Publications by authors named "Zhousheng Xiao"

34 Publications

Recent Advances of Osterix Transcription Factor in Osteoblast Differentiation and Bone Formation.

Front Cell Dev Biol 2020 15;8:601224. Epub 2020 Dec 15.

Key Laboratory of Brain and Neuroendocrine Diseases, College of Hunan Province, Hunan University of Medicine, Huaihua, China.

With increasing life expectations, more and more patients suffer from fractures either induced by intensive sports or other bone-related diseases. The balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is the basis for maintaining bone health. Osterix (Osx) has long been known to be an essential transcription factor for the osteoblast differentiation and bone mineralization. Emerging evidence suggests that Osx not only plays an important role in intramembranous bone formation, but also affects endochondral ossification by participating in the terminal cartilage differentiation. Given its essentiality in skeletal development and bone formation, Osx has become a new research hotspot in recent years. In this review, we focus on the progress of Osx's function and its regulation in osteoblast differentiation and bone mass. And the potential role of Osx in developing new therapeutic strategies for osteolytic diseases was discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.601224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769847PMC
December 2020

Design and development of FGF-23 antagonists: Definition of the pharmacophore and initial structure-activity relationships probed by synthetic analogues.

Bioorg Med Chem 2021 Jan 18;29:115877. Epub 2020 Nov 18.

Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505-0001, USA. Electronic address:

Hereditary hypophosphatemic disorders, TIO, and CKD conditions are believed to be influenced by an excess of Fibroblast Growth Factor-23 (FGF-23) which activates a binary renal FGFRs / α-Klotho complex to regulate homeostatic metabolism of phosphate and vitamin D. Adaptive FGF-23 responses from CKD patients with excess FGF-23 frequently lead to increased mortality from cardiovascular disease. A reversibly binding small molecule therapeutic has yet to emerge from research and development in this area. Current outcomes described in this work highlight efforts related to lead identification and modification using organic synthesis of strategic analogues to probe structure-activity relationships and preliminarily define the pharmacophore of a computationally derived hit obtained from virtual high-throughput screening. Synthetic strategies for the initial hit and analogue preparation, as well as preliminary cellular in vitro assay results highlighting sub micromolar inhibition of the FGF-23 signaling sequence at a concentration well below cytotoxicity are reported herein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2020.115877DOI Listing
January 2021

Osteoporosis: Mechanism, Molecular Target, and Current Status on Drug Development.

Curr Med Chem 2020 Mar 30. Epub 2020 Mar 30.

Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, 38163. United States.

Osteoporosis is a pathological loss of bone mass due to an imbalance in bone remodeling where osteoclast-mediated bone resorption exceeds osteoblast-mediated bone formation resulting in skeletal fragility and fractures. Anti-resorptive agents such as bisphosphonates and SERMs, and anabolic drugs that stimulate bone formation, including PTH analogues and sclerostin inhibitors, are current treatments for osteoporosis. Despite their efficacy, severe side effects and loss of potency may limit the long term usage of a single drug. Sequential and combinational use of current drugs, such as switching from an anabolic to an anti-resorptive agent, may provide an alternative approach. Moreover, there are novel drugs being developed against emerging new targets such as Cathepsin K and 17β-HSD2 that may have less side effects. This review will summarize the molecular mechanisms of osteoporosis, current drugs for osteoporosis treatment, and new drug development strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/0929867327666200330142432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665836PMC
March 2020

Therapeutic evidence of umbilical cord-derived mesenchymal stem cell transplantation for cerebral palsy: a randomized, controlled trial.

Stem Cell Res Ther 2020 02 3;11(1):43. Epub 2020 Feb 3.

Department of Neonatology, The First Affiliated Hospital of Xi'an Jiaotong University, No.277 West Yanta Road, Xi'an, 710061, Shaanxi, People's Republic of China.

