RIPK4 suppresses the TGF‐β1 signaling pathway in HaCaT cells

Receptor‐interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor‐β 1 (TGF‐β1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF‐β signaling during epidermal homeostasis‐related events and suppression of RIPK4 expression by TGF‐β1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor‐κB (NF‐κB), protein kinase C (PKC), wingless‐type MMTV integration site family (Wnt), and (mitogen‐activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad‐mediated TGF‐β1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad‐mediated TGF‐β1 signaling events through suppression of TGF‐β1‐induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3‐Smad4 interaction, nuclear localization, and TGF‐β1‐induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound‐scratch assay demonstrated that RIPK4 suppressed TGF‐β1‐mediated wound healing through blocking TGF‐β1‐induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF‐β1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF‐β1 signaling.     

GP73 promotes invasion and metastasis of bladder cancer by regulating the epithelial–mesenchymal transition through the TGF‐β1/Smad2 signalling pathway

This study investigated the effects of Golgi membrane protein 73 (GP73) on the epithelial–mesenchymal transition (EMT) and on bladder cancer cell invasion and metastasis through the TGF‐β1/Smad2 signalling pathway. Paired bladder cancer and adjacent tissue samples (102) and normal bladder tissue samples (106) were obtained. Bladder cancer cell lines (T24, 5637, RT4, 253J and J82) were selected and assigned to blank, negative control (NC), TGF‐β, thrombospondin‐1 (TSP‐1), TGF‐β1+ TSP‐1, GP73‐siRNA‐1, GP73‐siRNA‐2, GP73‐siRNA‐1+ TSP‐1, GP73‐siRNA‐1+ pcDNA‐GP73, WT1‐siRNA and WT1‐siRNA + GP73‐siRNA‐1 groups. Expressions of GP73, TGF‐β1, Smad2, p‐Smad2, E‐cadherin and vimentin were detected using RT‐qPCR and Western blotting. Cell proliferation, migration and invasion were determined using MTT assay, scratch testing and Transwell assay, respectively. Compared with the blank and NC groups, levels of GP73, TGF‐β1, Smad2, p‐Smad2, N‐cadherin and vimentin decreased, and levels of WT1 and E‐cadherin increased in the GP73‐siRNA‐1 and GP73‐siRNA‐2 groups, while the opposite results were observed in the WT1 siRNA, TGF‐β, TSP‐1 and TGF‐β + TSP‐1 groups. Cell proliferation, migration and invasion notably decreased in the GP73‐siRNA‐1 and GP73‐siRNA‐2 groups in comparison with the blank and NC groups, while in the WT1 siRNA, TGF‐β, TSP‐1 and TGF‐β + TSP‐1 groups, cell migration, invasion and proliferation showed the reduction after the EMT. These results suggest that GP73 promotes bladder cancer invasion and metastasis by inducing the EMT through down‐regulating WT1 levels and activating the TGF‐β1/Smad2 signalling pathway.     

Crosstalk between Smad2/3 and specific isoforms of ERK in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes

This study investigated the roles of ERK1 and ERK2 in transforming growth factor‐β1 (TGF‐β1)‐induced tissue inhibitor of metalloproteinases‐3 (TIMP‐3) expression in rat chondrocytes, and the specific roles of ERK1 and ERK2 in crosstalk with Smad2/3 were investigated to demonstrate the molecular mechanism of ERK1/2 regulation of TGF‐β1 signalling. To examine the interaction of specific isoforms of ERK and the Smad2/3 signalling pathway, chondrocytes were infected with LV expressing either ERK1 or ERK2 siRNA and stimulated with or without TGF‐β1. At indicated time‐points, TIMP‐3 expression was determined by real‐time PCR and Western blotting; p‐Smad3, nuclear p‐Smad3, Smad2/3, p‐ERK1/2 and ERK1/2 levels were assessed. And then, aggrecan, type II collagen and the intensity of matrix were examined. TGF‐β1‐induced TIMP‐3 expression was significantly inhibited by ERK1 knock‐down, and the decrease in TIMP‐3 expression was accompanied by a reduction of p‐Smad3 in ERK1 knock‐down cells. Knock‐down of ERK2 had no effect on neither TGF‐β1‐induced TIMP‐3 expression nor the quantity of p‐Smad3. Moreover, aggrecan, type II collagen expression and the intensity of matrix were significantly suppressed by ERK1 knock‐down instead of ERK2 knock‐down. Taken together, ERK1 and ERK2 have different roles in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes. ERK1 instead of ERK2 can regulate TGF‐β/Smad signalling, which may be the mechanism through which ERK1 regulates TGF‐β1‐induced TIMP‐3 expression.     

