共查询到20条相似文献,搜索用时 31 毫秒
1.
Feng S Lu X Reséndiz JC Kroll MH 《American journal of physiology. Cell physiology》2006,291(6):C1346-C1354
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 相似文献
2.
Zhou YW Oak SA Senogles SE Jarrett HW 《American journal of physiology. Cell physiology》2005,288(2):C377-C388
-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 Gs and G occurs and active Gs, measured as GTP-35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gs is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-S-bound) Gs, 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 相似文献
3.
Sade H Muraki K Ohya S Hatano N Imaizumi Y 《American journal of physiology. Cell physiology》2006,290(1):C77-C86
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 相似文献
4.
Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2 总被引:3,自引:0,他引:3
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 Gq, G13, and all Gi isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or Gq antibody, PLC-1 or PLC-3 antibody, and by expression of Gq or Gi minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G13 or Gq 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 Gq 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 Gq and Gi, 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 Gq and G13, resulting in Rho kinase- and PKC-dependent MLC20 phosphorylation. muscle contraction; signal transduction 相似文献
5.
Uezono Y Kaibara M Murasaki O Taniyama K 《American journal of physiology. Cell physiology》2004,287(4):C885-C894
We studied the functions of -subunits of Gi/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl currents in oocytes expressing 2-adrenoceptor and the protein kinase A-dependent Cl channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala2, D-Leu5]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing 2-adrenoceptor-CFTR and 5-HT1A receptor (5-HT1AR), -opioid receptor, or GABAB receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (Gt). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or -opioid receptor alone. They elicited Ca2+-activated Cl currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of Gt, suggesting that -subunits of Gi/o protein activate PLC-2 and then cause intracellular Ca2+ mobilization. Our results indicate that -subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca2+ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology 相似文献
6.
Bernstein BW Chen H Boyle JA Bamburg JR 《American journal of physiology. Cell physiology》2006,291(5):C828-C839
When neurons in culture are transiently stressed by inhibition of ATP synthesis, they rapidly form within their neurites rodlike actin inclusions that disappear when the insult is removed. Oxidative stress, excitotoxic insults, and amyloid -peptide oligomers also induce rods. Immunostaining of neurites indicates that these rods also contain the majority of the actin filament dynamizing proteins, actin-depolymerizing factor (ADF) and cofilin (AC). If the rods reappear within 24 h after the stress is removed, the neurite degenerates distal to the rod but with no increase in neuronal death. Here, rods were generated in cultured rat E18 hippocampal cells by overexpression of a green fluorescent protein chimera of AC. Surprisingly, we have found that, for a short period (60 min) immediately after initial rod formation, the loss of mitochondrial membrane potential (m) and ATP in neurites with rods is slower than in neurites without them. The m was monitored with the fluorescent dye tetramethylrhodamine methyl ester, and ATP was monitored with the fluorescent ion indicator mag-fura 2. Actin in rods is less dynamic than is filamentous actin in other cytoskeletal structures. Because m depends on cellular ATP and because ATP hydrolysis associated with actin filament turnover is responsible for a large fraction of neuronal energy consumption (50%), the formation of rods transiently protects neurites by slowing filament turnover and its associated ATP hydrolysis. actin dynamics; neurodegeneration; actin inclusions; neuroprotection; ischemia 相似文献
7.
CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake 总被引:1,自引:0,他引:1
Codina Juan; Li Jian; DuBose Thomas D. Jr. 《American journal of physiology. Cell physiology》2005,288(6):C1279
The carboxy terminus (CT) of the colonic H+-K+-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK2, we selected 84 amino acids in the CT of the -subunit of mouse colonic H+-K+-ATPase (CT-HK2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK2 with CD63, recombinant CT-HK2 and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK2 protein (but not CT-HK1) coprecipitated with CD63, confirming stable assembly of HK2 with CD63. In HEK-293 transfected with HK2 plus 1-Na+-K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK2/NK1 and 86Rb+ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK2-NK1 complex in the cell membrane. protein assembly; cell surface localization 相似文献
8.
Menon B Singh M Ross RS Johnson JN Singh K 《American journal of physiology. Cell physiology》2006,290(1):C254-C261
Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with 1 integrins. Herein we tested the hypothesis that MMP-2 impairs 1 integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of 1 integrins using adenoviruses expressing the human 1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of 1 integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the 1 integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH2-terminal kinase; cytochrome c 相似文献
9.
Smallwood ND Hausman BS Wang X Liedtke CM 《American journal of physiology. Cell physiology》2005,288(4):C906-C912
Direct binding of nonmuscle F-actin and the C2-like domain of PKC- (C2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid C2-like domain. Truncations of the C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His6 probe for expression of the His6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC- with nonmuscle F-actin. Peptides from the NH2-terminal region, but not the COOH-terminal region, of the C2-like domain blocked binding of activated PKC- to F-actin. The C2-like domain and three NH2-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC50 values ranging from 1.2 to 2.2 nmol (611 µM). NH2-terminal C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH2 terminus of the C2-like domain encodes a 1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the 1-sheet region of the C2-like domain in airway epithelial cells. truncation; protein kinase C-; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter 相似文献
10.
Laminin-10 and Lutheran blood group glycoproteins in adhesion of human endothelial cells 总被引:3,自引:0,他引:3
Vainionpää N Kikkawa Y Lounatmaa K Miner JH Rousselle P Virtanen I 《American journal of physiology. Cell physiology》2006,290(3):C764-C775
Laminin 5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of 5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin 5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed Mr 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins 1 and v3, whereas in the adhesion to laminin-10/11, Lu and integrin 1 were involved. In the cells adhering to the 5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that 5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to 5 laminins in collaboration with integrins 1 and v3. integrin; cycloheximide 相似文献
11.
Jerdeva GV Yarber FA Trousdale MD Rhodes CJ Okamoto CT Dartt DA Hamm-Alvarez SF 《American journal of physiology. Cell physiology》2005,289(4):C1052-C1068
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, 215 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 相似文献
12.
Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes 总被引:1,自引:0,他引:1
Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in -adrenergic receptor (-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). -AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed -AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited -AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with 1-integrins after -AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of 1-integrin signaling using laminin inhibited the increased association of MMP-2 with 1-integrins. -AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) -AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits -AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with 1 integrins and poly-ADP-ribose-polymerase cleavage. integrins; poly-ADP-ribose-polymerase 相似文献
13.
Oshikawa J Toya Y Fujita T Egawa M Kawabe J Umemura S Ishikawa Y 《American journal of physiology. Cell physiology》2003,285(3):C567-C574
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 相似文献
14.
Chanrachakul B Broughton Pipkin F Khan RN 《American journal of physiology. Cell physiology》2004,287(6):C1747-C1752
The 2-adrenergic receptor (2-AR) and the large-conductance Ca2+-activated K+ (BKCa) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both 2-AR and BKCa channel proteins in pregnant human myometrium decrease by 50% after the onset of labor. We present direct evidence in support of a structural and functional association between the 2-AR and the BKCa channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of 2-AR colocalizing with the BKCa channel. Coimmunoprecipitation studies indicate that BKCa and 2-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BKCa channel blocker, paxilline, significantly antagonized the relaxant effect of the 2-AR agonist ritodrine. These novel findings provide an insight into the coupling between the 2-AR and BKCa channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor. 2-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction 相似文献
15.
