Saos-2 cells were cultured in McCoy's 5A medium (Gibco, Grand Island, New York, USA) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco) and 15% (v/v) fetal bovine serum (Gibco). The cells were cultured either in basic cell culture medium or in osteogenic medium (OM) supplemented with 50 mg/ml AA (Sigma-Aldrich, St. Louis, Missouri, USA) and 7.5 mM β-GP (Sigma). The protocol was based on the study by Thouverey 9.

Cell cultures were washed with Phosphate buffered saline buffer (PBS) and stained with 0.5% (w/v) Alizarin Red-S (Sigma-Aldrich ) in PBS (pH = 5.0) for 30 minutes at room temperature. After washing four times with PBS, the stained cells were photographed. The cultured cells were then de-stained with 10% (w/v) cetylpyridinium chloride (Sigma-Aldrich, ) in PBS (pH = 7.0) for 60 minutes at room temperature. The AR-S concentration was determined by measuring the absorbance at 562 nm. The protocol was based on previously published studies 91011.

To verify the effect of CA1 expression on bio-mineralization, acetazolamide, a chemical inhibitor of CA, was used to treat Saos-2 cells. Acetazolamide (Sigma) was dissolved in dimethyl sulfoxide (DMSO, Solabio) at a concentration of 0.05% and was added to cell cultures that were grown to 70% confluence in OM. The cultures were grown in the presence of acetazolamide at a final concentration of 100 μM for nine days. Saos-2 cells were also cultured in OM with 0.05% DMSO as a control. The protocol was based on a study by Hall et al. 12.

To evaluate the proliferative capacity of Saos-2 cells in OM or in the presence of acetazolamide, the cells were seeded onto 96-well culture plates and incubated until they reached 70% confluence. The cultures were then incubated with OM in the presence of 10 μM, 100 μM or 1 mM acetazolamide for three, six and nine days. Cell proliferation was assessed using the MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) assay. Aliquots of 20 μl of MTT reagent of MTT Cell Proliferation Assay Kit (ATCC, Manassas, Virginia, USA) at a concentration of 5 mg/ml were added to the cell cultures. After four hours of treatment, 150 μl of DMSO was added to the culture and slightly mixed for 10 minutes. The absorbance was measured at 490 nm with a spectrophotometer.

Total RNA was extracted from the cultured Saos-2 cells using a total RNA Isolation Kit (Omega Bio-Tek, Norcross, Georgia, USA) according to the protocol of the manufacturer. Concentrations of total RNA were determined with a spectrophotometer. The cDNA was prepared with 500 ng of total RNA from each sample using oligo-dT primers and the PrimeScriptTM RT-PCR Kit (TaKaRa, Ostsu, Shiga, Japan). A quantitative PCR analysis was performed with a LightCycler 480 thermocycler (Roche, Basel, Switzerland.). Taqman real-time PCR was conducted in 10 μl reaction mixtures containing 5 μl of SYBR Green (ToYoBo, Osaka, Japan), 1 μl of each primer, 1 μl of cDNA and 2 μl of H₂O. The optimized thermal cycling program was as follows: step one, 95ºC for 180 seconds; step two, 95ºC for 10 seconds, 59ºC for 10 seconds and 72ºC for 25 seconds (45 cycles); and step three, a melting curve analysis from 59°C to 95ºC in 0.5ºC increments. The specificity of the individual amplification reactions was assessed by examining the melting curves to confirm the presence of a single gene-specific peak with the characteristic melting temperature of the expected product. The primers for ALP, BSP, OCN, OSX, Runx2 and CA1 are listed in Table 1. The PCR results were standardized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. For each sample, two reactions were performed at the same time. One reaction was performed to determine the mRNA level of the target gene and the second reaction was performed to determine the level of GAPDH. The experiment was performed in triplicate and the PCR products were confirmed by a melting curve analysis. The relative expression of mRNA was calculated using the comparative threshold cycle (Ct) method according to the following formula: Ratio = 2-ΔΔCt = 2ΔCt (sample), where ΔCt = Ct of target genes-Ct of the endogenous control gene (GAPDH). The relative target gene expression was normalized in comparison to the GAPDH mRNA. The copy numbers of the targeted mRNA in the samples were calculated automatically by the data analysis software. Levels of the transcripts are expressed as the median and range. Statistical differences were assessed using the Mann-Whitney U-test; P <0.05 was considered statistically significant.

