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1.
Calcif Tissue Int ; 75(5): 396-404, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15592796

ABSTRACT

beta-Catenin plays a dual role in cells: one at cell-cell junctions and one regulating gene transcription together with TCF (T-cell Factor) in the nucleus. Recently, a role for beta-catenin in osteoblast differentiation and gene expression has begun to be elucidated. Herein we investigated the effects of fluid shear stress (FSS) on beta-catenin signaling. FSS is a well-characterized anabolic stimulus for osteoblasts; however, the molecular mechanisms for the effects of this stimulation remain largely unknown. We found that 1 hour of laminar FSS (10 dynes/cm(2)) induced translocation of beta-catenin to the nucleus and activated a TCF-reporter gene. Analysis of upstream signals that may regulate beta-catenin signaling activity revealed two potential mechanisms for increased beta-catenin signaling. First, FSS induced a transient, but significant, increase in the phosphorylation of both glycogen synthase kinase 3beta (GSK-3beta) and Akt. Second, FSS reduced the levels of beta-catenin associated with N-cadherin, suggesting that less sequestration of beta-catenin by cadherins occurs in osteoblasts subjected to FSS. Functional analysts of potential genes regulated by beta-catenin signaling in osteoblasts revealed two novel observations. First, endogenous, nuclear beta-catenin purified from osteoblasts formed a complex with a TCF -binding element in the cyclooxygenase-2 promoter, and, second, overexpression of either a constitutively active beta-catenin molecule or inhibition of GSK-3beta activity increased basal cyclooxygenase-2 levels. Together, these data demonstrate for the first time that FSS modulates the activity of both GSK-3beta and beta-catenin and that these signaling molecules regulate cyclooxygenase-2 expression in osteoblasts.


Subject(s)
Cytoskeletal Proteins/metabolism , Osteoblasts/physiology , Signal Transduction , Trans-Activators/metabolism , 3T3 Cells , Animals , Animals, Newborn , Cell Line, Tumor , Cell Nucleus/metabolism , Cells, Cultured , Cytoskeletal Proteins/genetics , Electrophoretic Mobility Shift Assay , Fluorescent Antibody Technique, Indirect , Genes, Reporter , Glycogen Synthase Kinases/metabolism , Immunoblotting , Mice , Mutation , Phosphorylation , Precipitin Tests , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Skull/cytology , Stress, Mechanical , Time Factors , Trans-Activators/genetics , beta Catenin
2.
Biomaterials ; 25(3): 515-25, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14585701

ABSTRACT

Small intestinal submucosa (SIS) is a naturally occurring tissue matrix composed of extracellular matrix proteins and various growth factors. SIS is derived from the porcine jejunum and functions as a remodeling scaffold for tissue repair. While SIS has proven to be a useful biomaterial for implants in vivo, problems associated with endothelialization and thrombogenicity of SIS implants may limit its vascular utility. The goal of this study was to determine if the biological properties of SIS could be improved by growing human umbilical vein endothelial cells (HUVEC) on SIS and allowing these cells to deposit human basement membrane proteins on the porcine substrate to create what we have called "conditioned" SIS (c-SIS). Using an approach in which HUVEC were grown for 2 weeks on SIS and then removed via a technique that leaves behind an intact basement membrane, we hypothesized that the surface properties of SIS might be improved. We found that when re-seeded on c-SIS, HUVEC exhibited enhanced organization of cell junctions and had increased metabolic activity compared to cells on native SIS (n-SIS). Furthermore, HUVEC grown on c-SIS released lower amounts of the pro-inflammatory prostaglandin PGI2 into the media compared to cells grown on n-SIS. Additionally, we found that adhesion of resting or activated human platelets to c-SIS was significantly decreased compared to n-SIS suggesting that, in addition to improved cell growth characteristics, conditioning SIS with human basement membrane proteins might decrease its thrombogenic potential. In summary, conditioning of porcine SIS by human endothelial cells improves key biological properties of the material that may improve its usefulness as remodeling scaffold for tissue repair. Identification of critical modifications of SIS by human endothelial cells should help guide future efforts to develop more biocompatible vascular grafts.


