description) The research outlined in this application will investigate the signal transduction pathway by which Src tyrosine kinase modulates luteinizing hormone (LH)-stimulated steroid secretion and phosphodiesterase (PDE) activity using porcine thecal interstitial cells as a model.
The first aim will be to determine whether activation of Src tyrosine kinase activates a Ras-phosphatidylinositol-3-kinase (PI3-kinase)-protein kinase C zeta (PKC) signal transduction pathway in thecal cells. Porcine thecal cells will be challenged with LH or LH plus platelet derived growth factor, a known activator of Src. Src, Ras PI3-kinase and PKC, PDE activities, and progesterone secretion will be determined. In a second approach, porcine thecal cells will be infected using a retroviral gene transfer system with a vector encoding a temperature sensitive Src. Cells will be grown at the Src active and inactive temperatures, challenged with LH, and the above parameters determined.
The second aim will be to investigate the effects of constitutive activation and inhibition of Ras on LH-stimulated steroid secretion and PDE activity. The effects of activation of Ras will be investigated by infecting porcine thecal cells by retroviral gene transfer with expression vectors encoding a dominant negative Src, a constitutively active Ras, both vectors or empty vectors (to serve as controls). LH-responsiveness will be determined by cAMP and progesterone accumulation in culture media and PDE activity in response to a 24 hour challenge with LH. PI3-kinase and PKC activities will be measured in order to determine whether RAS activates these signal transduction components in porcine thecal cells. The effects of specific inhibition of Ras will be determined by infecting thecal cells with a temperature sensitive Src, a dominant negative Ras, or both. Again, LH-responsiveness, PDE, and PI3-kinase activities will be determined. These studies will provide insight into whether the Src-stimulated inhibition of gonadal steroidogenic cells to LH-stimulation is via a Ras-PI3-kinase-PKC signal transduction pathway. They will also shed light as to whether Src is modulating PDE activity by this pathway. Finally, as the components of this pathway are ubiquitous, the results obtained will provide new information regarding the role of Ras, PI3-kinase and PKC in regulation of gonadotropin responsiveness in steroidogenic cells. The results from these studies will be used to form the basis for future research into crosstalk between different signal transduction pathways and the regulation of steroid secretion, differentiation and growth in gonadal steroidogenic cells.
