Caveolin-1 functions as a scaffolding protein for phosphofructokinase in the metabolic organization of vascular smooth muscle

Using confocal microscopy, we have demonstrated a similar distribution of phosphofructokinase (PFK) with caveolin-1 (CAV-1) mainly at the periphery (membrane) in freshly isolated vascular smooth muscle (VSM) cells and in cultured A7r5 VSM cells. Co-immunoprecipitation analysis validated the interaction between the proteins. To further test the hypothesis that PFK and CAV-1 are colocalized, we used small interfering RNA (siRNA) to downregulate CAV-1 expression and disrupt the protein-protein interactions between PFK and CAV-1. Transfection of cultured A7r5 cells with CAV-1 siRNA resulted in a decreased level of immunoreactive CAV-1 and a consequent shift in the distribution of PFK with less localization of PFK to the periphery of the cells and increased immunoreactivity at the perinuclear region as compared to control. Analysis of the average PFK intensity across cultured A7r5 cells demonstrated a higher central:peripheral intensity ratio (CPI ratio) in siRNA-treated cells than in the control. These results validate the possible role of CAV-1 as a scaffolding protein for PFK as evidenced by the significant redistribution of PFK after CAV-1 downregulation. We therefore conclude that CAV-1 may function as a scaffolding protein for PFK and that this contributes to the compartmentation of glycolysis from other metabolic pathways in VSM.     

Caveolae and the organization of carbohydrate metabolism in vascular smooth muscle

We have previously found that glycolysis and gluconeogenesis occur in separate "compartments" of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [1999] Am J Physiol Cell Physiol. 277:C1250-C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [2000] Am J Physiol Cell Physiol. 278:C803-C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane-associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non-caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with beta methyl-cyclodextrin (CD) and examined the metabolism of [2-(13)C]glucose (a glycolytic substrate) and [1-(13)C]fructose 1,6-bisphosphate (FBP, a gluconeogenic substrate in PCMV) using (13)C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2-(13)C]glucose to [2-(13)C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1-(13)C]FBP to [3-(13)C]lactate was increased by CD treatment. Alternatively, the increased [3-(13)C]lactate production may reflect changes in FBP uptake, since conversion of [1-(13)C]FBP to [3-(13)C]glucose was also elevated in CD-treated cells. Thus, a link between caveolar organization and metabolic organization may exist.     

The third intracellular loop and carboxyl tail of neurokinin 1 and 3 receptors determine interactions with beta-arrestins

Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with -arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist Sar-Met-substance P induced rapid translocation of -arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only -arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with -arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced Ca²⁺ mobilization. The NK1R resensitized greater than twofold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with -arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with -arrestin 1 and determine the rate of resensitization. desensitization; endocytosis; tachykinins     

PKC-delta and CaMKII-delta 2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle

ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC- activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC- activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-₂, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-Ras^N17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC- and CaMKII-₂ coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells. protein kinase C-; calcium/calmodulin-dependent protein kinase II- ₂; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; adenovirus     

Beta-subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

Previous studies showed the presence of a significant fraction of Na⁺-K⁺-ATPase -subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na⁺-K⁺-ATPase with its signaling partners. Because both - and -subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na⁺-K⁺-ATPase, we have examined the relative distribution of two major subunit isoforms (₁ and ₁) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na₂CO₃ or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30–40% of ₁-subunits and 80–90% of ₁-subunits. Use of Na₂CO₃ was shown to inactivate Na⁺-K⁺-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained 75% of total myocyte Na⁺-K⁺-ATPase activity. Sealed isolated caveolae exhibited active Na⁺ transport. Confocal microscopy supported the presence of ,-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na⁺-K⁺ fluxes, and the sites where the pumping and signaling functions of Na⁺-K⁺-ATPase are integrated. Preferential concentration of ₁-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of ₁ and ₁ in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na⁺-K⁺-ATPase in cardiac caveolae. cardiac myocyte; caveolin; oligomer; ouabain; sodium pump     

Overexpression of caveolin-1 results in increased plasma membrane targeting of glycolytic enzymes: the structural basis for a membrane associated metabolic compartment