Subject(s)
Biocompatible Materials/chemistry , Endothelial Cells/metabolism , Intestinal Mucosa/pathology , Intestine, Small/pathology , Animals , Basement Membrane/metabolism , Biocompatible Materials/pharmacology , Blood Platelets/metabolism , Blotting, Western , Cell Adhesion , Cell Culture Techniques/methods , Cells, Cultured , Collagen/metabolism , Culture Media/pharmacology , Endothelium, Vascular/cytology , Epoprostenol/metabolism , Extracellular Matrix/metabolism , Fibronectins/metabolism , Humans , Inflammation , Jejunum/pathology , Microscopy, Confocal , Microscopy, Electron, Scanning , Phosphotyrosine/metabolism , Prostaglandins I/metabolism , Swine , Time Factors , Transplantation
3.
Bone ; 30(6): 931-6, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12052465

ABSTRACT

Nmp4 proteins are transcription factors that contribute to the expression of type I collagen and many of the matrix metalloproteinase genes. Numerous Nmp4 isoforms have been identified. These proteins, all derived from a single gene, have from five to eight Cys(2)His(2) zinc fingers, the arrangement of which directs specific isoforms to nuclear matrix subdomains. Nmp4 isoforms also have an SH3 binding domain, typical of cytoplasmic docking proteins. Although recent evidence indicates that Nmp4 proteins also reside in the osteoblast cytoplasm, whether they localize to specific organelles or structures is not well defined. The intracellular localization of a protein is a determinant of its function and provides insights into its mechanism of action. As a first step toward determining the functional relationship between the cytoplasmic and nuclear Nmp4 compartments, we mapped their location in the osteoblast cytoplasm. Immunocytochemical analysis of osteoblasts demonstrated that Nmp4 antibodies labeled the mitochondria, colocalized with Golgi protein 58K, and lightly stained the cytoplasm. Western analysis using Nmp4 antibodies revealed a complex profile of protein bands in the nuclear, mitochondrial, and cytosolic fractions. Several of these proteins were specific to defined intracellular domains. Consistent with the western analyses, reverse transcription-polymerase chain reaction (RT-PCR) analysis detected previously uncharacterized Nmp4 isoforms. These data necessarily enlarge the known Nmp4 family from nuclear matrix transcription factors to a more widely extended class of intracellular proteins.


Subject(s)
Intracellular Fluid/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Osteoblasts/metabolism , Transcription Factors/metabolism , 3T3 Cells/chemistry , 3T3 Cells/metabolism , Animals , Animals, Newborn , Cytoplasm/chemistry , Cytoplasm/metabolism , Golgi Apparatus/chemistry , Golgi Apparatus/metabolism , Intracellular Fluid/chemistry , Male , Mice , Mitochondria/chemistry , Mitochondria/metabolism , Nuclear Matrix-Associated Proteins/biosynthesis , Osteoblasts/chemistry , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Transcription Factors/biosynthesis
4.
Am J Physiol Cell Physiol ; 281(3): C1038-45, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11502582

ABSTRACT

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.


Subject(s)
Actins/physiology , Cytoskeleton/physiology , Dinoprostone/metabolism , Endothelium, Vascular/physiology , Epoprostenol/metabolism , Apoptosis/drug effects , Apoptosis/physiology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Colchicine/pharmacology , Cytochalasin D/pharmacology , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Kinetics , Marine Toxins/pharmacology , Microtubules/drug effects , Microtubules/physiology , Microtubules/ultrastructure , Nocodazole/pharmacology , Thiazoles/pharmacology , Thiazolidines , Umbilical Veins
5.
Am J Physiol Cell Physiol ; 278(5): C989-97, 2000 May.
Article in English | MEDLINE | ID: mdl-10794673

ABSTRACT

Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.


Subject(s)
Calcium/metabolism , Cytoskeleton/metabolism , Gene Expression Regulation , Osteoblasts/metabolism , 3T3 Cells , Actins/biosynthesis , Actins/genetics , Animals , Calcium Signaling/drug effects , Cyclooxygenase 2 , Cytoskeleton/drug effects , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Genes, fos , Isoenzymes/biosynthesis , Isoenzymes/genetics , Mice , Osteoblasts/drug effects , Prostaglandin-Endoperoxide Synthases/biosynthesis , Prostaglandin-Endoperoxide Synthases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stress, Mechanical , Thapsigargin/pharmacology , Type C Phospholipases/metabolism
6.
J Biol Chem ; 273(50): 33588-94, 1998 Dec 11.
Article in English | MEDLINE | ID: mdl-9837942

ABSTRACT

Circulating leukocytes are nonadherent but bind tightly to endothelial cells following activation. The increased avidity of leukocyte integrins for endothelial ligands following activation is regulated, in part, by interaction of the beta2 subunit cytoplasmic tail with the actin cytoskeleton. We propose a mechanism to explain how tethering of the actin cytoskeleton to leukocyte integrins is regulated. In resting leukocytes, beta2 integrins are constitutively linked to the actin cytoskeleton via the protein talin. Activation of cells induces proteolysis of talin and dissociation from the beta2 tail. This phase is transient, however, and is followed by reattachment of actin filaments to integrins that is mediated by the protein alpha-actinin. The association of alpha-actinin with integrins may stabilize the cytoskeleton and promote firm adhesion to and migration across the endothelium. Glutathione S-transferase-beta2 tail fusion protein/mutagenesis experiments suggest that the affinity of alpha-actinin binding to the beta2 tail is regulated by a change in the conformation of the tail that unmasks a cryptic alpha-actinin binding domain. Positive and inhibitory domains within the beta2 tail regulate alpha-actinin binding: a single 11-amino acid region (residues 736-746) is necessary and sufficient for alpha-actinin binding, and a regulatory domain between residues 748-762 inhibits constitutive association of the beta2 tail with alpha-actinin.


Subject(s)
Actinin/metabolism , CD18 Antigens/metabolism , Cytoskeleton/metabolism , Neutrophils/metabolism , Talin/metabolism , Amino Acid Sequence , Cell Adhesion , Cell Movement , Glutathione Transferase/genetics , Humans , Molecular Sequence Data , Neutrophil Activation , Neutrophils/cytology , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
7.
Am J Physiol ; 275(6 Pt 1): C1591-601, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9843721

ABSTRACT

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.


Subject(s)
Cytoskeleton/physiology , Integrins/physiology , Osteoblasts/physiology , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/enzymology , Actinin/pharmacology , Actins/physiology , Animals , Cell Adhesion/physiology , Cell Line , Cyclooxygenase 2 , Cytochalasin D/pharmacology , GTP-Binding Proteins/genetics , Gene Expression/drug effects , Integrin beta1/metabolism , Isoenzymes/metabolism , Membrane Proteins/genetics , Mice , Osteoblasts/drug effects , Peptide Fragments/pharmacology , Prostaglandin-Endoperoxide Synthases/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Signal Transduction , Stress, Mechanical , rhoB GTP-Binding Protein
8.
J Orthop Sci ; 3(6): 346-55, 1998.
Article in English | MEDLINE | ID: mdl-9811988

ABSTRACT

The skeleton's primary mechanical function is to provide rigid levers for muscles to act against as they hold the body upright in defiance of gravity. Many bones are exposed to thousands of repetitive loads each day. During growth and development, the skeleton optimizes its architecture by subtle adaptations to these mechanical loads. The mechanisms for adaptation involve a multistep process of cellular mechanotransduction including: mechanocoupling - conversion of mechanical forces into local mechanical signals, such as fluid shear stresses, that initiate a response by bone cells; biochemical coupling - transduction of a mechanical signal to a biochemical response involving pathways within the cell membrane and cytoskeleton; cell-to-cell signaling from the sensor cells (probably osteocytes and bone lining cells) to effector cells (osteoblasts or osteoclasts) using prostaglandins and nitric oxide as signaling molecules; and effector response - either bone formation or resorption to cause appropriate architectural changes. These architectural changes tend to adjust and improve the bone structure to its prevailing mechanical environment. Structural changes can be predicted, to some extent, by mathematical formulas derived from three fundamental rules: (1) bone adaptation is driven by dynamic, rather than static, loading; (2) extending the loading duration has a diminishing effect on further bone adaptation; (3) bone cells accommodate to a mechanical loading environment, making them less responsive to routine or customary loading signals.


