Immunomodulatory effects of progesterone and selective ligands of membrane progesterone receptors.

Progesterone (P4) and its analogues regulate various reproductive processes, such as ovulation, implantation, pregnancy maintenance and delivery. In these processes, an important role is played by the immune cells recruited to the female reproductive organs and tissues, where they are exposed to the action of P4. Progestins regulate cellular processes, acting through nuclear steroid receptors (nSRs), membrane P4 receptors (mPRs), and through the sensors. It remains unclear, what type of receptors is used by P4 and its derivatives to exert their effect on the immune cells and how similar their effects are in different types of these cells. We have previously synthesized new progesterone derivatives, among which two selective mPRs ligands, not interacting with nSRs were identified. The objective of this study was to examine the effects of P4 and new selective mPRs ligands on the expression of pro- and anti-inflammatory cytokines in activated human peripheral blood mononuclear cells (PBMCs), THP-1 monocyte cells, and Jurkat T cells. It was demonstrated that the action of P4 and selective ligands was unidirectional, but in different types of the immune cells, their effects were different, and sometimes even opposite. In PBMCs, exposure to these steroids resulted in the increase of mRNA and secreted protein levels of IL-1ß, TNFα, and IL-6 cytokines, as well as in the increase of INFγ mRNA level, decrease of IL-2 mRNA level, increase of TGFß mRNA level, and decrease of IL-4 mRNA and IL-10 secreted protein levels. In monocytes, similarly to PBMCs, expression of IL-1ß and TNFα mRNA was increased, but expression of IL-10 was also increased, and the TGFß expression statistically significantly remained the same. In Jurkat T cells, expression of IL-2 and TNFα mRNA decreased, while expression of IL-10 increased, and expression of TGFß did not change. Thus, progestins act on the immune cells through mPRs and have both pro- and anti-inflammatory effects, depending on the phenotypes of these cells. The data obtained are important for understanding the complexity of the immune system regulation by progestins, which depends on the type of the immune cells and individual characteristics of the immune system.

Agonistic and Antagonistic Effects of Progesterone Derivatives on the Transcriptional Activity of Nuclear Progesterone Receptor B in Yeast Model System.

Identification of progesterone selective agonists and antagonists that act through one of the nuclear progesterone receptor isoforms is of particular importance for the development of tissue-specific drugs in gynecology and anticancer therapy. Fourteen pregna-D'6- and pregna-D'3-pentarane progesterone derivatives with 16α,17α-cycloalkane groups and two progesterone 3-deoxyderivatives were examined for their ability to regulate transcriptional activity of human nuclear progesterone receptor isoform B (nPR-B) expressed in Saccharomyces cerevisiae yeast. Transcriptional activity of nPR-B was measured from the expression of the ß-galactosidase reporter gene with a hormone-responsible element in the promoter. Among the compounds tested, two were full progesterone agonists, four were partial agonists, one compound possessed both agonistic and antagonistic activity, one compound displayed only partial antagonistic activity, and eight compounds did not show any activity. Modifications of the pentarane structure, precisely, introduction of an additional double bound in the A or B rings and/or modification at the 6th position of progesterone, lead to a switch from the complete agonistic activity to partial agonistic or mixed activities. These modifications enable progestins to act as selective modulators of progesterone receptor. Steroids with reduced A-ring and 3-ketogroups lose their ability to regulate PR-B activity. Both 3-deoxycompounds, being selective ligands of progesterone membrane receptors, do not affect PR-B activity.

Selection of Progesterone Derivatives Specific to Membrane Progesterone Receptors.

The search of selective agonists and antagonists of membrane progesterone receptors (mPRs) is a starting point for the study of progesterone signal transduction mechanisms mediated by mPRs, distinct from nuclear receptors. According to preliminary data, the ligand affinity for mPRs differs significantly from that for classical nuclear progesterone receptors (nPRs), which might indicate structural differences in the ligand-binding pocket of these proteins. In the present work, we analyzed the affinity of several progesterone derivatives for mPRs of human pancreatic adenocarcinoma BxPC3 cell line that is characterized by a high level of mPR mRNA expression and by the absence of expression of nPR mRNA. The values were compared with the affinity of these compounds for nPRs. All tested compounds showed almost no affinity for nPRs, whereas their selectivity towards mPRs was different. Derivatives with an additional 19-hydroxyl group and removed 3-keto group had the highest selectivity for mPRs. These results suggest these compounds as the most selective progesterone analogs for studying the mechanisms of progestin action via mPRs.

