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PubMed1996

Pretreatment with H2O2 decreases the Ca2+ sensitivity of the exocytosis of glutamate in cerebrocortical synaptosomes.

The treatment of cerebrocortical synaptosomes with low concentrations of H2O2 induces a long-lasting inhibition of the Ca2+ -dependent release of glutamate induced by KCl or ionomycin, without interfering with the cytosolic calcium and without damaging the synaptosomes (Zoccarato, F., Valente, M., and Alexandre, A. (1995) J. Neurochem. 64, 2552-2558). We report now that the inhibition exerted by H2O2 decreases (from 50 +/- 9% to 25 +/- 11%) if exocytosis is triggered by high (80 mM) rather than by low (30 mM) KCl. Similarly the inhibition decreases when glutamate release is triggered by high rather than by low ionomycin. The decreased inhibition by H2O2 on increasing KCl is accompanied by an increase of [Ca2+]i. We conclude that the treatment with H2O2 decreases the CA2+ sensitivity of the synaptosomal exocytotic apparatus.

Animals↗

PubMed1997

Role of intracellular calcium in the angiotensin II-mediated tyrosine phosphorylation and dephosphorylation of PLC-gamma 1.

Angiotensin II induces the rapid temporal tyrosine phosphorylation and activation of phospholipase C-gamma 1 (PLC-gamma 1) and the elevation of intracellular calcium levels. To investigate the relationship of these intracellular signaling events, rat aortic smooth muscle cells were treated with the calcium chelator BAPTA-AM, the calcium channel blocker verapamil, the intracellular calcium antagonist TMB-8, and the calcium ionophore ionomycin. The effects of these agents on PLC-gamma 1 tyrosine phosphorylation were then measured. We found that treatment of these cells with the calcium inhibitors augmented the basal level of PLC-gamma 1 tyrosine phosphorylation, without changing the peak level of tyrosine phosphorylation induced by angiotensin II. The rapid dephosphorylation of PLC-gamma 1 that follows angiotensin II stimulation was prevented by these calcium antagonists. In contrast, angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 was inhibited by ionomycin. These results suggest that the angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 is calcium-independent, while the dephosphorylation is calcium-dependent.

Angiotensin II↗

PubMed1999

Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A2 activation.

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line results in differentiation toward a monocyte/granulocyte-like cell. This differentiation enables the cell to activate cytosolic phospholipase A2 (cPLA2) to release arachidonate upon stimulation. In contrast, undifferentiated cells are unable to release arachidonate even when stimulated with calcium ionophores. In the present research, a role for phospholipase D (PLD) in the regulation of cPLA2 was shown based on a number of observations. First, the ionomycin- and fMLP-stimulated production of arachidonate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol acts as an alternate substrate in place of water for PLD producing phosphatidylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA2 came from the observation that the stimulated production of diacylglycerol (for which phosphatidic acid is a major source) was greatly diminished in undifferentiated cells as compared to differentiated cells. Moreover, the normally deficient activation of cPLA2 in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence of GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) production which appears to activate PLD and cPLA2 in subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA2 by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PIP2 production.

Adenosine Triphosphate↗

PubMed2001

Rac and p38 kinase mediate 5-lipoxygenase translocation and cell death.

5-Lipoxygenase (5-LO) is a key enzyme involved in the synthesis of leukotrienes from arachidonic acid, and its activation is usually followed by translocation to the nuclear envelope. The details of mechanisms involved in the translocation of 5-LO are not well understood, though Ca(2+) is known to be essential. Here we show that ionomycin, a Ca(2+) ionophore, induces 5-LO translocation and necrotic cell death in Rat-2 fibroblasts, suggesting a potential relationship between activation of 5-LO and cell death. These effects were markedly attenuated in Rat2-Rac(N17) cells expressing a dominant negative Rac1 mutant. Pretreatment with SB203580, a specific inhibitor of p38 MAP kinase, or EGTA, a Ca(2+) chelator, likewise diminished ionomycin-induced 5-LO translocation and cell death, but PD98059, a MEK inhibitor, did not. Thus, Rac and p38 MAP kinase appear to be components in a Ca(2+)-dependent pathway leading to 5-LO translocation and necrotic cell death in Rat-2 fibroblasts.

