Cytoplasmic prostaglandin E2 synthase is dominantly expressed in cultured KAT-50 thyrocytes,cells that express constitutive prostaglandin-endoperoxide H synthase-2. Basis for low protaglandin E2 production

The recent identification and cloning of two glutathione-dependent prostaglandin E(2) synthase (PGES) genes has yielded important insights into the terminal step of PGE(2) synthesis. These enzymes form efficient functional pairs with specific members of the prostaglandin-endoperoxide H synthase (PGHS) family. Microsomal PGES (mPGES) is inducible and works more efficiently with PGHS-2, the inflammatory cyclooxygenase, while the cytoplasmic isoform (cPGES) pairs functionally with PGHS-1, the cyclooxygenase that ordinarily exhibits constitutive expression. KAT-50, a well differentiated thyroid epithelial cell line, expresses high levels of PGHS-2 but surprisingly low levels of PGE(2) when compared with human orbital fibroblasts. Moreover, PGHS-1 protein cannot be detected in KAT-50. We report here that KAT-50 cells express high basal levels of cPGES but mPGES mRNA and protein are undetectable. Thus, KAT-50 cells express the inefficient PGHS-2/cPGES pair, and this results in modest PGE(2) production. The high levels of cPGES and the absence of mPGES expression result from dramatic differences in the activities of their respective gene promoters. When mPGES is expressed in KAT-50 by transiently transfecting the cells, PGE(2) production is up-regulated substantially. These observations indicate that naturally occurring cells can express a suboptimal profile of PGHS and PGES isoforms, resulting in diminished levels of PGE(2) generation.     

Prostaglandin endoperoxide H synthase expression in human thyroid epithelial cells

KAT-50, an established human thyrocyte cell line, expresses constitutively high levels of prostaglandin endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Here, we examine primary human thyrocytes. We find that they, too, express PGHS-2 mRNA and protein under control culture conditions. A substantial fraction of the basal prostaglandin E(2) (PGE(2)) produced by these cells can be inhibited by SC-58125 (5 microM), a PGHS-2-selective inhibitor. Interleukin (IL)-1beta (10 ng/ml) induces PGHS-2 expression and PGE(2) production in primary thyrocytes. The induction of PGHS-2 and PGE(2) synthesis by IL-1beta could be blocked by glucocorticoid treatment. Unlike KAT-50, most of the culture strains also express PGHS-1 protein. Our observations suggest that both cyclooxygenase isoforms may have functional roles in primary human thyroid epithelial cells, and PGHS-2 might predominate under basal and cytokine-activated culture conditions.     

Interleukin-4 inhibits lipopolysaccharide-induced expression of prostaglandin H synthase-2 in human alveolar macrophages

Several studies have shown that interleukin-4 (IL-4) down-regulates synthesis of prostaglandin E₂ (PGE₂). We evaluated the mechanisms for this suppression in human alveolar macrophages (HAMs). Normal HAMs were obtained from healthy nonsmoking volunteers. The cells either remained unstimulated, or were exposed to 10 μg/ml of lipopolysaccharide (LPS) and/or various amounts of IL-4. LPS alone induced the synthesis of large amounts of PGE₂ and prostaglandin H synthase-2 (PGHS-2) protein. This effect of LPS was suppressed by increasing amounts of IL-4. Expression of LPS-induced PGHS-2 mRNA was also inhibited by IL-4. In addition, IL-4 inhibited expression of CD14, which is a receptor for LPS bound to the LPS-binding protein (LBP). We conclude that IL-4 down-regulates LPS-induced release of PGE₂, by reducing expression of the enzyme, PGHS-2. One potential mechanism for this effect of IL-4 is a reduced expression of CD14, which is the LPS-LBP receptor. © 1995 Wiley-Liss Inc.     

Cyclic AMP response element mediates dexamethasone induced suppression of prostaglandin H synthase-2 gene expression in human amnion derived WISH cells.

A human PGHS-2 promoter fragment (300 BP) linked to the luciferase reporter was used to study the regulation of PGHS-2 gene expression in human amnion-derived WISH cells. A cyclic AMP (cAMP) response element (CRE) was found to be important in the induction of PGHS-2 gene expression. This was demonstrated by showing that coexpression of CREB stimulated native but not CRE mutant promoter and that IL-1beta and PMA induced less activity with the mutant promoter as compared to the native promoter. The effect of dexamethasone on IL-1beta and PMA induced promoter activities was further examined. IL-1beta or PMA induced activity was blocked by dexamethasone, whereas IL-1beta or PMA induced mutant activity was not responsive to dexamethasone. Direct activation of CRE by a cAMP elevating agent, isoproterenol, was found to be inhibited significantly dexamethasone. These results suggest that CRE may mediate the induction of PGHS-2 by IL-1beta and PMA as well as the suppression of expression by dexamethasone in amnion-derived cells.     

Stable high level expression of human thyroid peroxidase in cultured Chinese hamster ovary cells

An expression plasmid containing both human thyroid peroxidase and mouse dihydrofolate reductase cDNAs was transfected into chinese hamster ovary cells. The stably transformed cells constitutively expressed immunoreactive thyroid peroxidase on the cell surface. These cells were further used to establish a subline producing a large amount of thyroid peroxidase by selecting clones resistant to methotrexate. The molecular weight of the expressed thyroid peroxidase was the same as purified human thyroid peroxidase. This expressed protein had peroxidase activity when determined by guaiacol oxidation. Furthermore, the expressed thyroid peroxidase was immunoreactive to sera of patients with autoimmune thyroid disease in which autoantibodies to thyroid peroxidase appeared.     

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.     

Evidence for differential synthesis of thyroxine and tri-iodothyronine by cultured human thyroid cells following exposure to thyrotrophin or dibutyryl cyclic AMP.

Cultured human thyroid cells treated with thyrotrophin (TSH) or dibutyryl cyclic AMP release more tri-iodothyronine (T3) and thyroxine (T4) than unsupplemented cells. Column chromatography was used to investigate the secretion of newly-synthesised 125-I labelled T3 and T4 from cells cultured with 125-I and TSH or dibutyryl cyclic AMP. Radioimmunoassays were used to determine total T3 and T4 release from cells cultured with unlabelled iodide.Iodothyronines released after TSH addition contained more 125-I than those released after dibutyryl cyclic AMP. This increase in 125-I was primarily in “new” T4. Release of “new” T3, however, was increased more by dibutyryl cyclic AMP than by TSH. Dibutyryl cyclic AMP and TSH were comparable in their stimulation of total T3 and total T4 release.Interpretation of these observations suggests that TSH and dibutyryl cyclic AMP may differ in some aspects of their in vitro effects on cellular iodination and iodothyronine coupling systems.     

Retinoid metabolism in cultured human retinal pigment epithelium.

Uptake, esterification and release of all-trans-retinol in primary cultures of human retinal epithelium were studied. Cultured cells were supplemented with 3H-labelled 11,12-all-trans-retinol, using fatty-acid-free albumin as the carrier. This led to incorporation of retinal and the formation of all-trans- and 11-cis-retinyl palmitate. The metabolism of the all-trans ester was monitored in a medium containing various concentrations of foetal-bovine serum (FBS). In 20% (v/v) FBS, the ester was hydrolysed, and all-trans-retinol was released into the culture medium. In the absence of FBS, little ester was hydrolysed and no retinol was found in the medium. Dialysed or heat-inactivated FBS or fatty-acid-free albumin was as effective as FBS in provoking ester hydrolysis and retinol release. The concentration-dependency of this effect on FBS was matched by the corresponding concentrations of albumin alone. A linear relationship was also found between interphotoreceptor retinoid-binding protein and retinoid release. Haemoglobin, which does not bind retinoids, is ineffective in this capacity. It is concluded that lipid-binding substances, mainly albumin, in FBS act as acceptors for retinol and drain the cultured cells of this molecule. The release of the retinol is coupled to the hydrolysis of retinyl esters in the cell, so that there is little or no net hydrolysis of ester if there is no acceptor for retinol in the culture medium. This effect explains why cultured human retinal epithelial cells are depleted of their stores of retinoids when maintained in medium supplemented with FBS.     

Generation of H2O2 is regulated by cytoplasmic free calcium in cultured porcine thyroid cells

Effects of calcium ionophore A23187 and BAY-K-8644, a calcium channel agonist, on cytoplasmic free calcium ([Ca2+]i) and H2O2 generation were studied in cultured porcine thyroid cells. We monitored continuously the effects of A23187 and BAY-K-8644 on [Ca2+]i and H2O2 generation, using the intracellularly trapped fluorescent Ca2+ indicator, fura-2, and homovanillic acid, respectively. A23187 and BAY-K-8644 induce an immediate increase in [Ca2+]i and H2O2 generation. The A23187- and BAY-K-8644-induced [Ca2+]i responses and H2O2 generation occur immediately, reach a maximum within several seconds, and then slowly decline. The minimum doses of A23187 or BAY-K-8644 to increase [Ca2+]i stimulate H2O2 generation. H2O2 generation is regulated by [Ca2+]i.     

Cytokeratin expression in human tongue epithelium.

