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     

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     

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     

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     

Nuclear translocation of calcineurin Abeta but not calcineurin Aalpha by platelet-derived growth factor in rat aortic smooth muscle

Calcineurin regulates the proliferation of many cell types through activation of the nuclear factor of activated T cells (NFAT). Two main isoforms of the calcineurin catalytic subunit [calcineurin A (CnA) and CnA] have been identified, although their expression and function are largely unknown in smooth muscle. Western blot analysis and confocal imaging were performed in freshly isolated and cultured rat aortic myocytes to identify these CnA isoforms and elucidate the effect of PDGF on their cellular distribution and interaction with NFAT isoforms. CnA and CnA isoforms displayed differential cellular distribution, with CnA being evenly distributed between the nucleus and cytosol and CnA being restricted to the cytosol. In contrast with the rat brain, we found no evidence for particulate/membrane localization of calcineurin. PDGF caused significant nuclear translocation of CnA and induced smooth muscle cell proliferation, with both effects being abrogated by the calcineurin inhibitor cyclosporin A, the novel NFAT inhibitors A-285222 and inhibitor of NFAT-calcineurin association-6, and the adenylyl cyclase activator forskolin. PDGF also caused cyclosporin A-sensitive translocation of NFATc3, with no apparent effect on either CnA or NFATc1 distribution. Moreover, 87% of nuclear CnA was found to colocalize with NFATc3, consistent with the finding that CnA bound more avidly than CnA to a glutathione S-transferase-NFATc3 fusion protein. Based on their differential distribution in aortic muscle, our results suggest that CnA and CnA are likely to have different cellular functions. However, CnA appears to be specifically activated by PDGF, and we postulate that calcineurin-dependent nuclear translocation of NFATc3 is involved in smooth muscle proliferation induced by this mitogen. nuclear factor of activated T cells c3; confocal imaging; cell proliferation; inhibitor of nuclear factor activated T cells-calcineurin association-6; A-285222     

Salutary effects of 17beta-estradiol on T-cell signaling and cytokine production after trauma-hemorrhage are mediated primarily via estrogen receptor-alpha

Although 17-estradiol (E2) administration following trauma-hemorrhage prevents the suppression in splenocyte cytokine production, it remains unknown whether the salutary effects of 17-estradiol are mediated via estrogen receptor (ER)- or ER-. Moreover, it is unknown which signaling pathways are involved in 17-estradiol's salutary effects. Utilizing an ER-- or ER--specific agonist, we examined the role of ER- and ER- in E2-mediated restoration of T-cell cytokine production following trauma-hemorrhage. Moreover, since MAPK, NF-B, and activator protein (AP)-1 are known to regulate T-cell cytokine production, we also examined the activation of MAPK, NF-B, and AP-1. Male rats underwent trauma-hemorrhage (mean arterial pressure 40 mmHg for 90 min) and fluid resuscitation. ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), 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 T cells were isolated, and their IL-2 and IFN- production and MAPK, NF-B, and AP-1 activation were measured. T-cell IL-2 and IFN- production was decreased following trauma-hemorrhage, and this was accompanied with a decrease in T-cell MAPK, NF-B, and AP-1 activation. PPT or 17-estradiol administration following trauma-hemorrhage normalized those parameters, while DPN administration had no effect. Since PPT, but not DPN, administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the T-cell suppression, it appears that ER- plays a predominant role in mediating the salutary effects of 17-estradiol on T cells following trauma-hemorrhage, and that such effects are likely mediated via normalization of MAPK, NF-B, and AP-1 signaling pathways. shock; MAPK; NF-B; activator protein-1; propyl pyrazole triol; diarylpropionitrile     

TGF-beta-regulated collagen type I accumulation: role of Src-based signals

Transforming growth factor- (TGF-) stimulates myofibroblast transdifferentiation, leading to type I collagen accumulation and fibrosis. We investigated the function of Src in TGF--induced collagen I accumulation. In human mesangial cells, PTyr416 Src (activated Src) was 3.3-fold higher in TGF--treated cells than in controls. Src activation by TGF- was blocked by rottlerin and by a dominant negative mutant of protein kinase C (PKC), showing that TGF- activates Src by a PKC-based mechanism. Pharmacological inhibitors and a dominant negative Src mutant prevented the increase in collagen type I secretion in cells exposed to TGF-. Similarly, on-target Src small interference RNA (siRNA) prevented type I collagen secretion in response to TGF-, but off-target siRNA complexes had no effect. It is well established in mesangial cells that upregulation of type I collagen by TGF- requires extracellular signal-regulated kinase 1/2 (ERK1/2), and we found that activation of ERK1/2 by TGF- requires Src. In conclusion, these results suggest that stimulation of collagen type I secretion by TGF- requires a PKC-Src-ERK1/2 signaling motif. mesangial cells; fibrosis; glomerulus; transforming growth factor-     

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its -subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin _IIb3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on _IIb3, we examined _IIb3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that -actinin, myosin heavy chain, and Syk coimmunoprecipitate with _IIb3 in resting platelets and that 120 dyn/cm² shear stress leads to their disassociation from _IIb3. Shear-induced disassociation of -actinin and myosin heavy chain from the ₃ tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to _IIb3. Syk's disassociation from ₃ is inhibited when VWF binding to either GpIb-IX-V or _IIb3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to _IIb3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing _IIb3 with ₃ truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates _IIb3 function and suggest that shear-induced _IIb3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the ₃-tail. platelets; mechanoreceptor; integrin; shear stress; signal transduction     

