Renal transplantation (RTx) is an effective therapy to improve clinical outcomes in pediatric patients with terminal chronic kidney disease. However, chronic immunosuppression with glucocorticoids (GCs) reduces bone growth and BMD. The mechanisms causing GC-induced growth impairment have not been fully clarified. Fibroblast growth factor 23 (FGF23) is a peptide hormone that regulates phosphate homeostasis and bone growth. In pathological conditions, FGF23 excess or abnormal FGF receptors (FGFR) activity leads to bone growth impairment. Experimental data indicate that FGF23 expression is induced by chronic GC exposure. Therefore, we hypothesize that GCs impair bone growth by increasing FGF23 expression, which has direct effects on bone growth plate. In a post hoc analysis of a multicentric randomized clinical trial of prepubertal RTx children treated with early GC withdrawal or chronic GC treatment, we observed that GC withdrawal was associated with improvement in longitudinal growth and BMD, and lower plasma FGF23 levels as compared with a chronic GC group. In prepubertal rats, GC-induced bone growth retardation correlated with increased plasma FGF23 and bone FGF23 expression. Additionally, GC treatment decreased FGFR1 expression whereas it increased FGFR3 expression in mouse tibia explants. The GC-induced bone growth impairment in tibiae explants was prevented by blockade of FGF23 receptors using either a pan-FGFR antagonist (PD173074), a C-terminal FGF23 peptide (FGF23180-205) which blocks the binding of FGF23 to the FGFR-Klotho complex or a specific FGFR3 antagonist (P3). Finally, local administration of PD173074 into the tibia growth plate ameliorated cartilage growth impairment in GC-treated rats. These results show that GC treatment partially reduces longitudinal bone growth via upregulation of FGF23 and FGFR3 expression, thus suggesting that the FGF23/Klotho/FGFR3 axis at the growth plate could be a potential therapeutic target for the management of GC-induced growth impairment in children.Intact plasma FGF23 levels were measured before RTx, and 1 week and 1 year after RTx ( Fig. 2A). Both groups had > 95% decrease in plasma FGF23 1 week after RTx, compared with baseline values, without differences between both groups. One year after RTx, SC patients had 3.2-fold higher plasma FGF23 levels compared with SW patients (1 year SC: 32.2 [24.0 to 45.7] pg/mL; 1 year SW: 10.1 [5.4 to 14.2] pg/mL; p < 0.001). When these FGF23 plasma levels were compared with a control group of healthy children ( Supplemental Table 3), SC patients had increased concentrations, but no significant differences compared with the ◼ 2 DELUCCHI ET AL. Fig. 4. Dexamethasone reduced growth of rat metatarsal explants, via fibroblast growth factor receptors (FGFRs). Prenatal rat metatarsal explants (extracted on E20) were cultured in the presence of dexamethasone (Dex; 1 nM); RU486 (RU; 25 μM), a glucocorticoid receptor antagonist; PD173074 (PD; 100 nM), a pan-FGFR antagonist; recombinant FGF23 (444 pM); or cell culture alone (Control), over ...

Epigenetic control is critical for the regulation of gene transcription in mammalian cells. Among the most important epigenetic mechanisms are those associated with posttranslational modifications of chromosomal histone proteins, which modulate chromatin structure and increased accessibility of promoter regulatory elements for competency to support transcription. A critical histone mark is trimethylation of histone H3 at lysine residue 27 (H3K27me3), which is mediated by Ezh2, the catalytic subunit of the polycomb group complex PRC2 to repress transcription. Treatment of cells with the active vitamin D metabolite 1,25(OH)2D3, results in transcriptional activation of the CYP24A1 gene, which encodes a 24‐hydroxylase enzyme, that is, essential for physiological control of vitamin D3 levels. We report that the Ezh2‐mediated deposition of H3K27me3 at the CYP24A1 gene promoter is a requisite regulatory component during transcriptional silencing of this gene in osteoblastic cells in the absence of 1,25(OH)2D3. 1,25(OH)2D3 dependent transcriptional activation of the CYP24A1 gene is accompanied by a rapid release of Ezh2 from the promoter, together with the binding of the H3K27me3‐specific demethylase Utx/Kdm6a and thereby subsequent erasing of the H3K27me3 mark. Importantly, we find that these changes in H3K27me3 enrichment at the CYP24A1 gene promoter are highly dynamic, as this modification is rapidly reacquired following the withdrawal of 1,25(OH)2D3.

In bone cells vitamin D dependent regulation of gene expression principally occurs through modulation of gene transcription. Binding of the active vitamin D metabolite, 1,25‐dihydroxy vitamin D3 (1,25(OH)2D3) to the vitamin D receptor (VDR) induces conformational changes in its C‐terminal domain enabling competency for interaction with physiologically relevant coactivators, including SRC‐1. Consequently, regulatory complexes can be assembled that support intrinsic enzymatic activities with competency to posttranslationally modify chromatin histones at target genomic sequences to epigenetically alter transcription. Here we examine specific transitions in representation and/or enrichment of epigenetic histone marks during 1,25(OH)2D3 mediated upregulation of CYP24A1 gene expression in osteoblastic cells. This gene encodes the 24‐hydroxylase enzyme, essential for biological control of vitamin D levels. We demonstrate that as the CYP24A1 gene promoter remains transcriptionally silent, there is enrichment of H4R3me2s together with its “writing” enzyme PRMT5 and decreased abundance of the istone H3 and H4 acetylation, H3R17me2a, and H4R3me2a marks as well as of their corresponding “writers.” Exposure of osteoblastic cells to 1,25(OH)2D3 stimulates the recruitment of a VDR/SRC‐1 containing complex to the CYP24A1 promoter to mediate increased H3/H4 acetylation. VDR/SRC‐1 binding occurs concomitant with the release of PRMT5 and the recruitment of the arginine methyltransferases CARM1 and PRMT1 to catalyze the deposition of the H3R17me2a and H4R3me2a marks, respectively. Our results indicate that these dynamic transitions of histone marks at the CYP24A1 promoter, provide a “chromatin context” that is transcriptionally competent for activation of the CYP24A1 gene in osteoblastic cells in response to 1,25(OH)2D3.

The architectural organization of the genome and regulatory proteins within the nucleus supports gene expression in a physiologically-regulated manner. In osteoblastic cells ligand activation induces a nuclear punctate distribution of the 1α,25-dihydroxy vitamin D3 (1α,25(OH)2D3) receptor (VDR) and promotes its interaction with transcriptional coactivators such as SRC-1, NCoA-62/Skip, and DRIP205. Here, we discuss evidence demonstrating that in osteoblastic cells VDR binds to the nuclear matrix fraction in a 1α,25(OH)2D3-dependent manner. This interaction occurs rapidly after exposure to 1α,25(OH)2D3 and does not require a functional VDR DNA binding domain. The nuclear matrix-bound VDR molecules colocalize with the also nuclear matrix-associated coactivator DRIP205. We propose a model where the rapid association of VDR with the nuclear matrix fraction represents an event that follows 1α,25(OH)2D3-dependent nuclear localization of VDR, but that precedes 1α,25(OH)2D3-dependent transcriptional upregulation at target genes.

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