Biomedical subjects

M Goppelt-Struebe

Publications and source records attributed to M Goppelt-Struebe.

At least 37 records · Page 2Linked to original sources

PubMed1998

Coordinate expression of cyclooxygenase-2 and renin in the rat kidney in renovascular hypertension.

Prostaglandins contribute to the regulation of renin synthesis and secretion. We tested the hypothesis that the inducible isoform of prostaglandin G/H synthase, cyclooxygenase-2, contributes to the stimulation of renin synthesis in renovascular hypertension. The expression of cyclooxygenase-2 and renin was investigated in the kidneys of rats with two-kidney, one-clip renovascular hypertension or sham operation. Systolic blood pressure was increased 2 weeks after clipping (153+/-7 versus 112+/-4 mmHg in controls, n=6 each, P<.05) and continued to rise until 4 weeks. Cyclooxygenase-2 mRNA levels were increased in clipped kidneys but remained unchanged or slightly decreased in nonclipped kidneys. Cyclooxygenase-2 protein was expressed mainly in the macula densa and occasionally in distal tubular cells not associated with the macula densa. Two weeks after clipping, the percentage of juxtaglomerular apparatus staining positive for cyclooxygenase-2 was 27.8+/-3.6 in clipped kidneys, 3.1+/-0.4 in contralateral kidneys, and 8.0+/-1.3 in controls; the percentages for immunoreactive renin staining in the afferent arteriole were 33.6+/-2 in clipped kidneys, 1.9+/-0.5 in contralateral kidneys, and 12.4+/-4.0 in controls, respectively. Similar parallel changes in renin and cyclooxygenase-2 staining were observed 4 weeks after clipping. The percentage of cyclooxygenase-2-positive juxtaglomerular apparatus correlated positively with the percentage that was renin positive (r=0.78, P<.05). Double immunostaining showed coexpression of cyclooxygenase-2 and renin protein in the same juxtaglomerular apparatus. Our data are consistent with a role for macula densa cyclooxygenase-2 in the regulation of renin in renovascular hypertension.

Animals↗

PubMed1998

Cyclooxygenase isoenzyme localization and mRNA expression in rat lungs.

Prostanoid generation may proceed via both isoforms of cyclooxygenase, Cox-1 and Cox-2. Cox-1 is thought to be ubiquitously expressed, whereas Cox-2 is mostly assumed to be dynamically regulated, responding to inflammatory stimuli. The cellular localization of Cox-1 and Cox-2 in the lung, an organ with high cyclooxygenase activity, is not known. In normal rat lungs the expression and localization of Cox-1 and Cox-2 were examined with immunogold-silver staining and the RT-PCR technique. Quantitative image analysis of the staining intensity was performed by measuring mean gray values of digitized epipolarization images. Expression of both Cox-1 and Cox-2 was readily detectable in rat lungs. Cox-1 immunoreactivity localized predominantly to bronchial epithelial cells, smooth muscle cells of large hilum veins, and (with lower expression) to alveolar macrophages and pulmonary artery endothelial cells. The most intense Cox-2 staining was noted in macrophage- and mast cell-like cells, detected in close vicinity to the bronchial epithelium and in the connective tissue surrounding the vessels. In addition, strong Cox-2 expression was found in smooth muscle cells of partially muscular vessels and large veins of the hilum. Bronchial epithelial cells displayed Cox-2 immunoreactivity with limited intensity. Alveolar macrophages and alveolar septal cells were only occasionally stained with anti-Cox-2 antibodies. Both Cox-1 and Cox-2 are constitutively expressed in several cell types of normal rat lung, but display clearly different patterns of cellular localization. Cox-2 may not be related only to lung inflammation, but is suggested to be implicated in regulatory processes under physiological conditions as well.

Animals↗

PubMed1997

Molecular mechanisms involved in the regulation of prostaglandin biosynthesis by glucocorticoids.

