Adenosine A1 receptors regulate bone resorption in mice...

Title

Adenosine A1 receptors regulate bone resorption in mice: Adenosine A1 receptor blockade or deletion increases bone density and prevents ovariectomy-induced bone loss in adenosine A1 receptor–knockout mice

Authors

Firas M. Kara, Stephen B. Doty, Adele Boskey, Steven Goldring, Mone Zaidi, Bertil B. Fredholm, Bruce N. Cronstein

Abstract

Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and developing new treatments, we studied the effect of adenosine A1 receptor blockade or deletion on bone density. The bone mineral density (BMD) in adenosine A1 receptor–knockout (A1R-knockout) mice was analyzed by dual x-ray absorptiometry (DXA) scanning, and the trabecular and cortical bone volume was determined by microfocal computed tomography (micro-CT). The mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analyzed by DXA scanning and micro-CT. A histologic examination of bones obtained from A1R-knockout and wild-type mice was carried out. Visualization of osteoblast function (bone formation) after tetracycline double-labeling was performed by fluorescence microscopy. Micro-CT analysis of bones from A1R-knockout mice showed significantly increased bone volume. Electron microscopy of bones from A1R-knockout mice showed the absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistologic analysis demonstrated that although osteoclasts were present in the A1R-knockout mice, they were smaller and often not associated with bone. No morphologic changes in osteoblasts were observed, and bone-labeling studies revealed no change in the bone formation rates in A1R-knockout mice. These results suggest that the adenosine A1 receptor may be a useful target in treating diseases characterized by excessive bone turnover, such as osteoporosis and prosthetic joint loosening.

Link to Article

http://dx.doi.org/10.1002/art.27219

Attenuated anabolic response to exercise in lamin A/C haploinsufficient mice

Authors

Gustavo Duque, Wei Li, Li Sze Yeo, Christopher Vidal, and Diane Fatkin

Abstract

The ability of exercise to decrease fat mass and increase bone mass occurs through mechanical biasing of mesenchymal stem cells away from adipogenesis and toward osteoblastogenesis. The mechanism explaining this effect remains poorly understood. Lamin A/C knockdown inhibits osteoblastogenesis while favors adipogenesis in vitro. In this study, we hypothesized that the presence of lamin A/C is required for the anabolic response of bone during exercise. Three-month-old female lamin A/C haploinsufficient (Lmna+/−) mice were exposed to strenuous maximal exercise protocol (2 sessions/week, 40 min/session) for 6 weeks. Wild type (WT) (exercise and sedentary) and sedentary Lmna+/− mice were used as controls. To determine changes in bone microarchitecture and cell numbers, distal femur was analyzed by microCT and histomorphometry respectively. Finally, levels of expression of nuclear β-catenin and sclerostin, two proteins involved in the anabolic response to exercise, were determined by immunofluorescence. Histomorphometry analysis showed a significant increase in bone volume fraction (BV/TV) in exercised vs. sedentary WT mice. In contrast, exercised Lmna+/− mice showed a significant reduction in microarchitecture as compared with sedentary Lmna+/− controls including trabecular and cortical thinning. In addition, we found a significant increase in bone cells number in exercised vs. sedentary WT mice whereas exercised Lmna+/− mice showed a significant reduction in osteoblasts and osteocytes number as compared with sedentary Lmna+/− controls. Finally, levels of activated β-catenin in osteoblasts and osteocytes were significantly decreased while sclerostin expression was increased in exercised Lmna+/− mice as compared with exercised WT controls. In summary, our data indicate that the presence of lamin A/C is required for the anabolic effect of exercise on bone thus suggesting a new important role of lamin A/C in bone biology.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.04.023

Connexin43 deficiency reduces the sensitivity of cortical bone to the effects of muscle paralysis

Authors

Susan K. Grimston, Daniel B. Goldberg, Marcus Watkins, Michael D. Brodt, Matthew J. Silva, Roberto Civitelli

Abstract

We have previously shown that the effect of mechanical loading on bone is partly dependent on connexin43 (Cx43). To determine whether Cx43 is also involved in the effect of mechanical unloading, we have used botulinum toxin A (BtxA) to induce reversible muscle paralysis in mice with a conditional deletion of the Cx43 gene in osteoblasts and osteocytes (cKO). BtxA injection in hind limb muscles of wild type (WT) mice resulted in significant muscle atrophy and rapid loss of trabecular bone. Bone loss reached a nadir of about 40% at 3 weeks post-injection, followed by a slow recovery. A similar degree of trabecular bone loss was observed in cKO mice. By contrast, BtxA injection in WT mice significantly increased marrow area and endocortical osteoclast number, and decreased cortical thickness and bone strength. These changes did not occur in cKO mice, whose marrow area is larger, osteoclast number higher, and cortical thickness and bone strength lower relative to WT mice in basal conditions. Changes in cortical structure occurring in WT mice had not recovered 19 weeks after BtxA injection, despite correction of the early osteoclast activation and a modest increase in periosteal bone formation. Thus, BtxA-induced muscle paralysis leads to rapid loss of trabecular bone and to changes in structural and biomechanical properties of cortical bone, neither of which are fully reversed after 19 weeks. Osteoblast/osteocyte Cx43 is involved in the adaptive responses to skeletal unloading selectively in the cortical bone, via modulation of osteoclastogenesis on the endocortical surface.

