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]

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]

TGFbeta superfamily signals are required for morphogenesis of the kidney mesenchyme progenitor population.

Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.

The TGFbeta superfamily plays diverse and essential roles in kidney development. Gdf11 and Bmp4 are essential for outgrowth and positioning of the ureteric bud, the inducer of metanephric mesenchyme. During nephrogenesis, Bmp7 is required for renewal of the mesenchyme progenitor population. Additionally, in vitro studies demonstrate inhibitory effects of BMPs and TGFbetas on collecting duct branching and growth. Here, we explore the predicted models of TGFbeta superfamily function by cell-specific inactivation of Smad4, a key mediator of TGFbeta signaling. Using a HoxB7cre transgene expressed in ureteric bud and collecting duct, we find that development of the collecting duct is Smad4 independent. By contrast, removal of Smad4 in nephrogenic mesenchyme using the Bmp7(cre/+) allele leads to disorganization of the nephrogenic mesenchyme and impairment of mesenchyme induction. Smad4-deficient metanephric mesenchyme does not display defects in inducibility in LiCl or spinal cord induction assays. However, in situ hybridization and lineage analysis of Smad4 null mesenchyme cells at E11.5 show that the nephrogenic mesenchyme does not aggregate tightly around the ureteric bud tips, but remains loosely associated, embedded within a population of cells expressing markers of both nephrogenic mesenchyme and peripheral stroma. We conclude that the failure of recruitment of nephrogenic mesenchyme leaves a primitive population of mesenchyme at the periphery of the kidney. This population is gradually depleted, and by E16.5 the periphery is composed of cells of stromal phenotype. This study uncovers a novel role for TGFbeta superfamily signaling in the recruitment and/or organization of the nephrogenic mesenchyme at early time-points of kidney development. Additionally, we present conclusive genetic lineage mapping of the collecting duct and nephrogenic mesenchyme.

PMID: 15342483 [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]

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]

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]

Smad4 (homolog of human DPC4) and Smad2 (homolog of human JV18-1): candidates for murine lung tumor resistance and suppressor genes.

Laboratory of Molecular Carcinogenesis, NIEHS, Research Triangle Park, NC 27709, USA.

In this study we investigated the mouse mad-related genes, Smad4/Dpc4 and Smad2 (homolog of JV18-1), as candidates for involvement in lung tumor resistance and suppression. These genes are located in a region of mouse chromosome 18 that is syntenic with human 18q21.1, where several genes that are mutated in various cancers have been mapped. A newly identified murine lung tumor resistance locus, Par2 has also been mapped to this region of chromosome 18. We found no mutations in the coding regions of either gene in 11 lung tumors from B6CF1 (C57BL/6 x BALB/c) mice by RT-PCR and SSCP/RFLP, suggesting that these genes are not mutated in lung carcinogenesis in this strain. Moreover, loss of heterozygosity in this region of chromosome 18 was not detected in 28 lung adenocarcinomas from B6CF1 mice, 17 lung adenocarcinomas from B6C3F1 mice or 18 lung adenocarcinomas from AB6F1 mice. These data provide evidence that a 'classical' tumor suppressor gene for mouse lung carcinogenesis in these strains does not reside in this region. In order to investigate Smad4/Dpc4 and Smad2 as candidates for the Par2 resistance locus mapped to this region, we also sequenced the coding regions of both genes in cDNA from normal lungs of A/J, BALB/c and C57BL/6 inbred strains of mice. No polymorphisms were detected in the coding region of Smad4. In Smad2, two sequence polymorphisms were identified that are not in the conserved regions of the gene. Northern blot analysis revealed no differential expression in normal lung tissue among the three strains for either gene. Thus, in this study we found no evidence that either Smad4 or Smad2 represents the Par2 lung tumor resistance locus or is a lung tumor suppressor gene in the B6CF1 mice.

PMID: 9328171 [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 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]

Pinch1 is required for normal development of cranial and cardiac neural crest-derived structures.

Department of Medicine, University of California at San Diego, La Jolla, CA 92093-0613, USA.

