Mitogen-stimulated Increases Research Articles

The Antimalarial Drug Artesunate Inhibits Primary Human Cultured Airway Smooth Muscle Cell Proliferation

Airway smooth muscle (ASM) cell hyperplasia contributes to airway wall remodeling (AWR) in asthma. Glucocorticoids, which are used as first-line therapy for the treatment of inflammation in asthma, have limited impact on AWR, and protracted usage of high doses of glucocorticoids is associated with an increased risk of side effects. Moreover, patients with severe asthma often show reduced sensitivity to glucocorticoids. Artesunate, a semisynthetic artemisinin derivative used to treat malaria with minimal toxicity, attenuates allergic airway inflammation in mice, but its impact on AWR is not known. We examined the effects of artesunate on ASM proliferation in vitro and in vivo. Primary human ASM cells derived from nonasthmatic donors were treated with artesunate before mitogen stimulation. Artesunate reduced mitogen-stimulated increases in cell number and cyclin D1 protein abundance but had no significant effect on ERK1/2 phosphorylation. Artesunate, but not dexamethasone, inhibited phospho-Akt and phospho-p70(S6K) protein abundance. Artesunate, but not dexamethasone, inhibited mitogen-stimulated increases in cell number, cyclin D1, and phospho-Akt protein abundance on ASM cells derived from asthmatic donors. In a murine model of allergic asthma, artesunate reduced the area of α-smooth muscle actin-positive cells and decreased cyclin D1 protein abundance. Our study provides a basis for the future development of artesunate as a novel anti-AWR agent that targets ASM hyperplasia via the PI3K/Akt/p70(S6K) pathway and suggests that artesunate may be used as combination therapy with glucocorticoids.

Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization.

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.

Negative regulation of protein translation by mitogen-activated protein kinase-interacting kinases 1 and 2.

Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP 57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation, and cell proliferation. Our results imply that activation of MNK by MAP kinase pathways does not constitute a positive regulatory mechanism to cap-dependent translation. Instead, we propose that the kinase activity of MNKs, eventually through phosphorylation of eIF4E, may serve to limit cap-dependent translation under physiological conditions.

Utilization of Biotin in Proliferating Human Lymphocytes

Lymphocytes are part of the immune system and respond to antigenic stimulation with proliferation. We sought to determine whether mitogen-stimulated, proliferating lymphocytes increase the cellular uptake of biotin and, if so, to identify mechanisms that mediate the increase. Lymphocytes were isolated from human peripheral blood; proliferation of lymphocytes was induced by incubation with pokeweed lectin, concanavalin A or phytohemagglutinin. Biotin uptake was quantitated by determination of [3H] uptake into the lymphocytes during incubation with [3H]biotin after establishing that [3H]biotin is not metabolized within the lymphocytes during the incubation period (<5%). Biotin uptake into proliferating lymphocytes increased to 278-722% of the control values for nonproliferating lymphocytes. Kinetic analysis of biotin transport provided evidence that the increase is mediated by an increased number of transporters on the cell surface rather than by an increase in transporter affinity. Cycloheximide, an inhibitor of protein synthesis, completely suppressed the mitogen-stimulated increase in biotin transport. This observation is consistent with the hypothesis that proliferating lymphocytes increase biotin uptake by increasing the synthesis of new transporters. Biotin affinity and structural specificity were similar in proliferating and nonproliferating lymphocytes, suggesting that mitogens induced an increase in the number of the same transporter molecule that mediates transport in unstimulated lymphocytes. Mitogen-stimulated lymphocytes exhibited 2.5 times greater activities of biotin-dependent beta-methylcrotonyl-CoA carboxylase compared with time 0 (at 72 h after addition of mitogen). This observation is consistent with the hypothesis that proliferating lymphocytes increase biotin uptake at least in part to provide adequate coenzyme for biotin-dependent carboxylases.

