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Transforming growth factor (TGF)-β is stored in the extracellular matrix as a latent complex with its prodomain. Activation of TGF-β1 requires the binding of α v integrin to an RGD sequence in the prodomain and exertion of force on this domain, which is held in the extracellular matrix by latent TGF-β binding proteins. Crystals of dimeric porcine proTGF-β1 reveal a ring-shaped complex, a novel fold for the prodomain, and show how the prodomain shields the growth factor from recognition by receptors and alters its conformation. Complex formation between α v β 6 integrin and the prodomain is insufficient for TGF-β1 release. Force-dependent activation requires unfastening of a ‘straitjacket’ that encircles each growth-factor monomer at a position that can be locked by a disulphide bond. Sequences of all 33 TGF-β family members indicate a similar prodomain fold. The structure provides insights into the regulation of a family of growth and differentiation factors of fundamental importance in morphogenesis and homeostasis. Key growth factor structure determined Members of the transforming growth factor-β (TGF-β) superfamily of growth factors are of fundamental importance in development and tissue homeostasis. TGF-β is secreted as an inactive complex that has to be activated to enable receptor binding and activation of downstream signalling events. Cell surface adhesion receptors of the integrin family are essential for the activation of TGF-β. Shi et al . present the structure of latent TGF-β and provide mechanistic insights into latency and force-dependent activation by integrins.

Retinitis pigmentosa (RP) is a genetically heterogenous group of eye diseases in which initial degeneration of rods triggers secondary degeneration of cones, leading to significant loss of daylight, color, and high-acuity vision. Gene complementation with adeno-associated viral (AAV) vectors is one strategy to treat RP. Its implementation faces substantial challenges, however; for example, the tremendous number of loci with causal mutations. Gene therapy targeting secondary cone degeneration is an alternative approach that could provide a much-needed generic treatment for many patients with RP. Here, we show that microglia are required for the upregulation of potentially neurotoxic inflammatory factors during cone degeneration in RP, creating conditions that might contribute to cone dysfunction and death. To ameliorate the effects of such factors, we used AAV vectors to express isoforms of the antiinflammatory cytokine transforming growth factor beta (TGF-β). AAV-mediated delivery of TGF-β1 rescued degenerating cones in 3 mouse models of RP carrying different pathogenic mutations. Treatment with TGF-β1 protected vision, as measured by 2 behavioral assays, and could be pharmacologically disrupted by either depleting microglia or blocking the TGF-β receptors. Our results suggest that TGF-β1 may be broadly beneficial for patients with cone degeneration, and potentially other forms of neurodegeneration, through a pathway dependent upon microglia.

Interleukin-6 (IL-6) is an important cytokine participating in multiple biologic activities in immune regulation and inflammation. IL-6 has been associated with cardiovascular remodeling. However, the mechanism of IL-6 in hypertensive cardiac fibrosis is still unclear. Angiotensin II (Ang II) infusion in mice increased IL-6 expression in the heart. IL-6 knockout (IL-6-/-) reduced Ang II-induced cardiac fibrosis: 1) Masson trichrome staining showed that Ang II infusion significantly increased fibrotic areas of the wild-type mouse heart, which was greatly suppressed in IL-6-/- mice and 2) immunohistochemistry staining showed decreased expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I in IL-6-/- mouse heart. The baseline mRNA expression of IL-6 in cardiac fibroblasts was low and was absent in cardiomyocytes or macrophages; however, co-culture of cardiac fibroblasts with macrophages significantly increased IL-6 production and expression of α-SMA and collagen I in fibroblasts. Moreover, TGF-β1 expression and phosphorylation of TGF-β downstream signal Smad3 was stimulated by co-culture of macrophages with cardiac fibroblasts, while IL-6 neutralizing antibody decreased TGF-β1 expression and Smad3 phosphorylation in co-culture of macrophage and fibroblast. Taken together, our results indicate that macrophages stimulate cardiac fibroblasts to produce IL-6, which leads to TGF-β1 production and Smad3 phosphorylation in cardiac fibroblasts and thus stimulates cardiac fibrosis.

