BIOLOGICAL ACTIVITY OF FGF-23 FRAGMENTS

Theresa Berndt

Theodore Craig

Daniel McCormick

Beate Lanske

Stephen O’Brien+2 authors

^{Department of Internal Medicine, Mayo Clinic College of Medicine, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA}

^{Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA}

^{Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA}

^{Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave., Boston, MA 02115}

^{Receptor Ligand Therapeutics, Endocrine and Renal Sciences, Genzyme Corp., 1 Mountain Rd, Framingham, Massachusetts 01701, USA}

Address correspondence to: Rajiv Kumar, MS 1-120, Mayo Clinic Rochester, Mayo College of Medicine, 200 First Street SW, Rochester, MN 55905, USA, Phone 507-284-0020, Fax 507-538-3954, ude.oyam@ramukr

SUMMARY

The phosphaturic activity of intact, full-length, fibroblast growth factor-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein, and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23, and FGF-23 176-251, significantly and equivalently increased fractional phosphate excretion (FE Pi)) from 14±3 to 32±5% and 15±2 to 33±2% (p <0.001), respectively. Chronic administration of FGF-23 176-251 reduced serum Pi and serum concentrations of 1α,25-dihydroxyvitamin D. Additionally, FGF-23 176-251 reduced serum Pi when administered intraperitoneally to hyperphosphatemic Fgf23^−/− mice. Shorter forms of FGF-23 (FGF-23 180-251 and FGF-23 184-251) retained phosphaturic activity. Further shortening of the FGF-23 carboxyl-terminal domain, however, abolished phosphaturic activity, as infusion of FGF-23 206-251 did not increase urinary phosphate excretion. Infusion of a short fragment of the FGF-23 molecule, FGF-23 180-205, significantly increased FE Pi in rats and reduced serum Pi in hyperphosphatemic Fgf23^−/− mice. The activity of FGF-23 180-251 was confirmed in OK cells in which the peptide reduced Na^{-dependent Pi uptake and enhanced internalization of the Na}^{-Pi IIa co-transporter. We conclude that carboxyl terminal fragments of FGF-23 are phosphaturic, and that a short, 26 amino acid fragment of FGF-23 retains significant phosphaturic activity.}

