Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages

The APO-1/Fasligand (FasL) and tumor necrosis factor- (TNF-) are twofunctionally related molecules that induce apoptosis ofsusceptible cells. Although the two molecules have been reported toinduce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-, raising thepossibility that TNF- may be involved in FasL-inducedapoptosis. Because TNF- gene expression is under the controlof nuclear factor-B (NF-B), we investigated whether FasL caninduce NF-B activation and whether such activation plays a role inFasL-mediated cell death in macrophages. Gene transfection studiesusing NF-B-dependent reporter plasmid showed that FasL did activateNF-B promoter activity. Gel shift studies also revealed that FasLmobilized the p50/p65 heterodimeric form of NF-B. Inhibition ofNF-B by a specific NF-B inhibitor, caffeic acid phenylethylester, or by dominant expression of the NF-B inhibitory subunitIB caused an increase in FasL-induced apoptosis and areduction in TNF- expression. However, neutralization of TNF- byspecific anti-TNF- antibody had no effect on FasL-inducedapoptosis. These results indicate that FasL-mediated cell deathin macrophages is regulated through NF-B and is independent ofTNF- activation, suggesting the antiapoptotic role of NF-Band a separate death signaling pathway mediated by FasL.

     

Combined modulation of the mesangial machinery for monocyte recruitment by inhibition of NF-kappa B

The activation of nuclear factor-B(NF-B) is required for the induction of many of the adhesionmolecules and chemokines involved in the inflammatory leukocyterecruitment to the kidney. Here we studied the effects of NF-Binhibition on the machinery crucial for monocyte infiltration of theglomerulus during inflammation. In mesangial cells (MC), the proteaseinhibitors MG-132 and N--tosyl-L-lysine chloromethyl ketone or adenoviral overexpression of IB- prevented the complete IB- degradation following tumor necrosis factor- (TNF-) stimulation. This resulted in a marked inhibition ofTNF--induced expression of mRNA and protein for the immunoglobulinmolecules intracellular adhesion molecule-1 and vascular cell adhesionmolecule-1 and the chemokines growth-related oncogene-, monocytechemoattractant protein-1, interleukin-8, or fractalkine in MC.Finally, the inhibition of IB- degradation or IB-overexpression suppressed the chemokine-induced transendothelialmonocyte chemotaxis toward MC and the chemokine-triggered firm adhesionof monocytic cells to MC. The inhibition of NF-B by pharmacologicalintervention or gene transfer may present a multimodal approach tocontrol the machinery propagating inflammatory recruitment of monocytesduring glomerular disease.

     

Differential requirement for NF-kappaB-inducing kinase in the induction of NF-kappaB by IL-1beta,TNF-alpha,and Fas

In this study, weexamined the role of the nuclear factor-B (NF-B)-inducing kinase(NIK) in distinct signaling pathways leading to NF-B activation. Weshow that a dominant-negative form of NIK (dnNIK) delivered byadenoviral (Ad5dnNIK) vector inhibits Fas-induced IBphosphorylation and NF-B-dependent gene expression in HT-29 and HeLacells. Interleukin (IL)-1- and tumor necrosis factor-(TNF-)-induced NF-B activation and B-dependent gene expressionare inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells.Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF--inducedapoptosis at an early time point. However, cytokine- andFas-induced signals to NF-B are finally integrated by the IBkinase (IKK) complex, since IB phosphorylation, NF-B DNAbinding activity, and IL-8 gene expression were strongly inhibited inHT-29 and HeLa cells overexpressing dominant-negative IKK(Ad5dnIKK). Our findings support the concept that cytokine signalingto NF-B is redundant at the level of NIK. In addition, this studydemonstrates for the first time the critical role of NIK and IKK inFas-induced NF-B signaling cascade.

