Peripheral Blood Monocyte-expressed ANXA2 Gene is Involved in Pathogenesis of Osteoporosis in Humans

Low bone mineral density (BMD) is a risk factor of osteoporosis and has strong genetic determination. Genes influencing BMD and fundamental mechanisms leading to osteoporosis have yet to be fully determined. Peripheral blood monocytes (PBM) are potential osteoclast precursors, which could access to bone resorption surfaces and differentiate into osteoclasts to resorb bone. Herein, we attempted to identify osteoporosis susceptibility gene(s) and characterize their function(s), through an initial proteomics discovery study on PBM in vivo, and multiscale validation studies in vivo and in vitro. Utilizing the quantitative proteomics methodology LC-nano-ESI-MSE, we discovered that a novel protein, i.e. ANXA2, was up-regulated twofold in PBM in vivo in Caucasians with extremely low BMD (cases) versus those with extremely high BMD (controls) (n = 28, p < 0.05). ANXA2 gene up-regulation in low BMD subjects was replicated at the mRNA level in PBM in vivo in a second and independent case-control sample (n = 80, p < 0.05). At the DNA level, we found that SNPs in the ANXA2 gene were associated with BMD variation in a 3rd and independent case-control sample (n = 44, p < 0.05), as well as in a random population sample (n = 997, p < 0.05). The above integrative evidence strongly supports the concept that ANXA2 is involved in the pathogenesis of osteoporosis in humans. Through a follow-up cellular functional study, we found that ANXA2 protein significantly promoted monocyte migration across an endothelial barrier in vitro (p < 0.001). Thus, elevated ANXA2 protein expression level, as detected in low BMD subjects, probably stimulates more PBM migration through the blood vessel walls to bone resorption surfaces in vivo, where they differentiate into higher number of osteoclasts and resorb bone at higher rates, thereby decreasing BMD. In conclusion, this study identified a novel osteoporosis susceptibility gene ANXA2, and suggested a novel pathophysiological mechanism, mediated by ANXA2, for osteoporosis in humans. Low bone mineral density (BMD) is a risk factor of osteoporosis and has strong genetic determination. Genes influencing BMD and fundamental mechanisms leading to osteoporosis have yet to be fully determined. Peripheral blood monocytes (PBM) are potential osteoclast precursors, which could access to bone resorption surfaces and differentiate into osteoclasts to resorb bone. Herein, we attempted to identify osteoporosis susceptibility gene(s) and characterize their function(s), through an initial proteomics discovery study on PBM in vivo, and multiscale validation studies in vivo and in vitro. Utilizing the quantitative proteomics methodology LC-nano-ESI-MSE, we discovered that a novel protein, i.e. ANXA2, was up-regulated twofold in PBM in vivo in Caucasians with extremely low BMD (cases) versus those with extremely high BMD (controls) (n = 28, p < 0.05). ANXA2 gene up-regulation in low BMD subjects was replicated at the mRNA level in PBM in vivo in a second and independent case-control sample (n = 80, p < 0.05). At the DNA level, we found that SNPs in the ANXA2 gene were associated with BMD variation in a 3rd and independent case-control sample (n = 44, p < 0.05), as well as in a random population sample (n = 997, p < 0.05). The above integrative evidence strongly supports the concept that ANXA2 is involved in the pathogenesis of osteoporosis in humans. Through a follow-up cellular functional study, we found that ANXA2 protein significantly promoted monocyte migration across an endothelial barrier in vitro (p < 0.001). Thus, elevated ANXA2 protein expression level, as detected in low BMD subjects, probably stimulates more PBM migration through the blood vessel walls to bone resorption surfaces in vivo, where they differentiate into higher number of osteoclasts and resorb bone at higher rates, thereby decreasing BMD. In conclusion, this study identified a novel osteoporosis susceptibility gene ANXA2, and suggested a novel pathophysiological mechanism, mediated by ANXA2, for osteoporosis in humans. Osteoporosis is a worldwide public health problem that is most prevalent in the elderly, and is particularly problematic in postmenopausal women. Osteoporosis is characterized by low bone mineral density (BMD) 1The abbreviations used are: BMDbone mineral densityPBMperipheral blood monocytePBMCperipheral blood mononuclear cellsHDMShigh definition mass spectrometryRMArobust multiarray averageHUVEChuman umbilical vein endothelial cellsMCP-1monocyte chemotactic protein 1FBSfetal bovine serumPPIApeptidyl-prolyl cis-trans isomerase A. 1The abbreviations used are: BMDbone mineral densityPBMperipheral blood monocytePBMCperipheral blood mononuclear cellsHDMShigh definition mass spectrometryRMArobust multiarray averageHUVEChuman umbilical vein endothelial cellsMCP-1monocyte chemotactic protein 1FBSfetal bovine serumPPIApeptidyl-prolyl cis-trans isomerase A. and increased risk of osteoporotic fractures. Osteoporotic hip fractures have a high associated morbidity and mortality, and contribute substantially to health care expenditures (1Harvey N. Dennison E. Cooper C. Osteoporosis: impact on health and economics.Nat. Rev. Rheumatol. 2010; 6: 99-105Crossref PubMed Scopus (436) Google Scholar). The annual cost of osteoporosis and fractures in the US elderly alone was estimated at $16 billion in 2002 and $22 billion in 2008 (2Blume S.W. Curtis J.R. Medical costs of osteoporosis in the elderly Medicare population.Osteoporos. Int. 2011; 22: 1835-1844Crossref PubMed Scopus (179) Google Scholar), and this is expected to increase substantially over the following decades. bone mineral density peripheral blood monocyte peripheral blood mononuclear cells high definition mass spectrometry robust multiarray average human umbilical vein endothelial cells monocyte chemotactic protein 1 fetal bovine serum peptidyl-prolyl cis-trans isomerase A. bone mineral density peripheral blood monocyte peripheral blood mononuclear cells high definition mass spectrometry robust multiarray average human umbilical vein endothelial cells monocyte chemotactic protein 1 fetal bovine serum peptidyl-prolyl cis-trans isomerase A. BMD is a quantitative trait determined by multiple factors. Heritability of BMD is estimated to be 0.5–0.9 (3Recker R.R. Deng H.W. Role of genetics in osteoporosis.Endocrine. 2002; 17: 55-66Crossref PubMed Scopus (103) Google Scholar). Extensive genetic and genomic studies, conducted over the past decade, have identified several genes that are associated with BMD variation in humans (4Xu X.H. Dong S.S. Guo Y. Yang T.L. Lei S.F. Papasian C.J. Zhao M. Deng H.W. Molecular genetic studies of gene identification for osteoporosis: the 2009 update.Endocr. Rev. 2010; 31: 447-505Crossref PubMed Scopus (68) Google Scholar). Collectively, however, these implicated genes explain no more than 10% of BMD variation in any individual human population. So the basis for the majority of variation in BMD that is genetically determined still awaits identification. Osteoporosis results from excessive bone loss, which is largely because of increased bone resorption by osteoclasts and/or decreased bone formation by osteoblasts. Functional studies, primarily based on mouse models or in vitro cell cultures, have been widely used to establish the relevance of well-known or novel genes to osteoblastogenesis and/or osteoclastogenesis and bone phenotypes (4Xu X.H. Dong S.S. Guo Y. Yang T.L. Lei S.F. Papasian C.J. Zhao M. Deng H.W. Molecular genetic studies of gene identification for osteoporosis: the 2009 update.Endocr. Rev. 2010; 31: 447-505Crossref PubMed Scopus (68) Google Scholar, 5Galli C. Zella L.A. Fretz J.A. Fu Q. Pike J.W. Weinstein R.S. Manolagas S.C. O'Brien C.A. Targeted deletion of a distant transcriptional enhancer of the receptor activator of nuclear factor-kappaB ligand gene reduces bone remodeling and increases bone mass.Endocrinology. 2008; 149: 146-153Crossref PubMed Scopus (81) Google Scholar, 6Wan Y. Chong L.W. Evans R.M. 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Consequently, clinical studies with feasible study designs for discovery of genes clinically significant to bone phenotypes/metabolism in vivo, followed by functional exploration and/or validation studies in vivo and in vitro, is highly desirable to identify and characterize the genes and their products for the risk of osteoporosis in humans. Bone-resorbing osteoclasts are hematopoietic in origin and are derived from cells of monocyte-macrophage lineage (9Roodman G.D. Regulation of osteoclast differentiation.Ann. N.Y. Acad. Sci. 2006; 1068: 100-109Crossref PubMed Scopus (155) Google Scholar). In the adult human peripheral skeleton such as the hip bone, the local marrow is not a significant source for generation of osteoclasts; virtually all osteoclasts functioning at these sites are primarily derived from monocytes migrating to bone via the peripheral circulation (10Parfitt A.M. 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It is extremely difficult to collect large numbers of osteoclasts in vivo in humans for experimentation; however, peripheral blood monocytes (PBM), which are osteoclast precursors, can be collected in large numbers with relative ease. Previous studies have identified several cytokines produced by PBM, (e.g. IL-1, IL-6, TNF-α), which are important for osteoclast differentiation, activation, and apoptosis (12Pacifici R. Estrogen, cytokines, and pathogenesis of postmenopausal osteoporosis.J. Bone Miner. Res. 1996; 11: 1043-1051Crossref PubMed Scopus (622) Google Scholar, 13Cohen-Solal M.E. Boitte F. Bernard-Poenaru O. Denne M.A. Graulet A.M. Brazier M. De Vernejoul M.C. Increased bone resorbing activity of peripheral monocyte culture supernatants in elderly women.J. Clin. Endocrinol. Metab. 