The anti-inflammatory properties of glucocorticoids are attributed in part, to their interference with prostaglandin synthesis. Phospholipases A2 and cyclooxygenases, the key enzymes of prostaglandin biosynthesis, are targets of glucocorticoid action; the molecular mechanisms, however, are not yet understood in detail. Obviously, glucocorticoids can act at different levels of gene regulation depending on cell type and inducing stimulus. The current knowledge of glucocorticoid interference with phospholipase A2 and cyclooxygenase expression is summarized. In comparison with other nonsteroidal anti-inflammatory drugs, glucocorticoids are unique inasmuch as they also inhibit cytokine synthesis and expression of other inflammation-related enzymes. Based on a more detailed understanding of glucocorticoid action, it may be possible to therapeutically exploit the anti-inflammatory effects and at the same time avoid the unwanted metabolic actions of these steroids.

Animals↗

PubMed1997

Monocytic-endothelial cell interaction: regulation of prostanoid synthesis in human coculture.

Coculture of a monocytic cell line (HL-60) and iliacal endothelial cells as an in vitro model of vascular inflammation was investigated for cooperative regulatory mechanisms of prostanoid synthesis under conditions of selective prestimulation. In coculture of endothelial cells and 12-O-tetradecanoylphorbol 13-acetate (TPA)-prestimulated monocytic HL-60 cells the capacity of prostanoid synthesis from arachidonic acid was strongly increased compared with monocultures. Concomitant with up-regulation of specific adhesion molecules, cyclooxygenase (COX) 2 mRNA was induced in endothelial cells in coculture independent of cell contact. HL-60 cells exhibited no alterations in mRNA expression of cyclooxygenases or thromboxane synthase. Coculture of TPA-prestimulated endothelial cells with unstimulated HL-60 cells led to a selectively increased capacity of thromboxane production. Under this condition HL-60 cells up-regulated COX1 and COX2 mRNA, whereas endothelial mRNA levels did not change. Our data demonstrate that the increase in prostanoid synthesis in coculture essentially depends on rapid induction of COX2 mRNA within 2 h.

Cell Communication↗

PubMed1997

Glucocorticoids inhibit oncostatin M-induced phospholipase A2 gene expression in human hepatoma cells.

The secreted phospholipase A2 (sPLA2) is released from hepatoma cells after stimulation with interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), and is considered to act as acute phase protein. In the present study, the regulation of sPLA2 secretion by two other members of the IL-6 cytokine family, oncostatin M (OSM) and leukaemia inhibitory factor (LIF), and the corticosteroid dexamethasone were investigated. Only a marginal increase in sPLA2 activity in cell culture supernatants of HepG2 cells was observed upon stimulation for 24 h with LIF, whereas OSM increased the activity about 10-fold and proved to be even more effective than the combination of IL-6 and TNF-alpha, the best known stimuli so far. sPLA2 activity was synergistically enhanced by OSM plus TNF-alpha (15-fold) or IL-1 beta (20-fold). Changes in sPLA2 activity were reflected at mRNA levels. Cytokine induction of sPLA2 mRNA was comparable to the induction of haptoglobin mRNA. The effect of dexamethasone on the expression of both genes, in contrast, was different: cytokine-induced haptoglobin mRNA expression was enhanced, whereas sPLA2 mRNA expression was partially inhibited by dexamethasone resulting in decreased sPLA2 activity. The strong induction by OSM in HepG2 cells thus confirmed sPLA2 as acute phase protein, whereas the effect of dexamethasone was comparable to the one observed in other cell types.

Antineoplastic Agents↗

PubMed1997

Cyclooxygenase-2 in the spinal cord: localization and regulation after a peripheral inflammatory stimulus.

Prostaglandins are known as mediators in spinal nociceptive processing after peripheral inflammation. Cyclooxygenase isozymes Cox-1 and Cox-2 as enzymes essential for prostaglandin biosynthesis were therefore investigated in rat spinal cord. mRNA and protein of both enzymes was detected in cervical and lumbar sections of the spinal cord of normal animals. Enzyme activity of Cox-1 and Cox-2 could be determined by the use of specific inhibitors. Cox-2-immunoreactivity (IR) was found in the spinal cord of untreated rats in neurons of laminae II-III, motoneurons of lamina IX and in glial cells. Most prominent staining was observed in the nuclear envelope. Under an acute peripheral inflammatory stimulus a transient 2-fold increase in Cox-2 mRNA was noticed bilaterally in the lumbar spinal cord by reverse transcription-polymerase chain reaction. This increase was reflected on a smaller scale at protein levels in Western blot analysis of spinal membrane preparations. In the chronic state of adjuvant-induced arthritis, Cox-2 levels were raised again significantly. Cox-2 might therefore be regarded as the Cox isozyme responsible for increased spinal prostanoid release in nociceptive processing under peripheral stimulation.

Animals↗

PubMed1997

Mechanisms of serotonin-induced Ca2+ responses in mesangial cells.

Smooth muscle cell-like mesangial cells play an important role in the regulation of glomerular blood flow and are involved in renal inflammatory reactions, thereby interacting with circulating cells. The platelet products serotonin (5-HT) and ATP induce similar, e.g. mitogenic, effects in mesangial cells, but differentially activate and induce inflammation-related genes. To get an insight into intracellular signaling steps, a very early step in the signaling cascade, the biphasic Ca2+ signal elicited by 5-HT and ATP in rat mesangial cells was investigated. Both phases of the Ca2+ signal, release from internal stores as well as influx of extracellular Ca2+, were dependent on phospholipase C activation as shown by the specific inhibitor U73122 (complete inhibition at 10 microM U73122). There was no evidence for voltage-gated L-type channels in these cells, suggesting that Ca2+ influx was mediated by Ca2+ release-activated channels. The L-type channel blocker verapamil, however, dose-dependently (0.1-10 microM) and specifically inhibited 5-HT-elicited Ca2+ signals by interference with binding of 5-HT to 5-HT2A receptors. 5-HT-mediated Ca2+ release was reduced by 80% when protein kinase C was activated by the phorbolester TPA (0.1 microM). Interaction of 5-HT2A receptors with phospholipase C was also inhibited by genistein (30% at 5 microM; 100% at 50 microM), an inhibitor of tyrosine kinases. Binding of 5-HT to its receptor reduced subsequent ATP-mediated Ca2+ signaling. The cross talk between the receptors was sensitive to genistein. ATP-mediated Ca2+ signaling was attributed to different types of P2y receptors and/or multiple G-proteins coupled, because the signal was partially inhibited by pertussis toxin (50%). In accordance, modulation of the ATP-mediated signaling by phosphorylation was less tightly controlled than 5-HT-mediated Ca2+ release. These data indicate that although the Ca2+ responses elicited by the two stimuli are comparable, interactions between receptors, G-proteins and target enzymes are regulated differentially.

Animals↗

PubMed1997

Modulation of phospholipase D stimulation in c-src transfected mesangial cells.

Stimulation of rat mesangial cells with platelet-derived growth factor BB (PDGF-BB) enhanced phospholipase D (PLD) activity in a concentration dependent manner. Mesangial cells overexpressing the tyrosine kinase pp60c-src (c-Src) were used to determine the effect of this non transforming protooncogene on PLD activation. Overexpression of c-Src interfered with PDGF-BB-mediated activation of PLD. This modulation was dependent on the tyrosine kinase activity of c-Src, since overexpression of tyrosine kinase-negative mutants of c-Src did not affect PLD activation. No effect of c-Src overexpression was observed, when PLD was activated by ATP or guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The results indicate that the tyrosine kinase c-Src specifically interfered with PDGF-mediated but not with ATP- or GTP gamma S-mediated PLD activation.

Adenosine Triphosphate↗

PubMed1997

Differential regulation of chemokines by leukemia inhibitory factor, interleukin-6 and oncostatin M.

M. Leukemia inhibitory factor (LIF). oncostatin M (OsM) and interleukin-6 (IL-6) are members of a cytokine family, which are produced by activated macrophages and glomerular mesangial cells. These cytokines have been implicated in the pathogenesis of glomerular inflammation, but their action on glomerular cells is presently unclear. Therefore, we examined the effects of IL-6, OsM and LIF on chemokine synthesis of rat mesangial cells in culture. While LIF as well as IL-6 up-regulated monocyte chemotactic protein-1 (MCP-1) mRNA expression, OsM showed no such effect. The induction of MCP-1 mRNA by LIF and IL-6 was transient, peaking at one to two hours and two to three hours, respectively, and returning to background levels within several hours. Induction of MCP-1 mRNA by LIF and IL-6 was strongly inhibited by dexamethasone. LIF activated STAT factors in mesangial cells, suggesting their involvement in signal transduction pathways that lead to LIF-stimulated up-regulation of MCP-1 mRNA. By contrast, LIF. IL-6 and OsM failed to affect the expression of the chemokines, macrophage inflammatory protein-2 (MIP-2) and RANTES. The rapid, transient and differential regulation of MCP-1 expression induced by LIF and IL-6 contrasted with uniformly powerful effects of the proinflammatory cytokines IL-1 beta and TNF alpha that induced all tested chemokines for prolonged time periods. These results suggest that the selective and transient induction of MCP-1 by LIF and IL-6 may play a role in the preferential attraction of monocytes to the injured glomerulus.

Animals↗

PubMed1997

Differential regulation of cyclo-oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells.

1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

5-Lipoxygenase-Activating Proteins↗

PubMed1996

Interference of corticosteroids with prostaglandin E2 synthesis at the level of cyclooxygenase-2 mRNA expression in kidney cells.

In the kidney, prostanoids play a role as vasoactive and immunomodulatory mediators. One of the main biosynthetic enzymes, the inducible cyclooxygenase-2 (EC 1.14.99.1, Cox-2), has been recognized as a target of glucocorticoids. Therefore, we investigated whether the physiologically active corticosteroid aldosterone in the kidney might also interfere with prostaglandin (PG) synthesis. In two cell types, an epithelial cell line of tubular origin (MDCK) and rat renal mesangial cells, PGE2, release, Cox activity and Cox mRNA expression were determined after stimulation with phorbol ester and IL-1 beta, respectively. An increase in PGE2 release and Cox activity was observed, which correlated with an increase in Cox-2 mRNA expression. In MDCK cells, both dexamethasone and aldosterone were equally effective, suppressing all parameters measured by approximately 60%. A similar effect of aldosterone was also seen in mesangial cells, whereas dexamethasone was far more potent (> 90% inhibition at 10(-6) M). Whole cell binding assays showed the same number of receptors for aldosterone in both cell types (approximately 70,000 receptors/cell) but more than ten times higher receptor numbers for dexamethasone in mesangial cells than in MDCK cells (90,000 vs. 6000 receptors/cell). Receptor affinities of the corticosteroids were comparable. Thus, interaction of the corticosteroids with their cognate receptors was not sufficient to explain their different potencies but indicated the involvement of more complex regulatory mechanisms. Pathophysiologically, inhibition of PGE2 synthesis by aldosterone may play a role in the induction of hypertension by high concentrations of aldosterone.

Adrenal Cortex Hormones↗

PubMed1996

Up-regulation of cyclooxygenase-2 mRNA in the rat spinal cord following peripheral inflammation.

Prostaglandins (PG) have been described as mediators in spinal nociceptive processing after peripheral inflammation. Enzymes essential for PG biosynthesis, cyclooxygenase isozymes COX-1 and COX-2, have not yet been investigated in the spinal cord. In two studies on rats with adjuvant-induced peripheral inflammation levels of mRNA expression of both COX isoforms were analyzed in the lumbar section of the spinal cord using reverse transcription-polymerase chain reaction (RT-PCR) technique. We could show that mRNA of both COX isoforms is expressed constitutively in the spinal cord with COX-2 as the predominant isoform. Six hours after induction of peripheral inflammation, levels of COX-2 mRNA expression were raised significantly in respect to untreated control rats and returned to baseline within 3 days after induction of inflammation. COX-2 might therefore be regarded as the COX isozyme responsible for spinal PG release in nociceptive processing under a peripheral inflammatory stimulus.

