TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells

Transforming growth factor-β (TGF-β) is upregulated at the time of arterial injury; however, the mechanism through which TGF-β enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-β stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-β has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-β mediates its proliferative effect in vascular SMCs. We found that TGF-β phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-β on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-β-induced Akt phosphorylation. Moreover, TGF-β/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-β/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-β/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-β on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.     

Thrombospondin-1-induced smooth muscle cell chemotaxis and proliferation are dependent on transforming growth factor-β2 and hyaluronic acid synthase

Angioplasty causes local vascular injury, leading to the release of thrombospondin-1 (TSP-1), which stimulates vascular smooth muscle cell (VSMC) migration and proliferation, important steps in the development of intimal hyperplasia. Transforming growth factor beta 2 (TGF-β2) and hyaluronic acid synthase (HAS) are two pro-stenotic genes upregulated in VSMCs by TSP-1. We hypothesized that inhibition of TGF-β2 or HAS would inhibit TSP-1-induced VSMC migration, proliferation, and TSP-1 signaling. Our data demonstrate that Inhibition of either TGF-β2 or HAS inhibited TSP-1-induced VSMC migration and proliferation. Activation of ERK 1 was decreased by TGF-β2 inhibition and unaffected by HAS inhibition. TGF-β2 and HAS are not implicated in TSP-1-induced thbs1 expression, while they are each implicated in TSP-1-induced expression of their own gene. In summary, TSP-1-induced VSMC migration and proliferation rely on intact TGF-β2 signaling and HAS function. TSP-1 activation of ERK 1 is dependent on TGF-β2. These data further expand our understanding of the complexity of TSP-1 cellular signaling and the involvement of TGF-β2 and HAS.     

MiR-21 inhibits c-Ski signaling to promote the proliferation of rat vascular smooth muscle cells

Previously, we reported that the decrease of endogenous c-Ski expression is implicated in the progression of vascular smooth muscle cell (VSMC) proliferation after arterial injury. However, the molecular mechanism of the down-regulation of c-Ski is not clear. In this study, a potential miR-21 recognition element was identified in the 3′-untranslated region (UTR) of rat c-Ski mRNA. A reporter assay revealed that miR-21 could recognize the miR-21 recognition element of c-Ski mRNA. In A10 rat aortic smooth muscle cells, overexpression of miR-21 significantly inhibited the expression of c-Ski protein and promoted cell proliferation, which could be blocked by inhibition of miR-21 or overexpression of c-Ski. Further investigation demonstrated that the effect of miR-21 on VSMC proliferation resulted from negative regulation of c-Ski to suppress p38–p21/p27 signaling, the downstream pathway of c-Ski in VSMCs. These results indicate that c-Ski is a target gene of miR-21. miR-21 specifically binds to the 3′-untranslated region of c-Ski and negatively regulates c-Ski expression to diminish the protective effects of c-Ski and stimulate VSMC proliferation in the progression of arterial injury.     

Perivascular gene transfer of dominant-negative N19RhoA attenuates neointimal formation via inhibition of TGF-β1-Smad2 signaling in rats after carotid artery balloon injury

Phenotypic differentiation of adventitial fibroblasts to myofibroblasts is an essential feature of vascular remodeling. Here, we carried out perivascular gene transfer of dominant-negative N19RhoA to investigate whether antagonism of RhoA signaling attenuates neointimal formation following rat carotid artery balloon injury and alters TGF-β1-Smad2-induced differentiation of adventitial fibroblasts to myofibroblasts. Perivascular delivery of an adenovirus coexpressing dominant-negative N19RhoA and humanized Renilla green fluorescent protein (hrGFP) (Ad-N19RhoA-hrGFP), as demonstrated by hrGFP staining, suppressed neointimal formation at 7 and 14 days post-injury. Ad-N19RhoA-hrGFP administration inhibited neointimal α-smooth muscle-actin and Calponin expression, as markers of myofibroblast differentiation and perivascular collagen deposition, at 14 days after balloon injury. Ad-N19RhoA-hrGFP administration also inhibited adventitial Smad2 phosphorylation, but did not alter local TGF-β1 and total-Smad2 expression after injury. Our results provide evidence that perivascular gene transfer of dominant-negative N19RhoA blocks TGF-β1-Smad2-induced differentiation of adventitial fibroblasts to myofibroblasts, which contributes to intimal hyperplasia after balloon injury.     

