Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Smad2 Protein”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2007

Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2-dependent TGF-beta signaling.

The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium-derived nitric oxide in endoglin(+/-) (Eng(+/-)) mice and cultured endothelial cells. Here, we find that TGF-beta1 leads to an increased vasodilatation in Eng(+/+) mice that is severely impaired in Eng(+/-) mice, suggesting the involvement of endoglin in the TGF-beta regulated vascular homeostasis. The endoglin-dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF-beta receptor ALK5 and its downstream substrate Smad2. In addition, Smad2-specific signaling is upregulated in endoglin-induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin-dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin-inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome-dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF-beta signaling, and leading to an increased eNOS expression in endothelial cells.

Animals↗
PubMed2004

Simultaneous triggering of protein activity and fluorescence.

Many areas of biology can benefit greatly from methods to spatially and temporally control protein activity. Here, we describe an approach that allows the simultaneous photo-triggering of the activity and the fluorescence of a protein. Smad2, a protein central to the transforming growth factor-beta (TGF-beta) signal transduction pathway, was modified with a fluorophore and a photocleavable moiety that acted as both a caging and a fluorescence quenching group. In its caged state, the protein formed a non-fluorescent heterodimer with the protein SARA. Irradiation with UV light and photocleavage of the caging group produced a fluorescent homotrimer. These in vitro experiments demonstrated that a photochemical trigger mimicking the critical biochemical event of serine phosphorylation involved in the TGF-beta signaling pathway could be obtained and that fluorescence could be used as a read-out of protein activity. This approach should prove particularly useful for the monitoring of a protein's activity and location inside of living cells.

Chromatography, Gel↗
PubMed1999

A quantitative analysis of signal transduction from activin receptor to nucleus and its relevance to morphogen gradient interpretation.

Previous work has shown that Xenopus blastula cells sense activin concentration by assessing the absolute number of occupied receptors per cell (100 and 300 molecules of bound activin activate Xbra and Xgsc transcription, respectively; a difference of only 3-fold). We now ask how quantitative differences in the absolute number of occupied receptors lead to the qualitatively distinct gene responses in the nucleus through SMAD2, a transducer of concentration-dependent gene responses to activin. We show that the injection of 0.2 or 0.6 ng of Smad2 mRNA activates Xbra or Xgsc transcription, respectively, involving, again, only a 3-fold difference. Furthermore, Xbra transcription is down-regulated by overexpression of SMAD2 as it is after activin signaling. We have developed a method to isolate nuclei from animal cap cells and subsequently have quantified the amount of nuclear SMAD2 protein. We find that the injection of 0.2 or 0.6 ng of Smad2 mRNA into an egg leads to only a 3-fold difference in the amount of SMAD2 protein in the nuclei of the blastula cells that express Xbra or Xgsc. We conclude that a 3-fold difference in the absolute number of occupied activin receptors can be maintained only as a 3-fold difference in the level of nuclear SMAD2 protein. Therefore, in this example of morphogen action, there appears to be no amplification of a key cytoplasmic transduction response, and a small but developmentally important change in extracellular signal concentration is relayed directly to the nucleus.

Activin Receptors↗
PubMedDate not supplied

A novel dominant negative Smad2 mutation in a TGFbeta resistant human carcinoma cell line.

An important biological mechanism contributing to the transformed phenotype of cancer cells is the ability to escape normal growth regulatory signals. In epithelial cells, transforming growth factor beta (TGFbeta) inhibits cell cycle progression in the G1-phase. The abnormal ability of cancer cells to escape TGFbeta-induced cell cycle inhibition may lead to deregulated mitosis, providing a growth advantage to these clones. The effects of TGFbeta are mediated through type I and type II receptors, which are transmembrane proteins possessing cytoplasmic serine/threonine kinase domains for signal propagation. TGFbeta binds to the type II receptor which then phosphorylates the cytoplasmic domain of the type I receptor. The receptor complex recruits and phosphorylates the downstream signaling proteins, Smad2 and Smad3, which then associate with Smad4. The Smad complex translocates to the nucleus to regulate target gene expression resulting in cell cycle inhibition. We have identified a new Smad2 mutation in a TGFbeta-resistant human carcinoma line. The mutant Smad2 protein exhibits decreased association with the receptor complex, is not phosphorylated in response to TGFbeta, fails to associate with Smad4 and does not localize to the nucleus. Expression of the mutant Smad2 protein in a TGFbeta sensitive carcinoma line induces resistance to this growth inhibitory factor and deregulates TGFbeta-responsive gene expression. These results indicate that this novel Smad2 mutant protein has dominant negative activity in cultured cells.

