Elsevier

Bone

Volume 46, Issue 1, January 2010, Pages 72-80
Bone

A microarray based identification of osteoporosis-related genes in primary culture of human osteoblasts

https://doi.org/10.1016/j.bone.2009.09.015Get rights and content

Abstract

Genetic factors influencing the pathogenesis of osteoporosis are still largely unknown. We employed genome-wide gene expression approach in order to discover novel genes involved in the pathogenesis of osteoporosis. To this end, primary cultures of osteoblasts isolated from osteoporotic and non-osteoporotic human bone tissue samples were prepared. One thousand six hundred six genes were found to be differentially expressed, indicating increased demand for protein synthesis and decreased cell proliferation rate in osteoblasts from osteoporotic tissue as compared to osteoblasts from non-osteoporotic tissue. At first, top four genes, based on the microarray data and potential role in bone metabolism, were further studied in bone tissue samples of 55 patients. PTN and COL15A1 were both downregulated in osteoporotic bone tissue (6.2- and 3.4-fold, respectively, both p < 0.05), while IBSP and CXCL2 were both upregulated (5.7-fold, p < 0.05, and 2.1-fold, p > 0.05). Further biostatistical analysis of the microarray data by gene set enrichment analysis suggested oxidative stress may have an important part in the pathogenesis of osteoporosis. Thus, secondly, we tested it by an in vitro assay on human osteosarcoma cell line cells treated with hydrogen peroxide. After 72 h of treatment with 500 μM hydrogen peroxide, the upregulation of the same genes involved in the response to oxidative stress as on the microarrays was observed: MT1G (metallothionein 1G, 22.1-fold, p < 0.05), TXNRD1 (thioredoxin reductase 1, 3.7-fold, p < 0.05), AOX1 (aldehyde oxidase 1, 24.5-fold, p < 0.05) and GSR (glutathione reductase, 4.7-fold, p < 0.05). Our results present a novel list of genes and metabolic pathways that may be associated with the pathogenesis of osteoporosis. PTN, CXCL2, COL15A1, IBSP, AOX1, MT1G, GSR and TXNRD1 are candidate genes for further studies in the assessment of the genetic susceptibility to osteoporosis. In addition, differences in protein synthesis, cell proliferation rate and response to oxidative stress may also be involved in the pathogenesis of osteoporosis.

Introduction

The increased life expectancy has led to higher incidence of chronic and cancer diseases as well as to degenerative disorders, including osteoporosis. Osteoporosis is a metabolic disease of the bones, characterized by low bone mineral density (BMD) and microarchitectural changes, both leading to an increased incidence of osteoporotic fractures. Enormous effort is put into research to discover the molecular mechanisms of pathogenesis of osteoporosis in order to obtain novel targets for the treatment or prevention of osteoporosis, early diagnostic markers for identification of individuals at higher risk for the development of osteoporosis and prognostic markers for an effective individualized therapy.

Osteoblasts (OBs) have a central role in bone metabolism. They differentiate from mesenchymal stem cells and are responsible for the synthesis of bone matrix and bone mineralization, synthesis of growth factors and hormones, and also for the regulation of osteoclastogenesis and bone resorption. In addition, aged and trapped in the newly formed bone, they differentiate into osteocytes forming an extensive bone tissue communication network able to respond to mechanical stress [1].

Current research results (studies) cannot explain the influence of genetic factors, described already 2 decades ago in twin studies [2]. The genes influencing the osteoporosis-related phenotypes, like bone mineral density, fracture risk, onset of menopause and bone geometry, remain still largely unknown [3], mainly due to the polygenic and multifactorial nature of osteoporosis. Various approaches are employed in current osteoporosis research. One of them is the analysis of differential expression using DNA microarrays. At the moment, there are only a small number of studies of osteoporosis using the combination of OBs and DNA microarrays. These studies usually analyze the effects of a substance, like glucocorticoids [4] or vitamin K2 [5], or transgenic animals resembling an artificial osteoporosis phenotype [6], on the gene expression profile. The drawback of these studies is that they analyze the mechanisms of response to a certain stimulus, that are not necessary the same as in the pathogenesis of osteoporosis. Furthermore, most commonly they are conducted on tumor cell lines [4], [5], [7] or animals [8], [9]. Some studies are performed on cells of the immune system, like osteoclast precursor cells—monocytes [10] and B cells [11]. Microarray studies of osteoporotic and non-osteoporotic tissues [12] are also a good approach; however, they are largely influenced by different biological processes in the bone tissue and the difficult interpretation of the results due to the mixed cell types analyzed at the same time.

The aim of our study was, first, to identify osteoporosis-related genes in human osteoblasts using genome-wide gene expression microarrays and, second, to evaluate the results obtained by quantifying the expression of some genes in human bone tissue samples and osteoblast cell line in order to assess their importance in the pathogenesis of osteoporosis.

Section snippets

Patients

The study group consisted of 63 unrelated patients undergoing hip arthroplasty due to the osteoarthritis (non-osteoporotic patients) or femoral neck fracture (osteoporotic patients). A subgroup of eight unrelated postmenopausal female patients, four with low and four with normal heel BMD (T-score), was selected for microarray analysis of their OBs. Patients were age (median age 72.5 years) and body mass index (BMI) (median BMI 28.5 kg/m2) matched, and they differed significantly in heel BMD

Results

We first performed genome-wide gene expression analysis using microarrays in order to obtain a list of genes that are potentially differentially expressed in osteoblasts from osteoporotic tissue. We then analyzed by qPCR the expression of top four genes (PTN, CXCL2, COL15A1 and IBSP) based on the microarray data and potential role in bone metabolism. Gene set enrichment analysis was performed in order to obtain a list of metabolic pathways that are potentially differentially regulated in

Discussion

Our results of microarray analysis of OBs from osteoporotic bones identify 208 downregulated and 144 upregulated genes. Reanalysis of four genes by qPCR revealed PTN, CXCL2, COL15A1 and IBSP as the new candidate genes for osteoporosis. Further in vitro study showed that oxidative stress plays an important role in the pathogenesis of osteoporosis and the genes encoding antioxidant enzymes aldehyde oxidase 1 (AOX1), glutathione reductase (GSR), thioredoxin reductase 1 (TXNRD1) and metallothionein

Acknowledgments

The authors acknowledge Natasa Faganeli for the heel BMD measurements, Paola Torricelli, Manja Cedilnik, Tanja Prijatelj, Marjana Barlic and Jana Dragojevic for technical assistance in the laboratory, and all the patients for taking part in the study.

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