Dynamics of TGF-β/Smad signaling

The physiological responses to TGF-β stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the non-canonical TGF-β signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-β signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-β biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-β signaling and how cells quantitatively decode variable doses of TGF-β stimulation.     

Sustained anti-inflammatory effects of TGF-β1 on microglia/macrophages

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3 days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7 dpr when transforming growth factor-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 for 24 h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72 h after removal of TGF-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 for 24 h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-β1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3 dpr, whereas it was almost disappeared on 7 dpr. The findings suggest that abundantly produced TGF-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.     

TGF-β1受体1和2在TGF-β1调节细胞增殖中的作用

转化生长因子β1(transforming growth factor-β1,TGF-β1)是一种多功能细胞因子,在细胞增殖、分化、伤口愈合和肿瘤生成转移等过程中均发挥重要调控作用。TGF-β1对细胞增殖的调节可因细胞类型、刺激剂量不同而不同,但其差异调节的机制还不清楚。现普遍认为,TGF-β1在TGFBR2/TGFBR1二聚体参与下通过经典的Smad信号通路抑制增殖,而通过非Smad信号通路促进细胞周期,但是机体是如何调控这种不同增殖调节作用转化的还不明确。TGFBR1和TGFBR2在细胞中的分布和比例变化可能是TGF-β1差异性调控细胞增殖作用的一个重要机制。     

Separation, purification, and sequence identification of TGF-β1 and TGF-β2 from bovine milk

Cox and Bürk (Eur. J. Biochem., 1991) reported the partial characterization of Milk Growth Factor (MGF) which stimulated the migration of fibroblasts. We have fractionated the partially purified sample by RP-HPLC and obtained the separation of two peaks of activity. The two active components were isolated as pure MGF-a and MGF-b by RP-HPLC and preparative SDS-PAGE. The purified MGF-a, consisting of a single band by gel electrophoresis and a single peak on an HPLC reversed-phase C-4 column, has the same specific activity as TGF-2 in the fibroblast migration assay. MGF-a was digested by endoprotease Asp-N and the cleaved peptides were analyzed by Edman degradation and plasma desorption mass spectrometry (PDMS). The whole sequence of MGF-a determined by automated sequenator and PDMS of S-pyridylethylated protein and selected fragments was found to be identical to that of TGF-2. MGF-b protein mixture separated by SDS-PAGE was electrophoretically transferred onto a Biometra Glassybond membrane, and the blotted MGF-b protein was directly sequenced on an automated sequenator. The identified 29 amino acids sequence of MGF-b was identical to the amino-terminal sequence of TGF-1. Our study demonstrates that MGF is composed of both TGF-1 and TGF-2. TGF-2 (85%) is the predominant form.     

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.     

Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (“ricin-like”) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-β1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-β1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-β1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-β1-stimulated invasion. Our results also indicate that signaling events involved in TGF-β1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.     

Induction of galectin-1 by TGF-β1 accelerates fibrosis through enhancing nuclear retention of Smad2

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.     

MFAP2 promotes HSCs activation through FBN1/TGF-β/Smad3 pathway

Liver fibrosis is a chronic inflammatory process characterized by the accumulation of extracellular matrix (ECM), which contributes to cirrhosis and hepatocellular carcinoma. Increasing evidence suggests that the activation of hepatic stellate cells (HSCs) under an inflammatory state leads to the secretion of collagens, which can cause cirrhosis. In this study, we analysed data from the Gene Expression Omnibus (GEO) databases to identify differentially expressed genes (DEGs) between quiescent and fibrotic HSCs. We found that Microfibril Associated Protein 2 (MFAP2) was elevated in carbon tetrachloride (CCl4)-induced liver fibrosis and Transforming Growth Factor-Beta 1 (TGF-β1)-activated HSCs. Knockdown of MFAP2 inhibited HSC proliferation and partially attenuated TGF-β-stimulated fibrogenesis markers. Bioinformatics analysis revealed that Fibrillin-1 (FBN1) was correlated with MFAP2, and the expression of FBN1 was significantly upregulated after MFAP2 overexpression. Silencing MFAP2 partially attenuated the activation of HSCs by inhibiting HSC proliferation and decreasing collagen deposits. In vitro results showed that the inhibition of MFAP2 alleviated hepatic fibrosis by inhibiting the activation and inducing the apoptosis of active HSCs in a CCl4-induced mouse model. In conclusion, our results suggest that MFAP2 is a potential target for the clinical treatment of liver fibrosis.     

