动态调节Rad GTPase可以控制骨形成的平衡
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摘要
小GTP结合蛋白Rad (Ras-related associated with diabetes)是小GTPases的RGK亚家族成员,其在心脏之外的细胞和生理功能仍有待阐明,本研究旨在探讨Rad对小鼠骨密度、破骨细胞分化和骨量的调节作用。本研究以Rad基因敲除小鼠为动物模型,野生(WT)小鼠为对照,通过微计算机断层摄影术(microscopic computed tomography,μCT)分析雄性和雌性小鼠的股骨小梁骨体积分数和骨小梁数量,以抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase, TRAP)染色和抗酒石酸酸性磷酸酶(TRAP)+多核细胞(multinucleated cell, MNC)计数检测破骨细胞的分化和表面积,使用组织形态计量学来考察骨形成速率。结果显示,与WT野生型小鼠相比,雌性Rad基因敲除小鼠的股骨表现出显著较低的小梁骨体积分数(BV/TV)。Rad缺失使小鼠股骨的皮质骨面积明显低于WT小鼠。抗酒石酸酸性磷酸酶(TRAP)染色和TRAP+MNCs计数表明Rad的缺失显著增强了体外破骨细胞的分化。与正常野生小鼠相比,Rad缺失使小鼠的破骨细胞表面积减少。在Rad基因敲除小鼠中矿物沉积率(MAR)显著降低,矿化表面百分比(MS/BS)升高,骨形成速率/骨表面(BFR/BS)下降。本研究初步结论表明,Rad GTPase在骨代谢的调节中起着重要的作用,在小鼠中敲除Rad可导致骨密度降低,对Rad作用和调节机制的研究可能会找到骨质疏松症治疗的潜在靶点。
The small GTP-binding protein Rad(Ras-related associated with diabetes) is a member of the RGK subfamily of small GTPases. Its cellular and physiological function outside the heart remain to be elucidated. This study aims to investigate the regulatory effect of Rad on mouse bone mineral density, osteoclast differentiation, and bone mass. Using Rad knockout mice as animal models and wild(WT) mice as controls, the femoral trabecular bone volume fraction and trabecular bone number were analyzed by micro-computed tomography(μCT) in male and female mice in this study. The differentiation and surface area of osteoclasts were detected by tartrate-resistant acid phosphatase(TRAP) staining and TRAP+ multinucleated cell(MNC) count. Histomorphometry was used to examine the rate of bone formation. The results showed that the femurs of female Rad knockout mice exhibited significantly lower trabecular bone volume fraction(BV/TV) compared to WT wild mice. The cortical bone area of Rad-deficient mouse mice was significantly lower than that of WT mice. TRAP staining and TRAP+ MNCs counts indicated that absence of Rad significantly enhanced the osteoclast differentiation in vitro. Compared to normal wild mice,Rad-deficient mice reduced osteoclast surface area. The rate of mineral deposition(MAR) decreased significantly, th e percentage of mineralized surface(MS/BS) increased, and bone formation rate/bone surface(BFR/BS) decreased in Rad knockout mice. The preliminary conclusions of this study indicated that Rad GTPase played an important role in the regulation of bone metabolism. Knocking out Rad in mice could lead to a decrease in bone mineral density. Studies of Rad's role and regulatory mechanisms might be potential targets for the treatment of osteoporosis.
The small GTP-binding protein Rad(Ras-related associated with diabetes) is a member of the RGK subfamily of small GTPases. Its cellular and physiological function outside the heart remain to be elucidated. This study aims to investigate the regulatory effect of Rad on mouse bone mineral density, osteoclast differentiation, and bone mass. Using Rad knockout mice as animal models and wild(WT) mice as controls, the femoral trabecular bone volume fraction and trabecular bone number were analyzed by micro-computed tomography(μCT) in male and female mice in this study. The differentiation and surface area of osteoclasts were detected by tartrate-resistant acid phosphatase(TRAP) staining and TRAP+ multinucleated cell(MNC) count. Histomorphometry was used to examine the rate of bone formation. The results showed that the femurs of female Rad knockout mice exhibited significantly lower trabecular bone volume fraction(BV/TV) compared to WT wild mice. The cortical bone area of Rad-deficient mouse mice was significantly lower than that of WT mice. TRAP staining and TRAP+ MNCs counts indicated that absence of Rad significantly enhanced the osteoclast differentiation in vitro. Compared to normal wild mice,Rad-deficient mice reduced osteoclast surface area. The rate of mineral deposition(MAR) decreased significantly, th e percentage of mineralized surface(MS/BS) increased, and bone formation rate/bone surface(BFR/BS) decreased in Rad knockout mice. The preliminary conclusions of this study indicated that Rad GTPase played an important role in the regulation of bone metabolism. Knocking out Rad in mice could lead to a decrease in bone mineral density. Studies of Rad's role and regulatory mechanisms might be potential targets for the treatment of osteoporosis.
引文
Devlin M.J.,and Rosen C.J.,2015,The bone-fat interface:basic and clinical implications of marrow adiposity,Lancet Diabetes Endocrinol.,3(2):141-147
Eddie R.C.,Beatriz G.,and Francesc V.,2016,p38 MAPK signaling in osteoblast differentiation,Frontiers Cell Dev.Biol.,4(21):40
Erlebacher A.,and Derynck R.,1996,Increased expression of TGF-beta 2 in osteoblasts results in an osteoporosis-like phenotype,Journal of Cell Biology,132(1):195-210
Levitan B.M.,Manning J.R.,Withers C.N.,Smith J.D.,Shaw R.M.,Andres D.A.,Sorrell V.L.,and Satin J.,2016,Rad-deletion phenocopies tonic sympathetic stimulation of the heart,Journal of Cardiovascular Translational Research,9(5-6):1-13
Minchenko D.O.,Kharkova A.P.,Hubenia O.V.,and Minchenko O.H.,2013,Insulin receptor,IRS1,IRS2,INSIG1,INSIG2,RRAD,and BAIAP2 gene expressions in glioma U87 cells with ERN1 loss of function:effect of hypoxia and glutamine or glucose deprivation,Endocrine Regulations,47(1):15-26
Mo Y.,Midorikawa K.,Zhang Z.,Zhou X.,Ma N.,Huang G.,Hiraku Y.,Oikawa S.,and Murata M.,2012,Promoter hypermethylation of Ras-related GTPase gene RRAD inactivates a tumor suppressor function in nasopharyngeal carcinoma.,Cancer Letters,323(2):147-154
Quinn J.M.,Elliott J.,Gillespie M.T.,and Martin T.J.,1998,Acombination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro,Endocrinology,139(10):4424-4427
Raggatt L.J.,and Partridge N.C.,2010,Cellular and molecular mechanisms of bone remodeling,Journal of Biological Chemistry,285(33):25103-25108
Rovsing L.,and Mller M.,2014,Photic stimulation of the suprachiasmatic nucleus via the non-visual optic system.Agene expression study in the blind Crx-/-mouse,Cell&Tissue Research,358(1):239-248
Satija N.K.,Sharma D.,Afrin F.,Tripathi R.P.,and Gangenahalli G.,2013,High throughput transcriptome profiling of lithium stimulated human mesenchymal stem cells reveals priming towards osteoblastic lineage,PLoS One,8(1):e55769
Wang Y.,Li G.,Mao F.,Li X.,Liu Q.,Chen L.,Lv L.,Wang X.,Wu J.,Dai W.,Wang G.,Zhao E.,Tang K.F.,and Sun Z.S.,2014,Ras-induced epigenetic inactivation of the RRAD(Ras-related associated with diabetes)gene promotes glucose uptake in a human ovarian cancer model,Journal of Biological Chemistry,289(20):14225-14238
Zart D.J.,Miya L.,Wolff D.A.,Makley J.T.,and Stevenson S.,1993,The effects of cisplatin on the incorporation of fresh syngeneic and frozen allogeneic cortical bone grafts,Journal of Orthopaedic Research,11(2):240-249
Zhang C.,Liu J.,Wu R.,Liang Y.,Lin M.,Liu J.,Chan C.S.,Hu W.,and Feng Z.,2014,Tumor suppressor p53 negatively regulates glycolysis stimulated by hypoxia through its target RRAD,Oncotarget,5(14):5535-5546
Zhang J.,Chang L.,Chen C.,Zhang M.,Luo Y.,Hamblin M.,Villacorta L.,Xiong J.W.,Chen Y.E.,Zhang J.,and Zhu X.,2011,Rad GTPase inhibits cardiac fibrosis through connective tissue growth factor,Cardiovascular Research,91(1):90-98
Eddie R.C.,Beatriz G.,and Francesc V.,2016,p38 MAPK signaling in osteoblast differentiation,Frontiers Cell Dev.Biol.,4(21):40
Erlebacher A.,and Derynck R.,1996,Increased expression of TGF-beta 2 in osteoblasts results in an osteoporosis-like phenotype,Journal of Cell Biology,132(1):195-210
Levitan B.M.,Manning J.R.,Withers C.N.,Smith J.D.,Shaw R.M.,Andres D.A.,Sorrell V.L.,and Satin J.,2016,Rad-deletion phenocopies tonic sympathetic stimulation of the heart,Journal of Cardiovascular Translational Research,9(5-6):1-13
Minchenko D.O.,Kharkova A.P.,Hubenia O.V.,and Minchenko O.H.,2013,Insulin receptor,IRS1,IRS2,INSIG1,INSIG2,RRAD,and BAIAP2 gene expressions in glioma U87 cells with ERN1 loss of function:effect of hypoxia and glutamine or glucose deprivation,Endocrine Regulations,47(1):15-26
Mo Y.,Midorikawa K.,Zhang Z.,Zhou X.,Ma N.,Huang G.,Hiraku Y.,Oikawa S.,and Murata M.,2012,Promoter hypermethylation of Ras-related GTPase gene RRAD inactivates a tumor suppressor function in nasopharyngeal carcinoma.,Cancer Letters,323(2):147-154
Quinn J.M.,Elliott J.,Gillespie M.T.,and Martin T.J.,1998,Acombination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro,Endocrinology,139(10):4424-4427
Raggatt L.J.,and Partridge N.C.,2010,Cellular and molecular mechanisms of bone remodeling,Journal of Biological Chemistry,285(33):25103-25108
Rovsing L.,and Mller M.,2014,Photic stimulation of the suprachiasmatic nucleus via the non-visual optic system.Agene expression study in the blind Crx-/-mouse,Cell&Tissue Research,358(1):239-248
Satija N.K.,Sharma D.,Afrin F.,Tripathi R.P.,and Gangenahalli G.,2013,High throughput transcriptome profiling of lithium stimulated human mesenchymal stem cells reveals priming towards osteoblastic lineage,PLoS One,8(1):e55769
Wang Y.,Li G.,Mao F.,Li X.,Liu Q.,Chen L.,Lv L.,Wang X.,Wu J.,Dai W.,Wang G.,Zhao E.,Tang K.F.,and Sun Z.S.,2014,Ras-induced epigenetic inactivation of the RRAD(Ras-related associated with diabetes)gene promotes glucose uptake in a human ovarian cancer model,Journal of Biological Chemistry,289(20):14225-14238
Zart D.J.,Miya L.,Wolff D.A.,Makley J.T.,and Stevenson S.,1993,The effects of cisplatin on the incorporation of fresh syngeneic and frozen allogeneic cortical bone grafts,Journal of Orthopaedic Research,11(2):240-249
Zhang C.,Liu J.,Wu R.,Liang Y.,Lin M.,Liu J.,Chan C.S.,Hu W.,and Feng Z.,2014,Tumor suppressor p53 negatively regulates glycolysis stimulated by hypoxia through its target RRAD,Oncotarget,5(14):5535-5546
Zhang J.,Chang L.,Chen C.,Zhang M.,Luo Y.,Hamblin M.,Villacorta L.,Xiong J.W.,Chen Y.E.,Zhang J.,and Zhu X.,2011,Rad GTPase inhibits cardiac fibrosis through connective tissue growth factor,Cardiovascular Research,91(1):90-98