Original Articles
Copyright ©2009 The WJG Press and Baishideng.
World J Gastroenterol. May 7, 2009; 15(17): 2097-2108
Published online May 7, 2009. doi: 10.3748/wjg.15.2097
Figure 2
Figure 2 An FXR responsive element is expressed in the CSE promoter. A: Analysis of the promoter of the human CSE gene, showing a putative IR-1 site at -699/-685 base pairs upstream of the transcriptional start site ATG; B: Schematic representation of reporter constructs containing four CSE-IR1 elements [pGL3 (CSE-IR1)4X] or the mutated CSE-IR1 (pGL3CSE-IR1mutated); C: HepG2 cells were transfected with pSG5-FXR and pSG5-RXR expression vectors and with the construct containing four copies of the CSE-IR1 [pGL3 (CSE-IR1)4X]. Forty-eight hours after transfection, cells were stimulated with 25 &mgr;mol/L of DCA, LCA, CA, CDCA and 6E-CDCA for 18 h. Luciferase activity is shown as the ratio of luciferase to β-galactosidase activities. aP < 0.05 versus not treated cells; D: Dose-dependent induction of Luciferase activity by 6E-CDCA. aP < 0.05 versus not treated cells; E: Mutagenesis of CSE-IR1 results in a loss of activation by FXR ligands. HepG2 cells were transfected with pSG5-FXR and pSG5-RXR expression vectors and with pGL3 or pGL3 (CSE-IR1)4X or pGL3CSE-IR1mutated. Forty-eight hours after transfection, cells were stimulated with 10 &mgr;mol/L of 6E-CDCA for 18 h. Luciferase activity is show as the ratio of luciferase to β-galactosidase activities. cP < 0.05 versus not stimulated pGL3 transfected cells. eP < 0.05 versus not stimulated pGL3 (CSE-IR1)4X transfected cells; F: Guggulsterone abolished the transactivation of the CSE-IR1 element. HepG2 cells co-transfected with pSG5-FXR and pSG5-RXR expression vectors and with pGL3 (CSE-IR1)4X were stimulated with 50 &mgr;mol/L of guggulsterone alone or in combination with 10 &mgr;mol/L of 6E-CDCA. aP < 0.05 versus not treated cells. gP < 0.05 versus 6E-CDCA stimulated cells. Data represent the mean ± SD of three experiments.