Increased messenger RNA from protein synthesis inhibited human fibroblasts

Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as "superinduction" and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)-cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal 3H-uridine labeled RNA, a 1.5- to 2-fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.     

Shiga toxin-1 regulation of cytokine production by human proximal tubule cells

BACKGROUND: Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) levels are elevated in kidneys of patients with post-diarrheal hemolytic uremic syndrome (D+HUS) and may contribute to renal dysfunction. The renal cellular sources of these inflammatory cytokines in D+HUS are largely unknown, however, the proximal tubule has emerged as a potentially important candidate. Since Shiga toxin-1 (Stx-1) has been implicated in the genesis of D+HUS, we examined the effect of Stx-1 on cytokine production by human proximal tubule cells. METHODS: Stx-1 cytotoxicity, protein synthesis inhibition, and effect on IL-1, IL-6, and TNF protein release and mRNA levels were determined. The effect of another protein synthesis inhibitor, cycloheximide (CHX), on these parameters was also evaluated. RESULTS: Stx-1 greatly increased TNF release and mRNA levels while CHX, at concentrations that produced similar inhibition of protein synthesis, had no effect on TNF production. In contrast, Stx-1 and CHX caused comparable elevations in IL-1 release and mRNA accumulation. Stx-1 and CHX also stimulated IL-6 mRNA accumulation, but only at concentrations that either were cytotoxic or substantially blocked protein synthesis. Finally, lipopolysaccharide, which is likely to be elevated in the circulation of patients with D+HUS, had no effect alone, but synergized with Stx-1 to increase IL-1 production. CONCLUSIONS: These results indicate that Stx-1 stimulates proximal tubule inflammatory cytokine production and that this effect is due partially to nonspecific induction of mRNA levels as well as activation of Stx-1-specific mechanisms.     

Cytokine regulation of granulocyte-macrophage colony stimulating factor and macrophage colony-stimulating factor production in human arterial smooth muscle cells

Smooth muscle cells (SMC) are the major cell type found in the walls of large blood vessels and appear to participate in local immune and inflammatory reactions, as well as in certain vascular diseases. We tested whether human arterial SMC can produce in vitro the colony stimulating factors (CSFs), granulocyte macrophage-CSF (GM-CSF) and macrophage CSF (M-CSF). Untreated internal mammary artery and aortic SMC produced no detectable GM-CSF but constitutively made M-CSF, measured by ELISA and radioimmunoassay, respectively. Interleukin-1 (IL-1) and, to a lesser extent, tumor necrosis factor alpha (TNF alpha) stimulated GM-CSF formation within 3 h; mRNA levels also increased particularly in the presence of the protein synthesis inhibitor, cycloheximide. IL-1, TNF alpha and, in addition, interferon-gamma (IFN-gamma) raised the M-CSF levels within 6 h; cycloheximide potentiated the effects of IL-1 and TNF alpha on mRNA levels. These results suggest that cytokine-stimulated human arterial SMC may be a source of the M-CSF found in atherosclerotic lesions. Since monocytes/macrophages can be activated by GM-CSF and M-CSF, while GM-CSF can also affect granulocyte function, SMC may participate in inflammatory reactions and vascular diseases by releasing these cytokines.     

CD11b/CD18-dependent stimulation of leukotriene B4 synthesis by human neutrophils (PMN) is synergistically enhanced by tumour necrosis factor alpha and low dose diacylglycerol