Serum and Glucocorticoid Regulation of Gene Transcription and Expression of the Prostaglandin H Synthase-1 and Prostaglandin H Synthase-2 Isozymes

Mitogenic stimulation has been shown to increase both prostaglandin H (PGH) synthase-1 (PGHS-I) and PGH synthase-2 (PGHS-2) mRNA levels, although the time course and magnitude of induction are different for the two genes. To investigate the mechanism for mRNA induction, we conducted nuclear run-off assays of these two genes in 3T3 cells and correlated mitogen-induced changes in PGHS gene transcription with changes in PGHS mRNA and PGHS isozyme expression. We also examined the mechanism for glucocorticoid inhibition of PGHS mRNA expression and the effects of glucocorticoids on PGHS isozyme expression. Serum stimulation of quiescent 3T3 cells led to a sequential increase in PGHS-2 gene transcription, PGHS-2 mRNA. and PGHS-2 enzyme levels. PGHS-2 gene transcription increased over 25-fold within 30 min of serum addition resulting in an over 70-fold increase in PGHS-2 mRNA by 1 h, and maximal PGHS-2 enzyme expression by 2 h. Increased PGHS-2 isozyme expression thus appears to depend on transcriptional activation of the gene. Transcription of the PGHS-2 gene declined after 30 min, and PGHS-2 mRNA levels declined similarly after 1 h, leading to a return of PGHS-2 levels to near basal levels by 6 h. Glucocorticoids, which previously have been shown to inhibit mitogen-stimulated increases in PGHS-2 levels, were found to inhibit serum-stimulated increases in PGHS-2 gene transcription by 70%, resulting in a 70% reduction in peak serum-stimulated PGHS-2 mRNA levels also. Western blotting with PGHS-2 specific antisera demonstrated that while dexamethasone simply reduced PGHS-2 mRNA levels, it completely suppressed expression of PGHS-2 protein. The coincidental reduction in PGHS-2 transcription, PGHS-2 mRNA, and enzyme levels by dexamethasone, provides further support for the hypothesis that control of transcription is one primary control mechanism for regulating PGHS-2 expression. That complete suppression of PGHS-2 enzyme expression occurs following partial suppression of PGHS-2 mRNA, however, suggests that other mechanisms may also contribute to the glucocorticoid effect. A small, but reproducible, increase in transcription of the PGHS-1 gene occurred 3 h following serum stimulation, coincident with a three- to fourfold increase in PGHS-1 mRNA; PGHS-1 mRNA remained elevated for at least 3 h. Dexamethasone reduced, but did not completely inhibit, the serum-stimulated increases in PGHS-1. However, changes in PGHS-1 mRNA were not accompanied by detectable changes in PGHS-1 protein in the presence or absence of dexamethasone.

Activation of phospholipase D by alpha-thrombin or epidermal growth factor contributes to the formation of phosphatidic acid, but not to observed increases in 1,2-diacylglycerol.

The receptor-mediated activation of a phosphatidylcholine-hydrolysing phospholipase D (PLD) has recently been described. We investigated the effect of alpha-thrombin and epidermal growth factor (EGF) on cellular PLD activity in order to determine the role of this enzyme in mitogen-induced increases in phosphatidic acid and sn-1,2-diacylglycerol. In the presence of ethanol, stimulation of [3H]myristic acid-labelled quiescent IIC9 cells with alpha-thrombin or EGF resulted in a rapid increase in radiolabelled phosphatidyl-ethanol which reached a plateau at 1 min, indicating the rapid and transient activation of PLD. We observed a concomitant decrease in the mitogen-stimulated increase of radiolabelled phosphatidic acid. In contrast, ethanol did not significantly effect the elevation of sn-1,2-diacylglycerol levels stimulated by alpha-thrombin or EGF as determined by measurement of sn-1,2-diacylglycerol mass or the appearance of [3H]1,2-diacylglycerol. A novel lipid, detected by two-dimensional t.l.c. analysis, was generated in [3H]myristic acid-labelled cells stimulated with alpha-thrombin, but not EGF, in the presence of ethanol. Treatment in vitro of cellular lipids isolated from [3H]myristic acid-labelled cultures with PLD in the presence of ethanol also resulted in the generation of this novel lipid species, supporting the role of this enzyme in its production. These data indicate that in quiescent IIC9 cells: (a) alpha-thrombin or EGF rapidly and transiently activates a PLD; (b) although this activation is responsible for part of the mitogen-induced increases in phosphatidic acid, it does not contribute to induced increases in sn-1,2-diacylglycerol; and (c) activation of this enzyme appears to be involved in the formation of a novel lipid generated in response to alpha-thrombin, but not EGF, in IIC9 fibroblasts.

