Induction of heme oxygenase-1 modulates cis-aconitase activity in lens epithelial cells.

Heme oxygenase-1 is the heme catabolic enzyme induced in human dermal fibroblasts by environmental stress. We report an increase of heme oxygenase-1 message in lens epithelial cells after exposure to UVA radiation, followed by a 10-fold increase of protein expression. The size of message was larger than previously demonstrated for fibroblasts. The relationship between heme oxygenase-1 activation and iron metabolism was investigated by measurement of activities of both cytosolic and mitochondrial cis-aconitase enzymes. A 2-fold increase in mitochondrial cis-aconitase activity in UVA-exposed cells coincided with the time of maximal heme oxygenase-1 expression. We propose that modulation of cis-aconitase activity at the translational level by an increase of cellular iron is an important consequence of heme oxygenase-1 activation. This might be a novel aspect of the protective role of heme oxygenase-1 in modulating the response of cells challenged with oxidative stress.

Interleukin-1beta activates protein kinase C zeta in renal mesangial cells. Potential role in prostaglandin E2 up-regulation.

Protein kinase C (PKC) plays a role in signal transduction mediated by interleukin-1beta (IL-1beta) leading to the increase in prostaglandin E2 (PGE2) production. In the present study we suggest that there are at least two distinct PKC isotypes involved in the signaling mechanism. Staurosporine potentiated the effect of IL-1beta on coxII mRNA expression while calphostin C totally inhibited mRNA expression. The down-regulation of PKC by growing mesangial cells in the presence of phorbol 12-myristate 13-acetate for 24 h failed to modify the up-regulated response in PGE2 formation by IL-1beta. Furthermore, incubation of mesangial cells with IL-1beta causes translocation of PKCzeta from cytosol to a presumed membrane compartment, and this translocation phenomenon was not inhibited by incubating the cells with staurosporine but was inhibited with calphostin C. Gel retardation assays also demonstrated that staurosporine did not inhibit the IL-1beta-stimulated binding of nuclear extracts to the NFkappaB motif. In contrast, calphostin C inhibited binding to the kappaB motif in a dose-dependent manner. Finally, antisense oligonucleotides to PKCzeta partially inhibited the IL-1beta-induced PGE2 formation while control sense oligonucleotides were without effect. Taken together, these data suggest that PKCzeta is involved in the IL-1beta signaling responses.

IL-1 beta regulates rat mesangial cyclooxygenase II gene expression by tyrosine phosphorylation.

The pro-inflammatory cytokine, interleukin-1 beta, induces the mRNA for prostaglandin endoperoxide synthase II gene in renal mesangial cells. This inductive effect is selective for prostaglandin endoperoxide synthase II and not prostaglandin endoperoxide synthase I. In the present experiments IL-1 beta increased COX II mRNA, and this was inhibited by genistein and herbimycin A, both inhibitors of protein tyrosine kinases. The dose dependent effect of genistein on inhibition of mRNA for COX II correlated with the inhibition of the release of PGE2 into the media. Induction of COX II by interleukin-1 beta was mimicked by incubating the cells in the presence of a protein tyrosine phosphatase inhibitor, vanadate. These experiments also illustrate selective induction of COX II mRNA without induction of COX I mRNA. Western analysis utilizing antiphosphotyrosine antibodies demonstrated in whole lysates of mesangial cells treated with interleukin-1 beta that the transient phosphorylation of several proteins occurred. Interleukin-1 beta induced the transient phosphorylation of a protein of about 39/40 kD. Similarly, vanadate also produced a rapid and transient phosphorylation of a protein of about 39/40 kD in addition to other proteins. Immunoprecipitation of mesangial cell lysates with agarose conjugated antiphosphotyrosine antibody and Western analysis of precipitated proteins with anti-ERK2 antibody demonstrate that the 39/40 kD protein phosphorylated on tyrosine is ERK2 and suggests participation of one of the MAP kinase family of extracellular receptor kinases in IL-1 beta stimulated induction of the COX II gene.

Human luteinizing hormone and chorionic gonadotropin are targeted to a regulated secretory pathway in GH3 cells.

