The protein kinase C system acts through the early growth response protein 1 to increase LHβ gene expression in synergy with steroidogenic factor-1

Expression of the LH beta gene has been shown to be modulated by both the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1, Egr-1. It is also well known that LH beta mRNA levels are increased after hormonal activation of the protein kinase C (PKC) signaling system, for example by GnRH; however, the mechanisms by which the PKC system exerts this effect has not been fully characterized. By transient transfection of the GH(3) cell line, we demonstrate that activation of the PKC system with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases activity of region -207/+5 of the rat LH beta gene promoter (similar to 2-fold) and markedly augments SF-1-induced stimulation (95-fold in the presence of both factors vs. 13-fold for SF-1 alone). Mutation of the two previously identified Egr-1 sites not only prevents Egr-1 effects on the LH beta gene promoter, but also eliminates the synergistic response to PMA and SF-1 together, findings that were confirmed in a longer construct spanning region -797/+5. In the gonadotrope-derived cell line, alpha T3-1, these mutations eliminate the GnRH responsiveness of the -207/+5 LH beta promoter construct. We next show that PMA treatment (GH, and alpha T3-1 cells) or GnRH treatment (alpha T3-1 cells) induces expression of Egr-1, as detected by Egr-1 interaction with Egr-1 DNA-binding sites in the rat LH beta gene promoter sequence. Furthermore, we demonstrate that PMA increases steady-state Egr-1 mRNA levels via increased Egr-1 transcription. We conclude that PMA-induced stimulation of LH beta gene expression is achieved, at least in part, by induction of Egr-1 expression.