The role of TGF‐β1/Smad2/3 pathway in platelet‐rich plasma in retarding intervertebral disc degeneration

Recent studies have suggested that platelet‐rich plasma (PRP) injections are an effective way to retard intervertebral disc degeneration, but the mechanism of action is unclear. Activated platelets release some growth factors, such as transforming growth factor‐β1 (TGF‐β1), which positively modulate the extracellular matrix of nucleus pulposus cells. The purpose of this study was to explore the mechanism underlying the PRP‐mediated inhibition of intervertebral disc degeneration. In an in vitro study, we found that the proliferation of nucleus pulposus cells was greatly enhanced with 2.5% PRP treatment. The TGF‐β1 concentration was much higher after PRP treatment. PRP administration effectively increased the collagen II, aggrecan and sox‐9 mRNA levels and decreased collagen X levels. However, Western blotting demonstrated that specifically inhibiting TGF‐β1 signalling could significantly prevent nucleus pulpous cellular expression of Smad2/3 and matrix protein. In a rabbit study, magnetic resonance imaging revealed significant recovery signal intensity in the intervertebral discs of the PRP injection group compared with the very low signal intensity in the control groups. Histologically, the PRP plus inhibitor injection group had significantly lower expression levels of Smad2/3 and collagen II than the PRP group. These results demonstrated that a high TGF‐β1 content in the platelets retarded disc degeneration in vitro and in vivo. Inhibiting the TGF‐β1/Smad2/3 pathway could prevent this recovery by inactivating Smad2/3 and down‐regulating the extracellular matrix. Therefore, the TGF‐β1/Smad2/3 pathway might play a critical role in the ability of PRP to retard intervertebral disc degeneration.     

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.     

Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex

Rho‐associated kinase (ROCK) plays a critical role in pressure overload‐induced left ventricular remodelling. However, the underlying mechanism remains unclear. Here, we reported that TGF‐β1‐induced ROCK elevation suppressed BMP‐2 level and strengthened fibrotic response. Exogenous BMP‐2 supply effectively attenuated TGF‐β1 signalling pathway through Smad6‐Smurf‐1 complex activation. In vitro cultured cardiomyocytes, mechanical stretch up‐regulated cardiac TGF‐β1, TGF‐β1‐dependent ROCK and down‐regulated BMP‐2, but BMP‐2 level could be reversed through blocking TGF‐β1 receptor by SB‐431542 or inhibition of ROCK by Y‐27632. TGF‐β1 could also activate ROCK and suppress endogenous BMP‐2 level in a dose‐dependent manner. Knock‐down BMP‐2 enhanced TGF‐β1‐mediated PKC‐δ and Smad3 signalling cascades. In contrast, treatment with Y‐27632 or SB‐431542, respectively suppressed ROCK‐dependent PKC‐δ and Smad3 activation, but BMP‐2 was only up‐regulated by Y‐27632. In addition, BMP‐2 silencing abolished the effect of Y‐27632, but not SB‐431542 on suppression of TGF‐β1 pathway. Further experiments showed that Smad6 Smurf1 interaction were required for BMP‐2‐evoked antagonizing effects. Smad6 overexpression attenuated TGF‐β1‐induced activation of PKC‐δ and Smad3, promoted TGF‐β RI degradation in BMP‐2 knock‐down cardiomyocytes, and could be abolished after knocking‐down Smurf‐1, in which Smad6/Smurf1 complex formation was critically involved. In vivo data showed that pressure overload‐induced collagen deposition was attenuated, cardiac function was improved and TGF‐β1‐dependent activation of PKC‐δ and Smad3 was reduced after 2 weeks treatment with rhBMP‐2(0.5 mg/kg) or Y‐27632 (10 mg/kg) in mice that underwent surgical transverse aortic constriction. In conclusion, we propose that BMP‐2, as a novel fibrosis antagonizing cytokine, may have potential beneficial effect in attenuating pressure overload‐induced cardiac fibrosis.     

