首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 78 毫秒
1.
Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodelling and repair after lung injury. In the present paper we hypothesize that secreted ATX (autotaxin), which exhibits lysoPLD (lysophospholipase D) activity, stimulates lung epithelial cell migration through LPA (lysophosphatidic acid) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag overexpressed conditional medium had higher LPA levels compared with control medium and stimulated cell migration through G(αi)-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKC (protein kinase C) δ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to the LPA receptor and integrin β4 complex on A549 cell surface. Finally, intratracheal administration of LPS (lipopolysaccharide) into the mouse airway induced ATX release and LPA production in BAL (bronchoalveolar lavage) fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodelling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated association of ATX with the epithelial cell-surface LPA receptor and integrin β4.  相似文献   

2.
Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A(1) (PLA(1)) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA(1). Addition of this recombinant PLA(1) significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA(1); 2) PLA(1) generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.  相似文献   

3.
《Matrix biology》2006,25(4):223-231
Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance α5β1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for α4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of α4β1-expressing CHOα4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOα4 and human A375 melanoma cells in an α4β1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for α4β1. LPA signaling combined with α4β1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.  相似文献   

4.
Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase-2, is a secreted lysophospholipase D (lysoPLD) that hydrolyzes extracellular lysophospholipids into the lipid mediator lysophosphatidic acid (LPA), a ligand for specific G protein-coupled receptors. ATX-LPA signaling is essential for development and has been implicated in a great diversity of (patho)physiological processes, ranging from lymphocyte homing to tumor progression. Structural and functional studies have revealed what makes ATX a unique lysoPLD, and how secreted ATX binds to its target cells. The ATX catalytic domain shows a characteristic bimetallic active site followed by a shallow binding groove that can accommodate nucleotides as well as the glycerol moiety of lysophospholipids, and by a deep lipid-binding pocket. In addition, the catalytic domain has an open tunnel of unknown function adjacent to the active site. Here, we discuss our current understanding of ATX structure-function relationships and signaling mechanisms, and how ATX isoforms use distinct mechanisms to target LPA production to the plasma membrane, notably binding to integrins and heparan sulfate proteoglycans. We also briefly discuss the development of drug-like inhibitors of ATX.  相似文献   

5.
Autotaxin (ATX) is a secreted phosphodiesterase that produces the lipid mediator lysophosphatidic acid (LPA). LPA acts through specific guanine-nucleotide-binding protein (G protein)-coupled receptors to stimulate migration, proliferation, survival and other functions in many cell types. ATX is important in vivo for processes as diverse as vasculogenesis, lymphocyte trafficking and tumour progression. However, the inner workings of ATX have long been elusive, in terms of both its substrate specificity and how localized LPA signalling is achieved. Structural studies have shown how ATX recognizes its substrates and may interact with the cell surface to promote specificity in LPA signalling.  相似文献   

6.
Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.  相似文献   

7.
Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.  相似文献   

8.
A transmembrane domain heterodimer, acting in concert with a membrane-proximal cytoplasmic domain clasp, is thought to maintain integrins in a low affinity state. To test whether helix-helix interactions between the alphaIIb and beta3 transmembrane domains regulate the activity of integrin alphaIIbbeta3, we synthesized a soluble peptide corresponding to the alphaIIb transmembrane domain, designated alphaIIb-TM, and we studied its ability to affect alphaIIbbeta3 activity in human platelets. alphaIIb-TM was alpha-helical in detergent micelles and phospholipid vesicles, readily inserted into membrane bilayers, bound to intact purified alphaIIbbeta3, and specifically associated with the transmembrane domain of alphaIIb, rather than the transmembrane domains of beta3, alpha2, and beta1, other integrin subunits present in platelets. When added to suspensions of gel-filtered platelets, alphaIIb-TM rapidly induced platelet aggregation that was not inhibited by preincubating platelets with the prostaglandin E(1) or the ADP scavenger apyrase but was prevented by the divalent cation chelator EDTA. Furthermore, alphaIIb-TM induced fibrinogen binding to platelets but not the binding of osteopontin, a specific ligand for platelet alphavbeta3. The peptide also induced fibrinogen binding to recombinant alphaIIbbeta3 expressed by Chinese hamster ovary cells, confirming that its effect was independent of platelet signal transduction. Finally, transmission electron microscopy of purified alphaIIbbeta3 revealed that alphaIIb-TM shifted the integrin from a closed configuration with its stalks touching to an open configuration with separated stalks. These observations demonstrate that transmembrane domain interactions regulate integrin function in situ and that it is possible to target intra-membranous protein-protein interactions in a way that can have functional consequences.  相似文献   

9.
Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.  相似文献   

10.
11.
The alpha M beta 2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the alpha M I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known beta 2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified beta 2 integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active beta 2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of beta 2 integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the alpha M beta 2 and alpha L beta 2 integrins in leukocytes, and pro-MMP-9 colocalized with alpha M beta 2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked beta 2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-beta 2 integrin complex-dependent manner.  相似文献   

