growth. PMID:24350258

  14. Demonstration of single crystal growth via solid-solid transformation of a glass.

    PubMed

    Savytskii, Dmytro; Knorr, Brian; Dierolf, Volkmar; Jain, Himanshu

    2016-01-01

    Many advanced technologies have relied on the availability of single crystals of appropriate material such as silicon for microelectronics or superalloys for turbine blades. Similarly, many promising materials could unleash their full potential if they were available in a single crystal form. However, the current methods are unsuitable for growing single crystals of these oftentimes incongruently melting, unstable or metastable materials. Here we demonstrate a strategy to overcome this hurdle by avoiding the gaseous or liquid phase, and directly converting glass into a single crystal. Specifically, Sb2S3 single crystals are grown in Sb-S-I glasses as an example of this approach. In this first unambiguous demonstration of an all-solid-state glass → crystal transformation, extraneous nucleation is avoided relative to crystal growth via spatially localized laser heating and inclusion of a suitable glass former in the composition. The ability to fabricate patterned single-crystal architecture on a glass surface is demonstrated, providing a new class of micro-structured substrate for low cost epitaxial growth, active planar devices, etc. PMID:26988919

  15. Altered expression of transforming growth factor-beta isoforms in bovine cystic ovarian disease.

    PubMed

    Matiller, V; Stangaferro, M L; Díaz, P U; Ortega, H H; Rey, F; Huber, E; Salvetti, N R

    2014-10-01

    Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors may contribute to follicular persistence. Transforming growth factor-beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)-induced COD. In the oestrous-synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous-synchronized control group with that in ACTH-induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH-induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease. PMID:25112788

  16. Demonstration of single crystal growth via solid-solid transformation of a glass

    NASA Astrophysics Data System (ADS)

    Savytskii, Dmytro; Knorr, Brian; Dierolf, Volkmar; Jain, Himanshu

    2016-03-01

    Many advanced technologies have relied on the availability of single crystals of appropriate material such as silicon for microelectronics or superalloys for turbine blades. Similarly, many promising materials could unleash their full potential if they were available in a single crystal form. However, the current methods are unsuitable for growing single crystals of these oftentimes incongruently melting, unstable or metastable materials. Here we demonstrate a strategy to overcome this hurdle by avoiding the gaseous or liquid phase, and directly converting glass into a single crystal. Specifically, Sb2S3 single crystals are grown in Sb-S-I glasses as an example of this approach. In this first unambiguous demonstration of an all-solid-state glass → crystal transformation, extraneous nucleation is avoided relative to crystal growth via spatially localized laser heating and inclusion of a suitable glass former in the composition. The ability to fabricate patterned single-crystal architecture on a glass surface is demonstrated, providing a new class of micro-structured substrate for low cost epitaxial growth, active planar devices, etc.

  17. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed Central

    Perlino, E.; Loverro, G.; Maiorano, E.; Giannini, T.; Cazzolla, A.; Napoli, A.; Fiore, M. G.; Ricco, R.; Marra, E.; Selvaggi, L.

    1998-01-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. Images Figure 1 Figure 5 Figure 6 Figure 8 PMID:9579831

  18. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed

    Perlino, E; Loverro, G; Maiorano, E; Giannini, T; Cazzolla, A; Napoli, A; Fiore, M G; Ricco, R; Marra, E; Selvaggi, L

    1998-04-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. PMID:9579831

  19. Demonstration of single crystal growth via solid-solid transformation of a glass

    PubMed Central

    Savytskii, Dmytro; Knorr, Brian; Dierolf, Volkmar; Jain, Himanshu

    2016-01-01

    Many advanced technologies have relied on the availability of single crystals of appropriate material such as silicon for microelectronics or superalloys for turbine blades. Similarly, many promising materials could unleash their full potential if they were available in a single crystal form. However, the current methods are unsuitable for growing single crystals of these oftentimes incongruently melting, unstable or metastable materials. Here we demonstrate a strategy to overcome this hurdle by avoiding the gaseous or liquid phase, and directly converting glass into a single crystal. Specifically, Sb2S3 single crystals are grown in Sb-S-I glasses as an example of this approach. In this first unambiguous demonstration of an all-solid-state glass → crystal transformation, extraneous nucleation is avoided relative to crystal growth via spatially localized laser heating and inclusion of a suitable glass former in the composition. The ability to fabricate patterned single-crystal architecture on a glass surface is demonstrated, providing a new class of micro-structured substrate for low cost epitaxial growth, active planar devices, etc. PMID:26988919

  20. Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

    PubMed Central

    Demers, G W; Espling, E; Harry, J B; Etscheid, B G; Galloway, D A

    1996-01-01

    Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals. PMID:8794328

  1. Cross-Talk Between Nitric Oxide and Transforming Growth Factor-β1 in Malaria

    PubMed Central

    Vodovotz, Yoram; Zamora, Ruben; Lieber, Matthew J.; Luckhart, Shirley

    2008-01-01

    Malaria has re-emerged as a global health problem, leading to an increased focus on the cellular and molecular biology of the mosquito Anopheles and the parasite Plasmodium with the goal of identifying novel points of intervention in the parasite life cycle. Anti-parasite defenses mounted by both mammalian hosts and Anopheles can suppress the growth of Plasmodium. Nonetheless, the parasite is able to escape complete elimination in vivo, perhaps by thwarting or co-opting these mechanisms for its own survival, as do numerous other pathogens. Among the defense systems used by the mammalian host against Plasmodium is the synthesis of nitric oxide (NO), catalyzed by an inducible NO synthase (iNOS). Nitric oxide produced by the action of an inducible Anopheles stephensi NO synthase (AsNOS) may be central to the anti-parasitic arsenal of this mosquito. In mammals, iNOS can be modulated by members of the transforming growth factor-β (TGF-β) cytokine superfamily. Transforming growth factor-β is produced as an inactive precursor that is activated following dissociation of certain inhibitory proteins, a process that can be promoted by reaction products of NO as well as by hemin. Ingestion by Anopheles of blood containing Plasmodium initiates parasite development, blood digestion which results in the accumulation of hematin (hemin) in the insect midgut, and induction of both AsNOS and TGF-β-like (As60A) gene expression in the midgut epithelium. Active mammalian TGF-β1 can be detected in the A. stephensi midgut up to 48h post-ingestion and latent TGF-β1 can be activated by midgut components in vitro, a process that is potentiated by NO and that may involve hematin. Further, mammalian TGF-β1 is perceived as a cytokine by A. stephensi cells in vitro and can alter Plasmodium development in vivo. Bloodfeeding by Anopheles, therefore, results in a juxtaposition of evolutionarily conserved mosquito and mammalian TGF-β superfamily homologs that may influence transmission

  2. Dynamics of Transforming Growth Factor Beta Signaling in Wound Healing and Scarring

    PubMed Central

    Finnson, Kenneth W.; McLean, Sarah; Di Guglielmo, Gianni M.; Philip, Anie

    2013-01-01

    Significance Wound healing is an intricate biological process in which the skin, or any other tissue, repairs itself after injury. Normal wound healing relies on the appropriate levels of cytokines and growth factors to ensure that cellular responses are mediated in a coordinated manner. Among the many growth factors studied in the context of wound healing, transforming growth factor beta (TGF-β) is thought to have the broadest spectrum of effects. Recent Advances Many of the molecular mechanisms underlying the TGF-β/Smad signaling pathway have been elucidated, and the role of TGF-β in wound healing has been well characterized. Targeting the TGF-β signaling pathway using therapeutic agents to improve wound healing and/or reduce scarring has been successful in pre-clinical studies. Critical Issues Although TGF-β isoforms (β1, β2, β3) signal through the same cell surface receptors, they display distinct functions during wound healing in vivo through mechanisms that have not been fully elucidated. The challenge of translating preclinical studies targeting the TGF-β signaling pathway to a clinical setting may require more extensive preclinical research using animal models that more closely mimic wound healing and scarring in humans, and taking into account the spatial, temporal, and cell-type–specific aspects of TGF-β isoform expression and function. Future Directions Understanding the differences in TGF-β isoform signaling at the molecular level and identification of novel components of the TGF-β signaling pathway that critically regulate wound healing may lead to the discovery of potential therapeutic targets for treatment of impaired wound healing and pathological scarring. PMID:24527343

  3. Mesenchymal stem cells promote tumor angiogenesis via the action of transforming growth factor β1

    PubMed Central

    LI, GUO-CAI; ZHANG, HONG-WEI; ZHAO, QING-CHUN; SUN, LI; YANG, JIAN-JUN; HONG, LIU; FENG, FAN; CAI, LEI

    2016-01-01

    Mesenchymal stem cells (MSCs) may influence the growth and metastasis of various human malignancies, including hepatocellular carcinoma (HCC). Therefore, the underlying mechanisms via which MSCs are able to affect malignancies require investigation. In the present study, the potential role of MSC in the angiogenesis of HCC was investigated. A total of 17 nude mouse models exhibiting human HCC were used to evaluate the effects of MSC on angiogenesis. A total of 8 mice were injected with human MSCs via the tail vein, and the remaining 9 mice were injected with phosphate-buffered saline as a control. A total of 35 days subsequent to the injection of MSCs, the microvessel density (MVD) of tumors was evaluated by immunostaining, using cluster of differentiation 31 antibody. The mRNA levels of transforming growth factor (TGF)β1, Smad2 and Smad7 were detected using reverse transcription-quantitative polymerase chain reaction. Protein expression levels of TGFβ1 and vascular endothelial growth factor (VEGF) in tumor tissues were analyzed using ELISA. Compared with controls, MVD in MSC-treated mice was significantly increased (28.00±9.19 vs. 18.11±3.30; P=0.006). The levels of TGFβ1 mRNA in the MSC-treated group were 2.15-fold higher compared with the control group (1.27±0.61 vs. 0.59±0.39; P=0.033), and MVD was higher in the group exhibiting increased TGFβ1 mRNA levels compared with the control group (26.50±9.11 vs. 19.44±6.14; P=0.038). In addition, a close correlation between the expression levels of TGFβ1 and VEGF was identified. The results of the present study suggested that MSCs may be capable of enhancing the angiogenesis of HCC, which may be partly due to the involvement of TGFβ1. PMID:26893697

  4. Analysis of Epithelial-Mesenchymal Transition Induced by Transforming Growth Factor β.

    PubMed

    Valcourt, Ulrich; Carthy, Jonathon; Okita, Yukari; Alcaraz, Lindsay; Kato, Mitsuyasu; Thuault, Sylvie; Bartholin, Laurent; Moustakas, Aristidis

    2016-01-01

    In recent years, the importance of the cell biological process of epithelial-mesenchymal transition (EMT) has been established via an exponentially growing number of reports. EMT has been documented during embryonic development, tissue fibrosis, and cancer progression in vitro, in animal models in vivo and in human specimens. EMT relates to many molecular and cellular alterations that occur when epithelial cells undergo a switch in differentiation that generates mesenchymal-like cells with newly acquired migratory and invasive properties. In addition, EMT relates to a nuclear reprogramming similar to the one occurring in the generation of induced pluripotent stem cells. Via such a process, EMT is gradually established to promote the generation and maintenance of adult tissue stem cells which under disease states such as cancer, are known as cancer stem cells. EMT is induced by developmental growth factors, oncogenes, radiation, and hypoxia. A prominent growth factor that causes EMT is transforming growth factor β (TGF-β).A series of molecular and cellular techniques can be applied to define and characterize the state of EMT in diverse biological samples. These methods range from DNA and RNA-based techniques that measure the expression of key EMT regulators and markers of epithelial or mesenchymal differentiation to functional assays of cell mobility, invasiveness and in vitro stemness. This chapter focuses on EMT induced by TGF-β and provides authoritative protocols and relevant reagents and citations of key publications aiming at assisting newcomers that enter this prolific area of biomedical sciences, and offering a useful reference tool to pioneers and aficionados of the field. PMID:26520123

  5. Effect of transforming growth factor beta on synthesis of glycosaminoglycans by human lung fibroblasts

    SciTech Connect

    Dubaybo, B.A.; Thet, L.A. )

    1990-09-01

    The processes of lung growth, injury, and repair are characterized by alterations in fibroblast synthesis and interstitial distribution of extracellular matrix components. Transforming growth factor beta (TGF-beta), which is postulated to play a role in modulating lung repair, alters the distribution of several matrix components such as collagen and fibronectin. We studied the effect of TGF-beta on the synthesis and distribution of the various glycosaminoglycans (GAGs) and whether these effects may explain its role in lung repair. Human diploid lung fibroblasts (IMR-90) were exposed to various concentrations of TGF-beta (0-5 nM) for variable periods of time (0-18 h). Newly synthesized GAGs were labeled with either (3H)glucosamine or (35S)sulfate. Individual GAGs were separated by size exclusion chromatography after serial enzymatic and chemical digestions and quantitated using scintillation counting. There was a dose-dependent increase in total GAG synthesis with maximal levels detected after 6 h of exposure. This increase was noted in all individual GAG types measured and was observed in both the cell associated GAGs (cell-matrix fraction) as well as the GAGs released into the medium (medium fraction). In the cell-matrix fraction, TGF-beta increased the proportion of heparan sulfate that was membrane bound as well as the proportion of dermatan sulfate in the intracellular compartment. In the medium fraction, TGF-beta increased the proportion of hyaluronic acid, chondroitin sulfate and dermatan sulfate released. We conclude that the role of TGF-beta in lung growth and repair may be related to increased synthesis of GAGs by human lung fibroblasts as well as alterations in the distribution of individual GAGs.

  6. Growth and phase transformations: Insights from hydrothermal coarsening experiements using nanocrystalline titanium dioxide

    NASA Astrophysics Data System (ADS)

    Penn, Rona Lee

    Controlling the morphology and size of crystallizing materials is of special interest in Materials Science as particle morphology can affect many materials characteristics, including magnetism, reactivity, and densification. High-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD) peak broadening (Scherrer) analysis, and determination of room temperature adsorption of organic acids by ion chromatography were used to study adsorption and hydrothermal coarsening and morphology development in nanocrystalline anatase (TiOsb2). HRTEM micrographs show that nanocrystalline anatase coarsens by two growth mechanisms: single crystal growth, via subnanometer-high surface steps, and a new growth mechanism known as oriented attachment, which involves topotactic joining of crystallites with subsequent coarsening to form single crystals of unique morphology. Addition of hydrochloric acid (pH = 3.0) to suspensions prior to hydrothermal treatment results in the formation of extremely elongate single crystals often comprised of more than 7 primary anatase crystallites. Both decreasing the pH of solutions below 3 and addition of organic acids resulted in a reduction in the frequency of observed attachments. Because attachment is only constrained by two dimensional match at the interface, attachment can lead to twinning and intergrowths on \\{112\\}, \\{101\\}, and (001). Slight misorientations between particles arising from surface topography at attaching surfaces, produces dislocations of edge, screw, and mixed character. Furthermore, anatase \\{112\\} twin boundaries consist of structural elements common to rutile and may serve as nucleation sites for the anatase-to-rutile transformation. These results have broad relevance for nucleation and growth models as they suggest that twinning, polytypism, and linear defects in macroscopic crystals can originate at oriented interfaces between primary nanocrystalline particles early in their crystallization history

  7. Transforming growth factor-beta activities in 'in vivo' lines of hormone-dependent and independent mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice.

    PubMed

    Elizalde, P V; Lanari, C; Kordon, E; Tezón, J; Charreau, E H

    1990-07-01

    We have determined the presence of transforming growth factor-beta (TGF-beta)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in 'in vivo' HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-beta from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-beta antibody. PMID:2145045

  8. Association between Fibrillin1 Polymorphisms (rs2118181, rs10519177) and Transforming Growth Factor β1 Concentration in Human Plasma

    PubMed Central

    Sepetiene, Ramune; Patamsyte, Vaiva; Zukovas, Giedrius; Jariene, Giedre; Stanioniene, Zita; Benetis, Rimantas; Tamosiunas, Abdonas; Lesauskaite, Vaiva

    2015-01-01

    Transforming growth factor (TGF)-β1 is a cytokine that participates in a broad range of cellular regulatory processes and is associated with various diseases including aortic aneurysm. Increased TGF-β1 levels are linked to Marfan syndrome (MFS) caused by fibrillin1 (FBN1) mutations and subsequent defects in signaling system. FBN1 single nucleotide polymorphisms (SNPs) rs2118181 and rs1059177 do not cause MFS but are associated with dilative pathology of aortic aneurysms (DPAAs). TGF-β1 and FBN1 SNPs rs2118181 and rs1059177 are potential biomarkers for early diagnosis of DPAA. We investigated the relationship between TGF-β1 levels in human blood plasma and FBN1 rs2118181 and rs1059177 in 269 individuals. The results showed a quantitative dependence of SNP genotype and TGF-β1 concentration. Presence of a single rs2118181 minor allele (G) increased the amount of TGF-β1 by roughly 1 ng/mL. Two copies of FBN1 rs1059177 minor allele (G) were required to have an additive effect on TGF-β1 levels. We found higher TGF-β1 concentrations in men compared with women (p = 0.001). A strong correlation between TGF-β1 levels and FBN1 SNPs suggests that a single nucleotide substitution in FBN1 sequence might reduce bioavailability or binding properties of fibrillin-1 and have an effect on TGF-β1 activation and cytokine concentration in blood plasma. By establishing the relationship between TGF-β1 and FBN1 SNPs rs2118181 and rs1059177, we provide evidence that their combination might be used as molecular biomarkers to identify patients at risk for sporadic ascending aortic aneurysm and aortic dissection. PMID:26322848

  9. Canonical Transforming Growth Factor-β Signaling Regulates Disintegrin Metalloprotease Expression in Experimental Renal Fibrosis via miR-29

    PubMed Central

    Ramdas, Vasudev; McBride, Martin; Denby, Laura; Baker, Andrew H.

    2014-01-01

    Fibrosis pathophysiology is critically regulated by Smad 2– and Smad 3–mediated transforming growth factor-β (TGF-β) signaling. Disintegrin metalloproteases (Adam) can manipulate the signaling environment, however, the role and regulation of ADAMs in renal fibrosis remain unclear. TGF-β stimulation of renal cells results in a significant up-regulation of Adams 10, 17, 12, and 19. The selective Smad2/3 inhibitor SB 525334 reversed these TGF-β–induced changes. In vivo, using ureteral obstruction to model renal fibrosis, we observed increased Adams gene expression that was blocked by oral administration of SB 525334. Similar increases in Adam gene expression also occurred in preclinical models of hypertension-induced renal damage and glomerulonephritis. miRNAs are a recently discovered second level of regulation of gene expression. Analysis of 3′ untranslated regions of Adam12 and Adam19 mRNAs showed multiple binding sites for miR-29a, miR-29b, and miR-29c. We show that miR-29 family expression is decreased after unilateral ureter obstruction and this significant decrease in miR-29 family expression was observed consistently in preclinical models of renal dysfunction and correlated with an increase in Adam12 and Adam19 expression. Exogenous overexpression of the miR-29 family blocked TGF-β–mediated up-regulation of Adam12 and Adam19 gene expression. This study shows that Adams are involved in renal fibrosis and are regulated by canonical TGF-β signaling and miR-29. Therefore, both Adams and the miR-29 family represent therapeutic targets for renal fibrosis. PMID:24103556

  10. Transforming Growth Factor-β1 Signaling Represses Testicular Steroidogenesis through Cross-Talk with Orphan Nuclear Receptor Nur77

    PubMed Central

    Park, Eunsook; Song, Chin-Hee; Park, Jae-Il; Ahn, Ryun-Sup; Choi, Hueng-Sik; Ko, CheMyong; Lee, Keesook

    2014-01-01

    Transforming growth factor- β1 (TGF-β1) has been reported to inhibit luteinizing hormone (LH) mediated-steroidogenesis in testicular Leydig cells. However, the mechanism by which TGF-β1 controls the steroidogenesis in Leydig cells is not well understood. Here, we investigated the possibility that TGF-β1 represses steroidogenesis through cross-talk with the orphan nuclear receptor Nur77. Nur77, which is induced by LH/cAMP signaling, is one of major transcription factors that regulate the expression of steroidogenic genes in Leydig cells. TGF-β1 signaling inhibited cAMP-induced testosterone production and the expression of steroidogenic genes such as P450c17, StAR and 3β-HSD in mouse Leydig cells. Further, TGF-β1/ALK5 signaling repressed cAMP-induced and Nur77-activated promoter activity of steroidogenic genes. In addition, TGF-β1/ALK5-activated Smad3 repressed Nur77 transactivation of steroidogenic gene promoters by interfering with Nur77 binding to DNA. In primary Leydig cells isolated from Tgfbr2flox/flox Cyp17iCre mice, TGF-β1-mediated repression of cAMP-induced steroidogenic gene expression was significantly less than that in primary Leydig cells from Tgfbr2flox/flox mice. Taken together, these results suggest that TGF-β1/ALK5/Smad3 signaling represses the expression of steroidogenic genes via the suppression of Nur77 transactivation in testicular Leydig cells. These findings may provide a molecular mechanism involved in the TGF-β1-mediated repression of testicular steroidogenesis. PMID:25140527

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

    PubMed

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

    2006-12-22

    Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling. PMID:17074765

  12. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: the role of T regulatory cells.

