Human placental lactogen (hPL) is a member of the growth hormone/prolactin gene family that acts as a """"""""growth hormone of pregnancy"""""""", regulating maternal and fetal metabolism and playing a critical role in the control of fetal growth. The overall goals of this proposal are to examine in depth the molecular mechanisms involved in the regulation of hPL gene expression in differentiated trophoblast cells, the upregulation of hPL gene expression during trophoblast differentiation, and the differentiation of cytotrophoblast cells to syncytiotrophoblast cells that express hPL.
Our specific aims are: to test the hypotheses that 1) modulation of hPL gene expression in differentiated syncytiotrophoblast cells is mediated by RARalpha, other nuclear hormone receptors, and the intermediate early gene products c-Fos and c-Jun, 2) upregulation of hPL gene expression during differentiation is mediated in part by RARalpha, other nuclear hormone receptors, c-Fos, c-Jun and other transcription factors that bind to the hPL promoter, and 3) transcription factors that mediate upregulation of hPL gene expression during differentiation are also involved in determination of trophoblast cell differentiation and the associated changes in gene expression. Initial studies will utilize DNase I footprinting, mobility shift assays, supershift assays and site-directed mutagenesis of the hPL promoter to identify the DNA elements on the hPL promoter that are critical in regulation of hPL gene expression in differentiated trophoblast cells and in upregulation during cytotrophoblast differentiation. The studies relating to differentiation will utilize an in vitro model system developed in our laboratories in which enzymatically dispersed cytotrophoblast cells from normal placentas differentiate into a syncytiotrophoblast phenotype that expresses abundant amounts of hPL and hCG. The cytotrophoblast cells will be transfected with expression vectors for nuclear hormone receptors, c-Fos, c-Jun and other transcription factors (wild type and dominant/negative) to determine the effects of the transcription factors on activation of hPL promoter activity as well as other genes upregulated during trophoblast differentiation (hCG, corticotrophin-releasing hormone and nitrous oxide synthetase, type III). These studies are of practical importance since abnormal plasma hPL concentrations have been noted in many pathologic conditions of pregnancy, such as pre-eclampsia, diabetes mellitus and other disorders associated with intrauterine growth retardation and fetal morbidity. In addition, studies of hPL gene expression during trophoblast development should provide new insight into the molecular mechanisms involved in cytotrophoblast differentiation. Detailed studies of the unique nuclear hormone response elements on the hPL promoter will also expand the general understanding of the molecular basis of nuclear hormone receptor action.
