Control of XPR1-dependent cellular phosphate efflux by InsP8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling

Abstract

Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP₈). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP₈ synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP₈) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP₈ levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP₈; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP₈ to the XPR1 N-terminus (K_d = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification.

Keywords: inositol; metabolism; signaling.

Fig. 1.

Reduced XPR1-mediated Pi efflux from HCT116 cells after either PPIP5K KO or XPR1 KO. (A) The proposed (17) cyclical pathway of PP-InsP turnover involving IP6K, PPIP5Ks (denoted by twin arrows because the enzyme has separate 1-kinase and 1-phosphatase activities), and diphosphoinositol polyphosphate phosphatases (DIPP). Carbons in InsP₆ are labeled at positions 1 and 5. (B) Net [*]Pi uptake by WT and PPIP5K KO HCT116 cells. (C) Graphic depicting the timeline of the [*]Pi efflux assays. The [*]Pi efflux data are normalized (except in D) to total cell [*]Pi at zero time determined after cell lysis. (D) [*]Pi efflux from WT and PPIP5K KO HCT116 cells plotted as decreasing amounts of cell [*]Pi; CPM, counts per minute. (E) Total Pi in WT and PPIP5K HCT116 KO cells. (F and G) [*]Pi influx into WT and PPIP5K HCT116 KO cells, respectively, incubated with extracellular Pi at 1 mM (red), 2.5 mM (green), or 5 mM (blue). (H and I) [*]Pi influx into WT and PPIP5K HCT116 KO cells, respectively, incubated with extracellular Pi at 1 mM (red), 2.5 mM (green), or 5 mM (blue). (J) Western analysis of XPR1 expression in WT and XPR1 KO HCT116 cells. (K) WT and XPR1 KO HCT116 cells were labeled with [*]Pi for 20 min, and then, total [*]Pi was determined. (L) [*]Pi efflux from WT and XPR1 KO HCT116 cells prelabeled as in K. Statistical significance is indicated as follows. *P < 0.05; ***P < 0.001.

Fig. 2.

Rescue of Pi efflux from PPIP5K KO HCT116 cells. (A) Graphic depicting the various procedures used to rescue Pi efflux from PPIP5K KO cells: inhibition of IP6K by either TNP or flavonoids; liposomal delivery of PCP analogs of PP-InsPs; and stable transfection of WT PPIP5K1 (InsP₇ kinase/InsP₈ phosphatase plus an intrinsically disordered region [IDR]), the D332A mutant (no kinase activity), or the “hyperkinase” R399A mutant (no phosphatase activity). (B) Total [*]Pi uptake after 20 min of labeling of WT cells, PPIP5K KO cells, and the KO cells stably transfected with either PPIP5K1 or one of the indicated mutants. (C) [*]Pi efflux from cells prelabeled with [*]Pi as described in B. (D) InsP₈ levels in WT cells plus or minus 24 h of treatment with 10 µM TNP. (E) Following TNP treatment (as in D), WT and PPIP5K KO cells were incubated with [*]Pi for 20 min, and then, total [*]Pi was determined. (F) Cells were first treated with TNP for 24 h and then, prelabeled with [*]Pi. (G) Cells were treated with TNP and prelabeled with [*]Pi as described in F, and then [*]Pi efflux was determined. (H) PPIP5K KO cells were incubated for 3 h with either empty liposomes or liposomes containing PCP-InsP₈ at the indicated dilution. Then, [*]Pi was added for 20 min, and total [*]Pi was determined. (I) Pi efflux from PPIP5K KO cells treated with liposomes (plus or minus PCP-InsP₈) and prelabeled with [*]Pi, all as described in H. Statistical significance is indicated as follows. *P < 0.05; **P < 0.02; ***P < 0.001; NS, not significant.

Fig. 3.

A regulatory interaction of InsP₈ with XPR1 contributes to the effects of PPIP5K KO on Pi fluxes and biomineralization by Saos-2 cells. (A–C) Representative (of three to four) thermograms for titration of the XPR1-SPX domain with (A) 5-InsP₇ (stoichiometry = 1.11 ± 0.05), (B) 1-InsP₇ (stoichiometry = 0.88 ± 0.01), or (C) InsP₈ (stoichiometry = 1.07 ± 0.05). (D) Western analysis of WT, XPR1 KO, and XPR1 KO + PPIP5K KO Saos-2 cells. (E) Saos-2 cells were incubated with [*]Pi for 20 min, and then, net [*Pi] uptake was determined. (F) [*]Pi efflux from WT and PPIP5K KO Saos-2 cells prelabeled as in E. (G) Alizarin Red S staining is shown for WT, PPIP5K KO cells, XPR1 KO cells, or PPIP5K KO + XPR1 KO cells cultured in osteoblastic medium for the indicated times. The area covered by each individual field of view is 0.15 mm². (H) Graphic describing InsP₈-mediated regulation of XPR1-dependent Pi efflux in the context of XPR1 restraining the availability of cellular Pi for stimulation of Pi-dependent matrix vesicle secretion for biomineralization as previously reported (28). Statistical significance is indicated as follows. ***P < 0.001.