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. 1997 Aug 1;100(3):538–549. doi: 10.1172/JCI119563

Regulation of sodium-dependent phosphate transport in osteoclasts.

A Gupta 1, X L Guo 1, U M Alvarez 1, K A Hruska 1
PMCID: PMC508220  PMID: 9239400

Abstract

Osteoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.

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Selected References

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  1. Alvarez J. I., Teitelbaum S. L., Blair H. C., Greenfield E. M., Athanasou N. A., Ross F. P. Generation of avian cells resembling osteoclasts from mononuclear phagocytes. Endocrinology. 1991 May;128(5):2324–2335. doi: 10.1210/endo-128-5-2324. [DOI] [PubMed] [Google Scholar]
  2. Biber J., Custer M., Werner A., Kaissling B., Murer H. Localization of NaPi-1, a Na/Pi cotransporter, in rabbit kidney proximal tubules. II. Localization by immunohistochemistry. Pflugers Arch. 1993 Aug;424(3-4):210–215. doi: 10.1007/BF00384344. [DOI] [PubMed] [Google Scholar]
  3. Blair H. C., Teitelbaum S. L., Ghiselli R., Gluck S. Osteoclastic bone resorption by a polarized vacuolar proton pump. Science. 1989 Aug 25;245(4920):855–857. doi: 10.1126/science.2528207. [DOI] [PubMed] [Google Scholar]
  4. Borisy G. G., Taylor E. W. The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein. J Cell Biol. 1967 Aug;34(2):525–533. doi: 10.1083/jcb.34.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brazy P. C., Mandel L. J., Gullans S. R., Soltoff S. P. Interactions between phosphate and oxidative metabolism in proximal renal tubules. Am J Physiol. 1984 Oct;247(4 Pt 2):F575–F581. doi: 10.1152/ajprenal.1984.247.4.F575. [DOI] [PubMed] [Google Scholar]
  6. Caverzasio J., Selz T., Bonjour J. P. Characteristics of phosphate transport in osteoblastlike cells. Calcif Tissue Int. 1988 Aug;43(2):83–87. doi: 10.1007/BF02555151. [DOI] [PubMed] [Google Scholar]
  7. Cheatham B., Vlahos C. J., Cheatham L., Wang L., Blenis J., Kahn C. R. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol. 1994 Jul;14(7):4902–4911. doi: 10.1128/mcb.14.7.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Custer M., Lötscher M., Biber J., Murer H., Kaissling B. Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry. Am J Physiol. 1994 May;266(5 Pt 2):F767–F774. doi: 10.1152/ajprenal.1994.266.5.F767. [DOI] [PubMed] [Google Scholar]
  9. Dodds R. A., Gowen M., Bradbeer J. N. Microcytophotometric analysis of human osteoclast metabolism: lack of activity in certain oxidative pathways indicates inability to sustain biosynthesis during resorption. J Histochem Cytochem. 1994 May;42(5):599–606. doi: 10.1177/42.5.8157931. [DOI] [PubMed] [Google Scholar]
  10. Garrett I. R., Boyce B. F., Oreffo R. O., Bonewald L., Poser J., Mundy G. R. Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo. J Clin Invest. 1990 Mar;85(3):632–639. doi: 10.1172/JCI114485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gupta A., Edwards J. C., Hruska K. A. Cellular distribution and regulation of NHE-1 isoform of the NA-H exchanger in the avian osteoclast. Bone. 1996 Feb;18(2):87–95. doi: 10.1016/8756-3282(95)00455-6. [DOI] [PubMed] [Google Scholar]
  12. Gupta A., Miyauchi A., Fujimori A., Hruska K. A. Phosphate transport in osteoclasts: a functional and immunochemical characterization. Kidney Int. 1996 Apr;49(4):968–974. doi: 10.1038/ki.1996.137. [DOI] [PubMed] [Google Scholar]
  13. Hayes G., Busch A., Lötscher M., Waldegger S., Lang F., Verrey F., Biber J., Murer H. Role of N-linked glycosylation in rat renal Na/Pi-cotransport. J Biol Chem. 1994 Sep 30;269(39):24143–24149. [PubMed] [Google Scholar]
  14. Hruska K. A., Rolnick F., Huskey M., Alvarez U., Cheresh D. Engagement of the osteoclast integrin alpha v beta 3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity. Endocrinology. 1995 Jul;136(7):2984–2992. doi: 10.1210/endo.136.7.7540546. [DOI] [PubMed] [Google Scholar]
  15. Kavanaugh M. P., Kabat D. Identification and characterization of a widely expressed phosphate transporter/retrovirus receptor family. Kidney Int. 1996 Apr;49(4):959–963. doi: 10.1038/ki.1996.135. [DOI] [PubMed] [Google Scholar]
