Absence Of TGF-beta Research Articles

Abstract 1055: Atxn2 Inhibition Enhances The Contractile Phenotype Of Vascular Smooth Muscle Cells

Introduction: Phenotype switching of vascular smooth muscle cells (VSMC) between their synthetic and differentiated contractile state is a critical player in controlling the stability of atherosclerotic plaque. Hundreds of genes associated with coronary artery disease (CAD) have been identified through Genome-Wide Association Studies (GWAS). However, the involvement of most identified genes in the pathogenesis of atherosclerosis remains unknown. ATXN2 was identified as a CAD GWAS candidate gene with unclear biological role in atherosclerosis. ATXN2 regulates the endocytic internalization of the EGF receptor, which is essential in the VSMC phenotype switching. Therefore, we aimed to investigate whether ATXN2 is involved in the VSMC phenotype switching. Methods and Results: Using immunofluorescence, we demonstrated that ATXN2 siRNA knockdown resulted in a 5-fold increase in the ratio of the contractile phenotype of human aortic VSMC that expressed a high alpha-smooth muscle cell actin content compared to controls. In addition, we found that ATXN2 inhibition reduced the VSMC proliferation and migration as assessed by the EdU cell proliferation assay and the wound healing migration assay, respectively. Next, we confirmed that ATXN2 siRNA knockdown increased the mRNA expression of contractile markers: calponin, alpha-smooth muscle cell actin, and smooth muscle protein 22 alpha using quantitative PCR. We found that the enhancing effect of VSMC contractile phenotype by ATXN2 siRNA was less pronounced in the absence of TGF-beta. Indeed, the enhancing effect of ATXN2 inhibition on the VSMC contractile phenotype was abolished by SMAD4 gene silencing, suggesting the involvement of TGF-beta signaling. Furthermore, we validated the expression signature of the ATXN2 gene in different phenotypic subsets of VSMC from human atherosclerotic plaque samples using single-cell RNA sequencing analysis. We detected that ATXN2 expression was negatively correlated with the contractile VSMC subset. In conclusion, our findings suggests ATXN2, CAD GWAS candidate gene, as a novel potential therapeutic target to enhance the VSMC contractile phenotype for stabilizing atherosclerotic plaque in coronary artery disease patients.

Effect of Swelling Ratio of Injectable Hydrogel Composites on Chondrogenic Differentiation of Encapsulated Rabbit Marrow Mesenchymal Stem Cells In Vitro

An injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) has been investigated as a cell and growth factor carrier for cartilage tissue engineering applications. In this study, hydrogel composites with different swelling ratios were prepared by cross-linking OPF macromers with poly(ethylene glycol) (PEG) repeating units of varying molecular weights from 1000 approximately 35000. Rabbit marrow mesenchymal stem cells (MSCs) and MPs loaded with transforming growth factor-beta1 (TGF-beta1) were encapsulated in the hydrogel composites to examine the effect of the swelling ratio of the hydrogel composites on the chondrogenic differentiation of encapsulated rabbit marrow MSCs both in the presence and in the absence of TGF-beta1. The swelling ratio of the hydrogel composites increased as the PEG molecular weight in the OPF macromers increased. Chondrocyte-specific genes were expressed at higher levels in groups containing TGF-beta1-loaded MPs and varied with the swelling ratio of the hydrogel composites. OPF hydrogel composites with PEG repeating units of molecular weight 35000 and 10000 with TGF-beta1-loaded MPs exhibited a 159 +/- 95- and a 89 +/- 31-fold increase in type II collagen gene expression at day 28, respectively, while OPF hydrogel composites with PEG repeating units of molecular weight 3000 and 1000 with TGF-beta1-loaded MPs showed a 27 +/- 10- and a 17 +/- 7-fold increase in type II collagen gene expression, respectively, as compared to the composites with blank MPs at day 0. The results indicate that chondrogenic differentiation of encapsulated rabbit marrow MSCs within OPF hydrogel composites could be affected by their swelling ratio, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for controlling the differentiation of stem cells.

