Interplay between primary familial brain calcification-associated SLC20A2 and XPR1 phosphate transporters requires inositol polyphosphates for control of cellular phosphate homeostasis
2020; Elsevier BV; Volume: 295; Issue: 28 Linguagem: Inglês
10.1074/jbc.ra119.011376
ISSN1083-351X
AutoresUriel López-Sánchez, Sandrine Tury, Gaël Nicolas, Miranda Wilson, Snejana Jurici, Xavier Ayrignac, Valérie Courgnaud, Adolfo Saiardi, Marc Sitbon, Jean‐Luc Battini,
Tópico(s)Genetic Syndromes and Imprinting
ResumoSolute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP–binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC. Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP–binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC. The solute carrier (SLC) SLC20 and SLC53 family genes encode three cell surface multitransmembrane proteins known as PiT1/SLC20A1, PiT2/SLC20A2, and polytropic retrovirus receptor 1 (XPR1)/SLC53A1, which were shown to transport phosphate (1Giovannini D. Touhami J. Charnet P. Sitbon M. Battini J.L. Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.Cell Rep. 2013; 3 (23791524): 1866-187310.1016/j.celrep.2013.05.035Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 2Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.Proc. Natl. Acad. Sci. U.S.A. 1994; 91 (8041748): 7071-707510.1073/pnas.91.15.7071Crossref PubMed Scopus (500) Google Scholar, 3Olah Z. Lehel C. Anderson W.B. Eiden M.V. Wilson C.A. 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We found that SLC20A2 and XPR1 regulated the rates of phosphate fluxes in a concerted and PP-IP-dependent manner and controlled intracellular phosphate and ATP levels, providing evidence for a specific SLC20A2, XPR1, and PP-IP interplay in phosphate homeostasis and metabolism, with new insights on the role of phosphate metabolic disorders in PFBC. To date, more than 100 variants have been found in the SLC20A2 gene of PFBC patients (36Ramos E.M. Carecchio M. Lemos R. Ferreira J. Legati A. Sears R.L. Hsu S.C. Panteghini C. Magistrelli L. Salsano E. Esposito S. Taroni F. Richard A.C. Tranchant C. Anheim M. et al.Primary brain calcification: an international study reporting novel variants and associated phenotypes.Eur. J. Hum. Genet. 2018; 26 (29955172): 1462-147710.1038/s41431-018-0185-4Crossref PubMed Scopus (33) Google Scholar). It is the major PFBC gene, as variants in this gene account for 66.5% of genetically explained cases. Although a majority of the variants were classified as pathogenic or likely pathogenic based on genetic arguments, very few studies have assessed these variants for phosphate transport (26Wang C. Li Y. Shi L. Ren J. Patti M. Wang T. de Oliveira J.R. Sobrido M.J. Quintans B. Baquero M. Cui X. Zhang X.Y. Wang L. Xu H. Wang J. et al.Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis.Nat. Genet. 2012; 44 (22327515): 254-25610.1038/ng.1077Crossref PubMed Scopus (258) Google Scholar, 37Larsen F.T. Jensen N. Autzen J.K. Kongsfelt I.B. Pedersen L. Primary brain calcification causal PiT2 transport-knockout variants can exert dominant negative effects on wild-type PiT2 transport function in mammalian cells.J. Mol. Neurosci. 2017; 61 (27943094): 215-22010.1007/s12031-016-0868-7Crossref PubMed Scopus (16) Google Scholar), and none has been evaluated for expression at the plasma membrane. 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With the exception of SLC20A2 p.ΔA145_V205, which most likely encoded a defective transporter, as it was undetectable by flow cytometry (Fig. 1A) or immunoblotting (Fig. 1B), all of the other SLC20A2 mutants were expressed at the expected size, were present at the plasma membrane as efficiently as WT SLC20A2, and did not affect cell surface expression of endogenous SLC20A1 and XPR1, as observed by flow cytometry using specific retroviral ligands derived from receptor-binding domains (RBDs) of gammaretrovirus envelope (Env) glycoproteins (1Giovannini D. Touhami J. Charnet P. Sitbon M. Battini J.L. Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.Cell Rep. 2013; 3 (23791524): 1866-187310.1016/j.celrep.2013.05.035Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 25Legati A. Giovannini D. Nicolas G. Lopez-Sanchez U. Quintans B. Oliveira J.R. Sears R.L. Ramos E.M. Spiteri E. Sobrido M.J. Carracedo A. Castro-Fernandez C. Cubizolle S. Fogel B.L. Goizet C. et al.Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export.Nat. Genet. 2015; 47 (25938945): 579-58110.1038/ng.3289Crossref PubMed Scopus (156) Google Scholar, 40Anheim M. Lopez-Sanchez U. Giovannini D. Richard A.C. Touhami J. N'Guyen L. Rudolf G. Thibault-Stoll A. Frebourg T. Hannequin D. Campion D. Battini J.L. Sitbon M. Nicolas G. XPR1 mutations are a rare cause of primary familial brain calcification.J. Neurol. 2016; 263 (27230854): 1559-156410.1007/s00415-016-8166-4Crossref PubMed Scopus (35) Google Scholar, 41Lopez-Sanchez U. Nicolas G. Richard A.C. Maltete D. Charif M. Ayrignac X. Goizet C. Touhami J. Labesse G. Battini J.L. Sitbon M. Characterization of XPR1/SLC53A1 variants located outside of the SPX domain in patients with primary familial brain calcification.Sci. Rep. 2019; 9 (31043717): 677610.1038/s41598-019-43255-xCrossref PubMed Scopus (7) Google Scholar). 