PKC zeta participates in activation of inflammatory response induced by enteropathogenic E. coli

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-B and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-B activation but that other signaling molecules such as PKC may be involved. The aim of this study was to determine whether PKC is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKC from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKC by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKC pseudosubstrate (MYR-PKC-PS), or transient expression of a nonfunctional PKC significantly suppressed EPEC-induced IB phosphorylation. Although PKC can activate ERK, MYR-PKC-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKC can regulate NF-B activation by interacting with and activating IB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKC and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKC-IKK complexes from infected intestinal epithelial cells and recombinant IB as a substrate showed a 2.5-fold increase in IB phosphorylation. PKC can also regulate NF-B by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKC-PS, suggesting that other signaling events may be involved in this particular arm of NF-B regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKC and ERK that lead to NF-B activation, thus ensuring the proinflammatory response. inflammation; enteropathogenic Escherichia coli; nuclear factor-B; protein kinase C; IB kinase; extracellular signal-regulated kinase     

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     

ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus

High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT₁R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT₁R (SYF/AT₁). ERK1/2 activation was reduced by 50% within these cells compared with wild-type controls (WT/AT₁). The remaining 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKC) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKC signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells compared with WT/AT₁ cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT₁ cells with a PKC pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKC pseudosubstrate-treated WT/AT₁ cells (P < 0.01) and was completely blocked in SYF/AT₁ cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, respectively. Src family tyrosine kinases; angiotensin II     

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     

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     

Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR ₇-subunit has a property of high Ca²⁺ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChR₇ existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR ₅- and ₂-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChR₇. Cholesterol depletion from plasma membranes with methyl--cyclodextrin redistributed nAChR₇ and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with -bungarotoxin, a specific antagonist of nAChR₇, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChR₇ has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChR₇ with AC within plasma membranes. In addition, nAChR₇ may regulate the AC activity via Ca²⁺ within lipid rafts. cholesterol; PC-12 cells     

Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-, -, and - (34), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC- inhibitor röttlerin, implicating a novel isozyme, likely PKC-. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC--dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex. protein kinase C; epithelial barrier function     

Galpha12 regulates epithelial cell junctions through Src tyrosine kinases

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that G₁₂ binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated G₁₂ increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of G₁₂ expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active G₁₂ (QL₁₂)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QL₁₂-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QL₁₂ phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QL₁₂-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [³H]mannitol flux was prevented by the inhibitors. Src activity was increased in QL₁₂-expressing MDCK cells as assessed by Src autophosphorylation, and -catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that G₁₂ regulates MDCK cell junctions, in part through Src tyrosine kinase pathways. G proteins; tight junction; adherens junction; Rho     

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     

Synergistic amplification of beta-amyloid- and interferon-gamma-induced microglial neurotoxic response by the senile plaque component chromogranin A

Activation of the microglial neurotoxic response by components of the senile plaque plays a critical role in the pathophysiology of Alzheimer's disease (AD). Microglia induce neurodegeneration primarily by secreting nitric oxide (NO), tumor necrosis factor- (TNF), and hydrogen peroxide. Central to the activation of microglia is the membrane receptor CD40, which is the target of costimulators such as interferon- (IFN). Chromogranin A (CGA) is a recently identified endogenous component of the neurodegenerative plaques of AD and Parkinson's disease. CGA stimulates microglial secretion of NO and TNF, resulting in both neuronal and microglial apoptosis. Using electrochemical recording from primary rat microglial cells in culture, we have shown in the present study that CGA alone induces a fast-initiating oxidative burst in microglia. We compared the potency of CGA with that of -amyloid (A) under identical conditions and found that CGA induces 5–7 times greater NO and TNF secretion. Coapplication of CGA with A or with IFN resulted in a synergistic effect on NO and TNF secretion. CD40 expression was induced by CGA and was further increased when A or IFN was added in combination. Tyrphostin A1 (TyrA1), which inhibits the CD40 cascade, exerted a dose-dependent inhibition of the CGA effect alone and in combination with IFN and A. Furthermore, CGA-induced mitochondrial depolarization, which precedes microglial apoptosis, was fully blocked in the presence of TyrA1. Our results demonstrate the involvement of CGA with other components of the senile plaque and raise the possibility that a narrowly acting agent such as TyrA1 attenuates plaque formation. Alzheimer's disease; oxidative burst; apoptosis; nitric oxide; tyrphostin A1     

