Control of bone mass and remodeling by PTH receptor signaling in osteocytes

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.     

Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

Deficiency of the G-protein alpha-subunit G(s)alpha in osteoblasts leads to differential effects on trabecular and cortical bone

The G-protein alpha-subunit G(s)alpha is required for the intracellular cAMP responses to hormones and other agonists. G(s)alpha is known to mediate the cAMP response to parathyroid hormone and other hormones and cytokines in bone and cartilage. To analyze the in vivo role of G(s)alpha signaling in osteoblasts, we developed mice with osteoblast/osteocyte-specific G(s)alpha deficiency (BGsKO) by mating G(s)alpha-floxed mice with collagen Ialpha1 promoter-Cre recombinase transgenic mice. Early skeletal development was normal in BGsKO mice, because formation of the initial cartilage template and bone collar was unaffected. The chondrocytic zones of the growth plates also appeared normal in BGsKO mice. BGsKO mice had a defect in the formation of the primary spongiosa with reduced immature osteoid (new bone formation) and overall length, which led to reduced trabecular bone volume. In contrast, cortical bone was thickened with narrowing of the bone marrow cavity. This was probably due to decreased cortical bone resorption, because osteoclasts were markedly reduced on the endosteal surface of cortical bone. In addition, the expression of alkaline phosphatase, an early osteoblastic differentiation marker, was normal, whereas the expression of the late osteoblast differentiation markers osteopontin and osteocalcin was reduced, suggesting that the number of mature osteoblasts in bone is reduced. Expression of the osteoclast-stimulating factor receptor activator of NF-kappaB ligand was also reduced. Overall, our findings have similarities to parathyroid hormone null mice and confirm that the differential effects of parathyroid hormone on trabecular and cortical bone are primarily mediated via G(s)alpha in osteoblasts. Osteoblast-specific G(s)alpha deficiency leads to reduced bone turnover.     

Bone morphogenetic protein receptor type Ia localization causes increased BMP2 signaling in mice exhibiting increased peak bone mass phenotype

Bone morphogenetic protein 2 (BMP2) is a growth factor that initiates osteoblast differentiation. Recent studies show that BMP2 signaling regulates bone mineral density (BMD). BMP2 interacts with BMP receptor type Ia (BMPRIa) and type II receptor leading to the activation of the Smad signaling pathway. BMPRIa must shuttle between distinct plasma membrane domains, enriched of Caveolin‐1 alpha and Caveolin‐1 beta isoforms, and receptor activation occurs in these domains. Yet it remains unknown whether the molecular mechanism that regulates BMP2 signaling is driving mineralization and BMD. Therefore, the B6.C3H‐1‐12 congenic mouse model with increased BMD and osteoblast mineralization was utilized in this study. Using the family image correlation spectroscopy, we determined if BMP2 led to a significant re‐localization of BMPRIa to caveolae of the alpha/beta isoforms in bone marrow stromal cells (BMSCs) isolated from B6.C3H‐1‐12 mice compared to the C57BL/6J mice, which served as controls. The control, C57BL/6J mice, was selected due to only 4 Mb of chromosome 1 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Using reporter gene assays, the B6.C3H‐1‐12 BMSCs responded to BMP2 with increased Smad activation. Furthermore, disrupting caveolae reduced the BMP2‐induced Smad signaling in BMSCs isolated from B6.C3H‐1‐12 and C57BL/6J. This study suggests for the first time a regulatory mechanism of BMPRIa signaling at the plasma membrane of BMSCs that (i) associated with genetic differences in the distal Chromosome 1 segment carried by the B6.C3H‐1‐12 congenic and (ii) contributes to increase BMD of the B6.C3H‐1‐12 compared to the C57BL/6J control mice. J. Cell. Physiol. 227: 2870–2879, 2012. © 2011 Wiley Periodicals, Inc.     

Loss of TGF-beta 1 leads to increased neuronal cell death and microgliosis in mouse brain

TGF-beta1 is a key regulator of diverse biological processes in many tissues and cell types, but its exact function in the developing and adult mammalian CNS is still unknown. We report that lack of TGF-beta1 expression in neonatal Tgfb1(-/-) mice results in a widespread increase in degenerating neurons accompanied by reduced expression of synaptophysin and laminin and a prominent microgliosis. Lack of TGF-beta1 also strongly reduces survival of primary neurons cultured from Tgfb1(-/-) mice. TGF-beta1 deficiency in adult Tgfb1(-/+) mice results in increased neuronal susceptibility to excitotoxic injury, whereas astroglial overexpression of TGF-beta1 protects adult mice against neurodegeneration in acute, excitotoxic and chronic injury paradigms. This study reveals a nonredundant function for TGF-beta1 in maintaining neuronal integrity and survival of CNS neurons and in regulating microglial activation. Because individual TGF-beta1 expression levels in the brain vary considerably between humans, this finding could have important implications for susceptibility to neurodegeneration.     

Cardiolipin deficiency leads to decreased cardiolipin peroxidation and increased resistance of cells to apoptosis

Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with ~ 45% of its normal content. ESI–MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or γ-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.     

Chemokine receptor 3 is a negative regulator of trabecular bone mass in female mice

Chemokines are secreted by a wide variety of cells; their functions are dependent on the binding to their chemokine receptors (CCRs) which induce directed chemotaxis in nearby responsive cells. Chemokines and their receptors can be induced under several different conditions. Based on data from clinical studies showing an increased expression of chemokine receptor 3 (CCR3) in circulating monocytes of human subjects with lower bone mineral density (BMD) as compared to those with high BMD, we predicted a role for CCR3 in the development of peak bone mass. We, therefore, first evaluated the expression pattern of Ccr3 in bone cells, in comparison to other CCRs, that have common ligands with CCR3. While Ccr1 and Ccr3 messenger RNA (mRNA) levels increased during both RANKL-induced osteoclast differentiation and AA-induced osteoblast differentiation, the levels of Ccr5 mRNA only increased during osteoblast differentiation. To examine if CCR3 influences osteoclast and/or osteoblast differentiation, we evaluated the consequence of blocking CCR3 function using neutralizing antibody on the expression of osteoclast and osteoblast differentiation markers. Treatment with CCR3 neutralizing antibody increased mRNA levels of Trap and cathepsin K in osteoclasts and osteocalcin in osteoblasts compared to cells treated with control IgG. Based on these in vitro findings, we next assessed the role of CCR3 in vivo by evaluating the skeletal phenotypes of Ccr3 knockout and corresponding control littermate mice. Disruption of CCR3 resulted in a significant increase in femur areal BMD at 5 and 8 weeks of age by dual-energy X-ray absorptiometry. Micro-CT analysis revealed a 25% increase in trabecular bone mass at 10 weeks of age caused by corresponding changes in trabecular number and thickness compared to wild type mice. Based on our findings, we conclude that disruption of CCR3 function favors bone mass accumulation, in part via enhancement of bone metabolism. Understanding the molecular pathways through which CCR3 acts to regulate osteoclast and osteoblast functions could lead to new therapeutic approaches to prevent inflammation-induced bone loss.     

Predicting changes in mechanical properties of trabecular bone by adaptive remodeling

Because changes in the mechanical properties of bone are closely related to trabecular bone remodeling, methods that consider the temporal morphological changes induced by adaptive remodeling of trabecular bone are needed to estimate long-term fracture risk and bone quality in osteoporosis. We simulated bone remodeling using simplified and pig trabecular bone models and estimated the morphology of healthy and osteoporotic cases. We then displayed the fracture risk of the remodeled models based on a cumulative histogram from high stress. The histogram showed more elements had higher stresses in the osteoporosis model, indicating that the osteoporosis model had a greater risk.     

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.     

Sleep deprivation impairs calcium signaling in mouse splenocytes and leads to a decreased immune response

Background

Sleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca^2 +) plays a critical signaling role. We performed live cell measurements of cytosolic Ca^2 + mobilization to understand the changes in Ca^2 + signaling that occur in splenic immune cells after various periods of sleep deprivation (SD).

Methods

Adult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72 h) and Ca^2 + intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca^2 + mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca^2 + channel gene expression was evaluated

Results

Splenocytes showed a progressive loss of intracellular Ca^2 + maintenance from endoplasmic reticulum (ER) stores. Transient Ca^2 + buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca^2 + channels.

Conclusions and general significance

These novel data suggest that SD impairs Ca^2 + signaling, most likely as a result of ER stress, leading to an insufficient Ca^2 + supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function.     

Dominant negative mutation of the TGF-beta receptor blocks hypoxia-induced pulmonary vascular remodeling.

The present study utilized a novel transgenic mouse model that expresses an inducible dominant negative mutation of the transforming growth factor (TGF)-beta type II receptor (DnTGFbetaRII mouse) to test the hypothesis that TGF-beta signaling plays an important role in the pathogenesis of chronic hypoxia-induced increases in pulmonary arterial pressure and vascular and alveolar remodeling. Nine- to 10-wk-old male DnTGFbetaRII and control nontransgenic (NTG) mice were exposed to normobaric hypoxia (10% O2) or air for 6 wk. Expression of DnTGFbetaRII was induced by drinking 25 mM ZnSO4 water beginning 1 wk before hypoxic exposure. Hypoxia-induced increases in right ventricular pressure, right ventricular mass, pulmonary arterial remodeling, and muscularization were greatly attenuated in DnTGFbetaRII mice compared with NTG controls. Furthermore, the stimulatory effects of hypoxic exposure on pulmonary arterial and alveolar collagen content, appearance of alpha-smooth muscle actin-positive cells in alveolar parenchyma, and expression of extracellular matrix molecule (including collagen I and III, periostin, and osteopontin) mRNA in whole lung were abrogated in DnTGFbetaRII mice compared with NTG controls. Hypoxic exposure had no effect on systemic arterial pressure or heart rate in either strain. These data support the hypothesis that endogenous TGF-beta plays an important role in pulmonary vascular adaptation to chronic hypoxia and that disruption of TGF-beta signaling attenuates hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, pulmonary arterial hypertrophy and muscularization, alveolar remodeling, and expression of extracellular matrix mRNA in whole lung.     

Computational simulation of simultaneous cortical and trabecular bone change in human proximal femur during bone remodeling

In this study, we developed a numerical framework that computationally determines simultaneous and interactive structural changes of cortical and trabecular bone types during bone remodeling, and we investigated the structural correlation between the two bone types in human proximal femur. We implemented a surface remodeling technique that performs bone remodeling in the exterior layer of the cortical bone while keeping its interior area unchanged. A micro-finite element (μFE) model was constructed that represents the entire cortical bone and full trabecular architecture in human proximal femur. This study simulated and compared the bone adaptation processes of two different structures: (1) femoral bone that has normal cortical bone shape and (2) perturbed femoral bone that has an artificial bone lump in the inferomedial cortex. Using the proposed numerical method in conjunction with design space optimization, we successfully obtained numerical results that resemble actual human proximal femur. The results revealed that actual cortical bone, as well as the trabecular bone, in human proximal femur has structurally optimal shapes, and it was also shown that a bone abnormality that has little contribution to bone structural integrity tends to disappear. This study also quantitatively determined the structural contribution of each bone: when the trabecular adaptation was complete, the trabecular bone supported 54% of the total load in the human proximal femur while the cortical bone carried 46%.     

Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling

Bone morphogenetic proteins (BMPs) function during various aspects of embryonic development including skeletogenesis. However, their biological functions after birth are less understood. To investigate the role of BMPs during bone remodeling, we generated a postnatal osteoblast-specific disruption of Bmpr1a that encodes the type IA receptor for BMPs in mice. Mutant mice were smaller than controls up to 6 months after birth. Irregular calcification and low bone mass were observed, but there were normal numbers of osteoblasts. The ability of the mutant osteoblasts to form mineralized nodules in culture was severely reduced. Interestingly, bone mass was increased in aged mutant mice due to reduced bone resorption evidenced by reduced bone turnover. The mutant mice lost more bone after ovariectomy likely resulting from decreased osteoblast function which could not overcome ovariectomy-induced bone resorption. In organ culture of bones from aged mice, ablation of the Bmpr1a gene by adenoviral Cre recombinase abolished the stimulatory effects of BMP4 on the expression of lysosomal enzymes essential for osteoclastic bone resorption. These results demonstrate essential and age-dependent roles for BMP signaling mediated by BMPRIA (a type IA receptor for BMP) in osteoblasts for bone remodeling.     

Inhibition of specific electron transport pathways leads to oxidative stress and decreased Candida albicans proliferation

Candida parapsilosis mitochondria contain three respiratory chains: the classical respiratory chain (CRC), a secondary parallel chain (PAR) and an “alternative” oxidative pathway (AOX). We report here the existence of similar pathways in C. albicans. To observe the capacity of each pathway to sustain yeast growth, C. albicans cells were cultured in the presence of inhibitors of these pathways. Antimycin A and KCN totally abrogated yeast growth, while rotenone did not prevent proliferation. Furthermore, rotenone promoted only partial respiratory inhibition. Lower concentrations of KCN that promote partial inhibition of respiration did not inhibit yeast growth, while partial inhibition of respiration with antimycin A did. Similarly, AOX inhibitor BHAM decreased O₂ consumption slightly but completely stunted cell growth. Reactive oxygen species production and oxidized glutathione levels were enhanced in cells treated with antimycin A or BHAM, but not rotenone or KCN. These findings suggest that oxidative stress prevents C. albicans growth.     

Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis.

Ligand induced activation of the beta-receptor for platelet-derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta-receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF-BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild-type beta-receptors were attenuated by inhibition of phosphatidylinositol 3'-kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF-BB-induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild-type beta-receptor was less affected. Moreover, the PDGF-BB-stimulated tyrosine phosphorylation of phospholipase C-gamma was increased in the mutant receptor cells compared with wild-type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C-gamma, and shifts the response to increased mitogenicity.     

Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis

Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from beta-oxidation and toward glycerolipid synthesis. Adenoviral-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have >2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.     

Co-ordination of TGF-beta and FGF signaling pathways in bone organ cultures

Transforming growth factor-beta (TGF-beta) is known to regulate chondrocyte proliferation and hypertrophic differentiation in embryonic bone cultures by a perichondrium dependent mechanism. To begin to determine which factors in the perichondrium mediate the effects of TGF-beta, we studied the effect of Insulin-like Growth Factor-1 (IGF-I) and Fibroblast Growth Factors-2 and -18 (FGF2, FGF18) on metatarsal organ cultures. An increase in chondrocyte proliferation and hypertrophic differentiation was observed after treatment with IGF-I. A similar effect was seen after the perichondrium was stripped from the metatarsals suggesting IGF-I acts directly on the chondrocytes. Treatment with FGF-2 or FGF-18 resulted in a decrease in bone elongation as well as hypertrophic differentiation. Treatment also resulted in a decrease in BrdU incorporation into chondrocytes and an increase in BrdU incorporation in perichondrial cells, similar to what is seen after treatment with TGF-beta1. A similar effect was seen with FGF2 after the perichondrium was stripped suggesting that, unlike TGF-beta, FGF2 acts directly on chondrocytes to regulate proliferation and hypertrophic differentiation. To test the hypothesis that TGF-beta regulates IGF or FGF signaling, activation of the receptors was characterized after treatment with TGF-beta. Activation was measured as the level of tyrosine phosphorylation on the receptor. Treatment with TGF-beta for 24h did not alter the level of IGFR-I tyrosine phosphorylation. In contrast, treatment with TGF-beta resulted in and increase in tyrosine phosphorylation on FGFR3 without alterations in total FGFR3 levels. TGF-beta also stimulated expression of FGF18 mRNA in the cultures and the effects of TGF-beta on metatarsal development were blocked or partially blocked by pretreatment with FGF signaling inhibitors. The results suggest a model in which FGF through FGFR3 mediates some of the effects of TGF-beta on embryonic bone formation.