     

PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

This work demonstrated the constitutive expressionof peroxisome proliferator-activated receptor (PPAR)- and PPAR-in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR- expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR- mRNA expression was not modified. 15-Deoxy-^12,14-prostaglandin J₂(15d-PGJ₂) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (80%) and inducible nitric oxide synthase (iNOS) mRNA expression (80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (50%). 15d-PGJ₂ decreasedLPS-induced interleukin (IL)-1 (25%) and tumor necrosis factor(TNF)- (40%) expression. Interestingly, troglitazone stronglydecreased TNF- expression (50%) but had no significant effect onIL-1 expression. 15d-PGJ₂ was able to inhibitDNA-binding activity of both nuclear factor (NF)-B and AP-1.Troglitazone had no effect on NF-B activation and was shown toincrease LPS-induced AP-1 activation. 15d-PGJ₂ andtroglitazone modulated the expression of LPS-induced iNOS, COX-2, andproinflammatory cytokines differently. Indeed, troglitazone seems tospecifically target TNF- and iNOS pathways. These results offer newinsights in regard to the anti-inflammatory potential of the PPAR-ligands and underline different mechanisms of action of15d-PGJ₂ and troglitazone in synovial fibroblasts.

     

The gamma-subunit of ENaC is more important for channel surface expression than the beta-subunit

The amiloride-sensitiveepithelial sodium channel (ENaC) plays a critical role in fluid andelectrolyte homeostasis and is composed of three homologous subunits:, , and . Only heteromultimeric channels made of ENaCare efficiently expressed at the cell surface, resulting in maximallyamiloride-sensitive currents. To study the relative importance ofvarious regions of the - and -subunits for the expression offunctional ENaC channels at the cell surface, we constructedhemagglutinin (HA)-tagged --chimeric subunits composed of -and -subunit regions and coexpressed them with HA-tagged - and-subunits in Xenopus laevis oocytes. The whole cellamiloride-sensitive sodium current (I_ami) andsurface expression of channels were assessed in parallel using thetwo-electrode voltage-clamp technique and a chemiluminescence assay.Because coexpression of ENaC resulted in largerI_ami and surface expression compared withcoexpression of ENaC, we hypothesized that the -subunit ismore important for ENaC trafficking than the -subunit. Usingchimeras, we demonstrated that channel activity is largely preservedwhen the highly conserved second cysteine rich domains (CRD2) of the- and -subunits are exchanged. In contrast, exchanging the wholeextracellular loops of the - and the -subunits largely reducedENaC currents and ENaC expression in the membrane. This indicates thatthere is limited interchangeability between molecular regions of thetwo subunits. Interestingly, our chimera studies demonstrated that theintracellular termini and the two transmembrane domains of ENaC aremore important for the expression of functional channels at the cellsurface than the corresponding regions of ENaC.

     

Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-beta 1

Transforming growth factor-(TGF-) is known to induce -smooth muscle actin (-SMA) infibroblasts and is supposed to play a role in myofibroblastdifferentiation and tumor desmoplasia. Our objective was to elucidatethe impact of TGF-1 on -SMA expression in fibroblasts in athree-dimensional (3-D) vs. two-dimensional (2-D) environment. Inmonolayer culture, all fibroblast cultures responded in a similarfashion to TGF-1 with regard to -SMA expression. In fibroblastspheroids, -SMA expression was reduced and induction by TGF-1 washighly variable. This difference correlated with a differentialregulation in the TGF- receptor (TGFR) expression, in particularwith a reduction in TGF-RII in part of the fibroblast types. Ourdata indicate that 1) sensitivity to TGF-1-induced -SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, suchas a general reduction/loss in TGF-RIII, contribute to an altered TGFR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFR typesI and II determine the sensitivity to TGF-1-induced -SMAexpression in the 3-D setting. We suggest that fibroblasts that can beinduced by TGF-1 to produce -SMA in spheroid culture reflect a"premyofibroblastic" phenotype.

