The role of TGF-beta signaling in the pathogenesis of fibrosis in scleroderma

Excessive extracellular matrix (ECM) deposition in the skin, lung, and other organs is a hallmark of systemic sclerosis (SSc). The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that transforming growth factor (TGF)-beta is a key mediator of tissue fibrosis as a consequence of ECM accumulation in pathological states such as systemic sclerosis. TGF-beta regulates diverse biological activities including cell growth, cell death or apoptosis, cell differentiation, and ECM synthesis. TGF-beta is known to induce the expression of ECM proteins in mesenchymal cells and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. This review focuses on the possible role of TGF-beta in the pathogenesis of fibrosis in SSc.     

Targeted delivery in scleroderma fibrosis

Systemic sclerosis (SSc) is considered one of the most challenging and difficult to treat among rheumatic disorders, due to its severity, multiorgan manifestation and different outcomes. It manifests fibrosis in different organs, mostly in skin and lungs. The skin fibrosis expression is considered the first sign of the disease and usually it is followed by internal organ fibrosis. An aberrant immune system activation seems to relate to the expression of the disease, but even environmental influences and dysregulation of many molecules signalling pathways are involved in the development of the disease. Current therapies are limited and characterized by multiple side effects: systemic route is the elective administration route, which decreases patient adherence to the therapy, as they are often already bothered by pain and disfigurement. Treatments available are organ-based, originally indicated for other conditions and there is no therapy available to reduce the fibroblast population size within existing fibrotic lesions. Disease-modifying therapies or immunomodulatory agents that are highly effective in other rheumatic diseases have shown disappointing results in SSc. There are thus no standardized and effective treatments for this disease, and there are even unanswered questions related to the insurgence of the pathology and all the mechanisms involved. An ideal approach could be considered “targeted therapy” that will be an increasingly attainable objective insofar as our understanding of the disease improves. The advantages in identifying the molecule and the signalling pathways involved in the pathology have helped to find some novel compounds for the therapy of scleroderma fibrosis or following innovative uses for already-approved drugs, corroborated by many clinical studies.     

CD19 regulates skin and lung fibrosis via Toll-like receptor signaling in a model of bleomycin-induced scleroderma

Mice subcutaneously injected with bleomycin, in an experimental model of human systemic sclerosis, develop cutaneous and lung fibrosis with autoantibody production. CD19 is a general "rheostat" that defines signaling thresholds critical for humoral immune responses, autoimmunity, and cytokine production. To determine the role of CD19 in the bleomycin-induced systemic sclerosis model, we investigated the development of fibrosis and autoimmunity in CD19-deficient mice. Bleomycin-treated wild-type mice exhibited dermal and lung fibrosis, hyper-gamma-globulinemia, autoantibody production, and enhanced serum and skin expression of various cytokines, including fibrogenic interleukin-4, interleukin-6, and transforming growth factor-beta1, all of which were inhibited by CD19 deficiency. Bleomycin treatment enhanced hyaluronan production in the skin, lung, and sera. Addition of hyaluronan, an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4, stimulated B cells to produce various cytokines, primarily through TLR4; CD19 deficiency suppressed this stimulation. These results suggest that bleomycin induces fibrosis by enhancing hyaluronan production, which activates B cells to produce fibrogenic cytokines mainly via TLR4 and induce autoantibody production, and that CD19 deficiency suppresses fibrosis and autoantibody production by inhibiting TLR4 signals.     

Activation of TGF-beta/Smad2 signaling is associated with airway remodeling in asthma

BACKGROUND: Transforming growth factor beta (TGF-beta) has been suggested to play an important role in the development of airway remodeling in asthma; this suggestion is based on evidence that expression levels of TGF-beta are correlated with unique parameters of airway remodeling, such as thickness of basement membrane. However, the relevant studies were inconclusive because they were unable to demonstrate active signaling mediated by the cytokine in the airways of asthmatic individuals. OBJECTIVE: We sought to determine whether TGF-beta signaling was active in the airways of asthmatic subjects and, if so, whether it was correlated with clinicopathologic features associated with the development of airway remodeling in asthma. METHODS: We examined the phosphorylation status of Smad2 in bronchial biopsy samples obtained from 40 asthmatic subjects as a marker of active TGF-beta signaling, and we studied its correlation with basement membrane thickness. RESULTS: Expression levels of phosphorylated Smad2 in bronchial biopsy specimens from asthmatic subjects were higher than those in specimens from normal subjects, and they were correlated with basement membrane thickness in asthma. CONCLUSION: The findings provide evidence that TGF-beta signaling was active in asthmatic airways and that the activity was associated with the development of airway remodeling in asthma.     

