Inflammation and functional iron deficiency regulate fibroblast growth factor 23 production

Abstract

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with chronic kidney disease (CKD), but the mechanisms are poorly understood. Here we tested whether inflammation and iron deficiency regulate FGF23. In wild-type mice, acute inflammation induced by single injections of heat-killed Brucella abortus or interleukin-1β (IL-1β) decreased serum iron within 6 h, and was accompanied by significant increases in osseous Fgf23 mRNA expression and serum levels of C-terminal FGF23, but no changes in intact FGF23. Chronic inflammation induced by repeated bacteria or IL-1β injections decreased serum iron, increased osseous Fgf23 mRNA, and serum C-terminal FGF23, but modestly increased biologically active, intact FGF23 serum levels. Chronic iron deficiency mimicked chronic inflammation. Increased osseous FGF23 cleavage rather than a prolonged half-life of C-terminal FGF23 fragments accounted for the elevated C-terminal FGF23 but near-normal intact FGF23 levels in inflammation. IL-1β injection increased Fgf23 mRNA and C-terminal FGF23 levels similarly in wildtype and Col4a3(ko) mice with CKD but markedly increased intact FGF23 levels only in the CKD mice. Inflammation increased Fgf23 transcription by activating Hif1α signaling. Thus, inflammation and iron deficiency stimulate FGF23 production. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD.

Keywords: FGF23; anemia; bone; hypoxia; inflammation; mineral metabolism.

Figure 1. Effects of diet-induced iron deficiency on FGF23 regulation

Serum levels of iron (A); ferritin (B); cFGF23 (C); iFGF23 (D); bone and renal expression of Fgf23 mRNA (E); representative immunoblot of bone FGF23 protein expression (F); and bone mRNA expression of the osteoblastic markers, osterix (Sp7) and osteocalcin (Bglap), the osteoclastic marker, cathepsin K (Ctsk), and renal mRNA expression of the vitamin D metabolizing enzymes, Cyp27b1 and Cyp24a1, and the sodium-phosphate co-transporters, Npt2a and Npt2c, in response to low or normal iron diets for 3 weeks (G). Data are presented as mean ± SEM, n ≥ 4/group, * p<0.05 vs. control (Ctr).

Figure 2. Effects of acute inflammation on iron and FGF23 regulation

Acute inflammation was induced by a single injection of Brucella abortus (BA) or IL1-β. Serum levels of iron (A); ferritin (B); cFGF23 (C); iFGF23 (D); bone and renal expression of Fgf23 mRNA (E); representative immunoblot of bone FGF23 protein expression (F); and bone mRNA expression of the osteoblastic markers, osterix (Sp7) and osteocalcin (Bglap), the osteoclastic marker, cathepsin K (Ctsk), and renal mRNA expression of the vitamin D metabolizing enzymes, Cyp27b1 and Cyp24a1, and the sodium-phosphate co-transporters, Npt2a and Npt2c, in response to BA (G). Serum levels of iron (H); ferritin (I); cFGF23 (J); iFGF23 (K); bone and renal expression of Fgf23 mRNA (L); representative immunoblot of bone FGF23 protein expression (M); and bone mRNA expression of Sp7, Bglap, and Ctsk, and renal mRNA expression of Cyp27b1, Cyp24a1, Npt2a and Npt2c in response to IL1-β (N). Data are presented as mean ± SEM, n ≥ 5/group, * p<0.05 vs. control (Ctr).

Figure 3. Effects of chronic inflammation on iron and FGF23 regulation

Serum levels of iron (A); ferritin (B); cFGF23 (C); iFGF23 (D); and representative immunoblot of bone FGF23 protein expression (E) 12 days after a single injection of Brucella abortus (BA). Data are presented as mean ± SEM, n ≥ 3/group,* p<0.05 vs. control (Ctr). Serum levels of iron (F); ferritin (G); cFGF23 (H); iFGF23 (I); representative immunoblot of bone FGF23 protein expression (J); organ-specific expression of Fgf23 mRNA (K); and bone mRNA expression of the osteoblastic markers, osterix (Sp7) and osteocalcin (Bglap), and the osteoclastic marker cathepsin K (Ctsk), and renal mRNA expression of the vitamin D metabolizing enzymes, Cyp27b1 and Cyp24a1, and the sodium-phosphate co-transporters, Npt2a and Npt2c, after 4 days of daily IL1-β injections (L). Data are presented as mean ± SEM, n ≥ 5/group, * p<0.05 vs. control (Ctr).

