Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States of America.
Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States of America; Center of Excellence for Inflammation, Infectious Disease and Immunity, East Tennessee State University, Johnson City, TN 37614, United States of America.
Life Sci. 2018 Oct 15;211:8-16. doi: 10.1016/j.lfs.2018.09.012. Epub 2018 Sep 5.
β-adrenergic receptor (β-AR) stimulation increases extracellular levels of ubiquitin (UB), and exogenous UB plays an important role in β-AR-stimulated myocardial remodeling with effects on heart function, fibrosis and myocyte apoptosis. Cardiac fibroblasts are vital for maintaining the normal function of the heart, and in the structural remodeling of the heart in response to injury. Here we hypothesized that extracellular UB modulates cardiac fibroblast phenotype and function via its interaction with CXC chemokine receptor type 4 (CXCR4).
Serum starved adult cardiac fibroblasts were used to identify CXCR4 as a receptor for UB. Fluorescent microscopy, co-immunoprecipitation, western blot, proliferation, migration and collagen contraction assays were performed to investigate the role of UB/CXCR4 axis on cell signaling, and modulation of fibroblast phenotype and function.
Using fluorescent microscopy and co-immunoprecipitation assay, we provide evidence that extracellular UB interacts with CXCR4. CXCR4 antagonist, AMD3100, inhibited interaction of UB with CXCR4. UB activated ERK1/2, not Akt. It enhanced VEGF-A expression, while decreasing β3 integrins expression. Two mutated UB proteins (V70A and F4A; unable to interact with CXCR4) failed to affect the expression of VEGF-A and β3 integrins. UB treatment inhibited migration of cells into the wound and FBS-stimulated cell proliferation. UB enhanced expression of α-smooth muscle actin (marker of myofibroblast differentiation) and contraction of fibroblast-populated collagen gel pads. Most of the effects of UB were negated by AMD3100.
The data presented here suggest that UB interacts with CXCR4, and UB/CXCR4 interaction affects intracellular signaling, and modulates fibroblast phenotype and function.
β-肾上腺素能受体（β-AR）刺激会增加泛素（UB）的细胞外水平，外源性 UB 在β-AR 刺激的心肌重构中起着重要作用，影响心脏功能、纤维化和心肌细胞凋亡。心肌成纤维细胞对于维持心脏的正常功能以及在心脏对损伤的结构重塑中至关重要。在这里，我们假设细胞外 UB 通过其与 CXC 趋化因子受体 4（CXCR4）的相互作用来调节心肌成纤维细胞表型和功能。
使用血清饥饿的成年心肌成纤维细胞来鉴定 CXCR4 作为 UB 的受体。荧光显微镜、共免疫沉淀、Western blot、增殖、迁移和胶原收缩测定用于研究 UB/CXCR4 轴对细胞信号转导的作用以及对成纤维细胞表型和功能的调节。
使用荧光显微镜和共免疫沉淀测定，我们提供了证据表明细胞外 UB 与 CXCR4 相互作用。CXCR4 拮抗剂 AMD3100 抑制了 UB 与 CXCR4 的相互作用。UB 激活了 ERK1/2，而不是 Akt。它增强了 VEGF-A 的表达，同时降低了β3 整合素的表达。两种突变的 UB 蛋白（V70A 和 F4A；无法与 CXCR4 相互作用）未能影响 VEGF-A 和β3 整合素的表达。UB 处理抑制了细胞向伤口的迁移和 FBS 刺激的细胞增殖。UB 增强了α-平滑肌肌动蛋白（成肌纤维细胞分化的标志物）的表达和纤维母细胞填充的胶原凝胶垫的收缩。UB 的大多数作用都被 AMD3100 否定了。
这里提出的数据表明 UB 与 CXCR4 相互作用，UB/CXCR4 相互作用影响细胞内信号转导，并调节成纤维细胞表型和功能。
