The Impaired Bone Anabolic Effect of PTH in the Absence of Endogenous FGF2 is Partially Due to Reduced ATF4 Expression

Parathyroid hormone (PTH) is currently the only approved anabolic agent for osteoporosis pharmacotherapy in the USA. However, the molecular and cellular mechanisms underlying which intermittent PTH stimulates bone formation are not fully established. Activating transcription factor 4 (ATF4) was recently identified to be a downstream target of PTH signaling in osteoblasts and FGF2 is able to rapidly increase ATF4 mRNA and protein expression in osteoblasts. Furthermore, ATF4 expression is markedly reduced in Fgf2−/− osteoblasts. In addition, FGF2 is required for the anabolic action of PTH on bone formation. Therefore, we hypothesize that the impaired anabolic effect of PTH in Fgf2−/− mice is partially due to reduced ATF4 expression. To test this hypothesis, we examined the ability of PTH to increase ATF4 expression in vitro and in vivo. In vitro data showed that PTH induced a significant increase in ATF4 mRNA expression as early as 15 min in Fgf2+/+ primary bone marrow stromal cells (BMSCs) but not in Fgf2−/− BMSCs. In vivo data showed that treatment with PTH (1–34) (40 μg/kg/d) treatment for 2 weeks in 21–23 months female mice increased lumbar vertebrae bone mineral density in Fgf2+/+ (13.8% increase). In contrast there was a 2.1% decrease in Fgf2−/− mice. Interestingly, basal ATF4 mRNA expression in tibiae was significantly lower in Fgf2−/− mice (46% decrease) compared to Fgf2+/+ mice. PTH treatment increased ATF4 mRNA by 97% (p\u3c 0.05) in Fgf2+/+ compared to 8% (p = 0.57) in Fgf2−/− mice. Immunohistochemistry of vertebrae showed less ATF4 staining in Fgf2−/− tissue, and treatment with PTH increased ATF4 staining in Fgf2+/+ but the increase was attenuated in Fgf2−/− tissue. In summary, reduced ATF4 expression may result in decreased osteoblast differentiation, and possibly contribute to the impaired stimulation of PTH on bone formation in Fgf2−/− mice

FGF2 crosstalk with Wnt signaling in mediating the anabolic action of PTH on bone formation

The mechanisms of the anabolic effect of parathyroid hormone (PTH) in bone are not fully defined. The bone anabolic effects of PTH require fibroblast growth factor 2 (FGF2) as well as Wnt signaling and FGF2 modulates Wnt signaling in osteoblasts. In vivo PTH administration differentially modulated Wnt signaling in bones of wild type (WT) and in mice that Fgf2 was knocked out (Fgf2KO). PTH increased Wnt10b mRNA and protein in WT but not in KO mice. Wnt antagonist SOST mRNA and protein was significantly higher in KO group. However, PTH decreased Sost mRNA significantly in WT as well as in Fgf2KO mice, but to a lesser extent in Fgf2KO. Dickhopf 2 (DKK2) is critical for osteoblast mineralization. PTH increased Dkk2 mRNA in WT mice but the response was impaired in Fgf2KO mice. PTH significantly increased Lrp5 mRNA and phosphorylation of Lrp6 in WT but the increase was markedly attenuated in Fgf2KO mice. PTH increased β-catenin expression and Wnt/β-catenin transcriptional activity significantly in WT but not in Fgf2KO mice. These data suggest that the impaired bone anabolic response to PTH in Fgf2KO mice is partially mediated by attenuated Wnt signaling. Keywords: PTH, FGF2, Wnt signaling, Bon

Prostaglandins differently regulate FGF-2 and FGF receptor expression and induce nuclear translocation in osteoblasts via MAPK kinase

We have previously reported that prostaglandin F-2alpha (PGF(2alpha)) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF(2alpha) or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF(2alpha) stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF(2alpha). Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF2a. Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF(2alpha) exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF(2alpha) stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process

Nuclear Isoforms of Fibroblast Growth Factor 2 Are Novel Inducers of Hypophosphatemia via Modulation of FGF23 and KLOTHO*

FGF2 transgenic mice were developed in which type I collagen regulatory sequences drive the nuclear high molecular weight FGF2 isoforms in osteoblasts (TgHMW). The phenotype of TgHMW mice included dwarfism, decreased bone mineral density (BMD), osteomalacia, and decreased serum phosphate (Pi). When TgHMW mice were fed a high Pi diet, BMD was increased, and dwarfism was partially reversed. The TgHMW phenotype was similar to mice overexpressing FGF23. Serum FGF23 was increased in TgHMW mice. Fgf23 mRNA in bones and fibroblast growth factor receptors 1c and 3c and Klotho mRNAs in kidneys were increased in TgHMW mice, whereas the renal Na+/Pi co-transporter Npt2a mRNA was decreased. Immunohistochemistry and Western blot analyses of TgHMW kidneys showed increased KLOTHO and decreased NPT2a protein. The results suggest that overexpression of HMW FGF2 increases FGF23/FGFR/KLOTHO signaling to down-regulate NPT2a, causing Pi wasting, osteomalacia, and decreased BMD. We assessed whether HMW FGF2 expression was altered in the Hyp mouse, a mouse homolog of the human disease X-linked hypophosphatemic rickets/osteomalacia. Fgf2 mRNA was increased in bones, and Western blots showed increased FGF2 protein in nuclear fractions from osteoblasts of Hyp mice. In addition, immunohistochemistry demonstrated co-localization of FGF23 and HMW FGF2 protein in osteoblasts and osteocytes from Hyp mice. This study reveals a novel mechanism of regulation of the FGF23-Pi homeostatic axis