Petchiether A attenuates obstructive nephropathy by suppressing TGF‐β/Smad3 and NF‐κB signalling

Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor‐β1 (TGF‐β1)/Smad3‐driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small‐molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF‐β1‐induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin‐1β and tumour necrosis factor‐α) and reducing extracellular matrix deposition (α‐smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF‐κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down‐regulated the expression of the fibrotic marker collagen I in TGF‐β1‐treated renal epithelial cells. Further, we found that petA dose‐dependently suppressed Smad3‐responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF‐β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF‐β/Smad3 signalling.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

Integrin–TGF‐β crosstalk in fibrosis,cancer and wound healing

Accumulating evidence indicates that there is extensive crosstalk between integrins and TGF‐β signalling. TGF‐β affects integrin‐mediated cell adhesion and migration by regulating the expression of integrins, their ligands and integrin‐associated proteins. Conversely, several integrins directly control TGF‐β activation. In addition, a number of integrins can interfere with both Smad‐dependent and Smad‐independent TGF‐β signalling in different ways, including the regulation of the expression of TGF‐β signalling pathway components, the physical association of integrins with TGF‐β receptors and the modulation of downstream effectors. Reciprocal TGF‐β–integrin signalling is implicated in normal physiology, as well as in a variety of pathological processes including systemic sclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cancer; thus, integrins could provide attractive therapeutic targets to interfere with TGF‐β signalling in these processes.     

Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) induces TGF‐β‐regulated apoptosis signal in human gingival epithelial cells via fibronectin/integrinβ1/FAK cascade

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor β (TGF‐β). However, the molecular mechanisms by which microbes induce the activation of TGF‐β remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF‐β receptor (TGF‐βR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF‐βR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF‐β by cooperating with integrin. Accordingly, we hypothesized that Omp29‐induced actin rearrangements via FAK activity would enhance the activation of TGF‐β, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF‐βR/smad2 signalling and decreased active TGF‐β protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF‐βR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinβ1 expression by siRNA transfection attenuated TGF‐βR/smad2 signalling activity and reduction of TGF‐β levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinβ1, which is associated with TGF‐β signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29‐induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinβ1/FAK signalling‐dependent TGF‐β release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF‐βR/smad2 pathway.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

Lipopolysaccharide enhances TGF‐β1 signalling pathway and rat pancreatic fibrosis

Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor‐beta1 (TGF‐β1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF‐β1 signalling and pancreatic fibrosis. Sprague‐Dawley rats fed with a Lieber‐DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP‐2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF‐β1 which was paralleled by an increased number of TLR4‐positive or TGF‐β1‐positive Mφs or PSCs in ALC‐fed rats. In vitro, TLR4 or TGF‐β1 production in Mφs or RP‐2 cells was up‐regulated by LPS. LPS alone or in combination with TGF‐β1 significantly increased type I collagen and α‐SMA production and Smad2 and 3 phosphorylation in serum‐starved RP‐2 cells. TGF‐β pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si‐TLR4 RNA or by inhibitors of MyD88/NF‐kB. Additionally, knockdown of Bambi with Si‐Bambi RNA significantly increased TGF‐β1 signalling in RP‐2 cells. These findings indicate that LPS increases TGF‐β1 production through paracrine and autocrine mechanisms and that LPS enhances TGF‐β1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF‐kB activation.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