Inhibition of calcium mobilization is an early event in opiate‐induced immunosuppression

Morphine administered as a subcutaneous implant inhibits the initial increase in cytoplasmic free-calcium [Ca2+]i induced by mitogens in mouse splenocytes. This effect was not reproduced by incubation of splenocytes with morphine (10(-8)-10(-4) M). Analysis of splenocyte subpopulations demonstrates that this effect was manifest in both B and T cells. However, within T cell subpopulations, CD4+ but not CD8+ cells were affected. Adrenalectomy abolished this effect of morphine in CD4+ T but not CD4-, CD8- spleen cells (most likely Thy 1.2- B cells). Moreover, simultaneous administration of the opiate antagonist naltrexone blocked the effect of morphine in CD4-, CD8- spleen cells, but not in CD4+ T cells. These data indicate that the effects of morphine on mitogen-stimulated increase in [Ca2+]i may be mediated through distinct glucocorticoid-dependent and -independent mechanisms. The morphine-induced inhibition of an increase in [Ca2+]i in immune cells reported here may be an early event mediating opiate-induced immunosuppression.

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca 2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca 2+]i in CD4 + T cells, but enhanced [Ca 2+]i in CD8 + T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca 2+]i in CD4 + T cells at 3 and 7 days, but not at 21 days. CD8 + T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca 2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4 + T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo . Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.

Transcriptional modulation of human IL-6 gene expression by verapamil.

Calcium channel-blocking agents interfere with the initial increase of cytosolic calcium that follows mitogenic stimulation of T lymphocytes. In cultures of mitogen-stimulated PBMC, verapamil also blocked T cell accumulation of cytoplasmic IL-2-encoding mRNA. In sharp contrast, the addition of verapamil to PHA and PMA-stimulated PBMC augmented the mitogen-stimulated increases in nuclear transcription of IL-6-encoding mRNA, steady state levels of IL-6 encoding mRNA, and release of IL-6 bioactivity. These experiments indicated that an increased IL-6 transcriptional rate rather than stabilization of transcripts accounted for the increased cytoplasmic IL-6 mRNA levels and subsequent expression of IL-6 bioactivity. These effects were not produced by nicardipine, another potent calcium channel blocker, or EGTA. We suggest that a non-calcium-dependent, IL-6 regulatory factor, absent or inactive in verapamil-treated cultures, inhibits IL-6 gene activation in mitogen-stimulated PBMC. Failure to express this inhibitory factor would result in IL-6 gene superinduction at a transcriptional level.

Differential lymphocyte growth-modifying effects of oxidants: Changes in cytosolic Ca +2

An increase in the concentration of cytosolic Ca +2 ([Ca +2] i) is among the earliest changes seen in mitogen-stimulated lymphocytes and is a consequence of signal transduction which usually results in the initiation of cell cycle progression. However, increased [Ca +2] i has also been correlated with cytoxicity. We have determined whether modulations of [Ca +2] i are involved in the functional inactivation of cells observed with sublethal concentrations of oxidants. Specifically, [Ca +2] i was measured in mouse splenic lymphocytes that were treated with different oxidants in order to determine if oxidative stress interferes with mitogen-stimulated increases in [Ca +2] i, if oxidants themselves modulated [Ca +2] i, and, if so, whether such Ca +2 modulations by oxidants had stimulatory or inhibitory effects on the response of lymphocytes to mitogens. The oxidants employed were copper phenanthroline (CuP; surface thiol oxidizer), N-ethyl maleimide (NEM; permeant thiol alkylator), hydrogen peroxide (H 2O 2; generates hydroxyl radical within the cell), and radiation (Cs 137; generates hydroxyl radical by radiolysis). Growth of all treated cells was equally inhibited upon stimulation with Con A or PMA/A23187, suggesting that all the oxidants inhibited cell functions required distal in activation to the transduction pathway utilized by Con A but bypassed by PMA/A23187. Doses of CuP, NEM, and radiation which fully inhibited Con A-stimulated proliferation had little effect on resting or mitogen-stimulated changes of [Ca +2] i, but H 2O 2 doses which fully inhibited proliferation increased [Ca +2] i in unstimulated cells and prevented the increase normally caused by Con A. Both intra- and extracellular Ca +2 contributed to the increased [Ca +2] i seen in unstimulated cells. An elevated [Ca +2] i was sufficient to reduce responsiveness, since pharmacologically increasing the [Ca +2] i with the ionophore A23187 rendered lymphocytes less responsive to Con A. Unlike A23187, H 2O 2 was unable to synergize with PMA, suggesting that the H 2O 2-induced increase of [Ca +2] i delivered predominantly negative signals to the cell. The results also suggest that [Ca +2] i utilization by Con A versus PMA-activated lymphocytes must be different. When cells were treated with H 2O 2 under conditions where intracellular and extracellular Ca +2 were chelated with BAPTA and EGTA, respectively, the response to Con A was restored. Under these conditions, higher concentrations of H 2O 2 were required to inhibit the response to Con A. Our results indicate that signal transduction may be compromised in cells treated with H 2O 2, but not in cells treated with CuP, NEM, or radiation. In addition, H 2O 2 inhibited the response to Con A via Ca +2-dependent and independent pathways, but CuP, NEM, and radiation inhibited the response to Con A via Ca +2-independent pathways only.

T cell receptor-mediated signaling occurs in the absence of inositol phosphate production.

Murine cytotoxic T lymphocytes (CTL) can lyse certain target cells in the presence or absence of extracellular Ca2+ (Ostergaard, H. L., Kane, K. P., Mescher, M. F., and Clark, W. R. (1987) Nature 330, 71-72). We have examined the effect of extracellular Ca2+ on the inositol phosphate response to antigen by CTL. In Ca2+-containing medium relevant antigen-bearing target cells and the mitogen concanavalin A induce a rapid accumulation of inositol phosphates in CTL. In the presence of 4 mM EGTA the antigen- and mitogen-stimulated increases in inositol mono-, bis-, and tris-phosphates cannot be detected, even with 10 mM LiCl added. Abrogation of the inositol phosphate response occurs whether CTL are preincubated in EGTA or EGTA is added with the stimulus. The results indicate that the killing of certain target cells by murine CTL may be independent of the involvement of the phosphatidylinositol pathway. Furthermore, since Ca2+-independent cytolysis remains antigen-specific, the data strongly support the existence of additional T cell receptor-mediated second messenger pathway(s) in CTL.

Pyridine nucleotide analog interference with metabolic processes in mitogen-stimulated human T lymphocytes

The differential metabolic effects of three nicotinamide analogs, 6-aminonicotinamide, 3-aminobenzamide, and 5-methylnicotinamide, were analyzed in mitogen-stimulated preparations of human T lymphocytes. Mitogen stimulation with the phorbol ester TPA and a monoclonal antibody to the T3 cell surface antigen caused an increase in cellular NAD and ATP levels and a marked increase in glucose metabolism as demonstrated by an increase in cellular levels of glucose 6-phosphate and a sevenfold increase in radioactive CO 2 formation from [1- 14C]glucose. 6-Aminonicotinamide had drastic inhibitory effects on the mitogen-stimulated increases in NAD and ATP levels as well as on the metabolism of glucose. Treatment of the mitogen-stimulated cells with 6-aminonicotinamide also caused a marked increase in cellular levels of 6-phosphogluconate, suggesting inhibition of the hexose monophosphate shunt at 6-phosphogluconate dehydrogenase. Radioactive CO 2 formation from [6- 14C]glucose showed that metabolism through the tricarboxylic acid cycle was not used to compensate for the inhibition of the hexose monophosphate shunt pathway. Treatment of cells with 3-aminobenzamide had the opposite effect of 6-aminonicotinamide in that cellular NAD levels increased, presumably due to inhibition of poly(ADP-ribose) polymerase. 3-Aminobenzamide did not interfere with ATP or glucose 6-phosphate levels and did not cause significant elevations of 6-phosphogluconate. Thus, 6-aminonicotinamide appears to have direct inhibitory effects on the synthesis of both pyridine nucleotides and poly(ADP-ribose), whereas 3-aminobenzamide has its major inhibitory effect on poly(ADP-ribose) synthesis. 5-Methylnicotinamide also interferes with the mitogen-stimulated increase in NAD levels but not as effectively as 6-aminonicotinamide. The alterations in pyridine nucleotide metabolism resulting from treatment with these nicotinamide analogs can produce drastic and diverse alterations in pathways of glucose utilization and energy generation.