Musculoskeletal Disease Center 151

Loma Linda, CA, United States

Musculoskeletal Disease Center 151

Loma Linda, CA, United States
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Aghajanian P.,Musculoskeletal Disease Center 151 | Hall S.,Musculoskeletal Disease Center 151 | Hall S.,Loma Linda University | Wongworawat M.D.,Musculoskeletal Disease Center 151 | And 3 more authors.
Journal of Bone and Mineral Research | Year: 2015

Vitamin C is an important antioxidant and cofactor that is involved in the regulation of development, function, and maintenance of several cell types in the body. Deficiencies in Vitamin C can lead to conditions such as scurvy, which, among other ailments, causes gingivia, bone pain, and impaired wound healing. This review examines the functional importance of Vitamin C as it relates to the development and maintenance of bone tissues. Analysis of several epidemiological studies and genetic mouse models regarding the effect of Vitamin C shows a positive effect on bone health. Overall, Vitamin C exerts a positive effect on trabecular bone formation by influencing expression of bone matrix genes in osteoblasts. Recent studies on the molecular pathway for Vitamin C actions that include direct effects of Vitamin C on transcriptional regulation of target genes by influencing the activity of transcription factors and by epigenetic modification of key genes involved in skeletal development and maintenance are discussed. With an understanding of mechanisms involved in the uptake and metabolism of Vitamin C and knowledge of precise molecular pathways for Vitamin C actions in bone cells, it is possible that novel therapeutic strategies can be developed or existing therapies can be modified for the treatment of osteoporotic fractures. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.


Kapur S.,Musculoskeletal Disease Center 151 | Amoui M.,Musculoskeletal Disease Center 151 | Kesavan C.,Musculoskeletal Disease Center 151 | Wang X.,Musculoskeletal Disease Center 151 | And 5 more authors.
Journal of Biological Chemistry | Year: 2010

This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ∼2,500 με, which was insufficient to produce a bone formation response in B6 mice, significantly increased bone formation parameters in leptin-deficient ob-/ob - mice and that a loading strain of ∼3,000 με also yielded greater osteogenic responses in Leprdeficient db-/db - mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [3H]thymidine incorporation and Erk1/2 phosphorylation in ob-/ob- osteoblasts and db-/db- osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced (but in osteoblasts of C3H (the mouse strain with poor bone mechanosensitivity) restored) their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts. In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which result in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice.


Linares G.R.,Musculoskeletal Disease Center 151 | Linares G.R.,Loma Linda University | Brommage R.,Lexicon Pharmaceuticals Inc. | Powell D.R.,Lexicon Pharmaceuticals Inc. | And 12 more authors.
Journal of Bone and Mineral Research | Year: 2012

Claudin 18 (Cldn-18) belongs to a large family of transmembrane proteins that are important components of tight junction strands. Although several claudin members are expressed in bone, the functional role for any claudin member in bone is unknown. Here we demonstrate that disruption of Cldn-18 in mice markedly decreased total body bone mineral density, trabecular bone volume, and cortical thickness in Cldn-18 -/- mice. Histomorphometric studies revealed that bone resorption parameters were increased significantly in Cldn-18 -/- mice without changes in bone formation. Serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b) and mRNA expression levels of osteoclast specific markers and signaling molecules were also increased. Loss of Cldn-18 further exacerbated calcium deficiency induced bone loss by influencing bone resorption, thereby resulting in mechanically weaker bone. In vitro studies with bone marrow macrophages revealed Cldn-18 disruption markedly enhanced receptor activator of NF-kB ligand (RANKL)-induced osteoclast differentiation but not macrophage colony-stimulating factor (MCSF)-induced bone marrow macrophage (BMM) proliferation. Consistent with a direct role for Cldn-18 in regulating osteoclast differentiation, overexpression of wild type but not PDZ binding motif deleted Cldn-18 inhibited RANKL-induced osteoclast differentiation. Furthermore, our findings indicate that Cldn-18 interacts with Zonula occludens 2 (ZO-2) to modulate RANKL signaling in osteoclasts. In conclusion, we demonstrate that Cldn-18 is a novel negative regulator of bone resorption and osteoclast differentiation. © 2012 American Society for Bone and Mineral Research.


Wergedal J.E.,Musculoskeletal Disease Center | Wergedal J.E.,Loma Linda University | Kesavan C.,Musculoskeletal Disease Center | Kesavan C.,Loma Linda University | And 5 more authors.
Endocrinology | Year: 2015

In this study, we evaluated the role of WNT16 in regulating bone size, an important determinant of bone strength. Mice with targeted disruption of the Wnt16 gene exhibited a 24% reduction in tibia cross-sectional area at 12 weeks of age compared with that of littermate wild-type (WT) mice. Histomorphometric studies revealed that the periosteal bone formation rate and mineral apposition rate were reduced (P < .05) by 55% and 32%, respectively, in Wnt16 knockout (KO) vs WT mice at 12 weeks of age. In contrast, the periosteal tartrate resistant acid phosphatase-labeled surface was increased by 20% in the KO mice. Because mechanical strain is an important physiological regulator of periosteal bone formation (BF), we determined whether mechanical loading-induced periosteal BF is compromised in Wnt16 KO mice. Application of 4800-μe strain to the right tibia using a 4-point bending loading method for 2 weeks (2-Hz frequency, 36 cycles per day, 6 days/wk) produced a significant increase in cross-sectional area (11% above that of the unloaded left tibia, P < .05, n = 6) in the WT but not in the KO mice (-0.2% change). Histomorphometric analyses revealed increases in the periosteal bone formation rate and mineral apposition rate in the loaded bones of WT but not KO mice. Wnt16 KO mice showed significant (20%-70%) reductions in the expression levels of markers of canonical (β-catenin and Axin2) but not noncanonical (Nfatc1 and Tnnt2) WNT signaling in the periosteum at 5 weeks of age. Our findings suggest that WNT16 acting via canonical WNT signaling regulates mechanical strain-induced periosteal BF and bone size. Copyright © 2015 by the Endocrine Society


Mohan S.,Musculoskeletal Disease Center 151 | Mohan S.,Loma Linda University | Hu Y.,Musculoskeletal Disease Center 151 | Edderkaoui B.,Musculoskeletal Disease Center 151 | Edderkaoui B.,Loma Linda University
Calcified Tissue International | Year: 2013

Studies on the identification of the genetic basis for sexual dimorphism in peak bone mass are obviously important for providing novel therapeutic approaches to prevent or treat metabolic bone diseases. Our goal in this study was to identify the bone microstructure that could lead to differences in volumetric bone mineral density (vBMD) and new candidate genes that regulate the gender effect on bone. We used a congenic line of mice that carry the BMD1-4 locus from CAST/EiJ (CAST) mice in a C57BL/6J (B6) background and show greater vBMD in female, but not male, congenics compared to age- and gender-matched B6 mice. To assess the vBMD variations between the two lines of mice, we performed μCT measurements and found no difference in cortical bone volume by tissue volume (BV/TV) between congenics and B6 mice. However, trabecular BV/TV was significantly greater in female, but not male, congenics compared to corresponding B6 mice, which was due to increased trabecular thickness but not reduced trabecular separation, suggesting that bone formation, but not bone resorption, is responsible for the trabecular bone phenotype observed in the female, but not male, congenics. To identify the gender candidate genes, we determined the polymorphisms between B6 and CAST within the BMD1-4 locus and performed gene expression profiling. We identified EF-hand calcium binding domain (Efcab2), consortin, connexin sorting protein (Cnst), and presenilin 2 (Psen2) as potential candidate genes that regulate bone mass by influencing trabecular thickness in a gender-specific manner. © 2012 Springer Science+Business Media New York.


Cheng S.,Musculoskeletal Disease Center 151 | Xing W.,Musculoskeletal Disease Center 151 | Xing W.,Loma Linda University | Pourteymoor S.,Musculoskeletal Disease Center 151 | And 3 more authors.
Endocrinology | Year: 2016

The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2genein chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (α50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length andosteoid surface perbonesurface in the primary spongiosa of thecKOmice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in themRNAlevels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice. Copyright © 2016 by the Endocrine Society.