33P-radiolabeled phosphate flux assays showed that overexpression of human SLC20A2 led to a significant increase in phosphate uptake, as expected, compared with control parental cells (Fig. 1C). In contrast, phosphate uptake remained close to basal levels with the SLC20A2 mutants, suggesting that PFBC variants altered critically phosphate uptake, with no detectable impact on endogenous SLC20A2 expression and phosphate transport. Surprisingly, the increase in phosphate uptake seen for WT SLC20A2 was concomitant with an increase in phosphate efflux (Fig. 1D). Similar observations were obtained with SLC20A1 (Fig. S1). In comparison, phosphate efflux with the SLC20A2 variants remained close to control levels. These results suggested a close interplay between phosphate uptake and efflux. Further ex vivo evaluation of the Y187C and ΔA145_V205 SLC20A2 variants with peripheral blood mononuclear cells (PBMC) isolated from PFBC patients showed a strong decrease in phosphate uptake, compared with PBMC from control healthy individual (Fig. 1E). However, phosphate efflux was similar to controls in patient PBMC (Fig. 1F). The SLC20A2-stimulated phosphate efflux may suggest a cellular adaptation that maintained phosphate homeostasis. Since SLC20A2 and XPR1 are both associated with PFBC and both participate in phosphate fluxes, we first tested whether they balanced each other when overexpressed or down-modulated. To this aim, we used human HAP1 cells in which either SLC20A2 or XPR1 genes were inactivated by genome editing. Knockout of SLC20A2 and XPR1 at the plasma membrane was confirmed by flow cytometry (Fig. 2A) and by cell resistance to infection by enhanced GFP (EGFP) lentiviral vectors pseudotyped with amphotropic murine leukemia virus (A-MLV) or xenotropic murine leukemia virus (X-MLV) Env, respectively (Fig. 2B). Expression of endogenous SLC20A1 and XPR1 in SLC20A2 KO cells remained unchanged (Fig. 2A), although infection assays suggested a slight increase for both transporters (Fig. 2B), while the irrelevant LDL receptor used by the vesicular stomatitis virus G (VSV-G) Env pseudotypes remained unaffected. We also generated SLC20A2-overexpressing HAP1 cells (SLC20A2+ cells) by transduction with a MLV-based retroviral vector carrying the SLC20A2 cDNA. SLC20A2+ cells presented 2.5-fold increases in both binding and infection by A-MLV pseudotype lentiviral vector (Fig. 2, A and B), a small but reproducible decrease in SLC20A1 expression, possibly due to compensation, and no change in XPR1. As expected, XPR1 KO cells showed a strong decrease in phosphate efflux and no impact on uptake, which confirmed the role of XPR1 as the main phosphate exporter (Fig. 2, C and D). Moreover, we found that overexpression of human SLC20A2 led to a significant increase in phosphate uptake, compared with parental cells (Fig. 2C), while depletion of SLC20A2 induced only a modest, although statistically significant, decrease of phosphate uptake (p < 0.05), most likely due to the compensatory activity of SLC20A1. Noticeably, the increase in phosphate uptake in SLC20A2+ cells was concomitant with an increase in phosphate efflux (Fig. 2D), although basal levels of XPR1 in these cells were unchanged (Fig. 2A). Consistent with this induced phosphate efflux, lack of SLC20A2 reduced phosphate efflux dramatically in SLC20A2 KO cells without affecting XPR1 expression at the plasma membrane. This SLC20A2-stimulated phosphate efflux depended on XPR1, since it was observed in HAP1 parental cells but not in XPR1 KO cells (Fig. 2E), despite increased phosphate uptake in both cell types. These data provided evidence that SLC20A2, described only as a sodium-dependent phosphate importer, also modulated XPR1-dependent phosphate efflux when overexpressed or depleted. Consistent with a cellular adaptation that maintains phosphate homeostasis, we found that the intracellular levels of both phosphate and ATP were similar in parental HAP1, SLC20A2+, and SLC20A2 KO cells (Fig. 2, F and G). In contrast, phosphate and ATP levels were significantly elevated in XPR1 KO cells, compared with parental cells (p < 0.001), consistent with a pivotal role for XPR1, and not SLC20A2, as a downstream effector of intracellular phosphate sensing required for phosphate homeostasis in HAP1 cells. Fluctuations of extracellular phosphate were shown to directly affect phosphate efflux (1Giovannini D. Touhami J. Charnet P. Sitbon M. Battini J.L. Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.Cell Rep. 2013; 3 (23791524): 1866-187310.1016/j.celrep.2013.05.035Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and to dictate intracellular levels of phosphate, ATP, and PP-IPs, namely, diphosphoinositol pentakisphosphate (IP7) as two isomers, i.e., 1PP-IP5 (1-IP7) and 5PP-IP5 (5-IP7), and diphosphoinositol tetrakisphosphate (IP8) (42Wils
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