REGULATION OF ORNITHINE DECARBOXYLASE EXPRESSION IN FIBROBLASTS

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Biosynthesis of the polyamines, spermidine and spermine, is regulated through the key enzyme ornithine decarboxylase (ODC) and is invariably enhanced upon activation of animal cells by a variety of stimuli. The level of ODC mRNA is regulated either transcriptionally or post- transcriptionally, depending on the cell type. The signal transduction pathways that control the ODC gene at the transcriptional level are varied, including those involving protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases, and steroid hormone receptors. We at present have identified two regions of the ODC promoter that are important for regulated transcription. Region A is centered at -45 bp relative to the site of transcriptional initiation, in a position adjacent to the TATAA box; this element shows strong sequence similarity to the cAMP-regulated element of the somatostatin gene ("CRE"), and binds the cAMP-regulated factor, CREB, when presented as purified recombinant protein or in cell extracts. Mutation of the CRE-like site strongly depresses basal level transcription driven by the ODC promoter. Region B is a 43-bp GC-rich region located between -92 bp and -134 bp. Deletion of this region, leaving Region A intact, has little or no effect on basal activity of the promoter, but interferes with regulation by PKA or phorbol esters. These results suggest that Region B plays an important role in regulation of ODC transcription by the PKA and PKC pathways. A major goal of this proposal is to test this hypothesis by detailed mutational analysis of Region B, correlating the influence of various mutations on regulation of the promoter with changes in protein binding. Proteins identified by this analysis will be isolated and characterized with regard to their regulatory properties, with the objective of understanding how these DNA-binding proteins interact functionally with the signal transduction pathways. The level of ODC mRNA is also upregulated in fibroblasts by the inflammatory mediator IL-1. The mechanism of this regulation will be examined in detail, and if it is at the transcriptional level, cytokine-responsive elements will be identified in the ODC gene. If these elements are different from those already found, proteins binding to them will be characterized. Regulation by other cytokines of interest, such as TNF- alpha and IL-6, will be examined in the same way.