Systems genetics and bioinformatics analyses using ESR1-correlated genes identify potential candidates underlying female bone development.

Estrogen receptor α (ESR1) is involved in E2 signaling and plays a major role in postmenopausal bone loss. However, the molecular network underlying ESR1 has not been explored. We used systems genetics and bioinformatics to identify important genes associated with Esr1 in postmenopausal bone loss. We identified ~2300 Esr1-coexpressed genes in female BXD bone femur, functional analysis of which revealed 'osteoblast signaling' as the most enriched pathway. PPI network led to the identification of 25 'female bone candidates'. The gene-regulatory analysis revealed RUNX2 as a key TF. ANKRD1 and RUNX2 were significantly different between osteoporosis patients and healthy controls. Sp7, Col1a1 and Pth1r correlated with multiple femur bone phenotypes in BXD mice. miR-3121-3p targeted Csf1, Ankrd1, Sp7 and Runx2. ß-estradiol treatment markedly increased the expression of these candidates in mouse osteoblast. Our study revealed that Esr1-correlated genes Ankrd1, Runx2, Csf1 and Sp7 may play important roles in female bone development.

Genetic interactions between polycystin-1 and Wwtr1 in osteoblasts define a novel mechanosensing mechanism regulating bone formation in mice.

Molecular mechanisms transducing physical forces in the bone microenvironment to regulate bone mass are poorly understood. Here, we used mouse genetics, mechanical loading, and pharmacological approaches to test the possibility that polycystin-1 and Wwtr1 have interdependent mechanosensing functions in osteoblasts. We created and compared the skeletal phenotypes of control Pkd1flox/+;Wwtr1flox/+, Pkd1Oc-cKO, Wwtr1Oc-cKO, and Pkd1/Wwtr1Oc-cKO mice to investigate genetic interactions. Consistent with an interaction between polycystins and Wwtr1 in bone in vivo, Pkd1/Wwtr1Oc-cKO mice exhibited greater reductions of BMD and periosteal MAR than either Wwtr1Oc-cKO or Pkd1Oc-cKO mice. Micro-CT 3D image analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone volume and cortical bone thickness in Pkd1/Wwtr1Oc-cKO mice compared to either Pkd1Oc-cKO or Wwtr1Oc-cKO mice. Pkd1/Wwtr1Oc-cKO mice also displayed additive reductions in mechanosensing and osteogenic gene expression profiles in bone compared to Pkd1Oc-cKO or Wwtr1Oc-cKO mice. Moreover, we found that Pkd1/Wwtr1Oc-cKO mice exhibited impaired responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing gene expression compared to control mice. Finally, control mice treated with a small molecule mechanomimetic, MS2 that activates the polycystin complex resulted in marked increases in femoral BMD and periosteal MAR compared to vehicle control. In contrast, Pkd1/Wwtr1Oc-cKO mice were resistant to the anabolic effects of MS2. These findings suggest that PC1 and Wwtr1 form an anabolic mechanotransduction signaling complex that mediates mechanical loading responses and serves as a potential novel therapeutic target for treating osteoporosis.

Genetic interactions between Polycystin-1 and TAZ in osteoblasts define a novel mechanosensing mechanism regulating bone formation in mice.

Molecular mechanisms transducing physical forces in the bone microenvironment to regulate bone mass are poorly understood. Here, we used mouse genetics, mechanical loading, and pharmacological approaches to test the possibility that polycystin-1 and TAZ have interdependent mechanosensing functions in osteoblasts. We created and compared the skeletal phenotypes of control Pkd1flox/+;TAZflox/+, single Pkd1Oc-cKO, single TAZOc-cKO, and double Pkd1/TAZOc-cKO mice to investigate genetic interactions. Consistent with an interaction between polycystins and TAZ in bone in vivo, double Pkd1/TAZOc-cKO mice exhibited greater reductions of BMD and periosteal MAR than either single TAZOc-cKO or Pkd1Oc-cKO mice. Micro-CT 3D image analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone volume and cortical bone thickness in double Pkd1/TAZOc-cKO mice compared to either single Pkd1Oc-cKO or TAZOc-cKO mice. Double Pkd1/TAZOc-cKO mice also displayed additive reductions in mechanosensing and osteogenic gene expression profiles in bone compared to single Pkd1Oc-cKO or TAZOc-cKO mice. Moreover, we found that double Pkd1/TAZOc-cKO mice exhibited impaired responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing gene expression compared to control mice. Finally, control mice treated with a small molecule mechanomimetic MS2 had marked increases in femoral BMD and periosteal MAR compared to vehicle control. In contrast, double Pkd1/TAZOc-cKO mice were resistant to the anabolic effects of MS2 that activates the polycystin signaling complex. These findings suggest that PC1 and TAZ form an anabolic mechanotransduction signaling complex that responds to mechanical loading and serve as a potential novel therapeutic target for treating osteoporosis.

