REGULATION OF HUMAN SP-A GENE EXPRESSION

The long term objective is to understand the molecular mechanisms involved in the regulation of human surfactant protein A (SP-A) gene expression. Towards this goal and based on current available information we propose a number of studies. Current knowledge suggests that there are two functional SP-A genes. cDNA sequences are available for both genes but genomic DNA sequence is available for only one. In this proposal we will first characterize the human SP-A locus, determine the number of genes, study their chromosomal organization, and determine their DNA sequence similarities/differences in order to assess potential similarities/differences in their regulation. Regulation of SP-A gene expression appears complex and unusual in certain regards. Knowledge obtained from the first aim will help design experiments to distinguish whether all SP-A genes are regulated similarly or whether the observed complexity is due to the fact that different SP-A genes respond differently to a given signal. Experiments in aims 2-4 comprise two groups. Since our preliminary data suggest differential regulation by glucocorticoids of SP-A genes in explant culture, in the first group of experiments (aim 2) we will study the basic mechanisms that underlie differential regulation of the SP-A genes in explants, by examining their expression in the absence of serum and hormones and by further characterizing the response to glucocorticoids. In the second group of experiments we will study molecular mechanisms that may be involved in tissue-specific and developmental stage-specific expression of SP-A genes (aim 3) as well as hormone-responsive factors that may be important for differential regulation of SP-A mRNA stability (aim 4). For this group of experiments emphasis will be placed on trans-acting factors for two major reasons. Firstly SP-A expression is tissue-specific. In preliminary experiments aimed at studying tissue-specific expression as a means of addressing SP-A gene regulation, we identified cis-acting elements that form tissue-specific and developmentally-specific DNA/protein complexes. Experiments are designed to further characterize these factors and assess their functional role in this process. Secondly, one of the mechanisms involved in the regulation of SP-A expression in response to glucocorticoids is mRNA stability and inhibitor studies suggest that a trans-acting factor is also involved in this process. Preliminary studies show differences in 3'untranslated region (3'UT) of the SP-A genes/alleles, including the presence or absence of an 11bp segment. Since elements in the 3'UT are often involved in mechanisms of mRNA stability we hypothesize that this 11bp segment provides or disrupts a binding site for a regulatory factor. Experiments in aim 4 will test this hypothesis. Information obtained from the proposed studies will provide a better definition of the SP-A locus and shed light on the molecular mechanisms of differential and tissue-specific regulation. Knowledge gained can be used to study molecular alterations in the regulation of SP-A gene expression in the disease state.