Increased Circulating FGF23 Does Not Lead to Cardiac Hypertrophy in the Male Hyp Mouse Model of XLH.

Serum levels of fibroblast growth factor 23 (FGF23) markedly increase with renal impairment, with FGF23 levels correlating with the presence of left ventricular hypertrophy (LVH) and mortality in patients with chronic kidney disease (CKD). FGF23 activates calcineurin/nuclear factor of activated T cell (NFAT) signaling and induces hypertrophy in murine cardiomyocytes. X-linked hypophosphatemia (XLH) is characterized by high circulating levels of FGF23 but, in contrast to CKD, is associated with hypophosphatemia. The cardiac effects of high circulating levels of FGF23 in XLH are not well defined. Thus, studies were undertaken to define the cardiac phenotype in the mouse model of XLH (Hyp mice). Echocardiographic and histological analyses demonstrated that Hyp left ventricles (LVs) are smaller than those of wild-type mice. Messenger RNA expression of cardiac hypertrophy markers was not altered in the LV or right ventricle of Hyp mice. However, the Hyp LVs had increased expression of the NFAT target genes NFATc1 and RCAN1. To determine whether phosphate alone can induce markers of hypertrophy, differentiated C2C12 myocytes were treated with phosphate. Phosphate treatment increased expression of cardiac hypertrophy markers, supporting a primary role for phosphate in inducing LVH. Although previous studies showed that increased circulating FGF23 and phosphate levels are associated with LVH, our results demonstrated that in XLH, high circulating levels of FGF23 in the setting of hypophosphatemia do not induce cardiac hypertrophy.

Hormonal Regulation of Osteocyte Perilacunar and Canalicular Remodeling in the Hyp Mouse Model of X-Linked Hypophosphatemia.

Osteocytes remodel their surrounding perilacunar matrix and canalicular network to maintain skeletal homeostasis. Perilacunar/canalicular remodeling is also thought to play a role in determining bone quality. X-linked hypophosphatemia (XLH) is characterized by elevated serum fibroblast growth factor 23 (FGF23) levels, resulting in hypophosphatemia and decreased production of 1,25 dihydroxyvitamin D (1,25D). In addition to rickets and osteomalacia, long bones from mice with XLH (Hyp) have impaired whole-bone biomechanical integrity accompanied by increased osteocyte apoptosis. To address whether perilacunar/canalicular remodeling is altered in Hyp mice, histomorphometric analyses of tibia and 3D intravital microscopic analyses of calvaria were performed. These studies demonstrate that Hyp mice have larger osteocyte lacunae in both the tibia and calvaria, accompanied by enhanced osteocyte mRNA and protein expression of matrix metalloproteinase 13 (MMP13) and genes classically used by osteoclasts to resorb bone, such as cathepsin K (CTSK). Hyp mice also exhibit impaired canalicular organization, with a decrease in number and branching of canaliculi extending from tibial and calvarial lacunae. To determine whether improving mineral ion and hormone homeostasis attenuates the lacunocanalicular phenotype, Hyp mice were treated with 1,25D or FGF23 blocking antibody (FGF23Ab). Both therapies were shown to decrease osteocyte lacunar size and to improve canalicular organization in tibia and calvaria. 1,25D treatment of Hyp mice normalizes osteocyte expression of MMP13 and classic osteoclast markers, while FGF23Ab decreases expression of MMP13 and selected osteoclast markers. Taken together, these studies point to regulation of perilacunar/canalicular remodeling by physiologic stimuli including hypophosphatemia and 1,25D. © 2017 American Society for Bone and Mineral Research.

Raf Kinases Are Essential for Phosphate Induction of ERK1/2 Phosphorylation in Hypertrophic Chondrocytes and Normal Endochondral Bone Development.