Animals↗

PubMed1996

Regulation of prostaglandin synthesis in Madin Darby canine kidney cells: role of prostaglandin G/H synthase and secreted phospholipase A2.

The renal epithelial cell line MDCK (Madin Darby canine kidney) was used as a model system to investigate the contribution of the secreted phospholipase A2 type II(sPLA2) and cyclooxygenases to prostaglandin E2 (PGE2) synthesis. Activation of protein kinase C by the phorbol ester TPA led to an enhanced PGE2 synthesis within 1 hour, which continued for more than 20 hours. Treatment of the cells with TPA increased the activities of sPLA2 and cyclooxygenase. Activation of cyclooxygenase was reflected by an increase in cyclooxygenase-2 mRNA. Coincubation of the cells with TPA and a specific sPLA2 inhibitor (BM 16.2224) almost completely inhibited sPLA2 activity in the cell culture supernatants. TPA-induced PGE2 synthesis was reduced by the inhibitor to about 50%. The inhibitor had no effect on cyclooxygenase activity or expression, indicating an involvement of sPLA2 in PGE2 synthesis. These experiments show that in resting cells, even in the presence of exogenous arachidonic acid, PGE2 synthesis was limited by the low abundance of cyclooxygenase. Enhanced expression and activity of cyclooxygenase, however, was not sufficient for increased prostaglandin synthesis. Availability of the precursor arachidonic acid seemed to be rate limiting in prostaglandin synthesis in stimulated MDCK cells.

Animals↗

PubMed1996

Modulation of prostaglandin G/H synthase expression in mesangial cells transfected by pp60c-src proto-oncogene.

The role of the protein tyrosine kinase pp60c-src in the expression of prostaglandin G/H synthase (PGHS), the key enzyme of prostaglandin synthesis, was investigated in rat renal mesangial cells. Transfection of mesangial cells with the proto-oncogene c-src resulted in nontransformed cells with constitutively enhanced pp60c-src kinase activity. As a control, mesangial cells were transfected with inactive pp60c-src, mutated in position 295 (lysine replaced by methionine). Expression of the constitutive isoform PGHS-1 was enhanced in cells overexpressing wild-type c-src compared to cells transfected with the kinase negative c-src mutant. Levels of other constitutively expressed proteins such as GAPDH and beta-actin were also enhanced. PGHS-2 was barely detectable in resting cells but was inducible by PDGF-AB, PDGF-BB, serotonin, FCS, and calcium ionophore A23187. Induction was diminished in pp60c-src kinase-overexpressing cells, independent of the stimulus used, suggesting interference at a late step in the signaling cascade. No induction of PGHS-2 mRNA was observed in mesangial cells transformed by the oncogene v-src. An increase in intracellular calcium levels is an early step in signal transduction by PDGF and serotonin in mesangial cells. c-src kinase overexpression reduced PDGF- and serotonin-mediated changes in Ca2+ signaling, indicating multiple targets of pp60c-src action. Overexpression of pp60c-src in mesangial cells thus affected basal protein expression, reflected by the enhanced PGHS-1 mRNA and protein expression. With regard to induction of PGHS-2, overexpression of pp60c-src reduced induction by stimuli coupled to different types of signaling pathways.

Animals↗

PubMed1996

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

OBJECTIVE AND DESIGN: Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. MATERIALS AND TREATMENT: As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). METHODS: Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. RESULTS: During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. CONCLUSIONS: These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Cell Differentiation↗

PubMed1996

Regulation of platelet-derived growth factor isoform-mediated expression of prostaglandin G/H synthase in mesangial cells.

