Burn-induced thymic apoptosis corresponds with altered TGF-beta(1) and Smad 2/3.

Department of Surgery, University of California Davis, 95616, USA.

Immune suppression is a common complication of injury. Transforming growth factor-beta(1) (TGF-beta(1)), a cytokine with diverse anti-inflammatory and anti-apoptotic effects, may play an important role. Smad 2 and Smad 3 are transcription factors that mediate the effects of TGF-beta(1). We hypothesized that burn-induced immunosuppression would be accompanied by increased apoptosis in lymphoid organs, a change likely associated with changes in TGF-beta(1) and Smad 2/3 expression. Mice were subjected to 18% body surface area flame burn. Lymph nodes, spleen, and thymus were harvested at multiple time points after injury. TGF-beta(1) and Smad 2/3 expression and levels of apoptosis were determined in whole tissue and in sorted T-cells by flow cytometry, RT-PCR, ELISA, and Western blot. TGF-beta(1) protein expression in the thymus increased from 1 to 7 days. Smad 2/3 protein expression was decreased at the same time points. This down-regulation was more dramatic in the non-T-cells than in the T-cells themselves. RT-PCR confirmed down-regulation of Smad 3 mRNA in the thymus from 3 to 6 h. Apoptosis in the thymus doubled at 1 day (6.4% control vs 12.8% burned), which corresponded with a marked decrease in thymus mass on subjective assessment. No changes were observed in other lymphoid organs. Burn injury in mice increases TGF-beta(1) expression in the thymus, while suppressing expression of its intracellular mediator, Smad 2/3. This response is most pronounced in the non-T-cell tissue, which suggests the thymic epithelial cells may be responsible for the increased thymic apoptosis. This TGF-beta(1) and Smad 2/3 counterregulation in response to injury may represent a potential mechanism for postinjury immune suppression. (c) 2002 Elsevier Science (USA).

PMID: 12069494 [PubMed - indexed for MEDLINE]

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

Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10021, USA. dbernard@popcbr.rockefeller.edu.

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.

PMID: 14701940 [PubMed - indexed for MEDLINE]

Loss of cooperative function of transforming growth factor-beta signaling proteins, smad3 with embryonic liver fodrin, a beta-spectrin, in primary biliary cirrhosis.

GI/Developmental Biology, Medicine, Surgical Sciences, Lombardi Cancer Center, Georgetown University & DVAMC, Washington, DC, USA.

Modulation of fibrogenesis, epithelial, and mesenchymal cell fates are prominent effects of transforming growth factor-beta (TGF-beta) signaling by Smad proteins. We have previously shown that Smad2 and Smad3 insufficiency leads to a loss of bile ducts. In addition, Smad3/4 activity is mediated by embryonic liver fodrin (ELF), a beta-Spectrin. In mouse elf(-/-) mutants and in liver explant cultures, loss of ELF function results in T lymphocytic proliferation and absent intrahepatic bile ducts. A similar phenotype is seen in a number of cholestatic diseases with progressive loss of intrahepatic bile ducts and fibrosis. However, the expression patterns of Smads or role of ELF in cholestatic and fibrotic liver diseases are not yet known. METHODS/RESULTS: We investigated the role of ELF in primary biliary cirrhosis (PBC), autoimmune hepatitis C, chronic viral hepatitis and in livers from mice deficient in Smad2/Smad3. We generated elf(+/-) mutant mice and analyzed for chronic liver disease and hepatocellular cancer (HCC) from 6 to 12 months. Perturbations in ELF expression were consistently seen only in PBC tissues. ELF expression was similarly aberrant in tissues from Smad2(+/-)/Smad3(+/-) mutant mice. Further studies indicated that ELF mislocalization is correlated with aberrant localization of Smad3 in some PBC tissues. Thirteen of 17 elf(+/-) mutant mice developed steatosis, fibrosis, hepatic dysplasia, with HCC in two mice. CONCLUSIONS: These results suggest that a compromised cytoarchitecture and polarized trafficking of TGF-beta signaling molecules, ELF and Smad3 are involved in the pathogenesis of PBC as well as HCC.

PMID: 15566516 [PubMed - indexed for MEDLINE]

Localization of activin and inhibin subunits, receptors and SMADs in the mouse mammary gland.

Department of Neurobiology and Physiology, Northwestern University, O.T. Hogan 4-150, 2153 N. Campus Dr., Evanston, IL 60208, USA.