Figure 4. Effects of acute inflammation on FGF23 production and cleavage in vivo and in vitro

Effects of IL1-β administration in mice pre-treated with or without furin inhibitors on bone expression of Fgf23 mRNA (A); serum levels of cFGF23 (B) and iFGF23 (C); and representative immunoblot of serum FGF23 protein probed with Immutopics (upper panel) and MyBiosource (lower panel) antibodies (D). Data are presented as mean ± SEM, n ≥ 3/group, p<0.05 * vs. untreated control, # vs. 50 ng/g IL1-β treated mice and & vs. furin treated mice. Effects of IL1-β on Fgf23 promoter activity (E); and on intracellular and extracellular FGF23 protein concentrations (F) in MC3T3-E1 cells in the presence or absence of furin inhibitor. Data are presented as mean ± SEM, n ≥ 5/group, p<0.05 * vs. untreated control and & vs. furin-treated cells.

Figure 5. Effects of acute inflammation on FGF23 production and cleavage in the Col4a3^ko mouse model of CKD

Effects of IL1-β on serum levels of BUN (A); iron (B); ferritin (C); bone expression of Fgf23 mRNA (D); and serum cFGF23 (E) and iFGF23 (F) in 6 week-old wild type (WT) and Col4a3 knockout (Col4a3^ko) mice. Data are presented as mean ± SEM, n ≥ 3/group, p<0.05 *vs. age-matched untreated WT, # vs. age-matched IL1-β treated WT and & vs. age-matched untreated Col4a3^ko mice.

Figure 6. The role of Hif1α in the regulation of FGF23 production and cleavage in vitro and in vivo

mRNA expression of Fgf23 (A) and Hif1α (B) and representative immunoblot for nuclear HIF1α protein (C) in MC3T3-E1 and BMSC cells that were stimulated to differentiate into osteoblasts and treated with IL1-β or deferoxamine (DFO). Effects of IL1-β on Fgf23 promoter activity in MC3T3-E1 cells in the presence or absence of the HIF1α inhibitor, 2ME2 (D); and representative immunoblots of nuclear HIF1α, cytoplasmic and secreted FGF23 (E). Data are presented as mean ± SEM, n ≥ 3/group, p<0.05 vs. * control (Ctr), &vs. IL-1β treated. Bone mRNA expression of Hif1α in response to iron deficiency, Brucella abortus (BA) and IL1-β injections (F). Effects of acute inflammation on bone mRNA Fgf23 expression (G) and serum levels of cFGF23 (H); iFGF23 (I) in wild-type mice injected with IL1-β or pre-treated with HIF inhibitors, 2ME2 and BAY 87-2243. Data are presented as mean ± SEM, n ≥ 3/group, p<0.05 * vs. untreated control, # vs. HIF1α inhibitor treated mice and & vs. 50 ng/g IL1-β injected mice. Effects of HIF1α induction by 2 prolyl hydroxylase inhibitors, FG-4592 and BAY 87-2243, on bone mRNA Fgf23 expression (J); serum levels of cFGF23 (K) and iFGF23 (L). Data are presented as mean ± SEM, n ≥ 3/group, p<0.05 * vs. untreated control, # vs. HIF1α agonist treated mice and & vs. furin inhibitor treated mice.

Figure 7. Schematic representation of FGF23 regulation by inflammation

Inflammation induces functional iron deficiency, which stabilizes HIF1α in osteocytes. Inflammatory cytokines also stimulate Hif1α transcription directly, leading to increased cytoplasmic HIF1α, which translocates to the nucleus and binds to HIF1β. Binding of HIF1 heterodimers to hypoxia response elements (HRE) on the Fgf23 promoter stimulates transcription, but the excess FGF23 protein is proteolytically cleaved within osteocytes. In the serum, physiological coupling of FGF23 production and cleavage during acute inflammatory states results in high circulating concentrations of C-terminal FGF23 fragments that can be detected with C-terminal FGF23 assays, but normal levels of biologically active FGF23 levels as demonstrated by intact FGF23 assays. In chronic inflammation, cFGF23 levels remain increased compared to baseline, but to a lesser extent than in acute inflammation. Sustained FGF23 overproduction during chronic inflammation also leads to increased serum levels of biologically active FGF23, perhaps due to saturation or partial down regulation of the FGF23 cleavage process within osteocytes.