Wang Ling, Li Da Jin, Wang Wen Jun, Zhu Ying
Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Shanghai Medical college, Fudan University, Shanghai 200011.
Fen Zi Xi Bao Sheng Wu Xue Bao. 2006 Aug;39(4):365-72.
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.
脱氢表雄酮（DHEA）是一种有望用于治疗绝经后骨质疏松症（PMO）的药物，但这种类固醇调节成骨细胞（OB）凋亡的分子机制和信号通路仍知之甚少。在本研究中，采用酶消化法体外培养成骨细胞，分别用DHEA（10⁻⁷mol/L）处理0小时、24小时、48小时、72小时。通过逆转录聚合酶链反应（RT-PCR）分析成骨细胞中雌激素受体α（ERα）、雌激素受体β（ERβ）和雄激素受体（AR）mRNA的表达。原代成骨细胞在进一步无血清培养24小时后，先用1μmol/L ICI 182,780（一种雌激素受体拮抗剂）、10μmol/L氟他胺（一种雄激素受体拮抗剂）或100μmol/L U0126（丝裂原活化蛋白激酶（MAPK）途径的特异性抑制剂）预处理1小时，然后在无血清培养基中用一系列浓度的DHEA（10⁻¹⁰ - 10⁻⁵mol/L）处理72小时，采用膜联蛋白-V-异硫氰酸荧光素/碘化丙啶（Annexin-V-FITC/PI）双标记技术通过流式细胞术（FCM）分析凋亡细胞。将成骨细胞在1μmol/L ICI 182,780或10μmol/L氟他胺中孵育25分钟，然后用不同浓度的DHEA再处理10分钟。通过蛋白质免疫印迹法（Western blot）分析细胞外信号调节激酶1/2（ERK1/2）的磷酸化状态。成骨细胞分别用DHEA（10⁻⁷mol/L）孵育24小时、48小时或72小时后，其ERβ和AR mRNA水平升高（分别为P<0.05，P<0.01），但ERα mRNA水平无变化。10⁻⁹ - 10⁻⁶mol/L的DHEA抑制血清剥夺诱导的成骨细胞早期凋亡（分别为P<0.05，P<0.01）。此外，这种抑制作用可被MAPK途径的特异性抑制剂U0126阻断。DHEA的作用既不被类固醇激素拮抗剂ICI 182,780阻断，也不被氟他胺阻断。蛋白质免疫印迹法显示，受体拮抗剂ICI 182,780和氟他胺均不能阻断DHEA诱导的成骨细胞中ERK的磷酸化，这与凋亡情况相似。DHEA可能通过一种涉及丝裂原活化蛋白激酶（MAPK）信号通路、磷酸化细胞外信号调节激酶1/2（pERK1/2）的DHEA特异性受体抑制成骨细胞凋亡，该过程独立于雄激素受体（ARs）或雌激素受体（ERs）。
