ATF3 gene induction by homocysteine involved activation of JNK/SAPK signaling pathway.

(A) HUVECs were treated with 3 mmol/L homocysteine for the indicated time, and their whole-cell extracts (40 μg) were subjected to Western blot with anti-JNK/SAPK (JNK), anti-phospho JNK/SAPK (JNK-○P), anti-p38 (p38), or anti-phospho p38 (p38-○P) antibody. (B) HUVECs were transfected with 2 μg of pLuc-1850 reporter plasmid and 1 or 2 μg of plasmids for dominant negative (dn) MKK4, dnMKK7, or both. The cells were incubated with 3 mmol/L homocysteine for 4 hours and then without homocysteine for 16 hours; then promoter activity was assayed as described in “Materials and methods.” Relative luciferase activity is expressed in arbitrary units; the results are the average of 3 independent experiments with an SE bar. Fold induction is the ratio of homocysteine-stimulated activity (solid columns) to that without homocysteine (open columns). Basic and pEGFP represent the reporter plasmid without ATF3 gene promoter and the empty vector for dnMKK4 and 7, respectively. Fold induction was compared with that of the control pEGFP vector; *P < .05, †P < .01, n = 3. (C,D) HUVECs were transfected with plasmids for the empty vector (i, vi), dnMKK4 (ii, vii), dnMKK7 (iii, viii), or both (iv, ix) and then stimulated with 3 mmol/L homocysteine. ATF3 expression and activation of JNK/SAPK were detected by immunostaining with anti-ATF3 (panel C) and anti-phospho JNK/SAPK (panel D) antibodies in the transfected cells, which were detected by green fluorescent protein (GFP) fluorescence (indicated by arrows in i-v). Cells were transfected with the empty vector but not treated by homocysteine (v, x). In the lower panels, ATF3-positive cells (panel C) or JNK-○P–positive cells (panel D) are expressed as a percentage of transfected GFP-positive cells. The data represent the means of 3 independent experiments with SE bars. Statistically significant inhibition from the control pEGFP vector; *P < .05, †P < .01, ‡P < .001, n = 3.