Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression

Abstract

S-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA(Sec) to the Um34 form. The formation of methylated tRNA(Sec) facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA(Sec) and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA(Sec) hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.

Keywords: Cell Adhesion; Endothelial Cell; Glutathione Peroxidase; Oxidative Stress; S-Adenosylhomocysteine; Selenoprotein; tRNA Methylation.

FIGURE 1.

Inhibition of cellular SAHH activity. A, ADA or siSAHH was used to decrease SAHH activity. Means are significantly different by Student's t test. ***, p < 0.0001 versus control, n = 3–4. Ctrl, control. B, the effects of ADA or siSAHH on SAHH protein expression was examined by Western blotting. Summary densitometry measurements, corrected for actin intensity, are shown below the immunoblot analyses (n = 3–4). *, p < 0.05 C, the effects of ADA or siSAHH on SAHH mRNA were measured by quantitative RT-PCR, using actin as an endogenous control. **, p < 0.001; ***, p < 0.0001 versus control; n = 3–4.

FIGURE 2.

Effect of SAH accumulation on GPx-1. A, GPx-1 activity was measured by an indirect coupled enzymatic assay. Means were significantly different by analysis of variance, followed by post hoc pairwise comparisons (n = 4–5). *, p < 0.05; **, p < 0.005. B, the effect of SAHH inhibition on the expression of GPx-1 was examined by Western blotting. A representative blot (top panel) and summary densitometry measurements, corrected for actin intensity (bottom panel), are shown for 24 and 48 h in the presence and absence of ADA or siSAHH (n = 3). *, p < 0.05; **, p < 0.005. C, mRNA levels from the same experiments were measured by quantitative RT-PCR using actin as an endogenous control. No significant difference was found between treatment groups.

FIGURE 3.

H₂O₂ levels are increased by SAHH inhibition. A, after 24 h of ADA exposure, H₂O₂ release from cells was measured by Amplex Red. The results represent means from three independent experiments that were analyzed by analysis of variance, followed by pairwise post hoc analysis. **, p < 0.005; ***, p < 0.0005 compared with control. B, intracellular H₂O₂ production was detected using the HyPer2 fluorescence ratio. The graph shows the means of three independent experiments, for which fluorescence ratios were measured for 3–5 cells/condition over 13 h. **, p < 0.005 compared with control. C, representative images of Hyper2-transfected cells 24 h following exposure to ADA-containing or control medium are shown.

FIGURE 4.

Adhesion molecules expression is increased by pharmacological or siRNA-mediated suppression of SAHH. A, the effect of 48 h of SAHH inhibition on the expression of ICAM-1 and VCAM-1 was evaluated by Western blotting. Top panel, a representative blot. Bottom panel, relative mean densitometry measurements, corrected for actin, for each protein and treatment condition. Mean densitometry measurements were compared by Student's t test (n = 3–5). *, p < 0.05; **, p < 0.005; ***, p < 0.0005. Ctrl, control. B, gene expression levels from the same experiments were measured by quantitative RT-PCR, using actin gene expression as a control. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

FIGURE 5.

Oxidative stress contributes to endothelial cell activation following SAHH inhibition. A, control cells (Ctrl) or cells exposed to ADA were incubated in the presence or absence of the antioxidant N-acetylcysteine (NAC, 8 mm). Western blot analysis was performed to evaluate ICAM-1 and VCAM-1 protein expression. A representative blot is shown (left panel) as well as mean densitometry measurements, corrected for actin, for each protein and treatment condition (right panel). Densitometry results were analyzed by analysis of variance, followed by pairwise post hoc analysis. *, p < 0.05; **, p < 0.005. B, left panel, cells with overexpression of a control gene (β-Gal) or GPx-1 were incubated in the presence or absence of ADA. Western blotting was used to evaluate ICAM-1 and VCAM-1 expression. Right panel, ICAM-1 and VCAM-1 expression was evaluated in the presence or absence of ADA and GPx-1 knockdown. Mean densitometry measurements, normalized for actin, were analyzed as in A.