Cultured cells and animal tissues were homogenized in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors on ice and centrifuged at 16000 × g for five minutes at 4°C. The supernatant was collected and the protein concentration was determined using a BCA Protein Assay Kit (Beyotime). Thirty micrograms of total protein were separated on a 12% SDS-polyacrylamide gel and transferred onto a PVDF membrane. The membrane was rinsed with wash solution and incubated with anti-human CA1 antibody (Santa Cruz, Santa Cruz, California, USA) at a dilution of 1:500 overnight at 4°C. The antibody was prepared by immunizing a mouse with a peptide comprising CA1 amino acids 33 to 80, which are mapped near the N-terminus. The manufacturer confirmed no cross-reactivity with other carbonic anhydrases. Immunosignals were visualized with a Protein Detector BCIP/NBT Western Blotting Kit (Beyotime) following the manufacturer's instructions. The quantification was conducted using ImageQuant 5.2 software. A separate membrane prepared in the same manner was probed with an anti-GAPDH antibody (Santa Cruz) to normalize the sample loading.

Peripheral blood samples were collected from patients with RA and AS. The diagnosis of RA was made according to the criteria of the American College of Rheumatology 13. The RA patients had high levels of C-reactive protein (30 to 100 mg/L, mean 24 mg/L), anti-cyclic citrullinated protein antibodies (anti-CCP; 300 to 3,000 U/ml) and rheumatoid factor (RF; 160 to 2,560 U/ml). AS patients had an average disease duration of seven years and were positive for HLA-B27 antigen. Their symptoms were consistent with the modified New York criteria for AS. Patients were selected from the same population living in the Shandong area of Northern China. They did not have any personal or family history of serious illness. Control individuals were from the same geographical area. Both patients and healthy controls gave their written consent to participate in the study and to allow their biological samples to be genetically analyzed. The Ethical Committee of the Shandong Academy of Medicinal Sciences approved this study.

Blood samples were put into Monovette tubes containing 3.8% sodium citrate. Genomic DNA was extracted from whole blood samples with the Omega E-Z 96 Blood DNA kit (Omega) according to the manufacturer's protocol. DNA samples were dissolved in deionized water. The concentration and quality of DNA samples were measured using a spectrophotometer at 260 nm (A260) and 280 nm (A280). An A260/A280 ratio of 1.7 to 1.9 was considered to be highly pure DNA. Electrophoresis was also used to examine the quality and integrity of DNA.

Tag SNPs across the CA1 were determined by searching the HapMap database. The tag SNPs were selected on the basis of linkage disequilibrium patterns observed in the Han Chinese in Beijing (CHB) samples that were genotyped as a part of the International HapMap Project. Only SNPs with a minor allele frequency of greater than 5% with a pair-wise r² ≥ 0.8 in the HapMap were considered. SNPs that were in exons and UTRs, as well as promoters and introns within 500 bp of exons were also selected. Candidate SNPs were submitted to Illumina for a design score. The Illumina Assay Design Tool filtered out SNPs that were not suitable for the Illumina platform. Finally, thirteen SNPs with a design score of '1' were selected; they span 23,000 bases of the chromosome. These SNPs included one tagSNP, one coding SNP, one SNP at the 5'UTR, nine SNPs in the introns and one SNP in the promoter region. These SNP sites and locations are described in Table 2.

Statistical analyses of the data were performed using SPSS V.16 software (SPSS, Chicago, Illinois, USA). The multiple comparisons were conducted using analysis of variance (ANOVA). T-test was used to assess statistical differences of two groups. P values less than 0.05 were considered significant.

A large amount of calcium deposits was observed in the cultured cells when the incubation was conducted in OM for six and nine days (Figure 1A). The absorbance of cetylpyridinium chloride was 3.46-fold higher in Saos-2 cells that were incubated with OM than in the cells that were cultured without induction for six days (P = 4.63*10-5). The absorbance was 4.06-fold higher after nine days than that of the control (P = 3.92*10-7) (Figure 1B). The formation of calcium nodules was significantly decreased in Saos-2 cells when acetazolamide was included in the OM compared with the cells that were stimulated with OM alone (Figure 1A). The absorbance of cetylpyridinium chloride was also considerably decreased by 1.28-fold in Saos-2 cells that were treated with acetazolamide for three days (P = 0.0003), 1.36-fold after six days (P = 0.0008) and 1.26-fold after nine days (P = 0.0001) when compared with cells that were cultured with OM alone (Figure 1B).

Saos-2 cells were incubated with OM in the absence or presence of acetazolamide at concentrations of 10 μM, 100 μM and 1 mM for three, six and nine days. The number of living cells was slightly increased in Saos-2 cells that were cultured in OM compared with the control group in normal medium for three, six and nine days, but there were no significant changes between the two cultures. The proliferation of Saos-2 cells was not significantly different between cells that were incubated with OM in the presence of 100 μM acetazolamide for three days, six days and nine days. When Saos-2 cells were incubated with OM in the presence of 1 mM acetazolamide, cell proliferation was significantly decreased on the third day (P = 0.03), the sixth day (P = 0.023) and the ninth day (P = 0.035) of the incubation when compared with cells that were cultured with OM alone. Therefore, 100 μM was considered to be the highest non-toxic concentration of acetazolamide and was used in the subsequent assays. The results are depicted in Figure 2.

The mRNA level of Runx2 was 8.05-fold (P = 3.57*10-7) higher in the stimulated cells than in cells without stimulation, ALP increased 2.91-fold (P = 0.000929), BSP increased 27.28-fold (P = 5.63*10^-5), OCN increased 2.48-fold (P = 0.04) and OSX increased 1.8-fold (P = 0.002), when Saos-2 cells were incubated in OM for nine days. After nine days of treatment with 100 μM acetazolamide in OM, the expression levels of ALP (P = 8.71*10^-5), BSP (P = 0.001), OCN (P = 0.007) and Runx2 (P = 5.74*10^-5) were significantly decreased in Saos-2 cells compared to their levels in OM alone, except for OSX (P = 0.026). These results are shown in Figure 3.

The transcription (P = 0.007) and translation (P = 0.009) of CA1 in the stimulated cells were significantly affected in a time-dependent manner for nine day's culture compared with the controls. After treatment with acetazolamide, CA1 transcription (P = 0.004) and translation (P = 0.012) were significantly decreased compared with the cells cultured in OM alone. These results are presented in Figure 4.

We genotyped twenty-nine SNPs in the CA1 gene. All SNPs yielded usable genotyping data and the study sample success rate was 99% [See Additional file 1]. The Illumina microarray data has been submitted to NCBI Gene Expression Omnibus (GEO), a public functional genomics data repository supporting MIAME-compliant data submissions. The record was approved and assigned GEO accession number (GSE39428). Differences in the allele and genotype frequencies between cases and controls were compared. Allelic frequency and genotype frequencies of all SNPs were in Hardy-Weinberg equilibrium in controls. SNP at rs34282382 showed significant differences in allelic frequency and genotypic frequency in the AS cohort. SNP at rs7841425 also showed significant differences in allelic frequency and genotypic frequency between AS cases and controls. Following multiple-test correction, rs7841425 still had a significant difference in allelic frequency and genotypic frequency. In addition, SNP at rs7827474 had a significant difference in allelic frequency and genotypic frequency between RA cases and controls. After multiple-test correction, rs7827474 still had a significant difference in allelic frequency and genotypic frequency between RA cases and controls, indicating that the SNP is significantly associated with an increased risk of RA. Other SNPs located in the CA1 gene were not significantly different in allelic or genotypic frequencies between the RA or AS patients and controls. Tables 3 and 4 contain the allele frequencies, genotype frequencies and the unadjusted and adjusted P-values after the statistical analysis of these genotyped SNPs. We also performed multivariate logistic regression analysis using software Plink v1.07 to test the correlations between the SNP and RA and AS. The P values for rs7827474 and rs7841425 after logistic regression are also shown in Tables 3 and 4.

We performed additional genotyping for tag SNP rs725605 in an independent case-control study using the TaqMan method. Allelic frequencies and gene frequencies of the SNP did not deviate from HWE in both cases and the controls. The allele frequency and gene frequency for SNP rs725605 demonstrated statistically significant evidence for association with AS (P = 0.022307, OR (%95 CI) = 1.332797 (1.041573 to approximately 1.705446)). The gene frequency for the SNPs also demonstrated statistically significant evidence for association with AS (P = 0.007731). On the other hand, the SNPs rs725605 did not show significant difference in allelic frequencies (P = 0.367743) and gene frequencies (P = 0.610868) between RA patients and controls.