PKC-epsilon is upstream and PKC-alpha is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium

Protein kinase C (PKC) is involved in the process of ischemic preconditioning (IPC), although the precise mechanism is still a subject of debate. Using specific PKC inhibitors, we investigated which PKC isoforms were involved in IPC of the human atrial myocardium sections and to determine their temporal relationship to the opening of mitochondrial potassium-sensitive ATP (mitoK_ATP) channels. Right atrial muscles obtained from patients undergoing elective cardiac surgery were equilibrated and then randomized to receive any of the following protocols: aerobic control, 90-min simulated ischemia/120-min reoxygenation, IPC using 5-min simulated ischemia/5-min reoxygenation followed by 90-min simulated ischemia/120-min reoxygenation and finally, PKC inhibitors were added 10 min before and 10 min during IPC followed by 90-min simulated ischemia/120-min reoxygenation. The PKC isoforms inhibitors investigated were V1–2 peptide, GO-6976, rottlerin, and LY-333531 for PKC-, -, - and -, respectively. To investigate the relation of PKC isoforms to mitoK_ATP channels, PKC inhibitors found to be involved in IPC were added 10 min before and 10 min during preconditioning by diazoxide followed by 90-min simulated ischemia/120-min reoxygenation in a second experiment. Creatine kinase leakage and methylthiazoletetrazolium cell viability were measured. Phosphorylation of PKC isoforms after activation of the sample by either diazoxide or IPC was detected by using Western blot analysis and then analyzed by using Scion image software. PKC- and - inhibitors blocked IPC, whereas PKC- and - inhibitors did not. The protection elicited by diazoxide, believed to be via mitoK_ATP channels opening, was blocked by the inhibition of PKC- but not - isoforms. In addition, diazoxide caused increased phosphorylation of PKC- to the same extent as IPC but did not affect the phosphorylation of PKC-, a process believed to be critical in PKC activation. The results demonstrate that PKC- and - are involved in IPC of the human myocardium with PKC- being upstream and PKC- being downstream of mitoK_ATP channels. cardioprotection; protein kinase C isoforms     

Pyruvate induces mitochondrial biogenesis by a PGC-1 alpha-independent mechanism

Oxidative cells increase mitochondrial mass in response to stimuli such as changes in energy demand or cellular differentiation. This plasticity enables the cell to adapt dynamically to achieve the necessary oxidative capacity. However, the pathways involved in triggering mitochondrial biogenesis are poorly defined. The present study examines the impact of altering energy provision on mitochondrial biogenesis in muscle cells. C2C12 myoblasts were chronically treated with supraphysiological levels of sodium pyruvate for 72 h. Treated cells exhibited increased mitochondrial protein expression, basal respiratory rate, and maximal oxidative capacity. The increase in mitochondrial biogenesis was independent of increases in peroxisomal proliferator activator receptor- coactivator-1 (PGC-1) and PGC-1 mRNA expression. To further assess whether PGC-1 expression was necessary for pyruvate action, cells were infected with adenovirus containing shRNA for PGC-1 before treatment with pyruvate. Despite a 70% reduction in PGC-1 mRNA, the effect of pyruvate was preserved. Furthermore, pyruvate induced mitochondrial biogenesis in primary myoblasts from PGC-1 null mice. These data suggest that regulation of mitochondrial biogenesis by pyruvate in myoblasts is independent of PGC-1, suggesting the existence of a novel energy-sensing pathway regulating oxidative capacity. oxidative metabolism; peroxisomal proliferator activator receptor- coactivator-1, mitochondria; muscle     

