Smad3 mediates the TGF-beta-induced contraction of type I collagen gels by mouse embryo fibroblasts

TGF-beta signals through TGF-beta receptors and Smad proteins. TGF-beta also augments fibroblast-mediated collagen gel contraction, an in vitro model of connective tissue remodeling. To investigate the importance of Smad2 or Smad3 in this augmentation process, embryo-derived fibroblasts from mice lacking expression of Smad2 or Smad3 genes were cast into native type I collagen gels. Fibroblast-populated gels were then released into 0.2% FCS-DMEM alone or with recombinant human TGF-beta1, beta2, beta3, or recombinant rat PDGF-BB. Gel contraction was determined using an image analyzer. All three isoforms of TGF-beta significantly augmented contraction of collagen gels mediated by fibroblasts with genotypes of Smad2 knockout (S2KO), Smad2 wildtype (S2WT), and Smad3 wildtype (S3WT), but not Smad3 knockout (S3KO) mice. PDGF-BB augmented collagen gel contraction by all fibroblast types. These results suggest that expression of Smad3 but not Smad2 may be critical in TGF-beta augmentation of fibroblast-mediated collagen gel contraction. Thus, the Smad3 gene could be a target for blocking contraction of fibrotic tissue induced by TGF-beta.     

Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.     

Transdifferentiation-dependent expression of alpha-SMA in hepatic stellate cells does not involve TGF-beta pathways leading to coinduction of collagen type I and thrombospondin-2.

Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.     

Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

Angiotensin II-induced differentiation of adipose tissue-derived mesenchymal stem cells to smooth muscle-like cells

Angiotensin II (Ang II) is involved in the development of cardiovascular disease and vascular remodeling. In this study, we demonstrate that treatment of human adipose tissue-derived mesenchymal stem cells (hADSCs) with Ang II increased the expression of smooth muscle-specific genes, including alpha-smooth muscle actin (alpha-SMA), calponin, h-caldesmon, and smooth muscle myosin heavy chain (SM-MHC), and also elicited the secretion of transforming growth factor-beta1 (TGF-beta1) and delayed phosphorylation of Smad2. The Ang II-induced expression of alpha-SMA and delayed phosphorylation of Smad2 were blocked by pretreatment of the cells with a TGF-beta type I receptor kinase inhibitor, SB-431542, small interference RNA-mediated depletion of endogenous Smad2, and adenoviral expression of Smad7. Furthermore, the Ang II-induced TGF-beta1 secretion, alpha-SMA expression, and delayed phosphorylation of Smad2 in hADSCs were abrogated by the MEK inhibitor U0126, suggesting a pivotal role of MEK/ERK pathway in the Ang II-induced activation of TGF-beta1-Smad2 signaling pathway. The smooth muscle-like cells which were differentiated from hADSCs by Ang II treatment exhibited contraction in response to 60mM KCl. These results suggest that Ang II induces differentiation of hADSCs to contractile smooth muscle-like cells through ERK-dependent activation of the autocrine TGF-beta1-Smad2 crosstalk pathway.     

Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.     

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.     

TGF-β1, TGF-β3, and PGE<Subscript>2</Subscript> regulate contraction of human patellar tendon fibroblasts

To understand the role of tendon fibroblast contraction in tendon healing, we investigated the contraction of human patellar tendon fibroblasts (HPTFs) and its regulation by transforming growth factor-beta1 (TGF-beta1), TGF-beta3, and prostaglandin E(2) (PGE(2)). HPTFs were found to wrinkle the underlying thin silicone membranes, demonstrating that these tendon fibroblasts are contractile. Using fibroblast populated collagen gels (FPCGs), exogenous addition of TGF-beta1 or TGF-beta3 was found to increase fibroblast contraction compared to non-treated fibroblasts in serum-free medium, whereas PGE(2) was found to decrease the tendon fibroblast contraction. Moreover, the tendon fibroblasts in collagen gels treated with TGF-beta1 contracted to a greater degree than those treated with TGF-beta3. Since the extent of fibroblast contraction is related to scar tissue formation, this differential effect of TGF-beta1 and TGF-beta3 on HPTF contraction supports the previous finding that TGF-beta1 induces scar tissue formation, whereas TGF-beta3 reduces its formation. Further, the reduced tendon fibroblast contraction by PGE(2) suggests that excessive presence of this inflammatory mediator in the wound site might retard tendon healing. Taken together, the results of this study suggest that regulation of human tendon fibroblast contraction may reduce scar tissue formation and therefore improve the mechanical properties of healing tendons.     

Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-cGMP pathway

Nitric oxide (NO) relaxes vascular smooth muscle in part through an accumulation of cGMP in the target cells. We hypothesized that a similar effect may also exist on collagen gel contraction mediated by human fetal lung (HFL1) fibroblasts, a model of wound contraction. To evaluate this, HFL1 cells were cultured in three-dimensional type I collagen gels and floated in serum-free DMEM with and without various NO donors. Gel size was measured with an image analyzer. Sodium nitroprusside (SNP, 100 microM) significantly augmented collagen gel contraction by HFL1 cells (78.5 +/- 0.8 vs. 58.3 +/- 2. 1, P < 0.01), whereas S-nitroso-N-acetylpenicillamine, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride, NONOate, and N(G)-monomethyl-L-arginine did not affect the contraction. Sodium ferricyanide, sodium nitrate, or sodium nitrite was not active. The augmentory effect of SNP could not be blocked by 1H-[1,2, 4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, whereas it was partially reversed by 8-(4-chlorophenylthio) (CPT)-cGMP. To further explore the mechanisms by which SNP acted, fibronectin and PGE(2) production were measured by immunoassay after 2 days of gel contraction. SNP inhibited PGE(2) production and increased fibronectin production by HFL1 cells in a concentration-dependent manner. CPT-cGMP had opposite effects on fibronectin and PGE(2) production. Addition of exogenous PGE(2) blocked SNP-augmented contraction and fibronectin production by HFL1 cells. Therefore, SNP was able to augment human lung fibroblast-mediated collagen gel contraction, an effect that appears to be independent of NO production and not mediated through cGMP. Decreased PGE(2) production and augmented fibronectin production may have a role in this effect. These data suggest that human lung fibroblasts in three-dimensional type I collagen gels respond distinctly to SNP by mechanisms unrelated to the NO-cGMP pathway.     

TGF-β1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-β1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-β1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-β1 and serum significantly augmented collagen gel contraction. TGF-β1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-β1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-β induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.     

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

Notch2 negatively regulates myofibroblastic differentiation of myoblasts

Myofibroblasts are one of the key cellular components involved in fibrosis of skeletal muscle as well as in other tissues. Transforming growth factor-beta1 (TGF-beta1) stimulates differentiation of mesenchymal cells into myofibroblasts, but little is known about the regulatory mechanisms of myofibroblastic differentiation. Since Notch2 was shown to be downregulated in TGF-beta1-induced non-muscle fibrogenic tissue, we investigated whether Notch2 also has a distinctive role in myofibroblastic differentiation of myogenic cells induced by TGF-beta1. TGF-beta1 treatment of C2C12 myoblasts led to expression of myofibroblastic marker alpha-smooth muscle actin (alpha-SMA) and collagen I with concomitant downregulation of Notch2 expression. Overexpression of active Notch2 inhibited TGF-beta1-induced expression of alpha-SMA and collagen I. Interestingly, transient knockdown of Notch2 by siRNA in C2C12 myoblasts and primary cultured muscle-derived progenitor cells resulted in differentiation into myofibroblastic cells expressing alpha-SMA and collagen I without TGF-beta1 treatment. Furthermore, we found Notch3 was counter-regulated by Notch2 in C2C12 cells. These findings suggest that Notch2 is inhibiting differentiation of myoblasts into myofibroblasts with downregulation of Notch3 expression.     

Antigen sensitization modulates alveolar macrophage functions in an asthma model

Fibroblast/myofibroblast expansion is critical in the pathogenesis of pulmonary fibrosis. To date, most research has focused on profibrotic mediators, whereas studies on antifibrotic factors are scanty. In this study, we explored the effects of acidic fibroblast growth factor (FGF-1) and FGF-1 plus heparin (FGF-1+H) on fibroblast growth rate, apoptosis, and myofibroblast differentiation. Heparin was used because it participates in FGF-1 signaling. Growth rate was evaluated by WST-1 colorimetric assay, DNA synthesis by [(3)H]thymidine incorporation, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved caspase 3. Expression of alpha-smooth muscle actin (alpha-SMA) was examined by immunocytochemistry, flow cytometry, real-time PCR, and immunoblotting. Despite the induction of DNA synthesis, FGF-1+H significantly reduced fibroblast growth rate. This correlated with a significant increase in apoptosis, evaluated by TUNEL (41.6 +/- 1.4% vs. 12.5 +/- 0.6% from controls; P < 0.01) and cleaved caspase 3 (295 +/- 32 vs. 200 +/- 19 ng/10(6) cells from controls; P < 0.05). Double immunostaining (alpha-SMA-TUNEL) revealed that the levels of induced apoptosis were similar in fibroblasts and myofibroblasts. FGF-1+H inhibited the effect of TGF-beta1 on myofibroblast differentiation. alpha-SMA-positive cells were reduced by immunocytochemistry from 44.5 +/- 6.5% to 10.9 +/- 1.9% and by flow cytometry from 30.6 +/- 2.5% to 7.7 +/- 0.6% (P < 0.01). Also, FGF-1+H significantly inhibited the TGF-beta1 induction of alpha-SMA quantified by real-time PCR and Western blot. This decrease was associated with a 35% reduction in TGF-beta1-induced collagen gel contraction. The effect of FGF-1+H was mediated by a significant decrease of TGF-beta1-induced Smad2 phosphorylation. FGF-1 alone exhibited similar but lower effects. These findings suggest that FGF-1 can have an antifibrogenic role, inducing apoptosis of fibroblasts and inhibiting myofibroblast differentiation.     

Effects of rhDecorin on TGF-beta1 induced human hepatic stellate cells LX-2 activation

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor beta (TGF-beta), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-beta, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-beta1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-beta1. Furthermore, the protein expression of smooth muscle-alpha-actin (alpha-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-beta1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.     

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7 days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7 days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7 days.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.