Late Developmental Plasticity in the T Helper 17 Lineage

Development of T helper (Th) 17 cells requires transforming growth factor (TGF)-beta and interleukin (IL)-6 and is independent of the Th1 pathway. Although T cells that produce interferon (IFN)-gamma are a recognized feature of Th17 cell responses, mice deficient for STAT4 and T-bet-two prototypical Th1 transcription factors-are protected from autoimmunity associated with Th17 pathogenesis. To examine the fate and pathogenic potential of Th17 cells and origin of IFN-gamma-producing T cells that emerge during Th17 immunity, we developed IL-17F reporter mice that identify cells committed to expression of IL-17F and IL-17A. Th17 cells required TGF-beta for sustained expression of IL-17F and IL-17A. In the absence of TGF-beta, both IL-23 and IL-12 acted to suppress IL-17 and enhance IFN-gamma production in a STAT4- and T-bet-dependent manner, albeit with distinct efficiencies. These results support a model of late Th17 developmental plasticity with implications for autoimmunity and host defense.

Regulation of connective tissue growth factor (CTGF) by hepatocyte growth factor in human tubular epithelial cells

Hepatocyte growth factor (HGF) is a pleiotropic protein with renoprotective functions, which have been attributed at least in part to its ability to counteract the profibrotic effects of transforming growth factor beta (TGF-beta). A major downstream mediator of TGF-beta is connective tissue growth factor (CTGF). However, the molecular mechanisms of CTGF regulation by HGF have not yet been investigated. CTGF expression was analysed in human primary tubular epithelial cells (hPTECs) and the cell line HKC-8 by western blotting. Morphological alterations were analysed by immunocytochemistry. HGF induced a transient expression of CTGF, which was maximal after 6 h and returned to baseline after 24 h. Coincubation with TGF-beta increased CTGF protein at 6 h, whereas HGF significantly decreased CTGF induction by TGF-beta after 24 h. Furthermore, HGF induced cell scattering associated with reorganization of focal adhesions and formation of lamellipodia and filopodia. The early induction of CTGF was linked to the HGF-mediated alterations of cell morphology. The PP2 inhibitor of Src-family kinases, which regulate focal adhesion turnover, reduced HGF-mediated upregulation of CTGF. In addition, inhibition of the Rho-kinase, which modulates the actin cytoskeleton, impaired CTGF expression. Combination of both inhibitors further decreased CTGF expression. Comparable inhibitory effects were obtained, when CTGF was induced by the combination of HGF and TGF-beta. We provide evidence for a dual effect of HGF on CTGF regulation in human tubular epithelial cells: transient upregulation of CTGF in the absence of TGF-beta, which was related to alterations of cell morphology, and interference with TGF-beta-mediated CTGF induction after prolonged incubation.

Activin A Induces Langerhans Cell Differentiation In Vitro and in Human Skin Explants

Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFβ. Ιn vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFβ. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFβ family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFβ. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFβ, responsible for LC differentiation during inflammatory/autoimmune conditions.

TGFβ‐mediated BIM expression and apoptosis are regulated through SMAD3‐dependent expression of the MAPK phosphatase MKP2

Transforming growth factor-beta (TGFbeta) induces the expression of the pro-apoptotic protein BIM, and mediates apoptosis in hepatocytes and B lymphocytes. BIM is regulated through a post-translational mechanism involving ERK-dependent phosphorylation and ubiquitin-mediated proteasomal degradation. Here, we show that TGFbeta induces BIM through its rapid inhibition of ERK, thereby preventing the phosphorylation and degradation of BIM. TGFbeta, through a SMAD3-dependent mechanism, transcriptionally induces the mitogen-activated protein kinase (MAPK) phosphatase MKP2, encoded by an immediate early gene, to attenuate ERK and promote the accumulation of BIM protein. Overexpression of MKP2 in hepatocytes modulates ERK-mediated phosphorylation of BIM and apoptosis in the absence of TGFbeta, whereas its ablation in pro-B cells, derived from MKP2-deficient mice, protects cells from TGFbeta-mediated apoptosis, and blocks TGFbeta-induced ERK inhibition and BIM induction. Furthermore, in pro-B cells derived from SMAD3-deficient mice, induction of MKP2 by TGFbeta, inhibition of ERK, induction of BIM and apoptosis do not occur. Our results indicate that MKP2 mediates TGFbeta-dependent apoptosis by linking SMAD3 to the modulation of ERK activity and mitochondrial-mediated pro-apoptotic events.