Smad protein and TGF-beta signaling in vascular smooth muscle cells

Transforming growth factor-beta1 (TGF-beta1) plays a role in vascular remodeling by stimulating vascular smooth muscle cell (SMC) growth and matrix-protein synthesis at sites of vascular injury. Smad proteins have been shown to mediate intracellular signaling of this growth factor. We investigated the expression and phosphorylation of Smads in cultured rat aortic smooth muscle cells. In addition, we evaluated the effects of overexpression of Smad proteins on TGF-beta signal transduction by adenovirus-mediated gene transfer. In rat SMC, Smad1, Smad2, Smad3, Smad4 and Smad5 were detected by immunoprecipitation. Using antisera against phosphorylated Smad2, we showed that TGF-beta1-induced Smad2 phosphorylation in a concentration- and time-dependent manner. Using adenovirus-mediated transfection method, we demonstrated that overexpression of Smad2 or Smad4 was associated with an increased production of TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1). However, the most prominent expression of PAI-1 was observed upon cotransfection of both Smad2 and Smad4. Both the proliferative effect of TGF-beta1 under serum-free conditions and its anti-proliferative effect under serum-rich conditions were suppressed by the adenovirus-mediated overexpression of Smad7. These results indicated that Smads proteins were expressed in vascular SMC and that they mediated TGF-beta signaling in those cells.     

SnoN和Smad2/3信号蛋白与糖尿病大鼠肾小管-间质纤维化的关系

目的 观察核转录共抑制因子SnoN和Smad2/3信号蛋白在糖尿病（DM）大鼠肾组织中的动态表达,并初步探讨它们与糖尿病肾病（DN）肾小管-间质纤维化的关系。方法 尾静脉注射链尿佐菌素（STZ）复制DM大鼠模型,随机分为DM2、4、8、12、16和24周组,每组均设鼠龄匹配的正常对照组(n=6)。免疫组化及Western blotting法检测肾组织SnoN、转化生长因子β1（TGF-β1）、Smad2/3、α-平滑肌肌动蛋白（α-SMA）、纤连蛋白（FN）的蛋白表达;RT-PCR检测SnoN的mRNA;生化方法测血糖、血肌酐及24h尿蛋白量。结果 DM4周起肾组织中SnoN蛋白表达明显减少(P < 0.01),DM各组SnoN的mRNA表达无变化;TGF-β1、Smad2/3及FN的蛋白表达随DM病程进展逐渐增多;DM12周始肾小管上皮细胞可见α-SMA阳性表达。结论 SnoN蛋白表达减少与TGF-β1/Smad信号通路共同参与了DN的发生发展过程。     

Inhibition of p38 mitogen-activated protein kinase and transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis in adriamycin-induced nephropathy

Inflammation and fibrogenesis are the two determinants of the progression of renal fibrosis, the common pathway leading to end-stage renal disease. The p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta1/Smad signaling pathways play critical roles in inflammation and fibrogenesis, respectively. The present study examined the beneficial renoprotective effect of combination therapy using the p38 MAPK pathway inhibitor (SB203580) and a TGF-beta receptor I (ALK5) inhibitor (ALK5I) in a mouse model of adriamycin (ADR) nephrosis. The p38 MAPK and TGF-beta1/Smad2 signaling pathways were activated in ADR-induced nephropathy in a sequential time course manner. Two weeks after ADR injection, the combined administration of SB203580 (1 mg/kg/24 hours) and ALK5I (1 mg/kg/24 hours) markedly reduced p38 MAPK and Smad2 activities. Moreover, the co-administration of SB203580 and ALK5I to ADR-injected mice resulted in a down-regulation of total and active TGF-beta1 production, reduced myofibroblast accumulation, and decreased expression of collagen type IV and fibronectin. In these mice, retardation in the development of glomerulosclerosis and interstitial fibrosis was observed. In conclusion, although p38 MAPK and TGF-beta1/Smad signaling pathways are distinct they coordinate the progression of renal fibrosis in ADR nephrosis. The co-administration of a p38 MAPK inhibitor and an ALK5 inhibitor may have potential applications in the treatment of renal fibrosis.     