     

Role of alpha(v)beta(3)-integrin in TNF-alpha-induced endothelial cell migration

Tumor necrosis factor- (TNF-), oneof the major inflammatory cytokines, is known to influence endothelialcell migration. In this study, we demonstrate that exposure of calfpulmonary artery endothelial cells to TNF- caused an increase in theformation of membrane protrusions and cell migration. Fluorescencemicroscopy revealed an increase in _v3focal contacts but a decrease in ₅₁ focalcontacts in TNF--treated cells. In addition, both cell-surface andtotal cellular expression of _v3-integrinsincreased significantly, whereas the expression of₅₁-integrins was unaltered. Only focalcontacts containing _v3- but not₅₁-integrins were present in membraneprotrusions of cells at the migration front. In contrast, robust focalcontacts containing ₅₁-integrins were present in cells behind the migration front. A blocking antibody to_v3, but not a blocking antibody to₅-integrins, significantly inhibited TNF--inducedcell migration. These results indicate that in response to TNF-,endothelial cells may increase the activation and ligation of_v3 while decreasing the activation andligation of ₅₁-integrins to facilitatecell migration, a process essential for vascular wound healing and angiogenesis.

     

NF-kappa B inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis

Toxins convertthe hepatocellular response to tumor necrosis factor- (TNF-)stimulation from proliferation to cell death, suggesting thathepatotoxins somehow sensitize hepatocytes to TNF- toxicity. Becausenuclear factor-B (NF-B) activation confers resistance to TNF-cytotoxicity in nonhepatic cells, the possibility that toxin-inducedsensitization to TNF- killing results from inhibition ofNF-B-dependent gene expression was examined in the RALA rathepatocyte cell line sensitized to TNF- cytotoxicity by actinomycinD (ActD). ActD did not affect TNF--induced hepatocyte NF-Bactivation but decreased NF-B-dependent gene expression. Expressionof an IB superrepressor rendered RALA hepatocytes sensitive toTNF--induced apoptosis in the absence of ActD. Apoptosis was blockedby caspase inhibitors, and TNF- treatment led to activation ofcaspase-2, caspase-3, and caspase-8 only when NF-B activation wasblocked. Although apoptosis was blocked by the NF-B-dependent factornitric oxide (NO), inhibition of endogenous NO production did notsensitize cells to TNF--induced cytotoxicity. Thus NF-Bactivation is the critical intracellular signal that determines whetherTNF- stimulates hepatocyte proliferation or apoptosis. Althoughexogenous NO blocks RALA hepatocyte TNF- cytotoxicity, endogenousproduction of NO is not the mechanism by which NF-B activationinhibits this death pathway.

     

Regulation and distribution of MAdCAM-1 in endothelial cells in vitro

Mucosal addressin cell adhesion molecule-1(MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein thatregulates lymphocyte trafficking to Peyer's patches and lymph nodes.Although it is widely agreed that MAdCAM-1 induction is involved inchronic gut inflammation, few studies have investigated regulation ofMAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)- using RT-PCR, blotting, adhesion, and immunofluorescence. TNF- induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38mitogen-activated protein kinase (MAPK), and nuclear factor(NF)-B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1is regulated via MAPKs, we examined mitogen/extracellularsignal-regulated kinase (MEK)-1/2 activation in SVEC. We found thatMEK-1/2 is activated by TNF- within minutes and is dependent on TKand p42/44 MAPKs. Similarly, TNF- activated NF-B through TK,p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was alsoshown to be frequently distributed to endothelial junctions both invitro and in vivo. Cytokines like TNF- stimulate MAdCAM-1 inhigh endothelium via TK, p38, p42/22 MAPKs, and NF-B/PARP.MAdCAM-1 expression requires NF-B translocation through both directp42/44 and indirect p38 MAPK pathways in high endothelial cells.

     

Upregulation of alpha(8)beta(1)-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-beta1

Using a novel pharmacological tool with¹²⁵I-echistatin to detect integrins on the cell, we haveobserved that cardiac fibroblasts harbor five different RGD-bindingintegrins: ₈₁,₃₁, ₅₁, _v1, and _v3.Stimulation of cardiac fibroblasts by angiotensin II (ANG II) ortransforming growth factor-1 (TGF-1) resulted in an increase ofprotein and heightening by 50% of the receptor density of₈₁-integrin. The effect of ANG II wasblocked by an AT₁, but not an AT₂, receptorantagonist, or by an anti-TGF-1 antibody. ANG II and TGF-1increased fibronectin secretion, smooth muscle -actin synthesis, andformation of actin stress fibers and enhanced attachment of fibroblaststo a fibronectin matrix. The ₈- and₁-subunits were colocalized by immunocytochemistry with vinculin or ₃-integrin at focal adhesion sites.These results indicate that ₈₁-integrinis an abundant integrin on rat cardiac fibroblasts. Its positivemodulation by ANG II and TGF-1 in a myofibroblast-likephenotype suggests the involvement of₈₁-integrin in extracellularmatrix protein deposition and cardiac fibroblast adhesion.

