Figure 7.
The two conserved ATF sites in the mkp1 promoter contribute to basal promoter activity and are required for promoter induction after NGF withdrawal in sympathetic neurons. A, A 1 kb DNA fragment containing the rat mkp1 promoter was cloned upstream of the Firefly luciferase gene in pGL3-basic. The two conserved ATF sites were then mutated using the QuikChange IIXL Site-Directed Mutagenesis Kit. The wild-type and mutant mkp1-LUC constructs (10 ng/μl) were microinjected into sympathetic neurons together with the Renilla luciferase construct pRL-TK (5 ng/μl). The injected cells were then refed with medium containing or lacking NGF, as indicated. After 16 h, a dual luciferase assay was performed and normalized activity was calculated relative to +NGF (gray bars). Statistical significance was calculated relative to +NGF (gray bars) for each construct. The data shown represents the mean of 4 experiments ± SEM. B, Sympathetic neurons were microinjected with wild-type or the 2xATF mutant mkp1-LUC (20 ng/μl), pRL-TK(5 ng/μl), and wild-type or kinase-dead MLK3 expression vectors (20 ng/μl) or pcDNA3 as a control. Cells were maintained in medium containing NGF for 16 h and then luciferase activity was measured. Luciferase activity was calculated relative to the level for mkp1-LUC coinjected with pcDNA3 (control), which was set as 1. Statistical significance was calculated relative to pcDNA3 (control) for each construct. The mean of three independent experiments ± SEM is shown. C, Sympathetic neurons were comicroinjected with mkp1-LUC (20 ng/μl) and pRL-TK(5 ng/μl) together with wtMLK3 or kdMLK3 (20 ng/μl) in the presence and absence of mkp1 siRNA (3 μm). Cells were maintained in medium containing NGF for 16 h and then luciferase activity was measured. Luciferase activity was calculated relative to the level for mkp1-LUC coinjected with the kdMLK3 construct and NTC siRNA. The mean of three independent experiments ± SEM is shown. D, Expression of the JIP-1 JBD reduces induction of an mkp1 reporter construct after NGF withdrawal. Sympathetic neurons were microinjected with mkp1-LUC (20 ng/μl), pRL-TK (5 ng/μl), and pcDNA3 or pcDJBD (50 ng/μl). Cells were maintained +NGF or −NGF for 16 h and then luciferase activity was measured. Luciferase activity was calculated relative to the level for mkp1-LUC injected with pcDNA3 +NGF, which was set as 1. Statistical significance was calculated relative to +NGF (gray bars) for each construct. The mean of three independent experiments ± SEM is shown. E, Sympathetic neurons were cultured for 6 d in vitro and then microinjected with mkp1-LUC (20 ng/μl), pRL-TK (5 ng/μl), and the c-Jun (H-79) or ATF2 (C-19) antibodies or rabbit Ig as a control (1 μg/μl) as indicated. Following injection, the cells were maintained in medium containing NGF (+) or anti-NGF antibody (−) for 16 h before luciferase activity was measured. For each experiment, the level of normalized luciferase activity for control IgG +NGF was set as 1 and other values were calculated relative to this. Statistical significance was calculated relative to +NGF (gray bars) for each antibody coinjection. The mean ± SEM. for five independent experiments is shown. Student's t test was used to determine whether inductions were significant: #p < 0.001; *p < 0.01; +p < 0.1, NS (not significant) p > 0.1.