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.     

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.     

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.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.     

Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

The DAN family: Modulators of TGF‐β signaling and beyond

Extracellular binding proteins or antagonists are important factors that modulate ligands in the transforming growth factor (TGF‐β) family. While the interplay between antagonists and ligands are essential for developmental and normal cellular processes, their imbalance can lead to the pathology of several disease states. In particular, recent studies have implicated members of the differential screening‐selected gene in neuroblastoma (DAN) family in disease such as renal fibrosis, pulmonary arterial hypertension, and reactivation of metastatic cancer stem cells. DAN family members are known to inhibit the bone morphogenetic proteins (BMP) of the TGF‐β family. However, unlike other TGF‐β antagonist families, DAN family members have roles beyond ligand inhibition and can modulate Wnt and vascular endothelial growth factor (VEGF) signaling pathways. This review describes recent structural and functional advances that have expanded our understanding of DAN family proteins with regards to BMP inhibition and also highlights their emerging roles in the modulation of Wnt and VEGF signaling pathways.     

Regulatory mechanisms of TGF‐β1‐induced fibrogenesis of human alveolar epithelial cells

Pulmonary fibrosis is characterized by an extensive activation of fibrogenic cells and deposition of extracellular matrix (ECM). Transforming growth factor (TGF)‐β1 plays a pivotal role in the pathogenesis of pulmonary fibrosis, probably through the epithelial‐ to‐mesenchymal transition (EMT) and ECM production. The present study investigates potential mechanism by which TGF‐β1 induces EMT and ECM production in the fibrogenesis of human lung epithelial cells during pulmonary fibrosis. The expression of EMT phenotype and other proteins relevant to fibrogenesis were measured and the cell bio‐behaviours were assessed using Cell‐IQ Alive Image Monitoring System. We found that TGF‐β1‐induced EMT was accompanied with increased collagen I deposition, which may be involved in the regulation of connective tissue growth factor (CTGF) and phosphoinositide 3‐kinase (PI3K) signalling pathway. Treatment with PI3K inhibitors significantly attenuated the TGF‐β1‐ induced EMT, CTGF expression and collagen I synthesis in lung epithelial cells. The interference of CTGF expression impaired the basal and TGF‐β1‐stimulated collagen I deposition, but did not affect the process of EMT. Our data indicate that the signal pathway of TGF‐β1/PI3K/CTGF plays an important role in the fibrogenesis of human lung epithelial cells, which may be a novel therapeutic approach to prevent and treat pulmonary fibrosis.     

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.