MODE OF ACTION OF STEROID HORMONES

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The long-term objective of this research is an understanding of actions of glucocorticoids on the immune system at molecular, cellular and physiological levels. Cells, tissues and functions of the immune system are major targets of glucocorticoids in their therapeutic functions as immunosuppressive and anti-inflammatory agents, and in the treatment of lymphocytic leukemias and lymphomas. Specific aims are directed towards three fundamental problems: (i) structural changes in glucocorticoid receptors that accompany "activation" (the transformation by which steroid-receptor complexes acquire affinity for nuclei and DNA); (ii) induction by glucocorticoids of mRNAs and proteins in rat thymus cells, in relation to rapid effects on glucose transport; (iii) interactions of glucocorticoids and gamma interferon on immune processes in intact mice. (i) The hypothesis that glucocorticoid receptors in the cell are cyclically phosphorylated and dephosphorylated, which emerged from earlier work on this project, will be tested by measuring 32P labeling of receptors in WEHI-7 mouse thymoma cells treated in various ways. Receptors purified with a monoclonal antibody will be analyzed for 32P content and location by peptide mapping, which will also be applied separately to identify peptides of the DNA-binding domain. The antibody will be used to identify nonbinding forms of receptors in energy-deprived cells. A kinetic model of receptor cycling will be tested with glucocorticoid antagonists. (ii) Glucocorticoids inhibit glucose transport in incubated rat thymus cells within 20 min, and exert other effects by 1 hour. Evidence suggests these effects are mediated by induced mRNAs coding for proteins that inhibit glucose transport, etc. Subtractive hybridization will be used to identify rare glucocorticoid-induced species of thymus mRNA and their respective proteins. Induced proteins will separately be sought by their putative ability to inhibit glucose transporters. (iii) Many potentially important effects of glycocorticoids on gamma interferon production and actions have been demonstrated in earlier work on this project with culture systems. Few have been demonstrated in animals. To bridge the gap between culture systems and whole animals, mice will be used to study the effects of adrenalectomy, exogenous gamma interferon and glycocorticoids on IgG-Fc receptor expression, B-cell (antibody) responses, and gamma interferon mRNA levels.