Ikko Kajihara

Impaired IL-17 Signaling Pathway Contributes to the Increased Collagen Expression in Scleroderma Fibroblasts

Among IL-17 families, IL-17A and IL-17F share amino acid sequence similarity and bind to IL-17R type A. IL-17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-17A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-17F levels did not increase. In contrast, the expression of IL-17R type A in SSc fibroblasts significantly decreased in comparison with that in normal fibroblasts, due to the intrinsic TGF-β1 activation in these cell types. Moreover, IL-17A, not IL-17F, reduced the protein expression of α1(I) collagen and connective tissue growth factor. miR-129-5p, one of the downregulated microRNAs in SSc fibroblasts, increased due to IL-17A and mediated the α1(I) collagen reduction. These results suggest that IL-17A signaling, not IL-17F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue growth factor and α1(I) collagen. IL-17A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-β1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-β and IL-17A may lead to a new therapeutic approach for this disease.

TGF-β–Mediated Downregulation of MicroRNA-196a Contributes to the Constitutive Upregulated Type I Collagen Expression in Scleroderma Dermal Fibroblasts

Previous reports indicated the significance of the TGF-β signaling in the pathogenesis of systemic sclerosis. We tried to evaluate the possibility that microRNAs (miRNAs) play a part in the type I collagen upregulation seen in normal fibroblasts stimulated with exogenous TGF-β and systemic sclerosis (SSc) fibroblasts. miRNA expression profile was evaluated by miRNA PCR array and real-time PCR. The protein expression of type I collagen was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. Several miRNAs were found to be downregulated in both TGF-β–stimulated normal fibroblasts and SSc fibroblasts compared with normal fibroblasts by PCR array. Among them, miR-196a expression was decreased in SSc both in vivo and in vitro by real-time PCR or in situ hybridization. In SSc fibroblasts, miR-196a expression was normalized by TGF-β small interfering RNA. miR-196a inhibitor leads to the overexpression of type I collagen in normal fibroblasts, whereas overexpression of the miRNA resulted in the downregulation of type I collagen in SSc fibroblasts. In addition, miR-196a was detectable and quantitative in the serum of SSc patients. Patients with lower serum miR-196a levels had significantly higher ratio of diffuse cutaneous SSc:limited cutaneous SSc, higher modified Rodnan total skin thickness score, and higher prevalence of pitting scars than those without. miR-196a may play some roles in the pathogenesis of SSc. Investigation of the regulatory mechanisms of type I collagen expression by miR-196a may lead to new treatments using miRNA.

The Downregulation of microRNA let-7a Contributes to the Excessive Expression of Type I Collagen in Systemic and Localized Scleroderma

Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3′-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

microRNA-92a expression in the sera and dermal fibroblasts increases in patients with scleroderma

OBJECTIVES microRNAs (miRNAs) play a part in various cellular activities. However, the role of miRNA in SSc is not fully understood. This study investigated the expression and role of miR-92a in SSc patients and evaluated the possibility that miR-92a is involved in the pathogenesis of this disease. METHODS Serum samples were obtained from 61 SSc patients. mRNAs were purified from serum and levels of miR-92a and miR-135 were measured with quantitative real-time PCR. miR-92a expression in dermal fibroblasts was also determined by quantitative real-time PCR. Immunoblotting was performed to detect MMP-1 protein. RESULTS The median serum levels of miR-92a, not miR-135, were significantly higher in SSc patients than normal subjects. The constitutive up-regulated miR-92a expression was also found in cultured dermal fibroblasts from SSc skin, which was decreased by the transfection with siRNA of TGF-β. Furthermore, the forced overexpression of miR-92a in normal dermal fibroblasts using miR-92a mimic resulted in the down-regulation of MMP-1 expression. CONCLUSION The increase of miR-92a in SSc may be due to the stimulation of intrinsic TGF-β activation seen in this disease. There is also a possibility that MMP-1 is the target of miR-92a and that increased miR-92a expression therefore plays a role in excessive collagen accumulation in SSc via the down-regulation of MMP-1. Clarifying the role of miRNAs in SSc may result in a better understanding of this disease and the development of new therapeutic approaches.

microRNA-7 down-regulation mediates excessive collagen expression in localized scleroderma.

Localized scleroderma (LSc), a connective tissue disorder restricted to the skin and subcutaneous tissue, is characterized by skin fibrosis due to an excessive deposition of types I collagen. The mechanism of such fibrosis is still unknown, but epigenetics may play some roles in the excessive collagen expression. In the present study, we investigated the mechanism of fibrosis seen in LSc, focusing on microRNA (miRNA). miRNA expression was determined by PCR array, real-time PCR, and in situ hybridization. The function of miRNA was evaluated using specific inhibitor. Immunoblotting was performed to detect α2(I) collagen protein. PCR array analysis using tissue miRNA demonstrated miR-7 level was significantly decreased in LSc skin as well as keloid tissue compared to normal skin in vivo. In situ hybridization also showed miR-7 expression in dermal fibroblasts was decreased in LSc dermis. The transfection of specific inhibitor for miR-7 into cultured normal dermal fibroblasts resulted in the up-regulation of α2(I) collagen protein in vitro. Also, the serum levels of miR-7 were significantly decreased in LSc patients compared with healthy controls, but serum miR-29a levels not. Systemic or local down-regulation of miR-7 may contribute to the pathogenesis of LSc via the overexpression of α2(I) collagen, and serum miR-7 may be useful as a disease marker. Investigation of the regulatory mechanisms of LSc by miRNA may lead to new treatments by the transfection into the lesional skin of this disease.

Increased accumulation of extracellular thrombospondin-2 due to low degradation activity stimulates type i collagen expression in scleroderma fibroblasts

The aim of the present study was to determine the expression and role of thrombospondin-2 (TSP-2) in systemic sclerosis (SSc). Both TSP-2 mRNA levels and protein synthesis in cell lysates were significantly lower in cultured SSc fibroblasts than in normal fibroblasts; however, the TSP-2 protein that accumulated in the conditioned medium of SSc fibroblasts was up-regulated, compared with that of normal fibroblasts, because of an increase in the half-life of the protein. In vivo serum TSP-2 levels were higher in SSc patients than in healthy control subjects, and SSc patients with elevated serum TSP-2 levels tended to have pitting scars and/or ulcers. TSP-2 knockdown resulted in the down-regulation of type I collagen expression and the up-regulation of miR-7, one of the miRNAs with an inhibitory effect on collagen expression. Expression levels of miR-7 were also up-regulated in SSc dermal fibroblasts both in vivo and in vitro. Taken together, these findings suggest that the increased extracellular TSP-2 deposition in SSc fibroblasts may contribute to tissue fibrosis by inducing collagen expression. Down-regulation of intracellular TSP-2 synthesis and the subsequent miR-7 up-regulation in SSc fibroblasts may be due to a negative feedback mechanism that prevents increased extracellular TSP-2 deposition and/or tissue fibrosis. Thus, TSP-2 may play an important role in the maintenance of fibrosis and angiopathy in patients with SSc.

Detection of hair-microRNAs as the novel potent biomarker: Evaluation of the usefulness for the diagnosis of scleroderma

BACKGROUND Serum microRNA levels are known as useful biomarkers for various diseases. Recent publication has indicated the existence of microRNAs in hair roots and hair shafts. OBJECTIVE In this study, we evaluated several methods for the extraction of hair microRNAs, and their usefulness for the diagnosis of scleroderma. METHODS A single hair root and 5 pieces of hair shafts were obtained from the occiput of each individual of 11 scleroderma patients and 13 normal subjects at the time of serum sampling. microRNA extraction from sera or hair roots was performed with commercially available kits. microRNAs were extracted from hair shafts using four different methods. microRNA expression was evaluated by PCR array and real-time PCR. RESULTS We demonstrated microRNAs in hair roots and hair shafts were detectable and quantitative using our method. We found the difference of microRNA levels in hair roots and hair shafts obtained from different places of head in each individual were within 2-fold, indicating the reproducibility of hair microRNA levels by our method. PCR array revealed microRNAs from sera, hair roots and hair shafts have different expression pattern, and can be independent biomarkers. Serum and hair root miR-196a levels were not significantly changed in scleroderma patients, while we found miR-196a levels in hair shafts were significantly decreased in scleroderma patients compared to those in normal subjects (p<0.05). CONCLUSION Hairs are more accessible than sera among human samples. microRNAs levels in hair roots or hair shafts may become effective and independent biomarkers.

