Figure 1.

The ability of U-STAT3 to regulate the RANTES promoter depends on a κB element. (A) Western and Northern analyses for STAT3 and RANTES expression in hTERT-HME1-derived cells. The cells were infected with retroviral constructs and stable pools were selected with G418. (C) hTERT-HME1 control cells; (WT) hTERT-HME1 cells expressing a high level of wild-type STAT3; (YF) hTERT-HME1 cells expressing a high level of Y705F-STAT3. Total cell lysates and total RNAs were analyzed. (B) Basal transcriptional activity of the human RANTES promoter in hTERT-HME1 cells. Luciferase constructs containing 5′- or 3′-deletions between bases −974 and −1 of the promoter were cotransfected with the pCH110 control plasmid and the cells were harvested 48 h later. The luciferase activity in each cell extract was normalized to the level of β-galactosidase activity (from pCH110) in the same extract. Values are means of triplicate determinations, and the bars show one standard error of the mean. (C) Inducible activity of human RANTES promoter fragments in YF cells. The reporter constructs were cotransfected with pCH110. The activities shown are relative to the activity of each fragment in hTERT-HME1 control cells. Values are means of triplicate determinations, and the bars show one standard error of the mean. (D) Inducible activity of promoter mutations in YF cells. The reporter constructs, containing mutations of individual promoter elements (marked by ×) of the 220-base-pair promoter fragment were transfected into the cells. Luciferase activities were determined and calculated relative to the values obtained in control cells as in C. (E) Y705F-STAT3 and p65 cooperate to drive the RANTES promoter. hTERT-HME1 cells were cotransfected with the pGL2-220 plasmid, in which the RANTES −1 to −220 promoter fragment drives luciferase expression, and pCH110, with or without pcDNA3.1-Y705F-STAT3 or pcDNA3.1-p65, expression plasmids for Y705F-STAT3 and p65, respectively. The cells, harvested 48 h later, were analyzed for luciferase activities, as described above. The reporter activities were normalized to activities in cells without cotransfection of p65 or Y705F-STAT3. Values are means of triplicate determinations, and the bars show one standard error of the mean. (F) U-STAT3 and p65 cooperate to activate the RANTES gene. hTERT-HME1 cells were transfected transiently with pcDNA3.1-p65 and/or pcDNA3.1-STAT3. After 48 h, total RNAs were isolated and analyzed by the Northern method.