Hypophosphatemia causes rickets by impairing hypertrophic chondrocyte apoptosis. Phosphate induction of MEK1/2-ERK1/2 phosphorylation in hypertrophic chondrocytes is required for phosphate-mediated apoptosis and growth plate maturation. MEK1/2 can be activated by numerous molecules including Raf isoforms. A- and B-Raf ablation in chondrocytes does not alter skeletal development, whereas ablation of C-Raf decreases hypertrophic chondrocyte apoptosis and impairs vascularization of the growth plate. However, ablation of C-Raf does not impair phosphate-induced ERK1/2 phosphorylation in vitro, but leads to rickets by decreasing VEGF protein stability. To determine whether Raf isoforms are required for phosphate-induced hypertrophic chondrocyte apoptosis, mice lacking all three Raf isoforms in chondrocytes were generated. Raf deletion caused neonatal death and a significant expansion of the hypertrophic chondrocyte layer of the growth plate, accompanied by decreased cleaved caspase-9. This was associated with decreased phospho-ERK1/2 immunoreactivity in the hypertrophic chondrocyte layer and impaired vascular invasion. These data further demonstrated that Raf kinases are required for phosphate-induced ERK1/2 phosphorylation in cultured hypertrophic chondrocytes and perform essential, but partially redundant roles in growth plate maturation.

Absence of vitamin D receptor (VDR)-mediated PPARγ suppression causes alopecia in VDR-null mice.

Vitamin D receptor (VDR) mutations in humans and mice cause alopecia. VDR-null (VDR-/-) mice exhibit lack of postmorphogenic hair cycles as a result of impaired keratinocyte stem cell (KSC) function. To identify the molecular basis for abnormal KSC function, RNA sequencing of wild-type (WT) and VDR-/- KSCs was performed. These studies demonstrated that >80% of differentially expressed genes are up-regulated in VDR-/- KSCs; thus, the VDR is a transcriptional suppressor in WT KSCs. Peroxisome proliferator-activated receptor γ (PPARγ), PPARγ coactivator 1ß (PGC1ß), and lipoprotein lipase (LPL) were among the up-regulated genes identified. Chromatin immunoprecipitation analyses demonstrated that these genes are direct VDR targets in WT keratinocytes. Notably, VDR occupancy of the PPARγ regulatory region precludes PPARγ occupancy of this site, based on the observation that PPARγ interacts with these sequences in VDR-/- but not WT keratinocytes. This contrasts with the VDR and PPARγ co-occupancy observed on PGC1ß and LPL gene regulatory regions identified. Studies in mice with keratinocyte-specific PPARγ haploinsufficiency were performed to identify the functional consequences of enhanced PPARγ expression. PPARγ haploinsufficiency normalized PPARγ mRNA levels in VDR-/- keratinocytes and restored anagen responsiveness in vivo in VDR-/- mice, resulting in hair regrowth. Thus, absence of VDR-mediated PPARγ suppression underlies alopecia in VDR-/- mice.-Saini, V., Zhao, H., Petit, E. T., Gori, F., Demay, M. B. Absence of vitamin D receptor (VDR)-mediated PPARγ suppression causes alopecia in VDR-null mice.

Hydrocortisone induces changes in gene expression and differentiation in immature human enterocytes.

It is known that functional maturation of the small intestine occurring during the weaning period is facilitated by glucocorticoids (such as hydrocortisone, HC), including an increased expression of digestive hydrolases. However, the molecular mechanisms are not well understood, particularly in the human gut. Here we report a microarray analysis of HC-induced changes in gene expression in H4 cells (a well-characterized human fetal small intestinal epithelial cell line). This study identified a large number of HC-regulated genes, some involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication. HC also regulates the expression of genes important for cell maturation such as development of cell polarity, tight junction formation, and interactions with extracellular matrices. Using human small intestinal xenografts, we also show that HC can regulate the expression of genes important for intestinal epithelial cell maturation. Our dataset may serve as a useful resource for understanding and dissecting the molecular mechanisms of intestinal epithelial cell maturation.