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.     

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.     

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.     

Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

Krüppel‐like factor 4 (KLF4) was closely associated with epithelial‐mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)‐enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF‐β1 pathway, whereas enforced KLF4 overexpression activated TGF‐β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF‐β1 pathway inhibition invalidated KLF4‐facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF‐β1/Smad/Snail pathway in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs.     

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.     

miR‐193a/b‐3p relieves hepatic fibrosis and restrains proliferation and activation of hepatic stellate cells

MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR‐193a/b‐3p in concanavalin A (ConA)‐induced liver fibrosis in mice was evaluated. According to the results, the expression of miR‐193a/b‐3p was down‐regulated in liver tissues after exposure to ConA. Lentivirus‐mediated overexpression of miR‐193a/b‐3p reduced ConA‐induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA‐induced liver fibrosis was restrained by the up‐regulation of miR‐193a/b‐3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor‐β1 (TGF‐β1) and activin A in liver tissues. Furthermore, miR‐193a/b‐3p mimics suppressed the proliferation of human HSCs LX‐2 via inducing the apoptosis of LX‐2 cells and lowering the levels of cell cycle‐related proteins Cyclin D1, Cyclin E1, p‐Rb and CAPRIN1. Finally, TGF‐β1 and activin A‐mediated activation of LX‐2 cells was reversed by miR‐193a/b‐3p mimics via repressing COL1A1 and α‐SMA expression, and restraining the activation of TGF‐β/Smad2/3 signalling pathway. CAPRIN1 and TGF‐β2 were demonstrated to be the direct target genes of miR‐193a/b‐3p. We conclude that miR‐193a/b‐3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR‐193a‐3p and miR‐193b‐3p may be new therapeutic targets for liver fibrosis.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

MicroRNA‐539 functions as a tumour suppressor in prostate cancer via the TGF‐β/Smad4 signalling pathway by down‐regulating DLX1

Prostate cancer (PCa) is the second leading cause of cancer‐related death in males, primarily due to its metastatic potential. The present study aims to identify the expression of microRNA‐539 (miR‐539) in PCa and further investigate its functional relevance in PCa progression both in vitro and in vivo. Initially, microarray analysis was conducted to obtain the differentially expressed gene candidates and the regulatory miRNAs, after which the possible interaction between the two was determined. Next, ectopic expression and knock‐down of the levels of miR‐539 were performed in PCa cells to identify the functional role of miR‐539 in PCa pathogenesis, followed by the measurement of E‐cadherin, vimentin, Smad4, c‐Myc, Snail1 and SLUG expression, as well as proliferation, migration and invasion of PCa cells. Finally, tumour growth was evaluated in nude mice through in vivo experiments. The results found that miR‐539 was down‐regulated and DLX1 was up‐regulated in PCa tissues and cells. miR‐539 was also found to target and negatively regulate DLX1 expression, which resulted in the inhibition of the TGF‐β/Smad4 signalling pathway. Moreover, the up‐regulation of miR‐539 or DLX1 gene silencing led to the inhibition of PCa cell proliferation, migration, invasion, EMT and tumour growth, accompanied by increased E‐cadherin expression and decreased expression of vimentin, Smad4, c‐Myc, Snail1 and SLUG. In conclusion, the overexpression of miR‐539‐mediated DLX1 inhibition could potentially impede EMT, proliferation, migration and invasion of PCa cells through the blockade of the TGF‐β/Smad4 signalling pathway, highlighting a potential miR‐539/DLX1/TGF‐β/Smad4 regulatory axis in the treatment of PCa.     

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.     

