Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor β (TGF-β) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-β on CTGF expression. We found that myoblasts treated with LPA and TGF-β1 produced an additive effect on CTGF expression. In the absence of TGF-β, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-β receptor type II (TGF-βRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-βRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-βRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-βRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-βRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-β are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.     

Expression of type I and II receptors for transforming growth factor β in the adult rat testis

 After having established the specificity of the antibodies for the rat testis by western blot analysis, the potential target cells for transforming growth factors (TGFβs) were identified by immunohistochemical detection of both type I (TβRI) and type II (TβRII) transducing receptors for TGFβs in the adult rat testis in situ. Leydig cells showed a strong TβRII immunoreactivity whereas the TβRI staining was weak. Only TβRII was detectable in Sertoli cells. In germ cells, staining for TβRI was stronger than for TβRII and the expression of both receptors depended on the seminiferous cycle stage. TβRI first appeared in pachytene spermatocytes and was absent in elongated spermatids from stage XIV onwards. Labelling for TβRII was observed as early as the spermatogonia stage; it increased in pachytene spermatocytes at the onset of TβRI and disappeared in elongating spermatids from stage XI onwards. These results show that TGFβs can affect somatic cells functions and suggest that these factors are involved in the control of meiosis and early spermiogenesis, exerting a direct effect on germ cells. Accepted: 18 June 1998     

Emerging roles of transforming growth factor β signaling in wet age-related macular degeneration

Age-related macular degeneration (AMD) is one of the major causes of irreversible blindness among aging populations in developed countries and can be classified as dry or wet according to its progression.Wet AMD,which is characterized by angiogenesis on the choroidal membrane,is uncommonly seen but more severe.Controlling or completely inhibiting the factors that contribute to the progression of events that lead to angiogenesis may be an effective strategy for treating wet AMD.Emerging evidence has shown that transforming growth factor-β(TGF-β) signaling plays a significant role in the progression of wet AMD.In this review,we described the roles of and changes in TGF-β signaling in the development of AMD and discussed the mechanisms of the TGF-β superfamily in choroidal neovascularization (CNV) and wet AMD,including the modulation of angiogenesis-related factors,inflammation,vascular fibrosis,and immune responses,as well as cross-talk with other signaling pathways.These remarkable findings indicate that TGF-β signaling is a potential target for wet AMD treatment.     

Interactions between interferon γ and retinoic acid with transforming growth factor β in the induction of immune recognition molecules

The cell-surface expression of major histocompatibility (MHC) antigens and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) is essential for target cell recognition by T lymphocytes. The expression of both classes of molecule is induced by various cytokines, notably interferon (IFN). Since transforming growth factor (TGF) has been recently reported to antagonise HLA-DR induction by IFN we have examined, using a number of murine and human cell lines, the effect of TGF on IFN-induced MHC class I and class II and ICAM-1 expression. All of the cell lines tested expressed elevated class I MHC following IFN treatment. Class II MHC induction was seen on most but not all of the cells, the exceptions being among a panel of human colorectal carcinoma cell lines. A striking difference between cells of different origin was noted in the response to TGF. TGF was found to antagonise IFN-induced class I and class II MHC expression on C3H 10T1/2 murine fibroblasts, early-passage BALB/c mouse embryo fibroblasts, a murine oligodendroglioma cell line, and on MRC5 human fibroblasts and two human glioblastoma cell lines. Class II MHC was much more strongly inhibited (sometimes completely) than class I MHC. TGF also inhibited induction of class I MHC expression by IFN. However, TGF did not inhibit class I or class II MHC induction by IFN in any of the nine colorectal carcinoma cell lines, although two of five of the lines tested were growth-inhibited by TGF. On the other hand, human ICAM-1 induction by IFN was not affected by simultaneous treatment with TGF in any of the cell lines. The down-regulation of IFN-induced MHC antigens by TGF is not, therefore, the result of a general antagonism of IFN. Retinoic acid has recently been reported to induce ICAM-1 expression on human tumour cells. We have confirmed this observation on MRC5, and the two human glioblastoma cell lines, however six colorectal carcinoma cell lines tested did not respond. In contrast to IFN-induced ICAM-1 expression, retinoic-acid-induced ICAM-1 expression was inhibited by TGF on two of the three responsive lines.     

Beclin 1 regulates neuronal transforming growth factor-β signaling by mediating recycling of the type I receptor ALK5

Background

Beclin 1 is a key regulator of multiple trafficking pathways, including autophagy and receptor recycling in yeast and microglia. Decreased beclin 1 levels in the CNS result in neurodegeneration, an effect attributed to impaired autophagy. However, neurons also rely heavily on trophic factors, and signaling through these pathways requires the proper trafficking of trophic factor receptors.

Results

We discovered that beclin 1 regulates signaling through the neuroprotective TGF-β pathway. Beclin 1 is required for recycling of the type I TGF-β receptor ALK5. We show that beclin 1 recruits the retromer to ALK5 and facilitates its localization to Rab11⁺ endosomes. Decreased levels of beclin 1, or its binding partners VPS34 and UVRAG, impair TGF-β signaling.

Conclusions

These findings identify beclin 1 as a positive regulator of a trophic signaling pathway via receptor recycling, and suggest that neuronal death induced by decreased beclin 1 levels may also be due to impaired trophic factor signaling.

