Dusan T. Kanazir

Psychosocial factors as strong predictors of mortality from cancer, ischaemic heart disease and stroke: The Yugoslav prospective study

We investigated the relation of psychosocial risk factors to mortality in a prospective study of 1353 inhabitants of Crvenka, 619 of whom died between 1966 and 1976. All 38 lung cancer deaths occurred in those with high scores for rationality and antiemotionality (R/A), a factor related to suppression of aggression. Compared with lower R/A, high R/A was also associated with a relative risk of mortality of 29 for other cancer, 4.3 for ischaemic heart disease and 6.5 for stroke. Standardising for R/A reduced the smoking/lung cancer association, virtually eliminated the smoking/other cancer and smoking/heart disease relationships and reduced the association of heart disease with blood cholesterol, blood sugar and hypertension. Long lasting hopelessness was also independently associated with cancer as was anger with heart disease, though not so strongly as for R/A. Psychosocial variables are important predictors of mortality and decisively modify the effect of physical risk factors such as smoking.

Cortisol dependent acute metabolic responses in rat liver cells.

Abstract Cortisol injected into the rat induces immediate metabolic changes in liver cells, occuring within time intervals shorter than that accounted for by the expression of specific genes. Among these extragenomic acute metabolic responses we describe: the enhanced rate of the synthesis of receptor protein for cortisol, the increase in overall capacity for protein synthesis and increased rate of phosphorylation of histones and nonhistone proteins. All these changes depend on cortisol and occur within the first 30 minutes after hormone administration. All these acute metabolic responses to cortisol might be correlated to the hormone-produced activation of the native macromolecular receptor-system. In an attempt to integrate extragenomic and genetic responses to cortisol a new concept and hypothesis of cortisol action is proposed, which can probably be extended to other steroids and extrapolated to other steroid responsive tissues.

Using yeast to study glucocorticoid receptor phosphorylation

The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process.

Adrenalectomy and dexamethasone treatment alter the patterns of basal and acute phase response-induced expression of acute phase protein genes in rat liver

Hormonal requirements for full hepatic expression of alpha2-macroglobulin (alpha2M), alpha1-acid glycoprotein (AGP), haptoglobin (Hp) and gamma-fibrinogen (Fb) were assessed at the level of mRNA. Prior to exposure to turpentine-induced inflammation, rats were either depleted of glucocorticoids by adrenalectomy or supplemented with an excess of dexamethasone. Adrenalectomy alone did not affect the basal level of acute phase protein (APP) expression except for alpha2M mRNA, the level of which was enhanced. In contrast, dexamethasone treatment alone promoted full induction of alpha2M, significant, but not maximal increase of AGP and Hp mRNAs and suppression of Fb. In adrenalectomized rats, acute phase (AP)-cytokines, released in response to inflammation, promoted full expression of Fb and Hp and increased the level of AGP mRNA whereas alpha2M mRNA remained at the basal level. Inflammation in dexamethasone pretreated rats elicited changes which, in comparison to mRNA values for dexamethasone unpretreated inflamed rats, were seen as overexpression of alpha2M, full expression of AGP and incomplete expression of Hp, whereas Fb mRNA remained at the basal level. These data suggest that glucocorticoids are the principal inducers of alpha2M and AP-cytokines of Fb. For full induction of AGP, additive actions of glucocorticoids and AP-cytokines are required whereas expression of Hp is predominantly controlled by AP-cytokines.

The structure and regulatory function(s) of cortisol receptor—1: Extragenomic effects dependent on the cortisol receptor activation