    PubMed

    Taylor, Alison; Verhagen, Johan; Blaser, Kurt; Akdis, Mübeccel; Akdis, Cezmi A

    2006-04-01

    Specific immune suppression and induction of tolerance are essential processes in the regulation and circumvention of immune defence. The balance between allergen-specific type 1 regulatory (Tr1) cells and T helper (Th) 2 cells appears to be decisive in the development of allergy. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals. In contrast, there is a high frequency of allergen-specific interleukin-4 (IL-4)-secreting T cells in allergic individuals. Allergen-specific immunotherapy can induce specific Tr1 cells that abolish allergen-induced proliferation of Th1 and Th2 cells, as well as their cytokine production. Tr1 cells utilize multiple suppressor mechanisms, such as IL-10 and transforming growth factor-beta (TGF-beta) as secreted cytokines and various surface molecules, such as cytotoxic T-lymphocyte antigen 4 and programmed death-1. IL-10 only inhibits T cells stimulated by low numbers of triggered T-cell receptors, which depend on CD28 costimulation. IL-10 inhibits CD28 tyrosine phosphorylation, preventing the binding of phosphatidylinositol 3-kinase p85 and consequently inhibiting the CD28 signalling pathway. In addition, IL-10 and TGF-beta secreted by Tr1 cells skew the antibody production from immunoglobulin E (IgE) towards the non-inflammatory isotypes IgG4 and IgA, respectively. Induction of antigen-specific Tr1 cells can thus re-direct an inappropriate immune response against allergens or auto-antigens using a broad range of suppressor mechanisms. PMID:16556256

  13. MEK inhibition prevents tumour-shed transforming growth factor-β-induced T-regulatory cell augmentation in tumour milieu.

    PubMed

    Hossain, Dewan M S; Panda, Abir K; Chakrabarty, Sreeparna; Bhattacharjee, Pushpak; Kajal, Kirti; Mohanty, Suchismita; Sarkar, Irene; Sarkar, Diptendra K; Kar, Santosh K; Sa, Gaurisankar

    2015-04-01

    Tumour progression is associated with immune-suppressive conditions that facilitate the escape of tumour cells from the regimen of immune cells, subsequently paralysing the host defence mechanisms. Induction of CD4(+)  CD25(+)  FoxP3(+) T regulatory (Treg) cells has been implicated in the tumour immune escape mechanism, although the novel anti-cancer treatment strategies targeting Treg cells remain unknown. The focus of this study is to define the interaction between tumour and immune system, i.e. how immune tolerance starts and gradually leads to the induction of adaptive Treg cells in the tumour microenvironment. Our study identified hyperactivated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -signalling as a potential target for reversing Treg cell augmentation in breast cancer patients. In more mechanistic detail, pharmacological inhibitors of MEK/ERK signalling inhibited transforming growth factor-β (TGF-β) production in tumour cells that essentially blocked TGF-β-SMAD3/SMAD4-mediated induction of CD25/interleukin-2 receptor α on CD4(+) T-cell surface. As a result high-affinity binding of interleukin-2 on those cells was prohibited, causing lack of Janus kinase 1 (JAK1)/JAK3-mediated signal transducer and activator of transcription 3 (STAT3)/STAT5 activation required for FoxP3 expression. Finally, for a more radical approach towards a safe MEK inhibitor, we validate the potential of multi-kinase inhibitor curcumin, especially the nano-curcumin made out of pure curcumin with greater bioavailability; in repealing tumour-shed TGF-β-induced Treg cell augmentation. PMID:25284464

  14. Transforming growth factor-beta mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells.

    PubMed

    Beck, Paul L; Rosenberg, Ian M; Xavier, Ramnik J; Koh, Theodore; Wong, Josée F; Podolsky, Daniel K

    2003-02-01

    In vitro studies suggest that transforming growth factor (TGF)-beta has potent effects on gastrointestinal mucosal integrity, wound repair, and neoplasia. However, the multiplicity of actions of this peptide on many different cell types confounds efforts to define the role of TGF-beta within the intestinal epithelium in vivo. To delineate these effects selective blockade of intestinal epithelial TGF-beta activity was undertaken through targeted expression of a dominant-negative (DN) TGF-beta RII to intestinal epithelial cells in vitro and in vivo. Stable intestinal epithelial cell (IEC)-6 lines overexpressing TGF-beta RII-DN (nucleotides -7 to 573) were established. Transgenic mice overexpressing TGF-beta RII-DN under the regulation of a modified liver fatty acid-binding promoter (LFABP-PTS4) were constructed. In vitro healing was assessed by wounding of confluent monolayers. Colitis was induced by the addition of dextran sodium sulfate (2.5 to 7.5% w/v) to their drinking water. Overexpression of TGF-beta RII-DN in intestinal epithelial cell-6 cells resulted in a marked reduction in cell migration and TGF-beta-stimulated wound healing in vitro. TGF-beta RII-DN transgenic mice did not exhibit baseline intestinal inflammation or changes in survival, body weight, epithelial cell proliferation, aberrant crypt foci, or tumor formation. TGF-beta RII-DN mice were markedly more susceptible to dextran sodium sulfate-induced colitis and exhibited impaired recovery after colonic injury. TGF-beta is required for intestinal mucosal healing and TGF-beta modulation of the intestinal epithelium plays a central role in determining susceptibility to injury. PMID:12547717

  15. Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

    SciTech Connect

    Ritvos, O.; Ranta, T.; Jalkanen, J.; Suikkari, A.M.; Voutilainen, R.; Bohn, H.; Rutanen, E.M.

    1988-05-01

    The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.

  16. Insulin-like growth factor binding protein-3 in preterm infants with retinopathy of prematurity

    PubMed Central

    Gharehbaghi, Manizheh Mostafa; Peirovifar, Ali; Sadeghi, Karim; Mostafidi, Haleh

    2012-01-01

    Background: Retinopathy of prematurity (ROP) is the main cause of visual impairment in preterm newborn infants. Objective: This study was conducted to determine whether insulin-like growth factor binding protein -3 (IGFBP-3) is associated with proliferative ROP and has a role in pathogenesis of the disease in premature infants. Materials and Methods: A total of 71 preterm infants born at or before 32 weeks of gestation participated in this study. Studied patients consisted of 41 neonates without vaso-proliferative findings of ROP as the control group and 30 preterm infants with evidence of severe ROP in follow up eye examination as the case group. Blood samples obtained from these infants 6-8 weeks after birth and blood levels of IGFBP-3 were measured using enzyme-linked immunosorbent assay (ELISA). Results: The mean gestation age and birth weight of the studied patients were 28.2±1.6 weeks and 1120.7±197 gram in the case group and 28.4±1.6 weeks and 1189.4±454 gram in the control group (P=0.25 and P=0.44 respectively). The infants in the case group had significantly lower Apgar score at first and 5 min after birth. Insulin-like growth factor binding protein -3 (IGFBP-3) was significantly lower in the patients with proliferative ROP than the patients without ROP [592.5±472.9 vs. 995.5±422.2 ng/ml (P=0.009)]. Using a cut-off point 770.45 ng/ml for the plasma IGFBP-3, we obtained a sensitivity of 65.9% and a specificity of 66.7% in the preterm infants with vasoproliferative ROP. Conclusion: Our data demonstrated that the blood levels IGFBP-3 was significantly lower in the patients with ROP and it is suspected that IGFBP-3 deficiency in the premature infants may have a pathogenetic role in proliferative ROP. PMID:23202391

  17. Fibroblast Growth Factor (FGF-2) and Its Receptors FGFR-2 and FGFR-3 May Be Putative Biomarkers of Malignant Transformation of Potentially Malignant Oral Lesions into Oral Squamous Cell Carcinoma

    PubMed Central

    Nayak, Seema; Goel, Madhu Mati; Makker, Annu; Bhatia, Vikram; Chandra, Saumya; Kumar, Sandeep; Agarwal, S. P.

    2015-01-01

    There are several factors like angiogenesis, lymphangiogenesis, genetic alterations, mutational factors that are involved in malignant transformation of potentially malignant oral lesions (PMOLs) to oral squamous cell carcinoma (OSCC). Fibroblast growth factor-2 (FGF-2) is one of the prototypes of the large family of growth factors that bind heparin. FGF-2 induces angiogenesis and its receptors may play a role in synthesis of collagen. FGFs are involved in transmission of signals between the epithelium and connective tissue, and influence growth and differentiation of a wide variety of tissue including epithelia. The present study was undertaken to analyze expression of FGF-2 and its receptors FGFR-2 and FGFR-3 in 72 PMOLs, 108 OSCC and 52 healthy controls, and their role in risk assessment for malignant transformation of Leukoplakia (LKP) and Oral submucous fibrosis (OSMF) to OSCC. Immunohistochemistry was performed using antibodies against FGF-2, FGFR-2 and FGFR-3. IHC results were validated by Real Time PCR. Expression of FGF-2, FGFR-2 and FGFR-3 was upregulated from PMOLs to OSCC. While 90% (9/10) of PMOLs which showed malignant transformation (transformed) expressed FGF-2, only 24.19% cases (15/62) of PMOLs which were not transformed (untransformed) to OSCC expressed FGF-2. Similarly, FGFR-2 expression was seen in 16/62 (25.81%) of untransformed PMOLs and 8/10 (80%) cases of transformed PMOLs. FGFR-3 expression was observed in 23/62 (37.10%) cases of untransformed PMOLs and 6/10 (60%) cases of transformed PMOLs. A significant association of FGF-2 and FGFR-2 expression with malignant transformation from PMOLs to OSCC was observed both at phenotypic and molecular level. The results suggest that FGF-2 and FGFR-2 may be useful as biomarkers of malignant transformation in patients with OSMF and LKP. PMID:26465941

  18. Interleukin-6, vascular endothelial growth factor and transforming growth factor beta 1 in canine steroid responsive meningitis-arteritis

    PubMed Central

    2013-01-01

    Background Steroid Responsive Meningitis-Arteritis (SRMA) is a common cause of inflammation of the canine central nervous system (CNS). To investigate if transforming growth factor beta 1 (TGF-β1), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) are involved in the production of excessive immunoglobulin A (IgA), the induction of acute phase proteins and in the development of a systemic necrotizing vasculitis, characteristic of SRMA, these three signalling proteins were evaluated. Results Cerebrospinal fluid (CSF) and serum samples of dogs during the acute phase of SRMA (SRMA) were tested for IL-6, VEGF and TGF- β1. Results were compared to those of dogs affected with SRMA during treatment (SRMA Th) and during relapse (SRMA R), to dogs with other meningoencephalomyelitides (ME), with miscellaneous non-inflammatory diseases of the CNS (CNS-Mix), with idiopathic epilepsy (IE), with systemic inflammatory diseases (Syst. Infl.) and with healthy dogs (Healthy). Concentrations of IL-6 and VEGF in CSF were significantly elevated in the SRMA group compared to the other disease categories (p < 0.05). The CSF concentrations of TGF-β1 were increased in SRMA group, but statistically significant differences were found only in comparison with Healthy and CNS-Mix groups. No differences were detected in the serum concentrations of TGF-β1 between the different groups. In untreated SRMA patients, a positive correlation (rSpear = 0.3549; P = 0.0337) between concentrations of TGF-β1 and IgA concentration was found in CSF, while concentrations of IL-6 and VEGF in CSF positively correlated with the degree of pleocytosis (rSpear = 0.8323; P < 0.0001 and rSpear = 0.5711; P = 0.0166, respectively). Conclusions Our results suggest that these three signalling proteins are biomarkers of disease activity in SRMA. VEGF might play an important role in the development of a systemic arteritis. TGF-β1 is considered to be involved in the excessive

  19. Androgen up-regulates vascular endothelial growth factor expression in prostate cancer cells via an Sp1 binding site

    PubMed Central

    2013-01-01

    Background Vascular Endothelial Growth Factor (VEGF) is regulated by a number of different factors, but the mechanism(s) behind androgen-mediated regulation of VEGF in prostate cancer are poorly understood. Results Three novel androgen receptor (AR) binding sites were discovered in the VEGF promoter and in vivo binding of AR to these sites was demonstrated by chromatin immunoprecipitation. Mutation of these sites attenuated activation of the VEGF promoter by the androgen analog, R1881 in prostate cancer cells. The transcription factors AR and Sp1 were shown to form a nuclear complex and both bound the VEGF core promoter in chromatin of hormone treated CWR22Rv1 prostate cancer cells. The importance of the Sp1 binding site in hormone mediated activation of VEGF expression was demonstrated by site directed mutagenesis. Mutation of a critical Sp1 binding site (Sp1.4) in the VEGF core promoter region prevented activation by androgen. Similarly, suppression of Sp1 binding by Mithramycin A treatment significantly reduced VEGF expression. Conclusions Our mechanistic study of androgen mediated induction of VEGF expression in prostate cancer cells revealed for the first time that this induction is mediated through the core promoter region and is dependent upon a critical Sp1 binding site. The importance of Sp1 binding suggests that therapy targeting the AR-Sp1 complex may dampen VEGF induced angiogenesis and, thereby, block prostate cancer progression, helping to maintain the indolent form of prostate cancer. PMID:23369005

  20. Presence of specific growth hormone binding sites in rainbow trout (Oncorhynchus mykiss) tissues: characterization of the hepatic receptor

    SciTech Connect

    Yao, K.; Niu, P.D.; Le Gac, F.; Le Bail, P.Y. )

    1991-01-01

    The present work outlines the presence of specific binding for chinook salmon growth hormone (sGH) in different tissue preparations of rainbow trout. Optimal incubation conditions (pH, Tris, MgCl{sub 2}) were determined. Specific binding was very sensitive to salt concentration during incubation. The specific binding reached a plateau after 15 and 25 hr of incubation at 12 and 4 {degree}. At 20 {degree}, specific and nonspecific binding were not stable. Specific binding dissociation was slower than association and was only partial. The binding was saturable (Bmax = 187 +/- 167 pmol), of high affinity (Ka = 2.4 +/- 0.8 10(9) M-1), and very specific for GH, properties which are in agreement with the characteristics of hormonal receptors. Sea bream and mammalian GH appeared 2- and 30-fold, respectively, less potent than cold sGH2 for displacing {sup 125}I-sGH2. Tissue preparations from ovary, testis, fat, skin, cartilage, gill, blood pellet, brain, spleen, kidney, and muscle showed significant saturable binding.

  1. Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo☆

    PubMed Central

    Tripathi, Vineeta Bhasker; Baskaran, Pranetha; Shaw, Christopher E.; Guthrie, Sarah

    2014-01-01

    Intracellular inclusions of the TAR-DNA binding protein 43 (TDP-43) have been reported in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD-TDP). Rare mutations in TARDBP have been linked to both ALS and FTD-TDP suggesting that TDP-43 dysfunction is mechanistic in causing disease. TDP-43 is a predominantly nuclear protein with roles in regulating RNA transcription, splicing, stability and transport. In ALS, TDP-43 aberrantly accumulates in the cytoplasm of motor neurons where it forms aggregates. However it has until recently been unclear whether the toxic effects of TDP-43 involve recruitment to motor axons, and what effects this might have on axonal growth and integrity. Here we use chick embryonic motor neurons, in vivo and in vitro, to model the acute effects of TDP-43. We show that wild-type and two TDP-43 mutant proteins cause toxicity in chick embryonic motor neurons in vivo. Moreover, TDP-43 is increasingly mislocalised to axons over time in vivo, axon growth to peripheral targets is truncated, and expression of neurofilament-associated antigen is reduced relative to control motor neurons. In primary spinal motor neurons in vitro, a progressive translocation of TDP-43 to the cytoplasm occurs over time, similar to that observed in vivo. This coincides with the appearance of cytoplasmic aggregates, a reduction in the axonal length, and cellular toxicity, which was most striking for neurons expressing TDP-43 mutant forms. These observations suggest that the capacity of spinal motor neurons to produce and maintain an axon is compromised by dysregulation of TDP-43 and that the disruption of cytoskeletal integrity may play a role in the pathogenesis of ALS and FTD-TDP. PMID:24423647

  2. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    SciTech Connect

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  3. Insulin-Like Growth Factor Binding Protein-4 as a Marker of Chronic Lupus Nephritis

    PubMed Central

    Han, Jie; Ye, Yujin; Singh, Sandeep; Zhou, Jinchun; Li, Yajuan; Ding, Huihua; Li, Quan-zhen; Zhou, Xin; Putterman, Chaim; Saxena, Ramesh; Mohan, Chandra

    2016-01-01

    Kidney biopsy remains the mainstay of Lupus Nephritis (LN) diagnosis and prognostication. The objective of this study is to identify non-invasive biomarkers that closely parallel renal pathology in LN. Previous reports have demonstrated that serum Insulin-like growth factor binding protein 4 (IGFBP-4) was increased in diabetic nephropathy in both animal models and patients. We proceeded to assess if IGFBP4 could be associated with LN. We performed ELISA using the serum of 86 patients with LN. Normal healthy adults (N = 23) and patients with other glomerular diseases (N = 20) served as controls. Compared to the healthy controls or other glomerular disease controls, serum IGFBP-4 levels were significantly higher in the patients with LN. Serum IGFBP-4 did not correlate well with systemic lupus erythematosus disease activity index (SLEDAI), renal SLEDAI or proteinuria, but it did correlate with estimated glomerular filtration rate (R = 0.609, P < 0.0001). Interestingly, in 18 patients with proliferative LN whose blood samples were obtained at the time of renal biopsy, serum IGFBP-4 levels correlated strongly with the chronicity index of renal pathology (R = 0.713, P < 0.001). IGFBP-4 emerges a potential marker of lupus nephritis, reflective of renal pathology chronicity changes. PMID:27019456

  4. Insulin-like growth factor (IGF) and IGF binding protein gene expression in multicystic renal dysplasia.

    PubMed

    Matsell, D G; Bennett, T; Armstrong, R A; Goodyer, P; Goodyer, C; Han, V K

    1997-01-01

    Multicystic dysplastic kidney disease is the most common form of renal dysplasia that leads to ESRD in children. This study describes the histopathological changes of multicystic dysplasia that occur from early fetal life to the postnatal period. At 14 wk gestation, early cystic enlargement of various segments of the nephron have been identified, in addition to a displaced metanephric blastema adjacent to zones of normal nephrogenesis. At later stages, the predominant features include cyst enlargement with marked fibromuscular collars, architectural disorganization, and replacement of the interstitium with a disarray of mesenchymal tissue. This study investigated the expression of the mRNA encoding the insulin-like growth factors (IGF) and IGF binding proteins (IGFBP) and have demonstrated IGF-II, IGFBP-2, and IGFBP-3 to be altered. Apart from their expression in the displaced metanephric blastema, both IGF-II and IGFBP-2 were overexpressed in abnormal tissue elements in all kidneys from fetal to postnatal life. IGF-II gene expression was localized to mesenchymal tissue, specifically in the periductal fibromuscular collars. IGFBP-2 mRNA was found to be expressed exclusively in the cyst epithelia of all cysts at all ages studied, whereas IGFBP-3 mRNA was absent from these epithelia. This study details the failure of normal IGF expression in the development of multicystic renal dysplasia and suggests a role for the IGF system in the progressive histopathological changes of this disorder. PMID:9013452

  5. Differential stability of TATA box binding proteins from archaea with different optimal growth temperatures

    NASA Astrophysics Data System (ADS)

    Kopitz, Annette; Soppa, Jörg; Krejtschi, Carsten; Hauser, Karin

    2009-09-01

    The TATA box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs), from below 0 °C to more than 100 °C. Thus, the archaeal TBP family is ideally suited to study the evolutionary adaptation of proteins to an extremely wide range of temperatures. We characterized the thermostability of one mesophilic and one thermophilic TBP by infrared spectroscopy. Transition temperatures ( Tms) of thermal unfolding have been determined using TBPs from Methanosarcina mazei (OGT 37 °C) and from Methanothermobacter thermautotrophicus (OGT 65 °C). Furthermore, the influence of protein and salt concentration on thermostability has been characterized. Together with previous studies, our results reveal that the Tms of archaeal TBPs are closely correlated with the OGTs of the respective species. Noteworthy, this is also true for the TBP from M. mazei representing the first characterized TBP from a mesophilic archaeon. In contrast, the only characterized eukaryotic TBP of the mesophilic plant Arabidopsis thaliana has a Tm more than 40 °C above the OGT.

  6. Cell-autonomous cytokinin-independent growth of tobacco cells transformed by Agrobacterium tumefaciens strains lacking the cytokinin biosynthesis gene.

    PubMed Central

    Black, R C; Binns, A N; Chang, C F; Lynn, D G

    1994-01-01

    Mutations at the cytokinin biosynthesis locus (tmr) of Agrobacterium tumefaciens usually result in strains that induce tumors exhibiting the rooty phenotype associated with high auxin-to-cytokinin ratios. However, tobacco (Nicotiana tabacum cv Havana 425) leaf disc explants responded to tmr- mutant strain A356 by producing rapidly growing, unorganized tumors, indicating that these lines can grow in a cytokinin-independent fashion despite the absence of a functional tmr gene. Several methods have been used to characterize the physiological and cellular basis of this phenotype. The results indicate that tmr- tumors have a physiologically distinct mechanism for cytokinin-independent growth in comparison to tumors induced by wild-type bacteria. The cytokinin-independent phenotype of the tmr- transformants appears to be cell autonomous in nature: only the transformed cells and their progeny were capable of cytokinin-independent growth. Specifically, the tmr- tumors did not accumulate cytokinin, and clonal analysis indicated the tmr- transformed cells were not capable of stimulating the growth of neighboring nontransformed cells. Finally, the cytokinin-independent phenotype of the tmr- transformants was shown to be cold sensitive, whereas the wild-type tumors exhibited a cold-resistant cytokinin-independent phenotype. Potential mechanisms for this novel form of cytokinin-independent growth, including the role of the dehydrodiconiferyl alcohol glucosides found in both tumor types, are discussed. PMID:8058843

  7. Growth habit and photo-synthetic activity of shoot cultures of Medicago sativa L. transformed with the oryzacystatin II gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vitro maintained shoot cultures of alfalfa (Medicago sativa L. cv. Zajeÿarska 83) that were transformed with the oryzacystatin II (OCII) gene and propagated on growth regulator-free medium were subjected to analysis of morphological characteristics and photosynthetic activity. The most striking f...

  8. From wavy hair to naked proteins: the role of transforming growth factor alpha in health and disease.

    PubMed

    Singh, Bhuminder; Coffey, Robert J

    2014-04-01

    Since its discovery in 1978 and cloning in 1984, transforming growth factor-alpha (TGF-α, TGFA) has been one of the most extensively studied EGF receptor (EGFR) ligands. In this review, we provide a historical perspective on TGFA-related studies, highlighting what we consider important advances related to its function in normal and disease states. PMID:24631356

  9. GAP JUNCTION COMMUNICATION MEDIATES TRANSFORMING GROWTH FACTOR-BETA ACTIVATION AND ENDOTHELIAL-INDUCED MURAL CELL DIFFERENTIATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-beta (TGF-beta) activation. We investigated whether gap junction channels are formed between endothelial cells and recrui...

  10. Increased susceptibility to atrial fibrillation secondary to atrial fibrosis in transgenic goats expressing transforming growth factor - B1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in people with significant morbidity and mortality. There is a strong association between atrial fibrosis and AF. Transforming growth factor B1 (TGF-B1) is an essential mediator of atrial fibrosis in animal models and human pat...

  11. Long Non-coding RNAs (LncRNA) Regulated by Transforming Growth Factor (TGF) β

    PubMed Central

    Richards, Edward J.; Zhang, Gu; Li, Zhu-Peng; Permuth-Wey, Jennifer; Challa, Sridevi; Li, Yajuan; Kong, William; Dan, Su; Bui, Marilyn M.; Coppola, Domenico; Mao, Wei-Min; Sellers, Thomas A.; Cheng, Jin Q.

    2015-01-01

    Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers. PMID:25605728

  12. Potassium Inhibits Dietary Salt-Induced Transforming Growth Factor-β Production

    PubMed Central

    Ying, Wei-Zhong; Aaron, Kristal; Wang, Pei-Xuan; Sanders, Paul W.

    2009-01-01

    Human and animal studies demonstrate an untoward effect of excess dietary NaCl (salt) intake on cardiovascular function and life span. The endothelium in particular augments the production of transforming growth factor (TGF)-β, a fibrogenic growth factor, in response to excess dietary salt intake. This study explored the initiating mechanism that regulates salt-induced endothelial cell production of TGF-β. Male Sprague-Dawley rats were given diets containing different amounts of NaCl and potassium for 4 days. A bioassay for TGF-β demonstrated increased (35.2%) amounts of active TGF-β in the medium of aortic ring segments from rats on the high-salt diet compared with rats maintained on a 0.3% NaCl diet. Inhibition of the large-conductance, calcium-activated potassium channel inhibited dietary salt-induced vascular production of TGF-β but did not affect production of TGF-β by ring segments from rats on the low-salt diet. Immunohistochemical and Western analyses demonstrated the α subunit of the calcium-activated potassium channel in endothelial cells. Increasing medium [K+] inhibited production of dietary salt-induced vascular production levels of total and active TGF-β but did not alter TGF-β production by aortic rings from rats on the 0.3% NaCl diet. Increasing dietary potassium content decreased urinary active TGF-β in animals receiving the high-salt diet but did not change urinary active TGF-β in animals receiving the low-salt diet. The findings demonstrated an interesting interaction between the dietary intake of potassium and excess NaCl and further showed the fundamental role of the endothelial calcium-activated potassium channel in the vascular response to excess salt intake. PMID:19738156

  13. Transforming growth factor-beta during carcinogenesis: the shift from epithelial to mesenchymal signaling.

    PubMed

    Matsuzaki, Koichi; Okazaki, Kazuichi

    2006-04-01

    Transforming growth factor-beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells, JNK/pSmad3L-mediated signaling is involved in invasion by activated mesenchymal cells. During sporadic human colorectal carcinogenesis, TGF-beta signaling confers a selective advantage on tumor cells by shifting from the TbetaRI/pSmad3C pathway characteristic of mature epithelial cells to the JNK/pSmad3L pathway, which is more characteristic of the state of flux shown by the activated mesenchymal cells. JNK acts as a regulator of TGF-beta signaling by increasing the basal level of pSmad3L available for action in the nuclei of the invasive adenocarcinoma, in the meantime shutting down TGF-beta-dependent nuclear activity of pSmad3C. Loss of epithelial homeostasis and acquisition of a migratory, mesenchymal phenotype are essential for tumor invasion. From the viewpoint of TGF-beta signaling, a key therapeutic aim in cancer would be restoration of the lost tumor suppressor function observed in normal colorectal epithelial cells at the expense of effects promoting aggressive behavior of the adenocarcinoma. Specific inhibitors of the JNK/pSmad3L pathway might prove useful in this respect. In the case of molecularly targeted therapy for human cancer, pSmad3L and pSmad3C could be assessed as biomarkers to evaluate the likely benefit from specific inhibition of the JNK/pSmad3L pathway. PMID:16741607

  14. Hypertrophic stimuli induce transforming growth factor-beta 1 expression in rat ventricular myocytes.

    PubMed Central

    Takahashi, N; Calderone, A; Izzo, N J; Mäki, T M; Marsh, J D; Colucci, W S

    1994-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) is a peptide growth factor that may play a role in the myocardial response to hypertrophic stimuli. However, the cellular distribution, mechanism of induction, and source of increased TGF-beta 1 in response to hypertrophic stimuli are not known. We tested the hypothesis that the cardiac myocyte responds to hypertrophic stimuli with the increased expression of TGF-beta 1. In adult rat ventricular myocardium freshly dissociated into myocyte and nonmyocyte cellular fractions, the preponderance of TGF-beta 1 mRNA visualized by Northern hybridization was in the nonmyocyte fraction. Abdominal aortic constriction (7 d) and subcutaneous norepinephrine infusion (36 h) each caused ventricular hypertrophy associated with 3.1-fold and 3.8-fold increases, respectively, in TGF-beta 1 mRNA in the myocyte fraction, but had no effect on the level of TGF-beta 1 mRNA in the nonmyocyte fraction. In ventricular myocytes, norepinephrine likewise caused a 4.1-fold increase in TGF-beta 1 mRNA associated with an increase in TGF-beta bioactivity. This induction of TGF-beta 1 mRNA occurred at norepinephrine concentrations as low as 1 nM and was blocked by prazosin, but not propranolol. NE did not increase the TGF-beta 1 mRNA level in nonmyocytes, primarily fibroblasts, cultured from neonatal rat ventricle. Thus, the cardiac myocyte responds to two hypertrophic stimuli, pressure overload and norepinephrine, with the induction of TGF-beta 1. These data support the view that TGF-beta 1, released by myocytes and acting in an autocrine and/or paracrine manner, is involved in myocardial remodeling by hypertrophic stimuli. Images PMID:7929822

  15. Transforming growth factor: beta signaling is essential for limb regeneration in axolotls.

    PubMed

    Lévesque, Mathieu; Gatien, Samuel; Finnson, Kenneth; Desmeules, Sophie; Villiard, Eric; Pilote, Mireille; Philip, Anie; Roy, Stéphane

    2007-01-01

    Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-beta). In the present study, the full length sequence of the axolotl TGF-beta1 cDNA was isolated. The spatio-temporal expression pattern of TGF-beta1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-beta signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-beta type I receptor, SB-431542, we show that TGF-beta signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-beta signaling are down-regulated. These data directly implicate TGF-beta signaling in the

  16. Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

    PubMed Central

    Lévesque, Mathieu; Gatien, Samuel; Finnson, Kenneth; Desmeules, Sophie; Villiard, Éric; Pilote, Mireille; Philip, Anie; Roy, Stéphane

    2007-01-01

    Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control

  17. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

    SciTech Connect

    Bashkin, P.; Doctrow, S.; Klagsbrun, M.; Svahn, C.M.; Folkman, J.; Vlodavsky, I. )

    1989-02-21

    Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of {sup 125}I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 x 10{sup 12} binding sites/mm{sup 2} ECM with an apparent k{sub D} of 610 nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 {mu}g/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 {mu}g/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descement's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

  18. Identification of common ligand binding determinants of the insulin and insulin-like growth factor 1 receptors. Insights into mechanisms of ligand binding.

    PubMed

    Mynarcik, D C; Williams, P F; Schaffer, L; Yu, G Q; Whittaker, J

    1997-07-25

    Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2-3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. cDNAs encoding alanine-substituted secreted recombinant IGF-1 receptors were expressed in 293 EBNA cells, and the ligand binding properties of the expressed proteins were evaluated. Mutation of Phe701 resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe714, produces a 140-fold reduction in affinity for insulin. Mutation of Asp8, Asn11, Phe58, Phe692, Glu693, His697, and Asn698 produces a 3.5-6-fold reduction in affinity for IGF-1. In contrast, alanine mutation of the corresponding amino acids of the insulin receptor with the exception of Asp12 produces reductions in affinity that are 50-fold or greater. The affinity of insulin for these mutants relative to wild type receptor was similar to that of their relative affinity for IGF-1 with two exceptions; the IC50 values for insulin binding to the mutants of Arg10, which has normal affinity for IGF-1, and His697, which has a 6-fold reduction in affinity for IGF-1, were both at least 2 orders of magnitude greater than for wild type receptor. The Kd values for insulin of the corresponding alanine mutants of the insulin receptor

  19. Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat prolactin promoter.

    PubMed

    Schweppe, R E; Gutierrez-Hartmann, A

    2001-03-01

    Ets factors play a critical role in oncogenic Ras- and growth factor-mediated regulation of the proximal rat prolactin (rPRL) promoter in pituitary cells. The rPRL promoter contains two key functional Ets binding sites (EBS): a composite EBS/Pit-1 element located at -212 and an EBS that co-localizes with the basal transcription element (BTE, or A-site) located at -96. Oncogenic Ras exclusively signals to the -212 site, which we have named the Ras response element (RRE); whereas the response of multiple growth factors (FGFs, EGF, IGF, insulin and TRH) maps to both EBSs. Although Ets-1 and GA binding protein (GABP) have been implicated in the Ras and insulin responses, respectively, the precise identity of the pituitary Ets factors that specifically bind to the RRE and BTE sites remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE) that bind to the EBSs contained in the RRE and BTE, we used EBS-RRE and BTE oligonucleotides in electrophoretic mobility shift assays (EMSAs), antibody supershift assays, western blot analysis of partially purified fractions and UV-crosslinking studies. EMSAs, using either the BTE or EBS-RRE probes, identified a specific protein-DNA complex, designated complex A, which contains an Ets factor as determined by oligonucleotide competition studies. Using western blot analysis of GH3 nuclear proteins that bind to heparin-Sepharose, we have shown that Ets-1 and GABP, which are MAP kinase substrates, co-purify with complex A, and supershift analysis with specific antisera revealed that complex A contains Ets-1, GABPalpha and GABPbeta1. In addition, we show that recombinant full-length Ets-1 binds equivalently to BTE and EBS-RRE probes, while recombinant GABPalpha/beta preferentially binds to the BTE probe. Furthermore, comparing the DNA binding of GH4NE containing both Ets-1 and GABP and HeLa nuclear extracts devoid of Ets-1 but containing GABP, we were able to show that the EBS

  20. Protective effect of transforming growth factor beta 1 on experimental autoimmune diseases in mice.

    PubMed

    Kuruvilla, A P; Shah, R; Hochwald, G M; Liggitt, H D; Palladino, M A; Thorbecke, G J

    1991-04-01

    Interleukin 1 (IL-1) and tumor necrosis factor alpha are thought to contribute to the inflammatory response associated with autoimmune diseases. Transforming growth factor beta 1 (TGF-beta 1) counteracts many effects of these cytokines and has various immunosuppressive properties. In the present study, it is shown that microgram amounts of TGF-beta 1, injected daily for 1-2 weeks, protect against collagen-induced arthritis (CIA) and relapsing experimental allergic encephalomyelitis (REAE), the animal models for rheumatoid arthritis and multiple sclerosis, respectively. When administered during induction of the disease, TGF-beta 1 prevents CIA but only delays the onset of REAE by 2-3 days. However, when administered during a remission. TGF-beta 1 prevents the occurrence of relapses in REAE. The results suggest that TGF-beta 1 has powerful anti-inflammatory effects, mimicking in some respects the beneficial effects of immunosuppressive drugs in these experimental models of autoimmune disease, but without discernable adverse effects. PMID:2011600

  1. Loss of transforming growth factor-beta 2 leads to impairment of central synapse function

    PubMed Central

    Heupel, Katharina; Sargsyan, Vardanush; Plomp, Jaap J; Rickmann, Michael; Varoqueaux, Frédérique; Zhang, Weiqi; Krieglstein, Kerstin

    2008-01-01

    Background The formation of functional synapses is a crucial event in neuronal network formation, and with regard to regulation of breathing it is essential for life. Members of the transforming growth factor-beta (TGF-β) superfamily act as intercellular signaling molecules during synaptogenesis of the neuromuscular junction of Drosophila and are involved in synaptic function of sensory neurons of Aplysia. Results Here we show that while TGF-β2 is not crucial for the morphology and function of the neuromuscular junction of the diaphragm muscle of mice, it is essential for proper synaptic function in the pre-Bötzinger complex, a central rhythm organizer located in the brainstem. Genetic deletion of TGF-β2 in mice strongly impaired both GABA/glycinergic and glutamatergic synaptic transmission in the pre-Bötzinger complex area, while numbers and morphology of central synapses of knock-out animals were indistinguishable from their wild-type littermates at embryonic day 18.5. Conclusion The results demonstrate that TGF-β2 influences synaptic function, rather than synaptogenesis, specifically at central synapses. The functional alterations in the respiratory center of the brain are probably the underlying cause of the perinatal death of the TGF-β2 knock-out mice. PMID:18854036

  2. Association of interleukin 10 and transforming growth factor β gene polymorphisms with chronic idiopathic urticaria.

    PubMed

    Tavakol, Marzieh; Movahedi, Masoud; Amirzargar, Ali Akbar; Aryan, Zahra; Bidoki, Alireza Zare; Heidari, Kimia; Soltani, Samaneh; Gharagozlou, Mohammad; Aghamohammadi, Asghar; Nabavi, Mohammad; Nasiri, Rasoul; Ahmadvand, Alireza; Rezaei, Nima

    2014-01-01

    Transforming growth factor β (TGF-β) and interleukin 10 (IL-10) are two anti-inflammatory cytokines that are implicated in the pathogenesis of urticaria. The goal of this study was to examine the possible association of polymorphisms of TGF-β and IL-10 genes with susceptibility to chronic idiopathic urticaria (CIU). This study was conducted on 90 patients with CIU. Polymerase chain reaction (PCR) was done to determine the genotype at 5 polymorphic sites; TGF-β (codon10C/T and codon25G/C) and IL-10 (-1082G/A, -819C/T, and -592C/A). The C allele at codon 25 of TGF-β was more prevalent in CIU patients compared to controls (OR = 9.5, 95% CI = 5.4-16.8, P<0.001). Genotypes of CT and CG at 10 and 25 codons of TGF-β gene, respectively, and AG, CT, and CA for loci of -1082, -819, and -592 of IL-10 gene were significantly higher in CIU patients (P<0.001). In haplotype analysis, frequency of TGF-β haplotypes differed between patients with CIU and controls; CC haplotype was overrepresented, while CG and TG haplotypes were underrepresented (P<0.001). These results suggest that TGF-β and IL-10 genetic variability could contribute to susceptibility to CIU. Additionally, patients with CIU seem to have genotypes leading to high production of TGF-β and IL-10. PMID:25580782

  3. Mitotic Asynchrony Induces Transforming Growth Factor-β1 Secretion from Airway Epithelium

    PubMed Central

    Alcala, Sarah E.; Benton, Angela S.; Watson, Alan M.; Kureshi, Suraiya; Reeves, Erica M. K.; Damsker, Jesse; Wang, Zuyi; Nagaraju, Kanneboyina; Anderson, Julia; Williams, Aaron M.; Lee, Amber J. Y.; Hayes, Kathleen; Rose, Mary C.; Hoffman, Eric P.

    2014-01-01

    We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to proinflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that mitotic asynchrony is associated with proinflammatory/fibrotic cytokine secretion (e.g., transforming growth factor [TGF]-β1), here we provide evidence supporting cause-and-effect. Under normal conditions, primary airway epithelial basal cell populations undergo mitosis synchronously and do not secrete proinflammatory or profibrotic cytokines. However, when pairs of nonasthmatic cultures were mitotically synchronized at 12 hours off-set and then combined, the mixed cell populations secreted elevated levels of TGF-β1. This shows that mitotic asynchrony is not only associated with but is also causative of TGF-β1 secretion. The secreted cytokines and other mediators from asthmatic cells were not the cause of asynchronous regeneration; synchronously mitotic nonasthmatic epithelia exposed to conditioned media from asthmatic cells did not show changes in mitotic synchrony. We also tested if resynchronization of regenerating asthmatic airway epithelia reduces TGF-β1 secretion and found that pulse-dosed dexamethasone, simvastatin, and aphidicolin were all effective. We therefore propose a new model for chronic inflammatory and fibrotic conditions where an underlying factor is mitotic asynchrony. PMID:24669775

  4. Transforming growth factor-β1 in carcinogenesis, progression, and therapy in cervical cancer.

    PubMed

    Zhu, Haiyan; Luo, Hui; Shen, Zhaojun; Hu, Xiaoli; Sun, Luzhe; Zhu, Xueqiong

    2016-06-01

    Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine that plays important roles in cervical tumor formation, invasion, progression, and metastasis. TGF-β1 functions as a tumor inhibitor in precancerous lesions and early stage cancers of cervix whereas as a tumor promoter in later stage. This switch from a tumor inhibitor to a tumor promoter might be due to various alterations in TGF-β signaling pathway, such as mutations or loss of expression of TGF-β receptors and SMAD proteins. Additionally, the oncoproteins of human papillomaviruses have been shown to stimulate TGF-β1 expression, which in turn suppresses host immune surveillance. Thus, in addition to driving tumor cell migration and metastasis, TGF-β1 is believed to play a key role in promoting human papillomavirus infection by weakening host immune defense. In this article, we will discuss the role of TGF-β1 in the expression, carcinogenesis, progression, and therapy in cervical cancers. A better understanding of this cytokine in cervical carcinogenesis is essential for critical evaluation of this cytokine as a potential prognostic marker and therapeutic target. PMID:27010470

  5. Overexpressed homeobox B9 regulates oncogenic activities by transforming growth factor-β1 in gliomas

    SciTech Connect

    Fang, Liping; Xu, Yinghui; Zou, Lijuan

    2014-03-28

    Highlights: • HOXB9 is overexpressed in gliomas. • HOXB9 over expression had shorter survival time than down expression in gliomas. • HOXB9 stimulated the proliferation, migration and sphere formation of glioma cells. • Activation of TGF-β1 contributed to HOXB9-induced oncogenic activities. - Abstract: Glioma is the leading cause of deaths related to tumors in the central nervous system. The mechanisms of gliomagenesis remain elusive to date. Homeobox B9 (HOXB9) has a crucial function in the regulation of gene expression and cell survival, but its functions in glioma formation and development have yet to be elucidated. This study showed that HOXB9 expression in glioma tissues was significantly higher than that in nontumor tissues. Higher HOXB9 expression was also significantly associated with advanced clinical stage in glioma patients. HOXB9 overexpression stimulated the proliferation, migration, and sphere formation of glioma cells, whereas HOXB9 knockdown elicited an opposite effect. HOXB9 overexpression also increased the tumorigenicity of glioma cells in vivo. Moreover, the activation of transforming growth factor-β1 contributed to HOXB9-induced oncogenic activities. HOXB9 could be used as a predictable biomarker to be detected in different pathological and histological subtypes in glioma for diagnosis or prognosis.

  6. Uptake of Host Cell Transforming Growth Factor-β by Trypanosoma cruzi Amastigotes in Cardiomyocytes

    PubMed Central

    Waghabi, Mariana C.; Keramidas, Michelle; Bailly, Sabine; Degrave, Wim; Mendonça-Lima, Leila; Soeiro, Maria de Nazaré C.; Meirelles, Maria de Nazareth L.; Paciornik, Sidnei; Araújo-Jorge, Tania C.; Feige, Jean-Jacques

    2005-01-01

    The cytokine transforming growth factor-β (TGF-β) plays various functions in the control of Trypanosoma cruzi infectivity and in the progression of Chagas’ disease. When we immunostained T. cruzi-infected cardiomyocytes (after either in vivo or in vitro infections) for TGF-β, we observed stronger immunoreactivity in parasites than in host cells. TGF-β immunoreactivity evolved during parasite cycle progression, with intense staining in amastigotes versus very faint staining in trypomastigotes. TGF-β was present on the surface of amastigotes, in the flagellar pocket, and in intraparasitic vesicles as revealed by electron microscopy. However, no ortholog TGF-β gene could be identified in the genome of T. cruzi by in silico analysis or by extensive polymerase chain reaction and reverse transcriptase-polymerase chain reaction studies. Immunoreactive TGF-β was most probably taken up by the parasite from the host cell cytoplasm because such an internalization process of biotinylated TGF-β could be observed in axenic amastigotes in vitro. These observations represent the first example of a novel mechanism by which a primitive unicellular protozoan can use host cell TGF-β to control its own intracellular life cycle. PMID:16192635

  7. Novel alleles of the transforming growth factor β-1 regulatory region and exon 1.

    PubMed

    Arrieta-Bolaños, E; Madrigal, J A; Shaw, B E

    2015-06-01

    Transforming growth factor β-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiologic and pathogenic processes with pleiotropic effects. Regulatory activity for this gene has been shown for 3.0 kb between positions -2665 and +423 from its translational start site. At least 17 TGFB1 regulatory region and exon 1 alleles have been defined on the basis of 18 polymorphic sites. Polymorphisms in TGFB1's regulatory region have been associated with differential levels of expression of this cytokine and to genetic risk in cancer and transplantation. In this report, we present 19 novel TGFB1 regulatory region and exon 1 alleles: p018-p036. Amplification of TGFB1's regulatory region was performed with an in-house protocol, and novel alleles were defined by either allele-specific amplification and/or molecular cloning of the amplicons, followed by sequencing in isolation. Three of these novel alleles (p018, p019, and p020) are shown to be formed by novel combinations of the aforementioned known polymorphic positions. Another 16 novel alleles are shown to carry additional known and unknown single-nucleotide polymorphisms. Polymorphism in TGFB1's regulatory region could have an impact on important processes, including embryogenesis, hematopoiesis, carcinogenesis, angiogenesis, fibrosis, immune responses, and transplantation, making its characterization necessary. PMID:25808355

  8. Transforming growth factor β1 inhibition protects from noise-induced hearing loss

    PubMed Central

    Murillo-Cuesta, Silvia; Rodríguez-de la Rosa, Lourdes; Contreras, Julio; Celaya, Adelaida M.; Camarero, Guadalupe; Rivera, Teresa; Varela-Nieto, Isabel

    2015-01-01

    Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor β (TGF-β) is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-β as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss (NIHL), we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-β1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-β1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-β1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage. PMID:25852546

  9. Transforming Growth Factor-β Signaling Guides the Differentiation of Innate Lymphoid Cells in Salivary Glands.

    PubMed

    Cortez, Victor S; Cervantes-Barragan, Luisa; Robinette, Michelle L; Bando, Jennifer K; Wang, Yaming; Geiger, Theresa L; Gilfillan, Susan; Fuchs, Anja; Vivier, Eric; Sun, Joe C; Cella, Marina; Colonna, Marco

    2016-05-17

    The signals guiding differentiation of innate lymphoid cells (ILCs) within tissues are not well understood. Salivary gland (SG) ILCs as well as liver and intestinal intraepithelial ILC1 have markers that denote tissue residency and transforming growth factor-β (TGF-β) imprinting. We deleted Tgfbr2 in cells expressing the ILC and NK marker NKp46 and found that SG ILCs were reduced in number. They lost distinct tissue markers, such as CD49a, and the effector molecules TRAIL and CD73. Expression of the transcription factor Eomes, which promotes NK cell differentiation, was elevated. Conversely, Eomes deletion in NKp46(+) cells enhanced TGF-β-imprinting of SG ILCs. Thus, TGF-β induces SG ILC differentiation by suppressing Eomes. TGF-β acted through a JNK-dependent, Smad4-independent pathway. Transcriptome analysis demonstrated that SG ILCs had characteristic of both NK cells and ILC1. Finally, TGF-β imprinting of SG ILCs was synchronized with SG development, highlighting the impact of tissue microenvironment on ILC development. PMID:27156386

  10. Transforming growth factor β1 inhibition protects from noise-induced hearing loss.

    PubMed

    Murillo-Cuesta, Silvia; Rodríguez-de la Rosa, Lourdes; Contreras, Julio; Celaya, Adelaida M; Camarero, Guadalupe; Rivera, Teresa; Varela-Nieto, Isabel

    2015-01-01

    Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor β (TGF-β) is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-β as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss (NIHL), we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-β1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-β1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-β1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage. PMID:25852546

  11. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway

    PubMed Central

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  12. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway.

    PubMed

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  13. Targeting the Transforming Growth Factor-β Signaling Pathway in Human Cancer

    PubMed Central

    Nagaraj, Nagathihalli S

    2009-01-01

    The transforming growth factor-β (TGF-β) signaling pathway plays a pivotal role in diverse cellular processes. TGF-β switches its role from tumor suppressor in normal or dysplastic cells to a tumor promoter in advanced cancers. It is widely believed that Smad-dependent pathway is involved in TGF-β tumor suppressive functions, whereas activation of Smad-independent pathways coupled with the loss of tumor suppressor functions of TGF-β is important for its pro-oncogenic functions. TGF-β signaling has been considered as a very suitable therapeutic target. The discovery of oncogenic actions of TGF-β has generated a great deal of enthusiasm for developing TGF-β signaling inhibitors for the treatment of cancer. The challenge is to identify the group of patients where targeted tumors are not only refractory to TGF-β-induced tumor suppressor functions but also responsive to tumor promoting effects of TGF-β. TGF-β pathway inhibitors including small and large molecules have now entered clinical trials. Preclinical studies with these inhibitors have shown promise in a variety of different tumor models. Here we emphasize on the mechanisms of signaling and specific targets of the TGF-β pathway that are critical effectors of tumor progression and invasion. This report also focuses on the therapeutic intervention of TGF-β signaling in human cancers. PMID:20001556

  14. Roles of transforming growth factor β in hyperoxia-induced lung injury.

    PubMed

    Liu, Y; Mao, Y F; Zheng, J; Liu, K; Han, C H; Liu, W W

    2016-01-01

    Hyperoxia induced lung injury (HILI) refers to the acute lung injury secondary to prolonged exposure to hyperoxia at elevated partial pressure. With the advent of efficient systems for delivery of high concentrations of oxygen in hospitals, the population at risk for this condition has been markedly increased. Although numerous studies have been conducted to investigate the pathogenesis of HILI, the specific mechanism is still poorly understood and some hypotheses have been proposed. Transforming growth factor β (TGF-β) is a secreted protein that controls proliferation, cellular differentiation and other functions in most cells and is a type of cytokine that plays a role in many diseases. In this mini-review, we summarize the role of TGF-β in HILI according to its relationships with reactive oxygen species (ROS), pro-inflammatory cytokines, cell apoptosis and pulmonary fibrosis. We hope it may help the understanding of pathogenesis of HILI and provide a greater understanding for the target therapy of HILI. PMID:27416690

  15. Role of Ras signaling in the induction of snail by transforming growth factor-beta.

    PubMed

    Horiguchi, Kana; Shirakihara, Takuya; Nakano, Ayako; Imamura, Takeshi; Miyazono, Kohei; Saitoh, Masao

    2009-01-01

    The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in some tumor cells. Here, we demonstrate the molecular mechanism whereby Snail, a key regulator of EMT, is induced by TGF-beta in tumor cells. Snail induction by TGF-beta was highly dependent on cooperation with active Ras signals, and silencing of Ras abolished Snail induction by TGF-beta in pancreatic cancer Panc-1 cells. Transfection of constitutively active Ras into HeLa cells led to induction of Snail by TGF-beta, while representative direct targets of TGF-beta, including Smad7 and PAI-1, were not affected by Ras signaling. Using mitogen-activated protein kinase inhibitors or Smad3 or Smad2 mutants, we found that phosphorylation at the linker region of Smad2/3 was not required for the induction of Snail by TGF-beta. Taken together, these findings indicate that Ras and TGF-beta-Smad signaling selectively cooperate in the induction of Snail, which occurs in a Smad-dependent manner, but independently of phosphorylation at the linker region of R-Smads by Ras signaling. PMID:19010789

  16. Effect of transforming growth factor-β3 on mono and multilayer chondrocytes.

    PubMed

    Sefat, Farshid; Youseffi, Mansour; Khaghani, Seyed Ali; Soon, Chin Fhung; Javid, Farideh

    2016-07-01

    Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface. PMID:27108397

  17. Immunocytochemical Localization of Latent Transforming Growth Factor-B1 Activation by Stimulated Macrophages

    SciTech Connect

    Chong, Hyonkyong; Vodovotz, Yoram; Cox, G.W.; Barcellos-Hoff, M.H.

    1998-09-22

    Transforming growth factor-{beta}1 (TGF-{beta}) is secreted in a latent form consisting of mature TGF-{beta} noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-{beta} from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-{beta} action. We have identified two events associated with latent TGF-{beta} (LTGF-{beta}) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-{beta} concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-{gamma} and lipopolysaccharide reportedly activate LTGF-{beta} via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-{beta} activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-{beta} epitopes. The induction of TGF-{beta} immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-{beta} activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-{beta} and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-{beta} activation provides an important tool for studies of its regulation.

  18. Transforming growth factor-beta 1 in experimental autoimmune neuritis. Cellular localization and time course.

    PubMed Central

    Kiefer, R.; Funa, K.; Schweitzer, T.; Jung, S.; Bourde, O.; Toyka, K. V.; Hartung, H. P.

    1996-01-01

    Experimental autoimmune neuritis (EAN) is a monophasic inflammatory disorder of the peripheral nervous system that resolves spontaneously by molecular mechanisms as yet unknown. We have investigated whether the immunosuppressive cytokine transforming growth factor-beta 1 (TGF-beta 1) might be endogenously expressed in the peripheral nervous system of Lewis rats with actively induced and adoptive transfer EAN. TGF-beta 1 mRNA was upregulated to high levels in sensory and motor roots, spinal ganglia, and sciatic nerve as revealed by quantitative Northern blot analysis and in situ hybridization histochemistry, with peak levels just preceding the first signs of clinical recovery. TGF-beta 1 mRNA was localized to scattered round cells and dense cellular infiltrates, but only rarely to Schwann cell profiles. Double labeling studies revealed macrophages and subpopulations of T cells as the major cellular source of TGF-beta 1 mRNA. TGF-beta 1 protein was visualized immunocytochemically and localized to infiltrating mononuclear cells with peak expression around the same time as mRNA, in addition to some constitutive expression in axons and Schwann cells. Our studies suggest that the spontaneous recovery observed in Lewis rat EAN might be mediated by the endogenous elaboration of TGF-beta 1 within the peripheral nerve, and that macrophages might control their own cytotoxicity by expressing TGF-beta 1. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8546208

  19. Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages

    NASA Technical Reports Server (NTRS)

    Chong, H.; Vodovotz, Y.; Cox, G. W.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

    1999-01-01

    Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

  20. Elevated Transforming Growth Factor β1 in Plasma of Primary Open-Angle Glaucoma Patients

    PubMed Central

    Kuchtey, John; Kunkel, Jessica; Burgess, L. Goodwin; Parks, Megan B.; Brantley, Milam A.; Kuchtey, Rachel W.

    2014-01-01

    Purpose. To test the hypothesis that primary open-angle glaucoma (POAG) patients have a systemic elevation of transforming growth factor β1 (TGFβ1). Methods. Plasma was prepared from blood samples drawn from patients of the Vanderbilt Eye Institute during clinic visits. Concentrations of total TGFβ1 and thrombospondin-1 (TSP1) in plasma were determined by ELISA. Statistical significance of differences between POAG and control samples was evaluated by Mann-Whitney test. Regression analysis was used to evaluate correlations between plasma TGFβ1 and patient age and between plasma TGFβ1 and TSP1. Results. Plasma samples were obtained from 148 POAG patients and 150 controls. Concentration of total TGFβ1 in the plasma of POAG patients (median = 3.25 ng/mL) was significantly higher (P < 0.0001) than in controls (median = 2.46 ng/mL). Plasma TGFβ1 was not correlated with age of patient (P = 0.17). Thrombospondin-1 concentration was also significantly higher (P < 0.0001) in POAG patients (median = 0.774 μg/mL) as compared to controls (median = 0.567 μg/mL). Plasma total TGFβ1 and TSP1 concentrations were linearly correlated (P < 0.0001). Conclusions. Plasma samples from POAG patients display elevated total TGFβ1 compared to controls, consistent with elevated systemic TGFβ1 in POAG patients. PMID:25061114

  1. Requirement of HDAC6 for Transforming Growth Factor-β1-induced Epithelial-Mesenchymal Transition*

    PubMed Central

    Shan, Bin; Yao, Tso-pang; Nguyen, Hong T.; Zhuo, Ying; Levy, Dawn R.; Klingsberg, Ross C.; Tao, Hui; Palmer, Michael L.; Holder, Kevin N.; Lasky, Joseph A.

    2008-01-01

    The aberrant expression of transforming growth factor (TGF)-β1 in the tumor microenvironment and fibrotic lesions plays a critical role in tumor progression and tissue fibrosis by inducing epithelial-mesenchymal transition (EMT). EMT promotes tumor cell motility and invasiveness. How EMT affects motility and invasion is not well understood. Here we report that HDAC6 is a novel modulator of TGF-β1-induced EMT. HDAC6 is a microtubule-associated deacetylase that predominantly deacetylates nonhistone proteins, including α-tubulin, and regulates cell motility. We showed that TGF-β1-induced EMT is accompanied by HDAC6-dependent deacetylation of α-tubulin. Importantly, inhibition of HDAC6 by small interfering RNA or the small molecule inhibitor tubacin attenuated the TGF-β1-induced EMT markers, such as the aberrant expression of epithelial and mesenchymal peptides, as well as the formation of stress fibers. Reduced expression of HDAC6 also impaired the activation of SMAD3 in response to TGF-β1. Conversely, inhibition of SMAD3 activation substantially impaired HDAC6-dependent deacetylation of α-tubulin as well as the expression of EMT markers. These findings reveal a novel function of HDAC6 in EMT by intercepting the TGF-β-SMAD3 signaling cascade. Our results identify HDAC6 as a critical regulator of EMT and a potential therapeutic target against pathological EMT, a key event for tumor progression and fibrogenesis. PMID:18499657

  2. Transforming growth factor beta (TGF-β) isomers influence cell detachment of MG-63 bone cells.

    PubMed

    Sefat, Farshid; Khaghani, Seyed Ali; Nejatian, Touraj; Genedy, Mohammed; Abdeldayem, Ali; Moghaddam, Zoha Salehi; Denyer, Morgan C T; Youseffi, Mansour

    2015-12-01

    Bone repair and wound healing are modulated by different stimuli. There is evidence that Transforming Growth Factor-beta (TGF-β) super-family of cytokines have significant effects on bone structure by regulating the replication and differentiation of chondrocytes, osteoblasts and osteoclasts. There is also significant evidence that interactions with extracellular matrix molecules influence cell behaviour. In this study cell surface attachment was examined via a trypsinization assay using various TGF-β isomers in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. Three TGF-β isomers (TGF-β1, 2 and 3), four combined forms (TGF-β(1+2), TGF-β(1+3), TGF-β(2+3) and TGF-β(1+2+3)) along with four different controls (BSA, HCl, BSA/HCl and negative control) were investigated in this study. The results indicated that treatment with TGF-β1, 2, 3 and HCl decreased cell attachment, however, this effect was significantly greater in the case of TGF-β3 (p<0.001) indicating perhaps that TGF-β3 does not act alone in cell detachment, but instead functions synergistically with signalling pathways that are dependent on the availability of hydrogen ions. Widefield Surface Plasmon Resonance (WSPR) microscope was also used to investigate cell surface interactions. PMID:26372305

  3. Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function

    SciTech Connect

    Hattersley, G.; Chambers, T.J. )

    1991-02-01

    Transforming growth factor (TGF) beta 1 is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-beta 1 on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an EC50 of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-beta 1 strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-beta 1 to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-beta 1 in osteoclast generation.

  4. Transforming Growth Factor {beta} Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors.

    PubMed

    Wrighton, Katharine H; Lin, Xia; Yu, Paul B; Feng, Xin-Hua

    2009-04-10

    Transforming growth factor-beta (TGFbeta) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGFbeta signaling is mediated via the TbetaRI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFbeta can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGFbeta can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFbeta/activin/nodal (ALK4/5/7) and BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFbeta induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGFbeta-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell type-specific manner. PMID:19224917

  5. MTMR4 attenuates transforming growth factor beta (TGFbeta) signaling by dephosphorylating R-Smads in endosomes.

    PubMed

    Yu, Junjing; Pan, Lei; Qin, Xincheng; Chen, Hua; Xu, Youli; Chen, Yeguang; Tang, Hong

    2010-03-12

    Homeostasis of Smad phosphorylation at its C-terminal SXS motif is essential for transforming growth factor beta (TGFbeta) signaling. Whereas it is known that TGFbeta signaling can be terminated by phosphatases, which dephosphorylate R-Smads in the nucleus, it is unclear whether there are any cytoplasmic phosphatase(s) that can attenuate R-Smad phosphorylation and nuclear translocation. Here we demonstrate that myotubularin-related protein 4 (MTMR4), a FYVE domain-containing dual-specificity protein phosphatase (DSP), attenuates TGFbeta signaling by reducing the phosphorylation level of R-Smads in early endosomes. Co-immunoprecipitation experiments showed that endogenous MTMR4 interacts with phosphorylated R-Smads, and that this interaction is correlated with dephosphorylation of R-Smads. Further analysis showed that overexpression of MTMR4 resulted in the sequestration of activated Smad3 in the early endosomes, thus reducing its nuclear translocation. However, both point mutations at the conserved catalytic site of the phosphatase (MTMR4-C407S) and small interference RNA of endogenous Mtmr4 expression led to sustained Smad3 activation. This work therefore suggests that MTMR4 plays an important role in preventing the overactivation of TGFbeta signaling by dephosphorylating activated R-Smads that have been trafficked to early endosomes. PMID:20061380

  6. Hepatic transforming growth factor-β 1 stimulated clone-22 D1 controls systemic cholesterol metabolism☆

    PubMed Central

    Jäger, Julia; Greiner, Vera; Strzoda, Daniela; Seibert, Oksana; Niopek, Katharina; Sijmonsma, Tjeerd P.; Schäfer, Michaela; Jones, Allan; De Guia, Roldan; Martignoni, Marc; Dallinga-Thie, Geesje M.; Diaz, Mauricio B.; Hofmann, Thomas G.; Herzig, Stephan

    2014-01-01

    Disturbances in lipid homeostasis are hallmarks of severe metabolic disorders and their long-term complications, including obesity, diabetes, and atherosclerosis. Whereas elevation of triglyceride (TG)-rich very-low-density lipoproteins (VLDL) has been identified as a risk factor for cardiovascular complications, high-density lipoprotein (HDL)-associated cholesterol confers atheroprotection under obese and/or diabetic conditions. Here we show that hepatocyte-specific deficiency of transcription factor transforming growth factor β 1-stimulated clone (TSC) 22 D1 led to a substantial reduction in HDL levels in both wild-type and obese mice, mediated through the transcriptional down-regulation of the HDL formation pathway in liver. Indeed, overexpression of TSC22D1 promoted high levels of HDL cholesterol in healthy animals, and hepatic expression of TSC22D1 was found to be aberrantly regulated in disease models of opposing energy availability. The hepatic TSC22D1 transcription factor complex may thus represent an attractive target in HDL raising strategies in obesity/diabetes-related dyslipidemia and atheroprotection. PMID:24634828

  7. Cardiac fibroblasts are predisposed to convert into myocyte phenotype: Specific effect of transforming growth factor. beta

    SciTech Connect

    Eghbali, M.; Tomek, R.; Woods, C.; Bhambi, B. )

    1991-02-01

    Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), acquire certain myocyte-specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-{beta}{sub 1}, (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-{beta}{sub 1} became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-{beta}{sub 1}, cell proliferation (as measured by ({sup 3}H)thymidine incorporation) was moderately increased. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-{beta}{sub 1} in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. The findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-{beta}{sub 1} seems to be a specific inducer of such conversion.

  8. Association of Transforming Growth Factor Alpha Polymorphisms with Nonsyndromic Cleft Lip and Palate in Iranian Population

    PubMed Central

    Ebadifar, Asghar; Hamedi, Roya; Khorram Khorshid, Hamid Reza; Saliminejad, Kioomars; Kamali, Koorosh; Aghakhani Moghadam, Fatemeh; Esmaeili Anvar, Nazanin; Ameli, Nazilla

    2015-01-01

    Background: Cleft lip with or without cleft palate (CL/P) is one of the most common congenital anomalies and the etiology of orofacial clefts is multifactorial. Transforming growth factor alpha (TGFA) is expressed at the medial edge epithelium of fusing palatal shelves during craniofacial development. In this study, the association of two important TGFA gene polymorphisms, BamHI (rs11466297) and RsaI (rs3732248), with CL/P was evaluated in an Iranian population. Methods: The frequencies of BamHI and RsaI variations were determined in 105 unrelated Iranian subjects with nonsyndromic CL/P and 218 control subjects using PCR and RFLP methods, and the results were compared with healthy controls. A p-value of <0.05 was considered statistically significant. Results: The BamHI AC genotype was significantly higher (p=0.016) in the patients (12.4%) than the control group (5.0%). The BamHI C allele was significantly higher (p=0.001; OR=3.4, 95% CI: 1.6–7.4) in the cases (8.0%) compared with the control group (2.5%). Conclusion: Our study showed that there was an association between the TGFA BamHI variation and nonsyndromic CL/P in Iranian population. PMID:26605011

  9. Transforming growth factor-beta. Murine glomerular receptors and responses of isolated glomerular cells.

    PubMed Central

    MacKay, K; Striker, L J; Stauffer, J W; Doi, T; Agodoa, L Y; Striker, G E

    1989-01-01

    Proliferation of resident glomerular cells and the accumulation of mesangial matrix are histologic abnormalities which are observed in the course of many progressive glomerular diseases. We explored the potential regulatory effects of transforming growth factor-beta (TGF-beta) on these processes. We found that cultured mouse glomerular endothelial, mesangial, and epithelial cells as well as isolated intact rat glomeruli possess high-affinity receptors for TGF-beta. We also found that, although TGF-beta consistently inhibited the proliferation of glomerular endothelial and epithelial cells, it acted as a bifunctional regulator of mesangial cell proliferation. TGF-beta significantly increased the production of collagen and fibronectin by glomerular mesangial cells whereas only fibronectin production was augmented in glomerular epithelial cells. The presence of TGF-beta receptors on intact glomeruli and on each glomerular cell type and the demonstrated responsiveness of these cells to TGF-beta combine to suggest that potentially important interactions may occur between resident glomerular cells and TGF-beta in vivo. Images PMID:2539392

  10. Integration of sexual trauma in a religious narrative: Transformation, resolution and growth among contemplative nuns

    PubMed Central

    Littlewood, Roland; Leavey, Gerard

    2013-01-01

    The psychological consequences of sexual abuse are generally serious and enduring, particularly when the perpetrator is known and trusted by the survivor. This paper explores the experiences of five contemplative nuns who were sexually abused by priests and the spiritual journeys that followed. In the context of an ethnographic study of contemplative practice, participant observation and in-depth interviews were used to examine the ways that the nuns sought to make sense of their experiences through a long process of solitary introspection. The pursuit of meaning was shaped by religious beliefs relating to forgiveness, sacrifice, and salvation. Thus, trauma was transformed into a symbolic religious narrative that shaped their sense of identity. They were able to restructure core beliefs and to manage their current relationships with priests more securely. They described regaining their spiritual well-being in ways that suggest a form of posttraumatic spiritual growth. We conclude by discussing the findings in the light of the existing literature on the interaction of trauma and spirituality. PMID:23296289

  11. Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO)

    SciTech Connect

    Shiang, R. ); Lidral, A.C.; Ardinger, H.H.; Murray, J.C.; Romitti, P.A.; Munger, R.G.; Buetow, K.H.

    1993-10-01

    Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. The authors carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3[prime] untranslated region of the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using X[sup 2] analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3[prime] untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P. 46 refs., 3 figs., 3 tabs.

  12. Generation of Mice With a Conditional Allele for the Transforming Growth Factor Beta3 Gene

    PubMed Central

    Doetschman, Thomas; Georgieva, Teodora; Li, Hongqi; Reed, Thomas D.; Grisham, Christina; Friel, Jacqueline; Estabrook, Mark A.; Gard, Connie; Sanford, L.P.; Azhar, Mohamad

    2013-01-01

    Summary The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFβ3 (Tgfb3) encodes one of the three ligands for TGF b receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3−/− mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3−/− mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3cko allele will now enable studies of TGFβ3 function in different cell or tissue types in embryonic development and during adulthood. genesis 50:59-66, 2012. PMID:22223248

  13. Integration of sexual trauma in a religious narrative: transformation, resolution and growth among contemplative nuns.

    PubMed

    Durà-Vilà, Glòria; Littlewood, Roland; Leavey, Gerard

    2013-02-01

    The psychological consequences of sexual abuse are generally serious and enduring, particularly when the perpetrator is known and trusted by the survivor. This paper explores the experiences of five contemplative nuns who were sexually abused by priests and the spiritual journeys that followed. In the context of an ethnographic study of contemplative practice, participant observation and in-depth interviews were used to examine the ways that the nuns sought to make sense of their experiences through a long process of solitary introspection. The pursuit of meaning was shaped by religious beliefs relating to forgiveness, sacrifice, and salvation. Thus, trauma was transformed into a symbolic religious narrative that shaped their sense of identity. They were able to restructure core beliefs and to manage their current relationships with priests more securely. They described regaining their spiritual well-being in ways that suggest a form of posttraumatic spiritual growth. We conclude by discussing the findings in the light of the existing literature on the interaction of trauma and spirituality. PMID:23296289

  14. A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of Insulin-like Growth Factor II*S⃞

    PubMed Central

    Alvino, Clair L.; McNeil, Kerrie A.; Ong, Shee Chee; Delaine, Carlie; Booker, Grant W.; Wallace, John C.; Whittaker, Jonathan; Forbes, Briony E.

    2009-01-01

    Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four “site 1” and six “site 2” analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val43, Phe28, and Val14 (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln18 affects only IGF-1R and not IR binding. Alanine substitutions at Glu12, Asp15, Phe19, Leu53, and Glu57 analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu12, Phe19, Leu53, and Glu57 that potentially engages the IGF-1R at one or more of the FnIII domains. PMID:19139090

  15. Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

    PubMed

    Baumkötter, Frederik; Schmidt, Nadine; Vargas, Carolyn; Schilling, Sandra; Weber, Rebecca; Wagner, Katja; Fiedler, Sebastian; Klug, Wilfried; Radzimanowski, Jens; Nickolaus, Sebastian; Keller, Sandro; Eggert, Simone; Wild, Klemens; Kins, Stefan

    2014-08-13

    Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function. PMID:25122912

  16. Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation.

    PubMed

    Graham, C H; Lysiak, J J; McCrae, K R; Lala, P K

    1992-04-01

    We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti

  17. Transforming Growth Factor Beta 1 Augments Calvarial Defect Healing and Promotes Suture Regeneration

    PubMed Central

    Shakir, Sameer; MacIsaac, Zoe M.; Naran, Sanjay; Smith, Darren M.; Bykowski, Michael R.; Cray, James J.; Craft, Timothy K.; Wang, Dan; Weiss, Lee; Campbell, Phil G.; Mooney, Mark P.; Losee, Joseph E.

    2015-01-01

    Background: Repair of complex cranial defects is hindered by a paucity of appropriate donor tissue. Bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 1 (TGFβ1) have been shown separately to induce bone formation through physiologically distinct mechanisms and potentially improve surgical outcome for cranial defect repair by obviating the need for donor tissue. We hypothesize that a combination of BMP2 and TGFβ1 would improve calvarial defect healing by augmenting physiologic osteogenic mechanisms. Methods/Results: Coronal suturectomies (3×15 mm) were performed in 10-day-old New Zealand White rabbits. DermaMatrix™ (3×15mm) patterned with four treatments (vehicle, 350 ng BMP2, 200 ng TGFβ1, or 350 ng BMP2+200 ng TGFβ1) was placed in suturectomy sites and rabbits were euthanized at 6 weeks of age. Two-dimensional (2D) defect healing, bone volume, and bone density were quantified by computed tomography. Regenerated bone was qualitatively assessed histologically. One-way analysis of variance revealed significant group main effects for all bone quantity measures. Analysis revealed significant differences in 2D defect healing, bone volume, and bone density between the control group and all treatment groups, but no significant differences were detected among the three growth factor treatment groups. Qualitatively, TGFβ1 treatment produced bone with morphology most similar to native bone. TGFβ1-regenerated bone contained a suture-like tissue, growing from the lateral edge of the defect margin toward the midline. Unique to the BMP2 treatment group, regenerated bone contained lacunae with chondrocytes, demonstrating the presence of endochondral ossification. Conclusions/Significance: Total healing in BMP2 and TGFβ1 treatment groups is not significantly different. The combination of BMP2+TGFβ1 did not significantly increase bone healing compared with treatment with BMP2 or TGFβ1 alone postoperatively at 4 weeks. We highlight the

  18. Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary.

    PubMed

    Bristol, Sarah K; Woodruff, Teresa K

    2004-03-01

    Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2

  19. Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

    NASA Technical Reports Server (NTRS)

    Perrone, Carmen E.; Fenwick-Smith, Daniela; Vandenburgh, Herman H.

    1995-01-01

    Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

  20. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    SciTech Connect

    Nakai, A.; Hirayama, C.; Ohtsuka, K.; Hirayoshi, K.; Nagata, K. )

    1990-06-01

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with (35S)methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of (32P)orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

  1. Assignment of human transforming growth factor-{beta} type I and type III receptor genes (TGFBR1 and TGFBR3) to 9q33-q34 and 1p32-p33, respectively

    SciTech Connect

    Johnson, D.W.; Qumsiyeh, M.; Marchuk, D.A.; Benkhalifa, M.

    1995-07-20

    Transforming growth factor-{Beta} (TGF-{beta}) is a multifunctional cytokine, known to modulate several tissue development and repair processes, including cell differentiation, cell cycle progression, cellular migration, adhesion, and extracellular matrix production. The TGF-{beta} receptors and cell surface binding proteins mediate the diverse effects of TGF-{beta}. An endothelial cell-specific TGF-{beta} binding protein, endoglin, is mutated in hereditary hemorrhagic telangiectasia type 1, an autosomal dominant disorder of vascular dysplasia. Mutations in other TGF-{beta} binding protein genes may also lead to disease. We have used PCR with a cell hybrid DNA panel, and fluorescence in situ chromosomal hybridization (FISH), to localize two other TGF-{beta} receptor genes. These are the TGF-{beta} type I and type II receptors (also known as ALK-5 and betaglycan, respectively). The corresponding gene loci are designated TGFBR1 and TGFBR3. 10 refs., 1 fig., 1 tab.

  2. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    SciTech Connect

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. )

    1991-01-01

    Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

  3. Insulin-like growth factor binding protein production and regulation in fetal rat lung cells.

    PubMed

    Price, W A; Moats-Staats, B M; D'Ercole, A J; Stiles, A D

    1993-04-01

    Insulin-like growth factor binding proteins (IGFBPs) are expressed in lung from early in gestation and may modulate IGF-stimulated fetal lung cell proliferation and/or differentiation. To begin to define IGFBP production and regulation in lung cells during development, we prepared primary cultures of 19 day gestation fetal rat lung fibroblasts and epithelial cells and identified IGFBPs secreted into medium. Ligand blot analysis of conditioned media (CM) from both cell types demonstrated IGFBP bands of approximately 39,000-45,000, 32,000, 24,000, and 22,000 M(r). These migration characteristics allowed the identification of the 39,000-45,000 M(r) bands as IGFBP-3 and the 24,000 M(r) band as IGFBP-4, while Western immunoblot analyses localized IGFBP-2 to the 32,000 M(r) band and IGFBP-5 to the 22,000 M(r) band. Polymerase chain reaction amplification of cDNAs generated by reverse transcription of fibroblast and epithelial cell RNA using specific oligodeoxynucleotide primers for IGFBPs 1 through 6, demonstrated the presence of amplified products for IGFBP-2, -3, -4, -5, and -6. In both cell types, IGFBP-2 and -3 production was sustained during 48 h of incubation in serum-free medium, whereas IGFBP-4 abundance increased only during the first 6 to 12 h of incubation. CM from fibroblasts and epithelial cells plated at low densities contained a high abundance of IGFBP-2 per microgram cellular DNA compared with cells at higher densities. In contrast, IGFBP-3 and -4 abundance normalized to cell DNA did not change with differing cell densities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7682822

  4. Mxi1 regulates cell proliferation through insulin-like growth factor binding protein-3

    SciTech Connect

    Ko, Je Yeong; Yoo, Kyung Hyun; Lee, Han-Woong; Park, Jong Hoon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Mxi1 regulates cell proliferation. Black-Right-Pointing-Pointer Expression of IGFBP-3 is regulated by Mxi1. Black-Right-Pointing-Pointer Inactivation of Mxi1 reduces IGFBP-3 expression in vitro and in vivo. -- Abstract: Mxi1, a member of the Myc-Max-Mad network, is an antagonist of the c-Myc oncogene and is associated with excessive cell proliferation. Abnormal cell proliferation and tumorigenesis are observed in organs of Mxi1-/- mice. However, the Mxi1-reltaed mechanism of proliferation is unclear. The present study utilized microarray analysis using Mxi1 mouse embryonic fibroblasts (MEFs) to identify genes associated with cell proliferation. Among these genes, insulin-like growth factor binding protein-3 (IGFBP-3) was selected as a candidate gene for real-time PCR to ascertain whether IGFBP-3 expression is regulated by Mxi1. Expression of IGFBP-3 was decreased in Mxi1-/- MEFs and Mxi1-/- mice, and the gene was regulated by Mxi1 in Mxi1 MEFs. Furthermore, proliferation pathways related to IGFBP-3 were regulated in Mxi1-/- mice compared to Mxi1+/+ mice. To determine the effect of Mxi1 inactivation on the induction of cell proliferation, a proliferation assay is performed in both Mxi1 MEFs and Mxi1 mice. Cell viability was regulated by Mxi1 in Mxi1 MEFs and number of PCNA-positive cells was increased in Mxi1-/- mice compared to Mxi1+/+ mice. Moreover, the IGFBP-3 level was decreased in proliferation defect regions in Mxi1-/- mice. The results support the suggestion that inactivation of Mxi1 has a positive effect on cell proliferation by down-regulating IGFBP-3.

  5. Thyroid Hormone Receptor Binds to a Site in the Rat Growth Hormone Promoter Required for Induction by Thyroid Hormone

    NASA Astrophysics Data System (ADS)

    Koenig, Ronald J.; Brent, Gregory A.; Warne, Robert L.; Reed Larsen, P.; Moore, David D.

    1987-08-01

    Transcription of the rat growth hormone (rGH) gene in pituitary cells is increased by addition of thyroid hormone (T3). This induction is dependent on the presence of specific sequences just upstream of the rGH promoter. We have partially purified T3 receptor from rat liver and examined its interaction with these rGH sequences. We show here that T3 receptor binds specifically to a site just upstream of the basal rGH promoter. This binding site includes two copies of a 7-base-pair direct repeat, the centers of which are separated by 10 base pairs. Deletions that specifically remove the T3 receptor binding site drastically reduce response to T3 in transient transfection experiments. These results demonstrate that T3 receptor can recognize specific DNA sequences and suggest that it can act directly as a positive transcriptional regulatory factor.

  6. Natural product (–)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1

    PubMed Central

    Lan, Lan; Appelman, Carl; Smith, Amber R.; Yu, Jia; Larsen, Sarah; Marquez, Rebecca T.; Liu, Hao; Wu, Xiaoqing; Gao, Philip; Roy, Anuradha; Anbanandam, Asokan; Gowthaman, Ragul; Karanicolas, John; De Guzman, Roberto N.; Rogers, Steven; Aubé, Jeffrey; Ji, Min; Cohen, Robert S.; Neufeld, Kristi L.; Xu, Liang

    2015-01-01

    Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21WAF-1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (–)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (–)-gossypol with the RNA binding pocket of MSI1. We further showed that (–)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (–)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (–)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy. PMID:25933687

  7. Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures.

    PubMed

    Tiwari, Rajesh Kumar; Trivedi, Mala; Guang, Zhang Chun; Guo, Guang-Qin; Zheng, Guo-Chang

    2007-02-01

    Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A(4)GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B(5) medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 (T +/L and T +/R) and the GM2 (T +/L and T -/R DNA) type clones in considerably higher amount whether as two T -/L but T +/R callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B(5) vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B(5) media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the T(L)-DNA and T(R)-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed. PMID:16972092

  8. Homodimerization Controls the Fibroblast Growth Factor 9 Subfamily's Receptor Binding and Heparan Sulfate-Dependent Diffusion in the Extracellular Matrix

    SciTech Connect

    Kalinina, J.; Byron, S; Makarenkova, H; Olsen, S; Eliseenkova, A; Larochelle, W; Dhanabal, M; Blais, S; Mohammadi, M; et. al.

    2009-01-01

    Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.

  9. A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

    PubMed

    Mercado, R; Hernández, J

    1992-09-18

    Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis. PMID:1424085

  10. Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures

    SciTech Connect

    Orcel, P.; Bielakoff, J.; De Vernejoul, M.C. )

    1990-02-01

    Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes.

  11. Transforming growth factor-beta1 upregulates myostatin expression in mouse C2C12 myoblasts.

    PubMed

    Budasz-Rwiderska, M; Jank, M; Motyl, T

    2005-06-01

    Myostatin (MSTN) and transforming growth factor-beta1 (TGF-beta1) belong to the same TGF-beta superfamily of proteins. They are involved in regulation of skeletal muscle growth and development as well as muscle catabolism. The aim of the present study was to investigate the relationship between MSTN and TGF-beta1 expression in proliferating and differentiating mouse C2C12 myoblasts cultured in normal and catabolic conditions and to evaluate the effect of exogenous TGF-beta1 as well as "knock down" of TGF-beta1 receptor type II on MSTN expression in proliferating and differentiating myogenic cells. The direct effect of TGF-beta1 on myostatin was also examined. Myostatin expression increased gradually with cell confluency in proliferating cultures, while the level of TGF-beta1, detected in the form of a 100 kDa small latent complex diminished. Myostatin expression was accompanied by a partial cell cycle arrest. Three forms of myostatin were found: a 52 kDa precursor, a 40 kDa latency associated propeptide, and a 26 kDa active peptide. A decrease in myostatin and TGF-beta1 levels was observed during the first three days of differentiation, which was subsequently followed by significant increase of their expression during next three to four days of differentiation. Catabolic state induced by dexamethasone significantly increased the level of all forms of myostatin as well as latent (100 kDa) and active (25 kDa) forms of TGF-beta1 in differentiating myoblasts in a dose dependent manner. Exogenous TGF-beta1 (2 ng/ml) significantly increased myostatin levels both in proliferating and differentiating C2C12 myoblasts, whereas silencing of the TGF-beta1 receptor II gene significantly lowered myostatin level in examined cells. The presented results indicate that TGF-beta1 may control myostatin-related regulation of myogenesis through up-regulation of myostatin, predominantly in the course of terminal differentiation and glucocorticoid-dependent catabolic stimulation. PMID

  12. Structural Insights into the Binding of Vascular Endothelial Growth Factor-B by VEGFR-1D2

    PubMed Central

    Iyer, Shalini; Darley, Paula I.; Acharya, K. Ravi

    2010-01-01

    The formation of blood vessels (angiogenesis) is a highly orchestrated sequence of events involving crucial receptor-ligand interactions. Angiogenesis is critical for physiological processes such as development, wound healing, reproduction, tissue regeneration, and remodeling. It also plays a major role in sustaining tumor progression and chronic inflammation. Vascular endothelial growth factor (VEGF)-B, a member of the VEGF family of angiogenic growth factors, effects blood vessel formation by binding to a tyrosine kinase receptor, VEGFR-1. There is growing evidence of the important role played by VEGF-B in physiological and pathological vasculogenesis. Development of VEGF-B antagonists, which inhibit the interaction of this molecule with its cognate receptor, would be important for the treatment of pathologies associated specifically with this growth factor. In this study, we present the crystal structure of the complex of VEGF-B with domain 2 of VEGFR-1 at 2.7 Å resolution. Our analysis reveals that each molecule of the ligand engages two receptor molecules using two symmetrical binding sites. Based on these interactions, we identify the receptor-binding determinants on VEGF-B and shed light on the differences in specificity towards VEGFR-1 among the different VEGF homologs. PMID:20501651

  13. Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis.

    PubMed Central

    Zhang, K.; Flanders, K. C.; Phan, S. H.

    1995-01-01

    Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF-beta 1 expression in this model of pulmonary fibrosis. Images Figure 2 Figure 3 Figure 4 PMID:7543734

  14. Redox-mediated activation of latent transforming growth factor-beta 1

    NASA Technical Reports Server (NTRS)

    Barcellos-Hoff, M. H.; Dix, T. A.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during

  15. Early defect of transforming growth factor β1 formation in Huntington’s disease

    PubMed Central

    Battaglia, Giuseppe; Cannella, Milena; Riozzi, Barbara; Orobello, Sara; Maat-Schieman, Marion L; Aronica, Eleonora; Busceti, Carla Letizia; Ciarmiello, Andrea; Alberti, Silvia; Amico, Enrico; Sassone, Jenny; Sipione, Simonetta; Bruno, Valeria; Frati, Luigi; Nicoletti, Ferdinando; Squitieri, Ferdinando

    2011-01-01

    Abstract A defective expression or activity of neurotrophic factors, such as brain- and glial-derived neurotrophic factors, contributes to neuronal damage in Huntington’s disease (HD). Here, we focused on transforming growth factor-β (TGF-β1), a pleiotropic cytokine with an established role in mechanisms of neuroprotection. Asymptomatic HD patients showed a reduction in TGF-β1 levels in the peripheral blood, which was related to trinucleotide mutation length and glucose hypometabolism in the caudate nucleus. Immunohistochemical analysis in post-mortem brain tissues showed that TGF-β1 was reduced in cortical neurons of asymptomatic and symptomatic HD patients. Both YAC128 and R6/2 HD mutant mice showed a reduced expression of TGF-β1 in the cerebral cortex, localized in neurons, but not in astrocytes. We examined the pharmacological regulation of TGF-β1 formation in asymptomatic R6/2 mice, where blood TGF-β1 levels were also reduced. In these R6/2 mice, both the mGlu2/3 metabotropic glutamate receptor agonist, LY379268, and riluzole failed to increase TGF-β1 formation in the cerebral cortex and corpus striatum, suggesting that a defect in the regulation of TGF-β1 production is associated with HD. Accordingly, reduced TGF-β1 mRNA and protein levels were found in cultured astrocytes transfected with mutated exon 1 of the human huntingtin gene, and in striatal knock-in cell lines expressing full-length huntingtin with an expanded glutamine repeat. Taken together, our data suggest that serum TGF-β1 levels are potential biomarkers of HD development during the asymptomatic phase of the disease, and raise the possibility that strategies aimed at rescuing TGF-β1 levels in the brain may influence the progression of HD. PMID:20082658

  16. Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue

    NASA Technical Reports Server (NTRS)

    Barcellos-Hoff, M. H.; Ehrhart, E. J.; Kalia, M.; Jirtle, R.; Flanders, K.; Tsang, M. L.; Chatterjee, A. (Principal Investigator)

    1995-01-01

    The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a

  17. Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts

    PubMed Central

    KOMATSU, YUKO; IBI, MIHO; CHOSA, NAOYUKI; KYAKUMOTO, SEIKO; KAMO, MASAHARU; SHIBATA, TOSHIYUKI; SUGIYAMA, YOSHIKI; ISHISAKI, AKIRA

    2016-01-01

    Bisphosphonates (BPs) are analogues of pyro-phosphate that are known to prevent bone resorption by inhibiting osteoclast activity. Nitrogen-containing BPs, such as zoledronic acid (ZA), are widely used in the treatment of osteoporosis and bone metastasis. However, despite having benefits, ZA has been reported to induce BP-related osteonecrosis of the jaw (BRONJ) in cancer patients. The molecular pathological mechanisms responsible for the development of BRONJ, including necrotic bone exposure after tooth extraction, remain to be elucidated. In this study, we examined the effects of ZA on the transforming growth factor-β (TGF-β)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs) and the migratory activity of hGFs, which are important for wound closure by fibrous tissue formation. The ZA maximum concentration in serum (Cmax) was found to be approximately 1.47 µM, which clinically, is found after the intravenous administration of 4 mg ZA, and ZA at this dose is considered appropriate for the treatment of cancer bone metastasis or bone diseases, such as Erdheim-Chester disease. At Cmax, ZA significantly suppressed i) the TGF-β-induced promotion of cell viability, ii) the TGF-β-induced expression of MF markers such as α-smooth muscle actin (α-SMA) and type I collagen, iii) the TGF-β-induced migratory activity of hGFs and iv) the expression level of TGF-β type I receptor on the surfaces of hGFs, as well as the TGF-β-induced phosphorylation of Smad2/3. Thus, ZA suppresses TGF-β-induced fibrous tissue formation by hGFs, possibly through the inhibition of Smad-dependent signal transduction. Our findings partly elucidate the molecular mechanisms underlying BRONJ and may prove to be beneficial to the identification of drug targets for the treatment of this symptom at the molecular level. PMID:27176567

  18. Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway

    PubMed Central

    Tao, Jie; Lan, Zhen; Hei, Hongya; Tian, Lulu; Pan, Wanma; Wang, Li; Zhang, Xuemei

    2016-01-01

    Hydrogen sulfide (H2S) has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS) and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-β1 (TGF-β1)-induced EMT in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-β1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-β receptor type I (TβR I) and TGF-β receptor type II (TβR II). In addition, NaHS attenuated TGF-β1-induced increase of β-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-β1-induced nuclear translocation of ββ-catenin. These effects of NaHS on fibronectin, E-cadherin and TβR I were abolished by the ERK inhibitor U0126 or β-catenin inhibitor XAV939, or β-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-β1-induced EMT in HK-2 cells through both ERK-dependent and β-catenin-dependent pathways. PMID:26760502

  19. Genetic variation in Transforming Growth Factor beta 1 and mammographic density in Singapore Chinese women

    PubMed Central

    Lee, Eunjung; Van den Berg, David; Hsu, Chris; Ursin, Giske; Koh, Woon-Puay; Yuan, Jian-Min; Stram, Daniel O.; Yu, Mimi C.; Wu, Anna H.

    2013-01-01

    Transforming growth factor-beta (TGF-β) plays a critical role in normal mammary development and morphogenesis. Decreased TGF-β signaling has been associated with increased mammographic density. Percent mammographic density (PMD) adjusted for age and body mass index (BMI) is a strong risk factor and predictor of breast cancer risk. PMD is highly heritable, but few genetic determinants have been identified. We investigated the association between genetic variation in TGFB1 and PMD using a cross-sectional study of 2,038 women who were members of the population-based Singapore Chinese Health Study cohort. We assessed PMD using a computer-assisted method. We used linear regression to examine the association between 9 tagging SNPs of TGFB1 and PMD and their interaction with parity, adjusting for age, BMI, and dialect group. We calculated ‘P-values adjusted for correlated tests’ (PACT) to account for multiple testing. The strongest association was observed for rs2241716. Adjusted PMD was higher by 1.5% per minor allele (PACT =0.04). When stratifying by parity, this association was limited to nulliparous women. For nulliparous women, adjusted PMD was higher by 8.6% per minor allele (PACT=0.003; P for interaction with parity=0.002). Three additional TGFB1 tagging SNPs, which were in linkage disequilibrium with rs2241716, were statistically significantly associated with adjusted PMD (PACT<0.05) for nulliparous women. However, none of these three SNPs showed statistically significant association after adjusting for rs2241716. Our data support that TGFB1 genetic variation may be an important genetic determinant of mammographic density measure that predicts breast cancer risk, particularly in nulliparous women. PMID:23333936

  20. Increased transforming growth factor beta 1 expression mediates ozone-induced airway fibrosis in mice

    PubMed Central

    Katre, Ashwini; Ballinger, Carol; Akhter, Hasina; Fanucchi, Michelle; Kim, Dae-Kee; Postlethwait, Edward; Liu, Rui-Ming

    2013-01-01

    Ozone (O3), a commonly encountered environmental pollutant, has been shown to induce pulmonary fibrosis in different animal models; the underlying mechanism, however, remains elusive. To investigate the molecular mechanism underlying O3-induced pulmonary fibrosis, 6- to 8-week-old C57BL/6 male mice were exposed to a cyclic O3 exposure protocol consisting of 2 days of filtered air and 5 days of O3 exposure (0.5 ppm, 8 h/day) for 5 and 10 cycles with or without intraperitoneal injection of IN-1233, a specific inhibitor of the type 1 receptor of transforming growth factor beta (TGF-β), the most potent profibrogenic cytokine. The results showed that O3 exposure for 5 or 10 cycles increased the TGF-β protein level in the epithelial lining fluid (ELF), associated with an increase in the expression of plasminogen activator inhibitor 1 (PAI-1), a TGF-β-responsive gene that plays a critical role in the development of fibrosis under various pathological conditions. Cyclic O3 exposure also increased the deposition of collagens and alpha smooth muscle actin (α-SMA) in airway walls. However, these fibrotic changes were not overt until after 10 cycles of O3 exposure. Importantly, blockage of the TGF-β signaling pathway with IN-1233 suppressed O3-induced Smad2/3 phosphorylation, PAI-1 expression, as well as collagens and α-SMA deposition in the lung. Our data demonstrate for the first time that O3 exposure increases TGF-β expression and activates TGF-β signaling pathways, which mediates O3-induced lung fibrotic responses in vivo. PMID:21689010

  1. Transforming growth factor-β mediates endothelial dysfunction in rats during high salt intake.

    PubMed

    Feng, Wenguang; Ying, Wei-Zhong; Aaron, Kristal J; Sanders, Paul W

    2015-12-15

    Endothelial dysfunction has been shown to be predictive of subsequent cardiovascular events and death. Through a mechanism that is incompletely understood, increased dietary salt intake promotes endothelial dysfunction in healthy, salt-resistant humans. The present study tested the hypothesis that dietary salt-induced transforming growth factor (TGF)-β promoted endothelial dysfunction and salt-dependent changes in blood pressure (BP). Sprague-Dawley rats that received diets containing 0.3% NaCl [low salt (LS)] or 8.0% NaCl [high salt (HS)] were treated with vehicle or SB-525334, a specific inhibitor of TGF-β receptor I/activin receptor-like kinase 5, beginning on day 5. BP was monitored using radiotelemetry in four groups of rats (LS, LS + SB-525334, HS, and HS + SB-525334) for up to 14 days. By day 14 of the study, mean daytime systolic BP and mean pulse pressure of the HS group treated with vehicle was greater than those in the other three groups; mean daytime systolic BP and pulse pressure of the HS + SB-525334 group did not differ from the LS and LS + SB-525334-treated groups. Whereas mean systolic BP, mean diastolic BP, and mean arterial pressure did not differ among the groups on the seventh day of the study, endothelium-dependent vasorelaxation was impaired specifically in the HS group; treatment with the activin receptor-like kinase 5 inhibitor prevented the dietary HS intake-induced increases in phospho-Smad2 (Ser(465/467)) and NADPH oxidase-4 in endothelial lysates and normalized endothelial function. These findings suggest that HS-induced endothelial dysfunction and the development of salt-dependent increases in BP were related to endothelial TGF-β signaling. PMID:26447221

  2. Dedifferentiation of Immortalized Human Podocytes in Response to Transforming Growth Factor-β

    PubMed Central

    Herman-Edelstein, Michal; Thomas, Merlin C.; Thallas-Bonke, Vicki; Saleem, Moin; Cooper, Mark E.; Kantharidis, Phillip

    2011-01-01

    OBJECTIVE Diabetic nephropathy is associated with dedifferentiation of podocytes, losing the specialized features required for efficient glomerular function and acquiring a number of profibrotic, proinflammatory, and proliferative features. These result from tight junction and cytoskeletal rearrangement, augmented proliferation, and apoptosis. RESEARCH DESIGN AND METHODS Experiments were performed in conditionally immortalized human podocytes developed by transfection with the temperature-sensitive SV40-T gene. Cells were then cultured in the presence of transforming growth factor (TGF)-β1 or angiotensin II in the presence or absence of a selective inhibitor of the TGF-β type I receptor kinase, SB-431542. Gene and protein expression were then examined by real-time RT-PCR and immunofluorescence, and correlated with changes observed in vivo in experimental diabetes. RESULTS Treatment of cells with TGF-β1 resulted in dynamic changes in their morphology, starting with retraction and shortening of foot processes and finishing with the formation of broad and complex tight junctions between adjacent podocytes. This dedifferentiation was also associated with dose- and time-dependent reduction in the expression of glomerular epithelial markers (nephrin, p-cadherin, zonnula occludens-1) and increased expression of mesenchymal markers (α−smooth muscle actin, vimentin, nestin), matrix components (fibronectin, collagen I, and collagen IV α3), cellular proliferation, and apoptosis. The induction of diabetes in mice was also associated with similar changes in morphology, protein expression, and proliferation in glomerular podocytes. CONCLUSIONS In response to TGF-β and other TGF-dependent stimuli, mature podocytes undergo dedifferentiation that leads to effacement of foot processes, morphologic flattening, and increased formation of intercellular tight junctions. This simplification of their phenotype to a more embryonic form is also associated with reentry of mature

  3. Transforming growth factor-β3 promotes facial nerve injury repair in rabbits

    PubMed Central

    WANG, YANMEI; ZHAO, XINXIANG; HUOJIA, MUHTER; XU, HUI; ZHUANG, YOUMEI

    2016-01-01

    The present study investigated the effects of transforming growth factor (TGF)-β3 on the regeneration of facial nerves in rabbits. A total of 20 adult rabbits were randomly divided into three equal groups: Normal control (n=10), surgical control (n=10) and TGF-β3 treatment (n=10). The total number and diameter of the regenerated nerve fibers was significantly increased in the TGF-β3 treatment group, as compared with in the surgical control group (P<0.01). Furthermore, in the TGF-β3 treatment group, the epineurial repair of the facial nerves was intact and the nerve fibers, which were arranged in neat rows, were morphologically intact with visible myelin swelling. However, in the surgical control group, the epineurial repair was incomplete, as demonstrated by: Atrophic nerve fibers, partially disappeared axons and myelin of uneven thickness with fuzzy borders. Electron microscopy demonstrated that the regenerated fibers in the TGF-β3 treatment group were predominantly myelinated, with clear-layered myelin sheath structures and axoplasms rich in organelles. Although typical layered myelin sheath structures were observed in the surgical control group, the myelin sheaths of the myelinated nerve fibers were poorly developed and few organelles were detected in the axoplasms. Neuro-electrophysiological examination demonstrated that, as compared with the surgical control group, the latency period of the action potentials in the TGF-β3 treatment group were shorter, whereas the stimulus amplitudes of the action potentials were significantly increased (P<0.01). The results of the present study suggest that TGF-β3 may improve the regeneration of facial nerves following trauma or injury. PMID:26997982

  4. Transforming Growth Factor-β2 Induces Bronchial Epithelial Mucin Expression in Asthma

    PubMed Central

    Chu, Hong Wei; Balzar, Silvana; Seedorf, Gregory J.; Westcott, Jay Y.; Trudeau, John B.; Silkoff, Phil; Wenzel, Sally E.

    2004-01-01

    The transforming growth factor (TGF)-β family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-β1 or TGF-β2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-β1 and TGF-β2 expression and their effects on mucin expression, and the role of TGF-β1 or TGF-β2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-β1, TGF-β2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-β1 or TGF-β2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-β1, TGF-β2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-β2 expression levels were higher than TGF-β1 in both asthmatics and normals. TGF-β2, but not TGF-β1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-β2, but not TGF-β1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-β2, but not TGF-β1, from epithelial cells. A neutralizing TGF-β2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-β2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-β2 up-regulation. PMID:15466377

  5. Transforming growth factor-beta2 induces bronchial epithelial mucin expression in asthma.

    PubMed

    Chu, Hong Wei; Balzar, Silvana; Seedorf, Gregory J; Westcott, Jay Y; Trudeau, John B; Silkoff, Phil; Wenzel, Sally E

    2004-10-01

    The transforming growth factor (TGF)-beta family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-beta1 or TGF-beta2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-beta1 and TGF-beta2 expression and their effects on mucin expression, and the role of TGF-beta1 or TGF-beta2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-beta1, TGF-beta2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-beta1 or TGF-beta2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-beta1, TGF-beta2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-beta2 expression levels were higher than TGF-beta1 in both asthmatics and normals. TGF-beta2, but not TGF-beta1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-beta2, but not TGF-beta1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-beta2, but not TGF-beta1, from epithelial cells. A neutralizing TGF-beta2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-beta2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-beta2 up-regulation. PMID:15466377

  6. Redox-mediated activation of latent transforming growth factor-beta 1.

    PubMed

    Barcellos-Hoff, M H; Dix, T A

    1996-09-01

    Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during

  7. Orofacial clefts, parental cigarette smoking, and transforming growth factor-alpha gene variants

    SciTech Connect

    Shaw, G.M.; Wasserman, C.R.; O`Malley, C.D.

    1996-03-01

    Results of studies determine whether women who smoke during early pregnancy are at increased risk of delivering infants with orofacial clefts have been mixed, and recently a gene-environment interaction between maternal smoking, transforming growth factor-alpha (TGFa), and clefting has been reported. Using a large population-based case-control study, we investigated whether parental periconceptional cigarette smoking was associated with an increased risk for having offspring with orofacial clefts. We also investigated the influence of genetic variation of the TGFa locus on the relation between smoking and clefting. Parental smoking information was obtained from telephone interviews with mothers of 731 (84.7% of eligible) orofacial cleft case infants and with mothers of 734 (78.2%) nonmalformed control infants. DNA was obtained from newborn screening blood spots and genotyped for the allelic variants of TGFa. We found that risks associated with maternal smoking were most elevated for isolated cleft lip with or without cleft palate, (odds ratio 2.1 [95% confidence interval 1.3-3.6]) and for isolated cleft palate (odds ratio 2.2 [1.1-4.5]) when mothers smoked {ge} 20 cigarrettes/d. These risks for white infants ranged from 3-fold to 11-fold across phenotypic groups. Paternal smoking was not associated with clefting among the offspring of nonsmoking mothers, and passive smoke exposures were associated with at most slightly increased risks. This study offers evidence that the risk for orofacial clefting in infants may be influenced by maternal smoke exposures alone as well as in combination (gene-environment interaction) with the presence of the uncommon TGFa allele. 56 refs., 5 tabs.

  8. Transforming growth factor-β and atherosclerosis: interwoven atherogenic and atheroprotective aspects

    PubMed Central

    Toma, Ian

    2016-01-01

    Age-related progression of cardiovascular disease is by far the largest health problem in the US and involves vascular damage, progressive vascular fibrosis and the accumulation of lipid-rich atherosclerotic lesions. Advanced lesions can restrict flow to key organs and can trigger occlusive thrombosis resulting in a stroke or myocardial infarction. Transforming growth factor-beta (TGF-β) is a major orchestrator of the fibroproliferative response to tissue damage. In the early stages of repair, TGF-β is released from platelets and activated from matrix reservoirs; it then stimulates the chemotaxis of repair cells, modulates immunity and inflammation and induces matrix production. At later stages, it negatively regulates fibrosis through its strong antiproliferative and apoptotic effects on fibrotic cells. In advanced lesions, TGF-β might be important in arterial calcification, commonly referred to as “hardening of the arteries”. Because TGF-β can signal through multiple pathways, namely the SMADs, a MAPK pathway and the Rho/ROCK pathways, selective defects in TGF-β signaling can disrupt otherwise coordinated pathways of tissue regeneration. TGF-β is known to control cell proliferation, cell migration, matrix synthesis, wound contraction, calcification and the immune response, all being major components of the atherosclerotic process. However, many of the effects of TGF-β are essential to normal tissue repair and thus, TGF-β is often thought to be “atheroprotective”. The present review attempts to parse systematically the known effects of TGF-β on both the major risk factors for atherosclerosis and to isolate the role of TGF-β in the many component pathways involved in atherogenesis. PMID:21626289

  9. Nonparallel changes of growth hormone (GH) and insulin-like growth factor-I, insulin-like growth factor binding protein-3, and GH-binding protein, after craniospinal irradiation and chemotherapy

    SciTech Connect

    Nivot, S.; Adan, L.; Souberbielle, J.; Rappaport, R.; Brauner, R.; Benelli, C.; Clot, J.P.; Saucet, C.; Zucker, J.M.

    1994-03-01

    The authors studied the GH-insulin-like growth factor-I (IGF-I) axis serially over 24-36 months in six patients with medulloblastoma who underwent surgical removal of the tumor followed by craniospinal irradiation therapy for 6 weeks and then chemotherapy for 42 weeks. Eighteen and 24 months after beginning irradiation there was a decline in the peak GH secretory response to acute stimulation with arginine/insulin hypoglycemia. Six months after irradiation and during chemotherapy there was a transient decline in IGF-I, IGF binding protein-3 (IGFBP-3), and GH-BP values (respective mean values of 56.1 {+-} 9.0 ng/mL, 1.1 {+-} 0.2 {mu}g/mL, and 7.6 {+-} 3.3% of radioactivity as compared to time 0 values: 139 {+-} 15 ng/mL, 2.2 {+-} 0.2 {mu}g/mL, and 20.0 {+-} 4.0%, P < 0.001), although provoked GH secretion was normal at this time. The IGF-I, IGFBP-3, and GH-BP returned to pretreatment ranges by 12-36 months after initiation of the study. There was also a decline in body mass index and serum protein values at 6 months after irradiation in ligand and immunoblot analysis there was a decline in IGFBP-3 and an abnormal electrophoretic mobility of IGFBP-2 that were both normalized at 36 months. In one patient they observed a high level of IGFBP-3 proteolysis at this time. This study demonstrates that before the decrease of GH secretion in patients receiving cranial irradiation there is a transient phase of GH insensitivity that may be characteristic of the acute therapeutic phase including the chemotherapy. This partial insensitivity may explain the early growth retardation observed in these patients. 28 refs., 4 figs., 1 tab.

  10. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor.

    PubMed

    Friedman, M; Nordberg, E; Höidén-Guthenberg, I; Brismar, H; Adams, G P; Nilsson, F Y; Carlsson, J; Ståhl, S

    2007-04-01

    Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed. PMID:17452435

  11. In silico investigation of pH-dependence of prolactin and human growth hormone binding to human prolactin receptor

    PubMed Central

    Wang, Lin; Witham, Shawn; Zhang, Zhe; Li, Lin; Hodsdon, Michael E.; Alexov, Emil

    2011-01-01

    Experimental data shows that the binding of human prolactin (hPRL) to human prolactin receptor (hPRLr-ECD) is strongly pH-dependent, while the binding of the same receptor to human growth hormone (hGH) is pH-independent. Here we carry in silico analysis of the molecular effects causing such a difference and reveal the role of individual amino acids. It is shown that the computational modeling correctly predicts experimentally determined pKa’s of histidine residues in an unbound state in the majority of the cases and the pH-dependence of the binding free energy. Structural analysis carried in conjunction with calculated pH-dependence of the binding revealed that the main reason for pH-dependence of the binding of hPRL-hPRLr-ECD is a number of salt- bridges across the interface of the complex, while no salt-bridges are formed in the hGH-hPRlr-ECD. Specifically, most of the salt-bridges involve histidine residues and this is the reason for the pH-dependence across a physiological range of pH. The analysis not only revealed the molecular mechanism of the pH-dependence of the hPRL-hPRLr-ECD, but also provided critical insight into the underlying physic-chemical mechanism. PMID:24683423

  12. Selectivity in glycosaminoglycan binding dictates the distribution and diffusion of fibroblast growth factors in the pericellular matrix

    PubMed Central

    Marcello, Marco

    2016-01-01

    The range of biological outcomes generated by many signalling proteins in development and homeostasis is increased by their interactions with glycosaminoglycans, particularly heparan sulfate (HS). This interaction controls the localization and movement of these signalling proteins, but whether such control depends on the specificity of the interactions is not known. We used five fibroblast growth factors with an N-terminal HaloTag (Halo-FGFs) for fluorescent labelling, with well-characterized and distinct HS-binding properties, and measured their binding and diffusion in pericellular matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FGF6 bound to HS, whereas Halo-FGF10 also interacted with chondroitin sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFs in the pericellular matrix was not homogeneous, and for FGF10 exhibited striking clusters. Fluorescence recovery after photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused more slowly, and FGF10 was immobile. The results demonstrate that the specificity of the interactions of proteins with glycosaminoglycans controls their binding and diffusion. Moreover, cells regulate the spatial distribution of different protein-binding sites in glycosaminoglycans independently of each other, implying that the extracellular matrix has long-range structure. PMID:27009190

  13. Notch1 signaling antagonizes transforming growth factor-β pathway and induces apoptosis in rabbit trophoblast stem cells.

    PubMed

    Tan, Tao; Lu, Bin; Zhang, Jing; Niu, Yuyu; Si, Wei; Wei, Qiang; Ji, Weizhi

    2014-04-15

    During mammalian development, placental growth needs to be tightly controlled by apoptosis. However, despite the potentially significant problems, the strategies used to balance growth and apoptosis have remained elusive. Here we report that activation of the Notch1 signal pathway inhibits transduction of transforming growth factor (TGF)-β signaling, which leads to cell cycle arrest and apoptosis in rabbit trophoblast stem cells (TSCs). The subcellular location of notch intracellular domain 1 (NICD1) appears to determine whether TGF-β signaling will be inhibited or not. Moreover, changes in NICD1 subcellular location are regulated by intracellular calcium distribution. Collectively, these results establish a potential mechanism whereby TSCs can balance growth and apoptosis, and thus guarantee the development of the fetus. PMID:24251382

  14. Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

    PubMed

    Moser, D R; Lowe, W L; Dake, B L; Booth, B A; Boes, M; Clemmons, D R; Bar, R S

    1992-11-01

    Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and

  15. Circulating insulin-like growth factor-binding protein 3 as prognostic biomarker in liver cirrhosis

    PubMed Central

    Correa, Carina Gabriela; Colombo, Bruno da Silveira; Ronsoni, Marcelo Fernando; Soares e Silva, Pedro Eduardo; Fayad, Leonardo; Silva, Telma Erotides; Wildner, Letícia Muraro; Bazzo, Maria Luiza; Dantas-Correa, Esther Buzaglo; Narciso-Schiavon, Janaína Luz; Schiavon, Leonardo de Lucca

    2016-01-01

    AIM: To investigate the prognostic significance of insulin-like growth factor-binding protein 3 (IGFBP-3) in patients with cirrhosis. METHODS: Prospective study that included two cohorts: outpatients with stable cirrhosis (n = 138) and patients hospitalized for acute decompensation (n = 189). Development of complications, mortality or liver transplantation was assessed by periodical phone calls and during outpatient visits. The cohort of stable cirrhosis also underwent clinical and laboratory evaluation yearly (2013 and 2014) in predefined study visits. In patients with stable cirrhosis, IGFBP-3 levels were measured at baseline (2012) and at second re-evaluation (2014). In hospitalized subjects, IGFBP-3 levels were measured in serum samples collected in the first and in the third day after admission and stored at -80 °C. IGFBP-3 levels were measured by immunochemiluminescence. RESULTS: IGFBP-3 levels were lower in hospitalized patients as compared to outpatients (0.94 mcg/mL vs 1.69 mcg/mL, P < 0.001) and increased after liver transplantation (3.81 mcg/mL vs 1.33 mcg/mL, P = 0.008). During the follow-up of the stable cohort, 17 patients died and 11 received liver transplantation. Bivariate analysis showed that death or transplant was associated with lower IGFBP-3 levels (1.44 mcg/mL vs 1.74 mcg/mL, P = 0.027). The Kaplan-Meier transplant-free survival probability was 88.6% in patients with IGFBP-3 ≥ 1.67 mcg/mL and 72.1% for those with IGFBP3 < 1.67 mcg/mL (P = 0.015). In the hospitalized cohort, 30-d mortality was 24.3% and was independently associated with creatinine, INR, SpO2/FiO2 ratio and IGFBP-3 levels in the logistic regression. The 90-d transplant-free survival probability was 80.4% in patients with IGFBP-3 ≥ 0.86 mcg/mL and 56.1% for those with IGFBP3 < 0.86 mcg/mL (P < 0.001). CONCLUSION: Lower IGFBP-3 levels were associated with worse outcomes in patients with cirrhosis, and might represent a promising prognostic tool that can be incorporated in

  16. Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli

    PubMed Central

    Khodadadi, Emad; Panjepour, Mojtaba; Abbasian, Mahdi; Broujeni, Zahra Khalili; Mofid, Mohammad Reza

    2015-01-01

    Background: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host. Materials and Methods: First the sequence encoding of IGFBP3 was designed based on the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis. Conclusion: DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3) expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa) are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein. PMID:25878991

  17. 14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

    PubMed

    Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

    2010-03-01

    The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells. PMID:20082218

  18. A heparin-binding growth factor secreted from breast cancer cells homologous to a developmentally regulated cytokine.

    PubMed

    Wellstein, A; Fang, W J; Khatri, A; Lu, Y; Swain, S S; Dickson, R B; Sasse, J; Riegel, A T; Lippman, M E

    1992-02-01

    We report purification of an 18-kDa heparin-binding growth factor secreted from human cancer cells which is homologous to a developmentally regulated, neurotrophic factor, heparin-binding growth-associated molecule/pleiotrophin (HB-GAM/PTN; Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724; Li, Y. S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694). We have purified the protein from tissue culture supernatants of human breast cancer cells (MDA-MB 231) and have used soft agar cloning of an epithelial cell line (SW-13) to detect its growth stimulating activity. A 32,000-fold purification was achieved by isoelectric focusing, heparin affinity chromatography, and reversed phase high pressure liquid chromatography. The molecular mass of the protein was confirmed by gel filtration chromatography in the presence of detergent and bioassay of the fractions. The N-terminal sequence was homologous to HB-GAM/PTN, and polymerase chain reaction amplification and DNA sequencing confirmed that the respective transcript was present in the cancer cells. We conclude that HB-GAM/PTN can function as a tumor growth factor in addition to its role during neuronal development. PMID:1733956

  19. Actin binding and proline rich motifs of CR16 play redundant role in growth of vrp1Delta cells.

    PubMed

    Meng, Lei; Rajmohan, Rajamuthiah; Yu, Shangjuan; Thanabalu, Thirumaran

    2007-05-25

    CR16, (Glucocorticoid-regulated) belongs to the verprolin family of proteins which are characterized by the presence of a V domain (verprolin) at the N-terminal. Expression of CR16 suppressed the growth and endocytosis defect of vrp1Delta strain without correcting the actin patch polarization defect. The V domain of CR16 is critical for suppression of the growth defect of vrp1Delta strain but not for localisation to cortical actin patches. Mutations in the actin binding motif alone did not abolish the activity of CR16 but the mutations in combination with deletion of N-terminal proline rich motif abolished the ability of CR16 to suppress the growth defect. This suggests that the V domain of CR16 has two functionally redundant motifs and either one of these motifs is sufficient for suppressing the growth defect of vrp1Delta strain. This is in contrast to the observation that both WIP and WIRE require the actin binding motif for their activity. PMID:17418095

  20. Identification of the sAPRIL Binding Peptide and Its Growth Inhibition Effects in the Colorectal Cancer Cells

    PubMed Central

    Liu, Fang; Li, Jing; He, Mei-rong

    2015-01-01

    Background A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer. Methods A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP. Results Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells. Conclusions sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL. PMID:25826583

  1. Cytokines modulate the sensitivity of human fibroblasts to stimulation with insulin-like growth factor-I (IGF-I) by altering endogenous IGF-binding protein production.

    PubMed

    Yateman, M E; Claffey, D C; Cwyfan Hughes, S C; Frost, V J; Wass, J A; Holly, J M

    1993-04-01

    Human dermal fibroblasts produce a number of insulin-like growth factor-binding proteins (IGFBPs) including the main circulating form, IGFBP-3. It has been suggested that the regulation of IGFBP secretion may play a major role in modulating insulin-like growth factor (IGF) bioactivity. We have quantified the effects of two cytokines, transforming growth factor-beta 1 (TGF-beta 1) and tumour necrosis factor-alpha (TNF-alpha) which have opposing actions on fibroblast IGFBP-3 production, and examined their subsequent role in IGF-I mitogenesis. TGF-beta 1 caused a dose-dependent increase in IGFBP-3 in serum-free fibroblast-conditioned media. TGF-beta 1 (1 microgram/l) resulted in immunoreactive IGFBP-3 levels reaching 286.5 +/- 22.4% of control after 20 h, the increase being confirmed by Western ligand blot. TNF-alpha caused a dose-dependent decrease in fibroblast IGFBP-3 secretion, 1 microgram TNF-alpha/l reducing IGFBP-3 levels to 32.1 +/- 11.% of control. This effect was not due to cytotoxicity and was not cell-density-dependent. Fibroblast proliferation was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric cytochemical bioassay. The addition of IGF-I resulted in dose-dependent growth stimulation after 48 h, the effective range being 20-100 micrograms/l. The IGF-I analogue Long-R3-IGF-I which has little affinity for the IGFBPs was approximately 20-fold more potent in this assay, and was unaffected by exogenous IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7684061

  2. Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type. beta

    SciTech Connect

    Brown, K.D.; Holley, R.W.

    1987-06-01

    A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type ..beta.. (TGF-..beta..), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. /sup 125/I-EGF binding was measured and the efflux of /sup 45/Ca/sup 2 +/ was measured in response to mitogen stimulation. A similar synergistic response has been demonstrated for TGF-..beta.. purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-..beta.. in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and the authors have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-..beta.. and insulin converge at a post-receptor stage.

  3. Type I (RI) and type II (RII) receptors for transforming growth factor-beta isoforms are expressed subsequent to transforming growth factor-beta ligands during excisional wound repair.

    PubMed Central

    Gold, L. I.; Sung, J. J.; Siebert, J. W.; Longaker, M. T.

    1997-01-01

    Transforming growth factor (TGF)-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) regulate cell growth and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-beta function is transmitted by a heteromeric complex of receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-beta receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to 21 days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-beta ligands. However, immunoreactivity for TGF-beta receptors, throughout repair, occurred 1 to 5 days later than TGF-beta isoform immunostaining. This implies that the presence of TGF-beta ligands may up-regulate TGF-beta receptors for function and/or may reflect a lag due to local processing of latent TGF-beta. As observed for the immunohistochemical localization of TGF-beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, fibroblasts, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only

  4. Insulin-like growth factor-binding protein-5 (IGFBP-5): a critical member of the IGF axis

    PubMed Central

    Beattie, James; Allan, Gordon J.; Lochrie, Jennifer D.; Flint, David J.

    2006-01-01

    The six members of the insulin-like growth factor-binding protein family (IGFBP-1–6) are important components of the IGF (insulin-like growth factor) axis. In this capacity, they serve to regulate the activity of both IGF-I and -II polypeptide growth factors. The IGFBPs are able to enhance or inhibit the activity of IGFs in a cell- and tissue-specific manner. One of these proteins, IGFBP-5, also has an important role in controlling cell survival, differentiation and apoptosis. In this review, we report on the structural and functional features of the protein which are important for these effects. We also examine the regulation of IGFBP-5 expression and comment on its potential role in tumour biology, with special reference to work with breast cancer cells. PMID:16526944

  5. Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.

    PubMed

    Chou, Wan-Chih; Prokova, Vassiliki; Shiraishi, Keiko; Valcourt, Ulrich; Moustakas, Aristidis; Hadzopoulou-Cladaras, Margarita; Zannis, Vassilis I; Kardassis, Dimitris

    2003-03-01

    We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads. PMID:12631740

  6. Transforming growth factor beta (TGFB) signaling is activated during porcine implantation: proposed role for latency-associated peptide interactions with integrins at the conceptus-maternal interface.

    PubMed

    Massuto, Dana A; Kneese, Eric C; Johnson, Gregory A; Burghardt, Robert C; Hooper, R Neil; Ing, Nancy H; Jaeger, Laurie A

    2010-02-01

    The process of implantation is mediated by a complex network of signaling and adhesive factors. In the pig, latent and active transforming growth factor beta (TGFB), TGFB receptors (TGFBR), and integrins (ITGs) are present during the peri-implantation period. TGFB signals via TGFBR and activates downstream effector SMAD proteins 2 and 3 (p-SMAD2/3). Latency-associated peptide (LAP), part of the latent TGFB complex, is known to bind to ITG heterodimers and activate TGFB. We hypothesize that active TGFBs and TGFBRs along with LAP and ITGs functionally interact at the conceptus-maternal interface to mediate events essential for conceptus development and attachment in pigs. Uteri and conceptuses from days 10, 12, 16, 20, and 24 pregnant gilts were immunostained for TGFB, LAP, and ITG subunits (ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, and ITGB8). Activation of TGFBRs was evaluated by the presence of phosphorylated downstream effector SMAD2/3. Binding of LAP to ITGs was also evaluated using porcine trophectoderm cells. Abundant active TGFB was detected at the apical surfaces of epithelia at the conceptus-maternal interface, and p-SMAD2/3 was detected at both conceptus attachment and nonattachment sites during implantation. Separate aggregates of LAP, ITGB1, ITGB5, and later ITGB3 were detected at the porcine conceptus-maternal interface, and binding of LAP to ITGs on apical surfaces was demonstrated. Results suggest that functional LAP-ITG adhesion complexes support conceptus attachment and promote TGFB activation leading to TGFB interaction with TGFBR supporting events of porcine implantation. PMID:19920116

  7. Characterization of insulin-like growth factor-binding proteins secreted by isolated sheep thyroid epithelial cells.

    PubMed

    Wang, J F; Becks, G P; Buckingham, K D; Hill, D J

    1990-06-01

    We have characterized the insulin-like growth factor-binding proteins (IGF-BPs) released by isolated sheep thyroid epithelial cells. Thyroid follicles were isolated with collagenase and cultured in Coon's modified F-12 M (0H medium) supplemented with insulin, cortisol, transferrin, glycyl-histidyl-lysine and somatostatin (5H medium) and TSH (6H medium). Conditioned 0H medium specifically bound both 125I-labelled IGF-I and -II, although binding capacity was reduced following acid-gel filtration to separate endogenous IGF-BP complexes, suggesting some destruction of BPs. The binding of 125I-labelled IGF-I or -II to conditioned (0H) medium was progressively displaced by increasing amounts of unlabelled homologous peptides, while fractionation on concanavalin A-Sepharose showed that the IGF-BPs consisted of both glycoprotein and non-glycoprotein components. The molecular sizes of the IGF-BPs were resolved by separation of 0H medium on SDS-PAGE and ligand blot analysis with 125I-labelled IGF-I or -II. Conditioned medium contained four specific binding species for IGF-II of 19, 30, 38 and 46 kDa; all but the smallest also binding radiolabelled IGF-I. Prior fractionation on concanavalin A-Sepharose showed that the 46 kDa binding species was a glycoprotein. Competition studies with increasing concentrations of unlabelled IGF-I or -II during ligand blotting suggested that the 46 and 30 kDa binding species had a greater affinity for IGF-II than IGF-I, while the 38 kDa had a greater relative affinity for IGF-I. Incubation of cells in 5H medium reduced the abundance of the 46 kDa binding protein, while incubation in 6H medium decreased the release of all binding protein species. Results show that isolated thyroid follicles released several forms of IGF-BP with differing relative affinities for IGF-I and -II. Gross changes seen in the presence of BPs between 0H, 5H and 6H media suggest acute hormonal control of release. PMID:1695663

  8. Binding between Insulin-like Growth Factor 1 and Insulin-like Growth Factor-binding Protein 3 Is Not Influenced by Glucose or 2-Deoxy-d-glucose

    PubMed Central

    Mireuta, Matei; Hancock, Mark A.; Pollak, Michael

    2011-01-01

    A recent report (Zhong, D., Xiong, L., Liu, T., Liu, X., Liu, X., Chen, J., Sun, S. Y., Khuri, F. R., Zong, Y., Zhou, Q., and Zhou, W. (2009) J. Biol. Chem. 284, 23225–23233) details that 2-deoxy-d-glucose (2-DG), a well known inhibitor of glycolysis and a candidate antineoplastic agent, also induces insulin-like growth factor 1 receptor (IGF-1R) signaling through the inhibition of insulin-like growth factor 1-insulin-like growth factor-binding protein 3 (IGF-1-IGFBP-3) complex formation. Zhong et al. hypothesized that disrupted IGF-1/IGFBP-3 binding by 2-DG led to increased free IGF-1 concentrations and, consequently, activation of IGF-1R downstream pathways. Because their report suggests unprecedented off-target effects of 2-DG, this has profound implications for the fields of metabolism and oncology. Using ELISA, surface plasmon resonance, and novel “intensity-fading” mass spectrometry, we now provide a detailed characterization of complex formation between IGF-1 and IGFBP-3. All three of these independent methods demonstrated that there was no effect of glucose or 2-DG on the interaction between IGF-1 and IGFBP-3. Furthermore, we show examples of 2-DG exposure associated with reduced rather than increased IGF-1R and AKT activation, providing further evidence against a 2-DG increase in IGF-1R activation by IGF-1-IGFBP-3 complex disruption. PMID:21388950

  9. Expression of transforming growth factor β receptor II in mesenchymal stem cells from systemic sclerosis patients

    PubMed Central

    Vanneaux, Valérie; Farge-Bancel, Dominique; Lecourt, Séverine; Baraut, Julie; Cras, Audrey; Jean-Louis, Francette; Brun, Cécilia; Verrecchia, Franck; Larghero, Jérôme; Michel, Laurence

    2013-01-01

    Objectives The present work aimed to evaluate the expression of transforming growth factor-β (TGF-β) receptors on bone marrow-derived multipotent mesenchymal stromal cells (MSCs) in patients with systemic sclerosis (SSc) and the consequences of TGF-β activation in these cells, since MSC have potential therapeutic interest for SSc patients and knowing that TGF-β plays a critical role during the development of fibrosis in SSc. Design This is a prospective research study using MSC samples obtained from SSc patients and compared with MSC from healthy donors. Setting One medical hospital involving collaboration between an internal medicine department for initial patient recruitment, a clinical biotherapeutic unit for MSC preparation and an academic laboratory for research. Participants 9 patients with diffuse SSc for which bone marrow (BM) aspiration was prescribed by sternum aspiration before haematopoietic stem cell transplantation, versus nine healthy donors for normal BM. Primary and secondary outcome measures TGF-β, TGF-β receptor types I (TBRI) and II (TBRII) mRNA and protein expression were assessed by quantitative PCR and flow cytometry, respectively, in MSC from both SSc patients and healthy donors. MSC were exposed to TGF-β and assessed for collagen 1α2 synthesis and Smad expression. As positive controls, primary cultures of dermal fibroblasts were also analysed. Results Compared with nine controls, MSC from nine SSc patients showed significant increase in mRNA levels (p<0.002) and in membrane expression (p<0.0001) of TBRII. In response to TGF-β activation, a significant increase in collagen 1α synthesis (p<0.05) and Smad-3 phosphorylation was upregulated in SSc MSC. Similar results were obtained on eight SSc-derived dermal fibroblasts compared to six healthy controls. Conclusions TBRII gene and protein expression defect in MSC derived from SSc patients may have pathological significance. These findings should be taken into account when considering

  10. Angiotensin II stimulated expression of transforming growth factor-beta1 in cardiac fibroblasts and myofibroblasts.

    PubMed

    Campbell, S E; Katwa, L C

    1997-07-01

    Angiotensin II (Ang II) stimulates pathologic myocardial fibrosis. Cardiac fibroblasts (CFb) and myofibroblasts mediate this response, perhaps in part by indirect production of specific cytokines. We sought to determine if Ang II could stimulate transforming growth factor-beta1 (TGF-beta1) gene expression and protein production in adult rat CFb and two cardiac myofibroblast cell types, scar myofibroblasts (MyoFb) and valvular interstitial cells (VIC). Confluent CFb, MyoFb, and VIC in serum-deprived (0.4% FCS) media were treated with Ang II (10(-7) m for CFb; 10(-9) m for MyoFb, VIC) for 24 h. Untreated cells served as controls. Culture media was collected and TGF-beta1 levels determined in triplicate using a sandwich ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to determine TGF-beta1 mRNA expression. Ang II increased CFb (P<0.02) and VIC (P<0.04) TGF-beta1 mRNA expression, while the increase in MyoFb was not statistically significant. MyoFb produced the highest TGF-beta1 levels under control conditions compared to VIC and CFb. Ang II stimulated further TGF-beta1 secretion in VIC and CFb, but not MyoFb. The AT1 receptor antagonist Losartan (10(-7) m) greatly attenuated Ang II-stimulated TGF-B1 secretion and decreased TGF-beta1 immunostaining in VIC. The AT2 receptor antagonist PD123177 (10(-7) m) also decreased secretion and immunostaining of TGF-beta1 in VIC, but to a lesser extent than Losartan. TGF-beta1 secretion by MyoFb was unaffected by Losartan and PD123177, although TGF-B1 immunostaining was absent or greatly decreased, respectively, compared to Ang II-treated MyoFb. Ang II stimulates TGF-beta1 gene expression and/or protein production in cardiac fibroblast-like cells which may act as an autocrine/paracrine stimulus to collagen formation. Furthermore, TGF-beta1 production and secretion in these cells can be modulated by specific Ang II receptor antagonists, suggesting a potential benefit in preventing

  11. Expression of transforming growth factors beta-1, beta 2 and beta 3 in human bladder carcinomas.

    PubMed Central

    Eder, I. E.; Stenzl, A.; Hobisch, A.; Cronauer, M. V.; Bartsch, G.; Klocker, H.

    1997-01-01

    We previously detected elevated transforming growth factor beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. Fibroblasts expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, fibroblasts released only 19% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by fibroblasts with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer. Images Figure 1 PMID:9192977

  12. Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts.

    PubMed

    Komatsu, Yuko; Ibi, Miho; Chosa, Naoyuki; Kyakumoto, Seiko; Kamo, Masaharu; Shibata, Toshiyuki; Sugiyama, Yoshiki; Ishisaki, Akira

    2016-07-01

    Bisphosphonates (BPs) are analogues of pyrophosphate that are known to prevent bone resorption by inhibiting osteoclast activity. Nitrogen-containing BPs, such as zoledronic acid (ZA), are widely used in the treatment of osteoporosis and bone metastasis. However, despite having benefits, ZA has been reported to induce BP-related osteonecrosis of the jaw (BRONJ) in cancer patients. The molecular pathological mechanisms responsible for the development of BRONJ, including necrotic bone exposure after tooth extraction, remain to be elucidated. In this study, we examined the effects of ZA on the transforming growth factor-β (TGF‑β)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs) and the migratory activity of hGFs, which are important for wound closure by fibrous tissue formation. The ZA maximum concentration in serum (Cmax) was found to be approximately 1.47 µM, which clinically, is found after the intravenous administration of 4 mg ZA, and ZA at this dose is considered appropriate for the treatment of cancer bone metastasis or bone diseases, such as Erdheim-Chester disease. At Cmax, ZA significantly suppressed i) the TGF‑β-induced promotion of cell viability, ii) the TGF‑β-induced expression of MF markers such as α-smooth muscle actin (α-SMA) and type I collagen, iii) the TGF‑β-induced migratory activity of hGFs and iv) the expression level of TGF‑β type I receptor on the surfaces of hGFs, as well as the TGF‑β-induced phosphorylation of Smad2/3. Thus, ZA suppresses TGF‑β-induced fibrous tissue formation by hGFs, possibly through the inhibition of Smad‑dependent signal transduction. Our findings partly elucidate the molecular mechanisms underlying BRONJ and may prove to be beneficial to the identification of drug targets for the treatment of this symptom at the molecular level. PMID:27176567

  13. Urinary active transforming growth factor β in feline chronic kidney disease.

    PubMed

    Lawson, J S; Syme, H M; Wheeler-Jones, C P D; Elliott, J

    2016-08-01

    The cytokine transforming growth factor beta 1 (TGF-β1) has been widely implicated in the development and progression of renal fibrosis in chronic kidney disease (CKD) in humans and in experimental models. The aims of this study were to assess the association between urinary active TGF-β1 and (a) development of CKD in a cross-sectional study, (b) deterioration of renal function over 1 year in a longitudinal study, and (c) renal histopathological parameters in cats. A human active TGF-β1 ELISA was validated for use in feline urine. Cross-sectional analysis revealed no significant difference in urinary active TGF-β1:creatinine ratio (aTGF-β1:UCr) between groups with differing renal function. Longitudinally, non-azotaemic cats that developed CKD demonstrated a significant (P = 0.028) increase in aTGF-β1:UCr approximately 6 months before the development of azotaemia, which remained elevated (P = 0.046) at diagnosis (approximately 12 months prior, 8.4 pg/mg; approximately 6 months prior, 22.2 pg/mg; at CKD diagnosis, 24.6 pg/mg). In the histopathology study, aTGF-β1:UCr was significantly higher in cats with moderate (P = 0.02) and diffuse (P = 0.005) renal fibrosis than in cats without fibrosis. Cats with moderate renal inflammation had significantly higher urinary active aTGF-β1 concentrations than cats with mild (P = 0.035) or no inflammatory change (P = 0.004). The parameter aTGF-β1:UCr was independently associated with Log urine protein:creatinine ratio in a multivariable analysis of clinicopathological parameters and interstitial fibrosis score in a multivariable analysis of histopathological features. These results suggest that urinary aTGF-β1 reflects the severity of renal pathology. Increases in urinary aTGF-β1 followed longitudinally in individual cats may indicate the development of CKD. PMID:27387717

  14. Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta.

    PubMed

    Rahimi, Rod A; Andrianifahanana, Mahefatiana; Wilkes, Mark C; Edens, Maryanne; Kottom, Theodore J; Blenis, John; Leof, Edward B

    2009-01-01

    Transforming growth factor-beta (TGF-beta) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-beta signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-beta has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-beta signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-beta. We show that TGF-beta activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2-dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-beta-mediated anchorage-independent growth without affecting TGF-beta transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-beta action. mTORC2 promotes TGF-beta-induced morphologic transformation and is required for TGF-beta-induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-beta-mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-beta and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. PMID:19117990

  15. Developmental regulation of early mouse embryogenesis. A. Onset of insulin and related growth factor binding. B. Expression of selected proto-oncogenes

    SciTech Connect

    Mattson, B.A.

    1987-01-01

    Experimental and clinical evidence suggest a role for insulin and related growth factors in fetal growth. It was therefore of interest to determine the onset of insulin binding to cells of early embryos. These studies are the first to demonstrate stage-specific insulin binding during mouse preimplantation embryogenesis. Insulin binding was detected at the morula, blastocyst, and blastocyst outgrowth stages using indirect immunofluorescence and light microscopic autoradiographic techniques. No evidence of insulin binding was seen on unfertilized oocytes, two-, four-, or eight-cell embryos. Results of competitive inhibition studies using unlabeled insulin-like growth factors (IGFs) suggest binding of /sup 125/I-insulin to type I IGF receptors as well. Further studies are needed to confirm the presence of an IGF receptor in preimplantation embryos.

  16. Thyrotropin inhibits while insulin, epidermal growth factor and tetradecanoyl phorbol acetate stimulate insulin-like growth factor binding protein secretion from sheep thyroid cells.

    PubMed

    Eggo, M C; Bachrach, L K; Brown, A L; Burrow, G N

    1991-01-01

    Six insulin-like growth factor binding proteins (IGFBP) have been identified in the conditioned medium from sheep thyroid cells cultured under serum-free conditions. IGFBPs of 32, 28, 23 and 19 kDa were secreted by cells cultured for 14 days in serum-free and hormone-free medium. The constitutive secretion of IGFBP was inhibited by thyrotropin (TSH, 0.3 mU per mL). The effect was most marked on the secretion of the 28 kDa BP. High insulin concentrations stimulated the secretion of this IGFBP. The stimulatory effects of insulin were inhibited by TSH. Growth hormone treatment decreased the secretion of the 28 kDa protein. Tetradecanoylphorbol-13 acetate (TPA) and epidermal growth factor (EGF) both of which stimulate thyroid cell growth but inhibit differentiated function, markedly stimulated IGFBP secretion and induced the appearance of a 46 and a 150 kDa IGFBP. The effects of EGF and TPA were not identical. A rat IGFBP-2 cDNA reacted with sheep thyroid RNA of approximate size 1.6 kb. TPA treatment increased IGFBP-2 mRNA. Other hormones used to enhance differentiation and growth in thyroid cells in culture i.e. transferrin, somatostatin, cortisol and glycyl-histidyl-lysine acetate had no marked effects on IGFBP secretion nor on TSH-dependent, insulin-mediated iodide uptake and organification and cell growth. We show a correlation between secretion of high molecular weight IGFBP with enhanced growth but decreased function. Conversely, we find a correlation between decreased secretion of the 28 kDa BP and increased growth and function. PMID:1722684

  17. Effect of intestinal ischemia-reperfusion on expressions of endogenous basic fibroblast growth factor and transforming growth factor betain lung and its relation with lung repair.

    PubMed

    Fu, Xiao-Bing; Yang, Yin-Hui; Sun, Tong-Zhu; Gu, Xiao-Man; Jiang, Li-Xian; Sun, Xiao-Qing; Sheng, Zhi-Yong

    2000-06-01

    AIM:To study the changes of endogenous transforming growth factor beta(TGFbeta) and basic fibroblast growth factor (bFGF) in lung following intestinal ischemia and reperfusion injury and their effects on lung injury and repair.METHODS:Sixty Wistar rats were divided into five groups, which underwent sham-operation, ischemia (45 minutes), and reperfusion (6, 24 and 48 hours, respectively) after ischemia (45 minutes). Immunohistochemical method was used to observe the localization and amounts of both growth factors.RESULTS:Positive signals of both growth factors could be found in normal lung, mainly in alveolar cells and endothelial cells of vein. After ischemia and reperfusion insult, expressions of both growth factors were increased and their amounts at 6 hours were larger than those of normal control or of 24 and 48 hours after insult.CONCLUSION:The endogenous bFGF and TGF beta expression appears to be upregulated in the lung following intestinal ischemia and reperfusion, suggesting that both growth factors may be involved in the process of lung injury and repair. PMID:11819596

  18. Role of LncRNA-activated by transforming growth factor beta in the progression of hepatitis C virus-related liver fibrosis.

    PubMed

    Fu, Na; Niu, Xuemin; Wang, Yang; Du, Huijuan; Wang, Baoyu; Du, Jinghua; Li, Ya; Wang, Rongqi; Zhang, Yuguo; Zhao, Suxian; Sun, Dianxing; Qiao, Liang; Nan, Yuemin

    2016-08-01

    Long non-coding RNA (LncRNA)-activated by transforming growth factor-beta (LncRNA-ATB) is a key regulator of transforming growth factor-beta (TGF-β) signaling pathway, and is positively correlated with the development of liver cirrhosis and vascular invasion of hepatocellular carcinoma (HCC). However, the role of LncRNA-ATB in hepatitis C virus (HCV)-related liver fibrosis remains largely unknown. In the present study, we confirmed a high expression level of LncRNA-ATB in the liver tissues and plasma samples of patients with HCV-related hepatic fibrosis, and the plasma level of LncRNA-ATB was significantly correlated with liver fibrosis stages. Furthermore, increased expression level of LncRNA-ATB was also present in activated hepatic stellate cells (HSCs), and knockdown of LncRNA-ATB inhibited the expression of alpha-smooth muscle actin (α-SMA) and alpha-1 type I collagen (Col1A1). LncRNA-ATB was found to share the common miRNA responsive element of miR-425-5p with TGF-β type II receptor (TGF-βRII) and SMAD2. Ectopic expression of LncRNA-ATB in HSCs could upregulate the protein expression of TGF-βRII and SMAD2 by inhibiting the endogenous miR-425-5p. Moreover, overexpression of miR-425-5p could partly abrogate the expression of TGF-βRII and SMAD2 induced by LncRNA-ATB. Hence, we conclude that LncRNA-ATB promotes HCV-induced liver fibrogenesis by activating HSCs and increasing collagen I production through competitively binding to miR-425-5p. LncRNA-ATB may be a novel diagnostic biomarker and a potential therapeutic target for HCV-related hepatic fibrosis. PMID:27585228

  19. Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor.

    PubMed Central

    Rodríguez-Tébar, A; Dechant, G; Götz, R; Barde, Y A

    1992-01-01

    Neurotrophin-3 (NT-3) has low-affinity (Kd = 8 x 10(-10) M), as well as high-affinity receptors (Kd = 1.8 x 10(-11) M) on embryonic chick sensory neurons, the latter in surprisingly high numbers. Like the structurally related proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), NT-3 also binds to the low-affinity NGF receptor, a molecule that we suggest to designate low-affinity neurotrophin receptor (LANR). NT-3 dissociates from the LANR much more rapidly than BDNF, and more slowly than NGF. The binding of labelled NT-3 to the LANR can be reduced by half using a concentration of BDNF corresponding to the Kd of BDNF to the LANR. In contrast, the binding of NT-3 to its high-affinity neuronal receptors can only be prevented by BDNF or NGF when used at concentrations several thousand-fold higher than those corresponding to their Kd to their high-affinity neuronal receptors. Thus, specific high-affinity NT-3 receptors exist on sensory neurons that can readily discriminate between three structurally related ligands. These findings, including the remarkable property of the LANR to bind three related ligands with similar affinity, but different rate constants, are discussed. PMID:1547788

  20. Alternative binding modes identified for growth and differentiation factor-associated serum protein (GASP) family antagonism of myostatin.

    PubMed

    Walker, Ryan G; Angerman, Elizabeth B; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B

    2015-03-20

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  1. Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

    SciTech Connect

    Ponec, M.; Weerheim, A. ); Havekes, L. ); Boonstra, J. )

    1987-08-01

    The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

  2. Biglycan is a novel binding partner of fibroblast growth factor receptor 3c (FGFR3c) in the human testis.

    PubMed

    Winge, S B; Nielsen, J; Jørgensen, A; Owczarek, S; Ewen, K A; Nielsen, J E; Juul, A; Berezin, V; Rajpert-De Meyts, E

    2015-01-01

    Regulation of spermatogonial maintenance in the human testis is currently not well understood. One pathway suggested to be involved is activated by fibroblast growth factor receptor 3 (FGFR3), which is expressed in a subset of spermatogonia. FGFR3-activating mutations have been identified in spermatocytic seminoma, thought to originate from clonal expansion of spermatogonia. In this study we aimed to characterize potential binding partners of FGFR3, and specifically its mesenchymal "c" splice isoform, in human spermatogonia. Based on expression patterns and homology to the binding site, we identified FGF1, FGF2, and FGF9 as the best candidates for natural ligands of FGFR3c in the testis. In addition, we screened non-FGF proteins and found that a proteoglycan biglycan (BGN) contains a sequence homologous to the FGFR3c binding site on FGF1, and is expressed in peritubular cells adjacent to FGFR3-expressing spermatogonia. Experiments in a cell-free system confirmed that BGN binds to FGFR3c and FGF1. In conclusion, our findings further clarify the complex regulation of FGFR3c in the human testis. We postulate that BGN is a factor secreted by peritubular cells to modulate FGFR3c signaling and thus contributes to the regulation of spermatogonial maintenance. PMID:25260943

  3. Alternative Binding Modes Identified for Growth and Differentiation Factor-associated Serum Protein (GASP) Family Antagonism of Myostatin*

    PubMed Central

    Walker, Ryan G.; Angerman, Elizabeth B.; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B.

    2015-01-01

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  4. Energetic characterization of the basic fibroblast growth factor-heparin interaction: identification of the heparin binding domain.

    PubMed

    Thompson, L D; Pantoliano, M W; Springer, B A

    1994-04-01

    Fibroblast growth factors (FGF's) interact on cell surfaces with "low-affinity" heparan sulfate proteoglycans (HSPG) and "high-affinity" FGF receptors (FGFR) to initiate cell proliferation. Previous reports have implicated the binding of heparin, or heparan sulfate, to FGF as essential for FGF-mediated signal transduction and mitogenicity. However, the molecular recognition events which dictate the specificity of this interaction have remained elusive. Amino acid residues on the surface of basic FGF (bFGF) were targeted as potential heparin contacts on the basis of the position of sulfate anions in the X-ray crystal structure of bFGF and of a modeled pentasaccharide heparin-bFGF complex. Each identified amino acid was replaced individually with alanine by site-directed mutagenesis, and the resulting mutant proteins were characterized for differences in binding to a low molecular weight heparin (approximately 3000) by isothermal titrating calorimetry and also for differences in [NaCl] elution from a heparin-Sepharose affinity resin. The combination of site-directed mutagenesis and titrating calorimetry permitted an analysis of the energetic contributions of individual bFGF residues in the binding of heparin to bFGF. The key amino acids which comprise the heparin binding domain on bFGF constitute a discontinuous binding epitope and include K26, N27, R81, K119, R120, T121, Q123, K125, K129, Q134, and K135. Addition of the observed delta delta G degrees of binding for each single site mutant accounts for 8.56 kcal/mol (> 95%) of the free energy of binding. The delta delta G degrees values for N27A, R120A, K125A, and Q134A are all greater than 1 kcal/mol each, and these four amino acids together contribute 4.8 kcal/mol (56%) to the total binding free energy. Amino acid residues K119 through K135 reside in the C-terminal domain of bFGF and collectively contribute 6.6 kcal/mol (76%) of the binding free energy. Although 7 out of the 11 identified amino acids in the heparin

  5. The coordinate cellular response to insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-2 (IGFBP-2) is regulated through vimentin binding to receptor tyrosine phosphatase β (RPTPβ).

    PubMed

    Shen, Xinchun; Xi, Gang; Wai, Christine; Clemmons, David R

    2015-05-01

    Insulin-like growth factor-binding protein-2 (IGFBP-2) functions coordinately with IGF-I to stimulate cellular proliferation and differentiation. IGFBP-2 binds to receptor tyrosine phosphatase β (RPTPβ), and this binding in conjunction with IGF-I receptor stimulation induces RPTPβ polymerization leading to phosphatase and tensin homolog inactivation, AKT stimulation, and enhanced cell proliferation. To determine the mechanism by which RPTPβ polymerization is regulated, we analyzed the protein(s) that associated with RPTPβ in response to IGF-I and IGFBP-2 in vascular smooth muscle cells. Proteomic experiments revealed that IGF-I stimulated the intermediate filament protein vimentin to bind to RPTPβ, and knockdown of vimentin resulted in failure of IGFBP-2 and IGF-I to stimulate RPTPβ polymerization. Knockdown of IGFBP-2 or inhibition of IGF-IR tyrosine kinase disrupted vimentin/RPTPβ association. Vimentin binding to RPTPβ was mediated through vimentin serine phosphorylation. The serine threonine kinase PKCζ was recruited to vimentin in response to IGF-I and inhibition of PKCζ activation blocked these signaling events. A cell-permeable peptide that contained the vimentin phosphorylation site disrupted vimentin/RPTPβ association, and IGF-I stimulated RPTPβ polymerization and AKT activation. Integrin-linked kinase recruited PKCζ to SHPS-1-associated vimentin in response to IGF-I and inhibition of integrin-linked kinase/PKCζ association reduced vimentin serine phosphorylation. PKCζ stimulation of vimentin phosphorylation required high glucose and vimentin/RPTPβ-association occurred only during hyperglycemia. Disruption of vimetin/RPTPβ in diabetic mice inhibited RPTPβ polymerization, vimentin serine phosphorylation, and AKT activation in response to IGF-I, whereas nondiabetic mice showed no difference. The induction of vimentin phosphorylation is important for IGFBP-2-mediated enhancement of IGF-I-stimulated proliferation during hyperglycemia, and it

  6. Structural Variability of Tropospheric Growth Factors Transforming Mid-latitude Cyclones to Severe Storms over the North Atlantic

    NASA Astrophysics Data System (ADS)

    Wild, Simon; Befort, Daniel J.; Leckebusch, Gregor C.

    2015-04-01

    The development of European surface wind storms out of normal mid-latitude cyclones is substantially influenced by upstream tropospheric growth factors over the Northern Atlantic. The main factors include divergence and vorticity advection in the upper troposphere, latent heat release and the presence of instabilities of short baroclinic waves of suitable wave lengths. In this study we examine a subset of these potential growth factors and their related influences on the transformation of extra-tropical cyclones into severe damage prone surface storm systems. Previous studies have shown links between specific growth factors and surface wind storms related to extreme cyclones. In our study we investigate in further detail spatial and temporal variability patterns of these upstream processes at different vertical levels of the troposphere. The analyses will comprise of the three growth factors baroclinicity, latent heat release and upper tropospheric divergence. Our definition of surface wind storms is based on the Storm Severity Index (SSI) alongside a wind tracking algorithm identifying areas of exceedances of the local 98th percentile of the 10m wind speed. We also make use of a well-established extra-tropical cyclone identification and tracking algorithm. These cyclone tracks form the base for a composite analysis of the aforementioned growth factors using ERA-Interim Reanalysis from 1979 - 2014 for the extended winter season (ONDJFM). Our composite analysis corroborates previous similar studies but extends them by using an impact based algorithm for the identification of strong wind systems. Based on this composite analysis we further identify variability patterns for each growth factor most important for the transformation of a cyclone into a surface wind storm. We thus also address the question whether the link between storm intensity and related growth factor anomaly taking into account its spatial variability is stable and can be quantified. While the

  7. Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    PubMed Central

    Diao, Jingyu; Pantua, Homer; Ngu, Hai; Komuves, Laszlo; Diehl, Lauri; Schaefer, Gabriele

    2012-01-01

    While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR. PMID:22855500

  8. A 4-deoxy analogue of N-acetyl-D-glucosamine inhibits heparan sulphate expression and growth factor binding in vitro

    SciTech Connect

    Wijk, Xander M.R. van; Oosterhof, Arie; Broek, Sebastiaan A.M.W. van den; Griffioen, Arjan W.; Dam, Gerdy B. ten; Rutjes, Floris P.J.T.; Delft, Floris L. van; Kuppevelt, Toin H. van

    2010-09-10

    Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -{beta}1-4GlcA-{alpha}1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-D-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect. 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.

  9. A Glycine-rich RNA-binding Protein Mediating Cold-inducible Suppression of Mammalian Cell Growth

    PubMed Central

    Nishiyama, Hiroyuki; Itoh, Katsuhiko; Kaneko, Yoshiyuki; Kishishita, Masamichi; Yoshida, Osamu; Fujita, Jun

    1997-01-01

    In response to low ambient temperature, mammalian cells as well as microorganisms change various physiological functions, but the molecular mechanisms underlying these adaptations are just beginning to be understood. We report here the isolation of a mouse cold-inducible RNA-binding protein (cirp) cDNA and investigation of its role in cold-stress response of mammalian cells. The cirp cDNA encoded an 18-kD protein consisting of an amino-terminal RNAbinding domain and a carboxyl-terminal glycine-rich domain and exhibited structural similarity to a class of stress-induced RNA-binding proteins found in plants. Immunofluorescence microscopy showed that CIRP was localized in the nucleoplasm of BALB/3T3 mouse fibroblasts. When the culture temperature was lowered from 37 to 32°C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37°C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37°C with prolongation of G1 phase of the cell cycle. These results indicate that CIRP plays an essential role in cold-induced growth suppression of mouse fibroblasts. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in mammalian cells. PMID:9151692

  10. Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an autocrine mechanism.

    PubMed Central

    Mead, J E; Fausto, N

    1989-01-01

    We investigated whether transforming growth factor alpha (TGF-alpha) is involved in hepatocyte growth responses both in vivo and in culture. During liver regeneration after partial hepatectomy in rats, TGF-alpha mRNA increased; it reached a maximum (approximately 9-fold higher than normal) at the peak of DNA synthesis. The message and the peptide were localized in hepatocytes and found in higher amounts in hepatocytes obtained from regenerating liver. TGF-alpha caused a 13-fold elevation of DNA synthesis in hepatocytes in primary culture and was slightly more effective than epidermal growth factor. TGF-beta blocked TGF-alpha stimulation when added either simultaneously with TGF-alpha or a day later. TGF-alpha message increased in hepatocytes stimulated to undergo DNA synthesis by TGF-alpha or epidermal growth factor, and the peptide was detected in the culture medium by RIA. In the regenerating liver, the increase in TGF-alpha mRNA during the first day after partial hepatectomy coincided with an increase in epidermal growth factor/TGF-alpha receptor mRNA and a decrease (already reported) in the number of these receptors. We conclude that TGF-alpha may function as a physiological inducer of hepatocyte DNA synthesis during liver regeneration by means of an autocrine mechanism and that its stimulatory effects in this growth process are balanced by the inhibitory action of TGF-beta 1. Images PMID:2922399

  11. Stimulation of glycolysis and amino acid uptake in NRK-49F cells by transforming growth factor beta and epidermal growth factor.

    PubMed Cent