Link to Article

http://dx.doi.org/10.1002/jbmr.425

Comparing histological, vascular and molecular responses associated with woven and lamellar bone formation induced by mechanical loading in the rat ulna

Authors

Jennifer A. McKenzie, Matthew J. Silva

Abstract

Osteogenesis occurs by formation of woven or lamellar bone. Little is known about the molecular regulation of these two distinct processes. We stimulated periosteal bone formation at the ulnar mid-diaphysis of adult rats using a single bout of forelimb compression. We hypothesized that loading that stimulates woven bone formation induces higher over-expression of genes associated with cell proliferation, angiogenesis and osteogenesis compared to loading that stimulates lamellar bone formation. We first confirmed that a single bout of 100 cycles of loading using either a rest-inserted (0.1Hz) or haversine (2Hz) waveform (15N peak force) was non-damaging and increased lamellar bone formation (LBF loading). Woven bone formation (WBF loading) was stimulated using a previously described, damaging fatigue loading protocol (2Hz, 1.3mm disp., 18N peak force). There were dramatic differences in gene expression levels (based on qRT-PCR) between loading protocols that produced woven and lamellar bone. In contrast, gene expression levels were not different between LBF loading protocols using a rest-inserted or haversine waveform. Cell proliferation markers Hist4 and Ccnd1 were strongly upregulated (5- to 17-fold) 1 and 3days after WBF loading, prior to woven bone formation, but not after LBF loading. The angiogenic genes Vegf and Hif1a were upregulated within 1h after WBF loading and were strongly up on days 1–3 (3- to 15-fold). In sharp contrast, we observed only a modest increase (<2-fold) in Vegfa and Hif1a expression on day 3 following LBF loading. Consistent with these relative differences in gene expression, vascular perfusion 3days after loading revealed significant increases in vessel number and volume following WBF loading, but not after LBF loading. Lastly, bone formation markers (Runx2, Osx, Bsp) were more strongly upregulated for woven (4- to 89-fold) than for lamellar bone (2-fold), consistent with the differences in new bone volume observed 10days after loading. In summary, robust early increases both molecularly and histologically for cell proliferation and angiogenesis precede woven bone formation, whereas lamellar bone formation is associated with only a modest upregulation of molecular signals at later timepoints.

Link to Article

http://dx.doi.org/10.1016/j.bone.2010.09.005

Distinct p53 Transcriptional Programs Dictate Acute DNA-Damage Responses and Tumor Suppression

Authors

Colleen A. Brady, Dadi Jiang, Stephano S. Mello, Thomas M. Johnson, Lesley A. Jarvis, Margaret M. Kozak, Daniela Kenzelmann Broz, Shashwati Basak, Eunice J. Park, Margaret E. McLaughlin, Anthony N. Karnezis, Laura D. Attardi

Abstract

The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knockin mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p5325,26, is severely compromised for transactivation of most p53 target genes, and, moreover, p5325,26 cannot induce G1-arrest or apoptosis in response to acute DNA damage. Surprisingly, p5325,26 retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p5325,26,53,54 mutant cannot induce senescence or inhibit tumorigenesis, like p53 nullizygosity. Thus, p53 transactivation is essential for tumor suppression but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression—a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.04.020

CXC receptor knockout mice: Characterization of skeletal features and membranous bone healing in the adult mouse

Authors

David S. Bischoff, Taylor Sakamoto, Kenji Ishida, Nalini S. Makhijani, Helen E. Gruber, Dean T. Yamaguchi

Abstract

The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR−/− knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR−/− mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR−/− mice at 6, 12, and 18weeks of age. Trabecular bone characteristics in mCXCR−/− (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by μCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR−/−. Decreased polar moment of inertia (J) in the tibias/femurs suggested that the mCXCR−/− long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR−/− and WT littermates were not completely healed at 12weeks; WT animals, however, had significantly more bone in-growth than mCXCR−/−. New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was >2× that seen in mCXCR−/−. Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR−/− mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular and cortical long bone; (4) decreased femur bone strength; and (5) significantly reduced intramembranous bone formation and number of BlV in new calvarial bone during bone repair.

Link to Article

http://dx.doi.org/10.1016/j.bone.2010.09.026