Estrogen receptor-alpha predominantly mediates the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage

Although 17-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)- or ER- and which signaling pathways are involved in such 17-estradiol effects. Utilizing ER-- or ER--specific agonists, this study examined the role of ER- and ER- in 17-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-B are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), the ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF- production and activation of MAPK and NF-B were measured. Macrophage IL-6 and TNF- production and MAPK activation were decreased, whereas NF-B activity was increased, following trauma-hemorrhage. PPT or 17-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER- plays the predominant role in mediating the salutary effects of 17-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-B signaling pathways. shock; mitogen-activated protein kinase; nuclear factor-B; propyl pyrazole triol; diarylpropionitrile     

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-kappaB through phosphorylation of IkappaBbeta

Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell     

RhoA regulation of NF-kappaB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells

Numerous studies have demonstrated a central role of renal tubular epithelial cells in the etiology of kidney injury and disease through the elaboration of inflammatory mediators. However, little is known about the cellular signaling mechanisms involved in this process. In this study we employed normal rat kidney epithelial (NRK52E) cells to identify a novel LPS-induced signaling pathway in which RhoA-mediated AP-1 activity promotes expression of cyclooxygenase-2 (COX-2) with consequent feedback inhibition of NF-B activation through IKK. Inhibition of RhoA signaling using either the RhoA kinase inhibitor Y-27632 or a dominant negative mutant of RhoA (RhoA-DN) dramatically extended the duration of p65-DNA binding, IB phosphorylation, and IKK activity following LPS treatment. Prolongation of events associated with NF-B activation was also observed in cells pretreated and/or cotransfected with the JNK inhibitor SP600125 or deletion mutants of MEKK1 (MEKK1-KD) or Jun (Jun-DN). Conversely, constitutive expression of RhoA prevented NF-B activation by LPS, and this effect was reversed by cotransfection with MEKK1-KD. In addition, we found that the RhoA/AP-1 signaling axis plays a necessary role in COX-2 expression by LPS and that this effect is independent of NF-B activation. Moreover, inhibition of COX-2 activity results in persistent p65-DNA binding, IB phosphorylation, and IKK activity, similar to that observed after prevention of RhoA/AP-1 axis signaling. These findings suggest that COX-2 links the RhoA/AP-1 signaling cascade to NF-B activation, thereby defining a novel integrated model for regulation of the inflammatory response of kidney epithelial cells to LPS and potentially other external stimuli. AP-1; cyclooxygenase-2; inflammation; lipopolysaccharide, nuclear factor-B; IB kinase     

Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-kappaB in endothelial cells

NF-B signaling pathway has been known to play a major role in the pathological process of atherogenesis. Unlike high shear stress, in which the NF-B activity is transient, our earlier studies have demonstrated a persistent activation of NF-B in response to low shear stress in human aortic endothelial cells. These findings partially explained why low shear regions that exist at bifurcations of arteries are prone to atherosclerosis, unlike the relatively atheroprotective high shear regions. In the present study, we further investigated 1) the role of NF-B signaling kinases (IKK and ) that may be responsible for the sustained activation of NF-B in low shear stress and 2) the regulation of these kinases by reactive oxygen species (ROS). Our results demonstrate that not only is a significant proportion of low shear-induced-kinase activity is contributed by IKK, but it is also persistently induced for a prolonged time frame. The IKK activity (both and ) is blocked by apocynin (400 µM), a specific NADPH oxidase inhibitor, and diphenyleneiodonium chloride (DPI; 10 µM), an inhibitor of flavin-containing oxidases like NADPH oxidases. Determination of ROS also demonstrated an increased generation in low shear stress that could be blocked by DPI. These results suggest that the source of ROS generation in endothelial cells in response to low shear stress is NADPH oxidase. The DPI-inhibitable component of ROS is the primary regulator of specific upstream kinases that determine the persistent NF-B activation selectively in low shear-induced endothelial cells. upstream B kinases; laminar shear stress; oxidative stress; atherogenesis; reactive oxygen species     

