FIGURE 5.

Ethanol enhances RANTES promoter activity via its two proximal HRE sites (HRE-1 and HRE-2) and its AP-1 site. A, Schematic of the human RANTES (975 bp) promoter region, containing three HRE sites and one AP-1 site. The mutations of specific nucleotides in the HRE and AP-1 sites of the RANTES promoter are denoted by asterisks (*). B, Effect of HREs and AP-1 mutations on RANTES (975 bp) promoter activity in HMEC-1 in response to ethanol (100 mM) treatment for 4 h. Mutant site promoters were constructed in the context of the full-length (975 bp) RANTES promoter. The RANTES HRE-M3 luciferase construct has a mutation in the HRE at nt −720 to −717. The RANTES HRE-M2 and HRE-M1 luciferase constructs have mutations in their HREs at nt −32 to −29 and nt −22 to −19, respectively. The RANTES AP-1-M luciferase construct has a mutation in the AP-1 binding site at nt −250 to −244. C, Effect of c-Jun, c-Fos, JunD, and JunB expression plasmids on RANTES promoter activity. HMEC-1 was transiently transfected with 1 μg of each expression plasmid for c-Jun, c-Fos, c-Jun and c-Fos, JunB, JunD, c-Jun and JunD, and a combination of c-Jun, c-Fos, and JunD. D, Effect of ethanol (100 mM) on AP-1 promoter luciferase activity in cells preincubated with the inhibitors GF109203X (PKC inhibitor), PP1 (Src kinase inhibitor), SB203580, or SP600125 for 30 min before ethanol treatment. Luciferase activity was normalized as described above. The data shown represent three independent experiments (means ± SD). E, ELISA of RANTES in plasma isolated from ethanol-fed mice expressed as fold-changes compared with control. Where indicated, ethanol-fed c-Jun^flox/flox mice were compared with ethanol-fed wt mice. The basal levels of RANTES in the plasma of control wt mice were 44.6 ± 6.1 ng/ml. The data represent plasma from eight mice in each category, and the assay was performed in duplicate (mean ± SD). Values of p are denoted as follows: ***, p < 0.001; **, p < 0.01; *, p < 0.05; and ns, p > 0.05.