Cholinergic receptor-mediated differential cytoskeletal recruitment of actin- and integrin-binding proteins in intact airway smooth muscle

We tested the hypothesis that cholinergic receptor stimulation recruits actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex in intact airway smooth muscle. We stimulated bovine tracheal smooth muscle with carbachol and fractionated the tissue homogenate into pellet (P) and supernatant (S) by ultracentrifugation. In unstimulated tissues, calponin exhibited the highest basal P-to-S ratio (P/S; 2.74 ± 0.47), whereas vinculin exhibited the lowest P/S (0.52 ± 0.09). Cholinergic receptor stimulation increased P/S of the following proteins in descending order of sensitivity: -actinin > talin metavinculin > -smooth muscle actin > vinculin calponin. Carbachol induced ERK1/2 phosphorylation by 300% of basal value. U0126 (10 µM) completely inhibited carbachol-induced ERK1/2 phosphorylation but did not significantly affect the correlation between -actinin P/S and carbachol concentration. This observation indicates that cytoskeletal/membrane recruitment of -actinin is independent of ERK1/2 mitogen-activated protein kinase activation. Metavinculin and vinculin are splice variants of a single gene, but metavinculin P/S was significantly higher than vinculin P/S. Furthermore, the P/S of metavinculin but not vinculin increased significantly in response to cholinergic receptor stimulation. Calponin and -actinin both belong to the family of calponin homology (CH) domain proteins. However, unlike -actinin, the calponin P/S did not change significantly in response to cholinergic receptor stimulation. These findings indicate differential cytoskeletal/membrane recruitment of actin- and integrin-binding proteins in response to cholinergic receptor stimulation in intact airway smooth muscle. -Actinin, talin, and metavinculin appear to be key cytoskeletal proteins involved in the recruitment process. actinin; mitogen-activated protein kinase; metavinculin; vinculin     

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     

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     

A gene for speed: contractile properties of isolated whole EDL muscle from an alpha-actinin-3 knockout mouse

The actin-binding protein -actinin-3 is one of the two isoforms of -actinin that are found in the Z-discs of skeletal muscle. -Actinin-3 is exclusively expressed in fast glycolytic muscle fibers. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of -actinin-3 in about 1 billion individuals worldwide. Recent genetic studies suggest that the absence of -actinin-3 is detrimental to sprint and power performance in elite athletes and in the general population. In contrast, -actinin-3 deficiency appears to be beneficial for endurance athletes. To determine the effect of -actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus (fast-twitch) muscles from a specially developed -actinin-3 knockout (KO) mouse. -Actinin-3-deficient muscles showed similar levels of damage to wild-type (WT) muscles following lengthening contractions of 20% strain, suggesting that the presence or absence of -actinin-3 does not significantly influence the mechanical stability of the sarcomere in the mouse. -Actinin-3 deficiency does not result in any change in myosin heavy chain expression. However, compared with -actinin-3-positive muscles, -actinin-3-deficient muscles displayed longer twitch half-relaxation times, better recovery from fatigue, smaller cross-sectional areas, and lower twitch-to-tetanus ratios. We conclude that -actinin-3 deficiency results in fast-twitch, glycolytic fibers developing slower-twitch, more oxidative properties. These changes in the contractile properties of fast-twitch skeletal muscle from -actinin-3-deficient individuals would be detrimental to optimal sprint and power performance, but beneficial for endurance performance. extensor digitorum longus     

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     

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     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

TNF-alpha-mediated apoptosis in vascular smooth muscle cells requires p73

Atherosclerosis, now considered an inflammatory process, is the leading cause of death in the Western world and is manifested by a variety of diseases in multiple organ systems. Because of its prevalence and associated morbidity, novel therapies directed at arresting this progressive process are urgently needed. The inflammatory mediator TNF-, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF- mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-, when added to these cells in the presence of serum-containing media, increases p73 expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH₂-terminally deleted p73 plasmid results in markedly decreased TNF--induced apoptosis. Thus p73 is likely a mediator of the apoptotic effect of TNF- in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies. atherosclerosis; inflammation; plaque     

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     

Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells

Knockout of transforming growth factor (TGF)-1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle -actin (SMA), SM22, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF- with an adenovirus expressing a soluble truncated TGF- type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF--Smad signaling for an early vs. late SMC marker gene in that SMA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs. embryoid body; Smad     