ClC-3 is required for LPA-activated Cl− current activity and fibroblast-to-myofibroblast differentiation

To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl(-) currents (I(Cl,LPA) and I(Cl,VRAC), respectively), cell differentiation, and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3, within a pcDNA3.1 vector, into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure I(Cl,LPA) and I(Cl,VRAC) currents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased I(Cl,LPA) and I(Cl,VRAC) activity in the presence of transforming growth factor-beta(1) (TGF-beta(1)) compared with controls, whereas ClC-3 overexpression resulted in increased I(Cl,LPA) activity in the absence of TGF-beta(1). ClC-3 knockdown also resulted in a reduction of alpha-smooth muscle actin (alpha-SMA) protein levels in the presence of TGF-beta(1), whereas ClC-3 overexpression increased alpha-SMA protein expression in the absence of TGF-beta(1). In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the I(Cl,LPA) current activity, and participates in the fibroblast-to-myofibroblast transition.

TβRIII suppresses non-small cell lung cancer invasiveness and tumorigenicity

The transforming growth factor-beta (TGF-beta) superfamily has essential roles in lung development, regulating cell proliferation, branching morphogenesis, differentiation and apoptosis. Although most lung cancers become resistant to the tumor suppressor effects of TGF-beta, and loss or mutation of one of the components of the TGF-beta signaling pathway, including TbetaRII, Smad2 and Smad4 have been reported, mutations are not common in non-small cell lung cancer (NSCLC). Here we demonstrate that the TGF-beta superfamily co-receptor, the type III TGF-beta receptor (TbetaRIII or betaglycan) is lost in the majority of NSCLC specimens at the mRNA and protein levels, with loss correlating with increased tumor grade and disease progression. Loss of heterozygosity at the TGFBR3 genomic locus occurs in 38.5% of NSCLC specimens and correlates with decreased TbetaRIII expression, suggesting loss of heterozygosity as one mechanism for TbetaRIII loss. In the H460 cell model of NSCLC, restoring TbetaRIII expression decreased colony formation in soft agar. In the A549 cell model of NSCLC, restoring TbetaRIII expression significantly decreased cellular migration and invasion through Matrigel, in the presence and absence of TGF-beta1, and decreased tumorigenicity in vivo. In a reciprocal manner, shRNA-mediated silencing of endogenous TbetaRIII expression enhanced invasion through Matrigel. Mechanistically, TbetaRIII functions, at least in part, through undergoing ectodomain shedding, generating soluble TbetaRIII, which is able to inhibit cellular invasiveness. Taken together, these results support TbetaRIII as a novel tumor suppressor gene that is commonly lost in NSCLC resulting in a functional increase in cellular migration, invasion and anchorage-independent growth of lung cancer cells.

Endogenous TNFα Suppression of Neovascularization in Corneal Stroma in Mice

To examine the role of tumor necrosis factor alpha (TNFalpha) in stromal neovascularization in injured cornea in vivo and in cytokine-enhanced vessel-like endothelial cell tube formation in vitro. An in vitro model of angiogenesis was used to examine the roles of TNFalpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta1 (TGFbeta1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNFalpha-null (Tnfalpha(-/-)) mice. At 7, 14, or 21 days of injury, experimental mice were killed, and the eyes were enucleated and subjected to histologic and immunohistochemical examination and real-time reverse transcription-polymerase chain reaction. HUVECs formed a vessel-like tube structure on the fibroblast feeder layer. Adding TGFbeta1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNFalpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGFbeta1 or VEGF in coculture. In vivo, cauterization of the central cornea induced the formation of CD31(+) new vessels surrounding the limbus in WT mice. More prominent central stromal neovascularization accompanied by increased expression of TGFbeta1 and VEGF was found in Tnfalpha(-/-) mice compared with WT mice. In addition to inhibiting TGFbeta1 and VEGF expression by fibroblasts, endogenous TNFalpha may counter the induction effects of TGFbeta1 and VEGF on vascular endothelial cells and may block neovascularization.

Epigenetic Control of the foxp3 Locus in Regulatory T Cells

Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4+ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3+CD25+CD4+ Tregs, but not in naïve CD25−CD4+ T cells. Partial DNA demethylation is already found within developing Foxp3+ thymocytes; however, Tregs induced by TGF-β in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-β–induced Foxp3+ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.