     

Vascular endothelial cells express isoforms of protein kinase A inhibitor

First published September 5, 2001;10.1152/ ajpcell.00256.2001.The expression and function of theendogenous inhibitor of cAMP-dependent protein kinase (PKI) inendothelial cells are unknown. In this study, overexpression of rabbitmuscle PKI gene into endothelial cells inhibited the cAMP-mediatedincrease and exacerbated thrombin-induced decrease in endothelialbarrier function. We investigated PKI expression in human pulmonaryartery (HPAECs), foreskin microvessel (HMECs), and brain microvesselendothelial cells (HBMECs). RT-PCR using specific primers for humanPKI, human PKI, and mouse PKI sequences detectedPKI and PKI mRNA in all three cell types. Sequencing and BLASTanalysis indicated that forward and reverse DNA strands for PKI andPKI were of >96% identity with database sequences. RNaseprotection assays showed protection of the 542 nucleotides in HBMEC andHPAEC PKI mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKImRNA. Western blot analysis indicated that PKI protein was detectedin all three cell types, whereas PKI was found in HBMECs. Insummary, endothelial cells from three different vascular beds expressPKI and PKI, which may be physiologically important inendothelial barrier function.

     

Protein kinase Calpha participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells

Protein kinase C (PKC) plays animportant role in activating store-operated Ca²⁺ channels(SOC) in human mesangial cells (MC). The present study was performed todetermine the specific isoform(s) of conventional PKC involved inactivating SOC in MC. Fura 2 fluorescence ratiometry showed that thethapsigargin-induced Ca²⁺ entry (equivalent to SOC) wassignificantly inhibited by 1 µM Gö-6976 (a specific PKC andI inhibitor) and PKC antisense treatment (2.5 nM for 24-48h). However, LY-379196 (PKC inhibitor) and2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether(HBDDE; PKC and  inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKC antisensesignificantly depressed thapsigargin-induced activation of SOC.However, LY-379196 and HBDDE did not affect the SOC responses. Ininside-out patches, application of purified PKC or I, but notII or , significantly rescued SOC from postexcision rundown.Western blot analysis revealed that thapsigargin evoked a decrease incytosolic expression with a corresponding increase in membraneexpression of PKC and . However, the translocation from cytosolto membranes was not detected for PKCI or II. These resultssuggest that PKC participates in the intracellular signaling pathwayfor activating SOC upon release of intracellular stores ofCa²⁺.

     

p38 MAPK mediates renal tubular cell TNF-{alpha} production and TNF-{alpha}-dependent apoptosis during simulated ischemia

Ischemia causes renal tubular cellloss through apoptosis; however, the mechanisms of this processremain unclear. Using the renal tubular epithelial cell lineLLC-PK₁, we developed a model of simulated ischemia(SI) to investigate the role of p38 MAPK (mitogen-activated proteinkinase) in renal cell tumor necrosis factor- (TNF-) mRNAproduction, protein bioactivity, and apoptosis. Resultsdemonstrate that 60 min of SI induced maximal TNF- mRNA productionand bioactivity. Furthermore, 60 min of ischemia induced renaltubular cell apoptosis at all substrate replacement time pointsexamined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF- mRNA production andTNF- bioactivity, and both p38 MAPK inhibition and TNF- neutralization (anti-porcine TNF- antibody) preventedapoptosis after 60 min of SI. These results constitute theinitial demonstration that 1) renal tubular cells produceTNF- mRNA and biologically active TNF- and undergoapoptosis in response to SI, and 2) p38 MAPKmediates renal tubular cell TNF- production and TNF--dependent apoptosis after SI.

