Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia

Abstract

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.

Figure 1

(A) Amino acid sequence of human FGF23. The signal peptide sequence is underlined. The consensus proteolytic cleavage site is boxed. (B) Expression profile of FGF23 in adult normal tissues analyzed by reverse transcriptase–PCR followed by Southern blotting. Template cDNAs are as follows: lanes 1, bone marrow; 2, brain; 3, colon; 4, heart; 5, kidney; 6, leukocyte; 7, liver; 8, lung; 9, lymph nude; 10, muscle; 11, ovary; 12, pancreas; 13, placenta; 14, prostate; 15, small intestine; 16, spleen; 17, testis; 18, thymus; 19, tonsil; 20, TIO tumor. (C) Western blotting with anti-His (c-term) antibody that recognizes the carboxyl-terminal tag sequence of recombinant FGF23 proteins secreted into media by CHO-FGF23 cells.

Figure 2

Effects of recombinant FGF23. The changes of serum phosphate (A) and calcium (B) concentrations in mice treated with purified recombinant protein (n = 6; filled columns) or vehicle (n = 6; open columns). FGF23 was i.p. injected three times with intervals of 5 h. Blood samples were collected at 12 and 24 h after the first injection. Each column represents the mean ± SEM. *, P < 0.05; **, P < 0.001 by Student's t test.

Figure 3

Biochemical changes in mice transplanted with CHO cells producing FGF23. Time course of changes of serum phosphate level (A), renal phosphate excretion index (B), and serum alkaline phosphatase activity (C) in nude mice bearing CHO-FGF23 cells (n = 6; ▴) were compared with those in mice with wild-type CHO cells (n = 6; ■) and control nude mice without CHO cells (n = 6; ○). Blood and 24-h urine samples were collected on days 1, 6, 20, and 45 after the implantation. Renal phosphate excretion index was urinary phosphate divided by serum phosphate and urinary creatinine. Data are means ± SEM at each time point. Significant difference from control group by one-way ANOVA followed by Dunnett's multiple comparison test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4

Expression of renal 1α- OHase in mice implanted with CHO cells or CHO-FGF23 cells. Vehicle-treated mice (n = 3), control CHO-implanted mice, and CHO-FGF23-implanted mice were killed on day 3 after implantation. Total RNAs (20 μg each) were used for the determination of expression level of 1α-OHase by Northern blot analysis. Expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) also was determined by using the same blot.

Figure 5

(A) Typical appearance of a mouse bearing CHO-FGF23 cell tumors. (B) Whole skeletal soft roentgenogram of a mouse bearing wild-type CHO cell tumors (Left) and a mouse bearing CHO-FGF23 cell tumors (Right) on day 45 after implantation. (C) Soft roentgenogram of femurs isolated from control mice (Left) and mice bearing CHO-FGF23 cell tumors (Right). (D) Histological appearance of proximal metaphysis of tibia from a control mouse (Upper) and a mouse bearing CHO-FGF23 (Lower) on day 45. The undecalcified tissue sections were subjected to the Villanueva bone stain and the Villanueva-Goldner stain to discriminate mineralized bone tissues (green) from osteoid tissues (pink).