Although membrane-associated glycolysis has been observed in a variety of cell types, the mechanism of localization of glycolytic enzymes to the plasma membrane is not known. We hypothesized that caveolin-1 (CAV-1) serves as a scaffolding protein for glycolytic enzymes and may play a role in the organization of cell metabolism. To test this hypothesis, we over-expressed CAV-1 in cultured A7r5 (rat aorta vascular smooth muscle; VSM) cells. Confocal immunofluorescence microscopy was used to study the distribution of phosphofructokinase (PFK) and CAV-1 in the transfected cells. Areas of interest (AOI) were analyzed in a central Z-plane across the cell transversing the perinuclear region. To quantify any shift in PFK localization resulting from CAV-1 over-expression, we calculated a periphery to center (PC) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found the PC index to be 1.92 +/- 0.57 (mean +/- SEM, N = 8) for transfected cells and 0.59 +/- 0.05 (mean +/- SEM, N = 11) for control cells. Colocalization analysis demonstrated that the percentage of PFK associated with CAV-1 increased in transfected cells compared to control cells. The localization of aldolase (ALD) was also shifted towards the plasma membrane (and colocalized with PFK) in CAV-1 over-expressing cells. These results demonstrate that CAV-1 creates binding sites for PFK and ALD that may be of higher affinity than those binding sites localized in the cytoplasm. We conclude that CAV-1 functions as a scaffolding protein for PFK, ALD and perhaps other glycolytic enzymes, either through direct interaction or accessory proteins, thus contributing to compartmented metabolism in vascular smooth muscle.     

Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes

In mammalian skeletal muscle,neuronal-type nitric oxide synthase (nNOS) is found to be enriched atneuromuscular endplates. Here we demonstrate the colocalization of thenicotinic acetylcholine receptor (nAChR, stained with -bungarotoxin)and nNOS (stained with a specific antibody) in murineC₂C₁₂ myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C₂C₁₂myotubes. An antibody to the ₁-subunit of the nAChR didnot coprecipitate nNOS, and an nNOS-specific antibody did notprecipitate the ₁-subunit of the nAChR. Treatment ofmice with bacterial LPS downregulated the expression of nNOS inskeletal muscle, and treatment of C₂C₁₂ cellswith bacterial LPS and interferon- markedly decreased nNOS mRNA andprotein expression. In contrast, mRNA and protein of the nAChR (-,-, and -subunits) remained unchanged at the mRNA and proteinlevels. These data demonstrate that nNOS and the nAChR are colocalizedin murine skeletal muscle and C₂C₁₂ cells but differ in their expressional regulation.

     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV-integrin alpha1/beta1/alphav and PDGF receptor pathways

Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1⁺) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1⁺ progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 6–90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1⁺ progenitors with antibodies against integrin ₁, _v, and ₁, but not ₃, inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor- significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin (₁, ₁, and _v)-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor- signaling pathways are involved in SMC differentiation. progenitor cells; extracellular matrix; growth factor receptors; platelet-derived growth factor     

Lung epithelial barrier function and wound healing are decreased by IL-4 and IL-13 and enhanced by IFN-gamma

To understand theeffects of cytokines on epithelial cells in asthma, we haveinvestigated the effects of interleukin (IL)-4, IL-13, and interferon(IFN)- on barrier function and wound healing in Calu-3 human lungepithelial cells. IL-4 and IL-13 treatment of Calu-3 cells grown onTranswell filters resulted in a 70-75% decrease in barrierfunction as assessed by electrophysiological and[¹⁴C]mannitol flux measurements. In contrast, IFN-enhanced barrier function threefold using these same parameters. Cellstreated concurrently with IFN- and IL-4 or IL-13 showed an initialdecline in barrier function that was reversed within 2 days, resulting in barrier levels comparable to control cells. Analysis of the tightjunction-associated proteins ZO-1 and occludin showed that IL-4 andIL-13 significantly reduced ZO-1 expression and modestly decreasedoccludin expression compared with controls. IFN-, quite unexpectedlygiven its enhancing effect on barrier function, reduced expression ofZO-1 and occludin to almost undetectable levels compared with controls.In wound-healing assays of cells grown on collagen I, IL-4 and IL-13decreased migration, whereas IFN- treatment enhanced migration,compared with control cells. Addition of IFN-, in combination withIL-4 or IL-13, restored migration of cells to control levels. Migrationdifferences observed between the various cytokine treatments wascorrelated with expression of the collagen I-binding₂₁-integrin at the leading edge of cellsat the wound front; ₂₁-integrinexpression was decreased in IFN--treated cells compared withcontrols, whereas it was highest in IL-4- and IL-13-treated cells.These results demonstrate that IL-4 and IL-13 diminish the capacity ofCalu-3 cells to maintain barrier function and repair wounds, whereasIFN- promotes epithelial restitution by enhancing barrier functionand wound healing.

