Fibroblast Growth Factor 23 (FGF23) and Alpha-Klotho Stimulate Osteoblastic MC3T3.E1 Cell Proliferation and Inhibit Mineralization

Abstract

Elevated serum levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) are found in patients with phosphate wasting diseases and chronic kidney disease-mineral and bone disorder (CKD-MBD). These diseases are associated with rickets and renal osteodystrophy, respectively. FGF23 is secreted from osteoblastic cells and signals through FGFRs, membrane coreceptor alpha-Klotho (Klotho), and, possibly, a circulating form of Klotho. Despite the absence of detectable Klotho on osteoblastic cells, studies have suggested that forced FGF23 expression in osteoblasts inhibited mineralization. Thus, we examined the effects of exogenously applied FGF23 on osteoblastic MC3T3.E1 cell proliferation and differentiation, with and without soluble Klotho. MC3T3.E1 cells were cultured in osteoblast differentiation medium, supplemented with FGF23 (0.1–1,000 ng/mL), Klotho (50 ng/mL), the combination FGF23 + Klotho, and FGF2 (100 ng/mL) as a control. Neither FGF23 nor Klotho exposure affected proliferation of day 4 growth phase cells or mineralization of day 14 cultures. In contrast, FGF23 + Klotho resulted in inhibition of mineralization and osteoblast activity markers at day 14, and a slight, reproducible induction of proliferation. Inhibition of FGFR1, but not FGFR2 or FGFR3, completely restored FGF23 + Klotho-induced inhibition of alkaline phosphatase (ALP) activity at day 7. ALP activity was partially restored by the MAPK inhibitor U0126 but not inhibitors p38 and P13K. Thus, soluble Klotho enables FGF23 signaling in MC3T3.E1 cells, likely through FGFR 1(IIIc). Elevated FGF23 actions, in part, appear to parallel FGF2 with lower potency. In addition to affecting bone via indirect phosphate wasting pathways, supraphysiological FGF23 and soluble Klotho may directly impact bone in diseases with elevated FGF23 levels.

FGF23 Suppresses Chondrocyte Proliferation in the Presence of Soluble α-Klotho both in Vitro and in Vivo

“…In line with this, FGF23 alone could not activate ERK or induce Egr1 expression in chondrogenic cells. Since there is an increasing amount of evidence that demonstrates the biological function of sKL in mice (12)(13)(14)(15), we assessed the functional interaction between FGF23 and sKL in chondrogenic cells. In the current study, we utilized ϳ130 kDa of sKL produced by ectodomain FIGURE 8.…”

“…Another levels of examples include the antagonistic effects of sKL on Wnt signaling and TGF-␤ signaling pathways (13,14). Of note is that sKL has also been implicated to mediate FGF23 signaling in cells which do not express membranebound ␣-Klotho (15). Combined together, these lines of evi-dence indicate that FGF23 may have a non-canonical function such that FGF23 can exert its signals in cells that do not express functional membrane-bound ␣-Klotho such as chondrocytes, which may be more relevant in the presence of sKL.…”

Molecular and Biochemical Aspects of Hypophosphatemic Rickets; an Updated Review

“…hgmd.org). 48 Pharmacological inhibition of FGFRs leads to the suppression of FGF23/Klotho signaling and succeeding elevation of 1,25OH 2 D 3 and phosphate. 37,49 As a result, elevated levels of vitamin D (hypervitaminosis D) cause a high level of FGF23 in serum while FGFR inhibitors block transcription of FGF23 in bone which is a dominant process responsible for impinging on systemic levels of FGF23 in patients.…”

“…The intracellular domain of membrane-bound Klotho is detectable in kidney and parathyroid gland, the 2 FGF23 target tissues. 48 The other 2 soluble types are SKL and CKL. However, just CKL has been identified in human.…”

Application of Low-Dose Laser Irradiation and 3D-Observation Methods for Detection and Assay of Features for Cell Differentiation Directions: A Pilot Study

“…In this aspect, the influence of FGFs and their receptors has also been discussed, 3,4 as well as of other components of the extracellular matrix (ECM), as metalloproteinases (MMPs), microtubule-activated protein-kinases (MAPKs), Nuclear Factor kappaB (NF-κB), as well as nuclear factor-induced kinases (NIKs) and IκB kinases (IKKs). 5,6 On the other hand, pro-inflammatory activation by the influence of NF-κB on the expression of appropriate genes in neutrophils has also been suggested. 7,8 Signals from Transforming Growth Factor-beta (TGF-β) have been found to up-regulate Wnt5A expression directly through the Smad-complex, as well as through Smad-induced CUX1 and MAP3K7-mediated NF-κB.…”