MCP-1 and CCR2 Gene Variants and the Risk for Osteoporosis and Osteopenia

Abstract

Aim: In this study, we investigated whether monocyte chemotactic protein 1 (MCP-1) and CC chemokine receptor 2 (CCR2) gene polymorphisms account for an increased risk of osteoporosis or osteopenia. Methods: Three hundred three postmenopausal women, 80 osteoporotic, 123 osteopenic, and 100 unrelated age-matched healthy controls, were included in the study. Genotyping of MCP-1 A2518G and CCR2 V64I gene polymorphisms were detected by PCR-RFLP. Results: We, for the first time, demonstrated the positive association of MCP-1 GG, CCR2 Val/Ile, and CCR2 Val+ genotype with osteoporosis risk. However, CCR2 Ile/Ile genotype frequencies were high in the control group compared with those of the patients with osteoporosis and osteopenia. Haplotype analysis confirmed the association of MCP-1/CCR2 gene variants with osteopenia and revealed that the frequency of MCP-1 A:CCR2 Val haplotype was significantly higher in patients when compared with controls. Conclusions: In conclusion, our findings have suggested that MCP-1 and CCR2 gene variants were risk factors for osteoporosis and osteopenia.

Introduction

Osteoporosis is a systemic skeletal disease in which loss of bone mass and the microarchitectural deterioration of bone tissue leads to fragility fractures (Zajickova and Zofkova, 2003; Raisz, 2005). Enhanced bone resorption may be related with osteoclastogenesis from precursor cells, enhanced fusion and activation of osteoclasts, and prolonged lifespan due to an inhibition of osteoclast apoptosis (Manolagas, 2000; Teitelbaum, 2000). There are multiple mechanisms that play roles in the regulation of bone remodeling, and these involve not only the osteoblastic and osteoclastic cell lineages but also other marrow cells, in addition to the interaction of systemic hormones, local cytokines, growth factors, and transcription factors (Raisz, 2005). They are speculated as candidates for involvement in bone loss in different types of inflammatory diseases through the recruitment of osteoclast precursors but their potential effects in osteoclast recruitment, development, or function are not well understood (Choi et al., 2000; Yu et al., 2004). Osteoblasts have also been shown as a source of chemokines in bones (Yu et al., 2004). Chemokines, particularly of two major (CXC, CC) families, are essential signals for the trafficking and localization of circulating hematopoietic cells. Monocyte chemotactic protein 1 (MCP-1, CCL2 or SCYA2) is the most investigated CCL chemokine family member (Van Coillie et al., 1999). It is expressed by various types of cells which include fibroblasts, endothelial cells (Yu and Graves, 1995), and osteoblasts (Simonet et al., 1997) and exhibits chemoattractant activity toward osteoclasts. It has been reported that an A2518G polymorphism in the regulatory region of the MCP-1 gene affects MCP-1 expression in response to inflammatory stimuli (Rovin et al., 1999). The cellular influence of MCP-1 is mediated by the CC chemokine receptor 2 (CCR2), a G-coupled receptor (Rollins, 1997). A 190 G/A single-nucleotide polymorphism (SNP) in the CCR2 gene, located on chromosome 3p21-p24, is the most researched CCR2 SNP in exon 1 (Murphy et al., 2000).This mutation results in the substitution of valine by isoleucine (V64I) in the transmembrane region of the protein (Attar et al., 2010). In light of this information, our aim in this study was to investigate some possible associations between MCP-1 A2518G and CCR2 V64I gene polymorphisms and the risk for osteoporosis.