The carboxylation status of osteocalcin has important consequences for its structure and dynamics.

BACKGROUND: The carboxylation status of Osteocalcin (Ocn) not only influences formation and structure in bones but also has important endocrine functions affecting energy metabolism and expenditure. In this study, the role of γ-carboxylation of the glutamate residues in the structure-dynamics-function relationship in Ocn is investigated. METHODS: Three forms of Ocn, differentially carboxylated at the Glu-17, 21 and 24 residues, along with a mutated form of Ocn carrying Glu/Ala mutations, are modeled and simulated using molecular dynamics (MD) simulation in the presence of calcium ions. RESULTS: Characterization of the global conformational dynamics of Ocn, described in terms of the orientational variations within its 3-helical domain, highlights large structural variations in the non-carboxylated osteocalcin (nOcn). The bi-carboxylated Ocn (bOcn) and tri-carboxylated (tOcn) species, in contrast, display relatively rigid tertiary structures, with the dynamics of most regions strongly correlated. Radial distribution functions calculated for both bOcn and tOcn show long-range ordering of the calcium ion distribution around the carboxylated glutamate (γGlu) residues, likely playing an important role in promoting stability of these Ocns. Additionally, the same calcium ions are observed to coordinate with neighboring γGlu, better shielding their negative charges and in turn stabilizing these systems more than do the singly coordinating calcium ions observed in the case of nOcn. bOcn is also found to exhibit a more helical C-terminal structure, that has been shown to activate its cellular receptor GPRC6A, highlighting the allosteric role of Ocn carboxylation in modulating the stability and binding potential of the active C-terminal. CONCLUSIONS: The carboxylation status of Ocn as well and its calcium coordination appear to have a direct influence on Ocn structure and dynamics, possibly leading to the known differences in Ocn biological function. GENERAL SIGNIFICANCE: Modification of Ocn sequence or its carboxylation state may provide the blueprint for developing high-affinity peptides targeting its cellular receptor GPRC6A, with therapeutic potential for treatment of metabolic disorders.

Molecular dynamics analysis of the binding of human interleukin-6 with interleukin-6 α-receptor.

Human interleukin-6 (hIL-6) is a multifunctional cytokine that regulates immune and inflammatory responses in addition to metabolic and regenerative processes and cancer. hIL-6 binding to the IL-6 receptor (IL-6Rα) induces homodimerization and recruitment of the glycoprotein (gp130) to form a hexameric signaling complex. Anti-IL-6 and IL-6R antibodies are clinically approved inhibitors of IL-6 signaling pathway for treating rheumatoid arthritis and Castleman's disease, respectively. There is a potential to develop novel small molecule IL-6 antagonists derived from understanding the structural basis for IL-6/IL-6Rα interactions. Here, we combine homology modeling with extensive molecular dynamics (MD) simulations to examine the association of hIL-6 with IL-6Rα. A comparison with MD of apo hIL-6 reveals that the binding of hIL-6 to IL-6Rα induces structural and dynamic rearrangements in the AB loop region of hIL-6, disrupting intraprotein contacts and increasing the flexibility of residues 48 to 58 of the AB loop. In contrast, due to the involvement of residues 59 to 78 in forming contacts with the receptor, these residues of the AB loop are observed to rigidify in the presence of the receptor. The binary complex is primarily stabilized by two pairs of salt bridges, Arg181 (hIL-6)- Glu182 (IL-6Rα) and Arg184 (hIL-6)- Glu183 (IL-6Rα) as well as hydrophobic and aromatic stacking interactions mediated essentially by Phe residues in both proteins. An interplay of electrostatic, hydrophobic, hydrogen bonding, and aromatic stacking interactions facilitates the formation of the hIL-6/IL-6Rα complex.

Novel Small Molecule Fibroblast Growth Factor 23 Inhibitors Increase Serum Phosphate and Improve Skeletal Abnormalities in Hyp Mice.