Incubation of rat renal mesangial cells with platelet-derived growth factor (PDGF) -AB or -BB led to a transient increase in prostaglandin G/H synthase-2 (PGHS-2) mRNA expression with a maximum after two hours. Expression of PGHS-1 mRNA remained unchanged during short term incubation, but was enhanced about twofold after 8 to 12 hours incubation with PDGF-AB or -BB. Enhanced PGHS activity was still observed after 24 hours. Nevertheless, PGE2 release from mesangial cells was not enhanced by PDGF, hinting to the availability of arachidonic acid as rate-limiting step. PDGF receptors are coupled to multiple signaling pathways, among them phospholipase C gamma PDGF-BB rapidly phoshorylated PLC gamma, while phosphorylation by PDGF-AB was barely detectable. The differential effect of PDGF-BB and PDGF-AB was also seen with respect to calcium signaling: PDGF-BB but not PDGF-AB induced release of Ca2+ from internal stores. Activation of PLC and the resulting transient release of Ca2+ were not considered to be essential for PGHS-2 mRNA induction as both PDGF isoforms were equally effective in mRNA induction. Both PDGF isoforms led to a Ca2+ influx resulting in a long lasting elevation of [Ca2+]i. Enhanced [Ca2+]i seemed to be related to PGHS-2 mRNA expression, because PDGF-induced PGHS-2 mRNA was significantly reduced under Ca2+ free conditions. Diacylglycerol, liberated by PLC, is an activator of protein kinase C (PKC). Down-regulation of PKC by overnight incubation with phorbol ester (0.1 microM) attenuated PGHS-2 mRNA induction by PDGF-AB and -BB. Involvement of PKC was substantiated by the PKC inhibitor H7, which interfered with PDGF-mediated PGHS-2 mRNA expression, while HA1004, a considerably specific inhibitor of protein kinases A and G, was without effect. Taken together, signaling pathways other than PLC gamma seem to be involved in activation of PKC and elevation of [Ca2+]i, which were shown to be essential elements of PDGF-mediated induction of PGHS-2 mRNA expression in mesangial cells.

Animals↗

PubMed1995

Synergistic induction of monocyte chemoattractant protein-1 (MCP-1) by platelet-derived growth factor and interleukin-1.

Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the recruitment of monocytic cells to the site of inflammation. Resting mesangial cells express barely detectable levels of MCP-1 mRNA. Treatment of rat mesangial cells with platelet products PDGF-AB, PDGF-BB or serotonin transiently induced MCP-1 expression with a maximum after 2 to 4 h and a decline to baseline after 6 to 8 h. Different kinetics were observed with interleukin-1 beta (IL-1 beta), which induced a long lasting elevation of MCP-1 mRNA for more than 20 h. Together, PDGF and IL-1 beta synergistically induced MCP-1 expression. The effect was most obvious after 16 to 20 h, when induction by PDGF alone had already faded, but still PDGF strongly enhanced IL-1 beta-induced MCP-1 mRNA expression. MCP-1 mRNA levels were regulated by changes in the stability of the mRNA: inhibition of protein synthesis by cycloheximide by itself induced MCP-1 mRNA expression and led to superinduction in the presence of PDGF. Message stabilization also contributed to the synergistic action of PDGF and IL-1 beta: the apparent half life of MCP-1 mRNA determined in the presence of actinomycin D was prolonged when both stimuli were added together. We could thus show that in mesangial cells different types of cytokines and growth factors synergize to enhance MCP-1, the secretion of which could lead to the recruitment of monocytic cells into the inflamed mesangium.

Animals↗

PubMed1995

Two step purification of human and murine leukotriene C4 synthase.

Leukotriene (LT) C4 synthase catalyzes the conjugation of LTA4 with reduced glutathione (GSH) to form LTC4. This enzyme was purified to homogeneity from Kirsten Sarcoma transformed murine mast cells and from the human monocytic cell line THP-1 by two steps: a microsomal extract was partially purified by affinity chromatography on S-hexyl GSH agarose. LTC4 synthase was separated from other GSH-binding proteins by preparative gel electrophoresis under non denaturing conditions. The proteins obtained from human and murine cells showed a single band on a SDS gel with a molecular mass of 14 to 17 kDa, depending on the gel system. N-terminal sequencing revealed high homology between the LTC4 synthases of both species.

Amino Acid Sequence↗