The long-term goals of this application are to characterize the mechanisms by which serotonin and progesterone regulate the production of interleukin-1 in the myometrial smooth muscle cells of the uterus. Preliminary studies conducted by the applicant demonstrate that depletion of serotonin from myometrial cell cultures results in a significant decrease in IL-1alpha mRNA. Conversely, replenishment of the indoleamine results in an induction of IL-1alpha mRNA. Selective serotonin receptor antagonists are able to block this induction while selective agonists fully mimic the effect of the native hormone. Nuclear run-on analysis demonstrates that the increase in steady state levels of IL-1alpha mRNA are accompanied by an increase in IL-1alpha transcription. Similarly, the addition of cycloheximide has neither a positive nor negative effect on induction, consistent with a direct effect on transcription as a major regulator of levels of IL-1alpha mRNA. The inductive effect of serotonin can be mimicked by phorbol esters, indicating the possible involvement of protein kinase C; regardless of the mode of induction, however, increases in IL-1alpha mRNA can be completely blocked by the addition of progesterone or glucocorticoids. We propose to isolate the 5'-flanking, and hence, putative promoter region of the rat IL-1alpha gene. Following sequencing, a detailed deletion and mutation analysis of this region will be conducted utilizing the promoter ligated into a reporter gene construct and transiently transfected into primary cultures of rat myometrial smooth muscle cells. These studies will establish the specific regions of the promoter that are necessary for serotonin and progesterone responsiveness. Similarly, studies will be conducted to elucidate the second messenger pathways utilized in these cells to accomplish the reciprocal regulation of IL-Ia mRNA by serotonin and progesterone. Included will be studies focused on elucidating the ability of IL-1 to auto-regulate levels of its own mRNA. Finally, we will investigate the production and regulation of uterine IL-Ia in vivo, using in situ hybridization and immunohistochemical techniques during pregnancy and post-partum involution. These studies should not only shed new light on this newly discovered mode of regulation of IL-1, but should also help us to better understand the role of IL- 1alpha in a number of important biological and pathological phenomenon that occur in the uterus during pregnancy and involution.
| Melendez, J A; Vinci, J M; Jeffrey, J J et al. (2001) Localization and regulation of IL-1alpha in rat myometrium during late pregnancy and the postpartum period. Am J Physiol Regul Integr Comp Physiol 280:R879-88 |
| Lan, L; Vinci, J M; Melendez, J A et al. (1999) Progesterone mediates decreases in uterine smooth muscle cell interleukin-1alpha by a mechanism involving decreased stability of IL-1alpha mRNA. Mol Cell Endocrinol 155:123-33 |
| Huang, T T; Vinci, J M; Lan, L et al. (1999) Serotonin-inducible transcription of interleukin-1alpha in uterine smooth muscle cells requires an AP-1 site: cloning and partial characterization of the rat IL-1alpha promoter. Mol Cell Endocrinol 152:21-35 |
| Dumin, J; Wilcox, B D; Otterness, I et al. (1998) Serotonin-mediated production of interstitial collagenase by uterine smooth muscle cells requires interleukin-1alpha, but not interleukin-1beta. J Biol Chem 273:25488-94 |