Excess fibroblast growth factor (FGF) 23 causes hereditary hypophosphatemic rickets, such as X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO). A small molecule that specifically binds to FGF23 to prevent activation of the fibroblast growth factor receptor/α-Klotho complex has potential advantages over the currently approved systemically administered FGF23 blocking antibody. Using structure-based drug design, we previously identified ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a small molecule antagonist for FGF23. Additional structure-activity studies developed a series of ZINC13407541 analogs with enhanced drug-like properties. In this study, we tested in a preclinical Hyp mouse homolog of XLH a direct connect analog [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] (8n), which exhibited the greatest stability in microsomal assays, and [(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] (13a), which exhibited increased in vitro potency. Using cryo-electron microscopy structure and computational docking, we identified a key binding residue (Q156) of the FGF23 antagonists, ZINC13407541, and its analogs (8n and 13a) in the N-terminal domain of FGF23 protein. Site-directed mutagenesis and bimolecular fluorescence complementation-fluorescence resonance energy transfer assay confirmed the binding site of these three antagonists. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and increased serum phosphate and 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations in Hyp mice. Long-term parenteral treatment with 8n or 13a also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in Hyp mice. The more potent 13a compound had greater therapeutic effects in Hyp mice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat excess FGF23-mediated disorders. SIGNIFICANCE STATEMENT: This study used structure-based drug design and medicinal chemistry approaches to identify and optimize small molecules with different stability and potency, which antagonize excessive actions of fibroblast growth factor 23 (FGF23) in hereditary hypophosphatemic rickets. The findings confirmed that these antagonists bind to the N-terminus of FGF23 to inhibit its binding to and activation of the fibroblast growth factor receptors/α-Klotho signaling complex. Administration of these lead compounds improved phosphate homeostasis and abnormal skeletal phenotypes in a preclinical Hyp mouse model.

Genetic Dissection of Femoral and Tibial Microarchitecture.

Our understanding of the genetic control of bone strength has relied mainly on estimates of bone mineral density. Here we have mapped genetic factors that influence femoral and tibial microarchitecture using high-resolution x-ray computed tomography (8-µm isotropic voxels) across a family of 61 BXD strains of mice, roughly 10 isogenic cases per strain and balanced by sex. We computed heritabilities for 25 cortical and trabecular traits. Males and females have well-matched heritabilities, ranging from 0.25 to 0.75. We mapped 16 genetic loci most of which were detected only in females. There is also a bias in favor of loci that control cortical rather than trabecular bone. To evaluate candidate genes, we combined well-established gene ontologies with bone transcriptome data to compute bone-enrichment scores for all protein-coding genes. We aligned candidates with those of human genome-wide association studies. A subset of 50 strong candidates fell into three categories: (1) experimentally validated genes already known to modulate bone function (Adamts4, Ddr2, Darc, Adam12, Fkbp10, E2f6, Adam17, Grem2, Ifi204); (2) candidates without any experimentally validated function in bone (eg, Greb1, Ifi202b), but linked to skeletal phenotypes in human cohorts; and (3) candidates that have high bone-enrichment scores, but for which there is not yet any functional link to bone biology or skeletal system disease (including Ifi202b, Ly9, Ifi205, Mgmt, F2rl1, Iqgap2). Our results highlight contrasting genetic architecture between sexes and among major bone compartments. The alignment of murine and human data facilitates function analysis and should prove of value for preclinical testing of molecular control of bone structure. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model.

Observations in transgenic α-Klotho (Kl) mice (KlTg) defined the antiaging role of soluble Klotho (sKL130). A genetic translocation that elevates sKL levels in humans is paradoxically associated with increased circulating fibroblast growth factor 23 (FGF23) levels and the potential of both membrane KL (mKL135) and sKL130 to act as coreceptors for FGF23 activation of fibroblast growth factor receptors (FGFRs). Neither FGF23 expression nor the contributions of FGF23, mKL135, and sKL130 codependent and independent functions have been investigated in KlTg mice. In the current study, we examined the effects of Kl overexpression on FGF23 levels and functions in KlTg mice. We found that mKL135 but not sKL130 stimulated FGF23 expression in osteoblasts, leading to elevated Fgf23 bone expression and circulating levels in KlTg mice. Elevated FGF23 suppressed 1,25(OH)2D and parathyroid hormone levels but did not cause hypophosphatemic rickets in KlTg mice. KlTg mice developed low aldosterone-associated hypertension but not left ventricular hypertrophy. Mechanistically, we found that mKL135 and sKL130 are essential cofactors for FGF23-mediated ERK activation but that they inhibited FGF23 stimulation of PLC-γ and PI3K/AKT signaling. Thus, increased longevity in KlTg mice occurs in the presence of excess FGF23 that interacts with mKL and sKL to bias FGFR pathways.