Transforming Growth Factor-�� Promotes Recruitment of Bone Marrow Cells and Bone Marrow-derived Mesenchymal Stem Cells through Stimulation of MCP-1 Production in Vascular Smooth Muscle Cells

Bone marrow-derived progenitor cells have recently been shown to be involved in the development of intimal hyperplasia after vascular injury. Transforming growth factor-β (TGF-β) has profound stimulatory effects on intimal hyperplasia, but it is unknown whether these effects involve progenitor cell recruitment. In this study we found that although TGF-β had no direct effect on progenitor cell recruitment, conditioned media derived from vascular smooth muscle cells (VSMC) stimulated with TGF-β induced migration of both total bone marrow (BM) cells and BM-mesenchymal stem cells (MSC) and also induced MSC differentiation into smooth muscle like cells. Furthermore, overexpression of the signaling molecule Smad3 in VSMC via adenovirus-mediated gene transfer (AdSmad3) enhanced the TGF-β''s chemotactic effect. Microarray analysis of VSMC stimulated by TGF-β/AdSmad3 revealed monocyte chemoattractant protein-1 (MCP-1) as a likely factor responsible for progenitor cell recruitment. We then demonstrated that TGF-β through Smad3 phosphorylation induced a robust expression of MCP-1 in VSMC. Recombinant MCP-1 mimicked the stimulatory effect of conditioned media on BM and MSC migration. In the rat carotid injury model, Smad3 overexpression significantly increased MCP-1 expression after vascular injury, consistent with our in vitro results. Interestingly, TGF-β/Smad3-induced MCP-1 was completely blocked by both Ro-32-0432 and rotterlin, suggesting protein kinase C-δ (PKCδ) may play a role in TGF-β/Smad3-induced MCP-1 expression. In summary, our data demonstrate that TGF-β, through Smad3 and PKCδ, stimulates VSMC production of MCP-1, which is a chemoattractant for bone marrow-derived cells, specifically MSC. Manipulation of this signaling system may provide a novel approach to inhibition of intimal hyperplasia.     

RB1CC1 protein positively regulates transforming growth factor-beta signaling through the modulation of Arkadia E3 ubiquitin ligase activity

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia.     

Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas

TGF-β1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-β1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-β signaling, and through these pathways it is able to enhance TGF-β induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-β1 combination on cell growth and on TGF-β and mTOR signalling events in lymphoma cells.Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-β1 induced apoptosis and restored TGF-β1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-β sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin + TGF-β1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin + TGF-β1 induced apoptosis was not completely eliminated by a caspase inhibitor.These results suggest that high mTOR activity contributes to TGF-β resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-β1.     

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.     

Activation of peroxisome proliferation–activated receptor-γ inhibits transforming growth factor-β1-induced airway smooth muscle cell proliferation by suppressing Smad–miR-21 signaling

The aims of the current study were to examine the signaling mechanisms for transforming growth factor-β1 (TGF-β1)-induced rat airway smooth muscle cell (ASMC) proliferation and to determine the effect of activation of peroxisome proliferation–activated receptor-γ (PPAR-γ) on TGF-β1-induced rat ASMC proliferation and its underlying mechanisms. TGF-β1 upregulated microRNA 21 (miR-21) expression by activating Smad2/3, and this in turn downregulated forkhead box O1 (FOXO1) mRNA expression. In addition, TGF-β1–Smad–miR-21 signaling also downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and thus de-repressed the PI3K–Akt pathway. Depletion of PTEN reduced the nuclear FOXO1 protein level without affecting its mRNA level. Inhibition of the PI3K–Akt pathway or proteasome function reversed PTEN knockdown-induced nuclear FOXO1 protein reduction. Our study further showed that loss of FOXO1 increased cyclin D1 expression, leading to rat ASMC proliferation. Preincubation of rat ASMCs with pioglitazone, a PPAR-γ activator, blocked TGF-β1-induced activation of Smad2/3 and its downstream targets changes of miR-21, PTEN, Akt, FOXO1, and cyclin D1, resulting in the inhibition of rat ASMC proliferation. Our study suggests that the activation of PPAR-γ inhibits rat ASMC proliferation by suppressing Smad–miR-21 signaling and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.     

