PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation

Abstract

The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia-and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3.

Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage

“…1C, right). In agreement with previously published work (Qi et al, 2015), caspase-3 activation can be observed during embryoid body formation as early as 2 days after leukemia inhibitory factor (LIF) withdrawal, consistent with an increased apoptotic rate. We, however, noticed higher cleaved caspase-3 levels in GSKJ-4-treated day 2 embryoid bodies than in untreated cells (Fig.…”

Inhibition of KDM6 activity during murine ES cell differentiation induces DNA damage

“…In analogy to embryonic development, apoptosis occurs in floating embryoid body cultures during a process termed cavitation, which forms a proamniotic-like cavity (He et al, 2010;Qi et al, 2012). Bnip3, a BH3-only proapoptotic protein, is of key importance for this process (Qi et al, 2015). As Bnip3 was among the downregulated genes in our global expression analysis (Table S1), we consider it unlikely that normal differentiation was switched to a global proapoptotic program upon inhibition of KDM6 proteins.…”

Pten facilitates epiblast epithelial polarization and proamniotic lumen formation in early mouse embryos

“…Subsequent studies indicate that the defect in axis specification in CD1 background mutant embryos is caused by abnormal behavior of anterior visceral endoderm (AVE) cells, whereby Pten mutant AVE cells initiate movement but fail to migrate to the same extent as that in wild‐type embryos (Bloomekatz et al, ). In another study using the embryoid body model, the authors conclude that Pten is required for apoptosis‐mediated cavitation by regulating the expression of HIF‐2α and Bnip3, but not by suppression of PI3K or Akt (Qi et al, ), though these intriguing mechanisms were not tested or confirmed in embryos.…”