Ensemble docking to difficult targets in early-stage drug discovery: Methodology and application to fibroblast growth factor 23.

Ensemble docking is now commonly used in early-stage in silico drug discovery and can be used to attack difficult problems such as finding lead compounds which can disrupt protein-protein interactions. We give an example of this methodology here, as applied to fibroblast growth factor 23 (FGF23), a protein hormone that is responsible for regulating phosphate homeostasis. The first small-molecule antagonists of FGF23 were recently discovered by combining ensemble docking with extensive experimental target validation data (Science Signaling, 9, 2016, ra113). Here, we provide a detailed account of how ensemble-based high-throughput virtual screening was used to identify the antagonist compounds discovered in reference (Science Signaling, 9, 2016, ra113). Moreover, we perform further calculations, redocking those antagonist compounds identified in reference (Science Signaling, 9, 2016, ra113) that performed well on drug-likeness filters, to predict possible binding regions. These predicted binding modes are rescored with the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) approach to calculate the most likely binding site. Our findings suggest that the antagonist compounds antagonize FGF23 through the disruption of protein-protein interactions between FGF23 and fibroblast growth factor receptor (FGFR).

Evidence for Osteocalcin Binding and Activation of GPRC6A in ß-Cells.

The possibility that G protein-coupled receptor family C member A (GPRC6A) is the osteocalcin (Ocn)-sensing G protein-coupled receptor that directly regulates pancreatic ß-cell functions is controversial. In the current study, we found that Ocn and an Ocn-derived C-terminal hexapeptide directly activate GPRC6A-dependent ERK signaling in vitro. Computational models probe the structural basis of Ocn binding to GPRC6A and predict that the C-terminal hexapeptide docks to the extracellular side of the transmembrane domain of GPRC6A. Consistent with the modeling, mutations in the computationally identified binding pocket of GPRC6A reduced Ocn and C-terminal hexapeptide activation of this receptor. In addition, selective deletion of Gprc6a in ß-cells (Gprc6a(ß)(-cell-cko)) by crossing Gprc6a(flox/flox) mice with Ins2-Cre mice resulted in reduced pancreatic weight, islet number, insulin protein content, and insulin message expression. Both islet size and ß-cell proliferation were reduced in Gprc6a(ß)(-cell-cko) compared with control mice. Gprc6a(ß)(-cell-cko) exhibited abnormal glucose tolerance, but normal insulin sensitivity. Islets isolated from Gprc6a(ß)(-cell-cko) mice showed reduced insulin simulation index in response to Ocn. These data establish the structural basis for Ocn direct activation of GPRC6A and confirm a role for GPRC6A in regulating ß-cell proliferation and insulin secretion.

Counter-regulatory paracrine actions of FGF-23 and 1,25(OH)2 D in macrophages.

Mechanisms underlying the association between fibroblastic growth factor 23 (FGF-23) and inflammation are uncertain. We found that FGF-23 was markedly up-regulated in LPS/INF-γ-induced proinflammatory M1 macrophages and Hyp mouse-derived peritoneal macrophages, but not in IL-4-induced M2 anti-inflammatory macrophages. NF-КB and JAK/STAT1 pathways mediated the increased transcription of FGF-23 in response to M1 polarization. FGF-23 stimulated TNF-α, but not IL-6, expression in M0 macrophages and suppressed Arginase-1 expression in M2 macrophages through FGFR-mediated mechanisms. 1,25(OH)2 D stimulated Arginase-1 expression and inhibited FGF-23 stimulation of TNF-α. FGF-23 has proinflammatory paracrine functions and counter-regulatory actions to 1,25(OH)2 D on innate immune responses.

Physiological mechanisms and therapeutic potential of bone mechanosensing.