Activin and inhibin, two closely related protein hormones, are members of the transforming growth factor beta (TGF beta) superfamily of growth factors. Activin and TGF beta have been associated with mouse mammary gland development and human breast carcinogenesis. TGF beta expression in the mammary gland has been previously described, and was found to be expressed in nonparous tissue and during pregnancy, down-regulated during lactation, and then up-regulated during involution. The expression pattern of activin subunits, receptors and cytoplasmic signaling molecules has not been thoroughly described in post-natal mammary gland development. We hypothesize that activin signaling components are dynamically regulated during mammary gland development, thereby permitting activin to have distinct temporal growth regulatory actions on this tissue. To examine the activin signal transduction system in the mammary gland, tissue from CD1 female mice was dissected from nonparous, lactating day 1, 10, and 20 and post-weaning day 4 animals. The expression of the activin receptors (ActRIIA, ActRIIB and ActRIB), the inhibin co-receptor (betaglycan), and ligand subunit (alpha, beta A and beta B), mRNA was measured by semi-quantitative RT-PCR in these tissues. In addition, the cellular compartmentalization of the activin signaling proteins, including the cytoplasmic signaling co-activators, Smads 2, 3 and 4, were examined by immunohistochemistry. Generally, mRNA abundance of activin signaling components was greatest in the nonparous tissue, and then decreased, whereas protein immunoreactivity for activin signaling components increased during lactation and decreased during involution. The alpha-subunit protein was detected in nonparous and lactating day 1 tissue only. Importantly, Smad 3, but not Smad 2, was detected in epithelial cell nuclei during all time points examined, indicating that activin signaling is mediated by Smad 3 at these times. These findings suggest that activin's growth regulatory role during lactation may be distinguished from that of TGF beta during post-natal mammary development. Future studies will focus on determining the exact role this ligand plays in mammary tissue differentiation and neoplasia.

PMID: 12782414 [PubMed - indexed for MEDLINE]

TGF-beta3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion.

Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90033, USA.

Transforming growth factor (TGF) -beta3 is known to selectively regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Previous studies suggested that the selective function of TGF-beta3 in MEE was conducted by TGF-beta receptors. Further studies were needed to demonstrate that the TGF-beta signaling mediators were indeed expressed and phosphorylated in the MEE cells. SMAD2 and SMAD3 were both present in the MEE, whereas SMAD2 was the only one phosphorylated during palatal fusion. SMAD2 phosphorylation was temporospatially restricted to the MEE and correlated with the disappearance of the MEE. No phosphorylated SMAD2 was found in MEE in TGF-beta3(-/-) mice, although nonphosphorylated SMAD2 was present. The results suggest that TGF-beta3 is required for initiating and maintaining SMAD2 phosphorylation in MEE. Phospho-SMAD3 was not detectable in palate during normal palatal fusion. Previous results suggested TGF-beta-induced cessation of DNA synthesis in MEE cells during palatal fusion in vitro. The present results provide evidence that inhibition of MEE proliferation in vivo was controlled by endogenous TGF-beta3. The number of 5-bromo-2'-deoxyuridine (BrdU) -labeled MEE cells was significantly reduced in TGF-beta3(+/+) compared with TGF-beta3(-/-) mice when the MEE seam formed (t-test, P < 0.05). This finding suggests that TGF-beta3 is required for inhibiting MEE proliferation during palatal fusion. The inhibition of MEE proliferation may be mediated by TGF-beta3-dependent phosphorylation of SMAD2. Copyright 2003 Wiley-Liss, Inc.

PMID: 12815624 [PubMed - indexed for MEDLINE]

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

Section of Rheumatology, University of Illinois at Chicago, 60607, USA.

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 possibility that Smads plays an important part in the pathogenesis of fibrosis, and may therefore represent targets for selective anti-fibrotic interventions.

PMID: 12839562 [PubMed - indexed for MEDLINE]

Identification and characterization of a PDZ protein that interacts with activin type II receptors.

Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.