[Progestins and Carcinogenesis].

Progesterone and its analogs may exert opposite effects on cell proliferation, apoptosis, and epithelial-mesenchymal transition, leading to higher cell motility and metastasis. Their ultimate effect is determined by a number of factors: the structure and concentration of the steroid, its affinity for various forms of steroid hormone receptors, activation of nongenomic mechanisms, the composition and proportion of different progesterone receptors and sensors, activity of various signaling pathways, the set of transcription factor coregulators, DNA accessibility in chromatin, activity of steroid-metabolizing enzymes, intercellular interactions within tissues, the hormonal status of the body, disease stage, and species-specific features. The review considers the factors that determine the role progestins play in proliferation and apoptosis of human tumor cells of various origins.

[Molecular basis and tissue specificity of the progestins action].

The review considers the effect of progestins on the function, proliferation, and apoptosis of cells of various organs in health and noncancerous disorders. Data are summarized to describe the mechanism of progestin action through various progesterone receptors and sensors and the regulation of their levels. The effects of progestins depend on the cell phenotype, including the composition and proportion of different receptors, activity of signaling pathways, and expression of transcription factor coregulators and steroid metabolism enzymes. The role paracrine regulation plays in the progestin effect is described. Particular attention is paid to the progestin effect on the tissues where progestins are thought or known to affect carcinogenesis or to stimulate or suppress the tumor growth, that is, to modulate cell proliferation, apoptosis, and the epithelial-mesenchymal transition.

Coordination in gene expression during atherogenesis.

General tendencies in the regulation of gene expression during atherogenesis were investigated using correlation analysis for 34 mRNA species of several functional groups. The contents of mRNA were measured by quantitative PCR in samples of human aortal intima containing no lesions or atherosclerotic lesions of types I (initial lesions), II (fatty streaks), and Va (fibroatheromas). The coupling between mRNA contents in lesions and the same mRNAs in intact tissue was found to descend in the course of the disease progression. The data are in accordance with the opinion that successive morphologic types of atherosclerotic lesions correspond to steps of atherogenesis. In addition, the contents of individual mRNA species could correlate with each other within the given sample type, the extent of this coupling rising along with the disease progression. The exception from this rule was a collapse in coupling for several functional groups of mRNA in lesions of type I. This collapse could indicate special position of these lesions in pathogenesis. Statistically significant correlations between mRNAs found in samples of all four types comprised in total about 50% of all possible correlations. 66% of these correlations were conservative, i.e. observed in at least two sample types. By coupling-strength, the studied mRNAs could be divided into four clusters whose composition significantly varied along with the disease progression. The disease progression was also associated with decline in number of regulatory factors that determine coordination in expression of the analyzed genes.

Changes in levels of gene expression in human aortal intima during atherogenesis.

Changes in the contents of 36 mRNAs species related to lipid turnover, inflammation, metabolism and the action of sex hormones in samples of aortal intima along the "intact tissue - lesions of type I - lesions of type II - lesions of type Va" sequence were analyzed using quantitative PCR. The expression of several mRNAs coding for components of the vesicular transfer and lipid turnover machinery was found to be resistant to atherogenesis or even decline in the course of atherogenesis. Decrease in expression was also recorded for steroid sulfatase, androgen receptor, and low density lipoprotein receptor mRNAs. However, the contents of the majority of other mRNA species increased gradually during disease progression. The earliest changes found as early as in lesions of type I were characteristic for estrogen sulfotransferase, apolipoprotein E, scavenger receptor SR-BI, collagen COL1A2, as well as chemokine CCL18 mRNAs. The contents of several mRNAs in intact tissue and atherosclerotic injuries had gender differences. Additionally, responses of two mRNAs, for aromatase and sterol regulatory element binding protein 2, to atherosclerotic lesion were also sex-differentiated. The contents of the majority of analyzed mRNAs in peripheral blood monocyte-derived macrophages were higher than in intact aorta. The correlations found in atherosclerotic lesions between mRNA species that predominant in macrophages and those expressed at comparable levels in macrophages and intact aorta or mainly in aorta suggest that the observed rise in the content of the majority of mRNAs during atherogenesis is determined by increase in expression in resident cells. The data suggest that the revealed absence of homeostatic regulation of expression of a number of genes associated with vesicular transfer and lipid turnover can serve as one of the reasons for lysosomal function insufficiency that leads to foam cell formation in atheroma. The observed sex differences in expression of a number of mRNAs suggest that estrogens in women perform their atheroprotective effects starting with predisposition to the disease and finishing with advanced stages of the pathologic process.

