Data from: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

urvival of trophoblast cells in the low oxygen environment of human placentation requires metalloproteinase-mediated shedding of HBEGF and downstream signaling. A matrix metalloproteinase (MMP) antibody array and quantitative RT-PCR revealed upregulation of MMP2 post-transcriptionally in human first trimester HTR-8/SVneo trophoblast cells and placental villous explants exposed to 2% O2. Specific MMP inhibitors established the requirement for MMP2 in HBEGF shedding and upregulation. Because α-amanitin inhibited the upregulation of HBEGF, differentially expressed genes were identified by next-generation sequencing of RNA from trophoblast cells cultured at 2% O2 for 0, 1, 2 and 4 h. Nine genes, all containing HIF-response elements, were upregulated at 1 h, but only HSPA6 (HSP70B’) remained elevated at 2–4 h. The HSP70 chaperone inhibitor VER 155008 blocked upregulation of both MMP2 and HBEGF at 2% O2, and increased apoptosis. However, both HBEGF upregulation and apoptosis were rescued by exogenous MMP2. Proximity ligation assays demonstrated interactions between HSP70 and MMP2, and between MMP2 and HBEGF, supporting the concept that MMP2-mediated shedding of HBEGF, initiated by HSP70, contributes to trophoblast survival at the low O2 concentrations encountered during the first trimester, and is essential for successful pregnancy outcomes. Trophoblast survival during human placentation, when oxygenation is minimal, required HSP70 activity, which mediated MMP2 accumulation and the transactivation of anti-apoptotic ERBB signaling by HBEGF shedding.

人类胎盘形成过程中，滋养层细胞（trophoblast cells）在低氧环境下的存活依赖于金属蛋白酶（metalloproteinase）介导的肝素结合表皮生长因子（HBEGF）脱落及其下游信号通路。采用基质金属蛋白酶（matrix metalloproteinase，MMP）抗体阵列与定量逆转录聚合酶链反应（quantitative RT-PCR）检测发现，在暴露于2%氧气环境的妊娠早期（first trimester）HTR-8/SVneo滋养层细胞（HTR-8/SVneo trophoblast cells）及胎盘绒毛外植体（placental villous explants）中，MMP2的表达在转录后水平（post-transcriptionally）上调。特异性MMP抑制剂实验证实，MMP2是介导HBEGF脱落与表达上调的关键因子。由于α-鹅膏蕈碱（α-amanitin）可抑制HBEGF的表达上调，因此我们对在2%氧气环境中分别培养0、1、2、4小时的滋养层细胞进行RNA下一代测序（next-generation sequencing），以筛选差异表达基因（differentially expressed genes）。培养1小时后，共9个均含有缺氧诱导因子反应元件（HIF-response elements）的基因出现表达上调，但在2至4小时时仅热休克蛋白70家族成员6（HSPA6，HSP70B’）的表达仍维持升高状态。HSP70分子伴侣抑制剂（HSP70 chaperone inhibitor）VER 155008可阻断2%氧气环境下MMP2与HBEGF的表达上调，并诱导细胞凋亡（apoptosis）。但外源性添加MMP2可挽救HBEGF表达上调与细胞凋亡表型。邻近连接试验（proximity ligation assays）结果显示，HSP70与MMP2之间、MMP2与HBEGF之间均存在相互作用，这支持了以下结论：由HSP70启动的MMP2介导的HBEGF脱落，可促进妊娠早期低氧环境中滋养层细胞的存活，且对良好妊娠结局（pregnancy outcomes）至关重要。在氧合水平极低的人类胎盘形成过程中，滋养层细胞的存活依赖于HSP70的活性：该活性可介导MMP2的积累，并通过HBEGF脱落反式激活抗凋亡的ERBB信号通路（ERBB signaling）。