Urotensin-2 promotes collagen synthesis via ERK1/2-dependent and ERK1/2-independent TGF-β1 in neonatal cardiac fibroblasts

U2 (urotensin-2) is the most potent vasoconstrictor in mammals which is involved in cardiac remodelling, including cardiac hypertrophy and cardiac fibrosis. Although the cellular mechanisms of the U2-induced vasoconstriction have been extensively studied, the signalling pathways involved in U2-induced TGF-β1 (transforming growth factor-β1) expression and collagen synthesis remain unclear. In this study, we show that U2 promoted collagen synthesis and ERK1/2 (extracellular signal-regulated kinase 1/2) activation in neonatal cardiac fibroblasts. The U2-induced collagen synthesis and TGF-β1 production were significantly but not completely inhibited by blocking ERK1/2. Both ERK1/2 inhibitor and TGF-β1 antibody could separately inhibit U2-induced collagen synthesis, and the synergistic inhibition effect was observed by blocking ERK1/2 and TGF-β1 simultaneously. These data suggest that U2 promotes collagen synthesis via ERK1/2-dependent and independent TGF-β1 pathway in neonatal cardiac fibroblasts.     

2-Aminoimidazoles inhibitors of TGF-β receptor 1

The 4-(5-fluoro-6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-1H-imidazol-2-ylamine 3 is a potent and selective inhibitor of TGF-βR1. Substitution of the amino group of 3 typically led to a slight decrease in the affinity for the receptor and in TGF-β-inducted PAI-luciferase reporter activity. However, 2-acetamidoimidazoles were identified as attractive candidates for further optimization as a result of their significant activity combined to their superior pharmacokinetic profile.     

The single-molecule mechanics of the latent TGF-β1 complex

Background

TGF-β1 controls many pathophysiological processes including tissue homeostasis, fibrosis, and cancer progression. Together with its latency-associated peptide (LAP), TGF-β1 binds to the latent TGF-β1-binding protein-1 (LTBP-1), which is part of the extracellular matrix (ECM). Transmission of cell force via integrins is one major mechanism to activate latent TGF-β1 from ECM stores. Latent TGF-β1 mechanical activation is more efficient with higher cell forces and ECM stiffening. However, little is known about the molecular events involved in this mechanical activation mechanism.

Results

By using single-molecule force spectroscopy and magnetic microbeads, we analyzed how forces exerted on the LAP lead to conformational changes in the latent complex that can ultimately result in TGF-β1 release. We demonstrate the unfolding of two LAP key domains for mechanical TGF-β1 activation: the α1 helix and the latency lasso, which together have been referred to as the “straitjacket” that keeps TGF-β1 associated with LAP. The simultaneous unfolding of both domains, leading to full opening of the straitjacket at a force of ∼40 pN, was achieved only when TGF-β1 was bound to the LTBP-1 in the ECM.

Conclusions

Our results directly demonstrate opening of the TGF-β1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-β1 to LTBP-1 is mandatory. Interfering with mechanical activation of latent TGF-β1 by reducing integrin affinity, cell contractility, and binding of latent TGF-β1 to the ECM provides new possibilities to therapeutically modulate TGF-β1 actions.     

