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The effects of sex hormones estradiol (E2), testosterone (Te), and 5α-dihydrotestosterone (DT) on cholesterol accumulation induced by modified low density lipoproteins (LDL) in macrophages differentiated from human peripheral blood monocytes and on the levels of mRNAs coding for proteins involved in lipid metabolism have been studied. All three hormones at physiological concentrations (1 nM) are capable of reducing cholesterol accumulation in cells. The treatment of cells with modified and native (not inducing cholesterol accumulation) LDL results in similar alterations in the expression of several mRNAs aimed primarily at homeostatic regulation of lipid metabolism. These alterations depend on the sex of macrophage donors and in some cases are even reversed in cells obtained from male and female donors. The cells not treated with modified LDL have no significant gender differences in the expression of the examined mRNAs. Hormones, either independently or in combination with the modified LDL, influence the levels of some mRNAs, and each hormone shows an individual range of effects. Correlation analysis of changes in mRNA content in the cells showed that the hormones may interfere with coordination of gene expression. Hormone action leads to: (1) reduced coupling of the content of individual mRNAs with their initial levels in the control cells; (2) reduced coupling of different mRNA levels; (3) regrouping of mRNAs between the clusters; and (4) changes in the number of factors that determine the correlation links between mRNAs. The data show that sex hormones may have impact on the level of expression of certain genes and, in particular, on the coordination of gene expression in macrophages.

Contents of mRNAs encoding endosome/lysosome components EEA1, Rab5a, Lamp1, Lamp2, p62 (SQSTM1), and CD63 were measured by quantitative PCR and compared in intact fragments of human aorta and in aorta fragments with atherosclerotic lesions of stage II (fatty streaks) of the same donors. During atherogenesis an increase was detected only in the level of p62 mRNA but not in other mRNAs. Nevertheless, correlation analysis revealed a profound rearrangement of inter-gene correlations: only 30% of correlations found in the fatty streaks coincided with the correlations in normal fragments. Thus, new constellations were formed in fatty streaks concurrently with disappearance of correlations between mRNAs under study and mRNAs encoding factors of lipid accumulation, reverse cholesterol transfer, and some lipid sensors/transcription regulators of lipid metabolism.

The potential role of estrogens in regulation of metabolism in arteries of men was studied. Contents of mRNAs of sex hormone receptors, of some enzymes of their metabolism, and of some potential markers of the hormone effects were determined by real-time polymerase chain reaction in fragments of 18-54-year-old men's large arteries with and without atherosclerotic lesions. Contents of estrogen receptor alpha (ERalpha) and transferrin receptor mRNAs were significantly different in undamaged fragments of the aorta and of the carotid and coronary arteries. Contents of some mRNAs in the carotid artery and aorta were found to correlate, which suggested a similarly directed regulation of their expressions. The levels of ERalpha and aromatase mRNAs negatively correlated with the blood plasma concentration of estradiol. Levels of steroid sulfatase and aromatase mRNAs were lower and the level of estrogen sulfotransferase mRNA was higher in blood vessel fragments with atherosclerotic lesions than in undamaged fragments. It is suggested that large arteries should be different in sensitivity to estrogens and that atherosclerotic lesions could lead to local suppression of the effect of estrogen on the cells of arteries.

The effects of obstructive cholestasis and sex hormones on the total content of prolactin receptor mRNA and ratio of mRNAs of its short and long isoforms have been studied in rat hepatocytes. Obstructive cholestasis caused insignificant changes in total content of prolactin receptor mRNA, but the proportion of mRNA of the long isoform increased. Comparison of prolactin receptor mRNA levels in gonadectomized and intact animals revealed opposite effects of male and female sex hormones on total mRNA, but both groups of these hormones increased the proportion of prolactin receptor short form mRNA. Changes in ratio of mRNA of receptor isoforms found in rat hepatocytes under obstructive cholestasis did not depend on levels of sex hormones. Obstructive cholestasis and sex hormones are suggested to regulate the content of long and short prolactin receptor isoforms in hepatocytes independently.

Two alternative variants of 5'-untranslated sequence of the rat growth hormone receptor (GHR) mRNA were previously described. These variants of mRNA appear to be produced by the splicing of primary transcripts initiated from alternative promoters. We employed the ribonuclease protection assay to reveal these two variants of the GHR mRNA in RNA preparations from the liver of male, normal female, and pregnant female rats. All tissues examined contain approximately the same amount of the GHR mRNA with type II 5'-untranslated sequence. To the contrary, the content of type I variant shows very pronounced sexual differences; its level is very low in the male liver, approximately equal to that of the type II variant in the female liver, and further increases in the pregnant female liver. These data suggest that the higher content of the GHR observed in the female liver and its elevation during gestation result from the accumulation of "female specific" type I variants of the GHR mRNA.

The human growth hormone receptor (GHR) gene was proposed to contain multiple 5'-noncoding exons (Leung et al., 1987). The exact number and structure of these exons are unknown. As a first step in investigating this point more closely, we decided to clone alternative 5'-noncoding sequences of human liver GHR mRNA. The ligation-mediated single-sided polymerase chain reaction (PCR) was applied for selective amplification of 5'-terminal sequences of human liver GHR cDNA. PCR products were cloned and sequenced. Eight different sequence variants diverging in the 5'-untranslated regions beginning 12 base pairs upstream from the initiating ATG codon were found. One variant seems to represent unspliced or partially spliced GHR mRNA. The remaining variants probably correspond to multiple alternatively spliced forms of GHR mRNA. Homologs for three of these variants were found among previously published 5'-noncoding sequences of GHR cDNA obtained from other species by conventional cDNA cloning. Most of the cloned human liver GHR cDNA variants contain one or more ATG preceding the main GHR open reading frame start of translation. Thus, the GHR genes appeared to be a striking example of a very complex transcription unit.