Role Of TGF-β Research Articles

Role of TGF-β1 in modulation of hepatic regeneration

Objective To investigate the role of TGF-β1 in regulation of liver regeneration after partial hepatectomy(PH).Methods The effect of TGF-β1 on the proliferation of primary hepatocytes was observed by MTT assay.The animal model of partial hepatectomy(PH)in rats was established.Serum collected from rats receiving partial hepatectomy could be used to stimulate the proliferation of IG12 and the expression of TGF-β1 in IG12 cells which could be detected by immuocytochemistry.The TGF-β1 expression characteristics of hepatocytes after PH were detected by immunohistochemistry and western blot.Results TGF-β1 could significantly arrest the proliferation of primary hepatocytes.Rat serum collected after PH could promote the proliferation of IG12,the result of MTT assay showed that the value of both 25ul group(A=1.109±0.033)and 15ul group(A=1.073±0.051)were higher than control (A=0.732±0.053,P＜0.05).Meanwhile,IG12 cells were stimulated by rat serum after PH,which showed a high expression of TGF-β1 by immunocytochemical assay.Immunohistochemical staining showed that TGF-β1 expression in hepatocytes of regenerated liver was decreased after PH,and then turned to be negative 3 day after PH.After liver regeneration accomplished,TGF-β1 positive signals was found again in hepatocytes.The same results were found by western blot assay.Conclusion TGF-β1 could significantly arrest the proliferation of primary hepatocytes.During the process of liver regeneration,the expression of TGF-β1 is gradually decreased,which is beneficial for the liver regeneration. Key words: Hepatectomy; TGF-β1; Hepatic regeneration

The role of TGFβ1 in multidrug resistance of gastric carcinoma cells and its possible mechanism

The role of TGFβ1 in multidrug resistance of gastric carcinoma cells and its possible mechanism

DAMGO-induced expression of chemokines and chemokine receptors: the role of TGF-β1

Studies from a number of laboratories suggest that modulation of cytokine expression plays an integral role in the immunomodulatory activity of opioids. Previously, our laboratory reported that activation of the mu-opioid receptor induced the expression of CCL2, CCL5, and CXCL10, as well as CCR5 and CXCR4. Previous work has also suggested the possibility that TGF-beta may participate in the opioid-induced regulation of immune competence, and in the present study, we set out to determine the role of this cytokine in the control of chemokine and chemokine receptor expression. We found that D-ala(2),N-Me-Phe(4)-Gly-ol(5)enkephalin (DAMGO), a highly selective mu-opioid agonist, induced the expression of TGF-beta1 expression at the protein and mRNA levels. In turn, the addition of TGF-beta1 was found to induce CCL5 and CXCR4 expression but not CCL2, CXCL10, or CCR5. Further analysis showed that pretreatment with neutralizing anti-TGF-beta1 blocked the ability of DAMGO to induce CCL5 or CXCR4. Similarly, pretreatment with cycloheximide prevented CCL5 or CXCR4 mRNA expression, consistent with the observation that DAMGO induction of chemokine and chemokine receptor expression requires newly synthesized TGF-beta1 protein. These results describe a common molecular basis for the activation of chemokine and chemokine receptor expression and may permit the development of strategies to inhibit certain undesirable immunological properties of micro-opioid agonists such as morphine and heroin.

Role of TGF-β1 and nitric oxide in the bystander response of irradiated glioma cells

The radiation-induced bystander effect (RIBE) increases the probability of cellular response and therefore has important implications for cancer risk assessment following low-dose irradiation and for the likelihood of secondary cancers after radiotherapy. However, our knowledge of bystander signaling factors, especially those having long half-lives, is still limited. The present study found that, when a fraction of cells within a glioblastoma population were individually irradiated with helium ions from a particle microbeam, the yield of micronuclei (MN) in the nontargeted cells was increased, but these bystander MN were eliminated by treating the cells with either aminoguanidine (an inhibitor of inducible nitric oxide (NO) synthase) or anti-transforming growth factor beta1 (anti-TGF-beta1), indicating that NO and TGF-beta1 are involved in the RIBE. Intracellular NO was detected in the bystander cells, and additional TGF-beta1 was detected in the medium from irradiated T98G cells, but it was diminished by aminoguanidine. Consistent with this, an NO donor, diethylamine nitric oxide (DEANO), induced TGF-beta1 generation in T98G cells. Conversely, treatment of cells with recombinant TGF-beta1 could also induce NO and MN in T98G cells. Treatment of T98G cells with anti-TGF-beta1 inhibited the NO production when only 1% of cells were targeted, but not when 100% of cells were targeted. Our results indicate that, downstream of radiation-induced NO, TGF-beta1 can be released from targeted T98G cells and plays a key role as a signaling factor in the RIBE by further inducing free radicals and DNA damage in the nontargeted bystander cells.