Novel effect of NF-kappaB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium

Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-B is key to cytoskeletal instability. In the present study, we hypothesized that NF-B activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-B, with or without exposure to oxidant (H₂O₂). Other cells were transfected with an IB mutant (an inhibitor of NF-B). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IB instability, NF-B subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-B inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-B iNOS NO pathway. Dominant mutant stabilization of IB to inactivate NF-B suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-B is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-B activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-B appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-B in cells. tubulin cytoskeleton; microtubules; oxidation/nitration; inducible nitric oxide synthase/peroxynitrite; inflammatory bowel disease; Caco-2 cells; gut barrier; nuclear factor-B/IB     

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-B signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-B activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF- plus cycloheximide (TNF-/CHX). Specific inhibition of NF-B by the recombinant adenoviral vector containing the IB superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-B activity and increased the sensitivity to TNF-/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-B activity and resulted in increased resistance to TNF-/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-/CHX-induced apoptosis, at least partially, through the activation of NF-B signaling in IECs. polyamines; -difluoromethylornithine; X-linked inhibitor of apoptosis protein; IB     

Differential effects of NF-{kappa}B and p38 MAPK inhibitors and combinations thereof on TNF-{alpha}- and IL-1{beta}-induced proinflammatory status of endothelial cells in vitro

Endothelial cells actively participate in inflammatory events by regulating leukocyte recruitment via the expression of inflammatory genes such as E-selectin, VCAM-1, ICAM-1, IL-6, IL-8, and cyclooxygenase (COX)-2. In this study we showed by real-time RT-PCR that activation of human umbilical vein endothelial cells (HUVEC) by TNF- and IL-1 differentially affected the expression of these inflammatory genes. Combined treatment with TNF- and IL-1 resulted in nonadditive, additive, and even synergistic induction of expression of VCAM-1, IL-8, and IL-6, respectively. Overexpression of dominant-negative inhibitor B protein blocking NF-B signaling confirmed a major role of this pathway in controlling both TNF-- and IL-1-induced expression of most of the genes studied. Although dexamethasone exerted limited effects at 1 µM, the thioredoxin inhibitor MOL-294, which regulates the redox state of NF-B, mainly inhibited adhesion molecule expression. Its most pronounced effect was seen on VCAM-1 mRNA levels, especially in IL-1-activated endothelium. One micromolar RWJ-67657, an inhibitor of p38 MAPK activity, diminished TNF-- and IL-1-induced expression of IL-6, IL-8, and E-selectin but had little effect on VCAM-1 and ICAM-1. Combined treatment of HUVEC with MOL-294 and RWJ-67657 resulted in significant blocking of the expression of E-selectin, IL-6, IL-8, and COX-2. The inhibitory effects were much stronger than those observed with single drug treatment. Application of combinations of drugs that affect multiple targets in activated endothelial cells may therefore be considered as a potential new therapeutic strategy to inhibit inflammatory disease activity. inflammatory gene expression; anti-inflammatory drugs; pharmacology; combination treatment     

An osteoclastic protein-tyrosine phosphatase may play a role in differentiation and activity of human monocytic U-937 cell-derived, osteoclast-like cells