Subjects and Methods

Subjects

The cohort of this study contained 303 Turkish postmenopausal women (80 osteoporotic, 123 osteopenic, and 100 healthy), 40-78 years of age, attending the Uskudar State Hospital in Istanbul between June 2009 and March 2010. During recruitment the WHO definitions and criteria for osteoporosis (World Health Organization Study Group, 1994) were used. The patients received a detailed, standardized questionnaire including questions regarding the osteoporosis risk factors, such as family history of osteoporosis, menopausal status and age, cigarette smoking, insufficient protein intake, alcohol consumption, medication use, and other medical conditions. Only patients with a clinical diagnosis of osteoporosis and osteopenia were recruited. The healthy group contained only individuals with normal bone mineral density (BMD). Exclusion criteria before enrollment included conditions, diseases, and/or treatments known to interfere with bone metabolism, such as malignancies, endocrinologic disorders (hypo- and hyperparathyroidism, hyperthyroidism, Cushing's syndrome), severe liver or gastroenteral diseases, skeletal diseases (Paget's disease, osteogenesis imperfecta, osteomalacia, and rheumatoid arthritis), and current pharmacological treatment with corticosteroids, anabolic androgenic steroids, estrogens, estrogen-related molecules, or anticonvulsants. Menopause was defined as amenorrhea of at least 1 year duration. The study was approved by the Local Ethical Committee of Istanbul University Medical Faculty (Protocol No. 2006/2145) and a written, informed consent was obtained from each participant prior to giving their blood sample.

BMD measurement

BMD was measured at the lumbar spine (L1-L4) and hip (femoral neck and total hip) by dual-energy X-ray absorptiometry (DXA; Lunar DPX; GE Lunar Corporation, Madison, WI).

Genotyping

Genotyping method of the MCP-1 A2518G and CCR2 V64I gene polymorphisms: Blood specimens were collected in tubes containing EDTA, and DNA was prepared from the leukocyte pellet by SDS lysis, ammonium acetate extraction, and ethanol precipitation (Miller et al., 1988). CCR2 V64I and MCP-1 A2518G genotypes were determined with the PCR-RFLP method according to previously published protocols (Szalai et al., 2001a; Abdi et al., 2002).

Statistical analyses

Statistical analyses were performed using the SPSS software package (revision 11.5; SPSS, Inc., Chicago, IL). Differences in the distribution of MCP-1 A2518G and CCR2 V64I genotypes or alleles between cases and controls were tested using the χ² statistic. Comparisons of haplotype frequencies between the patients and the control groups were carried out using the Haploview program (Barrett et al., 2005). Values of p<0.05 were considered statistically significant.

Results

Characteristics of patients and healthy controls are shown in Table 1. BMI, L1-L4 BMD, femoral neck BMD, wards BMD, total BMD, and trochanter BMD were significantly different for the two study groups (p<0.01).

Table 1.

Clinical Characteristics of Study Participants

Parameters	Osteopenia (n=123)	Osteoporosis (n=80)	Healthy controls (n=100)	P1	P2	P3
Age (year)	58.32±7.60	58.54±5.78	56.24±8.64	0.05	0.06	0.83
BMI (kg/m²)	30.28±4.79	27.84±4.22	33.43±4.81	0.00	0.00	0.00
Menopause age (years)	47.19±4.66	46.07±5.5	47.06±4.29	0.88	0.36	0.14
Family history (%)	44.4	39.7	47.5	0.73	0.42	0.51
Smoking (%)	8.9	8.8	20	0.01	0.03	0.96
L1-L4 BMD (g/cm²)	1.00±0.87	0.82±0.06	1.17±0.11	0.00	0.00	0.00
Femoral neck BMD (g/cm²)	0.84±0.08	0.76±0.09	0.96±0.09	0.00	0.00	0.00
Wards BMD (g/cm²)	0.67±0.09	0.58±0.10	0.81±0.10	0.00	0.00	0.00
Total BMD	0.90±0.08	0.81±0.09	1.05±0.09	0.00	0.00	0.00
Trochanter BMD (g/cm²)	0.73±0.08	0.66±0.08	0.85±0.01	0.00	0.00	0.00

P1, osteopenia vs. control; P2, osteoporosis vs. control; P3, osteopenia vs. osteoporosis; BMI, body mass index; BMD, bone mineral density.

The frequencies of MCP-1 A2518G and CCR2 V64I genotypes and their respective alleles among cases and controls are shown in Table 2. There was a statistically significant difference between the controls and the patients with osteoporosis for CCR2 V64I genotypes and (p=0.003).

Table 2.