Interference with transforming growth factor-beta/ Smad3 signaling results in accelerated healing of wounds in previously irradiated skin

Transforming growth factor (TGF)-beta regulates many aspects of wound repair including inflammation, chemotaxis, and deposition of extracellular matrix. We previously showed that epithelialization of incisional wounds is accelerated in mice null for Smad3, a key cytoplasmic mediator of TGF-beta signaling. Here, we investigated the effects of loss of Smad3 on healing of wounds in skin previously exposed to ionizing radiation, in which scarring fibrosis complicates healing. Cutaneous wounds made in Smad3-null mice 6 weeks after irradiation showed decreased wound widths, enhanced epithelialization, and reduced numbers of neutrophils and myofibroblasts compared to wounds in irradiated wild-type littermates. Differences in breaking strength of wild-type and Smad3-null wounds were not significant. As shown previously for neutrophils, chemotaxis of primary dermal fibroblasts to TGF-beta required Smad3, but differentiation of fibroblasts to myofibroblasts by TGF-beta was independent of Smad3. Previous irradiation-enhanced induction of connective tissue growth factor mRNA in wild-type, but not Smad3-null fibroblasts, suggested that this may contribute to the heightened scarring in irradiated wild-type skin as demonstrated by Picrosirius red staining. Overall, the data suggest that attenuation of Smad3 signaling might improve the healing of wounds in previously irradiated skin commensurate with an inhibition of fibrosis.     

肝纤维化大鼠转化生长因子β1／Smad蛋白的动态表达及意义

目的探讨转化生长因子TGF—β1／Smad信号通路在实验性肝纤维化发生中的作用。方法50只健康雄性SD大鼠分为2组：正常组和模型组,模型组大鼠利用40％CCl4油剂诱导形成肝纤维化模型,于6周及9周观测肝标本的病理,免疫组化法检测肝组织TGF—β1／Smad蛋白表达。结果①肝组织病理：与正常组比较,模型组大鼠肝组织都有不同程度的炎症和纤维化产生。模型组纤维化程度较正常对照组明显,差异有统计学意义（P〈0．05）;②TGF—β1／Smad基因蛋白：免疫组织化学检测显示,与正常对照组相比,模型组大鼠肝脏中TGF—β1、转化生长因子βI型受体（TβR—I）、Smad2、3、Smad,蛋白表达均显著增强（P〈0．01）,模型组大鼠肝脏TGF—β1、TβR-I、Smad。和Smad,之间存在正相关关系（P〈0．05或0．01）;模型组大鼠肝脏纤维化分级与TGF—β1、TβR—I、Smad2/3和Smad,之间存在正相关关系（P〈0．05或0．01）。结论肝组织TGF—β1／Smad蛋白表达水平与肝纤维化程度相关,TGF-β1／Smad信号的增强可能促进了肝纤维化的进展。     

Targeted disruption of mouse Dach1 results in postnatal lethality.