Opposite effect of corticosteroids and long‐acting β2‐agonists on serum‐ and TGF‐β1‐induced extracellular matrix deposition by primary human lung fibroblasts

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation and major structural lung tissue changes including increased extracellular matrix (ECM) deposition. Inhaled corticosteroids and long-acting beta(2)-agonists (LABA) are the basic treatment for both diseases, but their effect on airway remodeling remains unclear. In this study, we investigated the effect of corticosteroids and LABA, alone or in combination, on total ECM and collagen deposition, gene expression, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels by primary human lung fibroblasts. In our model, fibroblasts in 0.3% albumin represented a non-inflammatory condition and stimulation with 5% FCS and/or TGF-beta(1) mimicked an inflammatory environment with activation of tissue repair. FCS (5%) increased total ECM, collagen deposition, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels. In 0.3% albumin, corticosteroids reduced total ECM and collagen deposition, involving the glucocorticoid receptor (GR) and downregulation of collagen, heat shock protein 47 (Hsp47), and Fli1 mRNA expression. In 5% FCS, corticosteroids increased ECM deposition, involving upregulation of COL4A1 and CTGF mRNA expression. LABA reduced total ECM and collagen deposition under all conditions partly via the beta(2)-adrenergic receptor. In combination, the drugs had an additive effect in the presence or absence of TGF-beta(1) further decreasing ECM deposition in 0.3% albumin whereas counteracting each other in 5% FCS. These data suggest that the effect of corticosteroids, but not of LABA, on ECM deposition by fibroblasts is altered by serum. These findings imply that as soon as airway inflammation is resolved, long-term treatment with combined drugs may beneficially reduce pathological tissue remodeling.

Endogenous Transforming Growth Factor-β Receptor-mediated Smad Signaling Complexes Analyzed by Mass Spectrometry

ASmad proteins are the central feature of the transforming growth factor-beta (TGF-beta) intracellular signaling cascade. They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes. Changes in Smad proteins and their complexes upon treatment with TGF-beta were studied in mink lung epithelial (Mv1Lu) cell cultures. A time course of incubation with TGF-beta was carried out to determine the peak of appearance of phosphorylated Smad2. Immobilized monoclonal antibody against Smad2 was then used to isolate the naturally occurring complexes. Three strategies were used to identify changes in proteins partnering with Smad2: separation by one-dimensional SDS-PAGE followed by MALDI peptide mass fingerprinting, cleavable ICAT labeling of the protein mixtures analyzed by LC-MS/MS, and nano-LC followed by MALDI MS TOF/TOF. Smad2 forms complexes with many other polypeptides both in the presence and absence of TGF-beta. Some of the classes of proteins identified include: transcription regulators, proteins of the cytoskeletal scaffold and other tethering proteins, motility proteins, proteins involved in transport between the cytoplasm and nucleus, and a group of membrane adaptor proteins. Although some of these have been reported in the literature, most have not been reported previously. This work expands the repertoire of proteins known to participate in the TGF-beta signal transduction processes.

Transforming growth factor‐β1 enhances the antifibrinolytic and prothrombotic state of growing endothelial cells in a cell cycle‐specific manner

Cell cycle-dependent modulation of protein expression may influence the balance of antithrombotic and prothrombotic properties of endothelial cells. In the present study, we examined the regulation of prothrombotic and antithrombotic molecules by transforming growth factor-beta1 (TGF-beta1) during distinct phases of the cell cycle in human umbilical vein endothelial cells. In the absence of TGF-beta1, the expression of thrombomodulin, the plasminogen activators u-PA and t-PA, and their inhibitor PAI-1 was significantly increased in the S/G2 compared to the G1 phase. Treatment of endothelial cells with TGF-beta1, however, resulted in elevated expression of PAI-1 specifically in the S/G2 phase, while t-PA and u-PA increased to the same extent in both the G1 and S/G2 phase. These findings demonstrate that the expression of a subset of hemostatically relevant proteins is regulated during endothelial cell cycle and that TGF-beta1 can differentially modulate cell cycle-controlled protein expression.