Distribution of MCP-1 A2518G and CCR2 V64I Genotype Frequencies in Patients and Control Groups

Genotypes/alleles	Osteopenia n (%)	Osteoporosis n (%)	Healthy controls n (%)	p-Value
MCP-1 A2518G
AA	78 (63.4)	38 (47.5)	51 (51.0)	0.06^a; 0.64^b
GG	4 (3.3)	7 (8.8)	2 (2.0)	0.56^a; 0.042^b
AG	41 (47.0)	35 (43.8)	47 (47.0)	0.03^a; 0.664^b
	p: 0.111	p: 0.119
A	197 (80.0)	111 (69.37)	149 (74.5)
G	49 (19.91)	49 (30.62)	51 (25.5)	0.15^a; 0.28^b
CCR2 V64I
Val/Val	108 (87.8)	57 (71.3)	80 (80.0)	0.111^a; 0.171^b
Ile/Ile	2 (1.6)	0 (0)	7 (7.0)	0.045^a; 0.015^b
Val/Ile	13 (10.6)	23 (28.8)	13 (13.0)	0.574^a; 0.009^b
	p: 0.09	p: 0.003
Val	229 (93.08)	137 (85.6)	173 (86.5)
Ile	17 (6.91)	23 (14.37)	27 (13.5)	0.02^a; 0.811^b

Osteopenia vs. control.

Osteoporosis vs. control.

CCR2, CC chemokine receptor 2; MCP-1, monocyte chemotactic protein.

The control group was not in Hardy-Weinberg equilibrium for both MCP-1 and CCR2 genotypes (p=0.01 and p=0.00, respectively); patients with osteoporosis were consistent for MCP-1 (p=0.79) and CCR2 (p=0.13), and patients with osteopenia were consistent only for MCP-1 (p=0.61) but not consistent with CCR2 genotypes (p=0.04).

MCP-1 AG genotype frequencies were found low in patients with osteopenia and MCP-1 GG genotype was high in patients with osteoporosis compared with that of the control group (p=0.038, χ²=4.31, OR=0.56, 95% CI: 0.32-0.97; p=0.042, Fisher's exact test OR=4.69, 95% CI: 0.94-23.28, respectively).

The frequencies of CCR2 Val/Ile genotype and Val+ allele were high in patients with osteoporosis compared with that of the control group (p=0.042, Fisher's exact test OR=4.69, 95% CI: 0.94-23.28; p=0.009, χ²=6.89, OR=2.7, 95% CI: 1.26-5.76; p=0.015, Fisher's exact test OR=0.93, 95% CI: 0.88-0.98, respectively). In contrast, CCR2 Ile/Ile genotype frequencies were high in the control group compared with the patients with osteoporosis (p=0.015, Fisher's exact test OR=0.93, 95% CI: 0.88-0.98). In patients with osteopenia, carriers of the CCR2 Val+allele (Val/Val+Val/Ile genotype) frequency was high compared with the control group (p=0.045, Fisher's exact test OR=4.55, 95% CI: 0.92-22.43).

In addition to SNP analyses, haplotypes were evaluated for association with osteopenia and osteoporosis (Table 3). Haplotype analysis confirmed the association of MCP-1/CCR2 gene variants with osteopenia and revealed that the frequencies of MCP-1 A:CCR2 Val and MCP-1 A:CCR2 Ile haplotypes were significantly higher and lower, respectively, in patients when compared with controls.

Table 3.

The Frequencies of Haplotypes of MCP-1 and CCR2 Gene in Study Groups

	Frequency
	Overall	All patients	Control	χ²	p-Value
Haplotype associations in osteopenia
MCP-1 A:CCR2 Val	0.696	0.748	0.630	7.113	0.0077
MCP-1 G:CCR2 Val	0.205	0.182	0.235	1.752	0.1856
MCP-1 A:CCR2 Ile	0.080	0.052	0.115	5.594	0.018
MCP-1 G:CCR2 Ile	0.019	0.017	0.020	0.143	0.7051
Haplotype associations in osteoporosis
MCP-1 A:CCR2 Val	0.607	0.579	0.630	0.96	0.3272
MCP-1 G:CCR2 Val	0.254	0.277	0.235	0.828	0.3629
MCP-1 A:CCR2 Ile	0.115	0.114	0.115	0.0	0.988
MCP-1 G:CCR2 Ile	0.024	0.029	0.020	0.323	0.5695

Discussion

The osteoporosis candidate genes may be categorized according to the function(s) of the coded molecules, mostly included in the metabolism of bone cells (osteoblasts and osteoclasts), structure and turnover of collagen and minerals (calcium and phosphorus), and regulatory/hormonal (obviously, sex hormone) pathways. There are multiple gene polymorphisms that have been investigated in association studies with bone mass and fragility (Liu et al., 2006; Ralston and Crombrugghe, 2006) but new and more effective approaches are likely to arise from a better understanding of the regulation bone cell function (Raisz, 2005). We, for the first time, demonstrated the positive association of MCP-1 and CCR2 gene variants with osteoporosis or osteopenia risk. In our study, the frequencies of MCP-1 GG, CCR2 Val/Ile, and CCR2 Val+ genotype are more prevalent in patients with osteoporosis than in controls. On the other hand, the CCR2 Ile/Ile genotype is more prevalent in controls than in osteoporosis and osteopenia patients In addition, haplotype analysis confirmed the association of MCP-1/CCR2 gene variants with osteopenia and revealed that the frequency of MCP-1 A:CCR2 Val haplotype was significantly higher in patients when compared with controls.

At present, it is hard to explain by which mechanisms genotypes of chemokines result in osteoporosis or osteopenia. Nevertheless, we can make some plausible interpretations depending on the previous study findings. Inflammation can influence the balance between bone formation and resorption and patients with chronic inflammatory diseases seem to have increased risk of developing osteopenia (Hardy and Cooper, 2009). The relevance between the immune system and bone metabolism, or “osteoimmunology,” involves molecular and cellular interactions between osteoblasts, osteoclasts, lymphocytes, and the monocyte-macrophage lineage (Takayanagi, 2007). The association between inflammation and bone turnover appears to depend mainly on the production of cytokines. As estrogen levels decrease at menopause, there is an increase in the production of proinflammatory cytokines; this process may contribute to increased osteoclast activity and subsequent loss of bone density (Swanberg et al., 2010). However, cytokines play dual roles in bone homeostasis by different activities depending on the type of immune response (Dewhirst et al., 1985; Takayanagi et al., 2000; Takayanagi, 2007). Some of the studies reported that the −2518 A/G polymorphism of MCP-1 may be related with its gene expression. Rovin et al. (1999) reported the over-representation of −2518 G frequency in Asian and Mexican populations compared with a Caucasian population. In results from several studies, the G allele has generally been regarded to be associated with inflammation and different types of diseases such as Kawasaki disease, coronary artery disease, asthma, and lupus nephritis (Jibiki et al., 2001; Szalai et al., 2001a, 2001b; Tucci et al., 2004). The current study result is consistent with studies showing an increased risk of MCP-1 GG genotype and G allele in different type of diseases.

It was shown that polymorphisms in the CCR2 gene that alter macrophage recruitment have been reported to influence a number of diseases including AIDS (Doms and Peiper, 1997), multiple sclerosis (Miyagishi et al., 2003), breast cancer (Zafiropoulos et al., 2004), carotid atherosclerosis (Nyquist et al., 2009), and renal transplant rejection (Omrani et al., 2008). It has been speculated that its distribution is strongly dependent on ethnicity (Smith et al., 1997; Anzala et al., 1998; Struyf et al., 2000; Lewandowska et al., 2002). The CCR2 (V64I) mutation leads to the substitution of valine by isoleucine in the transmembrane region of the protein. Data on the effect of this SNP on the expression of CCR2 are controversial (Nakayama et al., 2004; Navratilova, 2006).

In conclusion, our study has suggested that MCP-1 polymorphism may increase the transcription of MCP-1 by increasing association of MCP-1 with its receptor, leading to the elevated biological activity of the MCP-1/CCR2 system. The increased activity of this system may contribute to inducing osteoclast activity and subsequent loss of bone density. However, further studies are required to evaluate the association of the MCP-1/CCR2 system with osteoporosis.

Footnotes

Disclosure Statement

No competing financial interests exist.

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