We have identified a mouse PDZ protein that interacts with the activin type IIA receptor (ActRIIA), which we named activin receptor-interacting protein 1 (ARIP1). By using yeast two-hybrid screening, we isolated a cDNA clone of ARIP1 from a mouse brain cDNA library. We detected two forms of ARIP1, ARIP1-long and ARIP1-short, which may be produced by alternative splicing. ARIP1-long had one guanylate kinase domain in the NH(2)-terminal region, followed by two WW domains and five PDZ domains (PDZ1-5). ARIP1-short had a deletion in the NH(2)-terminal region and lacked the guanylate kinase domain. Both forms interacted with ActRIIA through PDZ5. The COOH-terminal residues of ActRIIA (ESSL) agree with a PDZ-binding consensus motif, and ARIP1 recognized the consensus sequence. ARIP1 interacts specifically with ActRIIA among the receptors for the transforming growth factor beta family. Interestingly, ARIP1 also interacted with Smad3, which is an activin/transforming growth factor beta intracellular signaling molecule. The mRNA of ARIP1 was more abundant in the brain than in other tissues. Overexpression of ARIP1 controls activin-induced and Smad3-induced transcription in activin-responsive cell lines. These findings suggest that ARIP1 has a significant role in assembling activin signaling molecules at specific subcellular sites and in regulating signal transduction in neuronal cells.

PMID: 10681527 [PubMed - indexed for MEDLINE]

The gonadotropin releasing hormone (GnRH) receptor activating sequence (GRAS) is a composite regulatory element that interacts with multiple classes of transcription factors including Smads, AP-1 and a forkhead DNA binding protein.

Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.

Activin responsiveness of the murine GnRH receptor gene promoter is mediated at a regulatory element we termed the GnRH receptor activating sequence (GRAS). Here, we have sought to define the complex of transcription factors that interact at this element. Consistent with activin regulation at GRAS, gel shift analyses and yeast one-hybrid assays reveal Smad4 interaction at the 5' end of GRAS. While overexpression of Smad3 activates a GRAS reporter, Smad3 binding at GRAS was not detectable. A functional interaction of Smad3 at GRAS was, however, detectable in yeast expressing Smad4. Thus, Smad3 interaction at GRAS appears to be dependent on the presence of Smad4. Mutations located at the 3' end of GRAS do not affect Smad binding but eliminate functional activity. Thus, Smad binding alone cannot account for the functional attributes of GRAS. Consistent with this notion, we find that AP-1 binding is immediately juxtaposed to and, in fact, partially overlaps the Smad binding site. Finally, a recently identified member of the forkhead family of transcription factors, FoxL2, is also capable of interacting at GRAS. Furthermore, FoxL2 activation at GRAS is lost with mutation of either the 5' Smad binding site or a putative forkhead binding site located at the 3' end of the element. We suggest that GRAS is a composite regulatory element whose functional activity is dependent on the organization of a multi-protein complex consisting of Smads, AP-1 and a member of the forkhead family of DNA binding proteins.

PMID: 12943993 [PubMed - indexed for MEDLINE]

The DNA binding activities of Smad2 and Smad3 are regulated by coactivator-mediated acetylation.

Ludwig Institute for Cancer Research, Uppsala University, Biomedical Center, Box 595, Husargatan 3, S-751 24 Uppsala, Sweden.

Phosphorylation-dependent activation of the transcription factors Smad2 and Smad3 plays an important role in TGFbeta-dependent signal transduction. Following phosphorylation of Smad2 and Smad3, these molecules are translocated to the nucleus where they interact with coactivators and/or corepressors, including p300, CBP, and P/CAF, and regulate the expression of TGFbeta target genes. In the current study, we demonstrate that both Smad2 and Smad3 are acetylated by the coactivators p300 and CBP in a TGFbeta-dependent manner. Smad2 is also acetylated by P/CAF. The acetylation of Smad2 was significantly higher than that of Smad3. Lys(19) in the MH1 domain was identified as the major acetylated residue in both the long and short isoform of Smad2. Mutation of Lys(19) also reduced the p300-mediated acetylation of Smad3. By generating acetyl-Lys(19)-specific antibodies, we demonstrate that endogenous Smad2 is acetylated on this residue in response to TGFbeta signaling. Acetylation of the short isoform of Smad2 improves its DNA binding activity in vitro and enhances its association with target promoters in vivo, thereby augmenting its transcriptional activity. Acetylation of Lys(19) also enhanced the DNA binding activity of Smad3. Our data indicate that acetylation of Lys(19) induces a conformational change in the MH1 domain of the short isoform of Smad2, thereby making its DNA binding domain accessible for interactions with DNA. Thus, coactivator-mediated acetylation of receptor-activated Smad molecules could represent a novel way to regulate TGFbeta signaling.

PMID: 17074756 [PubMed - indexed for MEDLINE]

Arkadia enhances Nodal/TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover.

Mammalian Neurogenesis, Medical Research Council, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.