Approaches to the design of selective ligands for membrane progesterone receptor alpha.

A number of progesterone derivatives were assayed in terms of their affinity for recombinant human membrane progesterone receptor alpha (mPRα) in comparison with nuclear progesterone receptor (nPR). The 16α,17α-cycloalkane group diminished an affinity of steroids for mPRα without significant influence on affinity for nPR, thus rendering a prominent selectivity of ligands for nPR. On the contrary, substitution of methyl at C10 for ethyl or methoxy group moderately increased the affinity for mPRα and significantly lowered the affinity for nPR. A similar but even more prominent effect was observed upon substitution of the 3-oxo group for the 3-O-methoxyimino group. A significant preference towards mPRα was also rendered by the 17α-hydroxy group and additional C6-C7-double bond. The data suggest that the modes of ligand interaction with mPRα and nPR in the C3 region of the steroid molecule are different. One can speculate that combination of the above substitutions at C17, C10, C6, and C3 may give ligand(s) with high specificity towards mPRα over nPR.

Effect of sex hormones on levels of mRNAs coding for proteins involved in lipid metabolism in macrophages.

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 in normal human aorta and in stage II of atherogenesis: a hidden regulation.

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.

Comparative contents of mRNAs of sex steroid receptors and enzymes of their metabolism in arterial walls of men.

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.

Localization of a site for interaction with hepatic male-specific proteins in two rat estrogen sulfotransferase genes.

Using electromobility shift assay the interaction of fragments of two paralogous rat estrogen sulfotransferase (Ste) genes with proteins of nuclear extracts from male and female rat liver was studied. Male-specific DNA-protein complexes were revealed with labeled oligonucleotides corresponding to fragments +1150/+1449, +1358/+1449, +1397/+1449, and +1417/+1449 of intron 1 of the Ste1 gene. The removal of a 20 bp region corresponding to the sequence +1430/+1449, or even either 5;- or 3;-terminal 5 bp of this region abolished the selective interaction of the oligonucleotides with the male-specific protein(s). According to the results of the experiments on mutual competition of the oligonucleotides, the fragment of the Ste2 gene corresponding to the sequence +1397/+1449 of the Ste1 gene formed complexes with the same male-specific protein(s) as the fragment of the Ste1 gene did. The data suggest the mapped element to participate in gender differentiation of the expression of the Ste1 and Ste2 genes.

Pregna-D'-pentarane structure influences progesterone receptor affinity for DNA.

Electrophoretic mobility shift assay was used to determine whether pregna-D'-pentaranes allow progesterone receptor (PR) from rat uterine cytosol to bind hormone response element (HRE)-containing oligonucleotide duplexes and to measure the affinity of this interaction. The formation of DNA-protein complexes in low salt medium was progesterone-related ligand-, temperature-, and PR-dependent, and specific for HRE. The highest affinity of PR to DNA (equilibrium K(a) = 0.420 +/- 0.185 nM(-1)) was found in the presence of the partial agonist/antagonist RU486, while the lowest affinity (K(a) = 0.074 +/- 0.013 nM(-1)) was demonstrated with the full agonist 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone. With the exception of the strong full agonist R5020, there was a tendency toward correlation between the induced lower affinity of PR for DNA in the context of tyrosine aminotransferase HRE and the full agonistic activity of tested compounds.

[An androgenic program of expression of the particular estrogen-binding protein is present in male rats in all hepatocytes]. / Androgennaia programma ékspresii osobogo éstrogensviazyvaiushchego belka imeetsia u samtsov krys vo vsekh gepatotsitakh.