Uremic toxins are considered to have a determinant pathological role in the progression of chronic kidney disease. The aim of this study was to define the putative pathological roles of the renal renin-angiotensin-aldosterone system (RAAS) and renal tubular epithelial-to-mesenchymal transition (EMT) in kidney fibrosis induced by (indoxyl sulfate) IS and (p-cresol sulfate) PCS. Mouse proximal renal tubular cells (PKSV-PRs) treated with IS or PCS were used. Half-nephrectomized B-6 mice were treated with IS or PCS for 4 weeks. In the losartan treatment study, the study animal was administrated with IS+losartan or PCS+losartan for 4 weeks. IS and PCS significantly activated the intrarenal RAAS by increasing renin, angiotensinogen, and angiotensin 1 (AT1) receptor expression, and decreasing AT2 receptor expression in vitro and in vivo. IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression. The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. IS and PCS induced the phenotype of EMT-like transition in renal tubules by increasing the expression of fibronectin and α-smooth muscle actin and decreasing the expression of E-cadherin. Losartan significantly attenuated the expression of TGF-β1 and Snail, and decreased kidney fibrosis induced by IS and PCS in vivo. Activating the renal RAAS/TGF-β pathway has an important pathological role in chronic kidney injury caused by IS and PCS. IS and PCS may increase Snail expression and induce EMT-like transition.

Background Cigarette smoking contributes to lung remodelling in chronic obstructive pulmonary disease (COPD). As part of remodelling, peribronchiolar fibrosis is observed in the small airways of patients with COPD and contributes to airway obstruction. Epithelial to mesenchymal transition (EMT) appears to be involved in the formation of peribronchiolar fibrosis. This study examines the EMT process in human bronchial epithelial cells (HBECs) from non-smokers, smokers and patients with COPD as well as the in vitro effect of cigarette smoke extract (CSE) on EMT. Methods HBECs from non-smokers (n=5), smokers (n=12) and patients with COPD (n=15) were collected to measure the mesenchymal markers α-smooth muscle actin, vimentin and collagen type I and the epithelial markers E-cadherin, ZO-1 and cytokeratin 5 and 18 by real time-PCR and protein array. In vitro differentiated bronchial epithelial cells were stimulated with CSE. Results Mesenchymal markers were upregulated in HBECs of smokers and patients with COPD compared with non-smokers. In contrast, epithelial cell markers were downregulated. In vitro differentiated HBECs underwent EMT after 72 h of CSE exposure through the activation of intracellular reactive oxygen species, the release and autocrine action of transforming growth factor β1, the phosphorylation of ERK1/2 and Smad3 and by the downregulation of cyclic monophosphate. Conclusions The EMT process is present in bronchial epithelial cells of the small bronchi of smokers and patients with COPD and is activated by cigarette smoke in vitro.

Oro-facial fibrosis in systemic sclerosis (Scleroderma;SSc) has a major impact on mouth function, facial appearance, and patient quality of life. Lipotransfer is a method of reconstruction that can be used in the treatment of oro-facial fibrosis. The effect of this treatment not only restores oro-facial volume but has also been found to reverse the effects of oro-facial fibrosis. Adipose derived stem cells (ADSCs) within the engrafted adipose tissue have been shown to be anti-fibrotic in SSc and are proposed as the mechanism of the anti-fibrotic effect of lipotransfer. A cohort of 62 SSc patients with oro-facial fibrosis were assessed before and after stem cell enriched lipotransfer treatment. Clinical evaluation included assessment of mouth function using a validated assessment tool (Mouth Handicap in Systemic Sclerosis Scale-MHISS), validated psychological measurements and pre and post-operative volumetric assessment. In addition, to understand the mechanism by which the anti-fibrotic effect of ADSCs occur, SSc derived fibroblasts and ADSCs from this cohort of patients were co-cultured in direct and indirect culture systems and compared to monoculture controls. Cell viability, DNA content, protein secretion of known fibrotic mediators including growth factor- β1 (TGF β-1) and connective tissue growth factor (CTGF) using ELISA analysis and fibrosis gene expression using a fibrosis pathway specific qPCR array were evaluated. Mouth function (MHISS) was significantly improved (6.85±5.07) (p<0.0001) after treatment. All psychological measures were significantly improved: DAS 24 (12.1±9.5) (p<0.0001); HADS-anxiety (2.8±3.2) (p<0.0001), HADS-depression (2.0±3.1) (p<0.0001); BFNE (2.9 ± 4.3) (p<0.0001); VAS (3.56±4.1) (p<0.0001). Multiple treatments further improved mouth function (p<0.05), DAS (p<0.0001) and VAS (p = 0.01) scores. SSc fibroblast viability and proliferation was significantly reduced in co-culture compared to monoculture via a paracrine effect over 14 days (p < 0.0001). Protein secretion of transforming growth factor (TGF-β1) and connective tissue growth factor (CTGF) was significantly reduced in co-culture compared to monoculture (p < 0.0001). Multiple fibrosis associated genes were down regulated in SSc co-culture compared to monoculture after 14 days including Matrix metalloproteinase-8 (MMMP-8), Platelet derived growth factor-β (PDGF-β) and Integrin Subunit Beta 6 (ITG-β6). Autologous stem cell enriched lipotransfer significantly improved the effects of oro-facial fibrosis in SSc in this open cohort study. Lipotransfer may reduce dermal fibrosis through the suppression of fibroblast proliferation and key regulators of fibrogenesis including TG-β1 and CTGF. Our findings warrant further investigation in a randomised controlled trial.

