Smad‐induced alterations of matrix metabolism by a myristoyl tetra peptide

Regulation of extracellular matrix (ECM) components is essential for tissue homeostasis and function. We screened a small peptide that induces ECM protein synthesis for its usefulness in protecting keratinocytes. In this report, we demonstrate that myristoyl tetrapeptide Ala‐Ala‐Pro‐Val (mAAPV) stimulates the expression of ECM proteins and inhibits the expression of metalloproteinases (MMPs) that degrade ECM proteins in Hs68 human fibroblast cells. In order to elucidate the underlying molecular mechanisms for the effects of mAAVP, we investigated the changes in gene expression in the presence of mAAPV using a cDNA microarray. Treatment with mAAPV resulted in decreased expression of MMP‐related genes such as MMP1, MMP3, TIMP1 and TIMP3 and increased expression of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1 and COL6A3. The pattern of gene expression regulated by mAAPV was very similar to that of gene expression induced by transforming growth factor (TGF)‐β, indicating that the TGF‐β signaling pathway is crucial for simultaneous activation of several ECM‐related genes by mAAPV. We examined whether the activation of SMAD, a downstream protein of TGF‐β receptor, is involved in the signal transduction pathway induced by mAAPV. The results demonstrate that mAAVP directly activates SMAD2 and induces SMAD3 to bind to DNA. In conclusion, our results demonstrate that mAAPV both enhances the expression of collagen and inhibits its degradation via production of protease inhibitors that prevent enzymatic breakdown of the ECM. The results suggest that mAAPV would be a useful ECM‐protecting agent. Copyright © 2014 John Wiley & Sons, Ltd.     

In vitro cardiomyogenic differentiation of adipose‐derived stromal cells using transforming growth factor‐β1

Transplanting stem cells differentiated towards a cardiac lineage can regenerate cardiac muscle tissues to treat myocardial infarction. In this study, we tested the hypothesis that transforming growth factor‐β1 (TGF‐β1) induces cardiomyogenic differentiation of adipose‐ derived stromal cells (ADSCs) in vitro. Rat ADSCs were cultured with TGF‐β1 (10 ng ml^?1) for 2 weeks in vitro. ADSCs cultured without TGF‐β1 served as a control. The mRNA expression of cardiac‐specific gene was induced by TGF‐β1, while the control culture did not show cardiac‐specific gene expression. Immunocytochemical analyses showed that a small fraction of ADSCs cultured with TGF‐β1 for 2 weeks stained positively for cardiac myosin heavy chain (MHC) and α‐sarcomeric actin. Flow cytometric analyses showed that the proportion of cells expressing cardiac MHC increased with TGF‐β1. However, no mesenchymal differentiation (e.g., osteogenic and adipogenic differentiation) was detected other than cardiomyogenic differentiation. These results showed that TGF‐β1 induce ADSC cardiomyogenic differentiation in vitro, which could be useful for myocardial infarction stem cell therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

A novel molecular mechanism of microRNA‐21 inducing pulmonary fibrosis and human pulmonary fibroblast extracellular matrix through transforming growth factor β1–mediated SMADs activation

Pulmonary fibrosis (PF), characterized by the destruction of lung tissue architecture and the abnormal deposition of extracellular matrix (ECM) proteins, currently has no satisfactory treatment. The role of microRNA (miR)‐21 in PF has been reported; the current study attempted to investigate a novel molecular mechanism by which miR‐21 exerted its function. Consistent with previous studies, miR‐21 inhibition reduced ECM protein levels in bleomycin (BLM)‐induced mouse model of PF. In human pulmonary fibroblast (IMR‐90), miR‐21 inhibition reduced transforming growth factor β1 (TGFβ1)–induced ECM protein expression. Regarding a novel molecular mechanism, TGFβ1 combined with TGFβ1 receptor 1 (TGFβ1RI) to activate SMAD2/3, promote SMAD4 nucleus transformation, and thus regulate miR‐21 expression and ECM. SMAD3 and SMADs complex could bind to the promoter region of miR‐21 to promote miR‐21 expression. In conclusion, miR‐21 exerts promotive effects on BLM‐induced PF and TGFβ1‐induced ECM in IMR‐90; TGFβ1 combines with TGFβ1RI to activate SMAD2/3, promote SMAD4 nucleus transformation, promote miR‐21 expression, and thus to promote BLM‐induced PF and TGFβ1‐induced ECM in IMR‐90 cells.     

Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) induces TGF‐β‐regulated apoptosis signal in human gingival epithelial cells via fibronectin/integrinβ1/FAK cascade

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor β (TGF‐β). However, the molecular mechanisms by which microbes induce the activation of TGF‐β remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF‐β receptor (TGF‐βR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF‐βR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF‐β by cooperating with integrin. Accordingly, we hypothesized that Omp29‐induced actin rearrangements via FAK activity would enhance the activation of TGF‐β, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF‐βR/smad2 signalling and decreased active TGF‐β protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF‐βR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinβ1 expression by siRNA transfection attenuated TGF‐βR/smad2 signalling activity and reduction of TGF‐β levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinβ1, which is associated with TGF‐β signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29‐induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinβ1/FAK signalling‐dependent TGF‐β release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF‐βR/smad2 pathway.     

The IκB kinase inhibitor ACHP strongly attenuates TGFβ1‐induced myofibroblast formation and collagen synthesis

Excessive accumulation of a collagen‐rich extracellular matrix (ECM) by myofibroblasts is a characteristic feature of fibrosis, a pathological state leading to serious organ dysfunction. Transforming growth factor beta1 (TGFβ1) is a strong inducer of myofibroblast formation and subsequent collagen production. Currently, there are no remedies for the treatment of fibrosis. Activation of the nuclear factor kappa B (NF‐κB) pathway by phosphorylating IκB with the enzyme IκB kinase (IKK) plays a major role in the induction of fibrosis. ACHP {2‐Amino‐6‐[2‐(cyclopropylmethoxy)‐6‐hydroxyphenyl]‐4‐(4‐piperidinyl)‐3 pyridinecarbonitrile}, a selective inhibitor of IKK, prohibits the activation of the NF‐κB pathway. It is not known whether ACHP has potential anti‐fibrotic properties. Using adult human dermal and lung fibroblasts we have investigated whether ACHP has the ability to inhibit the TGFβ1‐induced transition of fibroblasts into myofibroblasts and its excessive synthesis of ECM. The presence of ACHP strongly suppressed the induction of the myofibroblast markers alpha‐smooth muscle actin (αSMA) and SM22α, as well as the deposition of the ECM components collagen type I and fibronectin. Furthermore, post‐treatment with ACHP partly reversed the expression of αSMA and collagen type I production. Finally, ACHP suppressed the expression of the three collagen‐modifying enzymes lysyl hydroxylase (PLOD1, PLOD2 and PLOD3) in dermal fibroblasts, but did not do so in lung fibroblasts. We conclude that the IKK inhibitor ACHP has potent antifibrotic properties, and that the NF‐κB pathway plays an important role in myofibroblast biology.     

Loss of tenascin X gene function impairs injury‐induced stromal angiogenesis in mouse corneas

To determine the contribution by tenascin X (Tnx) gene expression to corneal stromal angiogenesis, the effects were determined of its loss on this response in TNX knockout (KO) mice. In parallel, the effects of such a loss were evaluated on vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1) gene and protein expression in fibroblasts and macrophages in cell culture. Histological, immunohistochemical and quantitative RT‐PCR changes determined if Tnx gene ablation on angiogenic gene expression, inflammatory cell infiltration and neovascularization induced by central corneal stromal cauterization. The role was determined of Tnx function in controlling VEGF‐A or TGFβ1 gene expression by comparing their expression levels in ocular fibroblasts and macrophages obtained from wild‐type (WT) and body‐wide Tnx KO mice. Tnx was up‐regulated in cauterized cornea. In Tnx KO, macrophage invasion was attenuated, VEGF‐A and its cognate receptor mRNA expression along with neovascularization were lessened in Tnx KOs relative to the changes occurring in their WT counterpart. Loss of Tnx instead up‐regulated in vivo mRNA expression of anti‐angiogenic VEGF‐B but not VEGF‐A. On the other hand, TGFβ1 mRNA expression declined in Tnx KO cultured ocular fibroblasts. Loss of Tnx gene expression caused VEGF‐A expression to decline in macrophages. Tnx gene expression contributes to promoting TGFβ1 mRNA expression in ocular fibroblasts and VEGF‐A in macrophages, macrophage invasion, up‐regulation of VEGF‐A expression and neovascularization in an injured corneal stroma. On the other hand, it suppresses anti‐angiogenic VEGF‐B mRNA expression in vivo.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.     