Subject(s)
Adaptation, Physiological/physiology , Bone and Bones/physiology , Animals , Biomechanical Phenomena , Bone and Bones/anatomy & histology , Humans , Models, Biological , Osteocytes/metabolism , Signal Transduction , Stress, Mechanical
9.
Am J Respir Cell Mol Biol ; 17(4): 443-55, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9376119

ABSTRACT

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.


Subject(s)
Cell Adhesion Molecules/metabolism , Endothelium, Vascular/ultrastructure , Gap Junctions/ultrastructure , Protein-Tyrosine Kinases/metabolism , Talin/metabolism , Thrombin/pharmacology , Vinculin/metabolism , Animals , Cattle , Cell Adhesion Molecules/ultrastructure , Cells, Cultured , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Gap Junctions/drug effects , Gap Junctions/metabolism , Phosphorylation , Protein-Tyrosine Kinases/ultrastructure , Pulmonary Artery/cytology , Talin/ultrastructure , Vinculin/ultrastructure
10.
Am J Physiol ; 271(5 Pt 1): C1594-602, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8944643

ABSTRACT

Regulation of the attachment of actin filaments to the cell membrane at membrane-associated dense plaque (MADP) sites could allow smooth muscle cells to modulate their cytostructure in response to changes in external stress. In this study, changes in the tyrosine phosphorylation of the MADP protein paxillin were measured by Western blot during the contraction and relaxation of tracheal smooth muscle strips. Tyrosine phosphorylation of paxillin increased by three- to fourfold with a time course similar to force development during contractile stimulation with acetylcholine (ACh), 5-hydroxytryptamine, and KCl and decreased during washout of contractile stimuli and during relaxation induced by forskolin. Immunoprecipitation of muscle extracts with multiple rounds of anti-phosphotyrosine antibody removed approximately 20% of the total paxillin in resting muscles and approximately 60% of paxillin in muscles maximally stimulated with ACh. These results provide the first evidence associating the tyrosine phosphorylation of paxillin with the active contraction of smooth muscle or with any functional response of a fully differentiated tissue in vivo. The results are consistent with a role for MADP proteins in the regulation of force development in smooth muscle.


Subject(s)
Cytoskeletal Proteins/metabolism , Isometric Contraction , Muscle, Smooth/physiology , Phosphoproteins/metabolism , Acetylcholine/pharmacology , Analysis of Variance , Animals , Blotting, Western , Cytoskeletal Proteins/isolation & purification , Dogs , Genistein , In Vitro Techniques , Isoflavones/pharmacology , Isometric Contraction/drug effects , Kinetics , Muscle Relaxation , Muscle, Smooth/drug effects , Myosin Light Chains/metabolism , Paxillin , Phosphoproteins/isolation & purification , Phosphorylation , Phosphotyrosine/analysis , Serotonin/pharmacology , Trachea , Tyrosine
11.
J Immunol ; 157(1): 321-5, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8683133

ABSTRACT

The selectins are a family of carbohydrate-binding adhesion molecules involved in the regulation of leukocyte migration. Although there is strong homology between different selectins in their extracellular regions, there is none in the cytoplasmic tails, suggesting selectin-specific functions for these domains. Our previous work showed that the cytoplasmic tail of L-selectin interacts with the actin cytoskeleton via alpha-actinin and vinculin, and that truncation of the cytoplasmic tail of L-selectin blocked both association with alpha-actinin and vinculin and leukocyte adhesion. In the present study, the effects of truncation of the cytoplasmic tails of E- or P-selectin on cell adhesion and cell surface expression were examined, and possible interactions between alpha-actinin and the E- and P-selectin cytoplasmic tails were assessed. In contrast to previous observations demonstrating a requirement for the L-selectin cytoplasmic tail, truncation of the E- or P-selectin cytoplasmic domains had no effect on cell adhesion, or on cell surface expression, when assessed in transiently transfected COS cells. This lack of effect on cell surface expression and adhesion was also observed when transfections were performed with lower amounts of cDNA, which led to submaximal levels of expression. In addition, no interaction between alpha-actinin and the cytoplasmic tails of either E- or P-selectin could be detected under conditions in which binding of alpha-actinin to the L-selectin cytoplasmic tail could be readily demonstrated. Therefore, interactions between the cytoplasmic tail of E- or P-selectin and alpha-actinin or other cytoskeletal proteins are not necessary for leukocyte adhesion per se, but may facilitate downstream biologic events.


Subject(s)
Actinin/metabolism , Cytoplasm/metabolism , E-Selectin/metabolism , E-Selectin/pharmacology , Leukocytes/drug effects , P-Selectin/metabolism , P-Selectin/pharmacology , Amino Acid Sequence , Animals , Cell Adhesion/drug effects , Cells, Cultured , Chlorocebus aethiops , Cytoplasm/chemistry , E-Selectin/genetics , Humans , Kidney , Molecular Sequence Data , Mutagenesis, Site-Directed , P-Selectin/genetics , Protein Binding/immunology
12.
Blood Coagul Fibrinolysis ; 6(7): 609-26, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8562832

ABSTRACT

The serine protease, thrombin, evokes numerous endothelial cell responses which regulate hemostasis, thrombosis and vessel wall pathophysiology. One such response, the development of intercellular gap formation and vascular permeability is relevant to each of these processes and is a cardinal features of inflammation. Regulation of endothelial cell gap formation and therefore permeability is a function of a dynamic balance between competing adhesive, barrier-promoting tethering forces and contractile, tension-producing forces which result in barrier dysfunction. The key tethering events governing focal endothelial cell adhesion to the extracellular matrix and cell-cell interactions are poorly understood. In contrast, information is rapidly increasing regarding endothelial-specific contractile processes driven by the actomyosin molecular motor. The level of myosin light chain phosphorylation catalyzed by a unique myosin light chain kinase promotes productive actin-myosin interaction and governs the degree of centripetal tension produced. In this review the signal transducing and contractile mechanisms by which thrombin elicits endothelial cellular activation through its specific receptor are addressed. The pathways by which thrombin may alter the balance between contractile and tethering forces to promote endothelial cell gap formation are discussed.


Subject(s)
Capillaries/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Thrombin/pharmacology , Animals , Calcium/metabolism , Cell Adhesion , Cell Communication , Humans , Phospholipases/metabolism , Protein Kinases/metabolism
13.
J Cell Biol ; 129(4): 1155-64, 1995 May.
Article in English | MEDLINE | ID: mdl-7538138

ABSTRACT

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha-actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.


Subject(s)
Actinin/metabolism , Cell Adhesion Molecules/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/ultrastructure , Cell Adhesion Molecules/isolation & purification , Cell Compartmentation , Cell Membrane/metabolism , Humans , L-Selectin , Mice , Microscopy, Immunoelectron , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/isolation & purification , Precipitin Tests , Protein Binding
14.
Exp Cell Res ; 217(2): 534-40, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7698254

ABSTRACT

In this study we demonstrate that alpha-actinin is a prominent component of the focal adhesions of nonmuscle cells but that the alpha-actinin in focal adhesions is largely inaccessible to staining with antibodies against alpha-actinin. Our results explain a controversy that has existed in the literature. Investigators who microinject alpha-actinin into nonmuscle cells have routinely observed significant incorporation of alpha-actinin into focal adhesions as well as stress fibers. Immunofluorescence and immunoelectron microscopy have, however, indicated that alpha-actinin is located farther from the membrane than either talin or vinculin. Immunofluorescence studies of smooth muscle dense plaques and myotendinous junctions have also yielded conflicting results regarding the presence or absence of alpha-actinin at these sites. Here, we confirm that alpha-actinin immunofluorescence of fibroblasts yields weak or absent staining of focal adhesions. We also demonstrate that microinjected alpha-actinin readily incorporates into focal adhesions. However, various antisera against either the cell's endogenous alpha-actinin or against the microinjected chicken gizzard alpha-actinin fail to stain focal adhesions despite the presence of microinjected alpha-actinin at these sites. Furthermore, disassembly of stress fibers induced by dibutyrl cAMP demonstrates that alpha-actinin persists in focal adhesions in the absence of associated stress fibers, suggesting that alpha-actinin's association with focal adhesions is independent of stress fibers.


Subject(s)
Actinin/analysis , Antibodies , Cell Adhesion , Actinin/immunology , Animals , Antibodies/immunology , Cells, Cultured , Fibroblasts , Microinjections , Microscopy, Fluorescence , Rats
15.
Am J Physiol ; 268(3 Pt 1): C563-71, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7534979

ABSTRACT

Reorganization of cytoskeletal-membrane interactions during contractile stimulation may contribute to the regulation of airway smooth muscle contraction. We investigated the effect of contractile stimulation on the phosphorylation of the actin-membrane attachment proteins talin, vinculin, and paxillin. Stimulation of 32P-labeled canine tracheal smooth muscle strips with acetylcholine (ACh; 10(-3) M) resulted in a rapid 2.6-fold increase in phosphorylation of serine and/or threonine residues, compared with resting levels of 0.22 mol PO4(3-)/mol talin. After stimulation with ACh, phosphorylation of tyrosine residues on paxillin increased approximately threefold. Two-dimensional phosphopeptide mapping of in vivo labeled talin and paxillin indicated phosphorylation on a limited number of sites. Vinculin phosphorylation was undetectable in either resting or ACh-stimulated muscle. We conclude that phosphorylation of talin and paxillin occurs during ACh-stimulated contraction of tracheal smooth muscle and that distinct signaling pathways activate a serine/threonine kinase that phosphorylates talin and a tyrosine kinase that phosphorylates paxillin. The pharmacological activation of airway smooth muscle cells might involve the anchoring of contractile filaments to the membrane.


Subject(s)
Cytoskeletal Proteins/metabolism , Muscle Contraction/physiology , Phosphoproteins/metabolism , Talin/metabolism , Trachea/physiology , Acetylcholine/pharmacology , Animals , Binding Sites , Dogs , Immunosorbent Techniques , Muscle Contraction/drug effects , Muscle, Smooth/physiology , Paxillin , Phosphorylation , Phosphoserine/metabolism , Phosphothreonine/metabolism , Phosphotyrosine , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Vinculin/metabolism
16.
Proc Soc Exp Biol Med ; 205(4): 282-93, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8171051

ABSTRACT

The past ten years have seen significant progress in cell biology research aimed at understanding how cytoskeletal filaments interact with the plasma membrane. Considerable evidence suggests that both actin microfilaments and intermediate filaments attach to the membrane via the cytoplasmic domains of various membrane proteins including adhesion molecules. Interactions between the cytoskeleton and adhesion molecules appear to be essential for a variety of cellular functions, including cell-cell and cell-extracellular matrix (ECM) interactions, cell motility, receptor-ligand interactions, and receptor internalization. Recently, many of the detailed molecular mechanisms which mediate the associations between actin filaments and adhesion molecules have been identified. Among adhesion molecules that support the attachment of cytoskeletal filaments to their cytoplasmic domains are members of the integrin and cadherin families, the intracellular adhesion molecule-1 (ICAM-1, an immunoglobulin family member), and the glycoprotein Ib/IX complex in platelets. A general conclusion emerging from these studies is that physical associations between cytoskeletal filaments and transmembrane glycoproteins do not occur directly between the filaments and the cytoplasmic tails of adhesion molecules. Instead, these interactions appear to be indirect and involve a complex ensemble of intermediary linker proteins. The severe effects of cytoplasmic domain deletion and mutagenesis on adhesion-dependent functions support the view that receptor cytoplasmic domains play a vital role in regulating receptor function and in mediating communication across the membrane. Transfection studies with mutant and chimeric adhesion molecules, along with protein-binding studies, are clarifying the mechanisms which physically link the cytoskeleton to transmembrane proteins, regulate cytoskeletal organization, mediate signaling across the cell membrane, and regulate the ligand specificity and binding affinity of surface receptors.