Keywords: FGF-23, rat, phosphate, kidney, 1α,25(OH)₂D

SUMMARY

REFERENCES

References

1. Cancilla B, Davies A, Cauchi JA, Risbridger GP, Bertram JFFibroblast growth factor receptors and their ligands in the adult rat kidney. Kidney Int. 2001;60:147–155.[PubMed][Google Scholar]
2. Cancilla B, Ford-Perriss MD, Bertram JFExpression and localization of fibroblast growth factors and fibroblast growth factor receptors in the developing rat kidney. Kidney Int. 1999;56:2025–2039.[PubMed][Google Scholar]
3. Braun S, auf dem Keller U, Steiling H, Werner SFibroblast growth factors in epithelial repair and cytoprotection. Philos Trans R Soc Lond B Biol Sci. 2004;359:753–757.[Google Scholar]
4. Steiling H, Werner SFibroblast growth factors: key players in epithelial morphogenesis, repair and cytoprotection. Curr Opin Biotechnol. 2003;14:533–537.[PubMed][Google Scholar]
5. Eswarakumar VP, Lax I, Schlessinger JCellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 2005;16:139–149.[PubMed][Google Scholar]
6. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet. 2000;26:345–348.[PubMed]
7. White KE, Jonsson KB, Carn G, Hampson G, Spector TD, Mannstadt M, Lorenz-Depiereux B, Miyauchi A, Yang IM, Ljunggren O, Meitinger T, Strom TM, Juppner H, Econs MJThe autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting. J Clin Endocrinol Metab. 2001;86:497–500.[PubMed][Google Scholar]
8. Bowe AE, Finnegan R, Jan de Beur SM, Cho J, Levine MA, Kumar R, Schiavi SCFGF-23 inhibits renal tubular phosphate transport and is a PHEX substrate. Biochem Biophys Res Commun. 2001;284:977–981.[PubMed][Google Scholar]
9. White KE, Carn G, Lorenz-Depiereux B, Benet-Pages A, Strom TM, Econs MJAutosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. Kidney Int. 2001;60:2079–2086.[PubMed][Google Scholar]
10. Fukumoto S, Yamashita TFibroblast growth factor-23 is the phosphaturic factor in tumor-induced osteomalacia and may be phosphatonin. Curr Opin Nephrol Hypertens. 2002;11:385–389.[PubMed][Google Scholar]
11. Shimada T, Muto T, Urakawa I, Yoneya T, Yamazaki Y, Okawa K, Takeuchi Y, Fujita T, Fukumoto S, Yamashita TMutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo. Endocrinology. 2002;143:3179–3182.[PubMed][Google Scholar]
12. Yamazaki Y, Okazaki R, Shibata M, Hasegawa Y, Satoh K, Tajima T, Takeuchi Y, Fujita T, Nakahara K, Yamashita T, Fukumoto SIncreased circulatory level of biologically active full-length FGF-23 in patients with hypophosphatemic rickets/osteomalacia. J Clin Endocrinol Metab. 2002;87:4957–4960.[PubMed][Google Scholar]
13. Riminucci M, Collins MT, Fedarko NS, Cherman N, Corsi A, White KE, Waguespack S, Gupta A, Hannon T, Econs MJ, Bianco P, Gehron Robey PFGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. J Clin Invest. 2003;112:683–692.[Google Scholar]
14. Berndt TJ, Schiavi S, Kumar R“Phosphatonins“ and the regulation of phosphorus homeostasis. Am J Physiol Renal Physiol. 2005;289:F1170–1182.[PubMed][Google Scholar]
15. Schiavi SC, Kumar RThe phosphatonin pathway: new insights in phosphate homeostasis. Kidney Int. 2004;65:1–14.[PubMed][Google Scholar]
16. Schiavi SC, Moe OWPhosphatonins: a new class of phosphate-regulating proteins. Curr Opin Nephrol Hypertens. 2002;11:423–430.[PubMed][Google Scholar]
17. Benet-Pages A, Orlik P, Strom TM, Lorenz-Depiereux BAn FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet. 2005;14:385–390.[PubMed][Google Scholar]
18. Sitara D, Razzaque MS, Hesse M, Yoganathan S, Taguchi T, Erben RG, Juppner H, Lanske BHomozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol. 2004;23:421–432.[Google Scholar]
19. Sitara D, Razzaque MS, St-Arnaud R, Taguchi T, Erben RG, Lanske BGenetic ablation of vitamin D activation pathway reverses biochemical and skeletal anomalies in Fgf-23 null animals, In Press. American Journal of Pathology. 2006[Google Scholar]
20. Yamashita T, Yoshioka M, Itoh NIdentification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun. 2000;277:494–498.[PubMed][Google Scholar]
21. Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, Takeuchi Y, Fujita T, Nakahara K, Fukumoto S, Yamashita TFGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res. 2004;19:429–435.[PubMed][Google Scholar]
