MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway.

Melanoma-associated antigen D2 (MAGED2) plays an essential role in activating the cAMP/PKA pathway under hypoxic conditions, which is crucial for stimulating renal salt reabsorption and thus explaining the transient variant of Bartter's syndrome. The cAMP/PKA pathway is also known to regulate autophagy, a lysosomal degradation process induced by cellular stress. Previous studies showed that two members of the melanoma-associated antigens MAGE-family inhibit autophagy. To explore the potential role of MAGED2 in stress-induced autophagy, specific MAGED2-siRNA were used in HEK293 cells under physical hypoxia and oxidative stress (cobalt chloride, hypoxia mimetic). Depletion of MAGED2 resulted in reduced p62 levels and upregulation of both the autophagy-related genes (ATG5 and ATG12) as well as the autophagosome marker LC3II compared to control siRNA. The increase in the autophagy markers in MAGED2-depleted cells was further confirmed by leupeptin-based assay which concurred with the highest LC3II accumulation. Likewise, under hypoxia, immunofluorescence in HEK293, HeLa and U2OS cell lines demonstrated a pronounced accumulation of LC3B puncta upon MAGED2 depletion. Moreover, LC3B puncta were absent in human fetal control kidneys but markedly expressed in a fetal kidney from a MAGED2-deficient subject. Induction of autophagy with both physical hypoxia and oxidative stress suggests a potentially general role of MAGED2 under stress conditions. Various other cellular stressors (brefeldin A, tunicamycin, 2-deoxy-D-glucose, and camptothecin) were analyzed, which all induced autophagy in the absence of MAGED2. Forskolin (FSK) inhibited, whereas GNAS Knockdown induced autophagy under hypoxia. In contrast to other MAGE proteins, MAGED2 has an inhibitory role on autophagy only under stress conditions. Hence, a prominent role of MAGED2 in the regulation of autophagy under stress conditions is evident, which may also contribute to impaired fetal renal salt reabsorption by promoting autophagy of salt-transporters in patients with MAGED2 mutation.

Diacidic Motifs in the Carboxyl Terminus Are Required for ER Exit and Translocation to the Plasma Membrane of NKCC2.

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that the BS1 variant Y998X, which deprives NKCC2 from its highly conserved dileucine-like motifs, compromises co-transporter surface delivery through ER retention mechanisms. However, whether these hydrophobic motifs are sufficient for anterograde trafficking of NKCC2 remains to be determined. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of consensus di-acidic (D/E-X-D/E) motifs, 949EEE951 and 1019DAELE1023, located upstream and downstream of BS1 mutation Y998X, respectively. Di-acidic codes are involved in ER export of proteins through interaction with COPII budding machinery. Importantly, whereas mutating 949EEE951 motif to 949AEA951 had no effect on NKCC2 processing, mutating 1019DAE1021 to 1019AAA1021 heavily impaired complex-glycosylation and cell surface expression of the cotransporter in HEK293 and OKP cells. Most importantly, triple mutation of D, E and E residues of 1019DAELE1023 to 1019AAALA1023 almost completely abolished NKCC2 complex-glycosylation, suggesting that this mutant failed to exit the ER. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of 1019AAALA1023 was caused by defects in NKCC2 maturation. Accordingly, co-immunolocalization experiments revealed that 1019AAALA1023 was trapped in the ER. Finally, overexpression of a dominant negative mutant of Sar1-GTPase abolished NKCC2 maturation and cell surface expression, clearly indicating that NKCC2 export from the ER is COPII-dependent. Hence, our data indicate that in addition to the di-leucine like motifs, NKCC2 uses di-acidic exit codes for export from the ER through the COPII-dependent pathway. We propose that any naturally occurring mutation of NKCC2 interfering with this pathway could form the molecular basis of BS1.

New insights into the role of endoplasmic reticulum-associated degradation in Bartter Syndrome Type 1.