Background: Cerebral palsy (CP) is a syndrome of childhood movement and posture disorders. Clinical evidence is still limited and sometimes inconclusive about the benefits of human umbilical cord mesenchymal stem cells (hUC-MSCs) for CP. We conducted a randomized trial to evaluate the safety and efficacy of hUC-MSC transplantation concomitant with rehabilitation in patients with CP.

Methods: Eligible patients were allocated into the hUC-MSC group and control group. In addition to rehabilitation, the patients in the hUC-MSC group received four transfusions of hUC-MSCs intravenously, while the control group received a placebo. Adverse events (AEs) were collected for safety evaluation in the 12-month follow-up phase. Primary endpoints were assessed as activities of daily living (ADL), comprehensive function assessment (CFA), and gross motor function measure (GMFM) scales. In addition, cerebral metabolic activity was detected by F-FDG-PET/CT to explore the possible mechanism of the therapeutic effects. Primary endpoint data were analyzed by ANOVA using SPSS version 20.0.

Results: Forty patients were enrolled, and 1 patient withdrew informed consent. Therefore, 39 patients received treatments and completed the scheduled assessments. No significant difference was shown between the 2 groups in AE incidence. Additionally, significant improvements in ADL, CFA, and GMFM were observed in the hUC-MSC group compared with the control group. In addition, the standard uptake value of F-FDG was markedly increased in 3 out of 5 patients from the hUC-MSC group at 12 months after transplantation.

Conclusions: Our clinical data showed that hUC-MSC transplantation was safe and effective at improving the gross motor and comprehensive function of children with CP when combined with rehabilitation. Recovery of cerebral metabolic activity might play an essential role in the improvements in brain function in patients with CP. The therapeutic window, transfusion route, and dosage in our study were considerable for reference in clinical application.

Trial Registration: Chictr.org.cn, ChiCTR1800016554. Registered 08 June 2018-retrospectively registered. The public title was "Randomized trial of umbilical cord-derived mesenchymal stem cells for cerebral palsy."
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13287-019-1545-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998370PMC
February 2020

Genetic Dissection of Femoral and Tibial Microarchitecture.

JBMR Plus 2019 Dec 11;3(12):e10241. Epub 2019 Nov 11.

Department of Genetics, Genomics and Informatics University of Tennessee Health Science Center Memphis TN USA.

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 (, , , , ); (2) candidates without any experimentally validated function in bone (eg, , ), 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 ). 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. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbm4.10241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894729PMC
December 2019

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

JCI Insight 2019 12 5;4(23). Epub 2019 Dec 5.

Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.132820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962016PMC
December 2019

FGF23 induced left ventricular hypertrophy mediated by FGFR4 signaling in the myocardium is attenuated by soluble Klotho in mice.

J Mol Cell Cardiol 2020 01 21;138:66-74. Epub 2019 Nov 21.

Department of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, United States of America. Electronic address:

There is controversy regarding whether excess FGF23 causes left ventricular hypertrophy (LVH) directly through activation of fibroblast growth factor receptor 4 (FGFR4) in cardiomyocytes or indirectly through reductions in soluble Klotho (sK). We investigated the respective roles of myocardial FGFR4 and sKL in mediating FGF23-induced LVH using mouse genetic and pharmacological approaches. To investigate a direct role of myocardial FGFR4 in mediating the cardiotoxic effects of excess circulating FGF23, we administered rFGF23 to mice with cardiac-specific loss of FGFR4 (FGFR4 heart-cKO). We tested a model of sKL deficiency, hypertension and LVH created by the conditional deletion of FGFR1 in the renal distal tubule (FGFR1DT cKO mice). The cardioprotective effects of sKL in both mouse models was assessed by the systemic administration of recombinant sKL. We confirmed that FGF23 treatment activates PLCγ in the heart and induces LVH in the absence of membrane α-Klotho. Conditional deletion of FGFR4 in the myocardium prevented rFGF23-induced LVH in mice, establishing direct cardiotoxicity of FGF23 through activation of FGFR4. Recombinant sKL administration prevented LVH, but not HTN, in FGFR1DT cKO mice, consistent with direct cardioprotective effects. Co-administration of recombinant sKL with FGF23 in culture inhibited rFGF23-induced p-PLCγ signaling. Thus, FGF23 ability to include LVH represents a balance between FGF23 direct cardiac activation of FGFR4 and the modulating effects of circulating sKL to alter FGF23-dependent myocardial signaling pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yjmcc.2019.11.149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195870PMC
January 2020

Validation of a Novel Modified Aptamer-Based Array Proteomic Platform in Patients with End-Stage Renal Disease.

