GH与EGFR受体后信号交联在GH促进青春期大鼠生长板软骨细胞增殖中的作用

Epidermal growth factor receptor pathway participates in growth promotion of chondrocytes by growth hormone from sex hormone-inhibited adolescent rats

目的:观察促性腺激素释放激素类似物(GnRHa)处理后青春期大鼠生长板体外培养的软骨细胞内是否存在生长激素(GH)与表皮生长因子受体(EGFR)之间的信号交联(cross-talk)。方法:3周龄雌性SD大鼠开始注射GnRHa至7周龄;随后分离培养5-8只大鼠胫骨生长板软骨细胞,在GH作用前分别加入JAK2阻断剂AG490(1nmol/L、10nmol/L和100nmol/L)、EGFR酪氨酸激酶阻断剂AG1478(0.1nmol/L、1nmol/L和10nmol/L)和表皮生长因子(EGF)中和抗体(0.1mg/L、1mg/L和10mg/L);采用四氮甲基唑蓝(MTT)以及免疫组化法测定增殖细胞核抗原(PCNA),观察不同阻断剂对软骨细胞增殖的影响;采用Western blotting检测软骨细胞p-ERK1/2及p-EGFR的表达。结果:GH作用后具有浓度依赖性地促进软骨细胞增殖和增加p-ERK1/2及p-EGFR水平的作用,100μg/L时作用最为明显。AG490及AG1478几乎能完全抑制GH促进软骨细胞增殖和激活ERK1/2及EGFR的作用。而EGF中和抗体仅能部分抑制GH的作用。结论:GH通过激活JAK2而激活其下游的ERK通路,实现其直接促细胞增殖效应。GH亦能通过激活EGFR及其信号转导通路促进细胞增殖效应,表明GH通路和EGF通路间存在相互作用。

AIM： To investigate the possibility that epidermal growth factor receptor pathway participates in the growth promotion by growth hormone （GH） of growth plate chondrocytes cultured in vitro from adolescent rats treated with gonadotropin-releasing hormone analogue （GnRHa）. METHODS： The chondrocytes from tibial growth plate of 5-8 female Sprague-Dawley （SD） rats treated with GnRHa were cultured in monolayer. Specific pharmacological inhibitor of Janus kinase （JAK2 tyrphostin AG490, 1, 10, 100 nmol/L）, EGFR kinase inhibitor AG1478 （0.1, 1, 10 nmol/L） and neutralizing antibodies against EGF （0.1, 1, 10 mg/L） were added before GH stimulation. The proliferation of chondrocytes was investigated by the methods of MTT and immunohistochemical staining for PCNA. Phosphorylations of ERK1/2 and EGFR were detected by Western blotting. RESULTS： GH enhanced the proliferation of chondrocytes and the levels of ERK1/2 and EGFR phosphorylation in a dose dependent manner. The effect peaked at the concentration of 100 μg/L. Pretreatment with tyrphostin AG490 and AG1478 almost completely inhibited the proliferation of chondrocytes and phosphorylation of ERK1/2 and EGFR by GH. However, the neutralizing antibodies against EGF only partially inhibited the effects of GH.CONCLUSION： GH achieves its direct effect on the promotion of cell proliferation by the activation of JAK2 pathway and the downstream end of MAPK-ERK signaling molecules. The promotion of cell proliferation can be mediated by activation of EGFR pathway. The results suggest that there is signaling cross-talk between GH and EGF-EGFR pathway.