     

Effect of interleukin-1beta and tumor necrosis factor-alpha on gene expression in human endothelial cells

Interleukin-1(IL-1) and tumor necrosis factor- (TNF-) are two majorcytokines that rise to relatively high levels during systemicinflammation, and the endothelial cell (EC) response to these cytokinesmay explain some of the dysfunction that occurs. To better understandthe cytokine-induced responses of EC at the gene expression level,human umbilical vein EC were exposed to IL-1 or TNF- for varioustimes and subjected to cDNA microarray analyses to study alterations intheir mRNA expression. Of ~4,000 genes on the microarray, expressionlevels of 33 and 58 genes appeared to be affected by treatment withIL-1 and TNF-, respectively; 25 of these genes responded to bothtreatments. These results suggest that the effects of IL-1 andTNF- on EC are redundant and that it may be necessary to suppressboth cytokines simultaneously to ameliorate the systemic response.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappa B activation

Inflammatory mediators are involved in the early phase of acutepancreatitis, but the cellular mechanisms responsible for theirgeneration within pancreatic cells are unknown. We examined the role ofnuclear factor-B (NF-B) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokineexpression in pancreatic acinar cells in vitro. Supraphysiological, butnot physiological, concentrations of CCK-8 increased inhibitory B(IB-) degradation, NF-B activation, andmob-1 gene expression in isolatedpancreatic acinar cells. CCK-8-induced IB- degradation wasmaximal within 1 h. Expression ofmob-1 was maximal within 2 h. Neitherbombesin nor carbachol significantly increasedmob-1 mRNA or induced IB-degradation. Thus the concentration, time, and secretagogue dependenceof mob-1 gene expression and IB-degradation were similar. Inhibition of NF-B with pharmacologicalagents or by adenovirus-mediated expression of the inhibitory proteinIB- also inhibited mob-1 geneexpression. These data indicate that the NF-B signaling pathway isrequired for CCK-8-mediated induction ofmob-1 chemokine expression inpancreatic acinar cells. This supports the hypothesis that NF-Bsignaling is of central importance in the initiation of acute pancreatitis.

     

Decreased 5alpha-dihydrotestosterone catabolism suppresses T lymphocyte functions in males after trauma-hemorrhage

Trauma-hemorrhage producesprofound immunosuppression in males but not in proestrus females.Prior castration or flutamide treatment of males followingtrauma-hemorrhage prevents immunosuppression, implicating5-dihydrotestosterone for the immunosuppressive effects. 5-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests bothsynthesis and catabolism of 5-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5-dihydrotestosterone activity inT lymphocytes of males following trauma-hemorrhage is due to decreasedcatabolism. Accordingly, catabolism of 5-dihydrotestosterone wasassessed in splenic T lymphocytes by examining the activity andexpression of enzymes involved. Analysis showed increased synthesis anddecreased catabolism of 5-dihydrotestosterone in intact male Tlymphocytes following trauma-hemorrhage. In contrast, reduced5-reductase activity and increased expression of17-hydroxysteroid dehydrogenase oxidative isomers suggestinactivation of 5-dihydrotestosterone in precastrated males. Thusour study suggests increased synthesis and decreased catabolism of5-dihydrotestosterone as a reason for loss of T lymphocyte functionsin intact males following trauma-hemorrhage, as evidenced by decreasedrelease of interleukin-2 and -6.

     

TNF receptor I is required for induction of macrophage heat shock protein 70

Expression of heat shock proteins (HSP) is anadaptive response to cellular stress. Stress induces tumor necrosisfactor (TNF)- production. In turn, TNF- induces HSP70 expression.However, osmotic stress or ultraviolet radiation activates TNF-receptor I (TNFR-I) in the absence of TNF-. We postulated thatTNF- receptors are involved in the induction of HSP70 by cellularstress. Peritoneal M were isolated from wild-type (WT), TNF-knockout (KO), and TNFR (I or II) KO mice. Cells were culturedovernight and then heat stressed at 43 ± 0.5°C for 30 minfollowed by a 4-h recovery at 37°C. Cellular HSP70 expression wasinduced by heat stress or exposure to endotoxin [lipopolysaccharide(LPS)] as determined by immunoblotting. HSP70 expression induced byeither heat or LPS was markedly decreased in TNFR-I KO M, whereasTNFR-II KO M exhibited HSP70 expression comparable to that in WTmice. Expression of HSP70 after heat stress in TNF- KO M was alsosimilar to that in WT mice, suggesting that induction of HSP70 byTNFR-I occurs independently of TNF-. In addition, levels ofsteady-state HSP70 mRNA were similar by RT-PCR in WT and TNFR-I KO Mdespite differences in protein expression. Furthermore, the effect of TNFR-I appears to be cell specific, since HSP70 expression in splenocytes isolated from TNFR-I KO was similar to that in WT splenocytes. These studies demonstrate that TNFR-I is required for thesynthesis of HSP70 in stressed M by a TNF-independent mechanism andsupport an intracellular role for TNFR-I.

