Steps in integrin β1-chain glycosylation mediated by TGFβ1 signaling through ras

Ras is activated by transforming growth factor beta (TGFβ) in several cell types, but the biological consequences of this activation are largely unknown. We now show that ras mediates two stages in integrin β1-chain maturation: 1) glycosylation of the 86-kD core peptide, which is a TGFβ1-independent process, and 2) TGFβ1-mediated conversion of the 115-kD β1 integrin precursor into the mature 130-kD form. HD3 colon epithelial cells maintain elevated levels of integrin α2β1 heterodimers, strong binding to collagen I, and autocrine regulation by TGFβ1, which converts β1 integrin into the mature cell surface form. Each of three HD3 cell clones that stably express dominant negative ras (N17ras) exhibited abnormal glycosylation of the integrin β1-chain, decreased cell surface expression of the mature integrin β1, and impaired binding to collagen and laminin. Autocrine levels of TGFβ were not altered by expression of N17ras. The aberrant glycosylation of the integrin β1-chain was reversed by antisense oligonucleotides specific to the DNA sequence encoding the rasS17N mutation. Glycosylation of the 86-kD core peptide was delayed in the N17ras transfectants, but was not altered by either the addition of TGFβ1 or inhibition of autocrine TGFβ1. In contrast, conversion of the partially glycosylated β1 integrin precursor into the mature 130-kD isoform was accelerated by exogenous TGFβ1 and blocked by neutralizing antibody to autocrine TGFβ1 in control cell lines. Neither effect was seen in the N17ras transfectants, indicating that TGFβ1 modulates integrin β1-chain maturation by activating ras proteins. Cell fractionation studies demonstrated that this conversion takes place within the Golgi. J. Cell. Physiol. 181:33–44, 1999. © 1999 Wiley-Liss, Inc.     

The role of TGF‐β1 during skeletal muscle regeneration

The injury of adult skeletal muscle initiates series of well‐coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF‐β family members is TGF‐β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF‐β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function.     

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.     

TGFβ signaling in cartilage development and maintenance

Members of the transforming growth factor beta (TGFβ) superfamily of secreted factors play essential roles in nearly every aspect of cartilage formation and maintenance. However, the mechanisms by which TGFβs transduce their effects in cartilage in vivo remain poorly understood. Mutations in several TGFβ family members, their receptors, extracellular modulators, and intracellular transducers have been described, and these usually impact the development of the cartilaginous skeleton. Furthermore, genome‐wide association studies have linked components of the (TGFβ) superfamily to susceptibility to osteoarthritis. This review focuses on recent discoveries from genetic studies in the mouse regarding the regulation of TGFβ signaling in developing growth plate and articular cartilage, as well as the different modes of crosstalk between canonical and noncanonical TGFβ signaling. These new insights into TGFβ signaling in cartilage may open new prospects for therapies that maintain healthy articular cartilage. Birth Defects Research (Part C) 102:37–51, 2014. © 2014 Wiley Periodicals, Inc.     

Efficacy and specificity of antisense laminin chain-specific expression vectors in blocking laminin induction by TGFβ1: Effect of laminin blockade on TGFβ1-mediated cellular responses

Transforming growth factorβ1 (TGFβ1) elicits a multitude of cellular responses from the epithelial-derived human colon cancer Moser cells. TGFβ1 induces the expression of laminin and fibronectin, and previous studies show that the induction of fibronectin is functionally associated with the regulation of carcinoembryonic antigen (CEA) expression by TGFβ1 (Huang and Chakrabarty, 1994, J Biol Chem 269:28764–28768). In this study we constructed antisense laminin chain-specific expression vectors and determined their efficacy in blocking the expression and the induction of the large multichain laminin molecule by TGFβ1. We also determined the functional role of laminin in several TGFβ1-mediated responses: growth inhibition, downmodulation of anchorage-independent growth, and cellular invasion. Expression of either antisense laminin chain A, B1, or B2 RNA resulted in a downmodulation of endogenous laminin mRNA expression and blocked the induction of laminin protein by TGFβ1 without affecting the induction of other adhesion molecules such as fibronectin or CEA. It is concluded that antisense RNA directed to only one of the laminin chains was sufficient to disrupt the induction of the complex laminin molecule in quite a specific manner. Expression of antisense laminin RNA downregulated cellular adhesion to extracellular matrix (ECM) laminin and blocked the ability of TGFβ1 to upmodulate adhesion to ECM laminin. Expression of antisense laminin RNA, however, did not alter the downregulating effect of TGFβ1 on cellular proliferation, anchorage-independent growth, or cellular invasion, suggesting that the induction of laminin did not play a significant functional role in these TGFβ1-mediated cellular responses. It is likely that other adhesion pathways may be involved in mediating the action of TGFβ1 in this cell line. J. Cell. Physiol. 178:296–303, 1999. © 1999 Wiley-Liss, Inc.     

TGFβ‐stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro‐migratory TGFβ switch

During the course of breast cancer progression, normally dormant tumour‐promoting effects of transforming growth factor β (TGFβ), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro‐migratory TGFβ ‘switch’ in mammary epithelial cells in vitro, we found that TGFβ stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFβ receptor, ALK5, as evidenced by studies using short hairpin RNA‐resistant ALK5 mutants in ALK5‐depleted cells and in vitro kinase assays. Functionally, Smad1/5 co‐depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFβ‐stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFβ‐stimulated Smad1/5 phosphorylation, which occurs through a non‐canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro‐migratory TGFβ switch in mammary epithelial cells.     

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.     

Antagonistic Effects of TGFβ1 and BMP-6 on Skin Keratinocyte Differentiation

Several members of the transforming growth factor β (TGFβ) superfamily are expressed in the developing murine epidermis. Among these are TGFβ1, which is found in the basal layer, and bone morphogenetic protein (BMP)-6, located in the suprabasal layers. Although the role of TGFβ in cell growth has been studied extensively, little is known about the effects of these factors on keratinocyte differentiation. This study demonstrates that BMP-6 acts to positively regulate the differentiation of primary skin keratinocytes grown in culture. In contrast, TGFβ1 antagonizes keratinocyte differentiation blocking the upregulation of keratin markers by BMP-6. We show that the effects of BMP-6 on expression of keratin 1 (K1), a marker of differentiation, requires signaling through the Smad pathway. In addition, regulation of K1 levels by BMP-6 is modulated by the SEK signaling pathway. This suggests that regulation of keratinocyte differentiation by BMP-6 involves multiple signaling systems.