  16. Loghman-Adham M., Motock G. T., Wilson P., Levi M. Characterization of Na(+)-phosphate cotransporters in renal cortical endosomes. Am J Physiol. 1995 Jul;269(1 Pt 2):F93–102. doi: 10.1152/ajprenal.1995.269.1.F93. [DOI] [PubMed] [Google Scholar]
  17. Magagnin S., Werner A., Markovich D., Sorribas V., Stange G., Biber J., Murer H. Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5979–5983. doi: 10.1073/pnas.90.13.5979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nakamura I., Takahashi N., Sasaki T., Jimi E., Kurokawa T., Suda T. Chemical and physical properties of the extracellular matrix are required for the actin ring formation in osteoclasts. J Bone Miner Res. 1996 Dec;11(12):1873–1879. doi: 10.1002/jbmr.5650111207. [DOI] [PubMed] [Google Scholar]
  19. Okada T., Sakuma L., Fukui Y., Hazeki O., Ui M. Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. J Biol Chem. 1994 Feb 4;269(5):3563–3567. [PubMed] [Google Scholar]
  20. Rowe D. J., Hausmann E. The effects of calcitonin and colchicine on the cellular response to diphosphonate. Br J Exp Pathol. 1980 Jun;61(3):303–309. [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sasaki T., Debari K., Udagawa N. Cytochalasin D reduces osteoclastic bone resorption by inhibiting development of ruffled border-clear zone complex. Calcif Tissue Int. 1993 Sep;53(3):217–221. doi: 10.1007/BF01321841. [DOI] [PubMed] [Google Scholar]
  23. Selz T., Caverzasio J., Bonjour J. P. Regulation of Na-dependent Pi transport by parathyroid hormone in osteoblast-like cells. Am J Physiol. 1989 Jan;256(1 Pt 1):E93–100. doi: 10.1152/ajpendo.1989.256.1.E93. [DOI] [PubMed] [Google Scholar]
  24. Shioi A., Ross F. P., Teitelbaum S. L. Enrichment of generated murine osteoclasts. Calcif Tissue Int. 1994 Nov;55(5):387–394. doi: 10.1007/BF00299320. [DOI] [PubMed] [Google Scholar]
  25. Szczepanska-Konkel M., Yusufi A. N., VanScoy M., Webster S. K., Dousa T. P. Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane. J Biol Chem. 1986 May 15;261(14):6375–6383. [PubMed] [Google Scholar]
  26. Tezuka K., Tezuka Y., Maejima A., Sato T., Nemoto K., Kamioka H., Hakeda Y., Kumegawa M. Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts. J Biol Chem. 1994 Jan 14;269(2):1106–1109. [PubMed] [Google Scholar]
  27. Thelen M., Wymann M. P., Langen H. Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4960–4964. doi: 10.1073/pnas.91.11.4960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ullrich K. J., Murer H. Sulphate and phosphate transport in the renal proximal tubule. Philos Trans R Soc Lond B Biol Sci. 1982 Dec 1;299(1097):549–558. doi: 10.1098/rstb.1982.0151. [DOI] [PubMed] [Google Scholar]
  29. Verri T., Markovich D., Perego C., Norbis F., Stange G., Sorribas V., Biber J., Murer H. Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate. Am J Physiol. 1995 Apr;268(4 Pt 2):F626–F633. doi: 10.1152/ajprenal.1995.268.4.F626. [DOI] [PubMed] [Google Scholar]
  30. Werner A., Moore M. L., Mantei N., Biber J., Semenza G., Murer H. Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9608–9612. doi: 10.1073/pnas.88.21.9608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Werner A., Murer H., Kinne R. K. Cloning and expression of a renal Na-Pi cotransport system from flounder. Am J Physiol. 1994 Aug;267(2 Pt 2):F311–F317. doi: 10.1152/ajprenal.1994.267.2.F311. [DOI] [PubMed] [Google Scholar]
  32. Yusufi A. N., Szczepanska-Konkel M., Kempson S. A., McAteer J. A., Dousa T. P. Inhibition of human renal epithelial Na+/Pi cotransport by phosphonoformic acid. Biochem Biophys Res Commun. 1986 Sep 14;139(2):679–686. doi: 10.1016/s0006-291x(86)80044-4. [DOI] [PubMed] [Google Scholar]
  33. van Deurs B., Hansen S. H., Petersen O. W., Melby E. L., Sandvig K. Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium. Eur J Cell Biol. 1990 Feb;51(1):96–109. [PubMed] [Google Scholar]

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