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

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 Recep...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2020-02, Vol.117 (7), p.3568-3574
Main Authors: Li, Xingyao, Gu, Chunfang, Hostachy, Sarah, Sahu, Soumyadip, Wittwer, Christopher, Jessen, Henning J., Fiedler, Dorothea, Wang, Huanchen, Shears, Stephen B.
Format: Article
Language:English
Subjects:
Biological Sciences
Biomedical materials
Bones
Calcification
Calcification (ectopic)
Calorimetry
Cellular structure
Diet
Efflux
Epistasis
Flavonoids
Fluxes
Homeostasis
Inositol
Kinases
Metabolism
Mineralization
Mutation
N-Terminus
Organic compounds
Osteosarcoma
Restoration
Signaling
Teeth
Titration
Titration calorimetry
Transfection
Xenotropic
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1908830117