Studies from our laboratory during the past five years strongly suggest a novel physiological role for apolipoprotein AI (apoAI) in the regulation of the synthesis and secretion of human placental lactogen (hPL) during pregnancy. The stimulation of hPL synthesis in response to apoAI appears to be due to stimulation of hPL gene expression. Over the next five years, we propose to extend our cellular and intracellular studies of the actions of apoAI by examining the molecular mechanisms involved in apoAI-mediated hPL gene expression. Our specific goals are: (1). To determine the relative contributions of transcriptional and post-transcriptional mechanisms in the stimulation of hPL mRNA levels in response to apoAI. Nuclear run-on experiments will be used to determine the effects of apoAI on hPL mRNA transcription, and studies of the half-life of hPL mRNA will be used to determine the effect of apoAI on the degradation of hPL mRNA. (2). To delineate the DNA element(s) in the promoter of the hPL gene necessary for apoAI-mediated hPL expression. The DNA element(s) will be determined by examining the effects of different deletions in the hPL promoter on expression of CAT (chloramphenicol acetyltransferase) activity in choriocarcinoma cells transfected with vectors containing regions of the hPL promoter. (3). To determine the sequence of the cis-acting DNA element(s) of the hPL promoter involved in apoAI-mediated hPL gene expression. Mobility gel shift and DNAse I footprinting assays will be utilized to localize the specific nucleotide sequence of the DNA element(s). (4). To characterize the placental DNA binding protein(s) (transcription factor(s)) involved in regulation of apoAI-mediated hPL gene expression. Initial studies will determine the molecular weight(s) and N-terminal amino acid sequence(s) of the transcription factor(s). Since apoAI is the first factor known to stimulate a sustained increase in hPL synthesis, delineation of the cis- and trans-acting factors involved in apoAI-mediated gene expression should provide important insight into the regulation of hPL gene expression. Since recent studies also implicate apoAI as a regulatory factor in other cell types, these studies may also provide insight into the mechanisms by which apoAI affects gene expression in other cells.
