Inhibition of Transforming Growth Factor (TGF)-β1–Induced Extracellular Matrix with a Novel Inhibitor of the TGF-β Type I Receptor Kinase Activity: SB-431542
Nicholas J. LapingEugene T. GrygielkoAnil MathurSebastian ButterJennifer M. BombergerChristopher TweedW. MartinJames A. FornwaldRuth LehrJohn D. HarlingLaramie M. GasterJames F. CallahanBarbara A. Olson
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Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).Abstract TGF-β1 plays an important role in the pathogenesis of chronic renal diseases. Although the specific mechanism is unknown, a major factor is the potent fibrogenic activity of TGF-β1 in the chronic progression of renal diseases. TGF-β1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules. Recently it has been studied that, Smad proteins as intracellular mediators of TGF-β signaling pathways provide important insights into the mechanisms determining the specificity of TGF-β action in various renal cells. Some studies have proved that immunosuppressants can affect TGF-β expression, but the mechanisms are unclear. In this study, we investigated the effect of FK506 on mesangial cells via TGF-β and Smads signal pathways. Our results shows that FK506 effectively blocked the TGF-β/Smad signaling pathway by downregulation of TGF-β receptor, and played an important role in TGF-β1-induced Smad2 expression in mice mesangial cells. FK506 can inhibit the TGF-β1-stimulated cell proliferation via Smad-related pathways. And reduced the Smad2 protein and mRNA expression. Altogether, this study provided a theoretical proof for the protective and treating effect of FK506 on kidneys.
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Background Keloid is a unique proliferative disorder of fibroblasts resulting from derailment of the typical wound healing process. Due to lack of animal models for therapeutic testing, treatment of keloids remains a clinical challenge. Transforming growth factor (TGF)‐β1‐related signalling plays a key role in keloid formation. As tacrolimus (FK506) has been reported to inhibit the effects of TGF‐β1 on cultured fibroblasts, we hypothesized that FK506 may be useful in treating keloids. Objectives To explore the effects of FK506 on TGF‐β1‐stimulated keloid fibroblasts (KFs) in terms of proliferation, migration and collagen production and to investigate the regulatory pathways involved. Methods Fibroblasts derived from keloids were treated with TGF‐β1 with or without FK506. Relevant assays including 5‐bromo‐2′‐deoxyuridine incorporation assay, in vitro scratch assay, reverse transcription–polymerase chain reaction (PCR), quantitative PCR and Western blotting were performed. Results The proliferation and migration of KFs were significantly higher than those of normal fibroblasts. FK506 markedly inhibited KF proliferation, migration and collagen production enhanced by TGF‐β1. The increase in TGF‐β receptor I and II expression in TGF‐β1‐treated KFs was suppressed by FK506 treatment. TGF‐β1 increased the phosphorylation of Smad2/3 and Smad4 in KFs, and this enhancing effect was abrogated by FK506. In addition, FK506 significantly increased the expression of Smad7 which was suppressed by TGF‐β1 treatment. Conclusions Our results demonstrate that FK506 effectively blocks the TGF‐β/Smad signalling pathway in KFs by downregulation of TGF‐β receptors and suggest that FK506 may be included in the armamentarium for treating keloids.
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Members of transforming growth factor-β(TGF β) superfamily play an important roles in regulating cell growth, differentiation and tissue homeostasis. Smad proteins possess special structure and property and play important roles in transforming growth factor-β(TGF-β) signal transduction pathway. TGF-β activates the downstream Smad proteins,which initiate intracellular signaling pathways via its interaction with TGF-β receptor(TGF-βR) . Research on the varieties,structures and function of Smad proteins,and the complicated regulating mechanism of Smad proteins in TGF-β superfamily signaling transduction are very significative.
