Biomedical subjects
J D Baxter
Publications and source records attributed to J D Baxter.
Selective reduction of proadrenocorticotropin/endorphin proteins and messenger ribonucleic acid activity in mouse pituitary tumor cells by glucocorticoids.
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Thyroid hormone receptors. Alteration of hormone-binding specificity.
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Stimulation and inhibition of cellular functions by glucocorticoids. Correlations with rapid influences on chromatin structure.
The effect of media conditions on the glucocorticoid response has been examined in three types of cultured cells. In rat pituitary tumor cells (GC cells) growth hormone production was stimulated by glucocorticoids provided fresh culture media was present (enriched media conditions). In contrast, dexamethasone either failed to induce or deinduce growth hormone synthesis if added to cultures which had not received fresh media for 3 days (depleted media condition). With human skin fibroblasts, cortisol stimulated [3H]thymidine incorporation in the enriched condition but inhibited this response in the depleted condition. In mouse lymphoma (S49) cells the enriched media conditions significantly delayed the killing response to glucocorticoids (20% killing after 24 h versus 90% killing after 24 h for the depleted condition). Thus, the magnitude and in some cases, the direction of the glucocorticoid response are sensitive to the conditions to which the cells are exposed. In all three cell types the steroid also rapidly (detectable by 15 min, maximal by 2 h) altered chromatin structure as detected by a change in the number of initiation sites for Escherichia coli RNA polymerase assayed under cell-free conditions. This early nuclear response could be in a positive or negative direction and was also affected by the culture conditions; enriched media favored a positive or less negative effect on the initiation sites by the steroid, while depleted media favored a steroid-induced inhibition of this chromatin function. In S49 and GC cells the kinetics and magnitude of the change in chromatin closely followed receptor . glucocorticoid complex binding to nuclei while removal of dexamethasone from the culture media resulted in a rapid (t 1/2 = approximately 20 min) disappearance of the effect which paralleled loss of bound hormone from the nucleus. The glucocorticoid effect on chromatin was not observed in two lines of glucocorticoid-resistant mutant S49 cells. One line (R-) lacks detectable glucocorticoid receptors; the other line (Nti) has receptors that bind the hormone normally, but the receptor . glucocorticoid complexes bind more avidly to the nucleus. These results suggest that the receptor is involved in both the stimulatory and the inhibitory effects on chromatin. The findings in the Nti cells and of a slight lag between nuclear binding of receptors and initiation site alteration implies that some receptor property, in addition to nuclear binding per se, is responsible for the influence on chromatin. These results are discussed in terms of a model in which steroid hormones initiate their actions by influencing a reaction that modifies chromatin structure. The direction and magnitude of the reaction, and its effect on the expression of specific genes, are dictated by the metabolic state and differentiation of the cell.
Human growth hormone: complementary DNA cloning and expression in bacteria.
The nucleotide sequence of a DNA complementary to human growth hormone messenger RNA was cloned; it contains 29 nucleotides in its 5' untranslated region, the 651 nucleotides coding for the prehormone, and the entire 3' untranslated region (108 nucleotides). The data reported predict the previously unknown sequence of the signal peptide of human growth hormone and, by comparison with the previously determined sequences of rat growth hormone and human chorionic somatomammotropin, strengthens the hypothesis that these genes evolved by gene duplication from a common ancestral sequence. The human growth hormone gene sequences have been linked in phase to a fragment of the trp D gene of Escherichia coli in a plasmid vehicle, and a fusion protein is synthesized at high level (approximately 3 percent of bacterial protein) under the control of the regulatory region of the trp operon. This fusion protein (70 percent of whose amino acids are coded for by the human growth hormone gene) reacts specifically with antibodies to human growth hormone and is stable in E. coli.
Fluorescent rhodamine-labeled thyroid hormone derivatives: synthesis and binding to the thyroid hormone nuclear receptor.
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Glucocorticoid hormone action: an overview.
An overview of glucocorticoid action is presented including aspects of historic, clinical, and physiologic interest. Despite the diversity of effects, glucocorticoid hormone action at the cellular level simplifies to a rather universal model that involves binding to a soluble receptor, followed by interaction of the complex with the chromatin and modification of gene expression. This unitary concept has important implications in pathology, pharmacology, and therapeutics. Finally, a definition of a glucocorticoid in terms of its receptor is presented, which we feel is better than the traditional terminology based on specific biological effects. It is hoped that this review will also provide the reader with a framework in which the various contributions in the Monograph can be integrated.
Regulation of growth hormone messenger RNA.
In cultured rat pituitary cells, glucocorticoids regulate growth hormone production by modulating the number of growth hormone messenger RNA molecules. The effect is quite specific, since only a few other mRNAs are affected by the hormones. This response is demonstrated by assays involving cell-free mRNA translation and cDNA-RNA hybridization. Furthermore, the inducibility by the glucocorticoids is regulated by at least one other class of hormones, thyroid hormone. Thus, this system serves as a model for studying not only the glucocorticoid regulation of specific mRNA, but also the control of this regulation by other factors in the target tissue.
Mechanisms of glucocorticoid receptor function.
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Variations in cellular sensitivity to glucocorticoids: observations and mechanisms.
The spectrum of physiological, pathological, and genetic variations in sensitivity to glucocorticoids is reviewed. The receptor for these hormones is common to most mammalian tissues, and yet the responses are widely divergent. Although there may be differences in the receptors to account for some of this diversity, it is likely that it is largely due to cellular programming not involving the receptors. In addition to the intertissue differences in sensitivity, it is also clear that intra-tissue differences occur. The greatest amount of information has been accumulated with lymphoid cell systems and there are sensitivity differences to specific responses such as cell killing or effects on immunological functions. In these systems, there can be major variations in either the extent of the response (e. g., from mild growth inhibition to cellular killing) or whether any effect is observed. Further, dose requirements for certain responses can vary by several orders of magnitude. Within a given tissue there may be developmental changes in sensitivity that are not due to obvious changes in the receptor, and decreased sensitivity with aging that in some cases has been associated with changes in receptor binding activity. Finally, the cellular sensitivity can either be influenced by hormones and other factors that affect the ability of the glucocorticoid to elicit a particular response (in a synergistic or antagonistic manner), or the same function regulated by the glucocorticoid can be inducible by the steroid, appearing some time after administration of the steroid and disappearing after steroid removal. Genetic variations in sensitivity to glucocorticoids also occur. In humans these may be generalized, affecting glucocorticoid action in all responsive tissues, and could be important in the pathogenesis of certain diseases. Perhaps the most striking genetic alterations, however, are observed in cultured lymphoid and fibroblastic cells and in acute lymphoblastic leukaemia cells ordinarily growth inhibited or killed by the glucocorticoid. Mutant cell lines arise that are highly resistant and most of these have abnormalities in the glucocorticoid receptor. In some cases binding activity is totally lost, easily expalining the resistance. In other cases, there is a more modest reduction in binding or a change in receptor properties that give it increased or decreased nuclear and DNA binding activity. An analysis of these cell lines suggests that many of the defects are in some receptor property presently not understood that makes the receptor ineffective rather than the defect being due to the quantitative changes in receptor levels detected. The frequency of emergence of steroid-resistant cells can vary widely from about 10(-5) in S49 cells to less than 10(-8) in certain thymic cell lines...
Glucocorticoid receptors.
Glucocorticoid receptors are found in most mammalian tissues and have been studied in detail in a number of tissue culture systems. With cells that have not been exposed to steroids, the receptors are found in the cytoplasmic fraction from which they can be isolated and studied. Methods for studying glucocorticoid receptors depend on their high-affinity specific binding of radioactive steroids. The reversible interaction is intracellular. It follows Michaelian kinetics, at least in cell-free cytosol, and involves a thermodynamically homogeneous population of about 10 000 sites per cell. The receptor is an asymmetric, slightly acidic protein of about 100 000 daltons. It is very labile, especially in the unbound form. Binding activity depends on the integrity of thiol groups and perhaps on phosphorylation of amino acid residues. Although indirect, the evidence is overwhelmingly convincing that this protein is the physiologic glucocorticoid receptor. The time-kinetics of binding and dissociation are consistent with the sequence of events in glucocorticoid action. Various steroid analogs display binding characteristics predictable from their glucocorticoid activity. Loss of the binding protein from certain cultured cell lines is accompanied by unresponsiveness to glucocorticoids. The extensive tissue distribution of receptors parallels the extensive role of glucocorticoids in regulation. Finally, there is a strong correlation between nuclear binding of receptors and nuclear effects of the steroid. The glucocorticoid receptor can be distinguished from other glucocorticoid-binding proteins, based on their steroid specificity and physicochemical properties. There is no clear-cut demonstration that the receptor differs from tissue to tissue, and it is in fact very similar in various species. Unlike in other systems, receptor concentration does not seem to be regulated by its ligand or by other hormones. However, certain cases of hypo- as well as hypersensitivity to glucocorticoids appear to result from changes at the receptor level. The data indicate that the receptor can exist in inactive and active forms. The former predominate in the absence of steroid or when an angatonist is bound. Glucocorticoid agonists bind the active form, allowing it to be "activated" and subsequently bound to the nucleus. All of the receptors in isolated cytosol do not appear to be available for immediate occupancy by an agonist and this may be due to the time required for conversion of the receptors from inactive to active forms. The correlations between receptor binding and the glucocorticoid response indicate that the receptor is a rate-limiting factor in the magnitude and kinetics of the response, and this finding has important implications regarding mechanisms.
Glucocorticoids and the metabolic code.
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Nuclear binding of glucocorticoid receptors.
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Specific binding of [3H]-16 beta-hydroxydehydroepiandrosterone by rat kidney.
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Regulation of activity of chromatin receptors for thyroid hormone: possible involvement of histone-like proteins.
Thyroid hormone receptors lose their capability for high-affinity binding of the biologically active triiodothyronine after solubilization and separation from other chromatin proteins. The high-affinity triiodothyronine-binding capacity can be reconstituted by addition of a histone-containing extract of chromatin of purified core histones (H2A, H2B, H3, and H4); a number of other acidic or basic proteins tested were ineffective. The data support a model of the receptor in which a "core" receptor subunit that contains a thyroid hormone-binding site interacts with a regulatory subunit, which is possibly a histone or histone-like species. This interaction with the "core" subunit enables the resulting "holo" receptor to bind biologically active hormones. These data also suggest that histones or related proteins can modulate the activity of nonhistone chromosomal proteins that are involved in regulating the expression of specific genes.
Corticotropin and beta-endorphin: construction and analysis of recombinant DNA complementary to mRNA for the common precursor.
A cDNA fragment synthesized from mouse mRNA (ACTH/LPH mRNA) that codes for the precursor polypeptide containing corticotropin (ACTH), beta-lipotropin (LPH), and several other peptides has been cloned in bacteria. The mRNA was enriched for ACTH/LPH mRNA translational activity (to about 75%) prior to cDNA synthesis. It appears to contain about 1200 bases, of which approximately 450 bases are not translated. The cloned DNA fragment is complementary to the region of the mRNA coding for the protein fragment beta-LPH-(44--90); this contains all of the amino acids of [Met]-enkephalin (residues 61--65 of beta-LPH), most of the amino acids of beta-melanocyte-stimulating hormone, and all but the carboxy-terminal amino acid of beta-endorphin. Based on assignment of the amino acid sequence of mouse beta-LPH from the nucelic acid sequence, it appears that there is extensive homology of mouse beta-LPH with human and porcine beta-LPH. The data also establish the linkage between beta-melanocyte-stimulating hormone and beta-endorphin as a Lys-Arg sequence. It is hoped that this cloned DNA can be used as a probe to study the expression and structure of the ACTH/LPH gene.
Structure of genes for human growth hormone and chorionic somatomammotropin.
A 2.6-kilobase (kb) EcoRI restriction endonuclease fragment containing human growth hormone (hGH; somatotropin) gene sequences and a 2.8-kb EcoRI fragment containing human chorionic somatomammotropin (hCS; choriomammotropin) gene sequences have been identified by hybridization to cloned cDNA. Human DNA was cleaved with EcoRI and fractionated by preparative agarose gel electrophoresis; DNA in the size range 2--3 kb was ligated to lambda gt WES.lambda B DNA and viable recombinant bacteriophage were recovered by in vitro packaging. After infection of Escherichia coli and screening of phage plaques, single isolates of hGH and hCS gene sequences were obtained. Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence. Partial DNA sequence analysis of the hGH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment.
Thyroid hormone receptors and responses.
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