Skeletal loading is an important physiological regulator of bone mass. Theoretically, mechanical forces or administration of drugs that activate bone mechanosensors would be a novel treatment for osteoporotic disorders, particularly age-related osteoporosis and other bone loss caused by skeletal unloading. Uncertainty regarding the identity of the molecular targets that sense and transduce mechanical forces in bone, however, has limited the therapeutic exploitation of mechanosesning pathways to control bone mass. Recently, two evolutionally conserved mechanosensing pathways have been shown to function as "physical environment" sensors in cells of the osteoblasts lineage. Indeed, polycystin-1 (Pkd1, or PC1) and polycystin-2 (Pkd2, or PC2' or TRPP2), which form a flow sensing receptor channel complex, and TAZ (transcriptional coactivator with PDZ-binding motif, or WWTR1), which responds to the extracellular matrix microenvironment act in concert to reciprocally regulate osteoblastogenesis and adipogenesis through co-activating Runx2 and a co-repressing PPARγ activities. Interactions of polycystins and TAZ with other putative mechanosensing mechanism, such as primary cilia, integrins and hemichannels, may create multifaceted mechanosensing networks in bone. Moreover, modulation of polycystins and TAZ interactions identify novel molecular targets to develop small molecules that mimic the effects of mechanical loading on bone.

Osteoblast-specific deletion of Pkd2 leads to low-turnover osteopenia and reduced bone marrow adiposity.

Polycystin-1 (Pkd1) interacts with polycystin-2 (Pkd2) to form an interdependent signaling complex. Selective deletion of Pkd1 in the osteoblast lineage reciprocally regulates osteoblastogenesis and adipogenesis. The role of Pkd2 in skeletal development has not been defined. To this end, we conditionally inactivated Pkd2 in mature osteoblasts by crossing Osteocalcin (Oc)-Cre;Pkd2+/null mice with floxed Pkd2 (Pkd2flox/flox) mice. Oc-Cre;Pkd2flox/null (Pkd2Oc-cKO) mice exhibited decreased bone mineral density, trabecular bone volume, cortical thickness, mineral apposition rate and impaired biomechanical properties of bone. Pkd2 deficiency resulted in diminished Runt-related transcription factor 2 (Runx2) expressions in bone and impaired osteoblastic differentiation ex vivo. Expression of osteoblast-related genes, including, Osteocalcin, Osteopontin, Bone sialoprotein (Bsp), Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex), Dentin matrix protein 1 (Dmp1), Sclerostin (Sost), and Fibroblast growth factor 23 (FGF23) were reduced proportionate to the reduction of Pkd2 gene dose in bone of Oc-Cre;Pkd2flox/+ and Oc-Cre;Pkd2flox/null mice. Loss of Pkd2 also resulted in diminished peroxisome proliferator-activated receptor γ (PPARγ) expression and reduced bone marrow fat in vivo and reduced adipogenesis in osteoblast culture ex vivo. Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP), reciprocally acting as co-activators and co-repressors of Runx2 and PPARγ, were decreased in bone of Oc-Cre;Pkd2flox/null mice. Thus, Pkd1 and Pkd2 have coordinate effects on osteoblast differentiation and opposite effects on adipogenesis, suggesting that Pkd1 and Pkd2 signaling pathways can have independent effects on mesenchymal lineage commitment in bone.

Osteocyte-specific deletion of Fgfr1 suppresses FGF23.

Increases in fibroblastic growth factor 23 (FGF23 or Fgf23) production by osteocytes result in hypophosphatemia and rickets in the Hyp mouse homologue of X-linked hypophosphatemia (XLH). Fibroblastic growth factor (FGF) signaling has been implicated in the pathogenesis of Hyp. Here, we conditionally deleted FGF receptor 1 (FGFR1 or Fgfr1) in osteocytes of Hyp mice to investigate the role of autocrine/paracrine FGFR signaling in regulating FGF23 production by osteocytes. Crossing dentin matrix protein 1 (Dmp1)-Cre;Fgfr1null/+ mice with female Hyp;Fgfr1flox/flox mice created Hyp and Fgfr1 (Fgfr1Dmp1-cKO)-null mice (Hyp;Fgfr1Dmp1-cKO) with a 70% decrease in bone Fgfr1 transcripts. Fgfr1Dmp1-cKO-null mice exhibited a 50% reduction in FGF23 expression in bone and 3-fold reduction in serum FGF23 concentrations, as well as reductions in sclerostin (Sost), phosphate regulating endopeptidase on X chromosome (PHEX or Phex), matrix extracellular phosphoglycoprotein (Mepe), and Dmp1 transcripts, but had no demonstrable alterations in phosphate or vitamin D homeostasis or skeletal morphology. Hyp mice had hypophosphatemia, reductions in 1,25(OH)2D levels, rickets/osteomalacia and elevated FGF2 expression in bone. Compared to Hyp mice, compound Hyp;Fgfr1Dmp1-cKO-null mice had significant improvement in rickets and osteomalacia in association with a decrease in serum FGF23 (3607 to 1099 pg/ml), an increase in serum phosphate (6.0 mg/dl to 9.3 mg/dl) and 1,25(OH)2D (121±23 to 192±34 pg/ml) levels, but only a 30% reduction in bone FGF23 mRNA expression. FGF23 promoter activity in osteoblasts was stimulated by FGFR1 activation and inhibited by overexpression of a dominant negative FGFR1(TK-), PLCγ and MAPK inhibitors. FGF2 also stimulated the translation of an FGF23 cDNA transfected into osteoblasts via a FGFR1 and PI3K/Akt-dependent mechanism. Thus, activation of autocrine/paracrine FGF pathways is involved in the pathogenesis of Hyp through FGFR1-dependent regulation of FGF23 by both transcriptional and post-transcriptional mechanisms. This may serve to link local bone metabolism with systemic phosphate and vitamin D homeostasis.

Fibroblast growth factor-23: what we know, what we don't know, and what we need to know.

Traditional risk factors of cardiovascular morbidity and mortality such as hypertension, hypercholesterolemia and obesity are paradoxically associated with better outcomes in dialysis patients, and the few trials of interventions targeting modifiable traditional risk factors have yielded disappointing results in this patient population. Non-traditional risk factors such as inflammation, anemia and abnormalities in bone and mineral metabolism have been proposed as potential explanations for the excess mortality seen in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD), but without clear understanding of what the most important pathophysiologic mechanisms of these risk factors are, which ones might be ideal treatment targets and which therapeutic interventions may be effective and safe in targeting them. Among the novel risk factors, fibroblast growth factor-23 (FGF23) has recently emerged as one of the most powerful predictors of adverse outcomes in patients with CKD and ESRD. FGF23 is a hormone produced by osteoblasts/osteocytes in bone that acts on the kidney to regulate phosphate and vitamin D metabolism through activation of FGF receptor/α-Klotho co-receptor complexes. It is possible that elevated FGF23 may exert its negative impact through distinct mechanisms of action independent from its role as a regulator of phosphorus homeostasis. Elevated circulating FGF23 concentrations have been associated with left ventricular hypertrophy (LVH), and it has been suggested that FGF23 exerts a direct effect on the myocardium. While it is possible that 'off target' effects of FGF23 present in very high concentrations could induce LVH, this possibility is controversial, since α-klotho is not expressed in the myocardium. Another possibility is that FGF23's effect on the heart is mediated indirectly, via 'on target' activation of other humoral pathways. We will review the physiology and pathophysiology of FGF23, the outcomes associated with elevated FGF23 levels, and describe putative mechanisms of action responsible for its negative effects and potential therapeutic strategies to treat these.

Conditional mesenchymal disruption of pkd1 results in osteopenia and polycystic kidney disease.

Conditional deletion of Pkd1 in osteoblasts using either Osteocalcin(Oc)-Cre or Dmp1-Cre results in defective osteoblast-mediated postnatal bone formation and osteopenia. Pkd1 is also expressed in undifferentiated mesenchyme that gives rise to the osteoblast lineage. To examine the effects of Pkd1 on prenatal osteoblast development, we crossed Pkd1(flox/flox) and Col1a1(3.6)-Cre mice, which has been used to achieve selective inactivation of Pkd1 earlier in the osteoblast lineage. Control Pkd1(flox/flox) and Pkd1(flox/+), heterozygous Col1a1(3.6)-Cre;Pkd1(flox/+) and Pkd1(flox/null), and homozygous Col1a1(3.6)-Cre;Pkd1(flox/flox) and Col1a1(3.6)-Cre;Pkd1(flox/null) mice were analyzed at ages ranging from E14.5 to 8-weeks-old. Newborn Col1a1(3.6)-Cre;Pkd1(flox/null) mice exhibited defective skeletogenesis in association with a greater reduction in Pkd1 expression in bone. Conditional Col1a1(3.6)-Cre;Pkd1(flox/+) and Col1a1(3.6)-Cre;Pkd1(flox/flox) mice displayed a gene dose-dependent decrease in bone formation and increase in marrow fat at 6 weeks of age. Bone marrow stromal cell and primary osteoblast cultures from homozygous Col1a1(3.6)-Cre;Pkd1(flox/flox) mice showed increased proliferation, impaired osteoblast development and enhanced adipogenesis ex vivo. Unexpectedly, we found evidence for Col1a1(3.6)-Cre mediated deletion of Pkd1 in extraskeletal tissues in Col1a1(3.6)-Cre;Pkd1(flox/flox) mice. Deletion of Pkd1 in mesenchymal precursors resulted in pancreatic and renal, but not hepatic, cyst formation. The non-lethality of Col1a1(3.6)-Cre;Pkd1(flox/flox) mice establishes a new model to study abnormalities in bone development and cyst formation in pancreas and kidney caused by Pkd1 gene inactivation.