Abstract A large body of recent data is consistent with the idea that steroids, at least sex steroid hormones, regulate gene expression in eucariotic cells via a common two-step molecular mechanism. This two-step model has acquired the status of a “dogma” and has become a generally accepted theoretical framework for experimental research being currently conducted. However, this model (dogma) does not take into consideration the immediate extragenomic effects caused by steroids in responsive tissues, nor does it offer the tentative link between extragenomic and genomic events occurring in target cells. The results of our recent studies on cortisol specific receptor and its regulatory functions lead us to propose a new speculative model, the postulates of which are aimed at integrating both the extragenomic and genomic events occurring in target cells during the course of steroid hormone action. The key concepts of the proposed model are the following: the native cytoplasmic holoreceptor should be a multimer, consisting of several different subunits and comprising the denned “metabolic code” for various multiple cooperative metabolic functions. The “activation” of the receptor, caused by the binding of appropriate steroid hormone, results in the disaggregation of the receptor into the monomeric subunits, which play the role of regulatory proteins. The released subunits—various regulatory proteins—then exert their control at different levels of the mechanism of genetic expression, including the post-translational level. They modulate the preformed molecules and the activities of the regulatory mechanisms operative in respective target cells like for instance phosphorylation/dephosphorylation mechanism. These events result in modifications of translations on the preformed mRNAs and cell membrane transport. The resulting changes are immediate and underlie the steroid-induced extragenomic effects. The subunit(s), which binds the steroid hormone, so called “steroidophilic”—subunits, regulates the rate of transcription, i.e. selective and specific gene expression in target cells. This subunit is a key stone in the macromolecular organization of the receptor. It may be of an isoproteinic nature and may have different affinities towards other subunits thus determining the assembly and nature of other associated subunits, giving rise to the tissue specific receptor polymorphism and causing variations in the “metabolic code” of receptors. “The activation” of receptor, caused by steroids, and resulting in disaggregation of multimer into the monomeric subunits is the key event in the cell responses to steroids. It triggers, controls and links extragenomic events with gene expression. These extragenomic and genomic events are included in the “metabolic program” encoded in the molecular structure of the receptors. The steroids, as effectors trigger the receptor activation, which controls the sequential and selective metabolic events, underlying the specific physiological “responses” of steroid responsive tissues. In this paper we will present some of our recent data to further substantiate the proposed “model” of the integral steroid hormone action in target tissues.

Some properties of cortisol-receptor complex isolated from cytoplasm of rat liver and its effect on biosynthesis of nuclear RNA

Abstract The cortisol binding compound, from the cytoplasm of rat liver, was purified by gel filtration and precipitation with ammonium sulphate. The binding compound from rat liver cytoplasm, has been found to consist of two distinct protein fractions. The proteins eluted from DEAE-cellulose column chromatography at 0.04 M and 0.18 M concentrations of KCl, have two different isoelectric points, one at pH 4.85–5.00, and another at pH 5.85–6,10, but only the fraction eluted at 0.04 M KCl was found to be able to stimulate the incorporation of 32 P into RNA of incubated rat liver nuclei. The highest values of 32 P incorporation in the nucleic acids of incubated nuclei were obtained with partially purified hormone protein (s) complexes wich were precipitated with ammonium sulphate saturation between 20–25% and 25–30%.

The responses of rat liver glucocorticoid receptors and genes for tyrosine aminotransferase, alpha-2-macroglobulin and gamma-fibrinogen to adrenalectomy-, dexamethasone- and inflammation-induced changes in the levels of glucocorticoids and proinflammatory cytokines.

The responses of liver glucocorticoid receptor (GR) and genes coding for a glucocorticoid-inducible tyrosine aminotransferase (TAT) and two acute-phase proteins (APP) [α2-macroglobulin (α2-M) and γ-fibrinogen (Fb)] to changes in glucocorticoid (GC) and proinflammatory (AP) cytokine contents have been examined in rats after single or combined treatments with turpentine oil, dexamethasone (Dex) and adrenalectomy. Activation of two APP genes in turpentine-induced inflammation was accompanied by an increase in the level of GR mRNA and a preferential translocation of GR-GC complexes to the nucleoplasm, while the expression of TAT remained unaltered. Dex alone caused a decrease in the levels of GR and Fb mRNAs, activation of TAT and α2-M genes, a decrease in the affinity of hormone binding sites and redistribution of translocated GR-Dex complexes within the nuclei. Inflammation potentiated the effect which Dex alone exerted on the GR content and the number of GR binding sites but counteracted its influence on the affinity of GR binding sites and nuclear distribution of GR-Dex complexes. Adrenalectomy promoted a fall in TAT mRNA, no changes in the GR and Fb mRNA, a decrease in the affinity of GR hormone binding sites and redistribution of GR-hormone complexes within the nuclei. The AP cytokines released in response to inflammation exerted a counteracting effect on the adrenalectomy-induced changes in the affinity of hormone binding sites and nuclear distribution of GR-hormone complexes. They potentiated a fall of TAT mRNA but promoted full expression of the Fb gene. These results argue strongly for the influence of AP cytokines on the functional state of the GR and GC signaling pathways.

Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae

The glucocorticoid receptor (GR) signal transduction and transcriptional regulation are efficiently recapitulated when GR is expressed in Saccharomyces cerevisiae. In this report we demonstrate that the in vivo GR phosphorylation pattern, hormone dependency and interdependency of phosphorylation events were similar in yeast and mammalian cells. GR phosphorylation at S246 exhibited inhibitory effect on S224 and S232 phosphorylation, suggesting the conservation of molecular mechanisms that control this interdependence between yeast and mammalian cells. To assess the effects of GR phosphorylation the mutated GR derivatives T171A, S224A, S232A, S246A were overexpressed and their transcriptional activity was analysed. These receptor derivatives displayed significant hormone inducible transcription when overexpressed in S. cerevisiae. We have established an inducible methionine expression system, which allows the close regulation of the receptor protein levels to analyse the dependence of GR function on its phosphorylation and protein abundance. Using this system we observed that GR S246A mutation increased its activity across all of the GR concentrations tested. The activity of the S224A and S246A mutants was mostly independent of GR protein levels, whereas the WT, T171A and S232A mediated transcription diminished with declining GR protein levels. Our results suggest that GR phosphorylation at specific residues affects its transcriptional functions in a site selective manner and these effects were directly linked to GR dosage.

THE EFFECT OF CORTISOL ON BIOSYNTHESIS AND PHOSPHORYLATION OF HISTONES IN RAT LIVER

AbstractAdministration of corticosteroid hormones to rats results in an increased activity of several enzymes in liver cells. The induced enzymes are related to the pathway of gluconeogenesis. The mechanism of specific gene activation by hormone action is still not well understood. Recent studies have shown that the steroid hormones combine with specific receptor protein(s) in cytosol of target cells (Jensen et al, 1968; Sherman et al, 1970). The hormone-protein(s) receptor penetrates the liver nucleus and interacts with specific “acceptor sites” of chromatin. These interactions may result in new sites of transcription, i.e. in specific gene activation. It was also demonstrated earlier that the restriction of template activity of chromatin may be due to the presence of the regulatory proteins associated with chromatin (histones) which mask, or repress the function of genome, i.e. play role in gene activation.Although studies on the rate of biosynthesis of histones have given little insight into their phys...

The role of multiply phosphorylated S6 in ribosome degradation

Rat liver ribosomes, prepared 1-24 h after intraperitoneal cortisol injection, contain multiple phosphorylated S6 consisting of four distinct derivatives in addition to the original non-phosphorylated S6. 25 h following the hormone injection the extent of S6 phosphorylation, as judged by its electrophoretic pattern in two-dimensional gels, resembles that of untreated rats. Ribosomal subunits with gradually increased degree of S6 phosphorylation, isolated at different time intervals after cortisol injection, exhibit polyphenylalanine polymerization levels inversely proportional to the extent of S6 phosphorylation. In addition, they show an elevated misincorporation of leucine in a poly(U)-programmed in vitro system. The lower amount of polyphenylalanine synthesized by multiple phosphorylated ribosomes in vitro is likely due to an enhanced susceptibility of nascent polypeptide chains synthesized in the in vitro system to proteinases present in the pH 5 and S-100 fractions. Liver polysomes derived from cortisol-treated animals lose their highly phosphorylated derivatives when exposed to S-100 enzymes. The loss can be prevented by concomitant action of proteinase and RNAase inhibitors (phenylmethylsulfonyl fluoride and heparin) but not by an inhibitor of phosphatase (sodium fluoride). In the absence of RNAase and proteinase inhibitors only degradation of old 40 S subunits can be demonstrated. 25 h after the cortisol treatment degradation of liver ribosomes occurs simultaneously with S6 dephosphorylation and is preceded by polysomal breakdown.