Diagnostics (Basel) 2018 Oct 8;8(4). Epub 2018 Oct 8.

Department of Medicine-Nephrology, University of Tennessee Health Science Center, Memphis, TN 38103, USA.

End stage renal disease (ESRD) is characterized by complex metabolic abnormalities, yet the clinical relevance of specific biomarkers remains unclear. The development of multiplex diagnostic platforms is creating opportunities to develop novel diagnostic and therapeutic approaches. SOMAscan is an innovative multiplex proteomic platform which can measure >1300 proteins. In the present study, we performed SOMAscan analysis of plasma samples and validated the measurements by comparison with selected biomarkers. We compared concentrations of SOMAscan-measured prostate specific antigen (PSA) between males and females, and validated SOMAscan concentrations of fibroblast growth factor 23 (FGF23), FGF receptor 1 (FGFR1), and FGFR4 using Enzyme-Linked immunosorbent assay (ELISA). The median (25th and 75th percentile) SOMAscan PSA level in males and females was 4304.7 (1815.4 to 7259.5) and 547.8 (521.8 to 993.4) relative fluorescence units ( = 0.002), respectively, suggesting biological plausibility. Pearson correlation between SOMAscan and ELISA was high for FGF23 ( = 0.95, < 0.001) and FGFR4 ( = 0.69, < 0.001), indicating significant positive correlation, while a weak correlation was found for FGFR1 ( = 0.13, = 0.16). In conclusion, there is a good to near-perfect correlation between SOMAscan and standard immunoassays for FGF23 and FGFR4, but not for FGFR1. This technology may be useful to simultaneously measure a large number of plasma proteins in ESRD, and identify clinically important prognostic markers to predict outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/diagnostics8040071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316431PMC
October 2018

Role of Fibroblast Growth Factor-23 in Innate Immune Responses.

Front Endocrinol (Lausanne) 2018 12;9:320. Epub 2018 Jun 12.

Department of Medicine, University of Tennessee Health Science Center, Memphis, TN, United States.

Fibroblast growth factor-23 (FGF-23) is a bone-derived hormone that activates FGFR/α-Klotho binary complexes in the kidney renal tubules to regulate phosphate reabsorption and vitamin D metabolism. The objective of this review is to discuss the emerging data that show that FGF-23 has functions beyond regulation of mineral metabolism, including roles in innate immune and hemodynamic responses. Excess FGF-23 is associated with inflammation and adverse infectious outcomes, as well as increased morbidity and mortality, particularly in patients with chronic kidney disease. Enhancer elements in the FGF-23 promoter have been identified that mediate the effects of inflammatory cytokines to stimulate FGF-23 gene transcription in bone. In addition, inflammation induces ectopic expression of FGF-23 and α-Klotho in macrophages that do not normally express FGF-23 or its binary receptor complexes. These observations suggest that FGF-23 may play an important role in regulating innate immunity through multiple potential mechanisms. Circulating FGF-23 acts as a counter-regulatory hormone to suppress 1,25D production in the proximal tubule of the kidney. Since vitamin D deficiency may predispose infectious and cardiovascular diseases, FGF-23 effects on innate immune responses may be due to suppression of 1,25D production. Alternatively, systemic and locally produced FGF-23 may modulate immune functions through direct interactions with myeloid cells, including macrophages and polymorphonuclear leukocytes to impair immune cell functions. Short-acting small molecules that reversibly inhibit FGF-23 offer the potential to block pro-inflammatory and cardiotoxic effects of FGF-23 with less side effects compared with FGF-23 blocking antibodies that have the potential to cause hyperphosphatemia and soft tissue calcifications in animal models. In conclusion, there are several mechanisms by which FGF-23 impacts the innate immune system and further investigation is critical for the development of therapies to treat diseases associated with elevated FGF-23.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fendo.2018.00320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005851PMC
June 2018

Polycystin-1 interacts with TAZ to stimulate osteoblastogenesis and inhibit adipogenesis.