Key words:
Transforming growth factor-β; Smad; Signal transduction
R-SMAD
Smad2 Protein
Growth differentiation factor
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Role of transforming growth factor-β/Smad pathway and related factors in the pathogenesis of keloids
摘要： 【摘要】 转化生长因子（TGF）-β/Smad通路在瘢痕疙瘩成纤维细胞的分化和瘢痕疙瘩的疾病进展中起重要作用。在瘢痕疙瘩发病过程中，缺氧诱导因子1α、微小RNA、长链非编码RNA、激活转录因子3、炎症因子、赖氨酸氧化酶样蛋白2等是TGF-β/Smad通路常见的影响因子。本文综述瘢痕疙瘩发病机制中TGF-β/Smad通路和常见影响因子的最新研究进展。
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Qingxuan Jiangya Decoction (QXJYD) is a traditional Chinese medicine commonly used in the clinical treatment of hypertension. Earlier studies had shown that QXJYD could inhibit the elevation of blood pressure in spontaneously hypertensive rats (SHRs) and prevent remodeling of arterial vessels. This study examines the therapeutic efficacy of QXJYD against elevated blood pressure using the SHR model, as well as the mechanisms behind its antihypertensive activity and protection against renal fibrosis. The results showed that QXJYD significantly attenuated the increase in blood pressure in SHRs and mitigated the development of renal interstitial fibrosis. In addition, QXJYD also robustly decreased the excess accumulation of extracellular matrix and attenuated the elevated expression of MMPs. The antihypertensive effects and renal protection of QXJYD were determined to be strongly associated with inhibition of TGF‐ β 1/Smad signaling pathway.
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Objective:To observe the proliferation of JEG-3 choriocarcinoma cells and Smad 3,7 mRNA expression changes under the action of transforming growth factor β1(TGF-β1),explore the mechanism of TGF-β/Smads signal channel in occurrence and development of choriocarcinoma.Methods:The JEG-3 choriocarcinoma cells were treated with different concentrations and times of TGF-β1,the cell proliferation activity was detected by MTT method,the difference of Smad 3,7 mRNA expression was detected by RT-PCR.Results:TGF-β1 stimulated the proliferation of JEG-3 choriocarcinoma cell;the expression level of Smad 3 mRNA gradually increased with the increase of TGF-β1 concentration;after treated with a certain concentration of TGF-β1 for 12 hours,the expression level of Smad 3 mRNA gradually increased with time passing;under the same condition,there was no significant difference in expression level of Smad 7.Conclusion:TGF-β1 can stimulates the proliferation of JEG-3 choriocarcinoma cell;the reaction of Smad 3 mRNA to exogenous TGF-β1 is good,while Smad 7 mRNA has no response.
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AIM:To investigate the apoptotic effect of cepharanthine(CEP)on neonatal rat cardiomyocytes (NRCMs)and the underlying mechanisms.METHODS:MTT assay was used to detect the viability of the cells.CEP-induced apoptosis in NRCMs was evaluated by Hoechst 33342 staining and the expression of activated caspase-3.The phosphorylation levels of mitogen-activated protein kinases(MAPKs),such as extracellular signal-regulated kinase (ERK),c-jun N-terminal kinase(JNK)and p38 MAPK,were examined by Western blotting.The specific inhibitors of ERK and p38 MAPK were applied for identifying the roles of the corresponding signal pathways in CEP-induced apoptosis of cardiomyocytes.RESULTS:CEP inhibited the viability of NRCMs in a dose-and time-dependent manners.Positive nuclear fragmentation and activated caspase-3 were found in CEP-treated NRCMs.The phosphorylation levels of ERK and p38 MAPK were significantly elevated in CEP-treated NRCMs,but the change of JNK was not obvious.SB203580, an inhibitor of p38 MAPK,significantly alleviated the apoptotic effect induced by CEP.However,PD98059,an inhibitor of ERK1/2,did not significantly reduce the apoptotic effect.CONCLUSION:p38 MAPK is involved in CEP-induced apoptosis in NRCMs.
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As a crucial regulatory molecule in the context of vascular stenosis, transforming growth factor-β (TGF-β), plays a pivotal role in its initiation and progression. TGF-β, a member of the TGF-β superfamily, can bind to the TGF-β receptor and transduce extracellular to intracellular signals through canonical Smad dependent or noncanonical signaling pathways to regulate cell growth, proliferation, differentiation, and apoptosis. Restenosis remains one of the most challenging problems in cardiac, cerebral, and peripheral vascular disease worldwide. The mechanisms for occurrence and development of restenosis are diverse and complex. The TGF-β pathway exhibits diversity across various cell types. Hence, clarifying the specific roles of TGF-β within different cell types and its precise impact on vascular stenosis provides strategies for future research in the field of stenosis.
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Posterior capsule opacification (PCO) is the frequent complication after cataract surgery.Transforming growth factor-β (TGF-β) is the important growth factor to modulate the transdifferentiation and formation of extracellular cell matrix (ECM) of cells.TGF-β modulates the proliferation,epithelial-mesenchymal transition and the formation of ECM by Smad signaling pathway.The research progress of TGF-β/Smad signaling pathway in PCO is reviewed in this article.
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Smad2 Protein
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