Scleroderma Unit, Department of Medicine, Surgery and Neurosciences, University of Siena, Italy.
Medical Genetics, Department of Medical Biotechnologies, University of Siena, Italy.
Clin Exp Rheumatol. 2019 Jul-Aug;37 Suppl 119(4):69-75. Epub 2019 Jul 19.
Relaxin is a potent anti-fibrotic hormone that has been tested to ameliorate fibrosis in systemic sclerosis (SSc), but with controversial results. The aim of the study is to sequence relaxin receptor gene RXFP1 and to assess its mRNA expression and protein levels in the skin of SSc patients and healthy subjects.
Fibroblasts were isolated from unaffected/affected skin samples of (n=16) limited-cutaneous-SSc-(LcSSc) and from affected ones of (n=4) diffuse-cutaneous-SSc-(DcSSc) patients. Fibroblasts from healthy subjects were used as controls. Sequencing of exonic target regions of interest for RXFP1 gene was performed, coupled with mRNA transcript variant analysis. RXFP1 mRNA and protein levels were assessed by quantitative-real-time-PCR-(qRT-PCR) and by immunocytochemistry-(ICC). Alpha-smooth-muscle-actin-(α-SMA) synthesis induced by transforming-growth-factor-beta-1-(TGF-β1) stimulation was investigated in all fibroblasts with and without pre-treatment with serelaxin (a recombinant form of human relaxin-2 targeting the receptor RXFP1).
Sequencing of RXFP1 gene showed no relevant mutations in all fibroblast populations. The analysis of mRNA transcripts revealed the presence of 13 different mRNA isoforms of RXFP1 (7 coding and 6 non-coding) upregulated in LcSSc/DcSSc-affected samples and not in LcSSc-unaffected and in healthy ones. On the contrary, ICC demonstrated the absence of RXFP1 in LcSSc/DcSSc-affected fibroblasts and the presence in LcSSc-unaffected and in healthy ones. To prove these findings, serelaxin pre-incubation was unable to counteract TGF-β1-driven upregulation of α-SMA in LcSSc/DcSSc-affected fibroblasts only, but not in LcSSc-unaffected and healthy ones.
The absence/altered expression of relaxin receptor RXFP1 in the affected fibroblasts of SSc patients could explain the inefficacy of relaxin-based anti-fibrotic treatments in the disease.
松弛素是一种有效的抗纤维化激素，已被证实可改善系统性硬皮病（SSc）的纤维化，但结果存在争议。本研究旨在对松弛素受体基因 RXFP1 进行测序，并评估其在 SSc 患者和健康受试者皮肤中的 mRNA 表达和蛋白水平。
从局限性硬皮病（LcSSc）患者未受累/受累皮肤样本（n=16）和弥漫性硬皮病（DcSSc）患者受累皮肤样本（n=4）中分离成纤维细胞。健康受试者的成纤维细胞用作对照。对 RXFP1 基因的外显子靶区域进行测序，同时进行 mRNA 转录变体分析。通过定量实时-PCR（qRT-PCR）和免疫细胞化学（ICC）评估 RXFP1 mRNA 和蛋白水平。在所有成纤维细胞中，通过转化生长因子-β1（TGF-β1）刺激诱导α-平滑肌肌动蛋白（α-SMA）合成，并在有无预先用 serelaxin（一种针对受体 RXFP1 的人松弛素-2 重组形式）处理的情况下进行研究。
对 RXFP1 基因进行测序未发现所有成纤维细胞群体中存在相关突变。mRNA 转录本分析显示，LcSSc/DcSSc 受累样本中存在 13 种不同的 RXFP1 mRNA 异构体（7 种编码和 6 种非编码），而 LcSSc 未受累和健康样本中不存在。相反，ICC 显示 LcSSc/DcSSc 受累成纤维细胞中不存在 RXFP1，而 LcSSc 未受累和健康成纤维细胞中存在。为了证明这些发现，只有在 LcSSc/DcSSc 受累成纤维细胞中，松弛素预孵育不能拮抗 TGF-β1 驱动的 α-SMA 上调，但在 LcSSc 未受累和健康成纤维细胞中则不能。
SSc 患者受累成纤维细胞中松弛素受体 RXFP1 的缺失/表达改变可能解释了基于松弛素的抗纤维化治疗在该疾病中无效的原因。