Amino Acid Sequence↗
PubMed1999

Evidence that Smad2 is a tumor suppressor implicated in the control of cellular invasion.

The Smad2 protein plays an essential role in the transforming growth factor-beta (TGF-beta) signaling pathway. This pathway mediates growth inhibitory signals from the cell surface to the nucleus. Although Smad2 protein is significantly mutated in human cancers, there is no definitive evidence implicating Smad2 as a tumor-suppressor gene. Here we show that overexpression of the tumor-derived missense mutation Smad2.D450E, an unphosphorylable form of Smad2 found in colorectal and lung cancers, did not abolish the TGF-beta-mediated growth arrest, suggesting that resistance to the growth-inhibiting effects of TGF-beta exhibited by human tumors cannot be linked to the inactivation of Smad2 protein. In contrast, overexpression of Smad2.D450E induces cellular invasion, and this effect was enhanced by TGF-beta. A similar invasive phenotype was obtained in cells expressing another inactivating mutation in Smad2 (Smad2.P445H) found in colorectal cancer. These findings indicate that genetic defects in Smad2 are sufficient to confer the invasion-promoting effect of TGF-beta and reveal that TGF-beta acts through Smad2 to induce cellular invasion by a novel mechanism that is independent of Smad2 phosphorylation by the activated TGF-beta type I receptor.

Animals↗
PubMed2001

Transforming growth factor-beta induced collagenase-3 production in human osteoarthritic chondrocytes is triggered by Smad proteins: cooperation between activator protein-1 and PEA-3 binding sites.

OBJECTIVE: To examine the signaling pathways leading to transforming growth factor-beta (TGF-beta) induced collagenase-3 production in human osteoarthritic (OA) chondrocytes, as well as the transcription factors and their binding sites involved in the transcriptional control of collagenase-3 gene. METHODS: Identification of the TGF-beta signaling pathway was by Western immunoblotting using specific antibodies for the phosphorylated forms of p44/42 and p38 MAPK, SAPK/JNK, and the Smad2 protein. Electromobility shift assays (EMSA) were carried out for activator protein- (AP-1), polyomavirus enhancer A (PEA-3), activin-response-element-like, Smad-binding-element-like, and TGF-beta inhibitory element oligonucleotides. Supershift assays using antibodies to the Jun, Fos, and Smad families of proteins were used for identification of transcription factors. Chondrocyte transfections were also performed using the -133CAT collagenase-3 promoter plasmid (containing PEA-3, AP-1, and TATA sites) and mutated AP-1 and PEA-3 sites. RESULTS: The primary target of TGF-beta induced collagenase-3 in OA chondrocytes was the Smad2 protein, with significant phosphorylation within 5 min. Contrasting with the Smad2, the untreated OA chondrocytes already had detectable levels of the phosphorylated forms of p38 and p44/42 MAPK. Of the oligonucleotides tested, EMSA revealed that TGF-beta treated OA chondrocyte proteins bound only to the AP-1 and PEA-3. Supershifts with the AP-1 oligonucleotide showed the presence of the Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) proteins in the untreated and TGF-beta treated OA chondrocytes, whereas only Smad proteins (Smad2, 3, 4) were present in the AP-1 binding proteins from the TGF-beta treated chondrocytes. The AP-1 mutation decreased both basal (95%) and TGF-beta induced (99%) collagenase-3 production, whereas the PEA-3 mutation decreased the basal (15%) but more significantly (50%) the TGF-beta induced transcription. CONCLUSION: Smad proteins are the main cytoplasmic signaling pathways in TGF-beta stimulated collagenase-3 in OA chondrocytes. The AP-1 site appears critical for upregulation of collagenase-3 production, but TGF-beta stimulation requires both AP-1 and PEA-3 sites for optimal response.

Aged↗
PubMed1998

Abrogation of Smad3 and Smad2 or of Smad4 gene expression positively regulates murine embryonic lung branching morphogenesis in culture.