This study investigated if an osteoclastic protein-tyrosine phosphatase (PTP), PTP-oc, plays a role in the functional activity and differentiation of osteoclastic cells by determining the effects of overexpression of wild-type (WT)- or phosphatase-deficient (PD)-PTP-oc on bone resorption activity and differentiation of human promyelomonocytic U-937 cells, which could be induced to differentiate into "osteoclast-like" cells by phorbol ester/1,25(OH)₂D₃ treatment. U-937 cells overexpressing WT- or PD-PTP-oc were produced with a transposon-based vector. The size and depth of resorption pits created by WT-PTP-oc-overexpressing osteoclast-like cells were greater, while those by PD-PTP-oc-overexpressing osteoclast-like cells were less, than those created by control osteoclast-like cells. Overexpression of WT-PTP-oc also enhanced, while overexpression of PD-PTP-oc suppressed, their differentiation into osteoclast-like cells. Overexpression of WT-PTP-oc increased apoptosis and proliferation of U-937 cells, and overexpression of PD-PTP-oc reduced cell proliferation. Cells overexpressing WT-PTP-oc has also led to greater c-Src and NF- activation, whereas cells overexpressing PD-PTP-oc resulted in less c-Src and NF- activation. c-Src activation and NF- activation each correlated with resorption activity and differentiation into osteoclast-like cells. In summary, these results show that 1) PTP-oc regulates both the activity and the differentiation of osteoclast-like cells derived from U-937 cells; 2) PTP-oc enzymatic activity is important to these processes; 3) high PTP-oc enzymatic activity caused an increase in U-937 cell apoptosis and proliferation, leading to no significant changes in the number of viable cells; and 4) some of the PTP-oc actions are mediated in part by the c-Src and/or NF- pathways. osteoclast; resorption; nuclear factor-; c-Src     

Innate immune responses of human tracheal epithelium to Pseudomonas aeruginosa flagellin, TNF-alpha, and IL-1beta

We measured innate immune responses by primary human tracheal epithelial (HTE) cells grown as confluent, pseudostratified layers during exposure to inflammatory activators on apical vs. basolateral surfaces. Apical Pseudomonas aeruginosa strain PAK (but not flagellin mutant PAK·fliC), flagellin, and flagellin + PAK·fliC activated NF-B and IL-8 expression and secretion. In contrast, HTE cells were insensitive to LPS compared to flagellin. Flagellin activated NF-B in columnar but not basal cells. IL-1 + TNF- elicited responses similar to those of flagellin. Basolateral flagellin or IL-1 + TNF- caused 1.5- to 4-fold larger responses, consistent with the fact that NF-B activation occurred in both columnar and basal cells. MyD88 (toll receptor-associated adapter), IL-1 receptor (IL1R)1, and TNF- receptor (TNFR)1 were expressed in columnar and basal cells. ZO-1 was localized to tight junctions of columnar cells but not to basal cells. We infer the following. 1) Flagellin is necessary and sufficient to trigger inflammatory responses in columnar cells during accumulation of P. aeruginosa in the airway surface liquid (ASL); columnar cells express toll-like receptor 5 and MyD88, often associated with flagellin-activated cell signaling. 2) IL-1 + TNF- in the ASL also activate columnar cells, and these cells also express IL1R1 and TNFR1. 3) Apical flagellin, IL-1, and TNF- do not activate basal cells because tight junctions between columnar cells prevent access from the apical surface to the basal cells. 4) Exposure of basolateral surfaces to inflammatory activators elicits larger responses because both columnar and basal cells are activated, likely because both cell types express receptors for flagellin, IL-1, and TNF-. toll-like receptor; nuclear factor-B; interleukin-8; tumor necrosis factor; interleukin-1     

Neuropeptide substance P upregulates chemokine and chemokine receptor expression in primary mouse neutrophils

Neuropeptides play an important role in the active communication between the nervous and immune systems. Substance P (SP) is a prominent neuropeptide involved in neurogenic inflammation and has been reported to exert various proinflammatory actions on inflammatory leukocytes including neutrophils. The present study further investigated the modulatory effect of SP (1 µM) on chemokine production and chemokine receptor expression in primary mouse neutrophils. Our results showed that SP primed neutrophils for chemotactic responses not only to the CXC chemokine macrophage inflammatory protein (MIP)-2/CXCL2 but also to the CC chemokine MIP-1/CCL3. The activating effect of SP on neutrophils was further evidenced by upregulation of the CD11b integrin, the activation marker of neutrophils. SP induced both the mRNA and protein expression of the chemokines MIP-1/CCL3 and MIP-2/CXCL2 in neutrophils and upregulated the chemokine receptors CC chemokine receptor (CCR)-1 and CXC chemokine receptor (CXCR)-2. This stimulatory effect on chemokine and chemokine receptor expression in neutrophils was further found to be neurokinin-1 receptor (NK-1R) specific. Pretreatment with selective NK-1R antagonists inhibited SP-triggered activation of neutrophils and chemokine and chemokine receptor upregulation. Moreover, SP-induced chemokine upregulation was NF-B dependent. SP time dependently induced NF-B p65 binding activity, IB degradation, and NF-B p65 nuclear translocation in neutrophils. Inhibition of NF-B activation with its inhibitor Bay11-7082 (10 µM) abolished SP-induced NF-B binding activity and upregulation of MIP-1/CCL3 and MIP-2/CXCL2 in neutrophils. Together, these results suggest that SP exerts a direct stimulatory effect on the expression of chemokines and chemokine receptors in mouse neutrophils. The effect is NK-1R mediated, involving NF-B activation. chemokines and receptors; neuro-immune interaction; neurokinin-1 receptor; primary leukocytes; NF-B activation     

Selective regulation by delta-PKC and PI 3-kinase in the assembly of the antiapoptotic TNFR-1 signaling complex in neutrophils

TNF is implicated in the attenuation of neutrophil constitutive apoptosis during sepsis. Antiapoptotic signaling is mediated principally through the TNF receptor-1 (TNFR-1). In adherent neutrophils, when -integrin signaling is activated, TNF phosphorylates TNFR-1 and activates prosurvival and antiapoptotic signaling. Previously, we identified the -PKC isotype and phosphatidylinositol (PI) 3-kinase as critical regulators of TNF signaling in adherent neutrophils. Both kinases associate with TNFR-1 in response to TNF and are required for TNFR-1 serine phosphorylation, NF-B activation, and inhibition of apoptosis. The purpose of this study was to examine the role of -PKC and PI 3-kinase in the assembly of TNFR-1 signaling complex that regulates NF-B activation and antiapoptotic signaling. Coimmunoprecipitation studies established that PI 3-kinase, -PKC, and TNFR-1 formed a signal complex in response to TNF. -PKC recruitment required both -PKC and PI 3-kinase activity, whereas PI 3-kinase recruitment was -PKC independent, suggesting that PI 3-kinase acts upstream of -PKC. An important regulatory step in control of antiapoptotic signaling is the assembly of the TNFR-1-TNFR-1-associated death domain protein (TRADD)-TNFR-associated factor 2 (TRAF2)-receptor interacting protein (RIP) complex that controls NF-B activation. Inhibition of either -PKC or PI 3-kinase decreased TNF-mediated recruitment of RIP and TRAF2 to TNFR-1. In contrast, TRADD recruitment was enhanced. Thus -PKC and PI 3-kinase are positive regulators of TNF-mediated association of TRAF2 and RIP with TNFR-1. Conversely, these kinases are negative regulators of TRADD association. These results suggest that -PKC and PI 3-kinase regulate TNF antiapoptotic signaling at the level of the TNFR-1 through control of assembly of a TNFR-1-TRADD-RIP-TRAF2 complex. inflammation; tumor necrosis factor receptor-1-associated death domain protein; receptor interacting protein; tumor necrosis factor receptor-associated factor 2; antiapoptotic signaling     

TFF3 modulates NF-{kappa}B and a novel negative regulatory molecule of NF-{kappa}B in intestinal epithelial cells via a mechanism distinct from TNF-{alpha}

