Specific and Redundant Functions of Retinoid X Receptor/Retinoic Acid Receptor Heterodimers in Differentiation, Proliferation, and Apoptosis of F9 Embryonal Carcinoma Cells

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)α and retinoic acid receptor (RAR)α genes or the RXRα and RARγ genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRα^−/−, RARα^−/−, and RARγ^−/− cells. RXRα^−/−/ RARα^−/− cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRα^−/− cells. In contrast, RXRα^−/−/RARγ^−/− cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRα and RARγ represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype– and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.     

Role of Vitamin D3 Receptor in the Synergistic Differentiation of WEHI-3B Leukemia Cells by Vitamin D3 and Retinoic Acid

WEHI-3B D⁻ cells differentiate in response to 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)₂D₃ interact to produce synergistic differentiation of WEHI-3B D⁻ cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)₂D₃ receptor (VDR) and retinoid receptors, RARα and RXRα, was measured. No VDR was detected in untreated WEHI-3B D⁻ cells; however, RA and 1,25-(OH)₂D₃ when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARα and RXRα were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)₂D₃ produced synergistic differentiation of WEHI-3B D⁻ cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D⁺ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)₂D₃ in WEHI-3B D⁺ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)₂D₃ and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1,25-(OH)₂D₃.     

Reciprocal regulation of 17β-estradiol, interleukin-6 and interleukin-8 during growth and progression of epithelial ovarian cancer

Estrogens have been associated with risk for epithelial ovarian cancer (OVCA). Both IL-6 and IL-8 are also likely involved in the progression of OVCA. In order to discover the underline molecular mechanism, we investigated the modulation of estrogen and two cytokines in the growth and progression of epithelial OVCA. In these studies, the effect of 17β-estradiol (E₂) on the expression levels of IL-6, IL-8 and their receptors was investigated. The effect of IL-6 and IL-8 on activation of estrogen-responsive promoter as well as estrogen receptor (ER)α and ERβ expression was also analyzed. Gene expression profile analysis revealed that CAOV-3 and OVCAR-3 cells, which express ER, IL-6 and IL-8 receptors, are suitable model for this study. We found that E₂ not only enhanced IL-6 and IL-8 production via NF-κB signaling pathway, but also modulated their respective receptor expression. Tamoxifen (Txf), an ER antagonist, completely abolished E₂-stimulated cell growth and the expression of IL-6 and IL-8. IL-6/IL-8-induced cell proliferation was completely blocked by their specific neutralizing antibodies, which partially inhibited E₂-induced cell growth. In the absence of estrogen, both cytokines activated estrogen-responsive promoter, which was completely blocked by Txf, and caused a dose-dependent ERα increase and ERβ decrease. Pretreatment of OVCAR-3 with p38 MAPK, MEK1/2 or ErbB2 MAPK inhibitors, respectively, blocked IL-6-mediated induction of estrogen-responsive promoter while Src inhibitor blocked IL-8-induced activation of estrogen-responsive promoter. These results provide a novel mechanism that estrogens, IL-6 and IL-8 may form a common amplifying signaling cascade to modulate OVCA growth and progression. Estrogen-induced OVCA proliferation is partially occurring via enhanced IL-6 and IL-8 production and modulated their receptors, and IL-6/IL-8 could also promote OVCA growth through an ERα pathway.     

An RXR-Selective Analog Attenuates the RARα-Selective Analog-Induced Differentiation and Non-G1-Restricted Growth Arrest of NB4 Cells

NB4, a human acute promyelocytic leukemia cell line expressing the promyelocyte–retinoic acid receptor α (PML–RARα) hybrid protein was treated with RAR- and retinoid X receptor (RXR)-selective analogs to determine their effects on cell proliferation, retinoblastoma (RB) tumor-suppressor protein phosphorylation, and differentiation. An RAR- or just RARα-selective analog alone induced similar cell population growth arrest, cell cycle arrest without restriction to G₁, hypophosphorylation of RB, and myelomonocytic cell surface differentiation marker expression (CD11b). In addition, an RARα antagonist could inhibit the effects of the RARα agonist completely. The RARα-selective analog-elicited response was attenuated by simultaneous addition of various RXR-selective analogs. In contrast, each of the RXR-selective analogs was unable to induce any of the cellular responses analyzed. The growth arrest of NB4 cells is not G₁-restricted and occurs at all points in the cell cycle. Cells growth arrested by treatment with an RARα-selective analog show primarily hypophosphorylated RB. When these cells are sorted into G₁or S + G₂/M subpopulations by flow cytometry, hypophosphorylated RB protein was in G₁as well as S + G₂/M cells. This suggests that the hypophosphorylated RB protein may be mediating the growth arrest of NB4 cells at all points in the cell cycle. These results are consistent with an involvement of PML–RARα and/or RARα in the transduction of the retinoid signal in NB4 cells.     