Autocrine loop between TGF-beta1 and IL-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1 has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1 secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1 mRNA and secrete IL-1 peptide. Inhibition of TGF-₁ activity secreted from PSCs by TGF-₁-neutralizing antibody attenuated IL-1 secretion from PSCs. Exogenous TGF-₁ increased IL-1 expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative (dn)Smad2/3 expression reduced both basal and TGF-₁-stimulated IL-1 expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1 expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1 activity secreted from PSCs by IL-1-neutralizing antibody attenuated TGF-₁ secretion from PSCs. Exogenous IL-1 enhanced TGF-₁ expression and secretion by PSCs. IL-1 activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1 enhancement of TGF-₁ expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-₁ and IL-1 in activated PSCs through Smad3- and ERK-dependent pathways. fibrosis; cytokine; chronic pancreatitis     

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     

Cyclooxygenase-2 is required for activated pancreatic stellate cells to respond to proinflammatory cytokines

Cyclooxygenase-2 (COX-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of COX-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. COX-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-1, IL-1, and IL-6 enhanced COX-2 expression in activated PSCs, concomitantly increasing the expression of -smooth muscle actin (-SMA), a parameter of PSC activation. The COX-2 inhibitor NS-398 blocked culture activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of -SMA expression by TGF-1, IL-1, and IL-6 in activated PSCs. These data indicate that COX-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance COX-2 expression in PSCs. Adenovirus-mediated expression of dominant negative Smad2/3 inhibited the increase in expression of COX-2, -SMA, and collagen-1 mediated by TGF-1 in activated PSCs. Moreover, dominant negative Smad2/3 expression attenuated the expression of COX-2 and -SMA enhanced by IL-1 and IL-6. Anti-TGF- neutralizing antibody also attenuated the increase in COX-2 and -SMA expression caused by IL-1 and IL-6. IL-6 as well as IL-1 enhanced TGF-1 secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in COX-2 induction by TGF-1, IL-1, and IL-6. Furthermore, IL-1 and IL-6 promote PSC activation by enhancing COX-2 expression indirectly through Smad2/3-dependent pathway by increasing TGF-1 secretion from PSCs. transforming growth factor-; interleukin; Smad; autocrine; pancreatic fibrosis     

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     

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     

Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-kappa B signaling pathways in murine RAW 264.7 macrophages

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X₇. In this regard, after exposure to bacterial LPS, P2X₇ activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X₇ has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-B, we tested the hypothesis that LPS and nucleotides regulate NF-B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X₇-selective ligand 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-B DNA-binding activity and a sustained attenuation of levels of the NF-B inhibitory protein IB. Interestingly, a persistent reduction in IB levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IB protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IB levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-B/IB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-B and Ras/MEK/MAPK-associated pathways. nucleotide receptors; mitogen-activated protein kinases; nuclear factor-B; monocytes/macrophages; cytokines     

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     

Direct association of RhoA with specific domains of PKC-alpha

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)- and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC- involved in direct interaction with RhoA, His-tagged PKC- proteins of individual domains and different combinations of PKC- domains were used to perform in vitro binding assays with the fusion protein glutathione-S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC- in this study. The data indicate that RhoA directly bound to full-length PKC-, both in vitro (82.57 ± 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC- [PKC- (C1)] (70.48 ± 20.78% above control), PKC- (C2) (72.26 ± 29.96% above control), and PKC- (C4) (90.58 ± 26.79% above control), but not to PKC- (C3) (0.64 ± 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) (94.09 ± 12.13% and 85.10 ± 16.16% above control, respectively), but not to PKC- (C1, C2, and C3) or to PKC- (C2 and C3) (0.47 ± 1.26% and 7.45 ± 10.76% above control, respectively). RhoA bound to PKC- (C1 and C2) (60.78 ± 13.78% above control) only in vitro, but not in transfected cells, and PKC- (C2, C3, and C4) and PKC- (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-. The studies using cells transfected with truncated forms of PKC- indicate that PKC- (C1 and C2), PKC- (C1, C2, and C3), and PKC- (C2 and C3) did not associate with RhoA. Only full-length PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC- with RhoA may require the C4 domain. domains; histidine; fusion proteins     

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     

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     

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     

Dominant-negative PKC-epsilon impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini

We investigated the involvement of PKC- in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC- cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 µM, 2–15 min) significantly (P 0.05) increased PKC- recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC- association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC- in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and -hexosaminidase. The chemical inhibitor GF-109203X (10 µM, 3 h), which inhibits PKC-, -, -, and -, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö-6976 (10 µM, 3 h), which inhibits only PKC- and -. Transduction of lacrimal acini with Ad encoding syncollin-green fluorescent protein (GFP) resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation, whereas cotransduction of acini with Ad-DN-PKC- significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium, whereas Ad-DN-PKC- transduction suppressed its carbachol-stimulated release. We propose that DN-PKC- alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis. lacrimal gland; acinar epithelial cell; exocytosis; polymeric immunoglobulin A receptor