Trefoil factor 3 (intestinal trefoil factor) is a cytoprotective factor in the gut. Herein we compared the effect of trefoil factor 3 with tumor necrosis factor- on 1) activation of NF-B in intestinal epithelial cells; 2) expression of Twist protein (a molecule essential for downregulation of nuclear factor-B activity in vivo); and 3) production of interleukin-8. We showed that Twist protein is constitutively expressed in intestinal epithelial cells. Tumor necrosis factor- induced persistent degradation of Twist protein in intestinal epithelial cells via a signaling pathway linked to proteasome, which was associated with prolonged activation of NF-B. In contrast to tumor necrosis factor, trefoil factor 3 triggered transient activation of NF-B and prolonged upregulation of Twist protein in intestinal epithelial cells via an ERK kinase-mediated pathway. Unlike tumor necrosis factor-, transient activation of NF-B by trefoil factor 3 is not associated with induction of IL-8 in cells. To examine the role of Twist protein in intestinal epithelial cells, we silenced the Twist expression by siRNA. Our data showed that trefoil factor 3 induced interleukin-8 production after silencing Twist in intestinal epithelial cells. Together, these observations indicated that 1) trefoil factor 3 triggers a diverse signal from tumor necrosis factor- on the activation of NF-B and its associated molecules in intestinal epithelial cells; and 2) trefoil factor 3-induced Twist protein plays an important role in the modulation of inflammatory cytokine production in intestinal epithelial cells. trefoil factor 3; signal transduction     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch

We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via IB kinase (IKK)/nuclear factor-B (NF-B) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of ₅₁ integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C- inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca²⁺ pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca²⁺]_i rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC- activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC--PKC-IKK-NF-B signaling cascade. Another crucial factor, [Ca²⁺]_i increase, may at least be required to activate PKC needed for NF-B activation. nuclear factor-B; phosphatidylinositol 3-kinase; phospholipase C-; protein kinase C; intracellular Ca²⁺ concentration     

Involvement of PKCdelta and PKD in pulmonary microvascular endothelial cell hyperpermeability

The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (, I, II, and ), novel [, , , and µ (also known as PKD),], and atypical ( and /), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms , I, and and complete translocation of PKC and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKC and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms , I, and were dispensable with regard to these same phenomena. signal transduction; permeability; myristolated alanine-rich C kinase substrate; microvasculature; pulmonary endothelium     

Calcitonin gene-related peptide inhibits interleukin-1beta-induced endogenous monocyte chemoattractant protein-1 secretion in type II alveolar epithelial cells

As important multifunctional cells in the lung, alveolar epithelial type II (AEII) cells secrete numerous chemokines on various stimuli. Our previous data showed that AEII cells also express the neuropeptide calcitonin gene-related peptide (CGRP) and the proinflammatory factor interleukin (IL)-1 induces CGRP secretion in the A549 human AEII cell line. In the present study, the CGRP-1 receptor antagonist human (h)CGRP_8–37 (0.1–1 nM) greatly amplified the production of IL-1-induced monocyte chemoattractant protein (MCP)-1. The inhibition of CGRP expression by small interfering RNA significantly increased MCP-1 secretion on IL-1 stimulation. However, exogenous hCGRP (10–100 nM) suppressed IL-1-evoked MCP-1 secretion in MCP-1 promoter activity, and CGRP gene stably transfected cell clones significantly inhibited both the mRNA and protein levels of MCP-1 induced by IL-1. These data imply that AEII-derived CGRP suppressed IL-1-induced MCP-1 secretion in an autocrine/paracrine mode. Subsequent investigation revealed that CGRP inhibited IL-1-evoked NF-B activity by suppressing IB phosphorylation and degradation. Moreover, CGRP attenuated IL-1-induced reactive oxygen species (ROS) formation, the early event in proinflammatory factor signaling. We previously showed that the CGRP inhibitory effect was mediated by elevated intracellular cAMP and show here that analogs of cAMP, 8-bromoadenosine 3',5'-cyclic monophosphothioate and the Sp isomer of adenosine 3',5'-cyclic monophosphothioate, mimicked the CGRP suppressive effect on IL-1-induced ROS formation, NF-B activation, and MCP-1 secretion. Thus increased endogenous CGRP secretion in lung inflammatory disease might eliminate the excessive response by elevating the cAMP level through inhibiting the ROS-NF-B-MCP-1 pathway. reactive oxygen species; lung; inflammation     

