ESTROGEN REGULATION OF RAT UTERINE CREATINE KINASE B

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Maturation of the rat uterus is a dramatic example of sex steroid action. The prepubertal estrogen surge, or exogenous estrogen, causes profound remodeling of the immature organ. One of the earliest biochemical events in this process is a rapid but transient increase in creatine kinase B (CKB) synthesis in both the endometrium and myometrium. Little is known of the processes active in the estrogen regulation of this intracellular enzyme as models for estrogen regulation are primarily derived from studies on secretory proteins in oviparous vertebrates. Induction of these products show a much slower response to estrogen, and they are typically the principal products of specialized cell types. The variant features of creatine kinase B modulation suggest that alternative regulatory strategies may be active in its regulation by estrogen; providing a novel model in which our long term objective, understanding the molecular bases estrogen action can be investigated. Two aspects will be addressed in this proposal: 1. How are levels of uterine CKB mRNA altered? Levels of CKB mRNA increase within minutes of estrogen administration, peak at 3 hours and decline towards basal levels within 6 hours. Inhibitor data indicate that the initial increase is due to enhanced transcription, yet the subsequent decrease occurs during a period when general protein synthesis is increasing rapidly. We propose, and will test, the hypothesis that estrogen stimulates synthesis of an RN'ase leading to the selective degradation of CKB mRNA. 2. What is the mechanism by which estrogen stimulates CKB expression? Estrogen response elements are found in several estrogen regulated genes, but no perfect copies of these motifs are found in the CKB gene. These data suggest that different sequences, or multiple imperfect copies of the consensus motif, mediate receptor activation of CKB transcription. The alternatives will be tested by transfection studies in estrogen sensitive cell lines. Segments from the CKB gene will be evaluated for the ability to confer estrogen responsiveness on a chloramphenicol acetyl transferase reporter sequence. Further analysis of positive sequences will include in-vitro studies of receptor binding.