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     

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     

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     

Microtubule-dependent PKC-alpha localization in A7r5 smooth muscle cells

Using laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we investigated association of protein kinase C (PKC)- with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy with standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC- localization in the perinuclear region of the cell caused by phorbol 12,13-dibutyrate (PDBu; 10^–6M). Dual immunostaining suggested colocalization of PKC- and -tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy, which indicated that association of PKC- was heterogeneous in distribution and confined primarily to microtubules in the perinuclear region. FRET analysis further showed that association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting formation of tubulin-PKC- complexes in the cytosol. Confocal imaging indicated that perinuclear microtubular structure was more highly sensitive to colchicine dissolution than other regions of the cell. Topographic imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus that was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleevelike structure of microtubules increased approximately fivefold in PDBu-treated cells, suggesting a probable increase in microtubular mass. In light of PKC- localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that perinuclear microtubules form a specialized structure that may be more dynamically robust than in other regions of the cell. PKC- could contribute to this dynamic activity. Alternatively, perinuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center. cytoskeleton; protein kinase C-; translocation     

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     

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     

Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of epsilon-protein kinase C-dependent pathway

Certain angina and coronary artery disease forms do not respond to Ca²⁺ channel blockers, and a role for vasoactive eicosanoids such as PGF₂ in Ca²⁺ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF₂-induced coronary smooth muscle contraction is Ca²⁺ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca²⁺), PGF₂ (10^-5 M) caused transient [Ca²⁺]_i increase followed by maintained [Ca²⁺]_i increase and 34% cell contraction. Ca²⁺ channel blockers verapamil and diltiazem (10^-6 M) abolished maintained PGF₂-induced [Ca²⁺]_i increase but only partially inhibited PGF₂-induced cell contraction to 17%. Verapamil-insensitive PGF₂ contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and -PKC V1-2. PGF₂ caused Ca²⁺-dependent -PKC and Ca²⁺-independent -PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF₂-induced -but not -PKC translocation. PMA (10^-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca²⁺]_i increase and -PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca²⁺ influx, caused 36% cell contraction and [Ca²⁺]_i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause - or -PKC translocation. Thus a significant component of PGF₂-induced contraction of coronary smooth muscle is Ca²⁺ antagonist insensitive, involves Ca²⁺-independent -PKC activation and translocation, and may represent a signaling mechanism of Ca²⁺ antagonist-resistant coronary vasospasm. eicosanoids; calcium; vascular smooth muscle     

AKT phosphorylation is essential for insulin-induced relaxation of rat vascular smooth muscle cells

Insulin resistance, a major factor in the development of type 2 diabetes, is known to be associated with defects in blood vessel relaxation. The role of Akt on insulin-induced relaxation of vascular smooth muscle cell (VSMC) was investigated using siRNA targeting Akt (siAKTc) and adenovirus constructing myristilated Akt to either suppress endogenous Akt or overexpress constitutively active Akt, respectively. siAKTc decreased both basal and insulin-induced phosphorylations of Akt and glycogen synthase kinase 3, abolishing insulin-induced nitric oxide synthase (iNOS) expression. cGMP-dependent kinase 1 (cGK1) and myosin-bound phosphatase (MBP) activities, both downstream of iNOS, were also decreased. siAKTc treatment resulted in increased insulin and ANG II-stimulated phosphorylation of contractile apparatus, such as MBP substrate (MYPT1) and myosin light chain (MLC20), accompanied by increased Rho-associated kinase (ROK) activity, demonstrating the requirement of Akt for insulin-induced vasorelaxation. Corroborating these results, constitutively active Akt upregulated the signaling molecules involved in insulin-induced relaxation such as iNOS, cGK1, and MBP activity, even in the absence of insulin stimulation. On the contrary, the contractile response involving the phosphorylation of MYPT1 and MLC20, and increased ROK activity stimulated by ANG II were all abolished by overexpressing active Akt. In conclusion, we demonstrated here that insulin-induced VSMC relaxation is dependent on Akt activation via iNOS, cGK1, and MBP activation, as well as the decreased phosphorylations of MYPT1 and MLC20 and decreased ROK activity. angiotensin II; myosin-bound phosphatase substrate; inducible nitric oxide synthase; guanosine 3',5'-cyclic monophosphate-dependent kinase 1; Rho-associated kinase     

CCK-A receptor activates RhoA through G alpha 12/13 in NIH3T3 cells

Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G₁₃- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active -subunits; G₁₃ and G₁₂ but not G_q induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G_12/13 inhibited active _12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit G_q, had no effect. Cotransfection with plasmids coding for the G protein -subunit carboxy-terminal peptide from ₁₃ and, to a lesser extent ₁₂, also inhibited the effect of CCK, whereas the peptide from _q did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G₁₃, leading to rearrangement of the actin cytoskeleton. actin; cholecystokinin; Rho; Rho-kinase; stress fibers     

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