Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells
Figure 4
Inhibition of CN and endogenous CN-NFAT binding blocked Cox-2 gene promoter activation in primary ESC.
(A) ESC isolated from uteri of non-pregnant rats were transiently transfected with a series of luciferase reporter plasmids containing regions of the human Cox-2 promoter starting from −1900 down to −150 upstream of the transcription initiation site. The schematic representation of the proximal 1900 bp region of the human Cox-2 gene promoter, showing the positions of putative transcription factor response elements [22] is embedded. Cell cultures were co-transfected with Renilla plasmids to normalize for transfection efficiency. Transfected cells were treated for 4 h with vehicle (ns, open bars) or PIo (solid bars), and the luciferase activity determined in cell lysates. Transcriptional activity is expressed as the fold increase in luciferase activity above baseline levels from transfected, nonstimulated control cells. (B) ESC transfected with the −274 Cox-2 luciferase reporter construct were pretreated for 1 h with vehicle (open bars) or 200 ng/ml of CsA (solid bars) and treated for 4 h with PIo or 500 nM of Ang II as indicated. Data are presented as in A. (C) Primary stromal cells isolated from uteri of non-pregnant rats were transfected with 800 ng of expression constructs encoding either pEGFP-VIVIT (solid bars) or pEGFP-N1 as control (open bars). Transfected cells were stimulated as before for 4 h with PIo, Ang II, or left untreated (ns), and the luciferase activity determined in cell lysates. Data are presented as in A. (A–C) Results shown are from a representative experiment of three performed, and values are the means ±SD of triplicate determinations. *** P<0.001; ** P<0.01 (ANOVA).