Non-synonymous gene polymorphisms in the secretory signal peptide of human TGF-β1 affect cellular synthesis but not secretion of TGF-β1

We have demonstrated that gene polymorphisms within the N-terminal leader sequence of TGF-β1 contribute to the outcome of hepatic fibrogenesis. In addition, the polymorphism at codon 25 affects TGF-β1 production in peripheral blood leukocytes. Therefore, it is general assumed that these polymorphisms influence cellular secretion of this cytokine. In the present study, we analysed if this widespread hypothesis is true. We cloned FLAG-tagged CMV-driven human full-length TGF-β1 expression constructs of the different allelic variations (i.e. 10Leu/25Arg, 10Pro/25Pro and 10Pro/25Arg) and transfected them into the immortal hepatic stellate cell line LX-2 and Chinese Hamster Ovary cells. Surprisingly, the allelic variants carrying a proline either in codon 10 or 25 showed overall reduced expression as assessed by Western blot and quantitative ELISA. We conclude that the allelic variations within the signal sequence influence the expression and not secretion of TGF-β1. Detailed RNA structure prediction analysis further suggests that the individual variants form different secondary structures.     

TGF-β1/Smad 2/3信号通路在椎间盘退变中的作用

临床实践表明,富含血小板的血浆(PRP)注射是延缓椎间盘退变的有效方法,但其作用机制尚不清楚。已有研究表明,活化的血小板可释放转化生长因子-β1 (TGF-β1),它可以正向调节髓核细胞的细胞外基质。本研究旨在揭示TGF-β1/Smad 2/3信号通路在PRP缓解椎间盘退变过程中的作用机制。本研究通过制备新西兰白兔PRP,并使用PRP和TGF-β1抑制剂(SB431542)处理白兔髓核细胞,检测PRP对髓核细胞增殖、软骨形成标志物(CollagenⅡ和Aggrecan) m RNA的表达、Smad 2/3及相关髓核细胞功能蛋白表达的影响。结果显示,用2%PRP处理的髓核细胞的增殖显著增强。PRP处理显著增加髓核细胞中的CollagenⅡ和Aggrecan的mRNA水平。Western blotting结果显示,TGF-β1信号通路的特异性抑制剂可显著抑制Smad 2/3和基质蛋白的表达。在兔椎间盘退变模型中,PRP+抑制剂注射组的Smad 2/3和CollagenⅡ的表达水平显著低于PRP处理组。这些结果表明,PRP中由于TGF-β1含量高,可激活TGF-β1/Smad 2/3途径,并促进CollagenⅡ和其他基质成分的合成和分泌,从而延缓了椎间盘退变。     

Anti-Inflammatory Effects of Ginsenoside-Rh2 Inhibits LPS-Induced Activation of Microglia and Overproduction of Inflammatory Mediators Via Modulation of TGF-β1/Smad Pathway

Microglia activation plays an important role in neuroinflammation and contributes to several neurological disorders. Hence, inhibition of both microglia activation and pro-inflammatory cytokines may lead to the effective treatment of neurodegenerative diseases. In this study, we found that GRh2 inhibited the inflammatory response to lipopolysaccharide (LPS) and prevented the LPS-induced neurotoxicity in microglia cells. GRh2 significantly decreased the generation of nitric oxide production, and tumor necrosis factor-α, interleukin (IL)-6, IL-1β, cyclooxygenase-2 and inducible nitric oxide synthase in LPS-induced activated microglia cells. Furthermore, GRh2 (20 and 50 μM) significantly increased TGF-β1 expression and reduced the expression of Smad. These results suggest that GRh2 effectively inhibits microglia activation and production of pro-inflammatory cytokines via modulating the TGF-β1/Smad pathway.     

Autocrine secretion of TGF-β1 and TGF-β2 by pre-adipocytes and adipocytes: A potent negative regulator of adipocyte differentiation and proliferation of mammary carcinoma cells

Summary We have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells, but not DR27 mutant epithelial cells which lack TGF-β type II receptor. Inhibitory activity of CMs was partially abrogated by neutralizing anti-TGF-β1 and anti-TGF-β2 antibodies, and was removed following ultrafiltration through membranes of 10 000 M_r but not 30 000 M_r pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-β1-like and TGF-β2-like molecules. In addition, acid-treated CM as well as purified TGF-β inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that pre-adipocytes spontaneously activate their own secreted TGF-β, whereas mature adipocytes do not, and suggest that activation of TGF-β has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-β may be an important modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment.