Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells. (9/55562)

To investigate which retinoid receptors are critical in the regulation by all-trans-retinoic acid (RA) of the mucin genes MUC2, MUC5AC and MUC5B in cultured normal human tracheobronchial epithelial (NHTBE) cells, we used pan-RAR-, pan-RXR- and RAR- isotype (alpha, beta and gamma)-selective agonists and RARalpha- and RARgamma-selective antagonists (RAR is RA receptor and RXR is retinoid X receptor). RAR-, RARalpha- and RARgamma-selective agonists strongly induced mucin mRNAs in a dose-dependent manner, while the RARbeta-selective retinoid only weakly induced mucin gene expression at very high concentrations (1 microM). The pan-RXR-selective agonist by itself did not induce mucin gene expression, but acted synergistically with suboptimal concentrations of the pan-RAR agonist. A retinoid with selective anti-activator-protein-1 activity only marginally induced mucin gene expression. The RARalpha antagonist strongly inhibited mucin gene induction and mucous cell differentiation caused by RA and by the RARalpha- and RARgamma-selective retinoids. In contrast, the RARgamma antagonist only weakly inhibited RARalpha-selective-retinoid-induced mucin gene expression, but completely blocked mucin gene expression induced by the RARgamma-selective retinoid. Our studies indicate that RARalpha is the major retinoid receptor subtype mediating RA-dependent mucin gene expression and mucous cell differentiation, but that the RARgamma isotype can also induce mucin genes. Furthermore these studies suggest that RARbeta is probably not (directly) involved in RA-induced mucin gene expression.  (+info)

Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus. (10/55562)

Human UDP-glucuronosyltransferases (UGTs) are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues [Strassburg, Manns and Tukey (1998) J. Biol. Chem. 273, 8719-8726]. Previous work suggests that these enzymes play a protective role in chemical carcinogenesis [Strassburg, Manns and Tukey (1997) Cancer Res. 57, 2979-2985]. In this study, UGT1 and UGT2 gene expression was investigated in human oesophageal epithelium and squamous-cell carcinoma in addition to the characterization of individual UGT isoforms using recombinant protein. UGT mRNA expression was characterized by duplex reverse transcriptase-PCR analysis and revealed the expression of UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mRNAs. UGT1A1, UGT1A3, UGT1A4, UGT1A5 and UGT1A6 transcripts were not detected. UGT2 expression included UGT2B7, UGT2B10 and UGT2B15, but UGT2B4 mRNA was absent. UGT2 mRNA was present at significantly lower levels than UGT1 transcripts. This observation was in agreement with the analysis of catalytic activities in oesophageal microsomal protein, which was characterized by high glucuronidation rates for phenolic xenobiotics, all of which are classical UGT1 substrates. Whereas UGT1A9 was not regulated, differential regulation of UGT1A7 and UGT1A10 mRNA was observed between normal oesophageal epithelium and squamous-cell carcinoma. Expression and analysis in vitro of recombinant UGT1A7, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 demonstrated that UGT1A7, UGT1A9 and UGT1A10 catalysed the glucuronidation of 7-hydroxybenzo(alpha)pyrene, as well as other environmental carcinogens, such as 2-hydroxyamino-1-methyl-6-phenylimidazo-(4, 5-beta)-pyridine. Although UGT1A9 was not regulated in the carcinoma tissue, the five-fold reduction in 7-hydroxybenzo(alpha)pyrene glucuronidation could be attributed to regulation of UGT1A7 and UGT1A10. These data elucidate an individual regulation of human UGT1A and UGT2B genes in human oesophagus and provide evidence for specific catalytic activities of individual human UGT isoforms towards environmental carcinogens that have been implicated in cellular carcinogenesis.  (+info)

Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and lactoferrin receptor orf3 isogenic mutants. (11/55562)

Pathogenic members of the family Neisseriaceae produce specific receptors to acquire iron from their host's lactoferrin and transferrin. Recently, putative Moraxella catarrhalis lactoferrin receptor genes and a third open reading frame (lbpB, lbpA, and orf3) were cloned and sequenced. We describe the preliminary characterization of isogenic mutants deficient in LbpB, LbpA, or Orf3 protein.  (+info)