Interferon‐γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)‐1 and transforming growth factor (TGF) β2

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN‐α or IFN‐γ downregulated Fli1 and VE‐cadherin. In contrast, IFN‐α and IFN‐γ exerted opposite effects on the expression of α‐SMA, CTGF, ET‐1, and TGFβ2, with IFN‐α downregulating and IFN‐γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN‐γ‐mediated changes in Fli1, VE‐cadherin, CTGF, and ET‐1 levels, whereas upregulation of α‐SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN‐γ, including downregulation of α‐SMA and TGFβ2, suggesting that activation of the ET‐1 pathway plays a main role in the IFN‐γ responses in HDMECs. IFN‐γ induced expression of selected genes related to endothelial‐to‐mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN‐γ on EndoMT was mediated via TGFβ2 and ET‐1 signaling pathways. This study demonstrates distinct effects of IFN‐α and IFN‐γ on the biology of vascular endothelial cells. IFN‐γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT. J. Cell. Physiol. 228: 1774–1783, 2013. © 2013 Wiley Periodicals, Inc.     

Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation

Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.     

Using growth factor conditioning to modify the properties of human cell derived extracellular matrix

We have recently reported on a bench‐top approach for isolating extracellular matrix (ECM) from pure populations of cells grown in culture using sacrificial, open‐celled foams to concentrate and capture the ECM. To increase both the accumulation and the strength of the ECM harvested, cell‐seeded polyurethane (PU) foams were cultured in media supplemented with either transforming growth factor β‐1 (TGFβ1) or hepatocyte growth factor (HGF). At the end of a 3‐week culture period, ECM yield was significantly increased for samples conditioned in supplemented media. Control foams yielded 48 ± 12 mg of material for every gram of PU foam seeded. Yield values increased to 102 ± 21 and 243 ± 25 mg for HGF and TGFβ1‐treated samples, respectively. HGF supplementation increased the modulus by 59%, while TGFβ1 treatment increased the elastic modulus by 204%. TGFβ1‐stimulated material was organized into a network that was markedly denser than control material, with HGF‐stimulated network density intermediate to TGFβ1 and controls. Our study showed that TGFβ1‐treated samples were collagen enriched while HGF samples had an increased gylcosaminoglycan concentration. The results demonstrate that growth factor supplementation, particularly with TGFβ1, can significantly alter the biomechanical properties of cell‐derived ECM that may be used for therapeutic applications. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

TGFβ (transforming growth factor β) and keratocyte motility in 24 h zebrafish explant cultures

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFβ (transforming growth factor β) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFβ in fish keratocyte migration. Studying the involvement of TGFβ1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFβ levels increase 6.2‐fold in SFM (serum‐free medium) and 2.4‐fold in SFM+2% FBS (fetal bovine serum), while TGFβ1 and TGFβRII (TGFβ receptor II) mRNA levels increase ～3‐ and ～5‐fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFβ1 and culture media. The addition of 100 ng/ml exogenous TGFβ1 in SFM increases both measures [3.3‐fold (P=4.5 × 10^?5) and 26% (P=2.1 × 10^?2) respectively]. In contrast, 100 ng/ml of exogenous TGFβ1 in medium containing 2% FBS decreases migration distance by 2.1‐fold (P=1.7 × 10^?7), but does not affect sheet area. TGFβ1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8‐fold increase (P=1.5 × 10^?2) with 2% FBS. The variable response to TGFβ1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFβ1 and TGFβRII. Together, these results suggest that expression of TGFβ and its receptor are up‐regulated during zebrafish keratocyte explant culture and that TGFβ promotes fish keratocyte migration.     

Isolation and characterization of microsatellite DNA loci in the toad‐headed lizards,Phrynocephalus przewalskii complex

To assess mechanisms of hybridization and speciation, we isolated and characterized 12 dinucleotide microsatellite DNA loci for the toad‐headed lizards, Phrynocephalus przewalskii complex. A total of 48 specimens were examined and all loci were polymorphic with seven to 25 alleles per locus. The observed and expected heterozygosities ranged from 0.282 to 0.946 and 0.400 to 0.937, respectively. These loci are therefore suitable for a wide range of population level studies within the P. przewalskii complex.     

Regulatory mechanisms of TGF‐β1‐induced fibrogenesis of human alveolar epithelial cells

Pulmonary fibrosis is characterized by an extensive activation of fibrogenic cells and deposition of extracellular matrix (ECM). Transforming growth factor (TGF)‐β1 plays a pivotal role in the pathogenesis of pulmonary fibrosis, probably through the epithelial‐ to‐mesenchymal transition (EMT) and ECM production. The present study investigates potential mechanism by which TGF‐β1 induces EMT and ECM production in the fibrogenesis of human lung epithelial cells during pulmonary fibrosis. The expression of EMT phenotype and other proteins relevant to fibrogenesis were measured and the cell bio‐behaviours were assessed using Cell‐IQ Alive Image Monitoring System. We found that TGF‐β1‐induced EMT was accompanied with increased collagen I deposition, which may be involved in the regulation of connective tissue growth factor (CTGF) and phosphoinositide 3‐kinase (PI3K) signalling pathway. Treatment with PI3K inhibitors significantly attenuated the TGF‐β1‐ induced EMT, CTGF expression and collagen I synthesis in lung epithelial cells. The interference of CTGF expression impaired the basal and TGF‐β1‐stimulated collagen I deposition, but did not affect the process of EMT. Our data indicate that the signal pathway of TGF‐β1/PI3K/CTGF plays an important role in the fibrogenesis of human lung epithelial cells, which may be a novel therapeutic approach to prevent and treat pulmonary fibrosis.     

Possible strategies for anti-fibrotic drug intervention in scleroderma

There are no approved drugs for treating the fibrosis in scleroderma (systemic sclerosis, SSc). Myfibroblasts within connective tissue express the highly contractile protein α-smooth muscle actin (α-SMA) and are responsible for the excessive synthesis and remodeling of extracellular matrix (ECM) characterizing SSc. Drugs targeting myofibroblast differentiation, recruitment and activity are currently under consideration as anti-fibrotic treatments in SSc but thus far have principally focused on the transforming growth factor β (TGFβ), endothelin-1 (ET-1), connective tissue growth factor (CCN2/CTGF) and platelet derived growth factor (PDGF) pathways, which display substantial signaling crosstalk. Moreover, peroxisome proliferator-activated receptor (PPAR)γ also appears to act by intervening in TGFβ signaling. This review discusses these potential candidates for antifibrotic therapy in SSc.     

Inhibition of bleomycin‐induced pulmonary fibrosis by bone marrow‐derived mesenchymal stem cells might be mediated by decreasing MMP9, TIMP‐1, INF‐γ and TGF‐β

The study was aimed to investigate the mechanism and administration timing of bone marrow‐derived mesenchymal stem cells (BMSCs) in bleomycin (BLM)‐induced pulmonary fibrosis mice. Thirty‐six mice were divided into six groups: control group (saline), model group (intratracheal administration of BLM), day 1, day 3 and day 6 BMSCs treatment groups and hormone group (hydrocortisone after BLM treatment). BMSCs treatment groups received BMSCs at day 1, 3 or 6 following BLM treatment, respectively. Haematoxylin and eosin and Masson staining were conducted to measure lung injury and fibrosis, respectively. Matrix metalloproteinase (MMP9), tissue inhibitor of metalloproteinase‐1 (TIMP‐1), γ‐interferon (INF‐γ) and transforming growth factor β1 (TGF‐β) were detected in both lung tissue and serum. Histologically, the model group had pronounced lung injury, increased inflammatory cells and collagenous fibres and up‐regulated MMP9, TIMP‐1, INF‐γ and TGF‐β compared with control group. The histological appearance of lung inflammation and fibrosis and elevation of these parameters were inhibited in BMSCs treatment groups, among which, day 3 and day 6 treatment groups had less inflammatory cells and collagenous fibres than day 1 treatment group. BMSCs might suppress lung fibrosis and inflammation through down‐regulating MMP9, TIMP‐1, INF‐γ and TGF‐β. Delayed BMSCs treatment might exhibit a better therapeutic effect. Copyright © 2015 John Wiley & Sons, Ltd. Highlights are as follows:

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Increased circulating Th17 cells and elevated serum levels of TGF‐beta and IL‐21 are correlated with human non‐segmental vitiligo development

Although non‐segmental vitiligo (NSV) results from the autoimmune destruction of melanocytes, the detailed immune mechanisms have not yet been fully elucidated. Th17 cells have been identified to be implicated in human autoimmune diseases. In this study, the frequencies of peripheral blood Th17 cells and serum levels of IL‐17A and Th17 cell‐related cytokines were examined in 45 patients with active NSV compared to 45 race‐, gender‐, and age‐matched healthy controls. Our results showed increased circulating Th17 cell frequencies and elevated serum IL‐17A, TGF‐β1, and IL‐21 levels in patients with NSV. Meanwhile, the increased Th17 cell frequencies are positively correlated with serum TGF‐β1 level, and the body surface area of lesions is positively correlated with elevated TGF‐β1 and IL‐21 levels and Th17 cell frequencies. Furthermore, positive correlation was identified between Th17 and Th1 cell frequencies in patients with NSV. These results further indicate the potential involvement of Th17 cells and the collaborative contribution of Th17 and Th1 in NSV development, and suggest that the elevated serum TGF‐β1 and IL‐21 levels could contribute to enhanced Th17 cell differentiation in NSV.