Figure 4. EDN3 is associated with decreased levels of RA receptor mRNA and RA metabolic enzyme mRNA.

p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Relative Rarb (A), Rarg (B), Raldh2 (C), and Cyp26a1 (D) mRNA levels were measured by quantitative RT-PCR. Levels are normalized to β-actin and the -RA/-EDN3 condition was standardized to a value of 1. Solid lines denote a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No RA” and “RA”. EDN3 was associated with a net decrease in Rarb (A) and Rarg (B) mRNA transcripts in the presence of exogenous RA. In the absence of exogenous RA, EDN3 did not have an effect on RA receptor levels. (C) EDN3 was also associated with a decrease in Raldh2 abundance, but not when RA was present. (D) EDN3 also reduced Cyp26a1 mRNA levels, but only in the presence of RA. Consistent with autoregulation, RA treatment was responsible for a reduction in Raldh2 levels (C) and an increase in Cyp26a1 (D), in the absence of exogenous EDN3 However, associations of RA with Raldh2 and Cyp26a1 levels were lost when EDN3 was simultaneously present. There was a statistically significant interaction (ⓧ; p<0.001) between EDN3 and RA in their effect on Cyp26a1 levels at 3 days.