Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): proposed mechanism for modulation of endothelial cell response to VEGF

We have recently demonstrated that thrombin-activated FXIII (FXIIIA-subunit), a plasma transglutaminase, activates VEGFR-2 by crosslinking it with the alpha(v)beta(3) integrin on the surface of endothelial cells (EC), thereby stimulating angiogenesis. Tissue transglutaminase (tTG), which is functionally and structurally related to FXIIIA, is expressed by numerous cell types, among them EC. However, its role in EC function has not been fully characterized. In the present study, we investigated the potential involvement of tTG in angiogenesis. Using co-immunoprecipitation and immunofluorescent staining experiments, we observed that tTG forms a complex with VEGFR-2 on the cell surface and within the cytoplasm of EC. Stimulation of EC with VEGF resulted in translocation of the tTG-VEGFR-2 complex from the cytoplasm to the nucleus. In VEGF-treated cells, tTG-VEGFR-2 interaction resulted in incorporation of VEGFR-2 into high molecular weight crosslinked complex (es), as revealed by an antibody against gamma-glutamyl-epsilon-lysine isopeptide bond. tTG -VEGFR-2 association was inhibited by a specific VEGFR-2 protein tyrosine kinase inhibitor (PTKI ), as well as by cystamine, inhibitor of the transglutaminase activity of tTG, but not by bacitracin which inhibits the protein-disulfide isomerase (PDI) activity of tTG. Furthermore, cystamine completely abolished the VEGF-induced nuclear translocation of the tTG-VEGFR-2 complex. Blockade of the crosslinking activity of tTG by cystamine enhanced VEGF-induced migration of EC in Boyden chamber by 31% (P < 0.02), and prolonged VEGF-induced signaling response, as demonstrated by sustained activation of the MAP kinase ERK. Taken together, our findings suggest that endothelial cell tTG might be involved in modulation of the cellular response to VEGF by forming an intracellular complex with VEGFR-2, and mediating its translocation into the nucleus upon VEGF stimulation.     

Influence of Transglutaminase on the Functions of Mouse Peritoneal Macrophages

Influence of transglutaminase on the production of interleukin-1 (IL-1) and on the release of active oxygen from mouse peritoneal macrophages was examined using cystamine and methylamine, an enzyme inhibitor and a substrate inhibitor, respectively. Casein-elicited or lipopolysaccharide (LPS)-elicited macrophages have higher levels of transglutaminase activity in comparison with resident macrophages, and there exists a definite correlation between endocytosis of erythrocytes and transglutaminase activity in either group of macrophages. The release of IL-1 by resident macrophages stimulated with LPS in vitro was significantly inhibited by the treatment with both transglutaminase inhibitors. However, these inhibitors were not able to inhibit the release of IL-1 from casein-elicited macrophages stimulated with LPS in vitro. The production of active oxygen from LPS-elicited macrophages was inhibited in a dose-dependent manner by the treatment of macrophages with cystamine, but was not by the treatment with methylamine. However, the treatment of LPS-elicited macrophages with cystamine did not inhibit the uptake of glucose into macrophages. These results suggest that transglutaminase activity in mouse peritoneal macrophages is an important factor for macrophage functions.     

Impaired mitochondrial function results in increased tissue transglutaminase activity in situ

Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.     

Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2.

Rat pancreatic islets contain a Ca2+-activated and thiol-dependent transglutaminase (EC 2.3.2.13) comparable in activity with that found in rat liver, lung and spleen. The Ca2+-dependence of this enzyme is such that half-maximal velocity was obtained in the region of 40 microM. Preincubation of rat islets with primary-amine substrates of transglutaminase (monodansylcadaverine, methylamine, ethylamine, propylamine and cystamine) led to an inhibition of glucose-stimulated insulin release by these amines. Kinetic analysis of the competitive substrates methylamine, monodansylcadaverine, propylamine and ethylamine for their ability to inhibit islet transglutaminase activity indicated a potency that matched their ability to inhibit glucose-stimulated insulin release. When these amines were tested for their effects on glucose-stimulated protein synthesis and glucose utilization, the most potent inhibitor of insulin release, monodansylcadaverine, had no effect on either process at 100 microM. The amines cystamine, ethylamine, methylamine and propylamine had variable effects on these metabolic processes. For ethylamine, methylamine and propylamine, concentrations were found which inhibited glucose-stimulated insulin release in a manner which was found to be independent of their effects on either glucose oxidation or protein synthesis. Primary amines may therefore inhibit insulin release through their incorporation by islet transglutaminase into normal cross-linking sites. A role for protein cross-linking in the secretory mechanism is suggested.     

Vaccinia Virus Protein F1L Is a Caspase-9 Inhibitor

Apoptosis plays important roles in host defense, including the elimination of virus-infected cells. The executioners of apoptosis are caspase family proteases. We report that vaccinia virus-encoded F1L protein, previously recognized as anti-apoptotic viral Bcl-2 family protein, is a caspase-9 inhibitor. F1L binds to and specifically inhibits caspase-9, the apical protease in the mitochondrial cell death pathway while failing to inhibit other caspases. In cells, F1L inhibits apoptosis and proteolytic processing of caspases induced by overexpression of caspase-9 but not caspase-8. An N-terminal region of F1L preceding the Bcl-2-like fold accounts for caspase-9 inhibition and significantly contributes to the anti-apoptotic activity of F1L. Viral F1L thus provides the first example of caspase inhibition by a Bcl-2 family member; it functions both as a suppressor of proapoptotic Bcl-2 family proteins and as an inhibitor of caspase-9, thereby neutralizing two sequential steps in the mitochondrial cell death pathway.     

Unspecific activation of caspases during the induction of apoptosis by didemnin B in human cell lines

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD-fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis.     

Tissue transglutaminase differentially modulates apoptosis in a stimuli-dependent manner

Tissue transglutaminase is a unique member of the transglutaminase family as it not only catalyzes a transamidating reaction, but also binds and hydrolyzes GTP and ATP. Tissue transglutaminase has been reported to be pro-apoptotic, however, conclusive evidence is still lacking. To elucidate the role of tissue transglutaminase in the apoptotic process human neuroblastoma SH-SY5Y cells were stably transfected with vector only (SH/pcDNA), wild-type tissue transglutaminase (SH/tTG) and tissue transglutaminase that has no transamidating activity but retains its other functions (SH/C277S). In these studies three different apoptotic stimuli were used osmotic stress, staurosporine treatment and heat shock to delineate the role of tissue transglutaminase as a transamidating enzyme in the apoptotic process. In SH/tTG cells, osmotic stress and staurosporine treatments resulted in significantly greater caspase-3 activation and apoptotic nuclear changes then in SH/pcDNA or SH/C277S cells. This potentiation of apoptosis in SH/tTG cells was concomitant with a significant increase in the in situ transamidating activity of tissue transglutaminase. However, in the heat shock paradigm, which did not result in any increase in the transamidating activity in SH/tTG cells, there was a significant attenuation of caspase-3 activity, LDH release and apoptotic chromatin condensation in SH/tTG and SH/C277S cells compared with SH/pcDNA cells. These findings indicate for the first time that the effect of tissue transglutaminase on the apoptotic process is highly dependent on the type of the stimuli and how the transamidating activity of the enzyme is affected. Tissue transglutaminase facilitates apoptosis in response to stressors that result in an increase in the transamidating activity of the enzyme. However, when the stressors do not result in an increase in the transamidating activity of tissue transglutaminase, than tissue transglutaminase can ameliorate the apoptotic response through a mechanism that is independent of its transamidating function. Further, neither the phosphatidylinositol-3-kinase pathway nor the extracellular-regulated kinase pathway is downstream of the modulatory effects of wild-type tissue transglutaminase or C277S-tissue transglutaminase in the apoptotic cascade.     

Tissue Transglutaminase Is an In Situ Substrate of Calpain: Regulation of Activity

Abstract: Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the transamidation of specific polypeptide-bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium-activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine-5'- O -(3-thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH-SY5Y cells with 1 n M maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an ∼50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.     

Requirement of both the second and third BIR domains for the relief of X-linked inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by Smac

The inhibitor of apoptosis proteins (IAP) are endogenous caspase inhibitors in the metazoan and characterized by the presence of baculoviral IAP repeats (BIR). X-linked IAP (XIAP) contains three BIR domains and directly inhibits effector caspases such as caspase-7 via a linker_BIR2 fragment and initiator caspases such as caspase-9 via the BIR3 domain. A mitochondrial protein Smac/DIABLO, which is released during apoptosis, antagonizes XIAP-mediated caspase inhibition by interacting directly with XIAP. Here, using glutathione S-transferase pulldown and caspase activity assay, we show that Smac is ineffective in relieving either caspase-7 or caspase-9 inhibition by XIAP domain fragments. In addition, Smac forms a ternary complex with caspase-7 and linker_BIR2, suggesting that Smac/linker_BIR2 interaction does not sterically exclude linker_BIR2/caspase-7 interaction. However, Smac is effective in removing caspase-7 and caspase-9 inhibition by XIAP fragments containing both the BIR2 and BIR3 domains. Surface plasmon resonance measurements show that Smac interacts with the BIR2 or BIR3 domain in micromolar dissociation constants. On the other hand, Smac interacts with an XIAP construct containing both BIR2 and BIR3 domains in a subnanomolar dissociation constant by the simultaneous interaction of the Smac dimer with the BIR2 and BIR3 domains of a single XIAP molecule. This 2:1 Smac/XIAP interaction not only possesses enhanced affinity but also sterically excludes XIAP/caspase-7 interaction, demonstrating the requirement of both BIR2 and BIR3 domains for Smac to relieve XIAP-mediated caspase inhibition.     

Prevention of staurosporine-induced cell death in embryonic chick cardiomyocyte is more dependent on caspase-2 than caspase-3 inhibition and is independent of sphingomyelinase activation and ceramide generation

Apoptosis was induced in embryonic chick cardiomyocytes by staurosporine. Treatment of cardiomyocytes with the preferential caspase-2 inhibitor, z-VDVAD-fmk (100 microM), produced a significant (P < 0.05) although small reduction in the amount of cell death. Ac-DVED-cmk (100 microM), which preferentially inhibits caspase-3 but inhibits to a lesser extent caspase-6, -7, -8, and -10, produced a minimal decrease in cell death. The combination of the caspase-3 and -2 inhibitors produced an additive reduction in cell death after staurosporine (1 microM for 6 h) from 80.4 +/- 0.7 to 54.6 +/- 1.3%. The ability of staurosporine to activate caspase-3 was confirmed in these cardiomyocytes by measurement of caspase-3 activity. A role for ceramide formation, from sphingomyelin to induce caspase activation was unlikely, as there were no changes in cellular ceramide or sphingomyelin after staurosporine treatment of cardiomyocytes when sphingomyelin was labeled by [(3)H]palmitate for 24 h. Neither were there any changes in sphingomyelinase activity. While staurosporine effectively suppressed PKC activity, phorbol 12-myristate 13 acetate did not alter staurosporine-induced cell death or DNA fragmentation. These results demonstrate that, in this model of cardiac cell death, caspase-2 inhibition is of considerable importance, caspase-3 inhibition is of lesser significance but may produce additional effects in the combination with caspase-2 inhibition, and ceramide production from sphingomyelin is not operative in the pathway leading to caspase activation and cell death.     

Effect of Dicycloplatin,a Novel Platinum Chemotherapeutical Drug,on Inhibiting Cell Growth and Inducing Cell Apoptosis

Dicycloplatin, a new supramolecular platinum-based antitumor drug, has been approved by the State Food and Administration (SFDA) of China. In this study, we investigated the anticancer activity of dicycloplatin in cancer cells and signaling pathways involved in dicycloplatin-induced apoptosis. Dicycloplatin inhibited the proliferation of cancer cells and increased the percentage of apoptosis in a concentration-dependent manner. Besides, some apoptosis related events were observed after treatment with dicycloplatin, including increase of reactive oxygen species (ROS), collapse of mitochondrial membrane potential (Δψm), release of cytochrome c from the mitochondria to the cytosol, upregulation of p53, which were accompanied by activation of caspase-9, caspase-3, caspase-8, and poly (ADP-ribose) polymerase cleavage in a concentration-dependent manner. The role of apoptosis in dicycloplatin-mediated cell death was further confirmed by the concomitant treatment with caspase-8 or caspase-9 inhibitors, which inhibited apoptosis and PARP cleavage. Intracellular glutathione (GSH) was also found to inhibit the cytotoxic effect of dicycloplatin. In conclusion, these findings suggest that dicycloplatin induces apoptosis through ROS stress-mediated death receptor pathway and mitochondrial pathway which is similar to carboplatin.     

Protective role of Cop in Rip2/caspase-1/caspase-4-mediated HeLa cell death

CARD only protein (Cop) was recently identified as a protein with significant homology with the CARD of caspase-1. We have conducted functional studies on Cop and report on its role as an inhibitor of cell death in a broad range of cell death paradigms. A notable exception in the ability of Cop to inhibit cell death pertains to its inability to inhibit ER stress-mediated cell death. Furthermore, in addition to the known interaction of Cop and caspase-1, we demonstrated a novel interaction of Cop with caspase-4. We propose that Cop's action to prevent TNF-alpha-induced cell death may operate independently of the mitochondrial death pathway. Furthermore, Cop overexpression inhibits Bid cleavage. In summary, Cop inhibition of cell death, at least to a certain extent, results from its interference with the activation of caspase-1 and caspase-4. Understanding the mechanistic details modulating caspase cell death pathways should provide important information for the development of therapies for diseases featuring aberrant caspase activation. Cop, as an inhibitor of an important apical caspase cell death axis, may provide a tool for modulating pathological cell death.     

Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3 in vivo

Oxidants such as H(2)O(2) can induce a low level of apoptosis at low concentrations but at higher concentrations cause necrosis. Higher concentrations of H(2)O(2) also inhibit the induction of apoptosis by chemotherapy drugs. One theory is that, at higher concentrations, H(2)O(2) causes direct oxidative inactivation of caspase-3 activity, thus preventing the apoptotic pathway from being used. We find that treatment of recombinant caspase-3 with H(2)O(2) can partially reduce its enzymatic activity: However, the following findings show that this does not occur in the cell. (1) The inhibition by H(2)O(2) of VP-16-induced apoptosis and cellular caspase-3 activity can be overcome by adding inhibitors of poly(ADP-ribose) polymerase (PARP) at sub-stoichiometric concentrations. (2) Delayed addition of H(2)O(2) to VP-16-treated cells prevents additional caspase induction but does not inhibit the caspase activity that has already been generated. (3) H(2)O(2) is a poor inhibitor of caspase-3 activity in cell lysates. (4) Addition of H(2)O(2) to cells inhibits activation of caspase-9, which is required for activation of caspase-3. We conclude that inhibition of caspase-3 activity in the cell occurs indirectly at a step located upstream of caspase-3 activation. H(2)O(2) acts in part by inducing DNA strand breaks and activating PARP, thus depleting the cells of ATP. When this pathway is blocked, even high concentrations of H(2)O(2) can induce caspase-9 and -3 activation and cause apoptosis.     

Effects of glutathione-related compounds on increased caspase-3 and caspase-6-like activities in ricin-treated U937 cells

Both caspase-3 and -6-like activities increased in the cytosolic extract from ricin-treated U937 cells that were inhibited by glutathione disulfide (GSSG) in a dose-dependent manner, but reduced glutathione (GSH) had no effect. Interestingly, caspase-6 like activity was more sensitive to GSSG than caspase-3 like activity. The IC50 of GSSG against caspase-3 and caspase-6 like activities were estimated to be 2.8 mM and 0.8 mM, respectively. Cystine but not cysteine also showed similar inhibitory effect on caspase-3-like activity. The inhibitory effect of GSSG on these caspase-like activities was prevented by the addition of DTT to the assay mixture. These results suggest that an intact disulfide portion of GSSG is required for the effective inhibition of caspase activity.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse) (159.3 +/- 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse) (906 +/- 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Inhibition of gamma-glutamylcysteine synthetase by cystamine: an approach to a therapy of 5-oxoprolinuria (pyroglutamic aciduria).

γ-Glutamylcysteine synthetase is strongly inhibited by cystamine; thus, 20 μM cystamine inhibited the activity by 50%. Inhibition is rapid and the inhibited enzyme is reactivated by dithiothreitol suggesting that cystamine reacts with an enzyme sulfhydryl group. Inhibition by cystamine is not prevented by MgATP, L-α-aminobutyrate, or L-glutamate suggesting that cystamine may not interact at the active site. Little or no inhibition was observed with N,N′-diacetyl cystamine, L-cystine, glutathione disulfide, 2-hydroxyethyl disulfide, and thioglycolate disulfide, whereas thiocholine disulfide produced moderate inhibition. Cystamine or an inhibitory analog of cystamine might be useful in the therapy of the disease 5-oxoprolinuria in which there is an overproduction of γ-glutamylcysteine.     

BCL-2 protects peroxynitrite-treated thymocytes from poly(ADP-ribose) synthase (PARS)-independent apoptotic but not from PARS-mediated necrotic cell death

In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.     

Homocysteine-induced toxicity increases TG2 expression in Neuro2a cells

High levels of homocysteine promote cell damage mainly through induction of oxidative stress, endoplasmic reticulum (ER) stress, and activation of pro-inflammatory factors. The effects of homocysteine were here examined in the continuously dividing neuroblastoma cell line Neuro2a. Cell treatment with homocysteine (100–500 μM) for 4 h increased ROS production while reducing cell viability in a dose-dependent manner. Cell exposure to 250 μM homocysteine was able to induce transglutaminase 2 up-regulation and increased in situ transglutaminase activity. These effects were prevented by the incubation with the transglutaminase activity inhibitor cystamine. Homocysteine also induced NF-κB activation that seemed associated with transglutaminase 2 up-regulation since the specific NF-κB inhibition by SN50 was able to reduce transglutaminase expression and activity levels. In the light of these observations, it may be postulated that TG2 up-regulation is involved in cell response to homocysteine-induced stress, in which NF-κB activation plays also a pivotal role.     

C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement.

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.