Wang Y Tomar A George SP Khurana S 《American journal of physiology. Cell physiology》2007,292(5):C1775-C1786
While there is circumstantial evidence to suggest a requirement for phospholipase C-1 (PLC-1) in actin reorganization and cell migration, few studies have examined the direct mechanisms that link regulators of the actin cytoskeleton with this crucial signaling molecule. This study was aimed to examine the role that villin, an epithelial cell-specific actin-binding protein, and its ligand PLC-1 play in migration in intestinal and renal epithelial cell lines that endogenously or ectopically express human villin. Basal as well as epidermal growth factor (EGF)-stimulated cell migration was accompanied by tyrosine phosphorylation of villin and its association with PLC-1. Inhibition of villin phosphorylation prevented villin-PLC-1 complex formation as well as villin-induced cell migration. The absolute requirement for PLC-1 in villin-induced cell migration was demonstrated by measuring cell motility in PLC-1/ cells and by downregulation of endogenous PLC-1. EGF-stimulated direct interaction of villin with the Src homology domain 2 domain of PLC-1 at the plasma membrane was demonstrated in living cells by using fluorescence resonance energy transfer. These results demonstrate that villin provides an important link between the activation of phosphoinositide signal transduction pathway and epithelial cell migration. fluorescence resonance energy transfer; actin 相似文献
16.
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 相似文献
17.
Meyer TN Hunt J Schwesinger C Denker BM 《American journal of physiology. Cell physiology》2003,285(5):C1281-C1293
Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that G12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated G12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of G12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active G12 (QL12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QL12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QL12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QL12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QL12-expressing MDCK cells as assessed by Src autophosphorylation, and -catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that G12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways. G proteins; tight junction; adherens junction; Rho 相似文献
18.
Involvement of PKCdelta and PKD in pulmonary microvascular endothelial cell hyperpermeability 总被引:2,自引:0,他引:2
Tinsley JH Teasdale NR Yuan SY 《American journal of physiology. Cell physiology》2004,286(1):C105-C111
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 相似文献
19.
Fan H Zingarelli B Peck OM Teti G Tempel GE Halushka PV Spicher K Boulay G Birnbaumer L Cook JA 《American journal of physiology. Cell physiology》2005,289(2):C293-C301
Heterotrimeric Gi proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of Gi proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Gi2 or Gi1/3 protein in Gi2-knockout (Gi2/) or Gi1/3-knockout (Gi1/3/) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (M) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF- and IFN- in splenocytes from Gi2/ mice compared with wild-type (WT) mice. Also, LPS-induced TNF- was increased in splenocytes from Gi1/3/ mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-, IL-10, and thromboxane B2 (TxB2) production was decreased in M harvested from Gi2/ mice. Also, LPS-induced production of IL-10 and TxB2 was decreased in M from Gi1/3/ mice. In subsequent in vivo studies, TNF- levels after LPS challenge were significantly greater in Gi2/ mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated Gi2/ mice compared with WT mice. These data suggest that Gi proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli. Gi protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors 相似文献
20.
Nandrot EF Anand M Sircar M Finnemann SC 《American journal of physiology. Cell physiology》2006,290(4):C1256-C1262
v5-Integrin is the sole integrin receptor at the retinal pigment epithelium (RPE)-photoreceptor interface and promotes RPE phagocytic signaling to the tyrosine kinase Mer tyrosine kinase (MerTK) once a day in response to circadian photoreceptor shedding. Herein we identify a novel role for v5-integrin in permanent RPE-photoreceptor adhesion that is independent of v5's function in retinal phagocytosis. To compare retinal adhesion of wild-type and 5-integrin/ mice, we mechanically separated RPE and neural retina and quantified RPE protein and pigment retention with the neural retina. Lack of v5-integrin with normal expression of other RPE integrins greatly weakened retinal adhesion in young mice and accelerated its age-dependent decline. Unexpectedly, the strength of wild-type retinal adhesion varied with a diurnal rhythm that peaked 3.5 h after light onset, after the completion of phagocytosis, when integrin signaling to MerTK is minimal. Permanent v5 receptor deficiency attenuated the diurnal peak of retinal adhesion in 5-integrin/ mice. These results identify v5-integrin as the first RPE receptor that contributes to retinal adhesion, a vital mechanism for long-term photoreceptor function and viability. Furthermore, they indicate that v5 receptors at the same apical plasma membrane domain of RPE cells fulfill two separate functions that are synchronized by different diurnal rhythms. circadian rhythm; knockout; photoreceptors; retinal pigment epithelium 相似文献