Department of Orthopaedics and Trauma, Peking University People's Hospital, Beijing 100044, China.
Chin Med J (Engl). 2010 Jul;123(13):1748-53.
Calcitonin gene-related peptide (CGRP), a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that stimulates the migration of monocytes and plays important roles in regulating bone remolding during fracture repair. In this study, we investigated the effects of CGRP on proliferation and MCP-1 expression in cultured rat osteoblasts.
Primary rat osteoblasts were isolated from fetal rats calvariae. Cells were exposed to gradient concentrations (10(-9) to 10(-7) mol/L) of CGRP. Protein and mRNA levels of MCP-1 were quantified by Western blotting and semiquantitative reverse transcription-polymerase chain reaction, respectively. The protein level of MCP-1 was investigated and compared in cell culture media by enzyme linked immunosorbent assay (ELISA). Phospho-extracellular signal-regulated kinase (ERK) expression was detected by Western blotting. Cell proliferative activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and BrdU assay. The effects of MAPK/ERK kinase (MEK)-inhibitor U0126 on CGRP-induced MCP-1 expression in primary rat osteoblasts were examined.
CGRP effectively enhanced primary rat osteoblast proliferation and led to significant increases in the expression of MCP-1 mRNA and protein in time- and dose-dependent manners. CGRP activated the ERK pathway. Pretreatment of cultured rat osteoblasts with MEK inhibitor U0126 resulted in dose-dependent inhibitions of CGRP-induced MCP-1 mRNA and protein levels. Thus, CGRP promoted cell proliferation and stimulated MCP-1 expression in cultured rat osteoblasts.
These studies document novel links between CGRP and MCP-1 and illuminate the effects of CGRP in regulating bone remodeling.
降钙素基因相关肽（CGRP）是一种感觉神经肽，它影响成骨细胞的增殖和骨形成。然而，其机制尚未完全阐明。单核细胞趋化蛋白-1（MCP-1）是一种趋化因子，可刺激单核细胞迁移，并在骨折修复过程中调节骨重塑中发挥重要作用。在这项研究中，我们研究了 CGRP 对培养的大鼠成骨细胞增殖和 MCP-1 表达的影响。
从胎鼠颅骨中分离原代大鼠成骨细胞。细胞暴露于梯度浓度（10（-9）至 10（-7）mol/L）的 CGRP 中。通过 Western 印迹和半定量逆转录聚合酶链反应分别定量测定 MCP-1 的蛋白和 mRNA 水平。通过酶联免疫吸附试验（ELISA）在细胞培养物中检测和比较 MCP-1 的蛋白水平。通过 Western 印迹检测磷酸化细胞外信号调节激酶（ERK）的表达。通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐（MTT）和 BrdU 测定法测量细胞增殖活性。检查 MAPK/ERK 激酶（MEK）抑制剂 U0126 对 CGRP 诱导的原代大鼠成骨细胞中 MCP-1 表达的影响。
CGRP 有效增强了原代大鼠成骨细胞的增殖，并以时间和剂量依赖性方式导致 MCP-1 mRNA 和蛋白表达的显著增加。CGRP 激活了 ERK 途径。用 MEK 抑制剂 U0126 预处理培养的大鼠成骨细胞导致 CGRP 诱导的 MCP-1 mRNA 和蛋白水平呈剂量依赖性抑制。因此，CGRP 促进了培养的大鼠成骨细胞的增殖并刺激了 MCP-1 的表达。
这些研究记录了 CGRP 和 MCP-1 之间的新联系，并阐明了 CGRP 在调节骨重塑中的作用。
