1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine preosteoblastic MC3T3-E1 cells by direct regulation of cystathionine β-synthase

Abstract

High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY owing to its negative impact on collagen maturation. HCY is cleared by cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine preosteoblasts treated with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)(2) D(3) . Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)(2) D(3) treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine, and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)(2) D(3) -treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knockout osteoblasts, in which upregulation of cbs in response to 1,25(OH)(2) D(3) was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D(3) levels] and HCY levels. In conclusion, this study showed that cbs is a primary 1,25(OH)(2) D(3) target gene which renders HCY metabolism responsive to 1,25(OH)(2) D(3).

Fig. 1

Transsulfuration pathway. CBS, cystathionine β-synthase; CTH, cystathionine gamma-lyase the cofactor is vitamin B₆. Mutations in cbs or cth hamper the transsulfuration pathway and cause hyperhomocysteinemia. Also a failure in remethylation (dependent on folate, vitamin B₁₂) or deficiencies in vitamins B₆, B₁₂, and folate cause hyperhomocysteinemia, which is associated with impaired cross-link formation, connective tissue abnormalities and increased risk of osteoporosis.

Fig. 2

Transcript levels of cbs are up-regulated by 1,25(OH)₂D₃ in murine MC3T3-E1 and primary osteoblasts. (A) Transcript levels were determined by qRT-PCR at different time points after treatment with 1,25(OH)₂D₃ (10⁻⁸ M). Each data point represents the ratio of cbs levels normalized over β-actin, relative to the normalized cbs levels of vehicle-treated cells (1 h). The data are the mean ± SEM of four independent experiments. The overall up-regulation of cbs expression by 1,25(OH)₂D₃ was found to be significant according to ANOVA (p< .05). (B) Cbs expression measured by qRT-PCR in primary osteoblasts treated for 24 h with vehicle or 1,25(OH)₂D₃ (10⁻⁸ M). Osteoblasts originate from VDR wild-type (VDR wt; n= 6) and VDR knock-out (VDR ko; n= 5) mice. Bars represent the mean ± SEM of each group. The expression of cbs in 1,25(OH)₂D₃-treated VDR wt osteoblasts is significantly different from the other groups displayed, according to ANOVA followed by a Bonferroni multiple-comparison test (^a p< .05)

Fig. 3

Transcriptional activation of the cbs gene by VDR bound to a classical DR3-type VDRE. (A) ChIP-on-chip was used to determine transcription factor binding. The genomic location for the cbs gene is shown for chromosome 17 with genomic base pairs given in kilobases (k). Increased binding is indicated by enhanced read out (peaks) at the murine DNA sequence at chromosome 17 between nucleotide 31,359,000 and 31,361,000. The assay was performed on chromatin from vehicle- (Veh/Input) and 1,25(OH)₂D₃- (1.25/Input) treated MC3T3-E1 cells with antibodies against VDR, RXR, and H4-Ac. Data are displayed as log2 ratios (log2 R). Next to the murine DNA sequence, the results of classical ChIP experiments confirm the ChIP-on-chip data. Bars are the mean ± SEM of three independent experiments. (^a p< .05 according to Student’s t-test). (B) MC3T3-E1 cells were transfected with empty pGL3basic (neg) or pGL3basic containing the wild-type (wt) or mutated (mutDR3.1 or mutDR3.2) fragment of the second cbs intron (murine chromosome 17 between nucleotide 31,359,592 and 31,361,047) and with a β-galactosidase-expressing construct. After 24 h, cells were treated with 1,25(OH)₂D₃ (10⁻⁸ M) or vehicle and after another 24 h luciferase and β-galactosidase were measured. Luciferase activities were normalized to β-galactosidase activities. RLU, relative luciferase units. Bars are the mean ± SEM of at least three independent experiments. (C) Mutated fragments were obtained by substitution of 4 nucleotides (GGGTTG to ATACTG) within the 3′ hexamer of the VDRE (mutDR3.1) and the 5′ hexamer of the VDRE (AGTTCA to TAACCA (mutDR3.2). The relative luciferase activity (RLU) in 1,25(OH)₂D₃-treated cells transfected with the wt construct is significantly different from the other measurements displayed, according to ANOVA followed by a Bonferroni multiple-comparison test (^a p< .05).

Fig. 4

(A) Western blot analysis of CBS expression on total lysates of vehicle and 1,25(OH)₂D₃-treated murine pre-osteoblasts (MC3T3-E1) and human osteosarcoma cells (SaOS-2) at different time points, β-actin was used as loading control. (B) The enzymatic activity of CBS is depicted at 0 h and after 48 h and 72 h in vehicle and 1,25(OH)₂D₃-treated cells. The mean ± SEM from 6 independent experiments is shown. The overall changes in CBS enzymatic activity by 1,25(OH)₂D₃ were found to be significant according to ANOVA (p< .05).

Fig. 5

Changes in metabolite composition in 1,25(OH)₂D₃-treated MC3T3-E1 pre-osteoblasts. The concentrations of (A) HCY, (B) cystathionine, (C) cysteine and (D) GSH are depicted at 0 h and after 48 h and 72 h in vehicle and 1,25(OH)₂D₃-treated cells. Metabolites were measured in cell lysates. Each data point represents the mean ± SEM of three or four duplicate measurements. The overall changes in HCY, cystathionine, cysteine and GSH levels by 1,25(OH)₂D₃ were found to be significant according to ANOVA (p< .05).

Fig. 6

Changes of amino acid content in growth medium of vehicle or 1,25(OH)₂D₃-treated cells. The concentrations of (A) HCY, (B) cystathionine and (C) cysteine are depicted at baseline and after 48 h and 72 h of vehicle or 1,25(OH)₂D₃ treatment. Measurements were normalized by subtraction of the amino acid concentration present in fresh medium. Each data point represents the mean ± SEM of four duplicate measurements. The overall decrease of HCY levels and increase of cystathionine and cysteine levels by 1,25(OH)₂D₃ were found to be significant according to ANOVA (p< .05).

Fig. 7

Relation between 25(OH)D₃ and homocysteine obtained from a regression model on 1264 subjects with creatinine levels between 50 and 150 μM. Dotted lines represent pointwise 95% confidence intervals (CI) for the mean relation which is depicted for an “average” subject, i.e. having a creatinine level of 100 μM and being 75 years of age.