MAP3K4 signaling regulates HDAC6 and TRAF4 coexpression and stabilization in trophoblast stem cells^†

Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) promotes fetal and placental growth and development, with MAP3K4 kinase inactivation resulting in placental insufficiency and fetal growth restriction. MAP3K4 promotes key signaling pathways including JNK, p38, and PI3K/Akt, leading to activation of CREB-binding protein. MAP3K4 kinase inactivation results in loss of these pathways and gain of histone deacetylase 6 (HDAC6) expression and activity. Tumor necrosis factor receptor-associated factor 4 (TRAF4) binds MAP3K4 and promotes MAP3K4 activation of downstream pathways in the embryo; however, the role of TRAF4 and its association with MAP3K4 in the placenta is unknown. Our analyses of murine placenta single-cell RNA-Seq data showed that Traf4 is coexpressed with Map3k4 in trophoblast stem (TS) cells and labyrinth progenitors, whereas Hdac6 expression is higher in differentiated trophoblasts. We demonstrate that, like HDAC6, TRAF4 expression is increased in MAP3K4 kinase-inactive TS (TS^KI) cells and upon inhibition of MAP3K4-dependent pathways in WT TS cells. Moreover, Hdac6 shRNA knockdown in TS^KI cells reduces TRAF4 protein expression. We found that HDAC6 forms a protein complex with TRAF4 in TS cells and promotes TRAF4 expression in the absence of HDAC6 deacetylase activity. Finally, we examine the relationships among MAP3K4, TRAF4, and HDAC6 in the developing placenta, finding a previously unknown switch in coexpression of Traf4 with Map3k4 versus Traf4 with Hdac6 during differentiation of the placental labyrinth. Together, our findings identify previously unknown mechanisms of MAP3K4 and HDAC6 coregulation of TRAF4 in TS cells and highlight these MAP3K4, TRAF4, and HDAC6 associations during placental development.