Mouse Dach1 is a nuclear factor that is expressed during development in restricted areas of the central nervous system, neural crest, and limb buds. Its Drosophila homologue dachshund plays a role in differentiation of the eye imaginal disc, in leg morphogenesis, and in controlling neural differentiation in the mushroom bodies of the insect brain. Mouse Dach1 null homozygous survive pregnancy but become cyanotic after birth and subsequently die within 24 hr. In this report, the brain of Dach1 mutants was analyzed. Examination of mRNA expression of the central neuropeptides oxytocin, vasopressin, thyrotropin-releasing hormone, growth hormone releasing hormone, and somatostatin revealed no difference between wild-type and mutant newborn brains. Furthermore, no significant difference in cell proliferation as well as in the distribution of neurons, glia, radial glia, and neuronal progenitors was detected in the developing forebrain. Dach1-positive cells, which were visualized with Enhanced Green Fluorescent Protein (EGFP), show similar distribution and axonal projections in the cortex and hippocampus in mutants and wild-type controls. Neural stem cells derived from mutant and wild-type newborn brains display similar growth kinetics when cultivated in vitro.     

Smad signaling in the rat model of monocrotaline pulmonary hypertension

Mutations in the bone morphogenetic protein receptor type II (BMPrII) gene have been implicated in the development of familial pulmonary artery hypertension (PAH). The function of BMP signal transduction within the pulmonary vasculature and the role BMPrII mutations have in the development of PAH are incompletely understood. We used the monocrotaline (MCT) model of PAH to examine alterations in Smad signal transduction pathways in vivo. Lungs harvested from Sprague-Dawley rats treated with a single 60-mg/kg intraperitoneal (IP) injection of MCT were compared to saline-treated controls 2 weeks following treatment. Smad 4 was localized by immunohistochemistry to endothelial nuclei of the intra-acinar vessels undergoing remodeling. Smad 4, common to both BMP and transforming growth factor beta (TGFbeta) signaling, and BMP-specific Smad 1 were significantly decreased in western blot from whole lungs of treated animals, while no change was found for TGFbeta-specific Smad 2. MCT-treated rats also had increased expression of phosphorylated Smad 1 (P-Smad 1) but not phosphorylated Smad 2 (P-Smad 2). There was a decrease in the expression of the full BMPrII protein but not its short form variant in MCT-treated rat lungs. The type I receptor Alk1 had increased expression. Collectively, our data indicate that vascular remodeling in the MCT model is associated with alterations in BMP receptors and persistent endothelial Smad 1 signaling.     

Baicalin modulates microRNA expression in UVB irradiated mouse skin

This study aimed to evaluate the effects of baicalin on ultraviolet radiation B (UVB)-mediated microRNA (miRNA) expression in mouse skin.We determined miRNA expression profiles in UVB irradiated mice,baicalin treated irradiated mice,and untreated mice,and conducted TargetScan and Gene Ontology analyses to predict miRNA targets.Three miRNAs (mmu-miR-125a-5p,mmu-miR-146a,and mmu-miR-141) were downregulated and another three (mmu-miR-188-5p,mmu-miR-223 and mmu-miR-22) were upregulated in UVB irradiated mice compared with untreated mice.Additionally,these miRNAs were predicted to be related to photocarcinogenesis,hypomethylation and apoptosis.Three miRNAs (mmu-miR-378,mmu-miR-199a-3p and mmu-miR-181b) were downregulated and one (mmu-miR-23a) was upregulated in baicalin treated mice compared with UVB irradiated mice,and they were predicted to be related to DNA repair signaling pathway.These deregulated miRNAs are potentially involved in the pathogenesis of photodamage,and may aid treatment and prevention of UVB-induced dermatoses.     

miR-29b介导的TGF-β/Smad信号通路对大鼠肝纤维化进程的影响

目的:初步研究微小RNA-29b(mi R-29b)介导的TGF-β/Smad信号通路在肝星状细胞(HSC)活化中的作用及其对大鼠肝纤维化进程的影响。方法:构建肝纤维化大鼠模型并分离其HSC,同时通过体外获取并鉴定正常大鼠HSC。运用RT-qPCR和Western blot检测以上获取细胞中mi R-29b、TGF-β/Smad信号通路相关蛋白和肝纤维化标志蛋白的变化水平,并通过双萤光素酶报告基因检测系统鉴定mi R-29b对TGF-β1的直接靶向结合情况。结果:随着HSC活化加深,mi R-29b的表达量逐渐减少(P 0. 01),而HSC活性标志物I型胶原蛋白和α-平滑肌肌动蛋白的表达量逐渐增加(P 0. 01)。在TGF-β/Smad信号通路中,Smad2/3/4的表达显著增加,而Smad7的表达明显下降(P 0. 01)。双萤光素酶报告基因检测结果显示,mi R-29b可直接结合于TGF-β1 3’UTR的"UCUCUCCGU"序列,表明TGF-β1为mi R-29b的一个下游靶基因。结论:mi R-29b可参与抑制HSC的活化和迁移,进而抑制肝纤维化进程,而其生物学功能可能是通过直接靶向抑制TGF-β1进而调控TGF-β/Smad信号通路实现的。     