Regulation of transforming growth factor-beta (TGF-beta) signaling is critical in vertebrate development, as several members of the TGF-beta family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-beta pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous null (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.

PMID: 17341133 [PubMed - indexed for MEDLINE]

PMCID: PMC1808117

Inhibition of SMAD2 expression prevents murine palatal fusion.

Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90033-9062, USA.

Transforming growth factor (TGF)-beta 3 is known to regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Our previous studies showed that SMAD2, a TGF-beta signaling mediator, was expressed and phosphorylated primarily in the MEE and that SMAD2 phosphorylation in the MEE was temporospatially regulated by TGF-beta 3. The goal of this study was to examine the requirement for SMAD2 to complete the developmental events necessary for palatal fusion. SMAD2 expression was inhibited with Smad2 siRNA transfection into palatal tissues in vitro. The results showed that Smad2 siRNA transfection resulted in the maintenance of MEE cells in the palatal midline. Western blot and immunofluorescence analyses confirmed that the endogenous SMAD2 and phospho-SMAD2 levels were reduced following siRNA transfection. The SMAD3 level was not altered by the Smad2 siRNA transfection. The persistence of the MEE and the decreased SMAD2/phospho-SMAD2 levels were coincident with increased MEE cell proliferation. Addition of exogenous TGF-beta 3 increased p-SMAD2 level but not the total SMAD2 level. Therefore, exogenous TGF-beta 3 was not able to induce p-SMAD2 enough to rescue the palatal phenotype in the Smad2 siRNA group. The results indicated that the endogenous SMAD2 level is crucial in the regulation of disappearance of MEE during palatal fusion. (c) 2006 Wiley-Liss, Inc.

PMID: 16607645 [PubMed - indexed for MEDLINE]

Activin regulates estrogen receptor gene expression in the mouse ovary.

Department of Biochemistry, Molecular Biology and Cell Biology, and Center for Reproductive Science, Northwestern University, Evanston, Illinois 60208, USA.

Activin, a member of the transforming growth factor-beta superfamily, is an important modulator of follicle-stimulating hormone synthesis and secretion in the pituitary and plays autocrine/paracrine roles in the regulation of ovarian follicle development. From a microarray study on mouse ovarian granulosa cells, we discovered that the estrogen receptor beta (ERbeta) is inducible by activin. We previously demonstrated that estrogen suppresses activin gene expression, suggesting a feedback relationship between these two follicle-regulating hormones. The purpose of this study was to investigate fully activin A regulation of ER expression. Real time reverse transcription-PCR assays on cultured granulosa cells showed that both ERalpha and ERbeta mRNAs were induced by activin A at 4, 12, and 24 h in a dose-responsive manner. Western blots confirmed an increase in their protein levels. Consistent with increased ERalpha and ERbeta expression, activin A stimulated estradiol-induced estrogen response element promoter activity. Activin A stimulation of ER expression was a direct effect at the level of gene transcription, as it was not abolished by cycloheximide but was abolished by actinomycin D, and in transfected granulosa cells activin A stimulated ERalpha promoter activity. To investigate the effect of activin in vivo and, thus, its biological significance, we examined ER expression in inhibin transgenic mice that have decreased activin expression and discovered that these mice had decreased ERalpha and ERbeta expression in the ovary. We also found that ER mRNA levels were decreased in Müllerian inhibiting substance promoter (MIS)-Smad2 dominant negative mice that have impaired activin signaling through Smad2, and small interfering RNAs targeting Smad2 or Smad3 suppressed ERalpha promoter activation, suggesting that Smad2 and Smad3 are involved in regulating ER levels. Therefore, this study reveals an important role for activin in inducing the expression of ERs in the mouse ovary and suggests important interplay between activin and estrogen signaling.

PMID: 17951260 [PubMed - indexed for MEDLINE]

Transforming growth factor-beta- and Activin-Smad signaling pathways are activated at distinct maturation stages of the thymopoeisis.

