PKC regulates the production of fibroblast growth factor 23 (FGF23)

Abstract

Serine/threonine protein kinase C (PKC) is activated by diacylglycerol that is released from membrane lipids by phospholipase C in response to activation of G protein-coupled receptors or receptor tyrosine kinases. PKC isoforms are particularly relevant for proliferation and differentiation of cells including osteoblasts. Osteoblasts/osteocytes produce fibroblast growth factor 23 (FGF23), a hormone regulating renal phosphate and vitamin D handling. PKC activates NFκB, a transcription factor complex controlling FGF23 expression. Here, we analyzed the impact of PKC on FGF23 synthesis. Fgf23 expression was analyzed by qRT-PCR in UMR106 osteoblast-like cells and in IDG-SW3 osteocytes, and FGF23 protein was measured by ELISA. Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA), a PKC activator, up-regulated FGF23 production. In contrast, PKC inhibitors calphostin C, Gö6976, sotrastaurin and ruboxistaurin suppressed FGF23 formation. NFκB inhibitor withaferin A abolished the stimulatory effect of PMA on Fgf23. PKC is a powerful regulator of FGF23 synthesis, an effect which is at least partly mediated by NFκB.

Fig 1. Expression of Pkc isoforms in UMR106 osteoblast-like cells.

Original agarose gel photo showing Pkcα, -β, -γ, -δ, -ε, -ζ, -η, -θ or -ι specific cDNA in UMR106 cells. NC: non-template control.

Fig 2. PKC activator PMA induces FGF23 production in UMR106 osteoblast-like cells and in IDG-SW3 osteocytes.

Arithmetic means ± SEM (n = 6) of relative Fgf23 mRNA abundance normalized to Tbp in UMR106 osteoblast-like cells (A) or IDG-SW3 osteocytes (C), and FGF23 concentration in the cell culture supernatant of UMR106 cells (B) incubated without (white bars) or with (black bars) 0.1 μM PKC activator PMA. * p < 0.05 indicates significant difference. arb., arbitrary.

Fig 3. PKC inhibitors Calphostin C and Gö6976 decrease FGF23 expression levels in UMR106 osteoblast cells.

UMR106 cells were treated without and with PKC inhibitors Calphostin C (A) or Gö6976 (B, C) at the indicated concentrations. Arithmetic means ± SEM (n = 6) of the relative Fgf23 mRNA abundance in UMR106 cells (A, B). Gene expression was normalized to Tbp as housekeeping gene. Arithmetic means ± SEM (n = 6) of FGF23 protein concentration in the cell culture supernatant (C). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant difference. arb., arbitrary.

Fig 4. PKC inhibition abrogates the PMA-induced increase in Fgf23 gene expression in UMR106 osteoblast-like cells and in IDG-SW3 osteocytes.

Relative Fgf23 transcript levels in UMR106 cells (A,C,D) or in IDG-SW3 cells (B) incubated without or with PMA (0.1 μM, A-D) in the absence and presence of PKCα/β inhibitor Gö6976 (1 μM, A,B), pan PKC inhibitor Sotrastaurin (1 μM, C) or PKCβ inhibitor Ruboxistaurin (1 μM, D). Gene expression was normalized to Tbp as a housekeeping gene, and the values are expressed as arithmetic means ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant difference from vehicle (first bar). ###p < 0.001 indicates significant difference from the absence of PKC inhibitor (second bar vs. fourth bar). arb., arbitrary.

Fig 5. The PKC effect on Fgf23 gene expression is dependent on inflammation.

Relative Fgf23 transcript levels in UMR106 cells incubated without or with PMA (0.1 μM) in the absence and presence of NFκB inhibitor Withaferin A (0.5 μM) (A). Relative Tnfα (B) and Il6 (C) transcript levels in UMR106 cells incubated without or with PMA (0.1 μM). Gene expression was normalized to Tbp as a housekeeping gene, and the values are expressed as arithmetic means ± SEM (n = 6). **p < 0.01, and ***p < 0.001 indicate significant difference from vehicle (first bar). ###p < 0.001 indicates significant difference from the absence of withaferin A (second bar vs. fourth bar). arb., arbitrary.