脱氢表雄酮经不依赖于雌、雄激素受体的MAPK信号途径抑制成骨细胞凋亡

脱氢表雄酮(DHEA)已成为防治绝经后骨质疏松症(PMO)的新策略,但其调控成骨细胞(OB)凋亡的具体分子机制和信号转导途径尚不清楚。我们通过颅骨酶解法原代培养OB,体外模拟雌激素撤退现象,10-7mol/LDHEA分别作用0h、24h、48h、72h后,RT-PCR分析OB中ERα、ERβ和ARmRNA表达;原代OB去血清进一步培养24h,细胞以雌激素受体(ER)拮抗剂ICI182,780(1μmol/L)、雄激素受体(AR)拮抗剂Flutamide(10μmol/L)或U0126(100μmol/L)预处理后给予系列浓度DHEA(10-10-10-5mol/L)孵育72h,AnnexinV-FITC/PI双标记流式细胞仪分析细胞早期凋亡;原代OB以1μmol/LICI182,780或10μmol/LFlutamide预处理25min后给予不同浓度DHEA孵育10min,Westernblotting分析ERK1/2的磷酸化状态。结果表明OBs经10-7mol/LDHEA体外处理24h、48h、72h后,ERβ和ARmRNA水平升高(分别为P<0.05和P<0.01);而ERαmRNA水平无明显变化。10-9-10-6mol/LDHEA可显著抑制血清饥饿诱导的OBs早期凋亡(分别为P<0.05及P<0.01),该抑制效应可被U0126阻滞,ICI182,780或Flutamide则不能阻滞DHEA对OB的抗凋亡效应;Westernblot也显示ICI182,780或Flutamide都不能有效地阻滞DHEA对OB中ERKs磷酸化的诱导作用。因此可认为DHEA经ER或AR非依赖途径抑制OB凋亡;丝裂原活化蛋白激酶(MAPK)信号途径,磷酸化ERK1/2参与介导这一作用。

DHEA INHIBITS APOPTOSIS OF MURINE OB THROUGH MAPK SIGNALING PATHWAYS INDEPENDENT OF EITHER ARs OR ERs

Dehydroepiandrosterone (DHEA) is a promising agent for the treatment of post-menopausal osteoporosis (PMO), but the molecular mechanisms and signaling pathways by which this steroid modulates apoptosis of osteoblasts (OB) are still poorly understood. In this study,the OBs were cultured in vitro by the enzyme-digested method,treated with DHEA (10(-7) mol/L) for 0h, 24h, 48h, 72h, respectively. The expressions of ER alpha, ER beta and AR mRNA in OB were analyzed by RT-PCR. After the primary OBs were deprived of serum for a further 24h, and then pretreated with 1 micromol/L ICI 182,780 (an estrogen receptor antagonist), 10 micromol/L Flutamide (an androgen receptor antagonist) or 100 micromol/L U0126 (a specific inhibitor of MAPK pathway) for 1h, they were treated with a series of concentrations of DHEA (10(-10) - 10(-5) mol/L) in serum-free medium for 72h,the apoptotic cells were analyzed by FCM with the Annexin-V-FITC/PI dual labeling technique. The OBs were incubated in 1 micromol/L ICI 182,780 or 10 micromol/L Flutamide for 25 minutes,and then treated with different concentrations of DHEA for the further 10 minutes. The phosphorylation status of ERK1/2 was analyzed by Western blot. After the OBs were incubated with DHEA (10(-7)mol/L) for 24h, 48h or 72h, respectively, its ERbeta and AR mRNA level were increased (P<0.05, P<0.01, respectively), but the ER alpha mRNA level had no change. The 10(-9) - 10(-6) mol/L of DHEA inhibited OBs early apoptosis induced by the serum deprivation (P<0.05, P<0.01, respectively). The inhibiting effect, moreover, could be blocked by the specific inhibitor of MAPK pathway, U0126. The effects of DHEA were neither blocked by the steroid hormone antagonist ICI 182,780 nor by Flutamide. Western blot showed that neither receptor antagonist ICI 182,780 nor Flutamide could block the DHEA-induced ERKs phosphorylation in OBs,which was similar to the apoptosis. DHEA inhibits apoptosis in OBs presumably via a DHEA-specific receptor that involves mitogen activated protein kinase (MAPK) signal pathway, phospho-pERK1/2, independent of either ARs or ERs.