Smad genes are recently identified intracellular effectors for receptor signaling in the BMP/activin/TGF-beta pathway. Since TGF-beta ligands are known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad genes negatively regulate lung organogenesis. Antisense oligodeoxynucleotides were designed to attenuate Smad3 and Smad2 gene expression in embryonic (E11) mouse lungs over 4 days in culture. Endogenous Smad3 and Smad2 mRNA levels were suppressed by 97 and 91%, respectively, in cultured embryonic lungs when antisense oligodeoxynucleotide (40 microM) to Smad was added, compared to scrambled and sense sequence controls. The corresponding Smad3 and Smad2 protein amounts were also decreased respectively by 86 and 90% in lungs treated with Smad3 and Smad2 antisense oligodeoxynucleotide. Phenotypically, Smad antisense oligodeoxynucleotides resulted in a concentration-dependent increase in lung branching: embryonic lung branching was stimulated by up to 53% in culture with 40 microM antisense oligodeoxynucleotide, whereas both scrambled and sense controls showed no stimulatory effect. Thus, inhibition of endogenous Smad3 and Smad2 gene expression resulted in stimulation of embryonic lung branching similar to that caused by inhibition of TGF-beta type II receptor expression and signaling (J. Zhao et al., 1996, Dev. Biol. 180, 242-257). Abrogation of Smad4 (DPC4), the downstream mediator of Smad3 and Smad2 proteins, with antisense oligodeoxynucleotide, also resulted in increased branching morphogenesis. Furthermore, while TGF-beta alone inhibited lung branching morphogenesis in culture, addition of exogenous TGF-beta 1 could not overcome the stimulatory effect on lung branching of Smad antisense oligodeoxynucleotide treatment. By immunohistochemistry, Smad proteins were localized mainly to the epithelial cells lining the branching distal airways, indicating that Smad genes could regulate lung morphogenesis through mesoderm-endoderm interaction. Our results demonstrate, for the first time, that abrogation of Smad2 and Smad3 or of Smad4 gene expression stimulated early mouse embryonic lung branching morphogenesis in culture, possibly through reversing the negative influence of endogenous TGF-beta signaling upon lung branching morphogenesis.

Animals↗
PubMed2005

Restoration of Smad4 in BxPC3 pancreatic cancer cells attenuates proliferation without altering angiogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human malignancy in which the transforming growth factor beta (TGF-beta) signal transducer, Smad4, is commonly mutated or deleted. BxPC3 human pancreatic cancer cells exhibit a homozygous deletion of the Smad4 gene, yet are growth inhibited by TGF-beta1. In the present study, we sought to determine whether reintroduction of Smad4 into BxPC3 cells alters their behavior in vitro and in vivo. Sham transfected and Smad4 expressing BxPC3 cells exhibited similar responses to TGF-beta1 with respect to p21 upregulation, hypophosphorylation of the RB protein, Smad2 phosphorylation, and Smad2/3 nuclear translocation. TGF-beta1 did not alter p27 expression, and silencing of p21 with an appropriate siRNA markedly attenuated TGF-beta1-mediated growth inhibition. Nonetheless, the presence of Smad4 was associated in vitro with a more prolonged doubling time, enhanced sensitivity to the growth inhibitory actions of exogenous TGF-beta1, and a more flattened cellular morphology. In vivo, Smad4 expression resulted in delayed tumor growth and decreased cellular proliferation, without effects on either apoptosis or angiogenesis. These findings indicate that, in spite of the absence of Smad4, growth inhibition in BxPC3 cells by TGF-beta1 is dependent on p21 upregulation and maintenance of RB in a hypophosphorylated, active state. Moreover, the presence of a functional Smad4 attenuates the capacity of BxPC3 cells to proliferate in vivo. However, this effect is transient, indicating that Smad4 growth inhibitory actions are circumvented in the later stages of pancreatic tumorigenicity.

Active Transport, Cell Nucleus↗
PubMed2006

Deletion of Smad2 in mouse liver reveals novel functions in hepatocyte growth and differentiation.

Smad family proteins Smad2 and Smad3 are activated by transforming growth factor beta (TGF-beta)/activin/nodal receptors and mediate transcriptional regulation. Although differential functional roles of Smad2 and Smad3 are apparent in mammalian development, the relative functional roles of Smad2 and Smad3 in postnatal systems remain unclear. We used Cre/loxP-mediated gene targeting for hepatocyte-specific deletion of Smad2 (S2HeKO) in adult mice and generated hepatocyte-selective Smad2/Smad3 double knockouts by intercrossing AlbCre/Smad2(f/f) (S2HeKO) and Smad3-deficient Smad3ex8/ex8 (S3KO) mice. All strains were viable and had normal adult liver. However, necrogenic CCL4-induced hepatocyte proliferation was significantly increased in S2HeKO compared to Ctrl and S3KO livers, and transplanted S2HeKO hepatocytes repopulated recipient liver at dramatically increased rates compared to Ctrl hepatocytes in vivo. Using primary hepatocytes, we found that TGF-beta-induced G1 arrest, apoptosis, and epithelial-to-mesenchymal transition in Ctrl and S2HeKO but not in S3KO hepatocytes. Interestingly, S2HeKO cells spontaneously acquired mesenchymal features characteristic of epithelial-to-mesenchymal transition (EMT). Collectively, these results demonstrate that Smad2 suppresses hepatocyte growth and dedifferentiation independent of TGF-beta signaling. Smad2 is not required for TGF-beta-stimulated apoptosis, EMT, and growth inhibition in hepatocytes.

Animals↗
PubMed2000

Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA.

Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor beta (TGF-beta) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-beta. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.

Activin Receptors↗
PubMed2001

Mechanism for mutational inactivation of the tumor suppressor Smad2.

Transforming growth factor beta (TGF-beta) is a potent natural antiproliferative agent that plays an important role in suppressing tumorigenicity. In numerous tumors, loss of TGF-beta responsiveness is associated with inactivating mutations that can occur in components of this signaling pathway, such as the tumor suppressor Smad2. Although a general framework for how Smads transduce TGF-beta signals has been proposed, the physiological relevance of alterations of Smad2 functions in promoting tumorigenesis is still unknown. Here, we show that expression of Smad2.P445H, a tumor-derived mutation of Smad2 found in human cancer, suppresses the ability of the Smads to mediate TGF-beta-induced growth arrest and transcriptional responses. Smad2.P445H is phosphorylated by the activated TGF-beta receptor at the carboxy-terminal serine residues and associates with Smad3 and Smad4 but is unable to dissociate from the receptor. Upon ligand-induced phosphorylation, Smad2.P445H interacts stably with wild-type Smad2, thereby blocking TGF-beta-induced nuclear accumulation of wild-type Smad2 and Smad2-dependent transcription. The ability of the Smad2.P445H to block the nuclear accumulation of wild-type Smad2 protein reveals a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Animals↗
PubMed2000

Cloning and characterization of zebrafish smad2, smad3 and smad4.

smad genes encode transcription factors involved in the signal transduction of members of the TGFbeta superfamily. We report here the cloning, characterization and genomic mapping of smad2, smad3 and smad4 from the zebrafish, Danio rerio. In Xenopus, smad2 overexpression has been shown to interfere with gastrulation and dorsal cell fate specification. However, full-length zebrafish smad2, although functionally active in Xenopus explants, has no effect when overexpressed in zebrafish embryos. In contrast, an N-terminally truncated, constitutively active version of Smad2 protein causes severe dorsalization or partial secondary axis formation, pointing to a role of Smad2 during mesoderm and axis formation. The temporal and spatial expression patterns of zebrafish smad2, 3 and 4 were investigated by developmental RT-PCR and whole mount in-situ hybridization. All three genes show strong and ubiquitous maternal expression. Zygotic expression is weak and ubiquitous in the case of smad2, and strong and ubiquitious in the case of smad4, while smad3 shows a spatially restricted zygotic expression pattern. It is expressed in migrating neural crest cells of the trunk and a subset of cells in the diencephalon in close proximity to the expression domain of the Nodal-related protein Cyclops/Ndr2/Znr1, a potential signal upstream of Smad2/3 required for eye-field separation and floor plate specification. Overexpression of truncated smad2 in cyclops mutant embryos leads to a rescue of the eye and floorplate defects. These data suggest that Smad2 acts as a mediator of Nodal signals during zebrafish midline signaling, while Smad3 might be involved in later steps of eye field separation.