     

Induction of TNF-alpha and MnSOD by endotoxin: role of membrane CD14 and Toll-like receptor-4

Endotoxin (LPS) is a potent inducer oftumor necrosis factor- (TNF-) and manganese superoxide dismutase(MnSOD). Recent evidence suggests that LPS induction of TNF- andMnSOD mRNAs is mediated through distinct intracellular signaltransduction pathways. Membrane CD14 (mCD14) and Toll-like receptor-4(TLR4) mediate LPS induction of TNF- in macrophages. In the current study, we evaluated the role of mCD14 and TLR4 in LPS induction ofMnSOD using peritoneal macrophages from CD14 knockout (CD14-KO) miceand mice with the Tlr4 gene point mutation (C3H/HeJ) ordeletion (C57BL/10ScCr). We studied mCD14-dependent (1 and 10 ng/ml)and mCD14-independent (1,000 ng/ml) concentrations of LPS. Compared with control (BALB/c) macrophages, LPS at 1 and 10 ng/ml failed toinduce TNF- or MnSOD mRNA in CD14-KO macrophages. However, LPS at1,000 ng/ml induced TNF- and MnSOD mRNAs equally in macrophages fromCD14-KO and control mice. LPS (1, 10, or 1,000 ng/ml) failed to induceTNF- or MnSOD mRNA and failed to activate nuclear factor-B inC3H/HeJ or C57BL/10ScCr macrophages. Measurements of TNF- and MnSODenzyme activity paralleled TNF- and MnSOD mRNA levels. These datademonstrate that, like TNF-, induction of MnSOD by LPS is mediatedby mCD14 and TLR4 in murine macrophages.

     

Agonist-specific cross talk between ERKs and p38(mapk) regulates PGI(2) synthesis in endothelium

We have examined the mechanisms regulatingprostacyclin (PGI₂) synthesis after acute exposure of humanumbilical vein endothelial cells (HUVEC) to interleukin-1 (IL-1).IL-1 evoked an early (30 min) release of PGI₂ and[³H]arachidonate that was blocked by the cytosolicphospholipase A₂ (cPLA₂) inhibitorarachidonyl trifluoromethyl ketone. IL-1-mediated activationof extracellular signal-regulated kinase 1/2 (ERK1/2; p42/p44^mapk) coincided temporally with phosphorylation ofcPLA₂ and with the onset of PGI₂synthesis. The mitogen-activated protein kinase (MAPK) kinase (MEK)inhibitors, PD-98059 and U-0126, blocked IL-1-induced ERKactivation and partially attenuated cPLA₂phosphorylation and PGI₂ release, suggesting thatERK-dependent and -independent pathways regulate cPLA₂phosphorylation. SB-203580 treatment enhanced IL-1-induced MEK,p42/44^mapk, and cPLA₂ phosphorylation butreduced thrombin-stimulated MEK and p42/44^mapk activation.IL-1, but not thrombin, activated Raf-1 as assessed byimmune-complex kinase assay, as did SB-203580 alone. These results showthat IL-1 causes an acute upregulation of PGI₂generation in HUVEC, establish a role for theMEK/ERK/cPLA₂ pathway in this early release, and provideevidence for an agonist-specific cross talk between p38^mapkand p42/44^mapk that may reflect receptor-specificdifferences in the signaling elements proximal to MAPK activation.