Pinch1, an adaptor protein composed of 5 LIM domains, has been suggested to play an important role in multiple cellular processes. We found that Pinch1 is highly expressed in neural crest cells and their derivatives. To examine the requirement for Pinch1 in neural crest development, we generated neural crest conditional Pinch1 knockout mice using the Wnt1-Cre/loxP system. Neural crest conditional Pinch1 mutant embryos die perinatally from severe cardiovascular defects with an unusual aneurysmal common arterial trunk. Pinch1 mutants also exhibit multiple deficiencies in cranial neural crest-derived structures. Fate mapping demonstrated that initial migration of neural crest cells to the pharyngeal arch region occurs normally in the mutant embryos. However, in the cardiac outflow tract of mutants, neural crest cells exhibited hyperplasia and failed to differentiate into smooth muscle. Markedly increased apoptosis is observed in outflow tract cushions of mutants between embryonic days 11.5 and 13.5, likely contributing to the observed defects in cushion/valve remodeling and ventricular septation. Expression of transforming growth factor-beta(2), which plays a crucial role in outflow tract development, was decreased or absent in the outflow tract of the mutants. The decrease in transforming growth factor-beta(2) expression preceded neural crest cell death. Together, our results demonstrate that Pinch1 plays an essential role in neural crest development, perhaps in part through transforming growth factor-beta signaling.

PMID: 17272814 [PubMed - indexed for MEDLINE]

Generation of Cajal-Retzius neurons in mouse forebrain is regulated by transforming growth factor beta-Fox signaling pathways.

Department of Neuroscience and Physiology, State University of New York-Upstate Medical University, Syracuse, NY 13210, USA.

The generation of Cajal-Retzius (CR) neurons is restricted to discrete sites in the telencephalon. Most of these sites do not express Foxg1, a transcription factor that inhibits transforming growth factor (TGF)beta-dependent upregulation of p21. We tested the hypothesis that TGFbeta signaling triggers CR neurogenesis in Foxg1-deficient zones through p21 induction. In Foxg1(+/+) mice, p21 (a) was expressed in select cycling cells in CR neuron-producing areas and (b) was co-localized in newly generated CR neurons. Zones of CR neuronal production and p21 expression were expanded in the forebrains of Foxg1(Cre/Cre) mice. Manipulation of TGFbeta signaling in explants from cortical hems of wild-type mice altered p21 expression and the production of CR neurons. Furthermore, despite continued TGFbeta activity, p21 immunoreactivity diminished in CR neurons with distance from their generation site. This implicated a second pathway controlling p21 expression. We provide evidence that Foxo3a, which has been shown to translocate into the nucleus to act as a transcriptional co-activator of TGFbeta-dependent upregulation of p21, is strategically expressed to be involved in controlling p21 expression in CR neurons. Specifically, Foxo3a was nuclear in p21+/reelin+ cells in sites of CR neuronal generation, however, nuclear Foxo3a immunoreactivity was absent in p21-/reelin+ cells distal from sites of CR neurogenesis. Thus, TGFbeta and Foxo3a may work in concert to regulate expression of p21 during CR neuronal generation.

PMID: 18005957 [PubMed - indexed for MEDLINE]

PMCID: PMC2278013

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]

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]

Transforming growth factor beta-SMAD2 signaling regulates aortic arch innervation and development.

Department of Anatomy and Embryology, Leiden University Medical Center, PO Box 9602, 2300 RC Leiden, The Netherlands.

Aortic arch interruptions in humans and animal models are mainly caused by aberrant development of the fourth pharyngeal arch artery. Little is known about the maturation of this vessel during normal and abnormal development, which is the subject of this study. Tgfbeta2 knockout mice that present with fourth artery defects have been associated with defective neural crest cell migration. In this study, we concentrated on pharyngeal arch artery development during developmental days 12.5 to 18.5, focusing on neural crest cell migration using a Wnt1-Cre by R26R neural crest cell reporter mouse. Fourth arch artery maturation was studied with antibodies directed against smooth muscle alpha-actin and neural NCAM-1 and RMO-270. For diminished transforming growth factor beta (TGF-beta) signaling, SMAD2 and fibronectin have been analyzed. Neural crest migration and differentiation into smooth muscle cells is unaltered in mutants, regardless of the cardiovascular defect found; however, innervation of the fourth arch artery is affected. Absent staining for nuclear SMAD2, NCAM-1, and RMO-270 in the fourth artery in mutant coincides with severe defects of this segment. Likewise, fibronectin expression is diminished in these cases. From these data we conclude the following: (1) neural crest cell migration is not a common denominator in cardiovascular defects of Tgfbeta2-/- mice; (2) fourth arch artery maturation is a complex process involving innervation; and (3) TGF-beta2 depletion diminishes SMAD2-signaling in the fourth arch artery and coincides with reduced vascular NCAM-1 expression and neural innervation of this artery. We hypothesize that disturbed maturation of the fourth pharyngeal arch artery, and especially abrogated vascular innervation, will result in fourth arch interruptions.

