Role of DNA and Specific Cytoplasmic Receptors in Glucocorticoid Action

Abstract

Glucocorticoids induce tyrosine aminotransferase (EC 2.6.1.5) synthesis in cultured rat hepatoma cells. These steroids penetrate the cell membrane and bind to specific cytoplasmic receptor proteins. The resulting complex binds to the nucleus. This nuclear binding has nowlbeen studied in a cell-free preparation. The reaction appears to require a temperature-dependent modification of the steroid-receptor complex. There is a fixed number of nuclear sites that are half saturated at a complex concentration of 6 to 24 X 10-11 M. Treatment with deoxyribonuclease destroys nuclear-binding capacity. The complex also binds to purified HTC cell DNA with characteristics similar to the binding to isolated nuclei, and, as in intact cells,' receptors complexed with an anti-inducer steroid bind very poorly to DNA. These data suggest that the nuclear sites for binding steroid-receptor complexes are on the DNA. Since the extent of complex binding to purified DNA exceeds that observed with isolated nuclei, chromosomal proteins may act to restrict binding to certain reg'ions of the DNA. These studies suggest that steroid hormones stimulate the synthesis of specific proteins by affecting the transcription of structural or regulatory genes.Induction of tyrosine aminotransferase (EC 2.6.1.5) by steroid hormones in hepatoma tissue culture (HTC) cells is being studied to understand the mechanism of gene regulation in mammals (1). HTC cell cytosol contains specific steroid "receptor" proteins that appear to mediate the induction (2-5). When intact cells are exposed to inducer steroids, specifically bound hormone is predominantly found in the nuclear fraction (5). From other studies we concluded that this is due to accumulation of steroid-cytoplasmic receptor complexes in the nucleus (4). This mechanism, therefore, closely resembles that proposed for systems responsive to other steroid hormones [for review see Jensen et al. (6)]. To account for the effects of inducer, anti-inducer, and suboptimal inducer steroids, we have proposed (7) that the cytoplasmic receptor is an allosteric protein that has different conformations depending on the type of steroid with which it is complexed. In support of this idea we found that inducer-bound receptors differ from those that are uncomplexed or are bound by an anti-inducer (3, 4) and that, in intact cells, inducerreceptor complexes accumulate in the nucleus, whereas antiinducer-receptor complexes remain in the cytosol (4, 5).Thus, the biologic actions of steroids could depend on their capacity to alter the allosteric equilibrium between different conformations of the cytoplasmic receptors.In this report, some characteristics of the nuclear binding reaction, studied in a cell-free preparation, are presented.Abbreviation: HTC cells, hepatoma tissue culture cells.Inducer steroid-receptor complexes bind with high affinity to a limited number of DNA-containing nuclear sites. MATERIALS AND METHODSHTC cells, grown at 370 in suspension cultures to 2 to 10 X 105 cells/ml (8), were washed by centrifugat...

Glucocorticoid-Binding Proteins in Human Acute Lymphoblastic Leukemic Blast Cells

“…This is consistent with the observation that association with receptor is only the first of several steps between binding and biologic response (8). There is evidence that the receptor must undergo a temperature-dependent transformation after association with the steroid before uptake by the nucleus can occur (8,11,17,56,57), a step that does not occur with progesterone and cortexolene (8,10). Such steroids have therefore been termed "anti-inducers" (21).…”

“…The binding of steroid hormones to highly specific, high affinity cytoplasmic receptor proteins has been suggested as the first step in steroid hormone action in many systems (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). These steroid-binding proteins (SBP)1 appear to be the determinant of target tissue specificity (4,6,7,12).…”

Enhanced Effects of Prostaglandin E1 and Dibutyryl Cyclic AMP upon Human Lymphocytes in the Presence of Cortisol

“…Whether the observed increase in cyclic AMP levels is due to increased adenyl cyclase activity, inhibition of phosphodiesterase, or a more indirect mechaism remains to be tested and is the subject of further investigation in this laboratory. Since the potentiation of PGEi-induced rises in cyclic AMP levels occurred within minutes, the mechanism is unrelated to the events associated with glucocorticosteroid-stimulated synthesis of specific proteins, which involves increased transcription of messenger RNA as a result of steroidreceptor complex binding to DNA (23). It should be stressed that the further stimulation of PGE,-induced increases in cyclic AMP levels does not account for the inhibitory effect of cortisol when added alone to peripheral lymphocyte cultures.…”

“…Incubation of PHA-treated cultures with dibutyryl cyclic ANIP or Experiments were performed to determine the effects of PGE1 and cortisol, alone and in combination, upon lymphocyte cyclic AMP levels (Table III). Addition of 0.1 mM PGE1 resulted in a rapid elevation of intracellular cyclic AMP levels which peaked by [20][21][22][23][24][25][26][27][28][29][30] min, whereas at concentrations of 0.01-0.001 mMI PGE1, the response was variable. Cyclic ANIP levels rose 3.3±0.4- and with the combination of PGE1 plus cortisol.…”

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

“…In most cases studied these effects are at the transcriptional level. Baxter et al (2) demonstrated that glucocorticoid-receptor complexes or proteins associated with them bind to DNA. It has been postulated that these complexes recognize specific sequences on responsive genes and affect their transcription (for reviews, see references 30 and 43).…”