Animals↗

PubMed1994

Cyclosporin A and FK506 inhibit activation-induced cell death in the murine WEHI-231 B cell line.

The WEHI-231 B lymphoma cell line expresses the phenotype of immature B cells. Cross-linking of surface IgM induces programmed cell death (PCD) with typical features of apoptosis demonstrated by the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Activation of protein kinase C (PKC) by phorbol esters was reported to protect WEHI-231 cells against apoptosis induced by ligation of antigen receptors. It was therefore hypothesized that PCD could result from a defect in PKC response with an imbalance in the phosphoinositide pathway in favor of Ca2+ mobilization. In support of this hypothesis, we show here that apoptosis can be readily triggered by the calcium ionophore ionomycin. Furthermore, pretreatment of cells with cyclosporin A or FK506 which inhibit selectively the phosphoprotein calcineurin, a calcium-and calmodulin-dependent serine/threonine phosphatase, protects WEHI-231 cells against apoptosis induced by ionomycin or ligation of surface IgM. Unlike phorbol esters, cyclosporin A did not impair the rise of intracellular Ca2+ induced by cross-linking of antigen receptors. Altogether, the data indicate that the phosphorylation status of yet undefined key cellular substrates controls the cellular response to calcium-dependent apoptotic signals in this B cell lymphoma.

Animals↗

PubMed1994

Deregulated expression of c-fos disturbs proliferative responses of B cells to sIg cross-linking.

B cell activation by surface immunoglobulin (sIg) cross-linking is accompanied by transient expression of the c-fos protooncogene. This expression is strictly controlled in the B cells. To investigate a biological implication of the c-fos expression in the process of B cell activation by sIg cross-linking, we examined the proliferation of splenic B cells from H2-c-fos transgenic mice. Constitutive expression of the c-fos gene perturbs de novo synthesis of RNA and DNA in the B cells stimulated with anti-IgM antibody. Early events of signal transduction such as an increase in intracellular free calcium level and an induction of the endogenous immediate early genes (c-fos and c-myc) were apparently intact in those B cells. When the sIg stimulation of B cells was mimicked by the costimulation with 12-O-tetradecanoylphorbol 13-acetate and ionomycin, H2-c-fos B cells required higher concentrations of ionomycin for the optimal proliferative responses, suggesting that calcium-dependent signal transduction pathways are disturbed in those B cells. These results demonstrate a novel regulatory effect of c-fos protein on the proliferation of B cells mediated by sIg cross-linking.

Animals↗

PubMed2001

Fas/FasL-independent activation-induced cell death of T lymphocytes from HIV-infected individuals occurs without DNA fragmentation.

We assessed the effects of activation with phorbol myrystic acetate (PMA) and ionomycin on peripheral blood mononuclear cells (PBMC) from HIV-infected individuals by (51)Cr release, propidium iodide (PI) uptake, electron microscopy, and DNA analysis. Up to 70% (51)Cr release was induced from PBMC of HIV-infected individuals, versus up to 26% (51)Cr release from PBMC of non-HIV-infected volunteers. Flow cytometry identified mostly T cells undergoing activation-induced cell death (AICD). The kinetics of (51)Cr release and the effects of cold target inhibitors were consistent with cell-mediated cytotoxicity. Certain anti-CD3 antibodies or extracellular Ca(2+) chelation prevented AICD, but antagonistic anti-Fas antibodies, caspase inhibitors, and cycloheximide had no effect. The antioxidants thiourea and N-acetylcysteine reduced AICD, indicating a role for oxidative stress. Electron microscopy revealed plasma membrane disruption with nuclear integrity, while DNA analysis showed intact chromosomal DNA. This form of T cell AICD triggered by PMA and ionomycin differs from classical apoptosis in the absence of either caspase involvement or DNA fragmentation.

Acetylcysteine↗

PubMed1999

Ex vivo assessment of immunosuppression in undiluted whole blood from pigs dosed with tacrolimus (FK506).

To assess the duration of immunosuppression in FK506-dosed pigs, an undiluted whole blood assay was established to measure reactivities of T cells in their physiological milieu. PMA and ionomycin were shown to induce IL-2 production in swine blood. The IC50 of FK506 in inhibiting IL-2 production in whole blood and isolated PBMC stimulated with PMA and ionomycin measured 1.2 and 0.04 nM, respectively. These data underscore the influence of red blood cells and plasma proteins on drug potency. IL-2 levels were determined in blood drawn immediately before and 1, 24, 48, and 72 h after iv dosing. For pigs dosed with 0.05 mg/kg, 50% recovery of IL-2 production was observed at 16 h and 100% at 35 h after dosing. For pigs dosed with 0.15 mg/kg, 50% recovery was observed at 38 h and 100% at 72 h. Blood concentrations of FK506 at 50 and 100% recovery of IL-2 production measured 10.8 and 2.2 nM for pigs dosed with 0.05 mg/kg and 6.1 and 1.1 nM for pigs dosed with 0.15 mg/kg, respectively. These concentrations are severalfold higher than predicted from the IC50 of FK506 for inhibiting IL-2 production in the whole blood assay. These data suggest that the true potency of FK506 in blood after dosing is influenced by additional factors, which could include plasma protein binding, the presence of active or interfering metabolites, serum interfering factors, and sequestration of drug in blood cells. Our results demonstrate the utility of an undiluted whole blood assay for assessing the duration of immunosuppression in drug-dosed animals and emphasize the importance of assessing drug potency in the whole blood environment ex vivo.

Animals↗

PubMed2002

Glutamine protects activated human T cells from apoptosis by up-regulating glutathione and Bcl-2 levels.

Glutamine is the most abundant amino acid in the body. A decrease of plasma glutamine concentrations is found in catabolic stress and is related to susceptibility to infections. Glutamine is known to modulate lymphocyte activation; however, little is known about glutamine modulation of cell death of activated human T cells. Using Jurkat T cells, we investigated glutamine modulation of T-cell apoptosis activated by PMA plus ionomycin. We found that glutamine at various concentrations significantly enhanced IL-2 production, cell proliferation, and cell viability of Jurkat T cells. Glutamine also decreased the number of apoptotic cells stimulated with PMA plus ionomycin as demonstrated by flow cytometry. Meanwhile, glutamine down-regulated CD95 and CD95L expression, but up-regulated CD45RO and Bcl-2 expression in activated T cells. Further investigation of CD95-mediated caspase activities revealed that supplementation of glutamine significantly decreased caspase-3 and caspase-8 activities in activated T cells. Since oxidative stress is closely associated with induction of lymphocyte apoptosis, we found that glutamine significantly increased glutathione (GSH), but decreased reactive oxygen species levels in activated T cells. Blockade of intracellular GSH formation enhanced, but exogenous GSH supplementation decreased, activated T-cell apoptosis. Studying normal peripheral lymphoproliferation, we also found that the presence of glutamine increased lymphoproliferation as well as Bcl-2 and CD95 expression; but decreased CD95L and activation-induced T-cell death. Taken together, glutamine appeared to augment lymphoproliferation but suppressed activation-induced T-cell death in both Jurkat T cells and human peripheral T lymphocytes.

Apoptosis↗

PubMed2002

T-cell antigen receptor peptides inhibit signal transduction within the membrane bilayer.

Previous studies have shown that a synthetic peptide (core peptide, CP) corresponding to a 9-amino-acid region in the transmembrane domain of the alpha subunit of the T-cell antigen receptor (TCR) can suppress T-cell function in vitro and in vivo. The aim of these experiments was to determine the cellular site and molecular mechanism of CP inhibition in T cells. The cytochrome c-sensitive TCR-expressing hybridoma (2B4) was stimulated with pigeon cytochrome c antigen, anti-CD3 crosslinking, or PMA and ionomycin, in the presence or absence of CP, and the resulting IL-2 produced was measured in a bioassay using an IL-2-dependent cell line (CTLL-2). In the presence of CP, IL-2 production was inhibited following antigen-induced stimulation. By contrast, when stimulated with cross-linking antibodies to the CD3 complex or with PMA and ionomycin, both of which activate T cells downstream of the TCR antigen recognition site, CP had no effect on IL-2 production. These experiments suggest that CP interferes with TCR function by inhibiting T-cell activation at the transmembrane/receptor level. In addition, we show that CP inhibits early TCR signal transduction events such as TCR zeta chain phosphorylation following stimulation with either antigen or anti-CD3-crosslinking antibodies, although this is unlikely to be the mechanism leading to the reduced IL-2 production.

Amino Acid Sequence↗

PubMed1994

Ethanol inhibits early events in T-lymphocyte activation.

Ethanol has been reported to be immunosuppressive. We have studied the effects of ethanol on early activation events related to the proliferative response of human T lymphocytes. Ethanol inhibited T-cell proliferation in a dose-dependent manner. To define the target of this ethanol-mediated inhibition of T-cell function we examined its effect on the activation of T lymphocytes or induction of competence (acquisition of responsiveness to interleukin (IL)-2 or IL-4) by phytohemagglutinin (PHA) or the combination of phorbol dibutyrate (PDB)/ionomycin. Ethanol inhibited induction of competence with PHA by up to 50% when compared to control cells. In contrast to the effects on PHA-mediated activation of the cells, ethanol exerted no inhibitory action on the induction of competence by PDB/ionomycin. Ethanol also inhibited the induction of c-fos by PHA but not by PDB/ionomycin. To investigate the basis for these differences, the effects of ethanol on Ca2+ mobilization were examined. Ethanol inhibited PHA-induced Ca2+ mobilization in a dose-dependent manner. This inhibition was exerted mainly on transmembrane Ca2+ influx rather than on release of Ca2+ from intracellular stores. Ethanol did not affect Ca2+ mobilization induced by ionomycin. Co-incubation of ionomycin with PHA, during the induction of competence, abolished the inhibition exerted by ethanol when compared to cells treated with PHA alone. The inability of ethanol to exert complete inhibition on cell proliferation may be due to the activation of Ca(2+)-independent pathways by PHA, since combined treatment with ethanol and the intracellular Ca2+ chelator, BAPTA, did not completely inhibit the proliferative response. The inhibitory effects of ethanol on PHA-induced Ca2+ mobilization and subsequent induction of c-fos indicate that ethanol interferes with Ca(2+)-dependent pathways activated by PHA and this may provide the basis for its immunosuppressive action.

Calcium↗

PubMed1996

Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs.

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.

Animals↗

PubMed2000

Regulation of conjunctival goblet cell secretion by Ca(2+)and protein kinase C.

