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

Survival 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，滋养层细胞要在低氧环境下存活，需要金属蛋白酶介导肝素结合性表皮生长因子（HBEGF）的脱落以及下游信号转导。本研究采用基质金属蛋白酶（MMP）抗体芯片与定量逆转录聚合酶链反应（quantitative RT-PCR），发现在暴露于2%氧浓度的人类妊娠早期HTR-8/SVneo滋养层细胞及胎盘绒毛外植体中，基质金属蛋白酶2（MMP2）的表达在转录后水平上调。特异性MMP抑制剂实验证实，MMP2是HBEGF脱落及其表达上调所必需的。由于α-鹅膏蕈碱可抑制HBEGF的表达上调，研究人员对在2%氧浓度下分别培养0、1、2、4小时的滋养层细胞的RNA进行下一代测序，以鉴定差异表达基因。培养1小时后，共9个携带缺氧诱导因子（HIF）反应元件的基因出现表达上调，但至2~4小时仅热休克蛋白70家族成员6（HSPA6，即HSP70B’）的表达仍维持升高。热休克蛋白70（HSP70）分子伴侣抑制剂VER 155008可阻断2%氧浓度下MMP2与HBEGF的表达上调，并加剧细胞凋亡。而外源性添加MMP2则可挽救HBEGF的表达上调及细胞凋亡表型。邻近连接试验（PLA）证实了HSP70与MMP2、MMP2与HBEGF之间存在相互作用，这支持了以下观点：由HSP70启动的MMP2介导的HBEGF脱落，可促进滋养层细胞在妊娠早期所遭遇的低氧浓度环境下存活，且对成功妊娠至关重要。在人类胎盘形成的低氧阶段，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的脱落反式激活抗凋亡ERBB信号通路。