Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Electrofusion of rat adrenal and Leydig cells generated hybrids capable of synthesizing simultaneously both testosterone and corticosterone, under stimulation of lutropin or adrenocorticotropin. Evidence was obtained indicating that under such circumstances, heterologous lutropin receptor--adrenal adenylate cyclase complexes were formed.     

Ligand-dependent and -independent transactivation by thyroid hormone receptor beta 2 is determined by the structure of the hormone response element.

Chicken thyroid hormone receptor beta 2 (cTR beta 2) is likely to serve specific functions in gene regulation since it possesses a unique N-terminal domain and is expressed in very few tissues. We demonstrate here that TR beta 2 exhibits distinct transactivation properties which are dependent on the availability of ligand and on the structure of the hormone response element. First, a strong ligand-independent transactivation was observed with hormone response elements composed of direct repeats and everted repeats. Second, TR beta 2 was induced by triiodothyronine to transactivate more efficiently than TR beta 0 on palindromic and everted-repeat types of hormone response elements. However, coexpression of the retinoid X receptor reduced the strong transactivation by TR beta 2 but not by TR beta 0 via palindromic response elements, suggesting that TR beta 2 can transactivate as a homodimer. Finally, the N terminus of TR beta 2 contains two distinct transactivation regions rich in tyrosines, which are essential for transactivation. Our results thus show that the activity of the novel transactivating region of TR beta 2 is dependent on the organization of the half-sites in the response element.     

Functional interaction of hybrid response elements with wild-type and mutant steroid hormone receptors.

Steroid hormone receptors can be divided into two subfamilies according to the structure of their DNA binding domains and the nucleotide sequences which they recognize. The glucocorticoid receptor and the progesterone receptor (PR) recognize an imperfect palindrome (glucocorticoid responsive element/progesterone responsive element [GRE/PRE]) with the conserved half-sequence TGTYCY, whereas the estrogen receptor (ER) recognizes a palindrome (estrogen responsive element) with the half-sequence TGACC. A series of symmetric and asymmetric variants of these hormone responsive elements (HREs) have been tested for receptor binding and for the ability to mediate induction in vivo. High-resolution analysis demonstrates that the overall number and distribution of contacts with the N-7 position of guanines and with the phosphate backbone of various HREs are quite similar for PR and ER. However, PR and glucocorticoid receptor, but not ER, are able to contact the 5'-methyl group of thymines found in position 3 of HREs, as shown by potassium permanganate interference. The ER mutant HE84, which contains a single amino acid exchange, Glu-203 to Gly, in the knuckle of ER, creates a promiscuous ER that is able to bind to GRE/PREs by contacting this thymine. Elements with the sequence GGTCAcagTGTYCT that represent hybrids between an estrogen response element and a GRE/PRE respond to estrogens, glucocorticoids, and progestins in vivo and bind all three wild-type receptors in vitro. These hybrid HREs could serve to confer promiscuous gene regulation.     

Oligodeoxynucleotide base recognition by steroid hormone receptors

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E₂R) the relative binding order, in prior studies, was oligo(dG) > oligo(dT) ≧ oligo(dC) > > oligo(dA) > oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1–0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E₂R to chaotropic salts such as SCN^?, ClO₄^? and NO₃^? as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E₂R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37°C was not altered by addition of H2B. Treatment of uterine E₂R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.     

Modulation of transcription factor activity by a distant steroid modulatory element.

Variations in the biological activity of antisteroids, as determined by their percent agonist activity, is a well known but poorly understood phenomenon. For example, in tyrosine aminotransferase (TAT) induction by the antiglucocorticoid dexamethasone 21-mesylate in rat hepatoma tissue culture cells, the percent agonist activity varies with the density of cultured cells. A 21-basepair sequence of the rat TAT gene has now been isolated which confers all of the induction properties of the endogenous TAT gene to homologous and heterologous promoters and genes. We call this 21-basepair sequence, which acts in concert with a trans-acting factor identified by gel shift experiments, a glucocorticoid modulatory element. The changes in induction properties were found to be independent of the fold induction by dexamethasone, thus arguing that the GME does not synergize with the glucocorticoid response element. A model incorporating this new element is advanced which can explain the observed variations of TAT induction and may be generally applicable for the mechanism of action of other steroid hormones.     

Protection of steroid hormone receptors by protease inhibitors

Steroid hormone receptors are proteolyzed by different types of enzymes present in target tissues. Effective protease inhibitors protecting steroid hormone receptors in various target tissues were investigated. Progesterone receptor (PR) in hen oviduct and estrogen receptor (ER) in cow uterus were specifically protected by relatively low concentrations (0.5 mM) of leupeptin or antipain (inhibitors of serine and thiol proteases). It was indicated that two different types of enzymes which modify native glucocorticoid receptor (GR) are present in rat liver. One was inhibited by 1 mM leupeptin or 1 mM antipain, while the other was inhibited by 1 mM phosphoramidon (inhibitor of thermolysin like proteases) or 10 mM sodium molybdate. Native PR, ER, and GR were shown to have similar Stokes radii (44 Å).