Cilia-like structures and polycystin-1 in osteoblasts/osteocytes and associated abnormalities in skeletogenesis and Runx2 expression.

We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblast-related genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes.

Resveratrol prevents CsA inhibition of proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells through an ER/NO/cGMP pathway.

The purpose of this study was to investigate the in vitro effects of resveratrol (RSVL) and cyclosporin A (CsA) on proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures. Application of RSVL (10(-8) -10(-6) mol l(-1)) resulted in a dose-dependent increase in [3H]-thymidine incorporation, alkaline phosphatase (ALP) activity and calcium deposition of BMSCs cultures, which was accompanied with the increase of NO production and cGMP content. Concurrent treatment with the estrogen receptor antagonist ICI182,780 (10(-7) mol l(-1)) or the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (6 x 10(-3) mol l(-1)) abolished the RSVL (10(-6) mol l(-1))-induced increase in NO production and cGMP content and eliminated the RSVL-induced increase in proliferation and osteoblastic differentiation of BMSCs. In contrast, CsA (10(-6) -10(-5) mol l(-1)) dose-dependently decreased [3H]-thymidine incorporation, ALP activity and calcium deposition of BMSCs cultures, which was accompanied with the reduction of NO production in the conditioned media. Concurrent treatment with RSVL (10(-6) mol l(-1)) significantly reversed the CsA (3 x 10(-6) mol l(-1))-mediated decrease in NO production and restored the proliferation and differentiation potential of BMSCs. Our data suggest that (1) the NO/cGMP pathway may play an important role in both RSVL-induced and CsA-inhibited proliferation and osteoblastic differentiation of mouse BMSCs, and (2) RSVL may act through an ER/NO/cGMP pathway to reverse the inhibitory effect of CsA on BMSC cultures. Taken together, the data suggest that RSVL may prevent osteoporosis induced by CsA.

Selective Runx2-II deficiency leads to low-turnover osteopenia in adult mice.

Runx2 transcribes Runx2-II and Runx2-I isoforms with distinct N-termini. Deletion of both isoforms results in complete arrest of bone development, whereas selective loss of Runx2-II is sufficient to form a grossly intact skeleton with impaired endochondral bone development. To elucidate the role of Runx2-II in osteoblast function in adult mice, we examined heterozygous Runx2-II (Runx2-II(+/-)) and homozygous Runx2-II (Runx2-II(-/-))-deficient mice, which, respectively, lack one or both copies of Runx2-II but intact Runx2-I expression. Compared to wild-type mice, 6-week-old Runx2-II(+/-) had reduced trabecular bone volume (BV/TV%), cortical thickness (Ct.Th), and bone mineral density (BMD), decreased osteoblastic and osteoclastic markers, lower bone formation rates, impaired osteoblast maturation of BMSCs in vitro, and significant reductions in mechanical properties. Homozygous Runx2-II(-/-) mice had a more severe reduction in BMD, BV/TV%, and Ct.Th, and greater suppression of osteoblastic and osteoclastic markers than Runx2-II(+/-) mice. Non-selective Runx2(+/-) mice, which have an equivalent reduction in Runx2 expression due to the lack one copy of Runx2-I and II, however, had an intermediate reduction in BMD. Thus, selective Runx2-II mutation causes diminished osteoblastic function in an adult mouse leading to low-turnover osteopenia and suggest that Runx2-I and II have distinct functions imparted by their different N-termini.