Long non‐coding RNA SNAI3‐AS1 promotes the proliferation and metastasis of hepatocellular carcinoma by regulating the UPF1/Smad7 signalling pathway

Emerging evidence has indicated that deregulation of long non‐coding RNAs (lncRNAs) can contribute to the progression of human cancers, including hepatocellular carcinoma (HCC). However, the role and exact mechanism of most lncRNAs in tumours remains largely unknown. In the current study, we found a novel long non‐coding RNA termed SNAI3‐AS1 which was generally up‐regulated in HCC tissues compared with normal control. Higher expression of SNAI3‐AS1 was significantly correlated with shorter overall survival of HCC patients. Knockdown of SNAI3‐AS1 inhibited the proliferation and metastasis of HCC cells in vitro, whereas overexpression of SNAI3‐AS1 promoted the proliferation and metastasis of HCC cells. Further investigations showed that SNAI3‐AS1 could affect HCC tumorigenesis by binding up‐frameshift protein 1 (UPF1), regulating Smad7 expression and activating TGF‐β/Smad pathway. Functionally, SNAI3‐AS1 promoted HCC growth and metastasis by inducing tumour epithelial to mesenchymal transition (EMT). Taken together, these findings showed that SNAI3‐AS1 promotes the progression of HCC by regulating the UPF1 and activating TGF‐β/Smad pathway.     

TGF‐β Stimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways

The enzyme chondroitin polymerizing factor (ChPF) is primarily involved in extension of the chondroitin sulfate backbone required for the synthesis of sulfated glycosaminoglycan (sGAG). Transforming growth factor beta (TGF‐β) upregulates sGAG synthesis in nucleus pulposus cells; however, the mechanisms mediating this induction are incompletely understood. Our study demonstrated that ChPF expression was negatively correlated with the grade of degenerative intervertebral disc disease. Treatment of nucleus pulposus cells with TGF‐β induced ChPF expression and enhanced Smad2/3, RhoA/ROCK activation, and the JNK, p38, and ERK1/2 MAPK signaling pathways. Selective inhibitors of Smad2/3, RhoA or ROCK1/2, and knockdown of Smad3 and ROCK1 attenuated ChPF expression and sGAG synthesis induced by TGF‐β. In addition, we showed that RhoA/ROCK1 signaling upregulated ChPF via activation of the JNK pathway but not the p38 and ERK1/2 signaling pathways. Moreover, inhibitors of JNK, p38 and ERK1/2 activity also blocked ChPF expression and sGAG synthesis induced by TGF‐β in a Smad3‐independent manner. Collectively, our data suggest that TGF‐β stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1 and the three MAPK signaling pathways. J. Cell. Biochem. 119: 566–579, 2018. © 2017 Wiley Periodicals, Inc.     

Tetraspanin 1 as a mediator of fibrosis inhibits EMT process and Smad2/3 and beta‐catenin pathway in human pulmonary fibrosis

Tetraspanin 1(TSPAN1) as a clinically relevant gene target in cancer has been studied, but there is no direct in vivo or vitro evidence for pulmonary fibrosis (PF). Using reanalysing Gene Expression Omnibus data, here, we show for the first time that TSPAN1 was markedly down‐regulated in lung tissue of patient with idiopathic PF (IPF) and verified the reduced protein expression of TSPAN1 in lung tissue samples of patient with IPF and bleomycin‐induced PF mice. The expression of TSPAN1 was decreased and associated with transforming growth factor‐β1 (TGF‐β₁)‐induced molecular characteristics of epithelial‐to‐mesenchymal transition (EMT) in alveolar epithelial cells (AECs). Silencing TSPAN1 promoted cell migration, and the expression of alpha‐smooth muscle actin, vimentin and E‐cadherin in AECs with TGF‐β₁ treatment, while exogenous TSPAN1 has the converse effects. Moreover, silencing TSPAN1 promotes the phosphorylation of Smad2/3 and stabilizes beta‐catenin protein, however, overexpressed TSPAN1 impeded TGF‐β₁‐induced activation of Smad2/3 and beta‐catenin pathway in AECs. Together, our study implicates TSPAN1 as a key regulator in the process of EMT in AECs of IPF.