Cells in the center of a solid tissue primordium are usually limited by oxygen availability. However, the role of hypoxia in tissue morphogenesis has not been explored. In this study, we provide evidence that Bnip3 is up-regulated by hypoxia in the core cells of EBs and cooperates with AIF to induce apoptosis and cavitation in an HIF-dependent manner. Our data suggest a model in which hypoxia selectively stabilizes HIF-2α in the core cells via mitochondrial ROS production. HIF-2α translocates to the nucleus and dimerizes with constitutively expressed HIF-1β to activate Bnip3 transcription. AIF is also implicated in the regulation of ROS production, HIF-2α stabilization, and Bnip3 expression. In turn, Bnip3 induces apoptosis of the core cells and cavitation in cooperation with AIF.
AIF is a proapoptotic protein present in the intermembrane space of mitochondria and is released to the cytoplasm in response to death stimuli. The AIF gene is localized to the X chromosome. Targeted mutation of the AIF gene in ES cells failed to form chimeric mice after injected into host blastocysts (Joza et al., 2001
). EBs cultured from AIFy/−
ES cells had significantly reduced cell death and were unable to cavitate. Later, AIF was inactivated by crossing mice with a floxed AIF locus with mice expressing a β-actin Cre
transgene (Joza et al., 2005
; Brown et al., 2006
). This approach revealed that AIF was not essential for EB cavitation and the formation of the proamniotic cavity. These studies suggest that proapoptotic proteins other than AIF or in combination with AIF may be required for apoptosis-dependent cavitation of the early embryo. In the present study, we showed that the BH3-only proapoptotic protein Bnip3 is selectively up-regulated at both mRNA and protein levels during EB differentiation and is localized in the hypoxic core cells. shRNA-mediated silencing of Bnip3 inhibits apoptosis and delays cavitation. Moreover, knockdown of Bnip3 expression in the AIF-null background further decreases apoptosis and nearly blocks EB cavitation. These data suggest that Bnip3 cooperates with AIF to induce apoptosis and cavitation during embryonic epithelial morphogenesis. Bim, another BH3-only protein highly expressed in early embryos and differentiating EBs, has been shown to induce apoptosis of mammary gland cells and contribute to cavitation-mediated acinar morphogenesis (Reginato et al., 2005
; Mailleux et al., 2007
). However, it is not involved in EB cavitation because Bim silencing does not promote apoptosis of the core cells. Furthermore, knockdown of both Bim and Bnip3 has no additive/synergistic effects on EB cavitation.
A previous study in various cultured cells has demonstrated that Bnip3 is a hypoxia-responsive gene regulated by HIF-1 (Chinnadurai et al., 2008
). During EB morphogenesis, however, we detected very low levels of HIF-1α expression by immunoblotting, although HIF-1α, HIF-2α, and HIF-1β are similar at the mRNA level (unpublished data). In contrast, the HIF-2α protein is increased considerably over time and localized to the nucleus of the hypoxic core cells, closely correlating with the expression of Bnip3 and cleaved caspase-3. Importantly, genetic ablation of either HIF-2α or HIF-1β inhibited Bnip3 up-regulation and apoptosis. These results indicate that HIF-2α is selectively stabilized in the core cells during EB cavitation and is mainly responsible for Bnip3 induction. The stabilization of HIF-2α is likely a result of a gradual reduction of oxygen availability (chronic hypoxia) to the core cells as the size of EBs increases during differentiation. On the other hand, HIF-1α is preferably stabilized under acute hypoxic conditions (Holmquist-Mengelbier et al., 2006
). We could also consistently detect elevated HIF-1α protein expression in EBs cultured overnight in hypoxia pouches. Under this condition, hypoxia-induced Bnip3 expression is inhibited by ablation of HIF-1α in EBs (unpublished data). Our data support a notion that acute hypoxia induces Bnip3 expression mainly through HIF-1, whereas chronic hypoxia selectively stabilizes HIF-2α, which joins with HIF-1β in the nucleus and transactivates Bnip3 transcription.
