TGF—β及其信号分子SMAD3在伤口愈合过程中的作用

转化生长因子 β (TGF β)是一类分泌型多肽信号分子 ,在生物体发育及组织修复过程中发挥重要的调节作用。SMAD家族是一类新发现的TGF β信号的细胞质内介导者 ,它们可将TGF β信号直接从细胞膜转入细胞核内 ,并激活靶基因的转录。其中SMAD3和SMAD2作为受体激活的SMADs介导TGF βs和激活素 (activins)的细胞内信号。Smad 3基因敲除小鼠 (Smad 3ex8/ex8)可以存活至成年 ,其伤口愈合过程较正常对照鼠明显加快 ,并具有重上皮化加速和局部炎症反应减弱等特点。SMAD3在组织修复过程中的特殊作用途径可能成为治疗慢性难愈合伤口的靶点。     

Apigenin inhibits fibrous scar formation after acute spinal cord injury through TGFβ/SMADs signaling pathway

AimTo investigate the effect of apigenin on fibrous scar formation after mouse spinal cord injury (SCI).MethodsThe pneumatic impactor strike method was used to establish an SCI model. Mice were intraperitoneally injected with 5 mg/kg or 20 mg/kg apigenin daily for 28 days after SCI. The Basso Mouse Scale (BMS) score, hematoxylin–eosin staining, and immunohistochemical staining were used to assess the effect of apigenin on scar formation and motor function recovery. Western blotting and qRT‐PCR were used to detect the expression of fibrosis‐related parameters in spinal cord tissue homogenates. NIH‐3 T3 cells and mouse primary spinal cord fibroblasts, α‐Smooth muscle actin (α‐SMA), collagen 1, and fibronectin were used to evaluate apigenin''s effect in vitro. Western blotting and immunofluorescence techniques were used to study the effect of apigenin on TGFβ/SMADs signaling.ResultsApigenin inhibited fibrous scar formation in the mouse spinal cord and promoted the recovery of motor function. It reduced the expression of fibroblast‐related parameters and increased the content of nerve growth factor in vivo, decreasing myofibroblast activation and collagen fiber formation by inhibiting TGFβ‐induced SMAD2/3 phosphorylation and nuclear translocation in vitro.ConclusionApigenin inhibits fibrous scar formation after SCI by decreasing fibrosis‐related factor expression through TGFβ/SMADs signaling.     

Roles of TGF-β signaling pathway in tumor microenvirionment and cancer therapy

Transforming growth factor β (TGF- β) signaling pathway has pleiotropic effects on cell proliferation, differentiation, adhesion, senescence, and apoptosis. TGF-β can be widely produced by various immune or non-immune cells and regulate cell behaviors through autocrine and paracrine. It plays essential roles in biological processes including embryological development, immune response, and tumor progression. Few cell signalings can contribute to so many pleiotropic functions as the TGF- β signaling pathway in mammals. The significant function of TGF-β signaling in tumor progression and evasion leading it to draw great attention in scientific and clinical research. Understanding the mechanism of TGF- β signaling provides us with chances to potentiate the effectiveness and selectivity of this therapeutic method. Herein, we review the molecular and cellular mechanisms of TGF-β signaling in carcinomas and tumor microenvironment. Then, we enumerate main achievements of TGF-β blockades used or being evaluated in cancer therapy, providing us opportunities to improve therapeutical approaches in the tumor which thrive in a TGF-β-rich environment.     

罗格列酮对糖尿病大鼠心脏转化生长因子-β1/SMADs信号转导通路及心脏重塑的影响

目的观察罗格列酮对糖尿病大鼠心脏转化生长因子-β1(TGF-β1)/SMADs信号转导通路的影响及对心脏重塑的作用.方法30只雄性SD大鼠随机分成正常组、糖尿病组和罗格列酮组,每组10只.腹腔注射链脲菌素60 mg/kg建立糖尿病模型.12周后,多导生理记录仪测定大鼠心功能;测定大鼠体质量、心脏质量并计算心脏质量/体质量;电镜观察心肌超微结构改变;苦味酸酸性复红染色观察心肌间质纤维化程度;逆转录多聚酶链反应(RT-PCR)测定大鼠心脏SMAD3和SMAD7信使核糖核酸(mRNA)水平,免疫组化法分析大鼠心脏TGF-β1、SMAD3和SMAD7的蛋白水平.结果与正常组比较,糖尿病组心功能明显受损,心脏胶原含量显著增加(P＜0.01),心脏质量/体质量显著增加(P＜0.01),电镜超微结构显示心肌肌丝溶解、断裂,间质胶原增生,SMAD3 mRNA表达水平明显增加(P＜0.01),SMAD7mRNA表达水平明显降低(P＜0.01),TGF-β1和SMAD3蛋白表达水平均增加(P＜0.01),SMAD7蛋白表达水平降低(P＜0.01).应用罗格列酮干预后,心功能明显改善,胶原含量明显下降(P＜0.05),心脏质量/体质量下降(P＜0.05),超微结构破坏程度减轻,SMAD3 mRNA表达水平降低(P＜0.01),SMAD7 mRNA水平增加(P＜0.01),TGF-β1和SMAD3蛋白表达水平均降低(P＜0.05～0.01),SMAD7蛋白表达水平增加(P＜0.01).结论罗格列酮能够明显改善链脲菌素糖尿病大鼠心脏重塑,改善心功能,影响TGF-β1/SMADs信号通路活化可能是其作用机制之一.     

