T1730 Curcumin Down-Regulates Niemann-Pick C1-Like 1 Expression in Intestinal Epithelial Cells

Abstract

Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-HT) availability in the gut via reuptake of released serotonin. A decrease in SERT function and expression has been implicated in inflammatory bowel diseases and irritable colon. Hence, SERT is a potential pharmacological target in gastrointestinal disorders, however, very little is known regarding regulation of SERT in the human intestine. In this regard, EGF, a potent growth factor, is known to influence intestinal electrolyte and glucose transport processes and has protective effects on mucosa in models of colitis. Whether EGF regulates SERT function and expression in the human intestine is not known. The present studies were, therefore, designed to examine the regulation of SERT by EGF utilizing Caco2 cells grown on Transwell inserts as an In Vitro model. SERT activity was measured as Na+ and Cldependent 3[H] 5-HT uptake in fully differentiated cell monolayers. SERT mRNA abundance was determined by real time QRT-PCR. SERT promoter activity was assessed by luciferase assay in cells transiently transfected with promoter constructs. Treatment of Caco-2 cells with EGF from basolateral side (5-10ng/ml) for 24h significantly stimulated SERT activity (~50%, P<0.005). However, acute treatment with EGF (1-2 h) did not affect SERT function in Caco-2 cells. Corresponding with increased function at 24h, EGF treatment resulted in up-regulation of SERT mRNA levels (~1.8 fold) compared to control. For studying the regulation of SERT gene by EGF, a 873 bp (-871/+2) SERT promoter fragment was cloned upstream to the luciferase reporter gene and was found to be highly active when transfected in Caco2 cells. EGF treatment (10 ng/ml) for 8-24h resulted in significant stimulation in SERT promoter activity (~50-60%; P<0.05). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1nM) blocked the stimulatory effects of EGF on SERT promoter. Studies utilizing progressive deletion constructs of the SERT promoter indicated that the putative EGF responsive elements are in the -672/+472 region of the promoter. Conclusion: Our findings demonstrate transcriptional regulation of SERT by EGF via EGFR. These data define novel mechanisms of modulation of SERT function and expression in intestinal epithelium that may be relevant to therapeutic benefits of EGF in diarrheal diseases associated with altered 5-HT availability. [Supported by NIDDK/Dept of Veteran Affairs].