Effects of p38MAPK isoforms on renal mesangial cell inducible nitric oxide synthase expression

Several related isoforms of p38^MAPK have been identified and cloned in many species. Although they all contain the dual phosphorylation motif TGY, the expression of these isoforms is not ubiquitous. p38 and -2 are ubiquitously expressed, whereas p38 and - appear to have more restricted expression. Because there is evidence for selective activation by upstream kinases and selective preference for downstream substrates, the functions of these conserved proteins is still incompletely understood. We have demonstrated that the renal mesangial cell expresses the mRNA for all the isoforms of p38^MAPK, with p38 mRNA expressed at the highest level, followed by p38 and the lowest levels of expression by p382 and -. To determine the functional effects of these proteins on interleukin (IL)-1-induced inducible nitric oxide synthase (iNOS) expression, we transduced TAT-p38 chimeric proteins into renal mesangial cells and assessed the effects of wild-type and mutant p38 isoforms on ligand induced iNOS expression. We show that whereas p38 and - had minimal effects on iNOS expression, p38 and -2 significantly altered its expression. p38 mutant and p382 wild-type dose dependently inhibited IL-1-induced iNOS expression. These data suggest that p38 and 2 have reciprocal effects on iNOS expression in the mesangial cell, and these observations may have important consequences for the development of selective inhibitors targeting the p38^MAPK family of proteins. TAT proteins; p38 MAPK; inducible nitric oxide synthase; mesangial cell; interleukin-1     

Mechanism for normal splenic T lymphocyte functions in proestrus females after trauma: enhanced local synthesis of 17beta-estradiol

Trauma-hemorrhage and resuscitation (TH) produces profound immunodepression and enhances susceptibility to sepsis in males but not in proestrus females, suggesting gender dimorphism in the immune responses. However, the mechanism responsible for the maintenance of immune functions in proestrus females after TH is unclear. Splenic T lymphocytes express receptors for estrogen (ER), contain enzymes involved in estrogen metabolism, and are the major source of cytokine production; the metabolism of 17-estradiol was assessed in the splenic T lymphocytes of proestrus and ovariectomized mice by using appropriate substrates after TH. Analysis for aromatase and 17-hydroxysteroid dehydrogenases indicated increased 17-estradiol synthesis and low conversion into estrone in T lymphocytes of proestrus but not of ovariectomized mice. The effect of 17-estradiol on T lymphocyte cytokine release was reliant on ER expressions. This was apparent in the differences of ER expression, especially that of ER-, and an association between increased 17-estradiol synthesis and sustained release of IL-2 and IL-6 in T lymphocytes of proestrus females after TH. Because 17-estradiol is able to regulate cytokine genes, and the splenic T lymphocyte cytokine releases is altered after TH, continued synthesis of 17-estradiol in proestrus females appears to be responsible for the maintenance of T lymphocyte cytokine release associated with the protection of immune functions after TH. inflammation; immune suppression; steroid synthesis; T lymphocytes; cytokines     

Disruption of alpha-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis

Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of -actinin (ROD-GFP) that competitively displaces endogenous -actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas -actinin-GFP expression protected, cells from TNF--induced apoptosis. Further investigation revealed that activation of TNF--induced survival signals, specifically Akt phosphorylation and NF-B activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF--induced apoptosis. Thus we conclude that -actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF--induced survival signaling. tumor necrosis factor-; survival; cytoskeleton; nuclear factor-B     

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca²⁺-activated K⁺ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the -subunit of the BK channel (BK-), both - and ₁-subunits (BK-₁), or both - and ₄-subunits (BK-₄) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-₁ within a similar concentration range. Because methAEA could potentiate BK-, BK-₁, and BK-₄ with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-₁ did not change intracellular Ca²⁺ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB₁ antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB₂ agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener