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PubMed2001

Ovarian granulosa cell tumors express a functional membrane receptor for anti-Müllerian hormone in transgenic mice.

Anti-Müllerian hormone inhibits granulosa cell growth and function. Both anti-Müllerian hormone and its type II receptor are expressed in normal granulosa cells. We show by histologic and molecular analyses that ovarian tumors developing in transgenic mice, obtained by targeted oncogenesis using an anti-Müllerian hormone promoter-SV40 oncogene construct, are of granulosa-cell origin. Because tissue-specific, cell-surface molecules are of particular interest for the analysis and treatment of tumors, we examined the expression of anti-Müllerian hormone type II receptor in the ovaries of these transgenic mice. We demonstrate that the anti-Müllerian hormone type II receptor is expressed not only in normal ovarian follicles, but also in granulosa cell tumors. Using a cell line derived from one of these tumors, we show that the anti-Müllerian hormone type II receptor protein is present on the surface of tumor cells and binds anti-Müllerian hormone. Furthermore, we show that the anti-Müllerian hormone receptor is functional in the granulosa tumor cell line, with anti-Müllerian hormone treatment inducing selective activation of Smad1. In conclusion, in this study we present a new murine transgenic model of granulosa cell tumors of the ovary and, using this model, we demonstrate for the first time cell-surface expression of a highly tissue-specific molecule, anti-Müllerian hormone type II receptor, as well as the selective activation of Smad proteins by anti-Müllerian hormone, in granulosa tumor cells.

Animals↗
PubMed2001

Suppression of head formation by Xmsx-1 through the inhibition of intracellular nodal signaling.

It is well established that in Xenopus, bone morphogenetic protein (BMP) ventralizes the early embryo through the activation of several target genes encoding homeobox proteins, some of which are known to be necessary and sufficient for ventralization. Here, we used an inhibitory form of Xmsx-1, one of BMP's targets, to examine its role in head formation. Interestingly, ventral overexpression of a dominant Xmsx-1 inhibitor induced an ectopic head with eyes and a cement gland in the ventral side of the embryo, suggesting that Xmsx-1 is normally required to suppress head formation in the ventral side. Supporting this observation, we also found that wild-type Xmsx-1 suppresses head formation through the inhibition of nodal signaling, which is known to induce head organizer genes such as cerberus, Xhex and Xdkk-1. We propose that negative regulation of the BMP/Xmsx-1 signal is involved not only in neural induction but also in head induction and formation. We further suggest that the inhibition of nodal signaling by Xmsx-1 may occur intracellularly, through interaction with Smads, at the level of the transcriptional complex, which activates the activin responsive element.

Animals↗
PubMed2002

A novel Xenopus Smad-interacting forkhead transcription factor (XFast-3) cooperates with XFast-1 in regulating gastrulation movements.

In early Xenopus embryos, the prototypical XFast-1/Smad2/Smad4 complex ARF1 is induced at the Mix.2 ARE by activin overexpression. We have characterised ARF2, a related, but much more abundant, complex formed during gastrulation in response to endogenous TGFbeta family members and we have identified a novel Fast family member, XFast-3, as its transcription factor component. Endogenous ARF2 efficiently competes out ARF1 at early gastrulation, due to the ability of XFast-3 to interact with activated Smads with much higher affinity than XFast-1. We demonstrate that ARF1 and ARF2 are activated by distinct TGFbeta family members. Using morpholino antisense oligonucleotides to deplete levels of the constituent transcription factors XFast-1 and XFast-3 specifically, we demonstrate an important role for ARF1 and ARF2 in early Xenopus embryos in controlling the convergent extension movements of gastrulation.

3T3 Cells↗
PubMed2002

The roles of three signaling pathways in the formation and function of the Spemann Organizer.

Since the three main pathways (the Wnt, VegT and BMP pathways) involved in organizer and axis formation in the Xenopus embryo are now characterized, the challenge is to understand their interactions. Here three comparisons were made. Firstly, we made a systematic comparison of the expression of zygotic genes in sibling wild-type, VegT-depleted (VegT(-)), beta-catenin-depleted (beta-catenin(-)) and double depleted (VegT(-)/beta-catenin(-)) embryos and placed early zygotic genes into specific groups. In the first group some organizer genes, including chordin, noggin and cerberus, required the activity of both the Wnt pathway and the VegT pathway to be expressed. A second group including Xnr1, 2, 4 and Xlim1 were initiated by the VegT pathway but their dorsoventral pattern and amount of their expression was regulated by the Wnt pathway. Secondly, we compared the roles of the Wnt and VegT pathways in producing dorsal signals. Explant co-culture experiments showed that the Wnt pathway did not cause the release of a dorsal signal from the vegetal mass independent from the VegT pathway. Finally we compared the extent to which inhibiting Smad 1 phosphorylation in one area of VegT(-), or beta-catenin(-) embryos would rescue organizer and axis formation. We found that BMP inhibition with cm-BMP7 mRNA had no rescuing effects on VegT(-) embryos, while cm-BMP7 and noggin mRNA caused a complete rescue of the trunk, but not of the anterior pattern in beta-catenin(-) embryos.

Animals↗
PubMed2001

Smad regulation in TGF-beta signal transduction.

Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. Alternatively, nuclear R-Smads associate with ubiquitin ligases and promote degradation of transcriptional repressors, thus facilitating target gene regulation by TGF-beta. Smads themselves can also become ubiquitinated and are degraded by proteasomes. Finally, the inhibitory Smads (I-Smads) block phosphorylation of R-Smads by the receptors and promote ubiquitination and degradation of receptor complexes, thus inhibiting signalling.

Activin Receptors, Type I↗
PubMed2004

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin.

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K(d) 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

Activins↗
PubMed2006

Renal bone morphogenetic protein-7 protects against diabetic nephropathy.

Longstanding diabetes causes renal injury with early dropout of podocytes, albuminuria, glomerular and tubulointerstitial fibrosis, and progressive renal failure. The renal pathology seems to be driven, in part, by TGF-beta and is associated with a loss of renal bone morphogenic protein-7 (BMP-7) expression. Here, the hypothesis that maintenance of renal (especially podocyte) BMP-7 by transgenic expression reduces diabetic renal injury was tested. Diabetic mice that expressed the phosphoenolpyruvate carboxykinase promoter-driven BMP-7 transgene and nondiabetic, transgenic mice as well as diabetic and nondiabetic wild-type controls were studied for up to 1 yr. Transgenic expression of BMP-7 in glomerular podocytes and proximal tubules prevents podocyte dropout and reductions in nephrin levels in diabetic mice. Maintenance of BMP-7 also reduces glomerular fibrosis and interstitial collagen accumulation as well as collagen I and fibronectin expression. Diabetic wild-type mice develop progressive albuminuria, which is substantially reduced in transgenic mice. These effects of the BMP-7 transgene occur without changing renal TGF-beta levels. It is concluded that maintenance of renal BMP-7 during the evolution of diabetic nephropathy reduces diabetic renal injury, especially podocyte dropout. The findings also establish a role for endogenous glomerular BMP-7 as an autocrine regulator of podocyte integrity in vivo.

Animals↗
PubMed2001

The Smad transcriptional corepressor TGIF recruits mSin3.

The homeodomain protein TG-interacting factor (TGIF) represses transcription by histone deacetylase-dependent and -independent means. Heterozygous mutations in human TGIF result in holoprosencephaly, a severe genetic disorder affecting craniofacial development, suggesting that TGIF is critical for normal development. After transforming growth factorbeta (TGFbeta) stimulation, Smad proteins enter the nucleus and form transcriptional activation complexes or interact with TGIF, which functions as a corepressor. The relative levels of Smad corepressors and coactivators present within the cell may determine the outcome of a TGFbeta response. We show that TGIF interacts directly with the paired amphipathic alpha-helix 2 domain of the mSin3 corepressor, and TGIF recruits mSin3 to a TGFbeta-activated Smad complex. The mSin3 interaction domain of TGIF has been shown to be essential for repression of a TGFbeta transcriptional response. Thus, TGIF represents a targeting component of the mSin3 corepressor complex.

Animals↗
PubMed2004

[Multidrug resistant effect of alternative splicing form of MAD2 gene-MAD2beta on human gastric cancer cell].

OBJECTIVE: To study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901. METHODS: RNA was extracted from a multidrug resistance cell line SGC7901/ADR. The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction. Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine. Sensitivity to drug was detected by MTT assay. Cell cycle alteration and intracellular fluorescence intensity were determined by FACS. RESULTS: A fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta. pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells. CONCLUSION: MAD2beta could increase the multidrug resistance of SGC7901 cell line.

Adenocarcinoma↗
PubMed2004

[Studies on Smads at transcription level in liver fibrosis of mice with Schistosomiasis japonica].

OBJECTIVE: To study Smads involved in TGF-beta signal transduction at the transcription level during the development of liver fibrosis in BALB/c mice infected by Schistosoma japonicum. METHODS: BALB/c mice infected with cercariae of S. japonicum were used as liver fibrosis models. Liver specimens were harvested at 8, 12, 16 and 24 weeks after infection and normal control were sacrificed at the 24th week. A part of the liver specimens were preserved for pathologic examination and the other part was frozen for the detection of mRNA level of Smad 2, Smad 3, Smad 4 and Smad 7. RESULTS: The level of Smad 3 mRNA was significantly higher than that of control at the later stage, while the mRNA level of Smad 2 decreased at 12 and at 24 weeks, respectively. No significant difference in the mRNA level of Smad 4 and Smad 7 was observed between the infection group and the control. CONCLUSION: Smad 3 may induce the development of liver fibrosis in mice infected by S. japonicum while Smad 2 may induce the development of liver fibrosis at early stage and inhibit it at later stage.

Animals↗
PubMedDate not supplied

Comparison of transforming growth factor-beta/Smad signaling between normal dermal fibroblasts and fibroblasts derived from central and peripheral areas of keloid lesions.

BACKGROUND: Keloids are benign proliferations of fibroblasts, but their exact etiology and molecular pathogenesis are unknown. MATERIALS AND METHODS: Fibroblasts were isolated from the central and peripheral regions of keloids, and the growth behavior and molecular characteristics of the keloid fibroblasts were compared with those of age-adjusted normal dermal fibroblasts. RESULTS: Central (but not peripheral) keloid fibroblasts exhibited significantly increased growth and high levels of expression of transforming growth factor-beta (TGF-beta) receptor 1 and Smad 2/3. CONCLUSION: Proliferation of central keloid fibroblasts, which results in keloid formation, appears to mainly involve the TGF-beta/Smad pathway.

Actins↗
PubMed1996

Somatic in vivo alterations of the JV18-1 gene at 18q21 in human lung cancers.

The chromosome region 18q21 is frequently deleted in lung cancers. Recent identification of JV18-1 at this locus led us to examine whether or not it might also be altered in lung cancers, as is the case for the closely related DPC4 tumor suppressor gene. A missense somatic mutation and a 9-bp in-frame deletion were detected in the highly conserved region of JV18-1 among 57 lung cancer specimens taken directly from patients. The total alterations in JV18-1 and DPC4, however, are not sufficient to account for all 18q21 deletions in lung cancers. These findings suggest that although JV18-1 and DPC4 may play roles in a limited fraction of lung cancers, another tumor suppressor gene may also exist in this chromosome region.

Carcinoma, Non-Small-Cell Lung↗
PubMed2001

Possible involvement of protein kinases and Smad2 signaling pathways on osteoclast differentiation enhanced by activin A.

Bone tissues reportedly contain considerable amounts of activin A and follistatin, an activin A-binding protein. In the present study, we found that follistatin strongly inhibited osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts induced by 1alpha,25 dihydroxyvitamin D(3), prostaglandin E(2), and interleukin-1alpha. Antibody aganist activin A also inhibited the osteoclast formation. Furthermore, activin A synergistically stimulated osteoclast differentiation mediated by receptor activator NF-kappaB ligand (RANKL). RT-PCR analysis revealed that osteoblasts produced not only activin A but also follistatin. Western blot analysis of a panel of phosphorylated proteins revealed that activin A stimulated the phosphorylation of p44/42 mitogen activated protein (MAP) kinase (ERK1/2) and p38 MAP kinase in macrophage colony-stimulating factor-dependent bone marrow macrophages (M-BMMPhis). In addition, phosphorylation of Smad2 was observed in M-BMMPhis stimulated with activin A. These findings indicate that the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and Smad2 is involved in activin A-enhanced osteoclast differentiation induced by RANKL. Taken together, these results suggest that both activin A and follistatin produced by osteoblasts may play an important role in osteoclast differentiation through MAP kinases and Smad2 signaling pathways.

Activin Receptors↗
PubMed2004

Itch E3 ligase-mediated regulation of TGF-beta signaling by modulating smad2 phosphorylation.

Protein ubiquitination has been implicated in the intracellular biochemical events transduced by TGF-beta receptor via different mechanisms including the degradation of Smads or their binding proteins. Here we show that loss of Itch E3 ligase in mouse embryonic fibroblasts (MEFs) results in reduced susceptibility of TGF-beta-induced cell growth arrest and decreased phosphorylation of Smad2, without apparent alteration in protein levels for Smad2, Smad4, and Smad7 in Itch-/- MEFs. Itch promotes ubiquitination of Smad2 and augments Smad2 phosphorylation that requires an intact ligase activity of Itch. Moreover, Itch facilitates complex formation between TGF-beta receptor and Smad2 and enhances TGF-beta-induced transcription. This study reveals a previously unrecognized positive TGF-beta signaling pathway via proteolysis-independent ubiquitination.

Animals↗
PubMed1999

Ubiquitin-dependent degradation of TGF-beta-activated smad2.

SMAD proteins are phosphorylated by transforming growth factor-beta (TGF-beta) receptors and translocate to the nucleus, where they control transcription. Here we investigate the fate of activated Smad2. We show that receptor-mediated activation leads to multi-ubiquitination and subsequent degradation of Smad2 by the proteasome. Ubiquitination of Smad2 is a consequence of its accumulation in the nucleus. If degradation is averted, the phosphorylated Smad2 remains in the nucleus in an active state. By targeting Smad2 for destruction, TGF-beta ensures the irreversible termination of its own signalling function.

Animals↗
PubMed2002

Effects of salvianolic acid-B on TGF-beta 1 stimulated hepatic stellate cell activation and its intracellular signaling.

OBJECTIVE: To investigate hepatic stellate cells (HSC) responses at different differentiation stages on transforming growth factor-beta 1, and to elucidate the mechanisms of salvianolic acid - B (SA-B), a water soluble compound from Salvia miltiorrhiza, against hepatic fibrosis, relating to interference with TGF-beta 1 stimulated HSC activation and intracellular signal transduction via Smads. METHODS: HSC was isolated from rat by in situ perfusion of liver and 8.2% nycondenz gradient centrifugation, and primarily cultured on uncoated plastic for 1 d, 4 d and 7 d respectively, which represented quiescent, intermediate and activated phenotypes. The cells were stimulated with 100 pmol/L TGF-beta 1, cell phenotypes were observed under inverted microscope, alpha-actin expression was checked by Western blot, and collagen secretion was measured with [(3)H] proline incorporation and collangenase digestion, then HSC at one definite differentiation stage that responded most sensitively to TGF-beta 1 was selected as the cell model for the following study. 0.1 micromol/L - 1 mmol/L SA-B was incubated with HSC and the cell proliferation was measured by intracellular [(3)H] thymidine pulse. SA-B was also incubated with TGF-beta 1 stimulated HSC, the collagen secretion was measured as above, alpha-actin and plasmin activator inhibitor-1 (PAI-1) were checked with Western blot, and alpha1 (I) procollagen mRNA levels were analyzed with Northern blot. The cytoplasmic and nuclear proteins were extracted, and cytoplasmic and nuclear Smad2, 3 expression and phosphorylation levels were measured with Western blot. RESULTS: As culture duration prolonged, HSC phenotypes underwent activation gradually, accompanied by the increase of alpha-actin expression and collagen secretion. TGF-beta 1 increased the basal collagen levels at d1, d4 and d7 by 128.6%, 207.7% and 188.2% of the control respectively, while d4 HSC had the most sensitive response, and this intermediate HSC was used as cell model for the following study. Except 0.1 mmol/L-1 mmol/L SA-B caused parts of HSC death, 0.1 micromol/L-10 micromol/L SA-B had no influence on cell shape, but decreased HSC proliferation in a dose depend manner, by 76%, 60.1% and 47.8% of the control respectively. 1 micromol/L-10 micromol/L SA-B remarkably inhibited the collagen secretion of TGF-beta 1 stimulated HSC by 68.6% and 56.1% of the control, PAI-1 and alpha-actin expression, and down-regulated alpha 1 (I) pro-collagen gene expression. 0.1 micro mol/L approximately 10 micro mol/L SA-B decreased the cytoplasmic and nuclear Smad2, 3 protein expression, especially inhibited Smad2 phosphorylation and nuclear translocation. CONCLUSIONS: SA-B obviously inhibits intermediate HSC proliferation, decreases TGF-beta 1 stimulated HSC activation and matrix protein and gene expression, and inhibited stimulated HSC Smad2, 3 protein expression, phosphorylation and nuclear translocation. The inhibition of TGF-beta 1 signaling in HSC and its biological responses is the important mechanism of SA-B against hepatic fibrosis.

Actins↗
PubMed1999

c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads.

Smads are intracellular signaling mediators of the transforming growth factor-beta (TGF-beta) superfamily that regulates a wide variety of biological processes. Among them, Smads 2 and 3 are activated specifically by TGF-beta. We identified c-Ski as a Smad2 interacting protein. c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-beta-dependent manner. c-Ski contains two distinct Smad-binding sites with different binding properties. c-Ski strongly inhibits transactivation of various reporter genes by TGF-beta. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3 with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-beta signaling.

Acetyltransferases↗