Effects of analogues of prostaglandins E2 and F2α on uterine luminal fluid accumulation in the estrogen-treated ovariectomized rat: An indirect measure of cervical tone

Experiments were performed to determine if prostaglandins were able to reduce cervical tone in the rat. Cervical tone was assessed indirectly by measuring uterine luminal fluid accumulation in ovariectomized rats implanted subcutaneously with Silastic capsules containing crystalline estradiol-17β. When given subcutaneously in separate experiments, 16,16-dimethyl-prostaglandin E₂, methyl ester, and 15(S)-15-methyl-prostaglandin F_2α, analogous of prostaglandins E₂ and F_2α, respectively, caused the loss of uterine luminal fluid. Fluid accumulation in uterine horns ligated at the cervical end did not differ in control and treated rats, whereas in non-ligated horns the prostaglandin analogues reduced fluid accumulation, suggesting the cervix as their site of action. For both prostaglandin analogues, the effects on uterine luminal fluid accumulation were seen within 45 min of administration and were related to the dose administered. The effects of submaximal doses of the analogues were additive. These results suggest that prostaglandins are able to reduce cervical tone in the estrogen-treated rat.     

Changes in Ovaries and Uterus after Aglepristone Administration in the Third Week of Luteal Phase of Non-Pregnant Bitches

Objective

The mechanism of aglepristone action in the placentation time in the bitch remains unclear. The aim of this study was to describe the mechanism by which aglepristone influences ovaries and uterus and to measure the levels of steroid sex hormones in non-pregnant bitches.

Materials and Methods

Fourteen bitches assigned to a study (n=9) and control (n=5) group were given aglepristone and saline solution, respectively, on the 19th and 20th day after LH peak. On the 26th day after LH peak an ovariohysterectomy was performed. Blood samples were screened for estradiol and progesterone concentrations. Ovaries and uterine horns and bodies were isolated for histological and morphometrical diagnosis and immunohistochemistry analysis of α-estrogen and progesterone receptor expression.

Results

A decrease of progesterone (p<0.01) and no differences in total estrogen level in the study group were observed. There were no significant differences either in the histomorphometry or α-estrogen and progesterone receptors expression in ovaries. Increase in expression of progesterone receptors in endometrium without surface epithelium of horns (p<0.05), endometrial surface epithelium (p<0.05), myometrium of uterine body (p<0.01) and estrogen receptors in endometrium without surface epithelium of horns (p<0.05) was observed. Elevated estrogen receptors probably increased sensitivity of tissues to estrogens in the bloodstream and led to notable inflammation, haemorrhages, and hyperplasia in endometrium with mononuclear immune cell infiltration. The myometrium of horns and endometrium of uterine body of study bitches were significantly thicker than in the control group (p<0.05 and p<0.01). Furthermore myometrium of uterine body was thicker than myometrium of horns (p<0.001) and expression of progesterone receptors was higher in uterine body (p<0.01). No differences were observed between endometrium of horns and body within groups.

Conclusion

To the knowledge of the authors this is the first study, which describes the inflammatory effect developing in uterus in response to aglepristone administration, and attempts to elucidate its mechanisms.     

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation.

Measurements and Main Results

In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture.

Conclusions

We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.     

Bombesin enhances TGF-β growth inhibitory effect through apoptosis induction in intestinal epithelial cells

Mammalian intestinal epithelium undergoes continuous cell turn over, with cell proliferation in the crypts and apoptosis in the villus. Both transforming growth factor (TGF)-β and gastrin-releasing peptide (GRP) are involved in the regulation of intestinal epithelial cells for division, differentiation, adhesion, migration and death. Previously, we have shown that TGF-β and bombesin (BBS) synergistically induce cyclooxygenase-2 (COX-2) expression and subsequent prostaglandin E₂ (PGE2) production through p38^MAPK in rat intestinal epithelial cell line stably transfected with GRP receptor (RIE/GRPR), suggesting the interaction between TGF-β signaling pathway and GRPR. The current study examined the biological responses of RIE/GRPR cells to TGF-β and BBS. Treatment with TGF-β1 (40 pM) and BBS (100 nM) together synergistically inhibited RIE/GRPR growth and induced apoptosis. Pretreatment with SB203580 (10 μM), a specific inhibitor of p38^MAPK, partially blocked the synergistic effect of TGF-β and BBS on apoptosis. In conclusion, BBS enhanced TGF-β growth inhibitory effect through apoptosis induction, which is at least partially mediated by p38^MAPK.     

Differential Ligand Binding Affinities of Human Estrogen Receptor-α Isoforms

Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with K_d values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.     

Estrogen Responsiveness and the Estrogen Receptor during Development of the Murine Female Reproductive Tract

The developing uterus, vagina, and cervix of mice whose age ranged from 16 days of gestation to 90 days postnatal were examined for nuclear estrogen receptors (ERs) by autoradiographic and whole cell uptake techniques. ERs were present within mesenchymal cells of these organs throughout the entire period of development and maturation. By contrast, nuclear ER first became detectable by autoradiography in the epithelium of vagina and uterus at 5 and 6 days postnatal, respectively.
As a result of administration of the synthetic estrogen, diethylstilbestrol (DES), consecutively from 16 to 18 days of gestation, uterine and vaginal epithelial cell height was increased and epithelial secretory activity was elevated during the first 48 hr of postnatal life. Also, a single does of DES administered on the 2nd day after birth stimulated epithelial proliferation in the uterus as determined by ³H-thymidine incorporation. These typical estrogenic effects occurred in the absence of nuclear ER within the epithelium. Prenatal DES treatment accelerated the onset of ER activity within the epithelium by 2 to 3 days relative to controls. The possibility that certain effects of estrogen on epithelial differentiation may be mediated indirectly via ER positive mesenchymal cells is discussed.