J Clin Invest 2018 01 27;128(1):157-174. Epub 2017 Nov 27.

Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA.

The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. Here, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanical stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2-mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Thus, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI93725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749530PMC
January 2018

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

Chem Biol Drug Des 2018 02 3;91(2):491-504. Epub 2017 Nov 3.

UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, TN, USA.

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).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cbdd.13110DOI Listing
February 2018

A computationally identified compound antagonizes excess FGF-23 signaling in renal tubules and a mouse model of hypophosphatemia.

Sci Signal 2016 11 22;9(455):ra113. Epub 2016 Nov 22.

Department of Medicine, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38165, USA.

Fibroblast growth factor-23 (FGF-23) interacts with a binary receptor complex composed of α-Klotho (α-KL) and FGF receptors (FGFRs) to regulate phosphate and vitamin D metabolism in the kidney. Excess FGF-23 production, which causes hypophosphatemia, is genetically inherited or occurs with chronic kidney disease. Among other symptoms, hypophosphatemia causes vitamin D deficiency and the bone-softening disorder rickets. Current therapeutics that target the receptor complex have limited utility clinically. Using a computationally driven, structure-based, ensemble docking and virtual high-throughput screening approach, we identified four novel compounds predicted to selectively inhibit FGF-23-induced activation of the FGFR/α-KL complex. Additional modeling and functional analysis found that Zinc13407541 bound to FGF-23 and disrupted its interaction with the FGFR1/α-KL complex; experiments in a heterologous cell expression system showed that Zinc13407541 selectivity inhibited α-KL-dependent FGF-23 signaling. Zinc13407541 also inhibited FGF-23 signaling in isolated renal tubules ex vivo and partially reversed the hypophosphatemic effects of excess FGF-23 in a mouse model. These chemical probes provide a platform to develop lead compounds to treat disorders caused by excess FGF-23.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.aaf5034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544179PMC
November 2016

Joint mouse-human phenome-wide association to test gene function and disease risk.

Nat Commun 2016 Feb 2;7:10464. Epub 2016 Feb 2.

Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ∼5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ∼4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets--by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). We tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms10464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740880PMC
February 2016

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

FEBS Lett 2016 Jan 9;590(1):53-67. Epub 2016 Jan 9.

Department of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079529PMC
http://dx.doi.org/10.1002/1873-3468.12040DOI Listing
January 2016

Physiological mechanisms and therapeutic potential of bone mechanosensing.

Rev Endocr Metab Disord 2015 Jun;16(2):115-29

Department of Medicine, University of Tennessee Health Science Center, Memphis, TN, 38165, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079521PMC
http://dx.doi.org/10.1007/s11154-015-9313-4DOI Listing
June 2015

Membrane and integrative nuclear fibroblastic growth factor receptor (FGFR) regulation of FGF-23.

J Biol Chem 2015 Apr 9;290(16):10447-59. Epub 2015 Mar 9.

From the Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.609230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400353PMC
April 2015

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

PLoS One 2014 2;9(12):e114198. Epub 2014 Dec 2.

Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, 38165, United States of America.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114198PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4252138PMC
January 2016

Osteocyte-specific deletion of Fgfr1 suppresses FGF23.

PLoS One 2014 4;9(8):e104154. Epub 2014 Aug 4.

University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104154PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121311PMC
November 2015

Genistein promotion of osteogenic differentiation through BMP2/SMAD5/RUNX2 signaling.

Int J Biol Sci 2013 21;9(10):1089-98. Epub 2013 Nov 21.

1. Division of Gastroenterology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, 510080, China; ; 2. Institute of Clinical Pharmacology, Xiangya Medical College, Central South University, Changsha, Hunan, 410078, China;

To investigate the effects of Genistein on the osteogenic related gene expression profiles during osteoblastic differentiation of human bone marrow mesenchymal stem cell (hBMSC) cultures, the hBMSCs were cultured under osteogenic differentiation medium with the addition of Genistein (10(-8)∼10(-5) M) for 12 days. The cell proliferation was measured by BrdU incorporation, while the osteoblastic differentiation in hBMSC cultures was assessed by cellular alkaline phosphatase (ALP) activity. The cell apoptosis was determined by caspase 3/7 activation. GEArray Q series human osteogenesis gene array was used to analyze large-scale gene expression in Genistein-treated hBMSC cultures compared to the control group. Quantitative real-time RT-PCR, small interfering RNA (siRNA), and western blot analysis were used to confirm the microarray data in five representative transcripts. Genistein (10(-8)∼10(-6) M) dose- and time-dependently increased cell proliferation and cellular ALP activity, but had no significant effect on cell apoptosis in hBMSC cultures. The 96-gene array analysis indicated that 22 genes were upregulated more than 2-fold and 7 genes were downregulated at least 1.5-fold. The expressions of bone morphogenetic proteins (BMPs), small mothers against decapentaplegic homologs (SMADs), and Runt-related transcription factor 2 (RUNX2) were concomitantly increased under Genistein treatment while insulin-like growth factor 2 and inhibitory SMADs 6 and 7 expressions were significantly decreased. The results of the real-time RT-PCR had a correlation with the results of microarray analysis and were estrogen-receptor dependent. Specific gene siRNAs knock-down further confirmed the osteogenic effects of Genistein on BMP2, SMAD5 and RUNX2 protein expression. Genistein enhanced osteogenic differentiation in cultured hBMSCs mainly through the BMP-dependent SMADs and RUNX2 signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/ijbs.7367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858582PMC
July 2014

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

PLoS One 2012 21;7(9):e46038. Epub 2012 Sep 21.

Department of Medicine, University of Tennessee Health Science Center, Memphis, United States of America.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046038PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448720PMC
March 2013

Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia.

J Cell Sci 2012 Apr 22;125(Pt 8):1945-57. Epub 2012 Feb 22.

Department of Medicine, the University of Tennessee Health Science Center, Memphis, TN 38165, USA.

We investigated whether Kif3a in osteoblasts has a direct role in regulating postnatal bone formation. We conditionally deleted Kif3a in osteoblasts by crossing osteocalcin (Oc; also known as Bglap)-Cre with Kif3a(flox/null) mice. Conditional Kif3a-null mice (Kif3a(Oc-cKO)) had a 75% reduction in Kif3a transcripts in bone and osteoblasts. Conditional deletion of Kif3a resulted in the reduction of primary cilia number by 51% and length by 27% in osteoblasts. Kif3a(Oc-cKO) mice developed osteopenia by 6 weeks of age unlike Kif3a(flox/+) control mice, as evidenced by reductions in femoral bone mineral density (22%), trabecular bone volume (42%) and cortical thickness (17%). By contrast, Oc-Cre;Kif3a(flox/+) and Kif3a(flox/null) heterozygous mice exhibited no skeletal abnormalities. Loss of bone mass in Kif3a(Oc-cKO) mice was associated with impaired osteoblast function in vivo, as reflected by a 54% reduction in mineral apposition rate and decreased expression of Runx2, osterix (also known as Sp7 transcription factor 7; Sp7), osteocalcin and Dmp1 compared with controls. Immortalized osteoblasts from Kif3a(Oc-cKO) mice exhibited increased cell proliferation, impaired osteoblastic differentiation, and enhanced adipogenesis in vitro. Osteoblasts derived from Kif3a(Oc-cKO) mice also had lower basal cytosolic calcium levels and impaired intracellular calcium responses to fluid flow shear stress. Sonic hedgehog-mediated Gli2 expression and Wnt3a-mediated β-catenin and Axin2 expression were also attenuated in Kif3a(Oc-cKO) bone and osteoblast cultures. These data indicate that selective deletion of Kif3a in osteoblasts disrupts primary cilia formation and/or function and impairs osteoblast-mediated bone formation through multiple pathways including intracellular calcium, hedgehog and Wnt signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.095893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360919PMC
April 2012

Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells.

J Cell Biochem 2012 Mar;113(3):967-76

Institute of Clinical Pharmacology, Central South University, Changsha, Hunan, 410078, China.

Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARγ and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 µM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1 µM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcb.23426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264804PMC
March 2012

Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice.

FASEB J 2011 Jul 31;25(7):2418-32. Epub 2011 Mar 31.

Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38165, USA.

We investigated whether polycystin-1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1-Cre with Pkd1(flox/m1Bei) mice, in which the m1Bei allele is nonfunctional. We assessed in wild-type and Pkd1-deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1-Cre;Pkd1(flox/+)) and null mice (Pkd1(Dmp1-cKO)) exhibited a ∼ 40 and ∼ 90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2-II, Osteocalcin, Dmp1, and Phex. Anabolic load-induced periosteal lamellar MAR (0.58 ± 0.14; Pkd1(Dmp1-cKO) vs. 1.68 ± 0.34 μm/d; control) and increases in Cox-2, c-Jun, Wnt10b, Axin2, and Runx2-II gene expression were significantly attenuated in Pkd1(Dmp1-cKO) mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1(null/null) and Pkd1(m1Bei/m1Bei)-derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild-type controls. These data indicate that polycystin-1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.10-180299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219213PMC
July 2011

Inducible expression of Runx2 results in multiorgan abnormalities in mice.

J Cell Biochem 2011 Feb;112(2):653-65

The Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

Runx2 is a transcription factor controlling skeletal development, and is also expressed in extraskeletal tissues where its function is not well understood. Existing Runx2 mutant and transgenic mouse models do not allow the necessary control of Runx2 expression to understand its functions in different tissues. We generated conditional, doxycyline-inducible, triple transgenic mice (CMV-Cre;ROSA26-neo(flox/+)-rtTA;Tet-O-Runx2) to investigate the effects of wide spread overexpression of Runx2. Osteoblasts isolated from CMV-Cre;ROSA26-neo(flox/+)-rtTA; Tet-O-Runx2 mice demonstrated a dose-dependent effect of doxycycline to stimulate Runx2 transgene expression. Doxycycline administration to CMV-Cre;ROSA26-neo(flox/+)-rtTA;Tet-O-Runx2 mice induced Runx2 transgene expression in all tissues tested, with the highest levels observed in kidney, ovary, and bone. Runx2 overexpression resulted in deceased body size and reduced viability. With regard to bone, Runx2 overexpressing mice paradoxically displayed profound osteopenia and diminished osteogenesis. Induced expression of Runx2 in extraskeletal tissues resulted in ectopic calcification and induction of the osteogenic program in a limited number of tissues, including lung and muscle. In addition, the triple transgenic mice showed evidence of a myeloproliferative disorder and an apparent inhibition of lymphocyte development. Thus, overexpression of Runx2 both within and outside of the skeleton can have diverse biological effects. Use of tissue specific Cre mice will allow this model to be used to conditionally and inducibly overexpress Runx2 in different tissues and provide a means to study the post-natal tissue- and cell context-dependent functions of Runx2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079519PMC
http://dx.doi.org/10.1002/jcb.22968DOI Listing
February 2011

Kif3a deficiency reverses the skeletal abnormalities in Pkd1 deficient mice by restoring the balance between osteogenesis and adipogenesis.

PLoS One 2010 Dec 2;5(12):e15240. Epub 2010 Dec 2.

Institute of Clinical Pharmacology, Central South University, Changsha, Hunan, China.