     

Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells

Overexpression of aconstitutively active mutant of the mitogen-activated protein kinasekinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7cells disrupts morphogenesis, induces an invasive phenotype, and isassociated with a reduced rate of cell proliferation. The role ofcell-cell adhesion molecules and cell cycle proteins in theseprocesses, however, has not been investigated. We now report loss ofE-cadherin expression as well as a marked reduction of - and-catenin expression in transdifferentiated MDCK-C7 cells stablyexpressing caMEK1 (C7caMEK1) compared with epithelial mock-transfectedMDCK-C7 (C7Mock1) cells. At least part of the remaining -catenin wascoimmunoprecipitated with -catenin, whereas no E-cadherin wasdetected in -catenin immunoprecipitates. In both cell types, theproteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to atime-dependent accumulation of -catenin, including the appearance ofhigh-molecular-weight -catenin species. Quiescent as well asserum-stimulated C7caMEK1 cells showed a higher cyclin D expressionthan epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibitedextracellular signal-regulated kinase phosphorylation and cyclin Dexpression in C7caMEK1 cells and almost abolished their already reducedcell proliferation rate. We conclude that the transdifferentiated andinvasive phenotype of C7caMEK1 cells is associated with a diminishedexpression of proteins involved in cell-cell adhesion. Although-catenin expression is reduced, C7caMEK1 cells show a higherexpression of U-0126-sensitive cyclin D protein.

     

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     

beta-amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1

In patients withamyloid -related cerebrovascular disorders, e.g., Alzheimer'sdisease, one finds increased deposition of amyloid peptide (A) andincreased presence of monocyte/microglia cells in the brain. However,relatively little is known of the role of A in the trafficking ofmonocytes across the blood-brain barrier (BBB). Our studies show thatinteraction of A_1-40 with monolayer of human brainendothelial cells results in augmented adhesion and transendothelialmigration of monocytic cells (THP-1 and HL-60) and peripheral bloodmonocytes. The A-mediated migration of monocytes was inhibited byantibody to A receptor (RAGE) and platelet endothelial cell adhesionmolecule (PECAM-1). Additionally, A-induced transendothelialmigration of monocytes were inhibited by protein kinase C inhibitor andaugmented by phosphatase inhibitor. We conclude that interaction ofA with RAGE expressed on brain endothelial cells initiates cellularsignaling leading to the transendothelial migration of monocytes. Wesuggest that increased diapedesis of monocytes across the BBB inresponse to A present either in the peripheral circulation or in thebrain parenchyma may play a role in the pathophysiology of A-relatedvascular disorder.

     

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     

Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1 in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1 accumulation in different tumor cell lines. Hypoxia-induced HIF-1 accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F₀F₁-ATPase, prevented hypoxia-induced HIF-1 protein accumulation and had no effect on HIF-1 induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1 content was pronounced in cells exposed to hypoxia (1.5% O₂) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1 regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1 accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function. hypoxia-inducible factor 1; oxygen sensing     

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

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

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     

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     

Calcium/calmodulin-dependent protein kinase II-delta isoform regulation of vascular smooth muscle cell proliferation

There is accumulating evidence that Ca²⁺-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca²⁺ signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKII gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKII₂, a splice variant of the gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKII₂ protein and a significant decrease in CaMKII protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKII₂ in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKII₂ activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKII₂ is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation. calmodulin kinase II; phenotype modulation     

nPKC{varepsilon}, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y₂ purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP₃), diacylglycerol, Ca²⁺ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC, nPKC, nPKC, and nPKC; of these, only nPKC translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149–L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC, nPKC, and nPKC had the same levels of ATPS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC (14.6 and 23.5%, for ATPS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y₂-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC KO mice relative to its WT littermates. We conclude that nPKC is the effector isoform downstream of P2Y₂-R activation in the goblet cell secretory response. The translocation of nPKC observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung