Dr. Stanley has investigated the mechanism of insulin- and growth factor-activated gene transcription. Using a model system consisting of the prolactin promoter and pituitary tumor-derived GH4 cells, he identified a multifactor response element in the prolactin promoter that is a critical regulator of both basal and insulin/growth factor stimulated prolactin production. He later discovered that the transcription factor Elk-1 is the mediator of these responses with the aid of the accessory factor C/EBPa with which it interacts both physically and functionally. Current studies will determine how insulin/growth factor signaling events modify these factors and how that leads to increased gene transcription. Various inhibitor, gene interference, and immunoaffinity techniques will be used to identify signaling molecules important for insulin vs. growth factor activation. A genetic approach using mutagenesis of the insulin signaling pathway in GH4 cells is also being used to identify novel insulin signaling molecules. The functional domain(s) of Elk-1 will be defined by deletion analysis. The phosphorylation site(s) necessary for regulated transcription will be determined by mass spectrometry. The kinases and phosphatases that act at these sites will then be isolated. Further, it is clear that Elk-1 interacts with other transcription factors/transcriptional co-activators/co-repressors to alter gene expression. These transcription factors will be identified using two-hybrid analysis in yeast, affinity chromatography and cDNA expression libraries. Src affects insulin-increased prolactin gene expression in a complex fashion that is dependent on activation of PI 3-kinase. Experiments to explain this demonstrated that Src activated Rho. The activation of Rho led to alteration of the actin cytoskeleton and activation of PI 3-kinase. Future studies will determine how Src-induced alteration of the actin cytoskeleton activates PI 3-kinase signaling and how this interrelates to insulin signaling.

Recently, the Stanley laboratory initiated studies to determine how insulin and oxidative stress interact of regulate the expression of plasminogen activator inhibitor-1. They defined the insulin response element of the plasminogen activator inhibitor 1 promoter and determined that a Forkhead-related transcription factor mediated the effects of insulin at this element. They also determined that oxidative stress acted by increasing the phorphorylation of c-Jun. This led to increased plasminogen activator inhibitor 1 promoter activity through an AP-1 response element that was adjacent to the insulin response element. The response to oxidative stress was additive with that of insulin. Current studies focus on identifying the Forkhead-related transcription factor that mediates the effect of insulin. The activation of this factor by insulin (e.g. phosphorylation, de novo production, etc) can be determined. Interactions between this factor and AP-1 can also be examined. The insulin-signaling pathway to this factor will also be investigated. Finally, potential mediators of the stress response such as proinflammatory cytokines will be investigated for their effect on plasminogen activator inhibitor 1 production.