Stephen J. Higgins

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells.

Abstract Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells. Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid. Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed. Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.

Nuclear binding of glucocorticoid receptors: Relations between cytosol binding, activation and the biological response

Abstract To better understand the early steps in glucocorticoid hormone action from cytoplasmic receptor-steroid binding to nuclear binding of the resulting complexes, we have in the current studies examined in cultured hepatoma (HTC) cells the kinetics of the nuclear binding of glucocorticoid receptors, the activation of the receptor-steroid complex required for this binding and the nature of the nuclear binding sites. The quantitative relationships of these events have also been compared to the glucocorticoid response. When intact cells are incubated with dexamethasone at 0°C, cytosol binding of the steroid occurs readily but nuclear binding proceeds very slowly. After heating the cells at 37°C for 40s and then chilling them, the kinetics of the nuclear binding at 0°C are markedly increased. Thus, in the intact cell at 0°C. like cell-free systems, the heat-sensitive activation and not the nuclear binding itself appears to occur and be rate-limiting for nuclear binding of the complexes. Nuclear binding measured at 37°C was similar at 30–40 min, and 16 h, by which time induction of tyrosine aminotransferase is maximal. Thus, unlike the case with most other classes of hormones, in this system glucocorticoids do not affect the concentration of their receptors. Nuclear binding was linearly related to the content of cytoplasmic reccptor-glucocorticotd complexes and a Scatchard plot (nuclear-bound over cytosol-bound vs nuclear-bound dexamethasone) was parallel to the abscissa. These observations suggest that in the cell the nuclear acceptors are far from saturated with receptors. It has been proposed that there are multiple orders of affinity for binding receptors in the nucleus. Salt sensitivity of bound receptors has been used on one criterion. Indeed, whereas most of the nuclear-bound receptors can be extracted with 0.5 M KCl, about 20% resist this extraction. In the current studies, the proportion of salt-extractable and salt-resistant nuclear-bound receptors was found to be constant with time (to 16 h) and cytosol receptor-steroid complex concentration. Thus, the thermodynamics of receptor association with the salt-labile and salt-resistant nuclear-bound receptors appear similar and the evidence does not support the idea that these receptors have fundamental differences; the incomplete extraction of the receptors with salt may reflect some property of the solubilization process rather than two types of nuclear acceptors. The content of nuclear-bound receptors was found to be linearly related to the induction of tyrosine aminotransferase; this suggests that the number of glucocorticoid receptors, and not some distal event, is the limiting factor in the glucocorticoid hormone response and that “spare receptors” are not present. These data also do not support the idea that in these cells, a physiological “acceptor” that binds receptors with an affinity much higher than the observed nuclear-binding (“high-affinity operators”) mediates glucocorticoid action; these hypothetical sites would have been more saturated at the receptor concentrations achieved. Instead, the data are consistent with the possibility that a nuclear acceptor population with an affinity that does not vary greatly from the observed nuclear binding is responsible for the glucocorticoid response. The data are discussed in terms of a model in which receptors bind similarly to acceptors present in excess, many of which are located at sites where a physiological response is not elicited. By this model, the probability of obtaining a glucocorticoid response is proportional to the chance association of receptors with those acceptors located at important loci.

Measurement of specific binding of a ligand in intact cells: dexamethasone binding by cultured hepatoma cells.

Publisher Summary In recent years, hormone and drug receptors have received considerable attention. It is frequently advantageous to study the binding of the effector in intact cells under conditions required for the biological response to occur. Data obtained in this way can often be readily related to the drug or hormone effects. By studying the intact cell, one can avoid problems, such as receptor instability, as observed in cell-free systems, or lack of knowledge of the subcellular site of binding. A suggested approach to this problem is described in this chapter using, as an example, the binding of dexamethasone by cultured hepatoma (HTC) cells. In these cells, dexamethasone and other glucocorticoid steroids specifically induce the synthesis of tyrosine and aminotransferase and a few other proteins. Three procedures are described in the chapter. (1) Binding of intact cells under conditions in which the biological response occurs. This technique is especially useful when the affinity and association–dissociation rates are unknown or substantial dissociation of the ligand from its receptors could occur if cells were washed free of the medium in which they are incubated. (2) Binding under the same conditions followed by chilling the cells to slow the rate of receptor–ligand complex dissociation and washing them to reduce the amount of nonspecifically bound ligand. This technique is useful either when dissociation of specifically bound material is slow or when the cells can be treated (as by chilling) to decrease the dissociation. (3) Subcellular localization of the ligand specifically bound in the intact cell, again taking advantage of a slow dissociation at 0o. For all these procedures, the competition principle is employed in which incubations of cells with [ 3 H] ligand are performed in parallel to incubations with [ 3 H] ligand plus excess nonradioactive ligand. The latter inhibits specific, but not nonspecific, binding of the [ 3 H] ligand and thus allows determination of specific binding.

Nuclear acceptor sites for glucocorticoid receptors

Abstract Glucocorticoids increase the rate of synthesis of tyrosine transaminase in hepatoma tissue culture cells. The first steps in this hormonal action involve specific binding of steroid to a cytoplasmic receptor followed by interaction of the complex with the nucleus. To investigate the nature of nuclear acceptor sites, a cell-free system was designed in which nuclei isolated from hepatoma cells bind specifically the receptor-steroid complex. DNA appears to be involved in this process. Since binding of receptors to isolated nuclei resembles in many ways the corresponding interaction taking place in the intact cell, binding of receptors to pure DNA was studied in greater detail. Contrary to what is seen with whole nuclei, there is no evidence that DNA contains a limited number of sites for glucocorticoid receptors. It is concluded that DNA may be a necessary but not sufficient component of the chromatin acceptor sites.

Mechanism of Glucocorticoid Hormone Action and of Regulation of Gene Expression in Cultured Mammalian Cells

To understand the mechanism of gene expression in higher organisms we have been studying the hormonal regulation of specific protein synthesis in mammalian cells. Most of our work has involved cultured rat hepatoma (HTC) cells (Thompson et al., 1966) in which glucocorticoid hormones influence, as far as we know, only five functions (Table 1). Of these, the induction of tyrosine aminotransferase (TAT) synthesis has been the most extensively studied. A factor which promotes cell adhesiveness (Ballard and Tomkins, 1969, 1970) is also included, as well as under special circumstances, glutamine synthetase (Kulka et al., 1972). Finally, preliminary reports suggest that glucocorticoids increase the amount of phenylalanine t-RNA (Yang, Lippman and Thompson, unpublished) and decrease the activity of the phosphodiesterase which degrades adenosine-3′, 5′cyclic monophosphate (cAMP) (Manganiello and Vaughan, 1972).

NUCLEAR ACCEPTOR SITES FOR GLUCOCORTICOID RECEPTORS

Glucocorticoids increase the rate of synthesis of tyrosine transaminase in hepatoma tissue culture cells. The first steps in this hormonal action involve specific binding of steroid to a cytoplasmic receptor followed by interaction of the complex with the nucleus. To investigate the nature of nuclear acceptor sites, a cell-free system was designed in which nuclei isolated from hepatoma cells bind specifically the receptor-steroid complex. DNA appears to be involved in this process. Since binding of receptors to isolated nuclei resembles in many ways the corresponding interaction taking place in the intact cell, binding of receptors to pure DNA was studied in greater detail. Contrary to what is seen with whole nuclei, there is no evidence that DNA contains a limited number of sites for glucocorticoid receptors. It is concluded that DNA may be a necessary but not sufficient com-ponent of the chromatin acceptor sites.