AstraZeneca R & D Lund, Department of Bio & Molecular Sciences, Scheelevägen 2, 221 87 Lund, Sweden. alexander.rosendahl@astrazeneca.com

Members of the transforming growth factor (TGF)-beta family play pivotal roles in the control of differentiation, proliferation and tolerance in peripheral T cells. Recently, they have been implicated in thymic selection, but their role is so far not well characterized. In the present study, we demonstrate that specific thymocyte populations are under the influence of either the TGF-beta and/or Activin pathway, and transduce signals into the nucleus via phosphorylated Smad2 (pSmad2). Thymocytes in the medulla and in the subcapsular zone expressed nuclear translocated pSmad2, a hallmark of active TGF-beta/Activin receptor signaling. When analyzed at the cellular level, the pSmad2(+) cells were confined to the double-negative (DN) and single-positive (SP) subpopulations. Moreover, the most immature DN thymocytes (CD44(+)CD25(-) and CD44(+)CD25(+)) expressed higher levels of pSmad2 compared to the more mature DN. In vitro stimulation demonstrated that pure CD44(+)CD25(-), CD44(+)CD25(+) and CD44(+)CD25(+) thymocytes respond to ActivinA, while the mature CD4(+) and CD8(+) SP thymocytes respond to TGF-beta stimulation measured as enhanced phosphorylation of Smad2. Double staining of pSmad2(+) cells with either the Activin type I receptor, ALK4, or the TGF-beta type I receptor, ALK5, demonstrated that pSmad2(+) DN cells exhibited high levels of immunoreactivity to ALK4 and moderate levels of immunoreactivity to the TGF-beta-responsive ALK5 receptor. In sharp contrast, the SP pSmad2(+) cells were predominately ALK5(+). Collectively, our results demonstrate that early and late thymocytes express pSmad2 in the nuclei in vivo. The functional experiments in vitro suggest that members of the TGF-beta family (TGF-beta or Activin) may play important non-redundant roles during different stages of thymopoiesis.

PMID: 14645149 [PubMed - indexed for MEDLINE]

Resistance to TGF-beta1-mediated growth inhibition correlates with sustained Smad2 phosphorylation in primary murine splenocytes.

Department of Physiological Chemistry II, Biocenter, University of Würzburg, Würzburg, Germany.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that regulates cell growth and differentiation in many types of cells. TGF-beta1 is especially known to exert a variety of regulatory functions in the immune system, such as T cell differentiation and T cell function. Signal transduction of TGF-beta1 is mediated by phosphorylation of R-Smads upon receptor activation. Hetero-oligomers of R- and Co-Smads translocate into the nucleus and regulate transcription of specific target genes. Here we describe the effect of long-term exposure to TGF-beta1 on the effector function of differentially stimulated primary murine splenocytes and purified primary murine CD8(+) cytotoxic T cells. Long-term exposure to TGF-beta1 results in non-responsiveness to TGF-beta1-induced Smad2 phosphorylation. This is seen either by no phosphorylation or sustained phosphorylation of Smad2. Furthermore, we observed a strong correlation between sustained Smad2 phosphorylation and resistance to TGF-beta1-mediated growth inhibition. In contrast, splenocyte cultures strongly growth inhibited by TGF-beta1 showed no Smad2 phosphorylation. Lytic activity of these cultures, however, was found to be suppressed regardless of proliferation properties and Smad2 phosphorylation pattern. These findings may contribute to understanding the mechanisms of how TGF-beta1 suppresses immune responses and promotes tumor progression.

PMID: 11981827 [PubMed - indexed for MEDLINE]

FAST-2 is a mammalian winged-helix protein which mediates transforming growth factor beta signals.

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

The mechanisms by which transforming growth factor beta (TGF-beta) and related ligands regulate transcription remain poorly understood. The winged-helix (WH) transcription factor fork head activin signal transducer 1 (FAST-1) was identified as a mediator of activin signaling in Xenopus embryos (X. Chen, M. J. Rubock, and M. Whitman, Nature 383:691-696, 1996). We have cloned a novel WH gene from the mouse which shares many properties with FAST-1. We find that this gene, which we call FAST-2, is able to mediate transcriptional activation by TGF-beta. FAST-2 also interacts directly with Smad2, a cytoplasmic protein which is translocated to the nucleus in response to TGF-beta, and forms a multimeric complex with Smad2 and Smad4 on the activin response element, a high-affinity binding site for FAST-1. Analysis of the sequences of FAST-1 and FAST-2 reveals substantial protein sequence divergence compared to known vertebrate orthologs in the WH family. This suggests that FAST-2 represents a new WH gene related to FAST-1, which functions to mediate TGF-beta signals in mammals. We have also examined the structure of the FAST-2 gene and find that it overlaps with a kinesin motor protein gene. The genes are transcribed in opposite orientations, and their transcripts overlap in the 3' untranslated region.