Effects of lower fluence pulsed dye laser irradiation on production of collagen and the mRNA expression of collagen relative gene in cultured fibroblasts in vitro

Background Lower fluence of 585-nm flashlamp-pumped pulsed dye laser has been successfully used as a nonablative technique in the treatment of wrinkles. The objective of this study was to evaluate the effect of the pulsed dye laser （585 nm） on the production of collagen and the mRNA expression of collagen related gene in fibroblasts in vitro. Methods Cultured fibroblasts were treated with a 585-nm flashlamp-pumped pulsed dye laser （ fluence 3 J/cm^2, 4 J/cm^2, spot size 7 mm, pulse duration 450 12s）. The production of collagen and the mRNA expression of transforming growth factor （TGF）-β1, SMAD2, SMAD3, SMAD4, SMAD7 and type Ⅰ procollagen α1, α2 in fibroblasts were investigated by colorimetry or real time polymerase chain reaction. Results The production of collagen was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm^2 （P 〈0.001）. The mRNA expression of TGF- β1, SMAD2, SMAD3, SMAD4, SMAD7 and procollagen I was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm^2 （P 〈0.001）. No significant difference of mRNA expression of SMAD2, SMAD3, SMAD4, SMAD7 and type Ⅰ procollagen was found between controls and fibroblasts treated with pulsed dye laser with a fluence of 4 J/cm^2 （P 〉0.05）. Conclusions Lower fluence （3 J/cm^2） pulsed dye laser increased the collagen production in fibroblasts by up-regulating TGF-β1, SMAD2, SMAD3, SMAD4, SMAD7 and type Ⅰ procollagen mRNA expression. These may be the reason it can be effectively used in the treatment of wrinkles.     

TGF-β/SMADs信号通路在创伤愈合中的作用

转化生长因子-β（TGF-β）超家族是在脊椎动物和无脊椎动物中高度保守的一类细胞因子,其家族成员参与调节细胞增殖、分化、凋亡、黏附、骨骼形成、发育、炎症反应及创伤愈合等多种生命活动。SMAD家族成员是TGF-β信号的细胞内介导者,负责将TGF-β信号从细胞膜转入细胞核内,并激活靶基因的转录。创伤后TGF-β参与了皮肤愈合的炎症期、肉芽组织形成期和瘢痕形成期全过程。从信号转导通路不同环节干预和阻断,将对创伤愈合过程产生积极的作用。SMAD3在组织修复中的特殊作用可能成为今后探索难愈性创面的治疗热点。     

Molecular requirements for induction of CTGF expression by TGF-beta1 in primary osteoblasts

Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-β1 where it acts as a downstream mediator of TGF-β1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-β1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-β1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-β1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-β1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-β1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-β1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-β1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-β in some cell types, we determined its role in TGF-β1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-β1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-β1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To investigate the involvement of the TGF-β1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction, we cloned the rat CTGF proximal promoter (− 787 to + 1) containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using a combination of CTGF promoter deletion constructs and site-directed mutants, we demonstrated the unique requirement of both the TRE and SBE for CTGF induction by TGF-β1 in osteoblasts. Electro-mobility shift assays using specific probes containing the TRE, SBE or both showed TGF-β1 inducible complexes that can be ablated by mutation of the respective motif, confirming their requirement for TGF-β1 induced CTGF promoter activity. In conclusion, these studies demonstrate that CTGF induction by TGF-β1 in osteoblasts involves Smads 3 and 4, the Erk and Src signaling pathways, and requires both the TRE and SBE motifs in the CTGF proximal promoter.     

在支气管上皮细胞恶性转化过程中Smad7表达变化对TGF-βR、SMADs及STRAP蛋白的影响

目的研究永生化人支气管上皮细胞BEP2D恶性转化过程中,作为Sm ad蛋白家族的抑制分子,Sm ad7对TGF-βR、R Sm ads及STRAP蛋白的表达影响。方法培养BEP2D、BERP35T2细胞及稳定表达Sm ad7蛋白的细胞BS7(来源于BEP2D)、RS7(来源于BERP35T2),提取细胞蛋白,用W estern b lot方法比较稳定转染Sm ad7基因前后的BEP2D和BERP35T2细胞中TGF-βRⅠ、TGF-βRⅡ、Sm ad2/3、Sm ad4及STRAP蛋白表达差异。结果稳定转染Sm ad7基因后细胞中TGF-βRⅡ表达降低,Sm ad2和STRAP蛋白表达增高,Sm ad3、Sm ad4蛋白表达无变化。结论Sm ad7高表达导致TGF-β信号通路发生改变,其中TGF-βRⅡ表达降低,STRAP表达增高可能是诱使细胞发生恶性转化的机制之一。