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.

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.