Unopsonised zymosan particles bind to the CD11b/CD18 integrin on human neutrophils (PMN) and are phagocytosed. Binding stimulates the release of leukotriene (LT) B4. The present study examined the effect on this interaction of two agents that 'prime' PMN for augmented responses to a variety of agonists. The cell permeable diacyl glycerol, 1,2-dioctanoyl-glycerol (DiC8) and TNF alpha each increased CD11b/CD18 expression on PMN [maximal at 10-9 M TNF alpha or 10-8 M DiC8]. There was a decrease, however, in CD11b/CD18 expression above 10-8 M DiC8, which was not observed at high concentrations of TNF alpha. Pre-treatment with either DiC8 or TNF alpha dose-dependently augmented the zymosan-stimulated release of LTB4 from PMN. DiC8 and TNF alpha in combination, however, synergistically increased LTB4 release. In contrast, at concentrations above 10-8 M DiC8, whether in the presence or absence of TNF alpha, LTB4 release was inhibited and this was ameliorated by protein kinase C inhibitors. The response to neither TNF alpha nor DiC8 (below 10-8 M) was kinase inhibitor sensitive. Doses of DAG, which activate protein kinase C, inhibit CD11b/CD18-dependent responses by down-regulating receptor expression. In contrast, the mechanisms of TNF alpha and low dose DAG 'priming' are not clear but are independent of PKC activation. The synergy between these two priming agents, however, suggests independent, complementary signalling pathways that provide a novel, potentially important mechanism for the control of PMN CD11b/CD18 integrin-dependent activation.     

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.     

Abnormal G protein alpha(s) - and alpha(i2)-subunit mRNA expression in bipolar affective disorder

Disturbances of events associated with intracellular signaling pathways have been suspected of involvement in the development or progression of affective disorders. Often, heterotrimeric G proteins are located at the beginning of these pathways as modulators of extracellular messages. For this reason, messenger RNA expression of three G protein alpha-subunits and of phosphatidylinositol-3 kinase (PI-3 K) regulatory subunit p85 was examined in granulocytes from patients with bipolar or unipolar affective disorder and compared to healthy controls. Messenger RNA expression of the G protein subunit alpha(q) and of p85 was identical in unipolar and bipolar patients and in controls. Furthermore, mRNAs of G protein subunits alpha(s) and alpha(i2) were not different in unipolar patients as compared to healthy controls. Alpha(s) mRNA, however, was markedly increased in bipolar patients. This increase was observed in lithium-treated (more than 12 months) and in unmedicated patients. Elevated levels of alpha(i2) mRNA in unmedicated bipolar patients did not reach statistical significance, whereas mRNA in bipolar patients receiving lithium was significantly above controls. Finally, long-term medication of unipolar patients with lithium had no influence on alpha(i2) mRNA levels. The data reveal elevated mRNA levels of G alpha(s) as a robust feature of bipolar affective disorder. Moreover, despite responsiveness of alpha(i2) gene expression to cAMP-related events, no substantial upregulation of alpha(i2) mRNA was observed in bipolar patients. The lack of alpha(i2) mRNA upregulation, hence, could be an additional abnormality in these patients. Even though lithium was able to reinstate this upregulation, there was no feedback downregulation of alpha(s). This strongly supports the notion of major disturbances of the cAMP signaling system in bipolar illness.     

Prolonged heparin after uncomplicated coronary interventions: a prospective, randomized trial

BACKGROUND: Both ischemic and direct vascular injury (angioplasty) result in the elaboration of proinflammatory substances, including tumor necrosis factor alpha (TNF), which may regulate vascular smooth muscle cell (VSMC) proliferation and promote vessel stenosis. Interleukin-10 (IL-10) is a pleiotropic cytokine with potent antiinflammatory effects in many cells lines. We hypothesized that IL-10 could be used therapeutically to influence vascular remodeling by inhibiting TNF-induced VSMC proliferation. The purposes of this study were (1) to determine whether human myocardium produces endogenous TNF in response to ischemia-reperfusion, (2) to examine the effect of TNF on human arterial smooth muscle proliferation, and (3) to explore the potential therapeutic effect of IL-10 on unstimulated and TNF-stimulated VSMC proliferation. MATERIALS AND METHODS: Right atrial muscle was obtained from patients undergoing elective cardiac surgery. Atrial muscle was subjected to simulated ischemia and reperfusion in vitro and TNF was measured by immunoassay. Human aortic VSMCs were isolated and cultured. Proliferation assays were performed to determine the effect of TNF and IL-10 on VSMC growth. RESULTS: Ischemia-reperfusion resulted in an increase in atrial myocellular TNF (94.5 +/- 15.8 pg/g wet tissue versus control 12.9 +/- 4.4 pg/g wet tissue, P < 0.002). Compared with control, TNF stimulated concentration-dependent VSMC proliferation (P < 0.005). IL-10 alone did not influence VSMC growth. However, following TNF stimulation, IL-10 inhibited VSMC growth at a dose as low as 0.1 pg/ml (P < 0.005). CONCLUSIONS: Ischemia-reperfusion insult results in increased endogenous myocardial TNF accumulation. TNF stimulates VSMC growth which is abrogated by physiologically relevant levels of IL-10. This antiinflammatory cytokine may prove to be an effective therapeutic agent in regulating vessel wall remodeling following both ischemic and direct cardiovascular injury.     

Cell cycle-dependent tumor necrosis factor apoptosis

To determine if tumor necrosis factor (TNF)-mediated apoptosis affects cells at defined stages of the cell cycle, WEHI-164/2F (WEHI) cells were synchronized at G0-G1 after 3-day cultures in medium containing RPMI 1640 and 0.5% FCS (RPMI-0.5% FCS). The arrested WEHI cells (60-75% in G0-G1) showed increased sensitivity to TNF killing, measured as 48-h 3-(5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays, and 15-h apoptosis by propidium iodide staining and flow cytometry analysis. The TNF killing kinetics of G0-G1-arrested cells was similar to controls, and TNF did not accelerate or retard cell cycle progression of the arrested cells after feeding with fresh RPMI-0.5% FCS. However, TNF inhibited WEHI DNA synthesis as early as 1 h after treatment, and inhibition was proportionate to sensitivity to TNF-induced apoptosis. WEHI cells treated with TNF showed a higher percentage of cells in S phase with concomitant decrease in G0-G1 and G2-M. When cultured for 3-18 h in fresh RPMI-0.5% FCS to allow progression of the G0-G1-arrested cells toward the G1-S boundary, WEHI cells became more sensitive to TNF killing, especially at the 3-9 h time points. Moreover, TNF did not degrade [125I]5-iodo-2'-deoxyuridine-labeled WEHI DNA if the labeled cells were precultured for 9 h in fresh RPMI-0.5% FCS to allow them to pass S phase before the addition of TNF. These results show that TNF-induced apoptosis of WEHI cells is connected to cell cycle events; WEHI targets receive the TNF cytotoxic signal mainly at the G1-S boundary and begin to die by apoptosis as they exit from S phase.     

Comparison of the glucose-lowering properties of vanadyl sulfate and bis(maltolato)oxovanadium(IV) following acute and chronic administration

The possibility that progesterone or estradiol may regulate expression of G protein in the rat myometrium during the course of pregnancy has been investigated using 1) immunoblot analysis of Gi2 alpha, Gi3 alpha, and Gq alpha subunits and 2) hybridization blot analysis of subunit mRNA. Eighteen hours after administration, estradiol had significantly increased the levels of both Gi2 alpha subunit and Gi2 alpha mRNA (by 40% and 32%, respectively). In control pregnant rats, we observed similar changes at the end of pregnancy, when myometrial concentrations of estradiol had increased, i.e., a 41% increase in immunoreactive Gi2 alpha subunit that correlated with a parallel 45% increase in mRNA levels. In contrast, levels of immunoreactive Gi3 alpha subunit and mRNA, which decreased with advancing gestation, were not influenced by estradiol or progesterone administration. Progesterone administration resulted 30 h later in a significantly decreased level of Gq alpha immunoreactivity (32%) and Gq alpha mRNA (30%). In control rats, Gq alpha protein and mRNA were also significantly lower at midpregnancy under progesterone dominance vs. term. At this stage, a twofold increase in Gq alpha subunit correlated with a 40% increase in mRNA levels. These results demonstrate that myometrial Gi2 alpha and Gq alpha subunits are physiological targets for estradiol and progesterone, respectively, in vivo. Alterations of these G protein levels are discussed in relation to their mediating effects on adenylyl cyclase activity or the phospholipase C pathway during the course of pregnancy.