PMID: 15528466 [PubMed - indexed for MEDLINE]

Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1.

Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674, USA.

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.

PMID: 14764653 [PubMed - indexed for MEDLINE]

Divergence of epidermal growth factor - transforming growth factor beta signaling in embryonic orofacial tissue.

University of Louisville Birth Defects Center, Department of Molecular, Cellular and Craniofacial Biology, University of Louisville School of Dentistry, Louisville, Kentucky 40292, USA. vasker.bhattacherjee@louisville.edu

The epidermal growth factor (EGF) and transforming growth factor beta (TGFbeta) families of signaling molecules play a major role in growth and development of embryos. Abrogation of either signaling pathway results in defects in embryogenesis, including cleft palate. In the developing palate, both EGF and TGFbeta regulate cellular proliferation, extracellular matrix synthesis, and cellular differentiation but often in an opposing manner. Evidence from various adult cell types suggests the existence of cross talk between the EGF and TGFbeta signaling pathways, although it is unclear whether such cross talk exists in murine embryonic maxillary mesenchymal cells, from which the developing palate is derived. In this study, embryonic maxillary mesenchymal cells in culture were treated with EGF and TGFbeta, either singly or in combination, and the cells were subsequently examined for signaling interactions between these two pathways. Immunoblot analyses of nuclear extracts of embryonic maxillary mesenchymal cells revealed that TGFbeta-induced nuclear translocation of Smad 2 and Smad 3 proteins was not affected by EGF. Conversely, immunoblot analyses of whole-cell extracts of these cells indicated that EGF-induced phosphorylation of extracellular signal-regulated kinase proteins, ERK1 and ERK2, was not affected by TGFbeta. Expression of a transfected luciferase reporter gene driven by a promoter with Smad binding elements was induced by TGFbeta in these cells but was not affected by EGF. Last, TGFbeta was found to induce expression of the endogenous gelatinase B gene in embryonic maxillary mesenchymal cells; however, this effect was independent of any interaction of EGF. Collectively, data from this study suggest that the EGF and TGFbeta signal transduction pathways do not converge in murine embryonic maxillary mesenchymal cells.

PMID: 12880367 [PubMed - indexed for MEDLINE]

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]

Tgf-beta3-induced palatal fusion is mediated by Alk-5/Smad pathway.

Developmental Biology Program, Department of Pathology of University of Southern California, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA.

Cleft palate is among the most common birth defects in humans, caused by a failure in the complex multistep developmental process of palatogenesis. It has been recently shown that transforming growth factor beta3 (Tgf-beta3) is an absolute requirement for successful palatal fusion, both in mice and humans. However, very little is known about the mechanisms of Tgf-beta3 signaling during this process. Here we show that putative Tgf-beta type I receptors, Alk-1, Alk-2, and Alk-5, are all endogenously expressed in the palatal epithelium. Activation of Alk-5 in the Tgf-beta3 (-/-) palatal epithelium is able to rescue palatal fusion, whereas inactivation of Alk-5 in the wild-type palatal epithelium prevents palatal fusion. The effect of Alk-2 is similar, but less pronounced. The induction of fusion by activation of Alk-5 or Alk-2 is stronger in the posterior parts of the palates at the embryonic day 14 (E14), while their activation at E13.5 also restores anterior fusion, reflecting the natural anterior-posterior direction of palate maturation in vivo. We also show that Smad2 is endogenously activated in the palatal midline epithelial seam (MES) during the fusion process. By using a mutant Alk-5 receptor that is an active kinase but is unable to activate Smads, we show that activation of Smad-independent Tgf-beta responses is not sufficient to induce fusion of shelves deficient in Tgf-beta3. Based on these observations, we conclude that the Smad2-dependent Alk-5 signaling pathway is dominant in palatal fusion driven by Tgf-beta3.

PMID: 14729481 [PubMed - indexed for MEDLINE]