The hypoxia-inducible factor-1α activates ectopic production of fibroblast growth factor 23 in tumor-induced osteomalacia

Abstract

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome in which ectopic production of fibroblast growth factor 23 (FGF23) by non-malignant mesenchymal tumors causes phosphate wasting and bone fractures. Recent studies have implicated the hypoxia-inducible factor-1α (HIF-1α) in other phosphate wasting disorders caused by elevated FGF23, including X-linked hypophosphatemic rickets and autosomal dominant hypophosphatemia. Here we provide evidence that HIF-1α mediates aberrant FGF23 in TIO by transcriptionally activating its promoter. Immunohistochemical studies in phosphaturic mesenchymal tumors resected from patients with documented TIO showed that HIF-1α and FGF23 were co-localized in spindle-shaped cells adjacent to blood vessels. Cultured tumor tissue produced high levels of intact FGF23 and demonstrated increased expression of HIF-1α protein. Transfection of MC3T3-E1 and Saos-2 cells with a HIF-1α expression construct induced the activity of a FGF23 reporter construct. Prior treatment of tumor organ cultures with HIF-1α inhibitors decreased HIF-1α and FGF23 protein accumulation and inhibited HIF-1α-induced luciferase reporter activity in transfected cells. Chromatin immunoprecipitation assays confirmed binding to a HIF-1α consensus sequence within the proximal FGF23 promoter, which was eliminated by treatment with a HIF-1α inhibitor. These results show for the first time that HIF-1α is a direct transcriptional activator of FGF23 and suggest that upregulation of HIF-1α activity in TIO contributes to the aberrant FGF23 production in these patients.

Figure 1

Colocalization of HIF-1α and FGF23 in tumors from patients with TIO. (a, b) Serial five micron sections from paraffin-embedded tumor tissue stained with H&E or processed for immunohistochemistry using fluorescent tagged antibodies as indicated. In both tumors, abundant FGF23 (red) was detected in the spindle-shaped cells adjacent to blood vessels and was observed to colocalize (white arrows) with HIF-1α immunoreactivity (green). Osterix immunoreactivity (blue) was observed in populations of cells adjacent but distinct from those expressing FGF23 and HIF-1α (a, far right). FGF23, fibroblast growth factor 23; H&E, hematoxylin and eosin; HIF-1α, hypoxia-inducible factor-1α; TIO, tumor-induced osteomalacia.

Figure 2

HIF-1α expression in tumors from patients with TIO is inhibited by treatment with digoxin. Tumors resected from patients with proven TIO were cultured in vitro and HIF-1α protein was measured before and after treatment with digoxin. (a) HIF-1α protein expression analyzed by immunoblotting of cultured tumor tissue in the presence or absence of 200 or 400 μmol·L⁻¹ digoxin. (b, c) Intact Immunoreactive FGF23 concentrations measured in conditioned medium from two different tumors grown in organ culture in the absence (0) or presence of 200 or 400 μmol·L⁻¹ digoxin. FGF23, fibroblast growth factor 23; HIF-1α, hypoxia-inducible factor-1α; TIO, tumor-induced osteomalacia. *P<0.05.

Figure 3

Regulation of FGF23 gene expression by HIF-1α. (a) FGF23 promoter activity in Saos-2 and MC3T3-E1 osteoblasts co-transfected with a HIF-1α DNA (0.25 μg). (b) Effect of L-mimosine on HIF-1α, induced FGF23 promoter activity in MC3T3-E1 osteoblasts. (c) Effect of Bay87–2243 on HIF-1α induced FGF23 promoter luciferase activity in MC3T3-E1 osteoblasts. (d) ChIP analysis of HIF-1α occupancy at endogenous consensus elements within the FGF23 promoter in MC3T3-E1 cells. Lane 1. 100 bp DAN ladder; Lane 2. Input ChIP DAN; Lane 3. H3-positive control; Lane 4. Non-specific IgG; Lane 5. Untreated control; Lane 6. Co-transfection with HIF-1α; Lane 7. Extracts from Bay87–2243 treated cells showing inhibition of HIF-1α binding to the endogenous FGF23 promoter; Lane 8. Extracts from L-mimosine treated cells showing increased HIF-1α binding to the endogenous FGF23 promoter. Data are expressed as the mean±standard error (n=3) from three separate experiments. FGF23, fibroblast growth factor 23; HIF-1α, hypoxia-inducible factor-1α. *P<0.05, **P<0.005.