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake

The carboxy terminus (CT) of the colonic H⁺-K⁺-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK₂, we selected 84 amino acids in the CT of the -subunit of mouse colonic H⁺-K⁺-ATPase (CT-HK₂) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK₂ with CD63, recombinant CT-HK₂ and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK₂ protein (but not CT-HK₁) coprecipitated with CD63, confirming stable assembly of HK₂ with CD63. In HEK-293 transfected with HK₂ plus ₁-Na⁺-K⁺-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK₂/NK₁ and ⁸⁶Rb⁺ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK₂-NK₁ complex in the cell membrane. protein assembly; cell surface localization     

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     

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     

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     

Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-alpha

Angiotensin II (ANG II) has been etiologically linked to vascular disease; however, its role in the alterations of endothelial function that occur in vascular disorders is not completely understood. Matrix metalloproteinases (MMPs) and proinflammatory cytokines are involved in the pathological remodeling of blood vessels that occurs in vascular disease. In this study we evaluated the effects of ANG II on tumor necrosis factor (TNF)- and MMP-2 production in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with ANG II (0.1–10 µM) for 24 h, in the presence or absence of antagonists of ANG II type 1 (AT₁R) and type 2 (AT₂R) receptors, and the production and release of TNF- and MMP-2 were assessed. ANG II increased TNF- mRNA and protein expression and the release of bioactive TNF-. Moreover, ANG II induced MMP-2 release and reduced the secretion of tissue inhibitor of MMP (TIMP)-2 from endothelial cells. To elucidate whether endogenous TNF- could mediate the effects of ANG II on MMP-2 release, cells were pretreated with anti-TNF- neutralizing antibodies or pentoxifylline (an inhibitor of TNF- synthesis). TNF- inhibition prevented the secretion of MMP-2 induced by ANG II. Furthermore, AT₁R antagonism with candesartan prevented the formation of MMP-2 and TNF- and the reduction of TIMP-2 induced by ANG II. These results indicate that ANG II, via AT₁R, modulates the secretion of TNF- and MMP-2 from endothelial cells and that TNF- mediates the effects of ANG II on MMP-2 release. remodeling; vasoactive mediators; inflammation     

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     

Interleukin-1 beta induces posttranslational carboxymethylation and alterations in subnuclear distribution of lamin B in insulin-secreting RINm5F cells

We examined the effects of interleukin-1 (IL-1) treatment on the distribution and degradation of lamin B in the nuclear fraction from insulin-secreting RINm5F cells. Western blot analysis indicated that IL-1 treatment caused significant alterations in the redistribution of lamin B, specifically between the Triton X-100-soluble (membrane) and -insoluble (matrix) fractions of the nucleus. IL-1 treatment also increased the lamin carboxymethyltransferase activity and the relative abundance of the carboxymethylated lamin in the nuclear fraction. A significant increase in the relative abundance of lamin B degradation products was also observed in the nuclear fraction from the IL-1-treated cells. These findings are compatible with a measurable increase in the lamin-degrading caspase-6 activity in IL-1-treated cells. Confocal microscopic observation of IL-1-treated cells suggested a significant dissociation of lamin B from the nuclear lamina and its subsequent association with the DNA-rich elements within the nucleus. N^G-monomethyl-L-arginine, a known inhibitor of inducible nitric oxide synthetase (iNOS), markedly inhibited IL-1-induced iNOS gene expression, NO release, caspase-3 and caspase-6 activation, lamin B degradation, and loss of metabolic cell viability, indicating that the observed IL-1-induced effects on nuclear lamin B involve the intermediacy of NO. Together, our data support the hypothesis that IL-1 treatment results in significant increase in the carboxymethylation of lamin B, which would place lamin B in a strategic location for its degradation mediated by caspases. This could possibly lead to dissolution of the nuclear envelope, culminating in the demise of the effete -cell. pancreatic -cell; lamin carboxymethyltransferase; nitric oxide; nuclear matrix; caspases     

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