Molecular analysis of human endometrium: short-term tibolone signaling differs significantly from estrogen and estrogen + progestagen signaling.

Tibolone, a tissue-selective compound with a combination of estrogenic, progestagenic, and androgenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. The current study compares the endometrial gene expression profiles after short-term (21 days) treatment with tibolone to the profiles after treatment with estradiol-only (E(2)) and E(2) + medroxyprogesterone acetate (E(2) + MPA) in healthy postmenopausal women undergoing hysterectomy for endometrial prolapse. The impact of E(2) treatment on endometrial gene expression (799 genes) was much higher than the effect of tibolone (173 genes) or E(2) + MPA treatment (174 genes). Furthermore, endometrial gene expression profiles after tibolone treatment show a weak similarity to the profiles after E(2) treatment (overlap 72 genes) and even less profile similarity to E(2) + MPA treatment (overlap 17 genes). Interestingly, 95 tibolone-specific genes were identified. Translation of profile similarity into biological processes and pathways showed that ER-mediated downstream processes, such as cell cycle and cell proliferation, are not affected by E2 + MPA, slightly by tibolone, but are significantly affected by E(2). In conclusion, tibolone treatment results in a tibolone-specific gene expression profile in the human endometrium, which shares only limited resemblance to E(2) and even less resemblance to E2 + MPA induced profiles.

Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor (alpha) in the adult rodent heart.

Relatively little is known concerning the regulation of uncoupling proteins (UCPs) in the heart. We investigated in the adult rodent heart 1) whether changes in workload, substrate supply, or cytokine (TNF-alpha) administration affect UCP-2 and UCP-3 expression, and 2) whether peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the expression of either UCP-2 or UCP-3. Direct comparisons were made between cardiac and skeletal muscle. UCP-2, UCP-3, and PPARalpha expression were reduced when cardiac workload was either increased (pressure overload by aortic constriction) or decreased (mechanical unloading by heterotopic transplantation). Similar results were observed during cytokine administration. Reduced dietary fatty acid availability resulted in decreased expression of both cardiac UCP-2 and UCP-3. However, when fatty acid (the natural ligand for PPARalpha) supply was increased (high-fat feeding, fasting, and STZ-induced diabetes), cardiac UCP-3 but not UCP-2 expression increased. Comparable results were observed in rats treated with the specific PPARalpha agonist WY-14,643. The level of cardiac UCP-3 but not UCP-2 expression was severely reduced (20-fold) in PPARalpha-/- mice compared to wild-type mice. These results suggest that in the adult rodent heart, UCP-3 expression is regulated by PPARalpha. In contrast, cardiac UCP-2 expression is regulated in part by a fatty acid-dependent, PPARalpha-independent mechanism.

Metabolic effects of rexinoids: tissue-specific regulation of lipoprotein lipase activity.

Hypertriglyceridemia is a frequent complication accompanying the treatment of patients with either retinoids or rexinoids, [retinoid X receptor (RXR)-selective retinoids]. To investigate the cellular and molecular basis for this observation, we have studied the effects of rexinoids on triglyceride metabolism in both normal and diabetic rodents. Administration of a rexinoid such as LG100268 (LG268) to normal or diabetic rats results in a rapid increase in serum triglyceride levels. LG268 has no effect on hepatic triglyceride production but suppresses post-heparin plasma lipoprotein lipase (LPL) activity suggesting that the hypertriglyceridemia results from diminished peripheral processing of plasma very low density lipoproteins particles. Treatment of diabetic rats with rexinoids suppresses skeletal and cardiac muscle but not adipose tissue LPL activity. This effect is independent of changes in LPL mRNA. In C2C12 myocytes, LG268 suppresses the level of cell surface (i.e., heparin-releasable) LPL activity without altering LPL mRNA. This effect is very rapid (t(1/2) = 2 h) and is blocked by the transcriptional inhibitor actinomycin D. These studies demonstrate that RXR ligands can have dramatic effects on the post-translational processing of LPL and suggest that skeletal muscle may be an important target of rexinoid action. In addition, these data underscore that the metabolic consequences of RXR activation are distinct from either retinoic acid receptor or peroxisome proliferator-activated receptor activation.

Unloaded heart in vivo replicates fetal gene expression of cardiac hypertrophy.

The cardiac response to increased work includes a reactivation of fetal genes. The response to a decrease in cardiac work is not known. Such information is of clinical interest, because mechanical unloading can improve the functional capacity of the failing heart. We compared here the patterns of gene expression in unloaded rat heart with those in hypertrophied rat heart. Both conditions induced a re-expression of growth factors and proto-oncogenes, and a downregulation of the 'adult' isoforms, but not of the 'fetal' isoforms, of proteins regulating myocardial energetics. Therefore, opposite changes in cardiac workload in vivo induce similar patterns of gene response. Reactivation of fetal genes may underlie the functional improvement of an unloaded failing heart.

Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines.

Estrogen action in target cells: selective requirements for activation of different hormone response elements.

RENE1 cells, an estrogen receptor positive rat uterine endometrial cell line immortalized with the E1A oncogene, were analyzed for the presence of estrogen-dependent signal transduction pathways using the induction of transfected as well as endogenous genes. RENE1 cells express the estrogen receptor as analyzed by Northern blots and ligand binding assays (40 fmoles/mg protein). The receptor system appears functional, based on the induction of reporter constructs containing the consensus estrogen response element (ERE) in transient transfection assays and alterations in endogenous transcripts visualized by utilizing differential display methodology. However, neither transfected repoter constructs containing the c-fos ERE, nor the endogenous c-fos, c-jun, or c-myc genes are induced by estrogens in these cells despite being induced by estrogens in the uterus in vivo. In addition, estradiol did not induce endogenous c-fos expression or the activity of CAT reporters containing the c-fos ERE in a stable transfectant of RENE1 cells with a 3-fold elevation in estrogen receptor content. Under identical conditions, TPA and serum rapidly induce c-fos transcription in RENE1 cells, indicating that the lack of inducibility by estradiol is not due to a general inhibitor of transcription of these genes. These results suggest that RENE1 cells lack factors present in normal uterine cells which are required for the estrogenic induction of a specific subset(s) of EREs. These observations support the generally evolving hypothesis that steroid hormones may act through composite response elements via interactions with other transcription factors, in addition to functioning as homodimers at classical palindromic response elements.

Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene.

The extracellular nuclease of Serratia marcescens is regulated in a complex fashion. Unlike most catabolic enzymes, it appears not to be substrate regulated. However we have shown it to be regulated by an SOS-like system in S. marcescens. Additionally nuclease expression is regulated in a growth-phase-dependent manner. In this work we demonstrate that growth-phase-dependent regulation is at the transcriptional level. The putative LexA-binding site which mediates SOS regulation is shown to act as an operator site in vivo. The boundaries of a minimal promoter, still regulated by growth phase and SOS regulation, are defined along with the transcriptional start site. However, a region upstream of the nuclease promoter is shown to enhance significantly the expression of nuclease.

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum.

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis.

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation.

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.

Growth and differentiation of wild type amoebae of Physarum polycephalum in liquid culture.

A method for growing wild type amoebal strains of Physarum polycephalum in two-membered liquid culture is presented. The medium is a simple buffered salts solution. We found that a minimal level of divalent cation was required for growth. All amoebal strains tried to date have grown under our conditions in stationary culture. Growth under gyratory conditions was only successful at 60 rpm or less and consistent growth required a period of adaptation over several transfers. Differentiation of two apogamic strains, CL and CH1, were compared. Contrary to the results seen on agar plates, the time of onset for the first committed amoebae was identical for both strains in liquid culture. Attempts to demonstrate mating between two genetically compatible amoebal strains grown together in liquid culture were not successful.

Analysis of an inducer of the amoebal-plasmodial transition in the myxomycetes Didymium iridis and Physarum polycephalum.

Recent studies with amoebae of the Myxomycetes Didymium iridis and Physarum polycephalum reveal that competence to undergo sexual fusion or to differentiate to plasmodia apogamically can be induced by a diffusible factor. Suspension cultures of Didymium amoebae have proven to be a good source of the inducer activity. Cell-free culture supernatants accelerated mating of Didymium and Physarum amoebae and also apogamic differentiation of the Physarum strain Colonia . Characterization and purification of the inducer activity is being conducted with a semiquantitative bioassay based on the induction of zygote formation in suspensions of mating-compatible Didymium amoebae.

The effect of proteases on plasmodium formation in Didymium iridis.

An improved assay for quantitatively measuring the number of plasmodia formed with time is presented. Using this assay we have investigated the effects of three proteases, subtilisin PBN', subtilisin carlsberg and alpha-chymotrypsin. We have shown that 1) plasmodium formation is sensitive to protease treatment only during the first 2 h after mixing amoebae of compatible mating type but not after, 2) amoebae are protease sensitive when treated 1 h prior to mixing, 3) the two clones used have different sensitivities to protease treatment and 4) these effects are due to enzymatic activity and have little effect on viability. The meaning of these results in relation to recent evidence for a diffusible inducer of plasmodium formation is discussed.