Radioligand and immunohistochemical analysis was used for studying the expression of an unusual estrogen-binding protein (UEBP) in the regenerated liver of male rats poisoned with CCl4. As a result of CCl4 poisoning, most hepatocytes intensely producing UEBP and located in the central part of the liver lobules are destroyed (40 to 90% of the liver parenchymal cells). Dead cells are substituted by new hepatocytes formed due to proliferation of periportal hepatocytes in which UEBP is expressed at a low level. In regenerated liver, however, a high level of UEBP expression characteristic of the control animals is restored, as well as a gradient mode of distribution of UEBP-containing cells with the maximum UEBP concentration in hepatocytes surrounding the central vein. The results obtained with animals with either intact or removed testes were similar, except that the dynamics of liver regeneration was slower in the castrated rats. These data suggest the existence in all hepatocytes of the complete androgen-related program for UEBP expression. This program is inherited by daughter cells in the absence of an inducing hormone and is similar in all cells, independently of the extent of its phenotypic expression in hepatocytes with different localization within the liver lobule.

Inheritance of androgen program of male-specific expression of unusual estrogen-binding protein by daughter hepatocytes at rat liver regeneration.

A possibility of inheritance of androgen and basic genetic programs at the level of unusual estrogen-binding protein (UEBP) by daughter hepatocytes was investigated. Liver regeneration after partial (2/3) hepatectomy or after selective poisoning of hepatocytes of the central zone of hepatic lobules with CCl4 in adult rats were used as models of total and zonal proliferation of hepatocytes, respectively. UEBP content and the pattern of its tissue expression in the course of liver regeneration were monitored by radioligand and immunocytochemical technique. In animals of all groups possessing the androgen program of UEBP expression (intact, castrated and/or hypophysectomized males, and ovariectomized females treated with androgen) UEBP content was shown to be similarly high before initiation and after completion of liver regeneration. Unlike in males, in androgenized females a transient 4-fold increase of UEBP concentration on day 4 after partial hepatectomy was observed. In animals with a basic genetic program at the level of this protein (ovariectomized females, neonatally castrated males) only trace amounts of UEBP were observed in intact as well as in regenerated liver. The data were confirmed by immunocytochemical technique. A gradient mode of distribution of UEBP-contained cells within hepatic lobules with the highest specific staining around central veins was found by immunocytochemical technique in males. Specific staining of centrolobular and periportal hepatocytes was 7- to 10-fold in intact, and 4- to 6-fold in castrated and/or hypophysectomized males. In intact females specific staining was distributed uniformly at extremely low levels similar to that in periportal hepatocytes of males. Androgen administration to ovariectomized females stimulated a significant and stable increase of UEBP content in two layers of hepatocytes surrounding the central vein. Profiles of specific staining of hepatocytes within the hepatic lobules similar to that in control animals were observed after the completion of liver regeneration of different groups of rats. The results obtained suggest all the hepatocytes to be targets for androgen programming, natural in males or experimental in females, while the extent of expression of this program depends on the position of a hepatocyte within the liver lobules and the sex of the animal.

[The cellular localization of the unusual estrogen-binding protein in the liver of rats under different hormonal states]. / Kletochnaia lokalizatsiia osobogo éstrogensviazyvaiushchego belka v pecheni krys pri razlichnykh gormonal'nykh sostoianiiakh.

The localization of an unusual estrogen-binding protein (UEBP) was studied in the rat liver using indirect immunoperoxidase reaction in intact rats and after different hormonal influences. The distribution of the UEBR in normal males displays a form of a gradient with the maximum near the central veins. The gradient is absent in normal females. Castration or hypophysectomy of males, or their injection with estradiol or triiodyronine leads to a decrease in the UEBP concentration, though the gradient character of its distribution is persisting. There is a stable increase in the UEBP concentration in the first two layers of cells adjacent to the central veins in the female rat liver after ovariectomy and especially in combination with androgen injections. The revealed changes in the intensity of immunoperoxidase reaction after different hormonal influences correlate with the UEBP concentration in liver studies performed by the radioligand assay.