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The X-linked hypophosphatemic (Hyp) mouse carries a loss-of-function mutation in the phex gene and is characterized by hypophosphatemia due to renal phosphate (Pi) wasting, inappropriately suppressed 1,25-dihydroxyvitamin D [1,25(OH)₂D] production, and rachitic bone disease. Increased serum fibroblast growth factor-23 concentration is responsible for the disordered metabolism of Pi and 1,25(OH)₂D. In the present study, we tested the hypothesis that chronic inhibition of fibroblast growth factor-23-induced activation of MAPK signaling in Hyp mice can reverse their metabolic derangements and rachitic bone disease. Hyp mice were administered the MAPK inhibitor, PD0325901 orally for 4 wk. PD0325901 induced a 15-fold and 2-fold increase in renal 1α-hydroxylase mRNA and protein abundance, respectively, and thereby higher serum 1,25(OH)₂D concentrations (115 ± 13 vs. 70 ± 16 pg/ml, P < 0.05), compared with values in vehicle-treated Hyp mice. With PD0325901, serum Pi levels were higher (5.1 ± 0.5 vs. 3 ± 0.2 mg/dl, P < 0.05), and the protein abundance of sodium-dependent phosphate cotransporter Npt2a, was greater than in vehicle-treated mice. The rachitic bone disease in Hyp mice is characterized by abundant unmineralized osteoid bone volume, widened epiphyses, and disorganized growth plates. In PD0325901-treated Hyp mice, mineralization of cortical and trabecular bone increased significantly, accompanied by a decrease in unmineralized osteoid volume and thickness, as determined by histomorphometric analysis. The improvement in mineralization in PD0325901-treated Hyp mice was confirmed by microcomputed tomography analysis, which showed an increase in cortical bone volume and thickness. These findings provide evidence that in Hyp mice, chronic MAPK inhibition improves disordered Pi and 1,25(OH)₂D metabolism and bone mineralization.

Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Gremlin, a BMP antagonist, is expressed in osteoblasts and opposes BMP effects on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. To investigate the actions of gremlin on bone remodeling in vivo, we generated transgenic mice overexpressing gremlin under the control of the osteocalcin promoter. Gremlin transgenics exhibited bone fractures and reduced bone mineral density by 20-30%, compared with controls. Static and dynamic histomorphometry of femurs revealed that gremlin overexpression caused reduced trabecular bone volume and the appearance of woven bone. Polarized light microscopy revealed disorganized collagen bundles at the endosteal cortical surface. Gremlin transgenic mice displayed a 70% decrease in the number of osteoblasts/trabecular area and reduced mineral apposition and bone formation rates. In vivo bromodeoxyuridine labeling and marrow stromal cell cultures demonstrated an inhibitory effect of gremlin on osteoblastic cell replication, but no change on apoptosis was detected. Marrow stromal cells from gremlin transgenics displayed a reduced response to BMP on phosphorylated mothers against decapentaplegic 1/5/8 phosphorylation and reduced free cytosolic beta-catenin levels. In conclusion, transgenic mice overexpressing gremlin in the bone microenvironment have decreased osteoblast number and function leading to osteopenia and spontaneous fractures.

CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP/DDIT3), a member of the C/EBP family of transcription factors, plays a role in cell survival and differentiation. CHOP/DDIT3 binds to C/EBPs to form heterodimers that do not bind to consensus Cebp sequences, acting as a dominant-negative inhibitor. CHOP/DDIT3 blocks adipogenesis, and we postulated it could induce osteoblastogenesis. We investigated the effects of constitutive CHOP/DDIT3 overexpression in murine ST-2 stromal cells transduced with retroviral vectors. ST-2 cells differentiated toward osteoblasts, and CHOP/DDIT3 accelerated and enhanced the appearance of mineralized nodules, and the expression of osteocalcin and alkaline phosphatase mRNAs, particularly in the presence of bone morphogenetic protein-2. CHOP/DDIT3 overexpression opposed adipogenesis, and did not cause substantial changes in cell number. CHOP/DDIT3 overexpression did not modify C/EBPalpha or -beta mRNA levels but decreased C/EBPdelta after 24 d of culture. Electrophoretic mobility shift and supershift assays demonstrated that overexpression of CHOP/DDIT3 decreased the binding of C/EBPs to their consensus sequence by interacting with C/EBPalpha and -beta, confirming its dominant-negative role. In addition, CHOP/DDIT3 enhanced bone morphogenetic protein-2/Smad signaling. In conclusion, CHOP/DDIT3 enhances osteoblastic differentiation of stromal cells, in part by interacting with C/EBPalpha and -beta and also by enhancing Smad signaling.

Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Noggin is a glycoprotein that binds BMPs selectively and antagonizes BMP actions. Noggin expression in osteoblasts is induced by BMPs and noggin opposes the effects of BMPs on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. We investigated the direct in vivo effects of noggin on bone remodeling in transgenic mice overexpressing noggin under the control of the osteocalcin promoter. Noggin transgenics suffered long bone fractures in the first month of life. Total, vertebral, and femoral bone mineral densities were reduced by 23-29%. Static and dynamic histomorphometry of the femur revealed that noggin transgenic mice had decreased trabecular bone volume, number of trabeculae, and bone formation rate. Osteoblast surface and number of osteoblasts/trabecular area were not significantly decreased, indicating impaired osteoblastic function. Osteoclast surface and number were normal/decreased, there was no increase in bone resorption, and the tissue had the appearance of woven bone. Vertebral microcomputed tomography scanning confirmed decreased trabecular bone volume and trabecular number. In conclusion, transgenic mice overexpressing noggin in the bone microenvironment have decreased trabecular bone volume and impaired osteoblastic function, leading to osteopenia and fractures.

Bone morphogenetic proteins (BMP) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factor activity is regulated by binding proteins, and we previously showed that BMPs induce noggin, a glycoprotein that binds and blocks BMP action. Recently, additional BMP antagonists, such as gremlin, have been described, but there is no information about their expression or function in osteoblasts. We tested for the expression of gremlin and studied its induction by BMPs in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in gremlin messenger RNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on gremlin transcripts were independent of new protein synthesis. BMP-2 increased the rate of gremlin transcription as determined by nuclear run-on assays. Fibroblast growth factor-2 and platelet-derived growth factor BB also induced gremlin, but other hormones and growth factors had no effect. Gremlin prevented the stimulatory effects of BMP-2 on DNA, collagen, noncollagen protein synthesis, and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce gremlin transcription in Ob cells, a mechanism that probably limits BMP action in osteoblasts.

PTH induces the synthesis of insulin-like growth factor I (IGF-I) and regulates the expression of IGF-binding proteins (IGFBP) in osteoblast cultures. IGFBP-related protein-1 (IGFBP-RP-1), the product of the mac25 gene, binds IGF-I, IGF-II, and insulin. We tested the actions of PTH on the expression of mac25/IGFBP-RP-1 in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). PTH at 0.1-10 nM for 6-48 h increased mac25/IGFBP-RP-1 messenger RNA (mRNA) levels in Ob cells, an effect not altered by cycloheximide. PGE2 increased mac25/IGFBP-RP-1 mRNA levels, but indomethacin did not modify basal or PTH-stimulated mac25/IGFBP-RP-1 expression. The decay of mac25/IGFBP-RP-1 mRNA in transcriptionally arrested Ob cells was not modified by PTH, and PTH increased the rate of IGFBP-RP-1 transcription. GH, insulin, bone morphogenetic protein-2, fibroblast growth factor-2, platelet-derived growth factor BB, IGF-I, and IGF-II did not modify mac25/IGFBP-RP-1 expression, whereas transforming growth factor-beta1 was modestly stimulatory. In conclusion, PTH stimulates mac25/IGFBP-RP-1 transcription in osteoblasts, an effect that could be relevant to the actions of PTH in bone.

Glucocorticoids inhibit the synthesis of insulin-like growth factor I (IGF-I) and regulate the expression of IGF-binding proteins (IGFBPs) in osteoblast cultures. IGFBP-related protein-1 (IGFBP-rP1), the product of the mac25 gene, binds IGF-I, IGF-II, and insulin, and we postulated that glucocorticoids regulate IGFBP-rP1 synthesis in osteoblasts. We tested the expression of mac25/IGFBP-rP1 in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Cortisol treatment at 10 nM to 1 microM for 24-48 h caused a time- and dose-dependent increase in mac25/IGFBP-rP1 messenger RNA (mRNA) levels in Ob cells. Cycloheximide at 3.6 microM did not alter mac25/IGFBP-rP1 transcripts in control or cortisol-treated cells. Cortisol did not modify the decay of mac25/IGFBP-rP1 mRNA in transcriptionally arrested Ob cells and increased the rate of IGFBP-rP1 transcription as determined by nuclear run-on assays. Retinoic acid also increased mac25/IGFBP-rP1 mRNA levels, but 17beta-estradiol, testosterone, 5alpha-dihydrotestosterone, progesterone, and 1,25-dihydroxyvitamin D3 did not. In conclusion, cortisol stimulates mac25/IGFBP-rP1 expression in Ob cells by transcriptional mechanisms. As IGFBP-rP1 binds and possibly modifies the effects of IGFs and insulin, its increased expression could be relevant to the inhibitory actions of cortisol in bone.