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation

Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-β1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-β1 concentrations, but the mechanism by which low TGF-β1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-β1 concentrations not only promoted c-ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-β1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-β1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-β1/Smad pathway.     

Angiotensin II type 1 receptor-associated protein regulates carotid intimal hyperplasia through controlling apoptosis of vascular smooth muscle cells

Intimal hyperplasia is the main cause of restenosis after carotid artery injury, and the underlying mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). Angiotensin II Type 1 Receptor-Associated Protein (ATRAP) has been reported to withstand intimal hyperplasia by inhibiting VSMCs proliferation and migration; however, whether the beneficial effect of ATRAP associates with VSMCs apoptosis remains unclarified. We demonstrated that the adenoviral-mediated overexpression of ATRAP induced VSMC apoptosis, alleviating the balloon injury-induced neointima formation in rats. Under the condition of Angiotensin-II stimulation, ATRAP overexpression induced the apoptosis of rat VSMCs by depressing the PI3K-Akt signaling; whereas up-regulation of Akt by PTEN inhibitor abolished the apoptotic death. Thus, ATRAP regulates carotid intimal hyperplasia through controlling the PI3K-Akt signal-mediated VSMCs apoptosis.     

TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways

Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8 h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia–reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.     

Puerarin attenuates high-glucose-and diabetes-induced vascular smooth muscle cell proliferation by blocking PKCβ2/Rac1-dependent signaling

Oxidative stress has been implicated in several steps leading to the development of diabetic vascular complications. The purpose of this study was to determine the efficacy and the possible mechanism of puerarin on high-glucose (HG; 25 mM)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a carotid arterial balloon injury model of obese Zucker rats. Our data demonstrated that puerarin significantly inhibited rat VSMC proliferation as well as reactive oxygen species (ROS) generation and NADPH oxidase activity induced by HG treatment. Further studies revealed that HG treatment resulted in phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits, leading to NADPH oxidase activation. Puerarin treatment remarkably disrupted the phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits. Blocking PKCβ2 by infection with AdDNPKCβ2 also abolished HG-induced phosphorylation and membrane translocation of Rac1, p47phox, and p67phox subunits as well as ROS production and NADPH oxidase activation in VSMCs. In vivo neointimal formation of obese Zucker rats evoked by balloon injury was evidently attenuated by the administration of puerarin. These results demonstrate that puerarin may exert inhibitory effects on HG-induced VSMC proliferation via interfering with PKCβ2/Rac1-dependent ROS pathways, thus resulting in the attenuation of neointimal formation in the context of hyperglycemia in diabetes mellitus.     

5-(1,3-Benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives as potent and selective transforming growth factor-β type I receptor inhibitors

A series of 5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and for their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. As a representative compound, 16i was a potent and selective ALK5 inhibitor, exhibiting a good enzyme inhibitory activity (IC₅₀ = 5.5 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC₅₀ = 36 nM). Furthermore, the topical application of 3% 16i lotion significantly inhibited Smad2 phosphorylation in Mouse skin (90% inhibition compared with vehicle-treated animals).     

