Reciprocal Regulation of MAGED2 and HIF-1α Augments Their Expression under Hypoxia: Role of cAMP and PKA Type II.

Hypoxia stabilizes the transcription factor HIF-1α, which promotes the transcription of many genes essential to adapt to reduced oxygen levels. Besides proline hydroxylation, expression of HIF-1α is also regulated by a range of other posttranslational modifications including phosphorylation by cAMP-dependent protein kinase A (PKA), which stabilizes HIF-1α. We recently demonstrated that MAGED2 is required for cAMP generation under hypoxia and proposed that this regulation may explain the transient nature of antenatal Bartter syndrome (aBS) due to MAGED2 mutations. Consequently, we sought to determine whether hypoxic induction of HIF-1α requires also MAGED2. In HEK293 and HeLa cells, MAGED2 knock-down impaired maximal induction of HIF-1α under physical hypoxia as evidenced by time-course experiments, which showed a signification reduction of HIF-1α upon MAGED2 depletion. Similarly, using cobalt chloride to induce HIF-1α, MAGED2 depletion impaired its appropriate induction. Given the known effect of the cAMP/PKA pathway on the hypoxic induction of HIF-1α, we sought to rescue impaired HIF-1α induction with isoproterenol and forskolin acting upstream and downstream of Gαs, respectively. Importantly, while forskolin induced HIF-1α above control levels in MAGED2-depleted cells, isoproterenol had no effect. To further delineate which PKA subtype is involved, we analyzed the effect of two PKA inhibitors and identified that PKA type II regulates HIF-1α. Interestingly, MAGED2 mRNA and protein were also increased under hypoxia by a cAMP mimetic. Moreover, MAGED2 protein expression also required HIF-1α. Thus, our data provide evidence for reciprocal regulation of MAGED2 and HIF-1α under hypoxia, revealing therefore a new regulatory mechanism that may further explain the transient nature of aBS caused by MAGED2 mutations.

MAGED2 Is Required under Hypoxia for cAMP Signaling by Inhibiting MDM2-Dependent Endocytosis of G-Alpha-S.

Mutations in MAGED2 cause transient Bartter syndrome characterized by severe renal salt wasting in fetuses and infants, which leads to massive polyhydramnios causing preterm labor, extreme prematurity and perinatal death. Notably, this condition resolves spontaneously in parallel with developmental increase in renal oxygenation. MAGED2 interacts with G-alpha-S (Gαs). Given the role of Gαs in activating adenylyl cyclase at the plasma membrane and consequently generating cAMP to promote renal salt reabsorption via protein kinase A (PKA), we hypothesized that MAGED2 is required for this signaling pathway under hypoxic conditions such as in fetuses. Consistent with that, under both physical and chemical hypoxia, knockdown of MAGED2 in renal (HEK293) and cancer (HeLa) cell culture models caused internalization of Gαs, which was fully reversible upon reoxygenation. In contrast to Gαs, cell surface expression of the ß2-adrenergic receptor, which is coupled to Gαs, was not affected by MAGED2 depletion, demonstrating specific regulation of Gαs by MAGED2. Importantly, the internalization of Gαs due to MAGED2 deficiency significantly reduced cAMP generation and PKA activity. Interestingly, the internalization of Gαs was blocked by preventing its endocytosis with dynasore. Given the role of E3 ubiquitin ligases, which can be regulated by MAGE-proteins, in regulating endocytosis, we assessed the potential role of MDM2-dependent ubiquitination in MAGED2 deficiency-induced internalization of Gαs under hypoxia. Remarkably, MDM2 depletion or its chemical inhibition fully abolished Gαs-endocytosis following MAGED2 knockdown. Moreover, endocytosis of Gαs was also blocked by mutation of ubiquitin acceptor sites in Gαs. Thus, we reveal that MAGED2 is essential for the cAMP/PKA pathway under hypoxia to specifically regulate Gαs endocytosis by blocking MDM2-dependent ubiquitination of Gαs. This may explain, at least in part, the transient nature of Bartter syndrome caused by MAGED2 mutations and opens new avenues for therapy in these patients.