Abstract
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-MS(E), 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 3(rd) 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.
Highlights
From the §Center of Bioinformatics and Genomics, Tulane University School of Public Health and Tropical Medicine, New Orleans, LA 70112; ‡School of Medicine, University of Missouri-Kansas City, Kansas City, MO 64108; ¶College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, P
According to Kruskal-Wallis test using the raw data set and the data set normalized by the housekeeping protein beta actin, ANXA2 protein was found to be differentially expressed between low bone mineral density (BMD) and high BMD subjects (p Ͻ 0.05, Table II and supplemental Table S1)
ANXA2 Gene Single Nucleotide Polymorphism (SNP) were Associated with BMD Variation in a Caucasian Population—In 997 unrelated Caucasians (i.e. Sample 4), we found that among the total 15 studied SNPs located within the ANXA2 gene and covered by the Affymetrix array, three SNPs were nominally associated with hip BMD, as well as femoral neck BMD (Table IV)
Summary
Human Subjects—This study was approved by appropriate Institutional Review Boards. Signed informed-consent documents were obtained from all study participants before they enrolled in the study. Hip BMD (g/cm2) was measured for study subjects using Hologic 4500 W dual energy x-ray absorptiometry machines (Hologic Inc., Bedford, MA). It is worth noting that, according to World Health Organization’s diagnostic criteria, all subjects with low BMD in the three case-control samples were diagnosed as having osteopenia Sample 1 for Proteomics Discovery Study—Sample 1 included 28 unrelated postmenopausal Caucasian women, selected from an archived population database and composed of 14 subjects with extremely high hip BMD (Z-score: 2.13 Ϯ 0.22, mean Ϯ S.E.) and 14 subjects with extremely low hip BMD (Z-score: –1.39 Ϯ 0.21, mean Ϯ S.E.). Z-score is defined as the number of standard deviations a subject’s BMD differs from the average BMD of their age-, gender-, and ethnicity-matched population. Sample 2 for mRNA Expression Study—Sample 2 included 80 unrelated Caucasian women, selected from an archived population
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