Figure 8.

Effects of dominant-negative CREB and FOS/FRA-2 proteins on Star promoter activity in predifferentiated, graafian, and luteal granulosa cells. Granulosa cells were collected from ovaries at three phases of follicular development: prepubertal animals treated with E₂ (A and B), known to origin from tertiary follicles and represent steroidogenically predifferentiated cells (57), granulosa cells from animals treated with eCG for 48 h (C, graafian cells), and luteinizing granulosa cells (D–G) expressed from animals treated with eCG and hCG, as described in Materials and Methods. The cells were electroporation transfected with the indicated promoter-CAT constructs and expression plasmids, followed by 6 h incubations without or with FSH (100 ng/ml). When indicated the cells received high-dose LH (500 ng/ml). A and B, Effect of FSH and dominant-negative mutant proteins ACREB or AFOS on expression of −96/+6Star-CAT (−96Star) in predifferentiated cells. CAT activity was determined using 5 μg protein for a 1-h assay. The activity values (mean ± sd) are presented relative to the activity of −96Star in FSH treated cells. Activity levels were statistically significant when compared with the respective values obtained for the −96Star construct: a, P < 0.005; b, P < 0.001; c, P > 0.1. C1, Effect of LH and ACREB on −96/+6Star-CAT expression in graafian granulosa cells. C2, Western blot analyses of graafian granulosa cells exposed to 500 ng/ml LH for 6 h. Antisera to STAR was used to determine the antigen level in the treated cells (10 μg/lane). D, Star promoter trimming and lack of LH effect on activity of −123Star and −96Star CAT constructs in luteinizing granulosa cells; 10 h after transfection, the cells were treated with 500 ng/ml LH for 6 h. CAT activity was determined using 20 μg protein for a 7-h assay. The activity values (mean ± sd) are presented relative to the activity of −123Star in untreated cells. a, P < 0.005; b, P < 0.01; c, P > 0.1. E1 and E2, Western blot analyses of luteinizing granulosa cells exposed to LH for either 6 h (E₁) or 15 min (E₂). Antisera to STAR, CREB, and phospho-CREB (P-CREB) were used to determine the antigen levels in the treated cells (10 μg/lane). F and G, FOS-related protein, but not CREB, is required for activation of the −123Star construct in luteinizing granulosa cells. The latter cells were transfected with the promoter gene and plasmids carrying dominant-negative mutant proteins ACREB (F) or AFOS (G). CAT activity was determined using 20 μg protein for a 7-h assay. Activity values (mean ± sd) are presented relative to the activity of −123Star. Activity levels were statistically significant when compared with the respective values obtained for the −123Star construct. a, P < 0.005; c, P > 0.1.