Mitochondria have been suggested to play a key role in sensing changes of oxygen concentrations in cells and tissues (Brunelle et al., 2005
; Guzy et al., 2005
). AIF is a flavoprotein that is essential for maintaining the structural and functional integrity of mitochondrial complexes I and III (Vahsen et al., 2004
). Inactivation of AIF in ES cells reduces the content of complexes I and III and suppresses oxidative phosphorylation. However, it is unknown whether AIF participates in HIF regulation. In this study, we observed a significant reduction of HIF-2α in the absence of AIF. Treatment of normal EBs with the complex I inhibitor rotenone also destabilizes HIF-2α. The reduced HIF-2α protein expression likely results from inhibition of ROS production in the core cells because increased ROS production is detected in the interior of AIFy/+
but not AIFy/−
EBs, the ROS scavenger EUK134 reduces HIF-2α in AIFy/+
EBs, and H2
increases HIF-2α in AIFy/−
EBs. In addition, we have demonstrated that AIF not only cooperates with Bnip3 to induce apoptosis and cavitation but also regulates Bnip3 expression through HIFs. The latter is supported by the fact that ablation of AIF in EBs inhibits Bnip3 expression, whereas stable transfection of AIFy/−
EBs with constitutively active HIF-2α or HIF-1α restores Bnip3 expression. In all of these circumstances, the level of Bnip3 closely correlates with caspase-3 activation. Collectively, our results suggest a new mechanism of apoptosis induced by mitochondrial AIF under hypoxia. It is mediated through ROS production, HIF stabilization, and Bnip3 up-regulation. ROS produced in mitochondria could also induce apoptosis independent of Bnip3 (Simon et al., 2000
). This is in line with our findings that the ROS scavenger EUK134 further inhibits cavitation in Bnip3 knockdown EBs, whereas H2
treatment slightly increases the cavitation efficiency of HIF-1β−/−
EBs. Although AIF has been shown to directly induce nuclear condensation and chromatinolysis (Susin et al., 1999
), we could not detect nuclear translocation of AIF even in the apoptotic cells in the EB interior.
In addition to hypoxia, the core cells of EBs are subject to limited glucose and increased lactic acid. We did not observe any effect of lactic acidosis on Bnip3 expression, although acidosis has been suggested to stabilize Bnip3 and increase its association with mitochondria (Kubasiak et al., 2002
). Previous studies have shown that glucose deprivation can stabilize HIF-α through the AMP-activated protein kinase and mammalian target of rapamycin pathway (Hudson et al., 2002
; Lee et al., 2003
). In mouse ES cells, reduction in glucose levels in the culture medium causes apoptosis, and this is prevented by genetic ablation of HIF-2α, suggesting that HIF-2α–activated gene transcription inhibits hypoglycemia-induced apoptosis (Brusselmans et al., 2001
). In this study, we show that the reduced glucose concentration up-regulates Bnip3 expression, which is mediated by HIFs, as inactivation of HIF-1β abolishes the Bnip3 up-regulation. Therefore, our data provide a mechanistic insight into hypoglycemia-induced apoptosis.
The mechanisms whereby Bnip3 induces apoptosis are incompletely understood. It is suggested that Bnip3 triggers apoptosis by inserting into the outer mitochondrial membrane through its C-terminal transmembrane domain (Chen et al., 1999
; Kim et al., 2002
; Regula et al., 2002
). It opens the permeability transition pore and leads to loss of mitochondrial membrane potential. In cardiomyocytes, hypoxia-induced Bnip3 expression caused the opening of the permeability transition pore and DNA fragmentation, a hallmark of apoptotic cell death. However, the Bnip3-dependent apoptosis was not attenuated by caspase inhibition, suggesting that it is caspase independent (Kubasiak et al., 2002
). Similarly, in neurons, Bnip3 has been shown to induce endonuclease G release from mitochondria and its translocation to the nucleus, where it cleaves DNA (Zhang et al., 2007
). In fibroblasts, overexpression of Bnip3 caused loss of mitochondrial transmembrane potential, release of cytochrome C, and apoptosis, although caspase activation was not assessed (Kubli et al., 2007
). In the present study, we show that apoptosis of the core cells during EB cavitation is associated with mitochondrial cytochrome C release and caspase-3 activation, both of which are inhibited by shRNA-mediated silencing of Bnip3. Altogether, these findings suggest that Bnip3 can induce apoptosis in both a caspase-dependent and -independent manner based on cell types studied.