TGF-β 1 inhibits late-stage mast cell maturation

The objective of this study was to determine the effects of transforming growth factor (TGF)-beta1 on mast cell development. Mast cells were cultured from mouse bone marrow in interleukin (IL)-3 + stem cell factor, in the presence or absence of TGF-beta1. We assessed mast cell development by measuring the expression of kit, T1/ST2, FcvarepsilonRI, and Fcgamma receptors. Cell morphology was determined by histochemical staining. Alterations in FcvarepsilonRI subunit expression were measured by Western blot analysis. Adoptive transfer of cultured mast cells into mast cell-deficient W/W(v) mice was used to determine if the in vivo environment could reverse the inhibitory effects of TGF-beta1. TGF-beta1 decreased FcvarepsilonRI, c-kit, T1/ST2, and FcgammaR expression, and inhibited granule formation in developing mast cells. Accessory cells were not required for this inhibition. Smad3 deficiency did not alter the response of bone marrow cells to TGF-beta1. TGF-beta1 inhibited expression of the FcvarepsilonRI alpha subunit protein, without decreasing beta or gamma proteins. Mast cells derived in the presence of TGF-beta1 were functionally impaired, as IgE-mediated cytokine secretion was greatly reduced. The changes in granule formation and surface antigen expression were long-standing, as they were not reversed by transfer to W/W(v) mice. TGF-beta1 may contribute to mast cell homeostasis by inhibiting maturation from bone marrow precursors. The effects of TGF-beta1 result in greatly diminished expression of cell surface markers, reduced granulation, and lack of responsiveness to IgE-mediated activation. Thus TGF-beta1 can serve as a potent and multifunctional regulator of mast cell maturation.

Runx1/AML1/Cbfa2 Mediates Onset of Mesenchymal Cell Differentiation Toward Chondrogenesis

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.

SB-431542 inhibits TGF-β-induced contraction of collagen gel by normal and keloid fibroblasts

Transforming growth factor (TGF)-beta induces fibroblast contraction, which is implicated in wound healing and keloid formation. SB-431542 is a novel specific inhibitor of TGF-beta type I receptor kinase activity. We sought to determine whether SB-431542 inhibited TGF-beta-induced fibroblast contraction. We used an in vitro type I collagen gel contraction assay with normal or keloid dermal fibroblasts incorporated. TGF-beta induced contraction of collagen gels with normal dermal fibroblasts incorporated, which was efficiently suppressed by SB-431542. Keloid fibroblasts showed higher basal contraction of collagen gels in the absence of TGF-beta than normal fibroblasts, which was enhanced by addition of TGF-beta. SB-431542 suppressed both the basal and TGF-beta-enhanced contraction of collagen gels by keloid fibroblasts. These inhibitory effects of SB-431542 were associated with suppression of TGF-beta-induced alpha-smooth muscle actin (alpha-SMA) expression and phosphorylation of Smad2 in normal and keloid fibroblasts. SB-431542 can suppress TGF-beta-induced contraction of collagen gel by normal and keloid dermal fibroblasts. Importantly, SB-431542 can inhibit basal contraction of collagen gel by keloid fibroblasts. These results suggest that an inhibitor of TGF-beta type I receptor kinase activity may have therapeutic potential for excessive skin contraction as observed in keloid.

Influence of exogenous TGFbeta1 on the expression of smad2 and smad3 in rat bone marrow-derived mesenchymal stem cells.

The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorbeta1 (TGFbeta1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The effects of different concentrations of TGFbeta1 on cell proliferation and ALP activity were detected by MTT and PNPP in MSCs respectively. The expression of Smad2 and Smad3 and the influence of exogenous TGFbeta1, on them were also examined by immunocytochemistry and Western blot assays. The exogenous TGFbeta1 induced a dose-dependent decrease in cell proliferation and a dose-dependent increase in ALP activity, which plateaued at 5 ng/ml. Smad2 and Smad3 proteins were detected only in the cytoplasm in the absence of TGFbeta1, and TGFbeta1, could stimulate the translocation of them from the cytoplasm to the nucleus. The total amount of Smad2 protein remained unchanged before and after TGFbeta1 treatment (P > 0.05). The expression levels of Smad3 remained unchanged after 3 h and 6 h treatment (P > 0.05), but decreased markedly after 24 h treatment (P <0.05). It was concluded that TGFbeta1 is a latent osteoinductive factor involved in osteoblastic differentiation. Both Samd2 and Smad3 mediate TGFbeta1 signaling as downstream mediators in MSCs. The biological output of TGFbeta1 triggering the osteoblastic differentiation could be entirely determined by Smad3 in MSCs.