Pkd1 localizes to primary cilia in osteoblasts and osteocytes. Targeted deletion of Pkd1 in osteoblasts results in osteopenia and abnormalities in Runx2-mediated osteoblast development. Kif3a, an intraflagellar transport protein required for cilia function, is also expressed in osteoblasts. To assess the relationship between Pkd1 and primary cilia function on bone development, we crossed heterozygous Pkd1- and Kif3a-deficient mice to create compound Pkd1 and Kif3a-deficient mice. Pkd1 haploinsufficiency (Pkd1(+/Δ)) resulted in osteopenia, characterized by decreased bone mineral density, trabecular bone volume, and cortical thickness. In addition, deficiency of Pkd1 resulted in impaired osteoblastic differentiation and enhanced adipogenesis in both primary osteoblasts and/or bone marrow stromal cell cultures. These changes were associated with decreased Runx2 expression, increased PPARγ expression, and impaired hedgehog signaling as evidenced by decreased Gli2 expression in bone and osteoblast cultures. In contrast, heterozygous Kif3a(+/Δ) mice display no abnormalities in skeletal development or osteoblast function, but exhibited decreased adipogenic markers in bone and impaired adipogenesis in vitro in association with decreased PPARγ expression and upregulation of Gli2. Superimposed Kif3a deficiency onto Pkd1(+/Δ) mice paradoxically corrected the effects of Pkd1 deficiency on bone mass, osteoblastic differentiation, and adipogenesis. In addition, Runx2, PPARγ and Gli2 expression in bone and osteoblasts were normalized in compound double Pkd1(+/Δ) and Kif3a(+/Δ) heterozygous mice. The administration of sonic hedgehog, overexpression of Gli2, and the PC1 C-tail construct all increased Gli2 and Runx2-II expression, but decreased PPARγ2 gene expression in C3H10T1/2 cells. Our findings suggest a role for Pkd1 and Kif3a to counterbalance the regulation of osteogenesis and adipogenesis through differential regulation of Runx2 and PPARγ by Gli2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015240PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996304PMC
December 2010

Conditional disruption of Pkd1 in osteoblasts results in osteopenia due to direct impairment of bone formation.

J Biol Chem 2010 Jan 3;285(2):1177-87. Epub 2009 Nov 3.

Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

PKD1 (polycystin-1), the disease-causing gene for ADPKD, is widely expressed in various cell types, including osteoblasts, where its function is unknown. Although global inactivation of Pkd1 in mice results in abnormal skeletal development, the presence of polycystic kidneys and perinatal lethality confound ascertaining the direct osteoblastic functions of PKD1 in adult bone. To determine the role of PKD1 in osteoblasts, we conditionally inactivated Pkd1 in postnatal mature osteoblasts by crossing Oc (osteocalcin)-Cre mice with floxed Pkd1 (Pkd1(flox/m1Bei)) mice to generate conditional heterozygous (Oc-Cre;Pkd1(flox/+)) and homozygous (Oc-Cre;Pkd1(flox/m1Bei)) Pkd1-deficient mice. Cre-mediated recombination (Pkd1(Delta flox)) occurred exclusively in bone. Compared with control mice, the conditional deletion of Pkd1 from osteoblasts resulted in a gene dose-dependent reduction in bone mineral density, trabecular bone volume, and cortical thickness. In addition, mineral apposition rates and osteoblast-related gene expression, including Runx2-II (Runt-related transcription factor 2), osteocalcin, osteopontin, and bone sialoprotein, were reduced proportionate to the reduction of Pkd1 gene dose in bone of Oc-Cre;Pkd1(flox/+) and Oc-Cre;Pkd1(flox/m1Bei) mice. Primary osteoblasts derived from Oc-Cre;Pkd1(flox/m1Bei) displayed impaired differentiation and suppressed activity of the phosphatidylinositol 3-kinase-Akt-GSK3beta-beta-catenin signaling pathways. The conditional deletion of Pkd1 also resulted in increased adipogenesis in bone marrow and in osteoblast cultures. Thus, PKD1 directly functions in osteoblasts to regulate bone formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.050906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801246PMC
January 2010

Novel regulators of Fgf23 expression and mineralization in Hyp bone.