Mutations in Na-K-2Cl co-transporter, NKCC2, lead to type I Bartter syndrome (BS1), a life-threatening kidney disease. Yet, our knowledge of the molecular regulation of NKCC2 mutants remains poor. Here, we aimed to identify the molecular pathogenic mechanisms of one novel and three previously reported missense NKCC2 mutations. Co-immunolocalization studies revealed that all NKCC2 variants are not functional because they are not expressed at the cell surface due to retention in the endoplasmic reticulum (ER). Cycloheximide chase assays together with treatment by protein degradation and mannose trimming inhibitors demonstrated that the defect in NKCC2 maturation arises from ER retention and associated degradation (ERAD). Small interfering RNA (siRNA) knock-down experiments revealed that the ER lectin OS9 is involved in the ERAD of NKCC2 mutants. 4-phenyl butyric acid (4-PBA) treatment mimicked OS9 knock-down effect on NKCC2 mutants by stabilizing their immature forms. Importantly, out of the four studied mutants, only one showed an increased protein maturation upon treatment with glycerol. In summary, our study reveals that BS1 is among diseases linked to the ERAD pathway. Moreover, our data open the possibility that maturation of some ER retained NKCC2 variants is correctable by chemical chaperones offering, therefore, promising avenues in elucidating the molecular pathways governing the ERAD of NKCC2 folding mutants.

Differential Effects of STCH and Stress-Inducible Hsp70 on the Stability and Maturation of NKCC2.

Mutations in the Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in NKCC2 maturation and cell surface expression. Yet, the molecular mechanisms involved in this process remain obscure. Here, we report the identification of chaperone stress 70 protein (STCH) and the stress-inducible heat shock protein 70 (Hsp70), as two novel binding partners of the ER-resident form of NKCC2. STCH knock-down increased total NKCC2 expression whereas Hsp70 knock-down or its inhibition by YM-01 had the opposite effect. Accordingly, overexpressing of STCH and Hsp70 exerted opposite actions on total protein abundance of NKCC2 and its folding mutants. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, Hsp70 co-expression increased NKCC2 maturation. Interestingly, treatment by protein degradation inhibitors revealed that in addition to the proteasome, the ER associated degradation (ERAD) of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway. In summary, our data are consistent with STCH and Hsp70 having differential and antagonistic effects with regard to NKCC2 biogenesis. These findings may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.

Polyhydramnios, Transient Antenatal Bartter's Syndrome, and MAGED2 Mutations.

BACKGROUND: Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter's syndrome. METHODS: To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies. RESULTS: We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter's syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter's syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein. CONCLUSIONS: We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

OS9 Protein Interacts with Na-K-2Cl Co-transporter (NKCC2) and Targets Its Immature Form for the Endoplasmic Reticulum-associated Degradation Pathway.

Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome.

AUP1 Regulates the Endoplasmic Reticulum-Associated Degradation and Polyubiquitination of NKCC2.

Inactivating mutations of kidney Na-K-2Cl cotransporter NKCC2 lead to antenatal Bartter syndrome (BS) type 1, a life-threatening salt-losing tubulopathy. We previously reported that this serious inherited renal disease is linked to the endoplasmic reticulum-associated degradation (ERAD) pathway. The purpose of this work is to characterize further the ERAD machinery of NKCC2. Here, we report the identification of ancient ubiquitous protein 1 (AUP1) as a novel interactor of NKCC2 ER-resident form in renal cells. AUP1 is also an interactor of the ER lectin OS9, a key player in the ERAD of NKCC2. Similar to OS9, AUP1 co-expression decreased the amount of total NKCC2 protein by enhancing the ER retention and associated protein degradation of the cotransporter. Blocking the ERAD pathway with the proteasome inhibitor MG132 or the α-mannosidase inhibitor kifunensine fully abolished the AUP1 effect on NKCC2. Importantly, AUP1 knock-down or inhibition by overexpressing its dominant negative form strikingly decreased NKCC2 polyubiquitination and increased the protein level of the cotransporter. Interestingly, AUP1 co-expression produced a more profound impact on NKCC2 folding mutants. Moreover, AUP1 also interacted with the related kidney cotransporter NCC and downregulated its expression, strongly indicating that AUP1 is a common regulator of sodium-dependent chloride cotransporters. In conclusion, our data reveal the presence of an AUP1-mediated pathway enhancing the polyubiquitination and ERAD of NKCC2. The characterization and selective regulation of specific ERAD constituents of NKCC2 and its pathogenic mutants could open new avenues in the therapeutic strategies for type 1 BS treatment.

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.

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.

Golgi Alpha1,2-Mannosidase IA Promotes Efficient Endoplasmic Reticulum-Associated Degradation of NKCC2.

Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.