     

ROS directly activates transforming growth factor β type 1 receptor signalling in human vascular smooth muscle cells

BackgroundWidely used NAPDH oxidase (Nox) inhibitor, apocynin is a prodrug that needs to be converted to its pharmacologically active form by myeloperoxidase. In myeloperoxidase deficient non phagocytic cells such as vascular smooth muscle cells (VSMCs) apocynin stimulates the production of ROS. ROS is generated by the activation of many signalling pathways, thus we have used apocynin as a pharmacological tool to characterise the role of endogenous ROS in activating the transforming growth factor beta receptor (TGFBR1) without the activation of other pathways.MethodsThe in vitro study utilized human VSMCs. Western blotting and quantitative real time PCR were performed to assess signalling pathways and gene expression, respectively. Intracellular ROS levels was measured using fluorescence detection assay.ResultsTreatment with apocynin of human VSMCs stimulated ROS production and the phosphorylation of TGFBR1 and subsequent activation of TGFBR1 signalling leading to the formation of phosphorylated Smad2 which consequently upregulates the mRNA expression of glycosaminoglycan synthesizing enzyme.ConclusionsThese findings outline a specific involvement of ROS production in TGFBR1 activation. Furthermore, because apocynin stimulates Nox and ROS production, apocynin must be used with considerable care in vitro as its actions clearly extend beyond the stimulation of Nox enzymes and it has consequences for cellular signalling.General significanceApocynin can stimulate Nox leading to the production of ROS and the outcome is completely dependent upon the redox properties of the cell.     

Alk5-mediated transforming growth factor β signaling acts upstream of fibroblast growth factor 10 to regulate the proliferation and maintenance of dental epithelial stem cells

Mouse incisors grow continuously throughout life. This growth is supported by the division of dental epithelial stem cells that reside in the cervical loop region. Little is known about the maintenance and regulatory mechanisms of dental epithelial stem cells. In the present study, we investigated how transforming growth factor β (TGF-β) signaling-mediated mesenchymal-epithelial cell interactions control dental epithelial stem cells. We designed two approaches using incisor organ culture and bromodeoxyuridine (BrdU) pulse-chase experiments to identify and evaluate stem cell functions. We show that the loss of the TGF-β type I receptor (Alk5) in the cranial neural crest-derived dental mesenchyme severely affects the proliferation of TA (transit-amplifying) cells and the maintenance of dental epithelial stem cells. Incisors of Wnt1-Cre; Alk5(fl/fl) mice lost their ability to continue to grow in vitro. The number of BrdU label-retaining cells (LRCs) was dramatically reduced in Alk5 mutant mice. Fgf10, Fgf3, and Fgf9 signals in the dental mesenchyme were downregulated in Wnt1-Cre; Alk5(fl/fl) incisors. Strikingly, the addition of exogenous fibroblast growth factor 10 (FGF10) into cultured incisors rescued dental epithelial stem cells in Wnt1-Cre; Alk5(fl/fl) mice. Therefore, we propose that Alk5 functions upstream of Fgf10 to regulate TA cell proliferation and stem cell maintenance and that this signaling mechanism is crucial for stem cell-mediated tooth regeneration.     

Single-molecule imaging revealed enhanced dimerization of transforming growth factor β type II receptors in hypertrophic cardiomyocytes

Transforming growth factor β (TGF-β) signaling plays an important role in the pathogenesis of cardiac hypertrophy. However, the molecular mechanism of TGF-β signaling during the process of cardiac remodeling remains poorly understood. In the present study, by employing single-molecule fluorescence imaging approach, we demonstrated that in neonatal rat cardiomyocytes, TGF-β type II receptors (TβRII) existed as monomers at the low expression level, and dimerized upon TGF-β1 stimulation. Importantly, for the first time, we found the increased dimerization of TβRII in hypertrophic cardiomyocytes comparing to the normal cardiomyocytes. The enhanced TβRII dimerization was correlated with the enhanced Smad3 phosphorylation levels. These results provide new information on the mechanism of TGF-β signaling in cardiac remodeling.     

A systematic review and meta-analysis of the association of transforming growth factor β receptor I 6A/9A gene polymorphism with ovarian cancer risk

Abstract

Ovarian cancer is the leading cause of cancer-related death in women. This meta-analysis was conducted to evaluate the association of transforming growth factor β receptor I (TβR-I) 6A/9A gene polymorphism with ovarian cancer risk. The association literatures were identified from PubMed and Cochrane Library on 1 October 2013, and eligible reports were recruited and synthesized. Four reports were recruited into this meta-analysis for the association of TβR-I 6A/9A gene polymorphism with ovarian cancer risk. 6A allele and 6A/6A genotype of TβR-I were associated with the ovarian cancer risk (6A: OR?=?1.24, 95% CI: 1.02–1.51, p?=?0.03; 6A/6A: OR?=?2.30, 95% CI: 1.01–5.22, p?=?0.05). However, TβR-I 9A/9A genotype was not associated with the risk of ovarian cancer (OR?=?0.82, 95% CI: 0.66–1.02, p?=?0.08). In conclusion, TβR-I 6A allele and 6A/6A genotype are associated with the ovarian cancer risk. However, more studies should be performed to confirm this relationship in the future.     

Examination of structural changes in the transforming growth factor β receptor 1 (TGFβR1) gene in patients with chronic heart failure

We examined nine exons of transforming growth factor β receptor type 1 (TGFβR1) gene in patients with chronic heart failure with different types of heart remodelling. We identified two missense mutations (c.457G>A (p.V1531) and c.1285A>C (p.Y229S)) and two synonym substitutions (c.1125A>C (p.Y377Y) and c.516A>G (p.S172S)), as well as polymorphisms at splicing site c.1024+24G>A (rs334354). Substitutions c.1285A>C (p.Y229S) and c.516A>G (p.S172S) were not previously described.