Xing W.,Musculoskeletal Disease Center 151 | Xing W.,Loma Linda University | Kim J.,Musculoskeletal Disease Center 151 | Wergedal J.,Musculoskeletal Disease Center 151 | And 5 more authors.
Molecular and Cellular Biology | Year: 2010

Mutations of ephrin B1 in humans result in craniofrontonasal syndrome. Because little is known of the role and mechanism of action of ephrin B1 in bone, we examined the function of osteoblast-produced ephrin B1 in vivo and identified the molecular mechanism by which ephrin B1 reverse signaling regulates bone formation. Targeted deletion of the ephrin B1 gene in type 1α2 collagen-producing cells resulted in severe calvarial defects, decreased bone size, bone mineral density, and trabecular bone volume, caused by impairment in osterix expression and osteoblast differentiation. Coimmunoprecipitation of the TAZ complex with TAZ-specific antibody revealed a protein complex containing ephrin B1, PTPN13, NHERF1, and TAZ in bone marrow stromal (BMS) cells. Activation of ephrin B1 reverse signaling with soluble EphB2-Fc led to a time-dependent increase in TAZ dephosphorylation and shuttling from cytoplasm to nucleus. Treatment of BMS cells with exogenous EphB2-Fc resulted in a 4-fold increase in osterix expression as determined by Western blotting. Disruption of TAZ expression using specific lentivirus small hairpin RNA (shRNA) decreased TAZ mRNA by 80% and ephrin B1 reverse signaling-mediated increases in osterix mRNA by 75%. Knockdown of NHERF1 expression reduced basal levels of osterix expression by 90% and abolished ephrin B1-mediated induction of osterix expression. We conclude that locally produced ephrin B1 mediates its effects on osteoblast differentiation by a novel molecular mechanism in which activation of reverse signaling leads to dephosphorylation of TAZ and subsequent release of TAZ from the ephrin B1/NHERF1/TAZ complex to translocate to the nucleus to induce expression of the osterix gene and perhaps other osteoblast differentiation genes. Our findings provide strong evidence that ephrin B1 reverse signaling in osteoblasts is critical for BMS cell differentiation and bone formation. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Kim J.,Musculoskeletal Disease Center 151 | Xing W.,Musculoskeletal Disease Center 151 | Xing W.,Loma Linda University | Wergedal J.,Musculoskeletal Disease Center 151 | And 4 more authors.
Physiological Genomics | Year: 2010

Previous in vitro studies found that nuclear factor erythroid-derived 2-like 1 (NFE2L1) was involved in mediating ascorbic acid-induced osterix expression and osteoblast differentiation via binding to the antioxidant response element of the osterix promoter. To test the role of NFE2L1 in regulating bone formation in vivo, we disrupted NFE2L1 specifically in osteoblasts. Mice expressing Cre under the control of Col1α2 promoter were crossed with NFE2L1 loxP mice to generate Cre+ knockout (KO) and Cre- wild-type (WT) mice. Skeletal measurements by DEXA revealed 8-10% and 9-11% reduction in total body BMC and bone area in the KO mice from 3 to 8 wk of age. Peripheral quantitative computed tomography analyses found both periosteal and endosteal circumferences were reduced by 6% at the middiaphysis of the femurs from 8 wk old KO mice. Histomorphometric analyses revealed reduced bone formation was a cause for reduced bone size in the KO mice. Microcomputed tomography analysis of the metaphysis of the femur revealed that trabecular bone volume/total volume, and trabecular numbers were decreased by 30 and 53% in the NFE2L1 KO mice. Expression of osterix was decreased by 57% in the bones of NFE2L1 KO mice. In vitro nodule assay demonstrated that mineralized nodule area was reduced by 68% in the cultures of bone marrow stromal cells from NFE2L1 KO mice. Treatment of primary osteoblasts with ascorbic acid increased osterix expression by fourfold, whereas loss of NFE2L1 in osteoblasts diminished ascorbic acid stimulation of osterix expression by 50%. Our data provide the first in vivo experimental evidence that NFE2L1 produced by osteoblasts is involved in regulating osterix expression, osteoblast differentiation, and bone formation.


Stiffel V.,Musculoskeletal Disease Center 151 | Amoui M.,Musculoskeletal Disease Center 151 | Sheng M.H.-C.,Musculoskeletal Disease Center 151 | Sheng M.H.-C.,Loma Linda University | And 4 more authors.
Journal of Bone and Mineral Research | Year: 2014

Of the ephrin (Eph) receptors, mature osteoclasts express predominantly EphA4. This study sought to determine if EphA4 has a regulatory role in osteoclasts. Treatment of RAW/C4 cells with Epha4 small interfering RNAs (siRNAs) increased average size, Ctsk mRNA expression level, and bone resorption activity of the derived osteoclast-like cells. Activation of the EphA4 signaling in osteoclast precursors with EfnA4-fc chimeric protein reduced cell size and resorption activity of the derived osteoclasts. Homozygous Epha4 null mice had substantially less trabecular bone in femur and vertebra compared to wild-type controls. The bone loss was due to a decrease in trabecular number and an increase in trabecular spacing, but not to an increase in osteoclast-lined bone surface or an increase in the number of osteoclasts on bone surface. Dynamic histomorphometry and serum biomarker analyses indicate that bone formation in Epha4 null mice was reduced slightly but not significantly. Osteoclasts of Epha4 null mice were also larger, expressed higher levels of Mmp3 and Mmp9 mRNAs, and exhibited greater bone resorption activity than wild-type osteoclasts in vitro. Deficient Epha4 expression had no effects on the total number of osteoclast formed in response to receptor activator of NF-κB ligand nor on apoptosis of osteoclasts in vitro. It also did not affect the protein-tyrosine phosphorylation status of its ligands, EfnB2, EfnA2, and EfnA4, in osteoclasts. Deficient Epha4 expression in Epha4 null osteoclasts activated the β3-integrin signaling through reduced phosphorylation of the tyr-747 residue, which led to increased binding of the stimulatory talin and reduced binding of the inhibitory Dok1 to β3-integrin. This in turn activated Vav3 and the bone resorption activity of osteoclasts. In conclusion, we demonstrate for the first time that EphA4 is a potent negative regulator of osteoclastic activity, mediated in part through increased Dok1 binding to β3-integrin via an increase in EphA4-dependent tyr-747 phosphorylation. © 2014 American Society for Bone and Mineral Research.


Cheng S.,Musculoskeletal Disease Center 151 | Xing W.,Musculoskeletal Disease Center 151 | Xing W.,Loma Linda University | Pourteymoor S.,Musculoskeletal Disease Center 151 | And 2 more authors.
Journal of Bone and Mineral Research | Year: 2014

We have previously shown that the increase in osterix (Osx) expression during osteoblast maturation is dependent on the activity of the prolyl hydroxylase domain-containing protein 2 (Phd2), a key regulator of protein levels of the hypoxia-inducible factor family proteins in many tissues. In this study, we generated conditional Phd2 knockout mice (cKO) in osteoblast lineage cells by crossing floxed Phd2 mice with a Col1α2-iCre line to investigate the function of Phd2 in vivo. The cKO mice developed short stature and premature death at 12 to 14 weeks of age. Bone mineral content, bone area, and bone mineral density were decreased in femurs and tibias, but not vertebrae of the cKO mice compared to WT mice. The total volume (TV), bone volume (BV), and bone volume fraction (BV/TV) in the femoral trabecular bones of cKO mice were significantly decreased. Cross-sectional area of the femoral mid-diaphysis was also reduced in the cKO mice. The reduced bone size and trabecular bone volume in the cKO mice were a result of impaired bone formation but not bone resorption as revealed by dynamic histomorphometric analyses. Bone marrow stromal cells derived from cKO mice formed fewer and smaller nodules when cultured with mineralization medium. Quantitative RT-PCR and immunohistochemistry detected reduced expression of Osx, osteocalcin, and bone sialoprotein in cKO bone cells. These data indicate that Phd2 plays an important role in regulating bone formation in part by modulating expression of Osx and bone formation marker genes. © 2014 American Society for Bone and Mineral Research.

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