Adiponectin expression is decreased in the involved skin and sera of diffuse cutaneous scleroderma patients

Abstract:  In this study, we determined the adiponectin expression in the serum and lesional skin of patients with scleroderma (SSc). Serum adiponectin concentrations were measured in 32 patients with SSc, 10 patients with SLE, 12 patients with dermatomyositis patients and 13 healthy subjects with specific enzyme‐linked immunosorbent assays. Adiponectin mRNA was determined in skin tissues of five patients with diffuse cutaneous SSc (dcSSc), seven patients with limited cutaneous SSc (lcSSc) and seven healthy subjects with real‐time polymerase chain reaction. There was a significant reduction in serum adiponectin levels in patients with dcSSc. SSc patients with decreased serum adiponectin levels had higher total skin thickness score and higher incidence of pulmonary fibrosis. Adiponectin mRNA levels in skin tissues from patients with dcSSc were also reduced. Serum adiponectin levels may be a useful biomarker for fibrotic condition in patients with SSc. Clarifying the role of adiponectin in collagen diseases may lead to further understanding of the pathogenesis and new therapeutic approach.

Discoidin Domain Receptor 2–microRNA 196a–Mediated Negative Feedback against Excess Type I Collagen Expression Is Impaired in Scleroderma Dermal Fibroblasts

Systemic sclerosis (SSc) is characterized by excess collagen deposition in the skin, due to intrinsic transforming growth factor-β (TGF-β) activation. We tried to determine the expression and the role of discoidin domain receptor 2 (DDR2) in SSc. The expression of DDR2 mRNA and protein was significantly decreased in SSc dermal fibroblasts, which was recovered by knocking down TGF-β. The knockdown of DDR2 in normal fibroblasts induced microRNA-196a expression, which led to type I collagen downregulation, indicating that DDR2 itself has a negative effect on microRNA-196a expression and inducible effect on collagen expression. In SSc fibroblasts, however, the DDR2 knockdown did not affect TGF-β signaling and microRNA-196a expression. The microRNA-196a levels were significantly decreased in normal fibroblasts treated with TGF-β and in SSc fibroblasts. Taken together our data indicate that, in SSc fibroblasts, intrinsic TGF-β stimulation induces type I collagen expression, and also downregulates DDR2 expression. This probably acts as a negative feedback mechanism against excess collagen expression, as a decreased DDR2 expression is supposed to stimulate the microRNA-196a expression and further change the collagen expression. However, in SSc fibroblasts the microRNA-196a expression was downregulated by TGF-β signaling. DDR2-microRNA-196a pathway may be a previously unreported negative feedback system, and its impairment may be involved in the pathogenesis of SSc.

Serum levels of soluble CD163 in patients with systemic sclerosis

Macrophages may play a role in the pathogenesis of systemic sclerosis (SSc), and CD163-positive M2 macrophages are potentially important source for fibrosis-inducing cytokines. However, no link between M2 macrophages and SSc has been established. The aim is to evaluate the possibility that serum levels of soluble CD163 (sCD163) can be a useful marker for SSc, reflecting M2 activation of macrophages in this disease. Serum sCD163 levels of 43 patients with SSc, 10 patients with scleroderma spectrum disorder (SSD), and 12 healthy controls were measured with specific enzyme-linked immunosorbent assays. SSc patients had significantly higher serum sCD163 levels than healthy controls. The sCD163 levels in SSD patients were higher than healthy controls and lower than SSc patients. Significantly higher right ventricular systolic pressure and lower % DLco levels, and shorter duration of disease were seen in SSc patients with elevated serum sCD163 levels than those with normal levels. These results suggest that sCD163 levels may be increased in proportion to the progression of this disease, indicating the involvement of CD163 in the pathogenesis of SSc. Furthermore, serum sCD163 levels may be a marker of pulmonary hypertension at the early stage in patients with SSc.