Role of TGF-β1 and its receptors in breast carcinogenesis: Evaluation of gene expression patterns and clinical implications

Objective Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine that may play an important role in tumor development and progression.

Transmission ratio distortion and maternal effects confound the analysis of modulators of cystic fibrosis disease severity on 19q13

Two entities localised within in a 5 Mb interval on 19q13, that is the transforming growth factor beta 1 (TGFbeta1) and the cystic fibrosis modifier 1, have been reported to modulate disease severity of cystic fibrosis (CF), albeit the designation of the risk allele for TGFbeta1 differs between studies. We have analysed genotyping data at seven microsatellite loci and four single nucleotide polymorphisms targeting the 19q13 area from 37 nuclear CF families with two affected offspring exhibiting extreme clinical phenotypes for indicators of transmission-ration distortion, maternal genetic or maternal non-genetic effects. Evidence for a transmission-ratio distortion was obtained at D19S112 (P=0.0304) near the recently characterised myotonic dystrophy locus myotonic dystrophy protein kinase (DMPK). Maternal and paternal genotype distributions were significantly different at rs1982073 (Leu10Pro at TGFbeta1) whereby all CF sibs heterozygous at rs1982073 inherited the Leu10 allele from their mother (P=0.000132) in our sibling panel. To ask whether the improved survival in CF over the last decades has any influence on TGFbeta1 allele frequencies, we analysed unrelated F508del homozygotes who were stratified by birth cohort. Sensitivity with respect to the survivor bias was reflected by significantly higher incidence of mild cystic fibrosis transmembrane conductance regulator mutation genotypes in the early born patient cohort (P=0.0169), and an allelic imbalance was also observed at TGFbeta1 (P=0.0664). In conclusion, the role of TGFbeta1 as a CF modulator, suggested from studies with a case-control setting, needs to be interpreted with caution unless family-based analysis is carried out to identify parental genetic and non-genetic effects.

Accelerated senescence of human peritoneal mesothelial cells exposed to high glucose: the role of TGF-β1

Cellular senescence can be activated in response to noxious environmental stimuli. A senescent-like phenotype has been detected in the peritoneal mesothelium of mice exposed to high intraperitoneal glucose. We have sought to examine whether high glucose (HG) can induce the senescence program in human peritoneal mesothelial cells (HPMC) in vitro. Senescence of omentum-derived HPMC was induced by serial passages. Cells were cultured in media containing either 5?mM glucose, 30?mM glucose, or 5?mM glucose and 25?mM mannitol (M) for osmotic control. Compared with HPMC cultured in low glucose, the growth rate of cells exposed to HG was significantly decreased so that the cells reached fewer population doublings before entering senescence. Exposure to HG led to increased expression of senescence-associated β-galactosidase (SA-β-Gal) and of the cell cycle inhibitors p21Waf1 and p27Kip1. Late-passage HPMC exposed to HG displayed marked hypertrophy and released increased amounts of fibronectin and TGF-β1. These effects were absent from HPMC treated with equimolar M. Exposure of early-passage HPMC to exogenous recombinant TGF-β1 induced a senescence marker SA-β-Gal in a dose-dependent manner and mimicked other senescence-associated alterations induced by HG. The addition of anti-TGF-β1 neutralizing antibody partially reduced the activation of HG-induced SA-β-Gal. These results indicate that chronic exposure to elevated glucose may result in TGF-β1-mediated accelerated senescence of HPMC in vitro, which may hypothetically contribute to the peritoneal membrane dysfunction during peritoneal dialysis in vivo.

