Medical Student Research Fellowship for Summer 2003

Mentor: Carole R. Mendelson, Ph.D.
Department: Biochemistry
Room number: K3.106
Mail Code: 9038
Phone number: 214-648-2944
E-mail: cmende@biochem.swmed.edu

Two Projects

Project I title: Mechanisms in Tissue-Specific Regulation of Estrogen Biosynthesis in Humans

Human subjects IRB approved project number (where applicable): 1076 02000

Animal subjects IRB approved project number (where applicable): 0057-01-03-3

Project Type (patient-based research, animal-based research, or basic research; this characterization is only to permit a general classification for grouping similar types of projects) Basic

Brief Description of Project:
Approximately 450,000 babies are born prematurely in the U.S. each year. Consequences of preterm birth range from death and/or lifelong impairment to chronic lung disease. Despite extensive research, surprisingly little is known regarding the molecular mechanisms that lead to the initiation of term or preterm labor. For the first 95% of gestation, the uterus is kept in a quiescent state by progesterone, which regulates expression of genes that maintain uterine quiescence. While in most mammals, a drop in serum progesterone levels precedes the onset of labor, in humans, other primates and guinea pigs, circulating progesterone and uterine progesterone receptor (PR) levels remain high through term. We postulate that the initiation of spontaneous labor in humans, as well as other mammalian species, is caused by a complex and concerted series of biochemical and molecular events that negatively impact the ability of the PR to regulate target genes in the uterus that maintain myometrial quiescence. We have recently obtained evidence that PR function near term may be compromised by: (1) a decline in expression of certain coactivators that decrease PR transcriptional activity; (2) a decline in local concentrations of progesterone within the myometrium caused by increased expression of progesterone metabolizing enzymes, and; (3) an induction of inflammatory response pathways leading to activation of NF-kB, a transcription factor that antagonizes PR function. We also have obtained intriguing evidence that induction of inflammatory response pathways leading to inactivation of PR function and the initiation of labor is caused by augmented secretion of surfactant protein into amniotic fluid by the maturing fetal lung. Summer research will involve some aspect of this research.

Project II

In humans, the CYP19 gene, which encodes aromatase P450, is expressed in a number of tissues. The 5'-untranslated regions (UTR) of CYP19 mRNA transcripts in these tissues are encoded by different tissue-specific first exons, which are alternatively spliced onto a common site just upstream of the start of translation in exon II. In ovary, the 5'-UTR of CYP19 transcripts is encoded by exon IIa, which lies just upstream of exon II. In previous studies using mice carrying fusion genes with various amounts DNA flanking the 5'-end of ovary-specific exon IIa, we found that as little as 278 bp mediates ovary-specific CYP19 gene expression. For the present study, mice were created carrying fusion genes containing 278 or 214 bp of ovary-specific exon IIa 5'-flanking DNA linked to a mutated form of hGH (hGX), as reporter. Based on results from preliminary experiments, we know that the CYP19(IIa)-214:hGX fusion gene is expressed in the ovary; however, we have not defined the ovarian cell types in which the fusion gene is expressed, or determined whether expression is ovary-specific. Thus, northern analyses will be performed on the various lines of mice carrying the CYP19(IIa)-214:hGX fusion gene to analyze tissue-specific expression. Additionally, immunohistochemistry will be performed on ovaries from mice carrying the CYP19(IIa)-214:hGX versus the CYP19(IIa)-278:hGX fusion genes to determine the cell-specific pattern of expression. If time permits, some of the high copy lines can be used for in situ hybridization experiments to localize the cell types within the ovary where hGX transcripts are expressed. Lastly, the patterns of expression of the CYP19(IIa)-278:hGX and CYP19(IIa)-214:hGX fusion genes will be compared throughout the estrous cycle and compared to that of the endogenous mouse CYP19 gene.
The 214 bp region upstream of hCYP19 exon IIa contains cis-acting elements found to be critical for cAMP induction of CYP19 promoter activity in transfected ovarian cells. These cis-acting elements include a CRE-like sequence (CLS), which binds the cAMP-response element binding protein (CREB), two nuclear receptor half-sites, which bind steroidogenic factor-1 (SF-1) and liver receptor homolog 1 (LRH-1), and a GATA-4 site. Since CREB, SF-1 and GATA-4 are expressed in tissues other than the ovary, such as in the adrenal, their roles in ovary-specific gene expression remain unclear. Interestingly, the region between 214 and 278 bp contains a consensus cis-acting element for the hepatocyte nuclear factor 3 (HNF3) family of transcription factors, which have been reported to be expressed in the ovary. In concurrent studies, immunohistochemistry will be performed on mouse ovaries to determine the isoforms of HNF3 expressed, as well as the cell types where expression occurs. Electrophoretic mobility shift assays (EMSA) using granulosa cell nuclear extracts and antibodies to the relevant form of HNF3 will be utilized to determine whether HNF3 specifically binds to this site. If this is found to be the case, granulosa cells will be transiently transfected with luciferase (LUC) reporter constructs containing 278 bp of human CYP19(IIa) 5'-flanking DNA with or without mutations in the HNF3 site to determine its functional role in basal and cAMP-induced hCYP19 gene expression. In parallel studies, granulosa cells will be co-transfected with the CYP19(IIa):LUC reporter constructs and with expression vectors for HNF3.

Previous Research Activities or Publications with Medical Students:

Molsberry, R.L., Carr, B.R., Mendelson, C.R., and Simpson, E.R. hCG binding to human fetal testes as a function of gestational age. J. Clin. Endocrinol. Metab. 55:791-794, 1982.
Ng, V.L., Herndon, V.L., Mendelson, C.R., and Snyder, J.M. Characterization of rabbit surfactant associated proteins. Biochim. Biophys. Acta 754:218-226, 1983.
Anderson, C.M., and Mendelson, C.R. Regulation of the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in rat Leydig cells. Ann. N.Y. Acad. Sci. 438:259-268, 1984.
Durham, C.R., Zu, H., Masters, B.S.S., Simpson, E.R., and Mendelson, C.R. Regulation of aromatase in rat granulosa cells: Indication of synthesis of NADPH-cytochrome P-450 reductase by FSH and dibutyryl cyclic AMP. Mol. Cell. Endocrinol. 40:211-219, 1985.
Anderson, C.M., and Mendelson, C.R. Regulation of steroidogenesis in rat Leydig cells in culture: Effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side-chain cleavage and adrenodoxin. Arch. Biochem. Biophys. 238:378-387, 1985.
Lanoux, M.J., Cleland, W.H., Mendelson, C.R., Carr, B.R., and Simpson, E.R. Factors affecting the conversion of androstenedione to estrogens by human fetal hepatocytes in monolayer culture. Endocrinology 117:361-368, 1985.
Anderson, C.M., and Mendelson, C.R. Developmental and hormonal regulation of cholesterol side chain cleavage cytochrome P-450 in the fetal rabbit testis. Mol. Cell. Endocrinol. 55:121-130, 1988.
Young, P.P., and Mendelson, C.R. A CRE-like element plays an essential role in cyclic AMP regulation of the human surfactant protein-A2 (SP-A2) gene. Am. J. Physiol.: Lung Cell. Mol. Physiol., 271:L287-L299, 1996.
Young, P.P., and Mendelson, C.R. A GT box is essential for basal and cyclic AMP regulation of the human SP?A2 gene in alveolar type II cells: Evidence for the binding of lung nuclear factors distinct from Sp1. Mol. Endocrinol 11:1082-1093, 1997.
Muenster, M.R., Gao E., Wang, L., Smith M.E., Alcorn, J.L., and Mendelson, C.R. Role of transcription factor BTEB2 in developmental and cyclic AMP regulation of surfactant protein?A (SP?A) gene expression. The Endocrine Society Annual Meeting, 1998, oral presentation and travel grant award.
Mendelson, C.R., Young, P.P, Michael, L.F., Alcorn, J.L., and Gao, E.E. Transcriptional regulation of the surfactant protein-A gene. Chest, 111:96S-104S, 1997.
Mendelson, C.R., Young, P.P., Michael, L.F., Gao, E., and Alcorn, J.L. The surfactant protein A gene and its regulation. In: Lung Surfactant: Cellular and Molecular Processing. (Rooney, S.A., ed.) pp. 47-73, R.G. Landes, Co., 1998.
Mendelson, C.R., Gao, E., Li, J., Young, P.P., Michael, L.F., and Alcorn, J.L. Regulation of expression of surfactant protein?A. Biochim. Biophys. Acta 1408:132-149, 1998.
Mendelson, C.R., Michael, L.F., Young, P.P., Li, J., and Alcorn, J.L. Cyclic AMP and glucocorticoid regulation of surfactant protein-A gene expression. In: Endocrinology of the Lung: Development and Surfactant Synthesis (Mendelson, C.R., ed.), Humana Press, pp. 59-80, 2000.
Condon, J.C. Faust, J.M., Wilson, J.W., and Mendelson, C.R. The decline in coactivators in the pregnant myometrium at term may antagonize progesterone receptor function and contribute to the initiation of labor. Submitted to PNAS, 2003.

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