DEVELOPMENT OF RESPONSIVENESS TO GLUCOCORTICOID HORMONES

Abstract

Glucocorticoid (GC) hormones induce the activity of glutamine synthetase (GS) in the neural retina. This function of the hormone is mediated by the glucocorticoid receptors (GR), which upon binding the GC translocate from the cytosol to the nucleus, bind to specific acceptor sites in chromatin and evoke a differential increase in tissue-specific gene transcription, ultimately leading to high GS activity. The competence to respond to GC hormones is acquired in the neural retina of the chick embryo during the second week of embryogenesis. However, high levels of GRs, capable of translocating to the chromatin, are available to the retina at earlier stages of development. The mechanism(s) which endow the tissue with competence to respond to the hormonal signal are as yet unknown. Our studies were directed toward the elucidation of this biological phenomenon. We have shown that:(A)Acquisition of responsiveness is dependent on DNA synthesis and apparently requires a certain number of cell cycles before the cells gain competence to express this function.(B)Responsiveness is correlated with a loss of two-thirds of the specific cortisol binding sites, as shown by binding studies. We show that this is a developmental event which correlates with the age-dependent acquisition of responsiveness. This process requires the milieu of the intact cell (concentrated cytosol, 0.15 M KCl), and apparently reflects an early step in GR activation.The binding studies raised the working hypothesis that the GRs of the embryonic neural retina are heterogeneous in structure and function and react differently to the binding of the inducing ligand. The transformation of one GR form to the active regulator appears to be dependent on the inactivation (loss of binding) of another GR form acting as an inhibitory control unit. This working hypothesis is supported by studies on the molecular characterization of the GRs of the responsive retina: analytical polyacrylamide gel electrophoresis (PAGE) revealed that the GRs in the cytosol are dimers (or higher oligomers), made of nonidentical GR units; one of 108,000 Mr designated GR-B and another, not yet characterized, designated GR-X. GR-B is cleaved in the cytosol to the 61,000 Mr form designated GR-C.Isoelectric focusing of the unactivated, molybdate stabilized cytosol revealed two forms of inactive GRs, I and II. Activation (1 h at 30°C, in the presence of [3H]-triamcinolone acetonide and 0.15 M KCl) inactivates GR-I (loss of binding) and transforms GR-II to a basic nuclear binding protein, GR-III. The formation of GR-III correlates with the inactivation of GR-I. GR-I is an acidic protein, sensitive to isotonic KC1, and may be equivalent to GR-X, which has to date eluded characterization by PAGE.(C)The expression of the cellular functions involved in acquisition of responsiveness to GC hormones is correlated with the withdrawal of the cortisol-target cells from cell cycling.