730 Cyclic GMP Kinase II (CKII) Inhibits NHE3 by Affecting Trafficking via Phosphorylating NHE3 At Three Sites: Identification of a Multifunctional Phosphorylation Site

was measured by luciferase reporter activity assays after transiently transfecting Caco-2 cells with DRA promoter construct (-1183/+144). Treatment of Caco-2 cells with 10 μM concentration of ATRA significantly induced DRA mRNA levels within 8 h of incubation, which increased to ~3 fold by 24 h. A concomitant increase (140 ± 5 % of control P ,0.001) in DRA protein expression was also observed. In addition, ATRA treatment significantly increased DRA promoter activity (258.8 ± 3.4 % of control, P,0.001) indicating that ATRA effects are transcriptionally mediated. ATRA is known to mediate its effects via specific retinoic acid receptors (RAR's) α, β and γ. A remarkable increase (~11 fold) in mRNA levels of RAR-β receptor subtype were found in response to ATRA treatment with no change in levels of RAR'-α and γ. Treatment of cells with RAR-β agonist (CH-55, 1μM) mimicked ATRA effects on DRA promoter. RAR-β antagonist (LE-135, 1μM) blocked the stimulatory effects of ATRA on DRA promoter activity. Since, DRA expression is known to be modulated by hepatic nuclear factors (HNF's), the potential mediation of ATRA effects via HNFs was examined. A significant increase in HNF-1β mRNA levels (but not HNF-1α or HNF4) in response to ATRA treatment was observed. The ability of ATRA to induce DRA expression was blocked by silencing HNF-1β using siRNA. In summary, ATRA increased DRA expression via RAR-β through HNF-1β activation. We speculate that ATRA may act as a potential therapeutic tool under inflammatory conditions where DRA expression is altered (Supported by NIDDK and Dept. of Veteran Affairs).