Association between endogenous gene expression and growth regulation induced by TGF-β1 in human gastric cancer cells

To investigate the association between endogenous gene expression and growth regulation including proliferation and apoptosis induced by transforming growth factor-beta1 (TGF-beta1) in human gastric cancer (GC) cells. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the main components of the TGF-beta1/Smads signal pathway in human poorly differentiated GC cell line BGC-823. Localization of Smad proteins was also determined using immunofluorescence. Then, the BGC-823 cells were cultured in the presence or absence of TGF-beta1 (10 ng/mL) for 24 and 48 h, and the effects of TGF-beta1 on proliferation and apoptosis were measured by cell growth curve and flow cytometry (FCM) analysis. The ultrastructural features of BGC-823 cells with or without TGF-beta1 treatment were observed under transmission electron microscope. The apoptotic cells were visualized by means of the terminal deoxynucleotidyl transferase (TdT)-mediated dTUP in situ nick end-labeling (TUNEL) method. Meanwhile, the expression levels of endogenous p15, p21 and Smad7 mRNA and the corresponding proteins in the cells were detected at 1, 2 and 3 h after culture in the presence or absence of TGF-beta1 (10 ng/mL) by semi-quantitative RT-PCR and Western blot, respectively. The TGF-beta1/Smad signaling was found to be intact and functional in BGC-823 cells. The growth curve revealed the most evident inhibition of cell proliferation by TGF-beta1 at 48 h, and FCM assay showed G1 arrest accompanied with apoptosis induced by TGF-beta1. The typical morphological changes of apoptosis were observed in cells exposed to TGF-beta1. The apoptosis index (AI) in TGF-beta1-treated cells was significantly higher than that in the untreated controls (10.7+/-1.3% vs 0.32+/-0.06%, P<0.01). The levels of p15, p21 and Smad7 mRNA and corresponding proteins in cells were significantly up-regulated at 1 h, but gradually returned to basal levels at 3 h following TGF-beta1 (10 ng/mL) treatment. TGF-beta1 affects both proliferation and apoptosis of GC cells through the regulation of p15 and p21, and induces transient expression of Smad 7 as a negative feedback modulation of TGF-beta1 signal. Our results suggest a novel functional role of p21 as an accelerant of TGF-beta1-mediated apoptosis in GC cells.

Sphingosine Kinase 1 (SPHK1) Is Induced by Transforming Growth Factor-β and Mediates TIMP-1 Up-regulation

Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (approximately 2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK1 may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-beta. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-beta. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF-beta cooperated in TIMP-1 up-regulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-1 gene.

Hepatitis C viral proteins interact with Smad3 and differentially regulate TGF-beta/Smad3-mediated transcriptional activation.

Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine implicated as a pathogenic mediator in various liver diseases. Enhanced TGF-beta production and lack of TGF-beta responses are often observed during hepatitis C virus (HCV) infection. In this study, we demonstrate that TGF-beta-mediated transactivation is decreased in cells exogenously expressing the intact HCV polyprotein. Among 10 viral products of HCV, only core and nonstructural protein 3 (NS3) physically interact with the MH1 (Mad homology 1) region of the Smad3 and block TGF-beta/Smad3-mediated transcriptional activation through interference with the DNA-binding ability of Smad3, not the nuclear translocation. However, the interactive domain of NS3 extends to the MH2 (Mad homology 2) region of Smad3 and a distinction is found between effects mediated, respectively, by these two viral proteins. HCV core, in the presence or absence of TGF-beta, has a stronger suppressive effect on the DNA-binding and transactivation ability of Smad3 than NS3. Although HCV core, NS3, and the HCV subgenomic replicon all attenuate TGF-beta/Smad3-mediated apoptosis, only HCV core represses TGF-beta-induced G1 phase arrest through downregulation of the TGF-beta-induced p21 promoter activation. Along with this, HCV core, rather than NS3, exhibits a significant inhibitory effect on the binding of Smad3/Sp1 complex to the proximal p21 promoter in response to TGF-beta. In conclusion, HCV viral proteins interact with the TGF-beta signaling mediator Smad3 and differentially impair TGF-beta/Smad3-mediated transactivation and growth inhibition. This functional counteraction of TGF-beta responses provides insights into possible mechanisms, whereby the HCV oncogenic proteins antagonize the host defenses during hepatocarcinogenesis.