Adiponectin Is Involved in Connective Tissue Growth Factor-Induced Proliferation, Migration and Overproduction of the Extracellular Matrix in Keloid Fibroblasts.
Adiponectin Is Involved in Connective Tissue Growth Factor-Induced Proliferation, Migration and Overproduction of the Extracellular Matrix in Keloid Fibroblasts.
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DOI：
10.3390/ijms18051044
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发表时间：
2017-05-12
影响因子：
5.6
通讯作者：
He C
中科院分区：
文献类型：
--
作者：
Luo L;Li J;Liu H;Jian X;Zou Q;Zhao Q;Le Q;Chen H;Gao X;He C
脂联素是一种脂肪细胞源性激素，在代谢、炎症、血管稳态、细胞凋亡和免疫等方面具有多种生物学效应。最近，脂联素已被建议，以减轻人类皮肤纤维化的进展。结缔组织生长因子（CTGF）在瘢痕疙瘩中被诱导，并被认为参与瘢痕疙瘩纤维化的形成。然而，脂联素在瘢痕疙瘩中的作用尚不清楚。在这项研究中，我们探讨了脂联素对结缔组织生长因子诱导的细胞增殖，迁移和细胞外基质（ECM）的沉积及其相关的细胞内信号通路在瘢痕疙瘩成纤维细胞（KFs）的影响。我们还探讨了瘢痕疙瘩发病的可能机制。从瘢痕疙瘩患者的包皮活检和皮肤活检建立原代成纤维细胞培养物。通过逆转录-PCR（RT-PCR）、实时定量RT-PCR、免疫荧光染色和免疫组织化学分析来评估脂联素和脂联素受体（adipoRs）的表达。接下来，在存在或不存在脂联素的情况下，用CTGF处理KFs和正常真皮成纤维细胞（NF）。采用CCK-8细胞计数试剂盒和Transwell法检测细胞增殖和迁移。采用实时荧光定量RT-PCR和蛋白质印迹法检测I型胶原、纤维连接蛋白（FN）和α-平滑肌肌动蛋白（α-SMA）mRNA和蛋白质水平。检测针对adipoR基因的RNA干扰（RNAi）效果。Western blotting检测5′-磷酸腺苷（AMP）激活的蛋白激酶（AMPK）、丝裂原激活的蛋白激酶（MAPK）和磷脂酰肌醇3激酶-蛋白激酶（PI 3 K-Akt）的磷酸化水平，进一步研究其信号通路。此外，信号转导通路的抑制剂进行了研究。瘢痕疙瘩中脂联素和adipoRs的表达水平较正常皮肤组织明显降低。脂联素抑制CTGF诱导的KFs，但不抑制NF、增殖、迁移和ECM的产生。此外，脂联素抑制AMPK，p38和细胞外调节激酶（ERK）的磷酸化，但不是Jun N-末端激酶（JNK）或Akt，在CTGF处理的KFs。脂联素介导的信号传导途径的活性被靶向adipoR 1的小干扰RNA（siRNA）（但不靶向adipoR 2、T-钙粘蛋白或钙网蛋白的siRNA）、AMPK（化合物C）、p38（SB 203580）抑制剂和促分裂原活化蛋白激酶激酶（MEK）抑制剂（PD 98059）减弱。基于我们的研究结果，脂联素抑制CTGF诱导的KFs增殖、迁移和ECM过度产生。其中一个潜在的机制是adipoR 1、AMPK、p38和ERK信号通路的激活。因此，脂联素可能在瘢痕疙瘩的发生发展中起重要作用，提示其可能成为瘢痕疙瘩治疗的新靶点。
Adiponectin, an adipocyte-derived hormone, exerts pleiotropic biological effects on metabolism, inflammation, vascular homeostasis, apoptosis and immunity. Recently, adiponectin has been suggested to attenuate the progression of human dermal fibrosis. Connective tissue growth factor (CTGF) is induced in keloids and is thought to be participated in the formation of keloid fibrosis. However, the roles played by adiponectin in keloids remain unclear. In this study, we explored the effects of adiponectin on CTGF-induced cell proliferation, migration and the deposition of extracellular matrix (ECM) and their associated intracellular signalling pathways in keloid fibroblasts (KFs). We also explored possible mechanisms of keloid pathogenesis. Primary fibroblast cultures were established from foreskin biopsies and skin biopsies from patients with keloids. The expression of adiponectin and adiponectin receptors (adipoRs) was evaluated by reverse transcription-PCR (RT-PCR), quantitative real-time RT-PCR, immunofluorescence staining, and immunohistochemical analysis. Next, KFs and normal dermal fibroblasts (NFs) were treated with CTGF in the presence or absence of adiponectin. A cell counting kit-8 (CCK-8) and the Transwell assay were used to examine cell proliferation and migration. The level of the collagen I, fibronectin (FN) and α-smooth muscle actin (α-SMA) mRNAs and proteins were determined by quantitative real-time RT-PCR and western blotting. The effects of RNA interference (RNAi) targeting the adipoR genes were detected. Phosphorylation of adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase-protein kinase (PI3K-Akt) were examined by western blotting to further investigate the signalling pathways. Furthermore, inhibitors of signal transduction pathways were investigated. The expression levels of adiponectin and adipoRs were significantly decreased in keloids compared with those in normal skin tissue. Adiponectin suppressed the CTGF-induced KFs, but not NFs, proliferation, migration and ECM production. Moreover, adiponectin inhibited the phosphorylation of AMPK, p38 and extracellular-regulated kinase (ERK), but not that of Jun N-terminal kinase (JNK) or Akt, in CTGF-treated KFs. The activity of adiponectin-mediated signalling pathways was attenuated by small interfering RNAs (siRNAs) targeting adipoR1 (but not siRNAs targeting adipoR2, T-cadherin or calreticulin), AMPK (Compound C), p38 (SB203580) inhibitors, and mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059). Based on our results, adiponectin suppresses CTGF-induced KFs proliferation, migration and ECM overproduction. One of the underlying mechanisms is the activation of the adipoR1, AMPK, p38, and ERK signalling pathways. Therefore, adiponectin may play an important role in the progression of keloids, suggesting a potential novel target for keloid treatment.