RGS3 inhibits TGF‐β1/Smad signalling in adventitial fibroblasts

Recent evidence suggests that adventitial fibroblasts (AFs) are crucially implicated in atherosclerosis. However, the mechanisms by which AFs are dysfunctional and contribute to atherosclerosis remain unclear. This study aimed to investigate the role of regulator of G‐protein signalling 3 (RGS3) in the regulation of AFs using apoE knockout mouse as the model. Pathological changes in aortic arteries of apoE knockout mice fed with hyperlipid diet were examined by Movat staining. The expression of RGS3, α‐SMA, TGF‐β1, Smad2, and Smad3 in the adventitia was detected by immunohistochemistry. Adventitial fibroblasts were isolated from aortic arteries of apoE knockout mice and infected with RGS3 overexpression lentivirus or empty lentivirus. The expression of RGS3, α‐SMA, TGF‐β1, Smad2, and Smad3 in AFs was detected by real‐time polymerase chain reaction and Western blot analysis. We found that hyperlipidic diet caused significant aortic intima thickening and atherosclerotic plaques in 15‐week‐old apoE knockout mice. Compared to wild‐type mice, RGS3 expression was lower while α‐SMA, TGF‐β1, Smad2, and Smad3 expression was higher in the adventitia of apoE knockout mice. In addition, lentivirus mediated overexpression of RGS3 caused decreased expression of α‐SMA, TGF‐β1, Smad2, and Smad3 in AFs derived from apoE(?/?) mice. In conclusion, these results suggest that RGS3 may provide protection against pathological changes of AFs and the development of atherosclerosis by inhibiting TGF‐β1/Smad signalling. RGS3 may be a potential therapeutic target for atherosclerosis.     

MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.     

Role of endothelial‐to‐mesenchymal transition induced by TGF‐β1 in transplant kidney interstitial fibrosis

Chronic allograft dysfunction (CAD) induced by kidney interstitial fibrosis is the main cause of allograft failure in kidney transplantation. Endothelial‐to‐mesenchymal transition (EndMT) may play an important role in kidney fibrosis. We, therefore, undertook this study to characterize the functions and potential mechanism of EndMT in transplant kidney interstitial fibrosis. Proteins and mRNAs associated with EndMT were examined in human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor‐beta1 (TGF‐β1) at different doses or at different intervals with western blotting, qRT‐PCR and ELISA assays. Cell motility and migration were evaluated with motility and migration assays. The mechanism of EndMT induced by TGF‐β1 was determined by western blotting analysis of factors involved in various canonical and non‐canonical pathways. In addition, human kidney tissues from control and CAD group were also examined for these proteins by HE, Masson's trichrome, immunohistochemical, indirect immunofluorescence double staining and western blotting assays. TGF‐β1 significantly promoted the development of EndMT in a time‐dependent and dose‐dependent manner and promoted the motility and migration ability of HUVECs. The TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways were found to be associated with the pathogenesis of EndMT induced by TGF‐β1, which was also proven in vivo by the analysis of specimens from the control and CAD groups. EndMT may promote transplant kidney interstitial fibrosis by targetting the TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways, and hence, result in the development of CAD in kidney transplant recipients.     

Effect of budesonide on TGF‐β1‐enhanced VEGF production by lung fibroblasts

VEGF (vascular endothelial growth factor) is a potent proangiogenic cytokine, and vascular change is one of the characteristic features of airway remodelling. Since the glucocorticoids have shown antifibrosis properties, we sought to investigate whether budesonide, a widely used glucocorticoid in clinical practice, could attenuate TGF‐β1 (transforming growth factor‐β1)‐induced VEGF production by HFL‐1 (human lung fibroblasts). HFL‐1 fibroblasts were treated with various concentrations of budesonide (10^?11 M, 10^?9 M and 10^?7 M) in the absence or presence of TGF‐β1. Postculture media were collected for ELISA of VEGF at the indicated times. The cell lysates were subjected to Western blotting analysis to test TGF‐β1/Smad and MAP (mitogen‐activated protein) kinase signalling activation, respectively. The results suggested that budesonide pretreatment reduced the significant increase of VEGF release induced by TGF‐β1 in HFL‐1 fibroblasts in a dose‐dependent manner, and suppressed the increase of phospho‐Smad3 and phosphor‐ERK (extracellular signal‐regulated kinase) protein levels. In conclusion, budesonide may reduce TGF‐β1‐induced VEGF production in the lung, probably through the Smad/ERK signalling pathway and, thus, may provide new sight into the molecular mechanism underlying glucocorticoid therapy for airway inflammatory diseases.