Beneficial effect of quercetin on cholestatic liver injury

Bile duct obstruction and subsequent cholestasis are associated with hepatocellular injury, cholangiocyte proliferation, stellate cell activation, Kupffer cell activation, oxidative stress, inflammation and fibrosis. Flavonoids have been shown to confer beneficial health effects, including hepatoprotection. However, the molecular mechanism of flavonoid-mediated hepatoprotection is incompletely understood. In this study, we report the protective effect of quercetin on cholestatic liver injury. Cholestasis was produced by bile duct ligation (BDL) in male Sprague–Dawley rats for 3 weeks. Daily oral administration of quercetin was started 1 week before injury and lasted for 4 weeks. In comparison with the control group, the BDL group showed liver injury, as evidenced by histological changes, and elevation in serum biochemicals, ductular reaction, fibrosis, inflammation and oxidative stress. These pathophysiological changes were attenuated by daily quercetin supplementation. Quercetin alleviated BDL-induced transforming growth factor beta-1 (TGF-β1), interleukin-1 beta, connective tissue growth factor and collagen expression. The antifibrotic effect of quercetin was accompanied by reductions in α-smooth muscle actin-positive matrix-producing cells and Smad 2/3 activity critical to the fibrogenic potential of TGF-β1. Quercetin also attenuated BDL-induced oxidative stress, leukocyte accumulation, nuclear factor (NF)-κB activation and proinflammatory cytokine production. Further studies demonstrated an inhibitory effect of quercetin on MyD88 and TGF-β-activated kinase-1 critical for linking toll-like receptor (TLR) and NF-κB. Taken together, the hepatoprotective, anti-inflammatory and antifibrotic effects of quercetin seem to be multifactorial. The beneficial effects of daily quercetin supplementation are associated with antioxidative and anti-inflammatory potential as well as down-regulation of NF-κB and TGF-β/Smad signaling, probably via interference with TLR signaling.     

TGF-β1 inhibits the growth and metastasis of tongue squamous carcinoma cells through Smad4

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that regulates cell growth, differentiation, migration, apoptosis and extracellular matrix remodeling. TGF-β1 transduces signals from the cell membrane to the cell nucleus through serine/threonine kinase receptors and their downstream effectors, Smad molecules. Although many studies have been focused on TGF-β1-Smad signaling pathway, the role of TGF-β1/Smad in tongue squamous cell carcinoma is not fully understood. In the present study, we used a series of cell function assays to examine the role of TGF-β-Smad4 signaling in tongue squamous cell carcinoma. We observed the effects of TGF-β1 on the growth and metastatic potential of the tongue squamous cell carcinoma cell line Ts, which expresses lower level of Smad4 protein. We found that Smad4 could decrease TGF-β1-induced cell proliferation, and that Smad4 overexpression promoted Ts cell apoptosis. In Ts vector control cells, TGF-β1 increased the expression of TβRII, as well as MMP-2, and enhanced cell invasion through the basement membrane, and then induced cell metastasis. However in Ts cells stably expressing Smad4, Smad4 mediated TGF-β1-induced p21 expression promoted cell apoptosis and inhibited cell proliferation, delayed MMP-2 expression, and decreased cell metastasis. Therefore, TGF-β1 plays distinct roles in the Smad4-dependent and -independent signaling pathways.     

Thrombospondin-4 critically controls transforming growth factor β1 induced hypertrophic scar formation

Transforming growth factor β (TGF-β) is a growth factor presenting important functions during tissue remodeling and hypertrophic scar (HS) formation. However, the underlying molecular mechanisms are largely unknown. In this study, we identified thrombospondin-4 (TSP-4) as a TGF-β1 target that essentially mediates TGF-β1-induced scar formation both in vitro and in vivo. The expression of TSP-4 was compared on both mRNA and protein levels between hypertrophic scar fibroblasts (HSFs) and normal skin fibroblast (NFs) in response to TGF-β1 treatment. Two signaling molecules, Smad3 and p38, were assessed for their importance in regulating TGF-β1-mediated TSP-4 expression. The significance of TSP-4 in controlling TGF-β1-induced proliferation, invasion, migration, and fibrosis in HSFs was analyzed by knocking down endogenous TSP-4 using small hairpin RNA (shRNA) (TSP-4 shRNA). Finally, a skin HS model was established in rats and the scar formation was compared between rats treated with vehicle (saline), TGF-β1, and TGF-β1 + TSP-4 shRNA. The TSP-4 level was significantly higher in HSFs than in NFs and TGF-β1 more potently boosted TSP-4 expression in the former than in the latter. Both Smad3 and p38 essentially mediated TGF-β1-induced TSP-4 expression. TSP-4 shRNA significantly suppressed TGF-β1-stimulated proliferation, invasion, migration, or fibrosis of HSFs in vitro and drastically improved wound healing in vivo. TGF-β1, by activating both Smad3 and p38, induces TSP-4, which in turn not only presents a positive feedback regulation on the activation of Smad3 and p38, but also essentially mediates TGF-β1-induced HS formation. Targeting TSP-4 thus may benefit HS treatment.