Mol Endocrinol 2009 Sep 25;23(9):1505-18. Epub 2009 Jun 25.

The Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts associated with abnormal Fgf23 production and mineralization in Hyp mice. We found evidence that elevation of Fgf23 expression in osteocytes is associated with increments in Fgf1, Fgf7, and Egr2 and decrements in Sost, an inhibitor in the Wnt-signaling pathway, were observed in Hyp bone. beta-Catenin levels were increased in Hyp cortical bone, and TOPflash luciferase reporter assay showed increased transcriptional activity in Hyp-derived osteoblasts, consistent with Wnt activation. Moreover, activation of Fgf and Wnt-signaling stimulated Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the extracellular matrix protein Dmp1. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced posttranslational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regard to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH-altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2009-0085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737552PMC
September 2009

RUNX2 mutations in Chinese patients with cleidocranial dysplasia.

Mutagenesis 2009 Sep 10;24(5):425-31. Epub 2009 Jun 10.

Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China.

Cleidocranial dysplasia (CCD) is an autosomal dominant bone disease in humans caused by haploinsufficiency of the RUNX2 gene. The RUNX2 has two major isoforms derived from P1 and P2 promoters. Over 90 mutations of RUNX2 have been reported associated with CCD. In our study, DNA samples of nine individuals from three unrelated CCD families were collected and screened for all exons of RUNX2 and 2 kb of P1 and P2 promoters. We identified two point mutations in the RUNX2 gene in Case 1, including a nonsense mutation (c.577C>T) that has been reported previously and a silent substitution (c.240G>A). In vitro studies demonstrated that c.577C>T mutation led to truncated RUNX2 protein production and diminished stimulating effects on mouse osteocalcin promoter activity when compared with full-length Runx2-II and Runx2-I isoforms. These results confirm that loss of function RUNX2 mutation (c.577C>T) in Case 1 family is responsible for its CCD phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/mutage/gep025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2734498PMC
September 2009

Dose-dependent effects of Runx2 on bone development.

J Bone Miner Res 2009 Nov;24(11):1889-904

Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

Runx2 controls the commitment of mesenchymal cells to the osteoblastic lineage. Distinct promoters, designated P1 and P2, give rise to functionally similar Runx2-II and Runx2-I isoforms. We postulate that this dual promoter gene structure permits temporal and spatial adjustments in the amount of Runx2 isoforms necessary for optimal bone development. To evaluate the gene dose-dependent effect of Runx2 isoforms on bone development, we intercrossed selective Runx2-II(+/-) with nonselective Runx2-II(+/-)/Runx2-I(+/-) mice to create compound mutant mice: Runx2-II(+/-), Runx2-II(+/-)/Runx2-I(+/-), Runx2-II(-/-), Runx2-II(-/-)/Runx2-I(+/-), Runx2-II(-/-)/Runx2-I(-/-). Analysis of the different Runx2-deficient genotypes showed gene dose-dependent differences in the level of expression of the Runx2 isoforms. In addition, we found that Runx2-I is predominately expressed in the perichondrium and proliferating chondrocytes, whereas Runx2-II is expressed in hypertrophic chondrocytes and metaphyseal osteoblasts. Newborn mice showed impaired development of a mineralized skeleton, bone length, and widening of the hypertrophic zone that were proportionate to the reduction in total Runx2 protein expression. Osteoblast differentiation ex vivo was also proportionate to total amount of Runx2 expression that correlated with reduced Runx2 binding to the osteocalcin promoter by quantitative chromatin immunoprecipitation analysis. Functional analysis of P1 and P2 promoters showed differential regulation of the two promoters in osteoblastic cell lines. These findings support the possibility that the total amount of Runx2 derived from two isoforms and the P1 and P2 promoters, by regulating the time, place, and amount of Runx2 in response to changing environmental cues, impacts on bone development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1359/jbmr.090502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765932PMC
November 2009