Plasminogen activators (PAs) are specialized proteases which are highly specific for the inactive zymogen plasminogen, which, upon cleavage by PAs, forms the active serine protease plasmin. PAs, which are expressed by both normal and malignant cell types, are frequently regulated by steroid and peptide hormones. It has been suggested that PA expression may be related to regulation of cell growth and/or differentiation state. We have demonstrated that LICR-LON-HMy2 human myeloma lymphoblast cells (HMy2 cells) secrete the PA urokinase (UK). These cells are growth inhibited by glucocorticoids, and their extracellular UK activity is virtually eliminated following exposure to glucocorticoids. This inhibition of UK is unrelated to glucocorticoid induction of inhibitors to either UK or activated plasmin. We propose to examine the mechanisms of glucocorticoid inhibition of HMy2 UK. We will establish 1) whether inhibition of extracellular UK involves decreased biosynthesis and/or secretion of UK; 2) whether decreases in steady-state levels of mRNA coding for UK are involved; 3) whether decreased transcription of the UK gene occurs; and 4) whether decreased UK gene transcription is a primary effect of glucocorticoids or whether it is mediated by glucocorticoid-induced transcriptional factors. The molecular biology approaches will be facilitated by the use of the human UK cDNA plasmid pHUK-8, which has been supplied to us by Dr. F. Blasi (currently at NIH). Appropriate contingency plans for all possible outcomes are discussed. The experiments outlined in this proposal will establish the validity of using glucocorticoid inhibition of HMy2 UK in future studies to examine mechanisms by which steroid-receptors turn off constitutive gene transcription. Such future studies would contrast with the many systems currently being used to examine steroid induction of specific genes. In summary, this work will provide the groundwork for a number of significant long-range goals: 1) examining the mechanisms of glucocorticoid inhibition of constitutive gene transcription; 2) studying potential relationships between glucocorticoid inhibition of UK, inhibition of cell growth, and development of steroid resistance in myeloma lymphoblasts; and 3) development of a potentially important clinical assay for prediction of glucocorticoid responsiveness in myeloma patients.