The epithelium of the human tongue shows diverse morphological variations from one site to another and even within the epithelium of the same papilla. This complexity has led to confusion regarding tongue epithelium as being orthokeratinized, parakeratinized, or nonkeratinized. Cytokeratins have been shown to characterize different epithelia. The present paper describes cytokeratin expression by adult tongue epithelia and relates their distribution to morphology. Six healthy human tongue specimens were obtained after plastic surgery and cytokeratin expression was investigated immunohistochemically, using a panel of 15 antibodies for cytoskeletal proteins, and biochemically using two-dimensional gel electrophoresis. The results showed that the ventral and lateral surfaces of the tongue are related to the nonkeratinizing stratified squamous epithelia, esophageal type, whereas the dorsal surface showed mixed expression of cytokeratins. In the tip of filiform and on the surface of fungiform papillae, cytokeratins of terminal differentiation are expressed as skin type; and in the rest of the papillae as well as in interpapillary areas, the epithelium expresses esophageal type cytokeratins. Certain simple epithelial cytokeratins were found in taste buds. Cytokeratin 19 was also detected in the basal cell layer of all esophageal type epithelia in the tongue. The present results provide basis for studies on the biological events in epithelial differentiation during development and in pathology.     

Fromin vitro toin vivo. Progress in the use of cultured cells for human therapy

The use of cultured cells with the ultimate goal of using the cells or their products for human therapy has experienced an exponential growth during the last decade. Stable cell cultures have been established and genetically modified to obtain high quality products for protein replacement therapy or vaccines. Cells have also been directly isolated from the human organism and, after their expansionin vitro, been retransferred as skin grafts for treatment of burns or for cancer therapy by activated lymphocytes. With the explosive development of molecular biology techniques, it is now possible to genetically modifyex vivo, cells derived from the human body. These modifications should allow targeted expression of therapeutic genes into specific cells which will, upon retransfer to the body, exert their therapeutic action in a diseased organism.Abbreviations ADA adenosine deaminase - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN interferon - IL interleukin - TIL tumor infiltrating lymphocytes     

Evidence for oxytocin receptors in cultured bovine luteal cells.

Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Cultured cells from bovine corpora lutea (CL) at different stages (Days 3-5, 8-12, and 15-18 of the estrous cycle) were examined for OT receptors by a radioreceptor assay using the 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2(9)] -vasotocin. Binding specificity was demonstrated in displacement studies with various related peptides. Scatchard analysis revealed the presence of a binding site with an association constant of Ka = 2.6 x 10(9) M-1 and a capacity of 5.9 fmol/micrograms DNA. Additionally, in 50% of the experiments (n = 6) two different binding sites were observed. The Ka of the high-affinity site was 2.6 x 10(10) M-1; its capacity was 0.73 fmol/micrograms DNA. The low-affinity site had an apparent Ka of 4.9 x 10(8) M-1 and a capacity of 8.8 fmol/micrograms DNA. Observation of one versus two binding sites related neither to the assay conditions nor to the state of the individual CL used for the cell culture and therefore appeared to reflect individual variation within the OT receptor population. Significant binding of OT was observed at all luteal stages. OT binding was maximal at the mid-luteal stage (Days 8-12). We conclude that a direct action of OT on the bovine CL is mediated by the OT receptor, supporting the hypothesis that luteal OT plays an important physiological role in the regulation of progesterone release and/or other luteal functions in a paracrine or autocrine fashion.     

Oxytocin stimulation of RGS2 mRNA expression in cultured human myometrial cells

Regulators of G protein signaling (RGS proteins) interact with Galpha(q) and Galpha(i) and accelerate GTPase activity. These proteins have been characterized only within the past few years, so our understanding of their importance is still preliminary. We examined the effect of oxytocin on RGS2 mRNA expression to help determine the role of RGS proteins in oxytocin signaling in human myometrial cells in primary culture. Oxytocin increased RGS2 mRNA concentration maximally by 1 or 2 h in a dose-dependent and agonist-specific manner. RGS2 mRNA levels were also elevated by treatment with Ca(2+) ionophore, phorbol ester, or forskolin. Oxytocin's effects were completely inhibited by an intracellular Ca(2+) chelator and partially blocked by a protein kinase C inhibitor, indicating that intracellular Ca(2+) concentration is the primary signal for oxytocin elevation of RGS2 mRNA levels. Use of pharmacological inhibitors indicated that part of oxytocin-stimulated RGS2 mRNA expression is mediated by G(i)/tyrosine kinase activities. Although oxytocin does not stimulate increases in intracellular cAMP concentration, agents that elevate intracellular cAMP concentrations and cause myometrial relaxation may possibly cause heterologous desensitization to oxytocin via RGS2 expression. These results suggest that RGS2 may be important in regulating the myometrial response to oxytocin.