FIGURE 6.

Cell surface adhesion of leukocytes is enhanced by ADA exposure. A, flow cytometry was used to detect cell surface expression of ICAM-1 tagged with a fluorescent antibody following exposure to ADA for 48 and 72 h (n = 3). Means were compared at each time point between ADA treatment and no treatment by Student's t test. *, p < 0.05. MFI, mean fluorescence intensity. B, following a static adhesion assay, leukocytes that remained attached to the endothelial monolayer were labeled with a fluorescence-conjugated antibody (CD45) and counted by FACS. Results are shown as means from three independent assays, normalized to the control and analyzed as in A. *, p < 0.05.

FIGURE 7.

tRNA^[Ser]Sec hypomethylation and its effect on selenoproteome expression. A, structures of the mcm⁵U and mcm⁵Um isoforms of the wobble uridine (Um34). B, following aminoacylation with [³H]-serine, tRNA^[Ser]Sec isoforms were separated by reverse phase chromatography. A normal profile is shown in the left panel (control cells), where mcm⁵U elutes earlier than the mcm⁵Um isoform. In the right panel, cells exposed to ADA are lacking the peak corresponding to the later-eluting mcm⁵Um isoform. Note the differences in the y axis scales. cpm, counts per minute. C, duplicates of control cells (Ctrl) or cells exposed to ADA or siRNA treatment were labeled for 24 h with ⁷⁵Se. Proteins were extracted and separated by gel electrophoresis, and labeled selenoproteins were detected using a PhosphorImager. The protein marker sizes (in kilodalton) and selected selenoprotein bands are indicated on the left and right sides of the image, respectively. D, protein extracts were prepared from total cell lysates treated and untreated with ADA, and Western blot analyses were used to detect TrxR1 and TrxR2. A representative blot is shown. Right panel, relative mean densitometry measurements are shown, corrected for actin, for each protein and treatment condition. Mean densitometry measurements were compared by Student's t test (n = 3). *, p < 0.05; **, p < 0.005. E, TrxR1 and TrxR2 mRNA levels were measured in treated and untreated control samples by quantitative RT-PCR using actin as endogenous control.

FIGURE 8.

The effects of excess selenium on TrxR1 and GPx-1 expression. 48 h prior to ADA exposure sodium selenite was added to supplement medium that contained 37.5 nm selenium. Excess selenium concentrations were maintained throughout the subsequent 48-h ADA exposure. A, effect of excess selenium on TrxR enzyme activity and TrxR1 protein expression. Excess selenium had no effect on TrxR1 activity (n = 3) or expression (n = 4) in the absence of ADA. In the presence of ADA, the TrxR1 up-regulation of TrxR1 was not altered by excess selenium. B, effect of excess selenium on GPx-1 activity (n = 4–6) and GPx-1 protein expression (n = 4) in the presence and absence of ADA. Selenium did not significantly alter GPx-1 in the absence of ADA. In the presence of ADA, the suppression of GPx-1 was not decreased by excess selenium. C, effect of excess selenium on ICAM-1 and VCAM-1 protein. Excess selenium did not lessen ADA-induced up-regulation of the ICAM-1 or VCAM-1. For each graph, values that are not significantly different from no treatment or ADA are labeled a or b, respectively.

FIGURE 9.

The role of hypomethylation on endothelial dysfunction and activation. Excess SAH induces hypomethylation stress capable of decreasing the available tRNA^[Ser]Sec isoform mcm⁵Um. Lack of this tRNA^[Ser]Sec isoform decreases the expression of the antioxidant GPx-1 (as well as other selenoproteins). Loss of GPx-1 causes an increase in cellular oxidants, promoting oxidative stress. Oxidant stress contributes to increased expression of adhesion molecules that are responsible for an increased capacity to bind leukocytes, contributing to a proatherogenic environment. Other possible mechanisms (dashed arrow), such as DNA hypomethylation, may also be involved in the up-regulation of adhesion molecules such as ICAM-1 and VCAM-1.