Role of TGFβ1 and αvβ3‐integrin in the control of collagen type 1 turnover in response to angiotensin II and high glucose

Role of TGFβ1 and αvβ3‐integrin in the control of collagen type 1 turnover in response to angiotensin II and high glucose

Modulation of CD103 Expression on Human Colon Carcinoma-Specific CTL

Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103alpha(E)/beta(7) by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/beta(7) by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/beta(7) in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-beta1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8(+) T cells in the presence of TGF-beta1 ensures up-regulation of CD103/beta(7) in recall responses, at concentrations of TGF-beta1 significantly lower than those required by memory T cells primed in the absence of TGF-beta1. These results indicate a role of TGF-beta1 during T cell priming in modulating expression of CD103/beta(7) and controlling retention of human memory CD8(+) T cells into tumor epithelium.

Antisense targeting of TGF-β1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-β1 on bone formation remains elusive. In particular, the role of autocrine TGF-β1 on human osteoblasts is largely unknown. Here we show the effect of TGF-β1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-β1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-β1. Notably, TGF-β1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-β1. Moreover, TGF-β1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-β1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5.

Tissue toxicity induced by ionizing radiation to the normal intestine: Understanding the pathophysiological mechanisms to improve the medical management

At the present time, more than one-half of all cancer patients are treated with radiation therapy. Despite a good therapeutic index, radiotherapy can disable normal tissue injury to normal tissues in long-term cancer survivors. Thus, an important challenge to modern radiation therapy is to increase the tolerance of normal tissues, in order to improve the quality of life of the patients, and to enhance local tumor control using dose escalation and/or new biological radiosensitizers[1]. The recent progress made by 3D-conformal and intensity-modulated radiation therapy has reduced radiation-induced complications especially in dose-limiting organs like the intestine[2]. Yet, acute intestinal complications do occur but are generally transient, whereas low and mild grade chronic gastrointestinal side effects continue to influence the patient’s quality of life. Because the clinical evolution of delayed intestinal toxicity is progressive and inevitable, these complications are of much concern in clinical practice and further improvement in the management of such patients is required. The current serie published in the World Journal of Gastroenterology contains several articles reviewing the most recent research in the field of radiation-induced intestinal toxicity the current therapeutic advances according to the patient’s symptoms as well as constructive proposals for the improvement of the medical management of such patients. The proposed improvements depend upon the development of reliable diagnostic tests that are able to identify the underlying causes of the symptoms. The contribution by Lutgens et al[3] provides evidence that citrullinemia can be used in clinical and experimental settings as a biomaker of epithelial cell loss. The review by Kruse et al[4] addresses the recent advances in the use of micro array for the prediction of radiosensitivity and damage to normal tissues as well as their main limitations. The physiopathological, cellular and molecular mechanisms of radiation-induced toxicity have been discussed according to the cellular compartments of the intestine and their implication to acute and chronic intestinal toxicity. Tissue exposure to ionizing radiation stimulates the local production of reactive oxygen species which induce replicative and apoptotic death of epithelial and microvascular endothelial cells of the intestinal mucosa. However, the intestinal response to radiation injury cannot be restricted to a simple cell-killing process but depends upon continuous and integrated pathogenic processes involving cell differentiation and crosstalk between the various cellular components within the extracellular matrix. Otterson[5] reviews the impact of irradiation on gastrointestinal motility with special emphasis on the enteric nervous system. Molla et al[6], Wang et al[7] discuss the role of radiation-induced vascular damage in the acute and delayed intestinal response to ionizing radiation. The radiation-induced alterations of intracellular signaling pathways lead to the transactivation of specific target genes such as genes coding for paracrine factors including vasoactive factors, thrombogenic agents, pro/anti inflammatory mediators and growth factors. The review by Verrechia et al[8] focuses on the role of TGF-β1 in fibrogenesis. TGF-β1 is a potent fibrogenic growth factor that triggers alterations in the resident cell phenotypes that subsequently modify cell to cell interactions and tissue composition. Finally, the extracellular matrix itself may contribute to the self-perpetuation of these wound healing signals by the release of growth factors and by constant activation of cell phenotype via membrane-associated receptors. The long-term persistence of these phenotypical alterations may have an impact on the activity of neighboring cells (mesenchymal, endothelial, epithelial or immune cells) and may lead to a possible amplification of the wound healing signals that perpetuate fibrosis. This aspect has been well discussed by Haydont et al[9]. It is clear from these articles that the normal intestinal response to radiation injury cannot be restricted to a simple cell-killing process. Therefore, the previous concept of a primary cell target in which a single-cell type (whether it is epithelial or endothelial cell) dictated the whole tissue response to radiation injury should be replaced by the concept of coordinated multicellular response that may lead to either tissue recovery or to the development of complications[10]. The recent advances in this field should lead in the near future to the development of biologically-based therapeutic strategies which can be used clinical practice. These strategies would also be applicable to treat radiation injury in the event of radiation accidents or acts of terrorism and would help to improve the therapeutic management of other chronic intestinal diseases.