22. Shimada T, Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, Takeuchi Y, Fujita T, Fukumoto S, Yamashita TCloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci U S A. 2001;98:6500–6505.[Google Scholar]
23. Shimada T, Urakawa I, Yamazaki Y, Hasegawa H, Hino R, Yoneya T, Takeuchi Y, Fujita T, Fukumoto S, Yamashita TFGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa. Biochem Biophys Res Commun. 2004;314:409–414.[PubMed][Google Scholar]
24. Larsson T, Marsell R, Schipani E, Ohlsson C, Ljunggren O, Tenenhouse HS, Juppner H, Jonsson KBTransgenic mice expressing fibroblast growth factor 23 under the control of the alpha1(I) collagen promoter exhibit growth retardation, osteomalacia, and disturbed phosphate homeostasis. Endocrinology. 2004;145:3087–3094.[PubMed][Google Scholar]
25. Bai X, Miao D, Li J, Goltzman D, Karaplis ACTransgenic mice overexpressing human fibroblast growth factor 23 (R176Q) delineate a putative role for parathyroid hormone in renal phosphate wasting disorders. Endocrinology. 2004;145:5269–5279.[PubMed][Google Scholar]
26. Shimada T, Kakitani M, Yamazaki Y, Hasegawa H, Takeuchi Y, Fujita T, Fukumoto S, Tomizuka K, Yamashita TTargeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest. 2004;113:561–568.[Google Scholar]
27. Razzaque MS, Sitara D, Taguchi T, St-Arnaud R, Lanske BPremature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. Faseb J. 2006;20:720–722.[Google Scholar]
28. Bai XY, Miao D, Goltzman D, Karaplis ACThe autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency. J Biol Chem. 2003;278:9843–9849.[PubMed][Google Scholar]
29. Topaz O, Shurman DL, Bergman R, Indelman M, Ratajczak P, Mizrachi M, Khamaysi Z, Behar D, Petronius D, Friedman V, Zelikovic I, Raimer S, Metzker A, Richard G, Sprecher EMutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat Genet. 2004;36:579–581.[PubMed][Google Scholar]
30. Razzaque MS, Lanske BHypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice. Trends Mol Med. 2006;12:298–305.[PubMed][Google Scholar]
31. Yu X, Ibrahimi OA, Goetz R, Zhang F, Davis SI, Garringer HJ, Linhardt RJ, Ornitz DM, Mohammadi M, White KEAnalysis of the biochemical mechanisms for the endocrine actions of fibroblast growth factor-23. Endocrinology. 2005;146:4647–4656.[Google Scholar]
32. Yan X, Yokote H, Jing X, Yao L, Sawada T, Zhang Y, Liang S, Sakaguchi KFibroblast growth factor 23 reduces expression of type IIa Na+/Pi co-transporter by signaling through a receptor functionally distinct from the known FGFRs in opossum kidney cells. Genes Cells. 2005;10:489–502.[PubMed][Google Scholar]
33. Yamashita T, Konishi M, Miyake A, Inui K, Itoh NFibroblast growth factor (FGF)-23 inhibits renal phosphate reabsorption by activation of the mitogen-activated protein kinase pathway. J Biol Chem. 2002;277:28265–28270.[PubMed][Google Scholar]
34. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o MRegulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem. 2006;281:6120–6123.[Google Scholar]
35. Campos M, Couture C, Hirata IY, Juliano MA, Loisel TP, Crine P, Juliano L, Boileau G, Carmona AKHuman recombinant endopeptidase PHEX has a strict S1′ specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein. Biochem J. 2003;373:271–279.[Google Scholar]
36. Poduslo JF, Curran GL, Peterson JA, McCormick DJ, Fauq AH, Khan MA, Wengenack TMDesign and chemical synthesis of a magnetic resonance contrast agent with enhanced in vitro binding, high blood-brain barrier permeability, and in vivo targeting to Alzheimer’s disease amyloid plaques. Biochemistry. 2004;43:6064–6075.[Google Scholar]
37. Berndt TJ, Bielesz B, Craig TA, Tebben PJ, Bacic D, Wagner CA, O’Brien S, Schiavi S, Biber J, Murer H, Kumar RSecreted frizzled-related protein-4 reduces sodium-phosphate co-transporter abundance and activity in proximal tubule cells. Pflugers Arch. 2006;451:579–587.[PubMed][Google Scholar]
38. Chen P, Toribara T, Warnner HMicrodetermination of phosphorus. Anal Chem. 1956;28:1756–1758.[PubMed][Google Scholar]
39. Führ J, Kazmarczyk J, Krüttgen CDEine einfache colorimetrische Methode zur Inulin-Bestimmung für Nieren-clearance-untersuchungen bei StoffwechselGesunden und Diabetikern. Klin. Wochenschr. 1955;33:729–730.[PubMed][Google Scholar]
40. Kumar R, Harnden D, DeLuca HFMetabolism of 1,25-dihydroxyvitamin D3: evidence for side-chain oxidation. Biochemistry. 1976;15:2420–2423.[PubMed][Google Scholar]