Expression of B7 costimulatory molecules by salivary gland epithelial cells in patients with Sjogren's syndrome. (12/55562)

OBJECTIVE: To investigate the expression of B7 costimulatory molecules in the lymphoepithelial lesions of salivary gland (SG) biopsy tissues and in SG epithelial cell lines derived from patients with Sjogren's syndrome (SS). METHODS: B7.1 and B7.2 protein expression was studied by immunohistochemistry in minor SGs obtained from 11 patients with SS and 10 disease control patients with nonspecific sialadenitis and in cultured SG epithelial cell lines obtained from minor SGs from 15 SS patients and 15 control patients. B7.1 and B7.2 messenger RNA (mRNA) expression by SG epithelial cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In biopsy tissues from SS patients, but not control patients, ductal and acinar epithelial cells showed increased expression of both B7.1 and B7.2. Intense spontaneous B7.1 protein expression (as well as HLA-ABC, but not B7.2 or HLA-DR) was also found in 73% of SG epithelial cell lines from SS patients versus 13% of those from control patients (P < 0.01). Interferon-y treatment induced, or up-regulated, B7.1, B7.2, and HLA-DR expression in all SG epithelial cell lines tested. B7.1 and B7.2 expression by SG epithelial cell lines was also verified at the mRNA level by RT-PCR. CONCLUSION: Human SG epithelia are intrinsically capable of expressing B7 proteins upon activation. In SS patients, the expression of B7 molecules by SG epithelial tissues and by SG epithelial cell lines indicates the activated status of SG epithelial cells in this disorder and, possibly, their capacity for presenting antigens to T cells.  (+info)

Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligand-mediated prostaglandin E2 release. (13/55562)

OBJECTIVE: To assess the expression and function of purine receptors in articular chondrocytes. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to screen human chondrocyte RNA for expression of P1 and P2 purine receptor subtypes. Purine-stimulated prostaglandin E2 (PGE2) release from chondrocytes, untreated or treated with recombinant human interleukin-1alpha (rHuIL-1alpha), was assessed by radioimmunoassay. RESULTS: RT-PCR demonstrated that human articular chondrocytes transcribe messenger RNA for the P1 receptor subtypes A2a and A2b and the P2 receptor subtype P2Y2, but not for the P1 receptor subtypes A1 and A3. The P1 receptor agonists adenosine and 5'-N-ethylcarboxamidoadenosine did not change PGE2 release from chondrocytes. The P2Y2 agonists ATP and UTP stimulated a small release of PGE2 that was potentiated after pretreatment with rHuIL-1alpha. PGE2 release in response to ATP and UTP cotreatment was not additive, but release in response to coaddition of ATP and bradykinin (BK) or UTP and BK was additive, consistent with ATP and UTP competition for the same receptor site. The potentiation of PGE2 release in response to ATP and UTP after rHuIL-1alpha pretreatment was mimicked by phorbol myristate acetate. CONCLUSION: Human chondrocytes express both P1 and P2 purine receptor subtypes. The function of the P1 receptor subtype is not yet known, but stimulation of the P2Y2 receptor increases IL-1-mediated PGE2 release.  (+info)

Alternative splicing generates multiple mRNA forms of the acetylcholine receptor gamma-subunit in rat muscle. (14/55562)

The fetal type acetylcholine receptor, composed of the alphabeta gammadelta subunits, has shown a highly variable channel kinetics during postnatal development. We examine the hypothesis whether such a variability could result from multiple channel forms, differing in the N-terminus of the gamma-subunit. RT-PCR revealed, in addition to the full-length mRNA, three new forms lacking exon 4. One of them in addition lacks 19 nucleotides from exon 5, predicting a complete subunit, with a 43 residues shorter N-terminus. A third one lacking the complete exon 5 predicts a subunit without transmembrane segments. These forms, generated by alternative splicing, may account for the kinetic variability of the acetylcholine receptor channel.  (+info)

