EVIDENCE THAT FUNCTIONAL INTERACTIONS OF CREB AND SF-1 MEDIATE HORMONE-REGULATED EXPRESSION OF THE AROMATASE GENE IN GRANULOSA-CELLS AND CONSTITUTIVE EXPRESSION IN R2C CELLS

The proximal promoter of the rat aromatase CYP19 gene contains two fun ctional domains that can confer hormone/cAMP inducibility in primary c ultures of rat granulosa cells and constitutive expression in R2C Leyd ig cells. Region A contains a hexameric sequence that binds steroidoge nic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding facto rs X and Y had overlapping functions with CREB, and to determine if th e CREB and SF-I binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameri c SF-1 binding site were generated. Mutations within the CRE showed th at CREB but not factors X and Y mediated cAMP-dependent activity of ch imeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the conve rse) resulted in a loss of approximately 50% cAMP-dependent CAT activi ty. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-depend ent CAT activity. When these same constructs where transfected into R2 C Leydig cells, mutation of either the CREB or SF-1 binding sites resu lted in a greater than 90% loss of CAT activity. Western blot and immu nocytochemistry analyses revealed that the amount of phosphorylated CR EB increased in response to hormone/cAMP in granulosa cells and was hi gh in R2C Leydig cells, coinciding with expression of the transgenes a nd endogenous aromatase mRNA in each cell type. Therefore, in both cel l types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cel ls, whereas they interact synergistically to confer high basal transac tivation in R2C Leydig cells. Taken together, the results indicated th at the molecular mechanisms or pathways that activate CREE, SF-1 or th eir interaction are different in granulosa cells and R2C cells. (C) 19 97 Elsevier Science Ltd.