12.
Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.  相似文献   

13.
Regulation of integrin activation occurs by specific interactions among cytoplasmic proteins and integrin alpha and beta cytoplasmic tails. We report that the catalytic subunit of protein phosphatase 1 (PP1c) constitutively associates with the prototypic integrin alphaIIbbeta3 in platelets and in cell lines overexpressing the integrin. PP1c binds directly to the cytoplasmic domain of integrin alphaIIb subunit containing a conserved PP1c binding motif 989KVGF992. Anchored PP1c is inactive, while thrombin-induced platelet aggregation or fibrinogen-alphaIIbbeta3 engagement caused PP1c dissociation and concomitant activation as revealed by dephosphorylation of PP1c substrate, myosin light chain. Inhibition of ligand binding to activated alphaIIbbeta3 blocks PP1c dissociation and represses PP1c activation. These studies reveal a previously unrecognized role for integrins whereby the alpha subunit cytoplasmic tail localizes the machinery for initiating and temporally maintaining the regulatory signaling activity of a phosphatase.  相似文献   

14.
Autotaxin (ATX) is a multifunctional phosphodiesterase originally isolated from melanoma cells as a potent cell motility-stimulating factor. ATX is identical to lysophospholipase D, which produces a bioactive phospholipid, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC). Although enhanced expression of ATX in various tumor tissues has been repeatedly demonstrated, and thus, ATX is implicated in progression of tumor, the precise role of ATX expressed by tumor cells was unclear. In this study, we found that ATX is highly expressed in glioblastoma multiforme (GBM), the most malignant glioma due to its high infiltration into the normal brain parenchyma, but not in tissues from other brain tumors. In addition, LPA1, an LPA receptor responsible for LPA-driven cell motility, is predominantly expressed in GBM. One of the glioblastomas that showed the highest ATX expression (SNB-78), as well as ATX-stable transfectants, showed LPA1-dependent cell migration in response to LPA in both Boyden chamber and wound healing assays. Interestingly these ATX-expressing cells also showed chemotactic response to LPC. In addition, knockdown of the ATX level using small interfering RNA technique in SNB-78 cells suppressed their migratory response to LPC. These results suggest that the autocrine production of LPA by cancer cell-derived ATX and exogenously supplied LPC contribute to the invasiveness of cancer cells and that LPA1, ATX, and LPC-producing enzymes are potential targets for cancer therapy, including GBM.  相似文献   

15.
Integrin alpha(IIb)beta(3), an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts. Here, we tested the proposed structural rearrangements undergone by integrin alpha(IIb)beta(3) after cell activation, by using F?rster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(IIb) subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model.  相似文献   

16.
Autotaxin (ATX) catalyzes the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). Both ATX and LPA have been linked to pathophysiologies ranging from cancer to neuropathic pain. Inhibition of LPA production by ATX is therefore of therapeutic interest. Here we report the application of previously-developed, subsite-targeted pharmacophore models in a screening workflow that involves either docking or binary QSAR as secondary filters to identify ATX inhibitors from previously unreported structural types, four of which have sub-micromolar inhibition constants. Cell-based assays demonstrate that ATX inhibition and cytotoxicity structure–activity-relationships (SAR) exhibit selectivity cliffs, characterized by structurally similar compounds exhibiting similar biological activities with respect to ATX inhibition but very different biological activities with respect to cytotoxicity. Thus, general cytotoxicity should not be used as an early filter to eliminate candidate ATX inhibitor scaffolds from further SAR studies. Assays using two substrates of vastly different sizes demonstrate that the tools developed to identify compounds binding outside the central core of the active site did identify compounds acting at an allosteric site. In contrast, tools developed to identify active-site directed compounds did not identify active-site directed compounds. The stronger volume overlap imposed when selecting screening candidates expected to bind outside the active site is likely responsible for the stronger match between intended and actual target site.  相似文献   

17.
Staphylococcus aureus is a human pathogen that causes invasive and recurring infections. The ability to internalize into and persist within host cells is thought to contribute to infection. Here we report a novel role for the well‐characterized iron‐regulated surface determinant B (IsdB) protein which we have shown can promote adhesion of 293T, HeLa cells and platelets to immobilized bacteria independently of its ability to bind haemoglobin. IsdB bound to the active form of the platelet integrin αIIbβ3, both on platelets and when the integrin was expressed ectopically in CHO cells. IsdB also promoted bacterial invasion into human cells. This was clearly demonstrated with bacteria lacking fibronectin‐binding proteins (FnBPs), which are known to promote invasion in the presence of fibronectin. However, IsdB also contributed significantly to invasion by cells expressing FnBPs in the presence of serum. Thus IsdB appears to be able to interact with the broader family of integrins that bind ligands with the RGD motif and to act as a back up mechanism to promote interactions with mammalian cells.  相似文献   

18.
uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.  相似文献   

19.
整联蛋白是细胞表面的主要膜受体,具有介导细胞与细胞基质间的黏附及病毒吸附细胞等功能。αⅠ结构域型整联蛋白和非αⅠ结构域型整联蛋白与配体结合的方式各异。对整联蛋白-配体复合物分子结构的研究表明,整联蛋白利用其配体结合位点的αMIDAS(依赖金属离子的吸附位点)、βMIDAS与各种类型配体结合。  相似文献   

20.
Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k(1) ≥500 μm(-1) s(-1)) and is hydrolyzed slowly (k(2) = 0.024 s(-1)). Release of the first hydrolysis product is random and rapid (≥1 s(-1)), whereas release of the second is slow and rate-limiting (0.005-0.007 s(-1)). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号