Subject(s)
Cell Adhesion Molecules/metabolism , Cytoplasm/metabolism , Cytoskeleton/metabolism , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data
17.
J Immunol ; 151(7): 3795-807, 1993 Oct 01.
Article in English | MEDLINE | ID: mdl-8104223

ABSTRACT

Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily of adhesion molecules, rapidly change from a low avidity to a high avidity state on activation of neutrophils by various agonists. The control of CD18 integrin-dependent neutrophil adhesion and the mechanisms that regulate integrin avidity are poorly understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integrins are necessary for proper integrin function and suggest that interactions with intracellular proteins are involved. We have focused on identifying cytoskeletal proteins that interact with the cytoplasmic domain of the beta-subunit (beta 2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein alpha-actinin associates in vitro with a peptide corresponding to a portion of the CD18 cytoplasmic domain in solid phase binding assays and affinity chromatography experiments. The peptide sequence within the CD18 cytoplasmic domain that binds alpha-actinin is homologous with a region in the cytoplasmic domain of the integrin beta 1-subunit, which also binds alpha-actinin. We demonstrate that the association of alpha-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with alpha-actinin from stimulated human neutrophils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to coprecipitate with alpha-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-alpha. Very little CD18 coprecipitates with alpha-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with alpha-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between alpha-actinin and CD18 was transient, peaking 5-10 min after activation and decreasing to near resting levels by 20 min. CD18 did not coimmunoprecipitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an alpha-actinin-mediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, alpha-actinin has a major function as an actin membrane linker molecule, and integrin avidity may be affected by an association with the actin cytoskeleton involving alpha-actinin.


Subject(s)
Actinin/metabolism , Antigens, CD/metabolism , Neutrophils/physiology , Actinin/isolation & purification , Adult , Amino Acid Sequence , CD18 Antigens , Chromatography, Affinity , Cytoplasm/metabolism , Humans , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/pharmacology
19.
J Cell Biol ; 114(3): 481-91, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1907287

ABSTRACT

Alpha-actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin. The 27-kD fragment contains an actin-binding site and we have recently shown that the 53-kD fragment binds to the cytoplasmic domain of beta 1 integrin in vitro (Otey, C. A., F. M. Pavalko, and K. Burridge. 1990. J. Cell Biol. 111:721-729). We have explored the behavior of the isolated 27- and 53-kD fragments of alpha-actinin after their microinjection into living cells. Consistent with its containing a binding site for actin, the 27-kD fragment was detected along stress fibers within 10-20 min after injection into rat embryo fibroblasts (REF-52). The 53-kD fragment of alpha-actinin, however, concentrated in focal adhesions of REF-52 cells 10-20 min after injection. The association of this fragment with focal adhesions in vivo is consistent with its interaction in vitro with the cytoplasmic domain of the beta 1 subunit of integrin, which was also localized at these sites. When cells were injected with greater than 5 microM final concentration of either alpha-actinin fragment and cultured for 30-60 min, most stress fibers were disassembled. At this time, however, many of the focal adhesions, particularly those around the cell periphery, remained after most stress fibers had gone. By 2 h after injection only a few small focal adhesions persisted, yet the cells remained spread. Identical results were obtained with other cell types including primary chick fibroblasts, BSC-1, MDCK, and gerbil fibroma cells. Stress fibers and focal adhesions reformed if cells were allowed to recover for 18 h after injection. These data suggest that introduction of the monomeric 27-kD fragment of alpha-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact alpha-actinin molecules along stress fibers. The 53-kD fragment may interfere with endogenous alpha-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of alpha-actinin and that is needed to maintain stress fiber organization.


Subject(s)
Actinin/metabolism , Actins/metabolism , Cytoskeleton/metabolism , Animals , Cell Line , Chick Embryo , Cytoskeletal Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Immunohistochemistry , Microinjections , Microscopy, Fluorescence , Peptide Fragments/metabolism , Protein Binding , Rats , Vinculin
20.
J Cell Biol ; 111(2): 721-9, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2116421

ABSTRACT

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.


Subject(s)
Actinin/metabolism , Integrins/metabolism , Actinin/isolation & purification , Amino Acid Sequence , Animals , Cells, Cultured , Chick Embryo , Chromatography, Affinity , Cytoskeletal Proteins/isolation & purification , Cytoskeletal Proteins/metabolism , Fibroblasts/metabolism , Gizzard, Avian/metabolism , Immunoblotting , Kinetics , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Muscle, Smooth/metabolism , Peptides/chemical synthesis , Phospholipids/metabolism , Vinculin
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