Amino Acid Sequence↗
PubMed2002

Engagement of activin and bone morphogenetic protein signaling pathway Smad proteins in the induction of inhibin B production in ovarian granulosa cells.

In the mammalian ovary cell growth and differentiation is regulated by several members of the transforming growth factor beta (TGF beta) superfamily including activins, inhibins, growth differentiation factors and bone morphogenetic proteins (BMPs). The effects of TGF beta family members are mediated to the target cells via heteromeric complexes of type I and II serine/threonine kinase receptors which activate Smad signaling protein pathways in various cell types. We have previously shown that inhibin B, a hormonally important product from human granulosa cells, is up regulated by activin and BMPs. Here, we report the use of adenoviral gene transfer methodology to manipulate the TGF beta growth factor signaling system in primary cultures of human granulosa cells. These cells are exceedingly difficult to transfect by conventional transfection methods, but were virtually 100% infected with recombinant adenoviruses expressing green fluorescent protein (GFP). Adenoviruses expressing constitutively active forms of the seven known mammalian type I activin receptor-like kinase receptors (Ad-caALK1 through Ad-caALK7) cause activation of endogenous and adenovirally transferred Smad signaling proteins so that Ad-caALK1/2/3/6 and Ad-caALK4/5/7 induced phosphorylation of the Smad1 and Smad2 pathways, respectively. Activin A and BMP-2 activated the Smad1 and Smad2 pathways as well as inhibin B production as did all the Ad-caALKs. Furthermore, overexpression of adenoviral Smad1 and Smad2 proteins without exogenously added ligands induced inhibin B production. The inhibitory Smad7 protein suppressed BMP-2 and activin induced inhibin B production. Collectively, the present data demonstrate that adenoviral gene transfer provides an effective approach for dissecting the TGF beta signaling pathways in primary ovarian cells in vitro and more specifically indicate that the Smad1 and Smad2 pathways are involved in the regulation of inhibin B production by TGF beta family ligands in the ovary.

Activins↗
PubMed1997

The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling.

TGFbeta signaling is initiated when the type I receptor phosphorylates the MAD-related protein, Smad2, on C-terminal serine residues. This leads to Smad2 association with Smad4, translocation to the nucleus, and regulation of transcriptional responses. Here we demonstrate that Smad7 is an inhibitor of TGFbeta signaling. Smad7 prevents TGFbeta-dependent formation of Smad2/Smad4 complexes and inhibits the nuclear accumulation of Smad2. Smad7 interacts stably with the activated TGFbeta type I receptor, thereby blocking the association, phosphorylation, and activation of Smad2. Furthermore, mutations in Smad7 that interfere with receptor binding disrupt its inhibitory activity. These studies thus define a novel function for MAD-related proteins as intracellular antagonists of the type I kinase domain of TGFbeta family receptors.

Amino Acid Sequence↗
PubMed2003

Both SMAD2 and SMAD3 mediate activin-stimulated expression of the follicle-stimulating hormone beta subunit in mouse gonadotrope cells.

The activins are pleiotropic members of the TGFbeta superfamily. Within the anterior pituitary gland, activins stimulate FSH synthesis in an autocrine/paracrine fashion by stimulating transcription of the FSHbeta subunit gene. Here, the mechanisms mediating this effect were investigated in the murine gonadotrope cell line, LbetaT2. Recombinant activin A and activin B dose- and time-dependently stimulated endogenous FSHbeta mRNA expression. FSHbeta primary transcript and mRNA levels were increased within 30-60 min, but these effects were blocked by preincubation with the transcription inhibitor actinomycin-D, suggesting that the FSHbeta gene is a direct target of the activin signal transduction cascade. In other systems, activin signals are transduced through a heteromeric serine/threonine receptor complex, which includes the signaling activin type IB receptor [activin receptor-like kinase 4 (ALK4)]. Transfection of a constitutively active form of the receptor, ALK4T206D, stimulated FSHbeta mRNA levels. Overexpression of the inhibitory SMAD7 blocked this effect, as well as activin-stimulated FSHbeta transcription. Because SMAD7 functions by preventing access of SMAD2 and SMAD3 to ALK4, these data suggested that both activins and ALK4 require SMAD2 and/or SMAD3 to affect FSHbeta transcription. Consistent with this idea, activin A stimulated SMAD2 and SMAD3 phosphorylation and nuclear translocation within 5-10 min in LbetaT2 cells. Transient transfection of SMAD3, but not SMADs 1, 2, 4, 5, or 8, stimulated endogenous FSHbeta mRNA levels. The results of SMAD2 transfection studies were inconclusive, however, because of a persistent failure to overexpress the full-length SMAD2 protein specifically in LbetaT2 cells. To assess more directly roles for both SMAD2 and SMAD3 in activin-stimulated FSHbeta expression, RNA interference was used to decrease endogenous SMAD protein levels in LbetaT2 cells. Activin A- and ALK4T206D-stimulated transcription of the FSHbeta gene were significantly attenuated by the depletion of either SMAD2 or SMAD3. Collectively, these data suggest that activins use both SMAD2- and SMAD3-dependent mechanisms to stimulate FSHbeta transcription in mouse gonadotrope cells.

Activin Receptors, Type I↗
PubMed2006

Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.

Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-beta (TGFbeta) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFbeta1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFbeta superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFbeta1 and activin-A bioactivity, demonstrating its specificity. The TGFbetaR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFbeta1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFbeta superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.

Activins↗
PubMed2000

Smad protein expression and activation in transforming growth factor-beta refractory human squamous cell carcinoma cells.

In contrast to nonneoplastic keratinocytes, human squamous carcinoma cell lines are able to proliferate in the presence of transforming growth factor-beta (TGF-beta) in vitro. This has raised the question whether, how frequently, by which mechanism, and at which stage of development squamous carcinomas escape from TGF-beta control in vivo. We have developed a method to rapidly identify the most common molecular alterations in the TGF-beta signaling pathway by combining measurements of the levels and the activation state of Smad signaling intermediates with DNA-based diagnostic assays. In this report, we demonstrate the validity of this approach using a panel of seven squamous cell carcinoma (SCC) lines known to be refractory to TGF-beta-mediated cell cycle arrest. Each of the SCCs expressed the pathway-restricted Smad proteins, Smad2 and-3. Furthermore, treatment with TGF-beta induced phosphorylation of Smad2 in each of the SCCs with the exception of the two cell lines that carry inactivating mutations of the TGF-beta type II receptor. Three of the remaining SCC lines failed to express the common mediator Smad4, two on the basis of loss of transcription and one by a posttranscriptional mechanism. Thus, a mechanism for TGF-beta resistance was identified in five of the seven tumor cell lines. Interestingly, in the two remaining lines, no abnormalities of signaling intermediates were found, and TGF-beta was able to activate TGF-beta-responsive promoters. This suggests that the ability of these two cell lines to grow in the presence of TGF-beta is due to factors extraneous to the TGF-beta pathway itself. Application of our protein-based strategy to interrogate the TGF-beta signaling pathway should allow us to determine whether or not and, if so, how and at which stage human squamous cell carcinomas become TGF-beta resistant in vivo.

Carcinoma, Squamous Cell↗
PubMed2004

Cytoplasmic PML function in TGF-beta signalling.

Transforming growth factor beta (TGF-beta) is a pluripotent cytokine that controls key tumour suppressive functions, but cancer cells are often unresponsive to it. The promyelocytic leukaemia (PML) tumour suppressor of acute promyelocytic leukaemia (APL) accumulates in the PML nuclear body, but cytoplasmic PML isoforms of unknown function have also been described. Here we show that cytoplasmic Pml is an essential modulator of TGF-beta signalling. Pml-null primary cells are resistant to TGF-beta-dependent growth arrest, induction of cellular senescence and apoptosis. These cells also have impaired phosphorylation and nuclear translocation of the TGF-beta signalling proteins Smad2 and Smad3, as well as impaired induction of TGF-beta target genes. Expression of cytoplasmic Pml is induced by TGF-beta. Furthermore, cytoplasmic PML physically interacts with Smad2/3 and SARA (Smad anchor for receptor activation) and is required for association of Smad2/3 with SARA and for the accumulation of SARA and TGF-beta receptor in the early endosome. The PML-RARalpha oncoprotein of APL can antagonize cytoplasmic PML function and APL cells have defects in TGF-beta signalling similar to those observed in Pml-null cells. Our findings identify cytoplasmic PML as a critical TGF-beta regulator, and further implicate deregulated TGF-beta signalling in cancer pathogenesis.

Animals↗