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ABSTRACT: To assess the roles of Lrrk1 and Lrrk2, we examined skeletal phenotypes in Lrrk1 and Lrrk2 knockout (KO) mice. Lrrk1 KO mice exhibit severe osteopetrosis caused by dysfunction of multinucleated osteoclasts, reduced bone resorption in endocortical and trabecular regions, and increased bone mineralization. Lrrk1 KO mice have lifelong accumulation of bone and respond normally to the anabolic actions of teriparatide treatment, but are resistant to ovariectomy-induced bone boss. Precursors derived from Lrrk1 KO mice differentiate into multinucleated cells in response to M-CSF/RANKL treatment, but these cells fail to form peripheral sealing zones and ruffled borders, and fail to resorb bone. The phosphorylation of c-Src at Tyr-527 is significantly elevated whereas at Tyr-416 is decreased in Lrrk1 deficient osteoclasts. The defective osteoclast function is partially rescued by overexpression of the constitutively active form of Y527F c-Src. Immunoprecipitation assays in osteoclasts detected a physical interaction of Lrrk1 with Csk. Lrrk2 KO mice do not show obvious bone phenotypes. Precursors derived from Lrrk2 KO mice differentiate into functional multinucleated osteoclasts. Our finding of osteopetrosis in Lrrk1 KO mice provides convincing evidence that Lrrk1 plays a critical role in negative regulation of bone mass in part through modulating the c-Src signaling pathway in mice. © 2013 American Society for Bone and Mineral Research.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 03/2013; · 6.04 Impact Factor
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ABSTRACT: Claudin-18 (Cldn-18) is a negative regulator of RANKL-induced osteoclast differentiation and bone resorption (BR) in vivo. Since estrogen deficiency decreases bone mass in part by a RANKL-mediated increase in BR, we evaluated if estrogen regulates Cldn-18 expression in bone. We found that Cldn-18 expression was reduced in the bones of estrogen deficient mice while it was increased by estrogen treatment in osteoblasts in vitro. Cldn-18 knockout (KO) and littermate wild type (WT) mice were ovariectomized (OVX) or sham operated at 6 wks of age and the skeletal phenotype evaluated at 14 wks of age. PIXImus revealed that total body, femur and lumbar BMD were reduced by 8-13% (P<0.05) after 8 wks of OVX compared to sham in WT mice. As expected, total body, femur and lumbar BMD were reduced by 14-21% (P<0.05) in Cldn-18 KO sham mice compared to sham WT mice. However, OVX failed to induce significant changes in BMD of total body, femur or vertebra in the Cldn-18 KO mice. Micro-CT analysis of the distal femur revealed that trabecular (Tb) bone volume was decreased by 50% in the OVX WT mice compared to sham that was caused by a 26% decrease in Tb. number and a 30% increase in Tb. separation (all P<0.05). By contrast, none of the Tb. parameters were significantly different in OVX Cldn-18 KO mice compared to sham KO mice. Based on our findings, we conclude that the estrogen effects on osteoclasts may in part be mediated via regulation of Cldn-18 signaling.
AJP Endocrinology and Metabolism 01/2013; · 4.75 Impact Factor
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ABSTRACT: 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.
Calcified Tissue International 12/2012; · 2.38 Impact Factor
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ABSTRACT: While there is compelling evidence for an anabolic role of PAPP-A, an IGFBP protease, in muscle development, its effect on dynamic regulation of muscle regeneration has not been investigated. In this study, we evaluated the effect of transgenic PAPP-A overexpression in skeletal muscle of mice on myofiber formation in intact and crush-injured tibialus anterior muscle.
Skeletal muscle in transgenic mice overexpressing human PAPP-A in skeletal muscle was subjected to crush-injury. Myofiber formation and myogenic gene expression were then evaluated in injured or intact muscle of PAPP-A transgenic mice and wild-type mice.
In the intact muscle, aging PAPP-A transgenic (Tg.) mice (age of 12 months) showed more than a 2-fold increase in both myofiber size and number of nuclei per myofiber compared with their wild-type (Wt.) littermates. Myofibers with centered nuclei, a hallmark of muscle regeneration, were increased from <1% in Wt. mice to 65% in Tg. muscle. In the injured muscle, reduced inflammatory cell infiltration and increased new myofiber size and the area occupied by new myofibers were observed in PAPP-A transgenic mice compared to wild-type littermates. MyoD and creatine kinase in the injured muscle was also significantly increased in the Tg. mice. Although TNF-α induced PAPP-A expression in skeletal myoblast culture and its expression increased upon injury, abrogation of TNF-α signaling in TNF-α receptor knockout mice had no impact on the extent of injury induction of PAPP-A. We also found that TGF-β expression was significantly increased following muscle injury in vivo and treatment with recombinant TGF-β in vitro significantly enhanced PAPP-A expression in skeletal myoblasts.
Our findings demonstrate that exogenous PAPP-A can promote recovery of muscle injury in aging mice albeit the expression of endogenous PAPP-A had already been increased dramatically upon muscle injury.
Growth hormone & IGF research: official journal of the Growth Hormone Research Society and the International IGF Research Society 06/2012; 22(5):173-9. · 2.35 Impact Factor
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ABSTRACT: Transforming growth factor-beta induced (TGFBI) and periostin are two closely related proteins in structure as well as in function. A previous study found that periostin positively regulates bone size. Here, we hypothesize that TGFBI has a similar function in bone development. To test this hypothesis, we employed TGFBI-deficient mice, which were generated by targeted disruption of the TGFBI gene. We bred these mice with C57BL/6J mice to generate homozygous TGFBI-deficient (TGFBI(-/-)) mice and homozygous wild-type littermates. All mice were raised to 12 weeks of age. Bone mass parameters were determined by PIXImus and micro-CT, bone strength parameters by three-point bending, and bone formation and resorption parameters by histomorphometry. We found that targeted disruption of TGFBI led to reduced body size, bone mass, bone size, and bone strength. This indicates that, like periostin, TGFBI also positively regulates bone size and that changes in bone size affect bone strength. Furthermore, there was also a significant decrease in periosteal, but not endosteal, bone formation rate of cortical bone in TGFBI(-/-) mice, suggesting that the observed effect of TGFBI on bone mass and bone size was largely caused by the effect of TGFBI on periosteal bone formation.
Calcified Tissue International 05/2012; 91(1):81-7. · 2.38 Impact Factor
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ABSTRACT: BACKGROUND: RASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC). METHODS: Over-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes. RESULTS: The microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells. CONCLUSIONS: Taken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.
BMC Research Notes 05/2012; 5(1):239.
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Weirong Xing,
Kristen E Govoni,
Leah Rae Donahue,
Chandrasekhar Kesavan,
Jon Wergedal,
Carlin Long,
J H Duncan Bassett,
Apostolos Gogakos,
Anna Wojcicka,
Graham R Williams, Subburaman Mohan
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ABSTRACT: Understanding how bone growth is regulated by hormonal and mechanical factors during early growth periods is important for optimizing the attainment of peak bone mass to prevent or postpone the occurrence of fragility fractures later in life. Using genetic mouse models that are deficient in thyroid hormone (TH) (Tshr(-/-) and Duox2(-/-)), growth hormone (GH) (Ghrhr(lit/lit)), or both (Tshr(-/-); Ghrhr(lit/lit)), we demonstrate that there is an important period prior to puberty when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Daily administration of T3/T4 during days 5 to 14, the time when serum levels of T3 increase rapidly in mice, rescued the skeletal deficit in TH-deficient mice but not in mice lacking both TH and GH. However, treatment of double-mutant mice with both GH and T3/T4 rescued the bone density deficit. Increased body fat in the TH-deficient as well as TH/GH double-mutant mice was rescued by T3/T4 treatment during days 5 to 14. In vitro studies in osteoblasts revealed that T3 in the presence of TH receptor (TR) α1 bound to a TH response element in intron 1 of the IGF-I gene to stimulate transcription. In vivo studies using TRα and TRβ knockout mice revealed evidence for differential regulation of insulin-like growth factor (IGF)-I expression by the two receptors. Furthermore, blockade of IGF-I action partially inhibited the biological effects of TH, thus suggesting that both IGF-I-dependent and IGF-I-independent mechanisms contribute to TH effects on prepubertal bone acquisition.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 05/2012; 27(5):1067-79. · 6.04 Impact Factor
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ABSTRACT: The importance of the insulin-like growth factor (IGF)-I axis in the regulation of bone size and bone mineral density, two important determinants of bone strength, has been well established from clinical studies involving patients with growth hormone deficiency and IGF-I gene disruption. Data from transgenic animal studies involving disruption and overexpression of components of the IGF-I axis also provide support for a key role for IGF-I in bone metabolism. IGF-I actions in bone are subject to regulation by systemic hormones, local growth factors, as well as mechanical stress. In this review we describe findings from various genetic mouse models that pertain to the role of endocrine and local sources of IGF-I in the regulation of skeletal growth.
Current Osteoporosis Reports 04/2012; 10(2):178-86.
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ABSTRACT: 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-κB 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.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 03/2012; 27(7):1553-65. · 6.04 Impact Factor
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Weirong Xing,
Kristen Govoni,
Leah Rae Donahue,
Chandrasekhar Kesavan,
Jon Wergedal,
Carlin Long,
J H Duncan Bassett,
Apostolos Gogakos,
Anna Wojcicka,
Graham R Williams, Subburaman Mohan
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[hide abstract]
ABSTRACT: Understanding how bone growth is regulated by hormonal and mechanical factors during early growth periods is important for optimizing the attainment of peak bone mass to prevent or postpone the occurance of fragility fractures later in life. Using genetic mouse models that are deficient in thyroid hormone (TH) (Tshr(-/-) and Duox2(-/-) ), growth hormone (GH) (Ghrhr(lit/lit) ) or both (Tshr(-/- ) ;Ghrhr(lit/lit) ), we demonstrate that there is an important period prior to puberty when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Daily administration of T3/T4 during days 5 to 14, the time when serum levels of T3 increase rapidly in mice, rescued the skeletal deficit in TH-deficient mice but not in mice lacking both TH and GH. However, treatment of double-mutant mice with both GH and T3/T4 rescued the bone density deficit. Increased body fat in the TH-deficient as well as TH/GH double mutant mice was rescued by T3/T4 treatment during days 5-14. In vitro studies in osteoblasts revealed that T3 in the presence of TH receptor (TR) α1 bound to a TH response element in intron 1 of the IGF-I gene to stimulate transcription. In vivo studies using TRα and TRβ knockout mice revealed evidence for differential regulation of IGF-I expression by the two receptors. Furthermore, blockade of IGF-I action partially inhibited the biological effects of TH, thus suggesting that both IGF-I-dependent and independent mechanisms contribute to TH effects on prepubertal bone acquisition. © 2012 American Society for Bone and Mineral Research.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 01/2012; · 6.04 Impact Factor
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ABSTRACT: Post-traumatic stress disorder (PTSD) is an anxiety disorder that not only affects mental health, but may also affect bone health. However, there have been no studies to examine the direct relationship between PTSD and bone.
We employed electric shocks in mice to simulate traumatic events that cause PTSD. We also injected the anxiogenic drug FG-7142 prior to electric shocks. Electric shocks created lasting conditioned fear memory in all mice. In young mice, electric shocks elicited not only behavioral response but also skeletal response, and injection of FG-7142 appeared to increase both types of response. For example in behavioral response within the first week, mice shocked alone froze an average of 6.2 sec in 10 sec tests, and mice injected with FG-7142 froze 7.6 sec, both significantly different (P<0.05) from control mice, which only froze 1.3 sec. In skeletal response at week 2, shocks alone reduced 6% bone mineral content (BMC) in total body (P = 0.06), while shocks with FG-7142 injection reduced not only 11% BMC (P<0.05) but also 6% bone mineral density (BMD) (P<0.05). In addition, FG-7142 injection also caused significant reductions of BMC in specific bones such as femur, lumbar vertebra, and tibia at week 3. Strong negative correlations (R(2) = -0.56, P<0.05) and regression (y = 0.2527-0.0037 * x, P<0.01) between freezing behavior and total body BMC in young mice indicated that increased contextual PTSD-like behavior was associated with reduced bone mass acquisition.
This is the first study to document evidence that traumatic events induce lasting consequences on both behavior and skeletal growth, and electric shocks coupled with injection of anxiogenic FG-7142 in young mice can be used as a model to study the effect of PTSD-like symptoms on bone development.
PLoS ONE 01/2012; 7(8):e42684. · 4.09 Impact Factor
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ABSTRACT: This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption.
PLoS ONE 01/2012; 7(4):e35280. · 4.09 Impact Factor
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ABSTRACT: Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.
PLoS ONE 01/2012; 7(3):e32887. · 4.09 Impact Factor
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ABSTRACT: Down syndrome (trisomy 21) is associated with reduced bone density in humans, but it is unclear whether this is due to specific effects of chromosome 21 genes or lifestyle factors. Mouse models with aneuploidy of segments of mouse chromosome 16 that are homologous to human chromosome 21 can be used to elucidate the mechanism by which Down syndrome phenotypes arise. Ts1Rhr and Ms1Rhr mice are trisomic and monosomic, respectively, for the hypothesized "Down syndrome critical region" containing approximately 33 genes. We assessed the skeletons of these mice from 3 to 16 weeks of age using dual X-ray absorptiometry. Ts1Rhr mice were unexpectedly similar to normal controls, showing that a larger region of trisomy is necessary to recapitulate the Down syndrome phenotype. Ms1Rhr mice, in contrast, showed decreases in weight, bone mineral content, bone mineral density, and bone area from weaning to adulthood. Regional bone density was also decreased in the femur, tibia, and lower lumbar spine. The microarchitecture of 3 week old Ms1Rhr femurs was then analyzed using µCT. Volumetric density, total tissue volume, bone volume, and bone fraction were all reduced in both cortical and trabecular bone. Ms1Rhr trabeculae were thinner and had decreased connectivity. A 31.5% reduction in the level of insulin-like growth factor I in the serum was found, and we hypothesize that this is responsible for the bone density phenotype. We discuss bone-related genes in the region and propose that humans with distal chromosome 21 deletions may exhibit reduced bone density.
American Journal of Medical Genetics Part A 09/2011; 155A(10):2436-45. · 2.39 Impact Factor
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ABSTRACT: To establish a causal role for locally produced IGF-I in the mechanical strain response in the bone, we have generated mice with conditional disruption of the insulin-like growth factor (IGF) I gene in type 1α(2) collagen-expressing cells using the Cre-loxP approach. At 10 wk of age, loads adjusted to account for bone size difference were applied via four-point bending or axial loading (AL) in mice. Two wk of bending and AL produced significant increases in bone mineral density and bone size at the middiaphysis of wild-type (WT), but not knockout (KO), mice. In addition, AL produced an 8-25% increase in trabecular parameters (bone volume-tissue volume ratio, trabecular thickness, and trabecular bone mineral density) at the secondary spongiosa of WT, but not KO, mice. Histomorphometric analysis at the trabecular site revealed that AL increased osteoid width by 60% and decreased tartrate-resistance acidic phosphatase-labeled surface by 50% in the WT, but not KO, mice. Consistent with the in vivo data, blockade of IGF-I action with inhibitory IGF-binding protein (IGFBP4) in vitro completely abolished the fluid flow stress-induced MC3T3-E1 cell proliferation. One-way ANOVA revealed that expression levels of EFNB1, EFNB2, EFNA2, EphB2, and NR4a3 were different in the loaded bones of WT vs. KO mice and may, in part, be responsible for the increase in bone response to loading in the WT mice. In conclusion, IGF-I expressed in type 1 collagen-producing bone cells is critical for converting mechanical signal to anabolic signal in bone, and other growth factors cannot compensate for the loss of local IGF-I.
AJP Endocrinology and Metabolism 08/2011; 301(6):E1191-7. · 4.75 Impact Factor
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ABSTRACT: Mouse genetic studies reveal that ascorbic acid (AA) is essential for osteoblast (OB) differentiation and that osterix (Osx) was a key downstream target of AA action in OBs. To determine the molecular pathways for AA regulation of Osx expression, we evaluated if AA regulates Osx expression by regulating production and/or actions of local growth factors and extracellular matrix (ECM) proteins. Inhibition of actions of IGFs by inhibitory IGFBP-4, BMPs by noggin, and ECM-mediated integrin signaling by RGD did not block AA effects on Osx expression in OBs. Furthermore, blockade of components of MAPK signaling pathway had no effect on AA-induced Osx expression. Because AA is required for prolyl hydroxylase domain (PHD) activity and because PHD-induced prolyl-hydroxylation targets proteins to proteosomal degradation, we next tested if AA effect on Osx expression involves activation of PHD to hydroxylate and induce ubiquitin-proteosome-mediated degradation of transcriptional repressor(s) of Osx gene. Treatment of OBs with dimethyloxallyl glycine and ethyl 3, 4-dihydroxybenzoate, known inhibitors of PHD, completely blocked AA effect on Osx expression and OB differentiation. Knockdown of PHD2 expression by Lentivirus-mediated shRNA abolished AA-induced Osx induction and alkaline phosphatase activity. Furthermore, treatment of OBs with MG115, inhibitor of proteosomal degradation, completely blocked AA effects on Osx expression. Based on these data, we conclude that AA effect on Osx expression is mediated via a novel mechanism that involves PHD2 and proteosomal degradation of a yet to be identified transcriptional repressor that is independent of BMP, IGF-I, or integrin-mediated signaling in mouse OBs.
Physiological Genomics 04/2011; 43(12):749-57. · 2.73 Impact Factor
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ABSTRACT: Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men.
AJP Endocrinology and Metabolism 04/2011; 301(1):E40-8. · 4.75 Impact Factor
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ABSTRACT: The present study assesses the effect of the stem cell antigen-1 positive (Sca-1(+) ) cell-based human growth hormone (hGH) ex vivo gene transfer strategy on endosteal bone mass in the mouse.
Sublethally irradiated recipient mice were transplanted with Sca-1(+) cells transduced with lentiviral vectors expressing hGH or β-galactosidase control genes. Bone parameters were assessed by micro-computed tomography and histomorphometry.
This hGH strategy drastically increased hGH mRNA levels in bone marrow cells and serum insulin-like growth factor-I (IGF-I) (by nearly 50%, p < 0.002) in hGH recipient mice. Femoral trabecular bone volume of the hGH mice was significantly reduced by 35% (p < 0.002). The hGH mice also had decreased trabecular number (by 26%; p < 0.0001), increased trabecular separation (by 38%; p < 0.0002) and reduced trabecular connectivity density (by 64%; p < 0.001), as well as significantly more osteoclasts (2.5-fold; p < 0.05) and greater osteoclastic surface per bone surface (2.6-fold; p < 0.01).
Targeted expression of hGH in cells of marrow cavity through the Sca-1(+) cell-based gene transfer strategy increased circulating IGF-I and decreased endosteal bone mass through an increase in resorption in recipient mice. These results indicate that high local levels of hGH or IGF-I in the bone marrow microenvironment enhanced resorption, which is consistent with previous findings in transgenic mice with targeted bone IGF-I expression showing that high local IGF-I expression increased bone remodeling, favoring a net bone loss. Thus, GH and/or IGF-I would not be an appropriate transgene for use in this Sca-1(+) cell-based gene transfer strategy to promote endosteal bone formation. Published 2011 John Wiley & Sons, Ltd.
The Journal of Gene Medicine 02/2011; 13(2):77-88. · 2.48 Impact Factor
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ABSTRACT: Cortical bone dimensions are important determinants of bone strength. Gender differences in cortical bone size caused by greater periosteal expansion in males than in females during the pubertal growth spurt are well established both in humans and in experimental animal models. However, the mechanism by which gender influences cortical bone size is still a matter of investigation. The role of androgens and estrogen in pubertal bone growth has been examined in human disorders as well as animal models, such as gonadectomized or sex steroid receptor knockout mice. Based on the findings that growth hormone (GH) and insulin-like growth factor I (IGF-I) are major regulators of postnatal skeletal growth, we and others have predicted that sex hormones interact with the GH/IGF-I axis to regulate cortical bone size. However, studies conflict as to whether estrogen and androgens impact cortical bone size through the canonical pathway, through GH without IGF-I mediation, through IGF-I without GH stimulation, or independent of GH/IGF-I. We review recent data on the impact of sex steroids and components of the GH/IGF axis on sexual dimorphism in bone size. While the GH/IGF-I axis is a major player in regulating peak bone size, the relative contribution of GH/IGF-dependent mechanisms to sex differences in cortical bone size remains to be established.
Calcified Tissue International 01/2011; 88(1):1-8. · 2.38 Impact Factor
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ABSTRACT: 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 Lepr-deficient db(-)/db(-) mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [(3)H]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.
Journal of Biological Chemistry 11/2010; 285(48):37607-18. · 4.77 Impact Factor