Retinoic acid stimulates the expression of 11beta-hydroxysteroid dehydrogenase type 2 in human choriocarcinoma JEG-3 cells. (15/55562)

The syncytiotrophoblasts of the human placenta express high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme responsible for the inactivation of glucocorticoids. It has been proposed that the placental 11beta-HSD2 serves as a barrier to protect the fetus from high levels of maternal cortisol. To examine the hypothesis that nutritional signals regulate the expression of 11beta-HSD2 in placental syncytiotrophoblasts, we investigated the effects of retinoic acids (RAs), the major metabolites of vitamin A, on the expression of 11beta-HSD2 using human choriocarcinoma JEG-3 cells as a model. This trophoblast-like cell line displays a number of functional similarities to the syncytiotrophoblast. Treatment for 24 h with all-trans RA (1-1000 nM) resulted in a dose-dependent increase in 11beta-HSD2 activity with a maximal effect (increase to 3-fold) at 100 nM. The effect of all-trans RA (100 nM) was also time-dependent in that the effect was detectable at 6 h and reached its maximum by 48 h. Similar increases in 11beta-HSD2 activity were observed when the cells were treated with 9-cis RA. Results from semi-quantitative reverse transcription-polymerase chain reaction demonstrated that there was a corresponding increase in 11beta-HSD2 mRNA after RA treatment. Moreover, treatment with actinomycin D (100 ng/ml) abrogated the increase in 11beta-HSD2 mRNA induced by RA, indicating an effect on transcription. In conclusion, the present study has demonstrated for the first time that RA, at physiological concentrations, induces 11beta-HSD2 gene expression and enzyme activity in JEG-3 cells. If this occurs in vivo, the present finding suggests that high expression of 11beta-HSD2 in the human placenta may be maintained, at least in part, by dietary intake of vitamin A.  (+info)

Ontogeny of expression of a receptor for platelet-activating factor in mouse preimplantation embryos and the effects of fertilization and culture in vitro on its expression. (16/55562)

Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent ether phospholipid. It is one of the preimplantation embryo's autocrine growth/survival factors. It may act via a G protein-linked receptor on the embryo; however, the evidence for this is conflicting. The recent description of the intracellular form of the PAF:acetlyhydrolase enzyme as having structural homology with G proteins and Ras also suggests this as a potential intracellular receptor/transducer for PAF. This study used reverse transcription-polymerase chain reaction to examine the ontogeny of expression of the genes for these proteins in the oocyte and preimplantation-stage embryo. Transcripts for the G protein-linked PAF receptor were detected in the late 2-cell-stage embryo and in all stages from the 4-cell stage to blastocysts. They were also present in unfertilized oocytes and newly fertilized zygotes but only at relatively low levels. The incidence of expression was generally low and variable in late zygotes and early 2-cell embryos. Expression past the 2-cell stage was alpha-amanitin sensitive. The results indicated that mRNA for this receptor is a maternal transcript that was degraded during the zygote-2-cell stage. New expression of the receptor transcript required activation of the zygotic genome. Fertilization of embryos in vitro caused this transcript not to be expressed in the zygote. Culture of zygotes (irrespective of their method of fertilization) caused expression from the zygotic genome to be retarded by more than 24 h. This retardation did not occur if culture commenced at the 2-cell stage. The transcripts for the subunits of intracellular PAF:acetylhydrolase were not detected in oocytes or at any stage of embryo development examined, despite their being readily detected in control tissue. This study confirms the presence of the G protein-linked PAF receptor in the 2-cell embryo and describes for the first time its normal pattern of expression during early development. The adverse effects of in vitro fertilization (IVF) and embryo culture on the expression of this transcript may be a contributing factor for the poor viability of embryos produced in this manner. The reduced expression of PAF-receptor mRNA following IVF predicts that such embryos may have a deficiency in autocrine stimulation and also suggests that supplementation of growth media with exogenous PAF would be only partially beneficial. The effect of IVF and culture may also explain the conflicting literature.  (+info)