MicroRNA-30d/JAG1 axis modulates pulmonary fibrosis through Notch signaling pathway

Pulmonary fibrosis (PF) is a fibroproliferative disease which can finally end up fatal lung failure. PF is characterized by abnormal proliferation of fibroblast, dysregulated fibroblast differentiation to myofibroblast and disorganized collagen and extracellular matrix (ECM) production, deposition and degradation. JAG1/Notch signaling has been reported to play a key role in tissue fibrosis including PF. Herein, we confirmed the abnormal upregulation of JAG1 mRNA expression and protein levels in PF tissue specimens; JAG1 knockdown reduced TGF-β1-induced α-SMA and Collagen I protein levels. From the aspect of miRNA regulation, we searched for candidate miRNAs which might target JAG1 to inhibit its expression. Among the selected miRNAs, miR-30d expression was downregulated in PF tissues; miR-30d overexpression attenuated TGF-β1-induced primary normal human lung fibroblast (NHLF) proliferation, as well as α-SMA and Collagen I protein levels. Through directly binding to the 3′-UTR of JAG1, miR-30d significantly inhibited JAG1 mRNA expression and protein level. Furthermore, JAG1 overexpression partially reversed the effect of miR-30d on NHLF proliferation and α-SMA and Collagen I proteins upon TGF-β1 stimulation; miR-30d could suppress TGF-β1 function on NHLFs through blocking JAG1/Notch signaling. Rescuing miR-30d expression to suppress TGF-β1-induced activation of JAG1/Notch signaling may present a promising strategy for PF treatment.     

Targeted therapy comes of age in scleroderma

Systemic sclerosis (SSc, also known as scleroderma) has the highest case-specific mortality among the rheumatic diseases; however, advances in understanding of pathogenesis and an appreciation of the clinical heterogeneity of this disease, together with therapeutic advances in other areas, have raised the possibility of substantial improvements in its management. Key areas of advance include the development of clinical and laboratory methods for early detection of complications and the integration of vascular, immunomodulatory and antifibrotic therapies. Disease models have facilitated the identification of key mediators or processes that could be targeted therapeutically and have provided a platform for preclinical testing of novel candidate therapies. This review will consider current clinical aspects of SSc and the emergence of targeted therapy that is linked to key pathogenic processes or that targets pivotal mediators.     

Glycoconjugate metabolism in a cystic fibrosis knockout mouse model

Cystic fibrosis knockout mice (cftr(-/-)) die prematurely of obstruction of the intestine which may result from accumulation of dehydrated glycoconjugate-containing mucus. We noted an increase in the specific activity of [(14)C]glucosamine-labeled high-molecular weight glycoconjugates, probably mucin, in the lumen of the intestine of cftr(-/-) (homozygous) mice compared to cftr(+/+) (wild-type) and cftr(+/-) (heterozygous) mice and a decrease in the turnover of glycoconjugates of several organs of the cftr(-/-) mice. No difference in the anionic composition of secreted intestinal glycoconjugates was detected and no difference in the amount of mucin 1 (Muc1) was found in the small intestine, colon, pancreas, and lungs of the different genotypes. In addition, the spleen of the cftr(-/-) mice was significantly smaller than that of control mice and the small intestine and colon were, respectively, longer and shorter compared to control mice. These results indicate modified glycoconjugate metabolism in cystic fibrosis knockout mice and morphologic changes to the spleen and intestine where the latter modifications are possibly related to the intestinal malabsorption associated with cystic fibrosis.