LH is a dimeric glycoprotein hormone that is stored in the anterior pituitary and is released in response to GnRH, while the placental hormone, human CG (hCG), sharing the same alpha-subunit and a related beta-subunit, is secreted constitutively. In search of a determinant that allows sorting of LH into a regulated secretory pathway, the genes encoding the common alpha- and LH/CG beta-subunits were expressed in the GH3 rat pituitary tumor cell line, which contains a regulated secretory pathway. Steady state labeling and subsequent chase experiments showed that not only LH but also hCG can be sorted to a regulated secretory pathway; after an initial period of constitutive secretion, the mature forms of both hormones containing processed oligosaccharides were stored intracellularly, and their release was stimulated by either forskolin or KCl depolarization. In Chinese hamster ovary cells, which lack a regulated pathway and are devoid of storage granules, only hormones containing unprocessed N-linked oligosaccharides were found. In GH3 cells the LH beta-subunit was partially retained in an endoglycosidase H-sensitive form, presumably in the endoplasmic reticulum; the enzyme-resistant fraction was secreted through a regulated secretory pathway. A large fraction of the hCG beta-subunit was released constitutively, although some mature hCG beta-subunit accumulated in secretory granules and was released by forskolin. The common alpha-subunit was secreted constitutively with little intracellular accumulation of the mature forms. We conclude that the LH beta-subunit contains sufficient information to direct LH to a regulated pathway, and alpha:LH beta assembly is not a prerequisite for this targeting. The sorting of hCG to a regulated pathway in GH3 cells presumably reflects a structural similarity between LH and hCG. In addition, we have shown that GH3 cells can recognize the N-linked oligosaccharides on the gonadotropin subunits as substrates for sulfation.

Regulation of prostaglandin endoperoxide synthase gene expression in rat mesangial cells by interleukin-1 beta.

In primary cultures of rat mesangial cells from passage 3 to 6, interleukin-1 beta (IL-1) induced a time-dependent increase in prostaglandin E2 (PGE2) formation and release into the extracellular medium. This increase was associated with a dramatic upregulation of the steady-state levels of mRNA for the prostaglandin endoperoxide synthetase (PES)-2 gene transcript as demonstrated by Northern analysis. In contrast, there did not appear to be a significant increase in the mRNA levels for a 2.8-kb transcript for the PES-1 gene. At 18 h of exposure to IL-1, the steady-state level of message for PES-2 remained elevated at 50% of the 2-h time point. Culturing the cells in the presence of cycloheximide and IL-1 demonstrated a superinduction of the PES-2 message without any change in PES-1 message. The tumor-promoting phorbol ester, phorbol myristate acetate (PMA), was also associated with an upregulation of the message for the PES-2 gene and did not influence the levels of the message for the PES-1 gene as demonstrated by Northern analysis. Dexamethasone (Dex) inhibited to control levels the induction by PMA, but the induction of the message by IL-1 was only inhibited 30%. Despite 70% of the message being present by 2 h of induction, Dex was capable of totally inhibiting the inductive effect of IL-1 with respect to PGE2 biosynthesis. Immunocytochemical studies demonstrated a dramatic induction of PES-2 protein by IL-1, which was inhibited by Dex. The data suggest that Dex inhibits the translation of the PES-2 protein.

Changes in thermodependence of the tyrosine transaminating activity in mitochondria of Drosophila melanogaster larvae due to environmental stress.

1. The form of Arrhenius plots of enzyme in mitochondria isolated from Drosophila melanogaster larvae exposed to heat shock, ethanol, or ethanol and heat shock, solubilized with charged detergents was analysed. 2. Heat shock and ethanol caused different changes in membrane microenvironment of the tyrosine transaminating activity, which found expression in different forms of Arrhenius plots, and different values of activation energy of enzyme. 3. The Arrhenius plots of the enzyme from mitochondria of larvae exposed both to ethanol and heat shock, solubilized with charged detergents, were similar to those observed for mitochondria from organisms exposed only to ethanol.

Anoxia-induced changes in the resting potential of the cerebellar cells in tissue culture.

Membrane potentials of Purkyne cells, granular cells, astrocyte cells and oligodendrocyte cells were measured in the cerebral tissue culture under normal conditions and after anoxia. An increment of the membrane potential value with age of culture was found. After anoxia the resting potential decreased with exception of 2-week Purkyne cells and granular cells. The highest anoxia-induced decrease of the membrane potential of all the cells studied was observed in 3-week tissue culture.