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin’s pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor β family (TGFβ) expression showed significantly higher levels of Tgfβ3 transcript between nude and Balb/c mice but no differences were detected for Tgfβ1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFβ3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFβ3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.

Background Comparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors. Preliminary densitometry analysis of laminin-1, α-smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts. Methods In vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored. Results While normal fibroblasts produced components of interstitial matrix and TGF-β1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α6β4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells. Conclusions Our results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix.

Studies have demonstrated low expression of miR-145 associated with cell proliferation and migration in a wide variety of tumors. Here, we studied the expression of miR-145 in relation to the occurrence and development of breast cancer. Total RNA from breast cancer tissue and corresponding adjacent normal tissue was extracted and used to detect miR-145 expression by quantitative real-time polymerase chain reaction (qRT-PCR). We also transfected breast cancer cells with hsa-miR-145 mimics, hsa-miR-145 inhibitor, mimics negative control (mimics NC) or inhibitor negative control (inhibitor NC). Cell proliferation was analyzed by colony formation assays and methyl thiazolyl tetrazolium assays. Cell proliferation in breast cancer cells was decreased after overexpression of miR-145 and increased following miR-145 suppression. Cell migration and invasion were assessed using Transwell and wound healing assays, respectively, and were also decreased after overexpression of miR-145 and increased after miR-145 suppression in breast cancer cells. Finally, western blot assays showed that overexpression of miR-145 inhibited expression of transforming growth factor-β1 (TGF-β1). Collectively, these data suggest that miR-145 may inhibit TGF-β1 protein expression which may in turn contribute to tumor formation.

Histone deacetylase (HDAC) inhibitors are promising anti-fibrosis drugs; however, nonselective inhibition of class I and class II HDACs does not allow a detailed elucidation of the individual HDAC functions in renal fibrosis. In this study, we investigated the effect of MS-275, a selective class I HDAC inhibitor, on the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO) and activation of cultured renal interstitial fibroblasts. The UUO model was established by ligation of the left ureter and the contralateral kidney was used as a control. At seven days after UUO injury, kidney developed fibrosis as indicated by deposition of collagen fibrils and increased expression of collagen I, fibronectin and alpha-smooth muscle actin (alpha-SMA). Administration of MS-275 inhibited all these fibrotic responses and suppressed UUO-induced production of transforming growth factor-beta1 (TGF-beta), increased expression of TGF-beta receptor I, and phosphorylation of Smad-3. MS-275 was also effective in suppressing phosphorylation and expression of epidermal growth factor receptor (EGFR) and its downstream signaling molecule, signal transducer and activator of transcription-3. Moreover, class I HDAC inhibition reduced the number of renal tubular cells arrested in the G2/M phase of the cell cycle, a cellular event associated with TGF-beta1overproduction. In cultured renal interstitial fibroblasts, MS-275 treatment inhibited TGF-beta induced phosphorylation of Smad-3, differentiation of renal fibroblasts to myofibroblasts and proliferation of myofibroblasts. These results demonstrate that class I HDACs are critically involved in renal fibrogenesis and renal fibroblast activation through modulating TGF-beta and EGFR signaling and suggest that blockade of class I HDAC may be a useful treatment for renal fibrosis.