PMID: 9858566 [PubMed - indexed for MEDLINE]

PMCID: PMC83900

Formation of the definitive endoderm in mouse is a Smad2-dependent process.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

TGFbeta growth factors specify cell fate and establish the body plan during early vertebrate development. Diverse cellular responses are elicited via interactions with specific cell surface receptor kinases that in turn activate Smad effector proteins. Smad2-dependent signals arising in the extraembryonic tissues of early mouse embryos serve to restrict the site of primitive streak formation and establish anteroposterior identity in the epiblast. Here we have generated chimeric embryos using lacZ-marked Smad2-deficient ES cells. Smad2 mutant cells extensively colonize ectodermal and mesodermal populations without disturbing normal development, but are not recruited into the definitive endoderm lineage during gastrulation. These experiments provide the first evidence that TGFbeta signaling pathways are required for specification of the definitive endoderm lineage in mammals and identify Smad2 as a key mediator that directs epiblast derivatives towards an endodermal as opposed to a mesodermal fate. In largely Smad2-deficient chimeras, asymmetric nodal gene expression is maintained and expression of pitx2, a nodal target, is also unaffected. These results strongly suggest that other Smad(s) act downstream of Nodal signals in mesodermal populations. We found Smad2 and Smad3 transcripts both broadly expressed in derivatives of the epiblast. However, Smad2 and not Smad3 mRNA is expressed in the visceral endoderm, potentially explaining why the primary defect in Smad2 mutant embryos originates in this cell population.

PMID: 10862745 [PubMed - indexed for MEDLINE]

TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage.

Genetics of Development and Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.

PMID: 11285272 [PubMed - indexed for MEDLINE]

PMCID: PMC2185521

Functional interaction between Smad, CREB binding protein, and p68 RNA helicase.

Department of Molecular, Cellular, and Craniofacial Biology, University of Louisville Birth Defects Center, ULSD, Louisville, KY 40292, USA. dennis.warner@louisville.edu

The transforming growth factors beta control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFbeta superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFbeta-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFbeta-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFbeta-mediated cellular responses in developing orofacial tissue.

PMID: 15464984 [PubMed - indexed for MEDLINE]

Global analysis of Smad2/3-dependent TGF-beta signaling in living mice reveals prominent tissue-specific responses to injury.

Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA.

Smad2 and Smad3 (Smad2/3) proteins are key signaling molecules for TGF-beta and some related family members regulating the transcription of several hundred genes. TGF-beta have key roles in development, tissue homeostasis, and the pathogenesis of many human diseases, including cancer, fibrotic disorders, developmental defects, and neurodegeneration. To study the temporal and spatial patterns of Smad2/3-dependent signaling in normal and pathological conditions in the living organism, we engineered transgenic mice with a Smad-responsive luciferase reporter construct (SBE-luc mice). Using bioluminescent imaging, we assessed Smad2/3 signaling activity noninvasively in living mice. At baseline, this activity was highest in brain, intestine, heart, and skin, and correlated with biochemical measurements of reporter activity. Primary astrocytes cultured from SBE-luc mice showed specific activation of the reporter in response to Smad2/3-activating TGF-beta family members. Treatment of mice with the endotoxin LPS resulted in a fast and vigorous, but transient activation of the reporter in the intestine. Although the response was similarly rapid in brain, it remained increased, indicating important but different cellular responses to endotoxin challenge in these organs. Traumatic brain injury with a needle stab resulted in local activation of Smad2/3-dependent genes and a severalfold increase in bioluminescence in living mice. SBE-luc mice can therefore be used to study temporal, tissue-specific activation of Smad2/3-dependent signaling in living mice in normal or pathological conditions as well as for the identification of endogenous or synthetic modulators of this pathway.

PMID: 15972691 [PubMed - indexed for MEDLINE]

RhoA modulates Smad signaling during transforming growth factor-beta-induced smooth muscle differentiation.

Department of Cell Biology, Georgetown University Medical School, Washington, DC 20057, USA. sc229@Georgetown.edu

We recently reported that transforming growth factor (TGF)-beta induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-beta actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-beta-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-beta. We demonstrate here that RhoA signaling is critical to TGF-beta-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle alpha-actin, SM22alpha, and calponin in TGF-beta-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-beta-treated cells. RhoA signaling was activated as early as 5 min following TGF-beta addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smad-dependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-beta-induced SMC differentiation.

PMID: 16317010 [PubMed - indexed for MEDLINE]

PMCID: PMC1831550