Role of TGF-β1 in vascular smooth muscle cell apoptosis induced by Angiotensin II

Both Angiotensin II and transforming growth factor beta-1 (TGF-β1) are important mediators of vascular smooth muscle cell function and have been reported to mediate the balance between proliferation and apoptosis. Some crosstalk between Angiotensin II and TGF-β1 in end-organ hypertension has been established. However, whether TGF-β1 is able to mediate Angiotensin II-induced vascular cell damage remains unknown. Vascular smooth muscle cells were obtained from rat thoracic aorta and cultured in 10% foetal calf serum. In all experiments, medium was changed to a low-serum (0.4% foetal calf serum) or serum-free one with or without Angiotensin II. Apoptosis was assessed by DNA fragmentation, DNA synthesis was measured as bromo-deoxyuridine uptake. TGF-β1 production was determined by Enzyme-linked Immunosorbent Assay (ELISA) from cell conditioned media, RT-PCR from cell lysates and confocal immunostaining of fixed cells. Angiotensin II induced apoptosis in the absence of DNA synthesis when coincubated at 1 μM. Neither the specific anti-TGF-β1 monoclonal antibody (50 μg/ml) nor the novel activin-like kinase (ALK)-4/5/7 synthetic inhibitor SB-431542 (4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1 H-imidazol-2-yl)benzamide) at 10 μM were able to inhibit this effect. Angiotensin II induced expression of TGF-β1 without further secretion of this cytokine. This effect was not affected by incubation with the AT1 inhibitor irbesartan (10 μM). A pharmacological approach to TGF-β1 inhibition would be unable to reverse the apoptotic effect of Angiotensin II on vascular smooth muscle cells.

The role of TGF-β1 as a determinant of foreign body reaction to alloplastic materials in rat fibroblast cultures: comparison of different commercially available polypropylene meshes for hernia repair

Background Animal experiments on hernia repair demonstrated better biocompatibility of light-weight polypropylene meshes. However, implanted medical devices trigger a variety of adverse tissue responses, such as inflammation, fibrosis, infection and thrombosis, but the mechanisms involved in such responses remain largely unknown. This study aimed to determine the effect of transforming growth factor β1 (TGF-β1) on host tolerance by quantification of foreign body reaction in cultured fibroblasts depending on the amount and composition of implanted material used for hernia repair. Materials and methods An NRK-49F fibroblast culture was incubated in the presence of 4 commercially available meshes approved for hernia repair. A mesh-free cell suspension served as a control group, in which the influence of TGF-β1 on fibroblasts was evaluated. Levels of TGF-β1 in the supernatant were dynamically measured in a time interval of 6 to 96 h and cell proliferation rates were assessed colorimetrically using MTT test. Results A dose-dependent suppression of fibroblasts proliferation by TGF-β1 was observed. All meshes suppressed the secretion of TGF-β1 and conversely increased significantly cell proliferation in comparison to the control group ( p < 0.01) in the first 24 to 48 h of incubation. That effect was more pronounced in meshes partially containing absorbable material when compared to samples of pure polypropylene meshes ( p < 0.05) and to the control group ( p < 0.001). Conclusion Our experiment revealed that early biological reaction of connective tissue cells towards polypropylene meshes and their variants depended much more on the composition and type of the material than on its absolute amount. The assumption that material weight reduction alone might affect the foreign body reaction of mesh implants could not be confirmed by our in vitro study.

Correction: A “traffic control” role for TGFβ3: orchestrating dermal and epidermal cell motility during wound healing

Correction: A “traffic control” role for TGFβ3: orchestrating dermal and epidermal cell motility during wound healing

A “traffic control” role for TGFβ3: orchestrating dermal and epidermal cell motility during wound healing

A “traffic control” role for TGFβ3: orchestrating dermal and epidermal cell motility during wound healing

The Healing Effects of TGF-β

Proper wound healing requires orderly migration of multiple cell types. In humans, epidermal keratinocytes (HKs) move across the wound to form a new continuous epithelial layer. Dermal cells like dermal fibroblasts (DFs) and human dermal microvascular endothelial cells (HDMECs) move in to restore the extracellular matrix and help build new blood vessels. But how do these cells know when and where to do their work? New work from Bandyopadhyay et al. suggests that one important factor is that cells at the wound are first exposed to plasma (blood liquid without the cells), then serum (the cell-free liquid that remains after blood clotting), and then again to plasma. The authors observed the effects of serum and plasma on the various cell types in an assay in which cells left visible tracks on a surface of extracellular matrix coated with colloidal gold or in an in vitro wound-healing assay. Plasma stimulated migration of dermal cells (DFs and HDMECs) but had little effect on epidermal HKs. Conversely, serum stimulated migration of HKs but had little effect on DFs and HDMECs. The authors suspected transforming growth factor-β (TGF-β) as a factor that could account for these differential responses because it is relatively abundant in serum and scarce or undetectable in plasma. Cells responded to serum from which the TGF-β3 isoform had been removed with antibodies as though they were responding to plasma. The authors also found that type II TGF-β receptors were abundant in dermal cells but less prevalent in epidermal cells. Exogenous expression of TGF-βRII in HKs caused the cell to respond to serum with reduced, rather than enhanced, motility. When short inhibitory RNA was used to decrease expression of TGF-βRII in dermal cells, their migratory responses to plasma and serum were reversed. The authors propose that the sequential exposure of cells at a wound to plasma, then serum, and then plasma again coordinates the movement of the various cell types to allow tissue remodeling and healing. Wounds that fail to heal are a problem in certain disease states and in the elderly, and better understanding of the orchestration of the normal healing process may permit more effective use of growth-factor therapies to enhance healing of refractory wounds in the clinic. B. Bandyopadhyay, J. Fan, S. Guan, Y. Li, M. Chen, D. T. Woodley, W. Li, A "traffic control" role for TGFβ3: Orchestrating dermal and epidermal cell motility during wound healing. J. Cell Biol. 172 , 1093-1105 (2006). [Abstract] [Full Text]

A "traffic control" role for TGFbeta3: orchestrating dermal and epidermal cell motility during wound healing.

Cell migration is a rate-limiting event in skin wound healing. In unwounded skin, cells are nourished by plasma. When skin is wounded, resident cells encounter serum for the first time. As the wound heals, the cells experience a transition of serum back to plasma. In this study, we report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. The on-and-off switch is operated by transforming growth factor (TGF) β3 levels, which are undetectable in plasma and high in serum, and by TGFβ receptor (TβR) type II levels, which are low in epidermal cells and high in dermal cells. Depletion of TGFβ3 from serum converts serum to a plasmalike reagent. The addition of TGFβ3 to plasma converts it to a serumlike reagent. Down-regulation of TβRII in dermal cells or up-regulation of TβRII in epidermal cells reverses their migratory responses to serum and plasma, respectively. Therefore, the naturally occurring plasma→serum→plasma transition during wound healing orchestrates the orderly migration of dermal and epidermal cells.

Opposing actions of TGFβ1 and FGF2 on growth, differentiation and extracellular matrix accumulation in prostatic stromal cells

TGFβ1 and FGF2 are autocrine growth factors in prostatic stroma and are elevated in benign prostatic hyperplasia (BPH), a disease characterized by enlargement of the stromal compartment of the prostate. TGFβ1 has a biphasic effect on proliferation of prostatic stromal cells, inducing proliferation at low doses ( < 1 ng/ml), but inhibiting growth above 1 ng/ml. This study investigated the role of TGFβ1 and FGF2 on growth factor bioavailability and extracellular matrix (ECM) accumulation synthesis in cultured prostatic stromal cells. Real-Time-PCR showed that TGFβ1 expression is auto-inductive, whereas FGF2 is auto-repressive. FGF2 also induced TGFβ1 secretion in the absence of increased TGFβ1 mRNA expression. TGFβ1 and FGF2 have opposing actions on Type 1 collagen expression, a finding confirmed by Western blotting. The bioavailability of TGFβ1 regulated by FGF2 may represent part of a negative feedback mechanism controlling stromal growth, differentiation and ECM. Dysregulation of this pathway in favour of TGFβ1 bioactivity may exacerbate BPH.

Induction of human LTBP-3 promoter activity by TGF-β1 is mediated by Smad3/4 and AP-1 binding elements

Latent TGF-β binding proteins (LTBPs) are extracellular matrix glycoproteins, which are essential for the targeting and activation of TGF-βs. LTBP-3 regulates the bioavailability of TGF-β especially in the bone. To understand the regulation of LTBP-3 expression, we have isolated and characterized the promoter region of human LTBP-3 gene. The GC-rich TATA-less promoter contained several transcription initiation sites and putative binding sites for multiple sequence specific transcription factors including Sp1, AP-1, c-Ets, MZF-1, Runx1 and members of the GATA-family. Reporter gene analyses of the promoter indicated that it was more active in MG-63 than in Saos-2 osteosarcoma cells, suggesting that it is regulated as the endogenous gene. TGF-β1 stimulated the transcriptional activity of LTBP-3 promoter in MG-63 cells, while certain other bone-derived growth factors and hormones were ineffective. TGF-β1 increased LTBP-3 mRNA levels accordingly. Analyses of deletion constructs of the promoter and mutational deletion of specific transcription factor binding sites indicated that Smad3/4 and AP-1 binding sites mediated the TGF-β1 response. The involvement of AP-1 activity was further indicated by decreased TGF-β responsiveness of the LTBP-3 promoter in the presence of a MEK/Erk signaling pathway inhibitor. Our results suggest an important new role for TGF-β1 in the regulation of its binding protein, LTBP-3.

Transforming Growth Factor-β1-induced Expression of Smooth Muscle Marker Genes Involves Activation of PKN and p38 MAPK

Differentiated vascular smooth muscle cells (SMCs) exhibit a work phenotype characterized by expression of several well documented contractile apparatus-associated proteins. However, SMCs retain the ability to de-differentiate into a proliferative phenotype, which is involved in the progression of vascular diseases such as atherosclerosis and restenosis. Understanding the mechanisms involved in maintaining SMC differentiation is critical for preventing proliferation associated with vascular disease. In this study, the molecular mechanisms through which transforming growth factor-beta1 (TGF-beta1) induces differentiation of SMCs were examined. TGF-beta1 stimulated actin re-organization, inhibited cell proliferation, and up-regulated SMC marker gene expression in PAC-1 SMCs. These effects were blocked by pretreatment of cells with either HA1077 or Y-27632, which inhibit the kinases downstream of RhoA. Moreover, TGF-beta1 activated RhoA and its downstream target PKN. Overexpression of active PKN alone was sufficient to increase the transcriptional activity of the promoters that control expression of smooth muscle (SM) alpha-actin, SM-myosin heavy chain, and SM22alpha. In addition, PKN increased the activities of serum-response factor (SRF), GATA, and MEF2-dependent enhancer-reporters. RNA interference-mediated inhibition of PKN abolished TGF-beta1-induced activation of SMC marker gene promoters. Finally, examination of MAPK signaling demonstrated that TGF-beta1 increased the activity of p38 MAPK, which was required for activation of the SMC marker gene promoters. Co-expression of dominant negative p38 MAPK was sufficient to block PKN-mediated activation of the SMC marker gene promoters as well as the serum-response factor, GATA, and MEF2 enhancers. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-beta1 activates PKN to promote differentiation of SMCs.