1998; 83: 1687-1690Crossref PubMed Scopus (30) Google Scholar, 14Cohen-Solal M.E. Graulet A.M. Denne M.A. Gueris J. Baylink D. de Vernejoul M.C. 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Utilizing the quantitative proteomics and a of and integrative studies, the was to identify genes important to osteoporosis in vivo in and to the functional by which they contribute to the of identification of genes important to osteoporosis was based on studies at and and based on evidence from multiple independent study study was by were from all study they in the subjects were as of and that bone mass or bone were and and of bone as In subjects or bone such as were from this The of the above was to the of any and/or that bone thereby for genetic factors. 1 and used for PBM gene expression studies, we or the such as 1 of or such as systemic and such as hematopoietic and (e.g. BMD was for study subjects BMD a BMD from the and The were The of variation of was The involved study independent case-control and random population of the study are in It is to all subjects with low BMD in the case-control were as < or osteoporosis Herein, is as the number of a BMD from the average BMD of their and of the study are the hip BMD was as is as the number of a BMD from the average BMD of their and population. the subjects in low BMD were as or In the hip BMD was as is as the number of a BMD from the average BMD of their and population. the subjects in low BMD were as or in a new 1 postmenopausal from an population and of subjects with extremely high hip BMD and subjects with extremely low hip BMD is as the number of a BMD from the average BMD of their and population. from an population The sample subjects with extremely high hip BMD and subjects with extremely low hip BMD adult The sample subjects with extremely high hip BMD and subjects with extremely low hip BMD adult 1 and peripheral blood was collected from by was used as The blood were for PBM by peripheral blood mononuclear cells were from blood density with PBM were from a monocyte following the The a highly and to and cells from monocytes and of and the of PBM this was Y. H. R.R. Deng H.W. A novel pathophysiological for osteoporosis suggested by an in vivo gene expression study of PubMed Scopus Google Scholar). PBM were a San was to protein was a protein were in with by by and by were to of which was and to with and PBM were a of of by a of 2006; PubMed Scopus Google Scholar), through with was by the protein were into a and by A with and with at a of a of as 10% at at at at at at The was by The following were for for and for The was to high for mass of PBM were the were with on the and of of were and a of all and was the was derived from the of the and mass The was to be above protein protein was by the following number of and mass for mass for protein, protein, to identified at in the were as of the most were used to the as protein level was in and from were used to protein expression in PBM on PBM in the were used to the of protein expression to identify subjects with low BMD versus high BMD. was from PBM ANXA2 mRNA expression were determined Y. H. R.R. Deng H.W. A novel pathophysiological for osteoporosis suggested by an in vivo gene expression study of PubMed Scopus Google Scholar). used the B. F. and of high density level PubMed Scopus Google to the into mRNA expression the the most results and the with J. M. L.A. to of gene expression from 2006; PubMed Scopus Google Scholar). on expression by the mRNA expression was conducted subjects with low BMD versus high BMD a DNA was from peripheral blood a DNA DNA was by a DNA was and DNA in the which and of ANXA2 were The was the R. F. A. B. J. A. R. D. A. R. B. R. B. S. Y. M. C. T. C. J. M. K. H. K. J. K. C. A.M. S. J. M. A. D. S. B. C.A. on DNA 2010; PubMed Scopus Google by the human DNA DNA generation and DNA DNA and R. F. A. B. J. A. R. D. A. R. B. R. B. S. Y. M. C. T. C. J. M. K. H. K. J. K. C. A.M. S. J. M. A. D. S. B. C.A. on DNA 2010; PubMed Scopus Google Scholar). was used as a reference to identify in were conducted to are significant in subjects with low versus high BMD. 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In the 3rd case-control study sample a of in the ANXA2 gene were identified by these SNPs and were for with hip BMD. found that to SNPs and significant in low BMD and high BMD subjects (p < that SNPs and are potential sites of Thus, these SNPs may have potential effects on ANXA2 in ANXA2 gene in low high BMD subjects in sample subjects with low BMD were as or were used to the in bone mineral in a new In Caucasians we found that the SNPs the ANXA2 gene and by the SNPs and were associated with hip as well as BMD in of A at at and at have an average in hip BMD of and with and After for multiple of the i.e. associated with hip and BMD that of the above is a potential of Thus, this may have potential effects on ANXA2 SNPs in ANXA2 gene associated with BMD at hip and in sample The was used to the of SNPs with BMD. bone mineral is the in are with effects on hip and BMD for in are p for multiple for in are p for multiple is the in are with effects on hip and BMD in are p for multiple Y. Y. the discovery a and to multiple Scholar). in a new that ANXA2 protein was in the of human PBM cell ANXA2 protein is by human ANXA2 protein was to on monocyte in the and in the are for and ANXA2 to monocyte this the effects of ANXA2 on migration were in ANXA2 into the and promoted monocyte with the at the of increase with the a significant was not in ANXA2 into the and promoted monocyte migration (p < 0.001). of ANXA2 produced the in the migration increase with the Collectively, these that ANXA2 protein a significant in monocyte The to identify osteoporosis risk genes and characterize their a novel of and integrative quantitative proteomics methodology to in vivo PBM from and to genes that are to osteoporosis in humans. gene(s) identified were by mRNA expression for the gene(s) these were subsequently in an independent study of the ANXA2 at mRNA and protein expression in PBM in low versus high BMD subjects, strongly supports the functional relevance of ANXA2 to BMD regulation in vivo in humans. the of ANXA2 to bone we the ANXA2 and osteoporosis at the DNA In case-control and population we identified SNPs in the ANXA2 gene that are associated with hip BMD in Collectively, all this evidence supports the concept that ANXA2 is a susceptibility gene for osteoporosis in humans. monocyte into In the adult human peripheral skeleton such as the hip bone, osteoclastogenesis PBM have across endothelial surfaces from blood to bone (9Roodman G.D. Regulation of osteoclast differentiation.Ann. N.Y. Acad. Sci. 2006; 1068: 100-109Crossref PubMed Scopus (155) Google Scholar). The migration in this study, was to the endothelial barrier blood and bone, as through to sites of bone resorption on the of bone they and ANXA2 was found to be by human PBM, is important to the of ANXA2 protein in monocyte in vitro migration suggested ANXA2, in the blood of may monocyte migration to bone resorption ANXA2, at bone resorption sites in may monocyte migration from the circulation and to bone to differentiate into may to more PBM to bone. a significant for ANXA2 protein in monocyte evidence that ANXA2 on the cell of human PBM may contribute to activity of monocytes C. R. M. by human expression by PubMed Scopus Google Scholar, and by is on expression of PubMed Scopus Google Scholar). In to the of ANXA2 in monocyte evidence from in vitro studies that ANXA2 is important to osteoclast formation and bone in human marrow cultures, ANXA2 promoted osteoclast and differentiation, and increased osteoclast formation and bone resorption (9Roodman G.D. Regulation of osteoclast differentiation.Ann. N.Y. Acad. Sci. 2006; 1068: 100-109Crossref PubMed Scopus (155) Google Scholar, F. H. N. Zhao Roodman G.D. stimulates expression through Bone Miner. Res. PubMed Scopus Google Scholar, C. Y. Roodman G.D. increases osteoclast formation by the of osteoclast in human marrow Clin. PubMed Scopus Google Scholar, H. N. R. Roodman G.D. and of the receptor on human marrow 2006; PubMed Scopus Google Scholar, S. R. C. J. Roodman G.D. and identification of as an factor that increases osteoclast formation and bone PubMed Google Scholar). functional evidence from the in vivo and in vitro studies and in vitro studies, we a novel pathophysiological mechanism, by ANXA2, for osteoporosis in humans. increased expression of ANXA2 protein on probably via to PBM migration from the blood to sites of bone ANXA2 protein PBM bone resorption sites local ANXA2 Elevated ANXA2 at bone resorption sites to the sites by in an of osteoclast at the sites of bone ANXA2 monocyte differentiation, osteoclast and bone Collectively, the above to contribute to elevated in bone resorption and increased bone loss, and and the of this is the study in which ANXA2 has been identified in human PBM and implicated significant in in bone phenotypes in humans. by evidence from of genetic protein < = < = and by functional evidence from in vivo and in vitro studies, novel into the pathogenesis of osteoporosis in humans. The that expression proteomics methodology a new to identify gene(s) for The is particularly significant that genes are identified through functional that are associated with and clinical in humans. The novel and integrative study in this study, be to in humans. has potential to identify genes associated with these characterize their and the pathophysiological mechanisms the of these with

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