Conjunctival goblet cells secrete mucus in response to cholinergic (muscarinic) agonists, but the underlying signaling pathways activated in this tissue are not well understood. Cholinergic agonists usually activate phospholipase C to produce inositol 1,4,5 trisphosphate and diacylglycerol. Inositol 1,4,5 trisphosphate increases the intracellular Ca(2+)concentration ([Ca2(+)](i)) while diacylglycerol activates protein kinase C (PKC). PKC and Ca(2+), either by itself or with calmodulin, activate cellular functions. Goblet cell glycoprotein secretion, our index of mucin secretion, was measured from pieces of rat conjunctiva with an enzyme-linked lectin assay using the lectin Ulex europaeus agglutinin I (UEA-I). UEA-I selectively recognizes high molecular weight glycoproteins secreted by the goblet cells. Increasing the [Ca(+)](i)with the Ca(2+)ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca(2+)and by the Ca(2+)/calmodulin-dependent protein kinase inhibitors KN93 and W7 as well as their inactive analogs KN92 and W5. Activation of classical and novel PKC isozymes by phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate stimulated goblet cell glycoprotein secretion. When ionomycin and PMA were added simultaneously, secretion was additive. PKC isozymes were identified by Western blotting analyses with antibodies specific to nine of the 11 PKC isozymes (PKCgamma and zeta were not tested). All nine PKC isozymes were identified in the conjunctival epithelium. The cellular location of the PKC isozymes was determined by immunofluorescence microscopy. Goblet cells contained the classical PKC isozymes PKCalpha, -betaI and -betaII, the novel PKC isozymes PKCepsilon, -theta;, and - mu, and the atypical PKC isozyme PKCzeta. We were unable to determine if PKC activation is required for cholinergic-agonist induced secretion because the PKC inhibitors chelerythrine and staurosporine alone greatly increased secretion. We conclude that Ca(2+)plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca(2+)/calmodulin-dependent protein kinases. We also conclude that activated PKC can stimulate goblet cell secretion and that seven different PKC isoforms are present in the goblet cells.

Animals↗

PubMed1996

Trypanosoma cruzi: involvement of intracellular calcium in multiplication and differentiation.

The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1993

Gonadotropin action on brook trout sperm duct epithelium: ion transport stimulation mediated by cAMP and Ca2+.

The isolated sperm duct epithelium from the brook trout Salvelinus fontinalis serves as a model for gonadotropin (GtH) action and is the only example of direct GtH stimulation of epithelial ion transport. In response to purified salmonid carbohydrate-rich GtH added to either the luminal or blood side of the epithelium, the duct actively secretes K+ (measured as 86Rb+ fluxes) and actively reabsorbs Na+ (measured by 22Na+ fluxes or as short circuit current, Isc). As a consequence of the ion transport, the seminal plasma has low Na+ concentration and high K+ content that in turn keeps developing sperm quiescent prior to spawning. All of the increase in Na+ transport in response to GtH addition can also be evoked by 1.0 mM db-cAMP + 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), indicating that GtH action on Na+ transport is mediated by cAMP. In contrast, 86Rb+ efflux is only partially stimulated by db-cAMP + IBMX. K+ secretion can be stimulated fully by GtH or with the addition of the Ca2+ ionophore ionomycin (1 microM) in combination with db-cAMP + IBMX. Further, the cAMP-stimulated portion of K+ secretion is resistant to the K+ channel blocker Ba2+ (2.0 mM, added to the luminal side) while the ionomycin-stimulated K+ secretion is Ba(2+)- and quinidine (0.1 mM, luminal side)-sensitive. We conclude that GtH acts by two intracellular messengers in this system. The stimulation of Na+ active reabsorption and a Cl(-)-dependent K+ secretion are both mediated by cAMP. A second, Ba(2+)-sensitive K+ secretion is evoked by intracellular Ca2+ and likely represents a group of Ca(2+)-activated K+ channels on the apical membrane of the epithelium.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Effects of Ca2+ on erythrocyte membrane skeleton-bound phosphofructokinase, ATP levels, and hemolysis.

Erythrocyte Ca2+ overload is known to occur in several different disease states, and to affect the erythrocyte membrane deformability. We show here that an increase in intracellular Ca2+ concentration in erythrocytes, induced by ionomycin, caused a reduction in ATP levels. Concomitant to the fall in ATP, a marked activation of phosphofructokinase (PFK) (EC 2.7.1.11), the rate-limiting enzyme in glycolysis, in the membrane skeleton fraction occurred. The increase in the membrane skeleton-bound PFK activity was most probably mediated by Ca2+, as direct addition of Ca2+ to the membrane skeleton fraction from the erythrocyte induced an enhancement of the bound PFK activity. Time-response curves revealed that erythrocyte hemolysis did not occur during the first 30 min of incubation with ionomycin, when the membrane skeleton-bound PFK was activated. Longer incubation time resulted in solubilization of the membrane skeleton-bound PFK and a concomitant hemolysis of the erythrocytes. These results suggest that the Ca2+-induced activation of membrane skeleton-bound PFK, and thereby glycolysis, the sole source of energy in erythrocytes, may be a defense mechanism to surmount the damage induced by high Ca2+ levels.

Adenosine Triphosphate↗

PubMed1999

Regulation of HSD17B1 and SRD5A1 in lymphocytes.

We previously reported lymphocyte expression of genes encoding enzymes required for steroid metabolism; however, only 17beta-HSD and 5alpha-reductase showed significant enzyme activity. We now investigate regulation of lymphocyte expression for genes encoding 17beta-HSD and 5alpha-reductase. Cultured human T and B lymphoid cell lines and peripheral blood mononuclear cells were treated with known regulators of steroidogenic gene expression including forskolin, PMA, ionomycin, various steroids, interleukin (IL)-4, and IL-6. Treatment with 10 or 50 microM forskolin resulted in a 20-60% reduction of expression for HSD17B1 (encoding 17beta-HSD I) in T and B lymphoid cell lines and peripheral blood mononuclear cells, although such a change was not observed in the expression of SRD5A1 (encoding 5alpha-reductase I). No significant changes were found when cells were treated for 24 h with various concentrations of PMA or ionomycin. Incubation with 10(-9) to 10(-7) M androstenedione or estradiol increased expression of HSD17B1, while testosterone decreased the expression of this gene. SRD5A1 expression was increased in the presence of 5alpha-DHT although no consistent changes were observed when the cells were treated with testosterone. Other steroids, including dexamethasone, progesterone, and 6-hydroxypregnanolone, produced no effects on expression of either HSD17B1 or SRD5A1. Treatment with 0.1-10 ng/ml of IL-4 or IL-6 also did not effect significant changes in gene expression. These data implicate the involvement of the cAMP-protein kinase signal transduction pathway in regulating lymphocyte expression of HSD17B1. Furthermore, it appears that lymphocyte HSD17B1 and SRD5A1 are regulated to some extent by specific steroids.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Inflammatory cytokines and the reactivation of Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma (KS) is a complex proliferative lesion long suspected of being dependent on exogenous paracrine signaling molecules to stimulate its proliferative, angiogenic, and inflammatory components. In particular, both clinical and experimental observations have pointed to a potential role for inflammatory cytokines as permissive factors for KS development, but KS pathogenesis is also critically dependent on infection by an exogenous herpesvirus, the KS-associated herpesvirus (KSHV). To examine the possible links between inflammatory cytokines and KSHV replication, we tested for the ability of such cytokines to induce lytic viral reactivation in the latently infected BCBL-1 cell line. Interferon-gamma consistently activated KSHV replication, whereas tumor necrosis factor, interleukin-1, interleukin-2, interleukin-6, granulocyte-macrophage colony stimulating factor, and basic fibroblast growth factor did not. Glucocorticoids also failed to induce lytic KSHV growth in these cells, but ionomycin, a calcium ionophore, induced replication and strongly augmented the known inductive effects of phorbol esters. Interferon-alpha had a dose-dependent inhibitory effect on KSHV induction by ionomycin. The identification of interferon-gamma as an activator and interferon-alpha as an inhibitor of KSHV induction in vitro correlates well with in vivo observations and demonstrates for the first time that inflammatory cytokines can directly modulate KSHV replication.

Cytokines↗