The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9

Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co-immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.     

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.     

Angiogenesis

Extracellular matrix (ECM) is essential for all stages of angiogenesis. In the adult, angiogenesis begins with endothelial cell (EC) activation, degradation of vascular basement membrane, and vascular sprouting within interstitial matrix. During this sprouting phase, ECM binding to integrins provides critical signaling support for EC proliferation, survival, and migration. ECM also signals the EC cytoskeleton to initiate blood vessel morphogenesis. Dynamic remodeling of ECM, particularly by membrane-type matrix metalloproteases (MT-MMPs), coordinates formation of vascular tubes with lumens and provides guidance tunnels for pericytes that assist ECs in the assembly of vascular basement membrane. ECM also provides a binding scaffold for a variety of cytokines that exert essential signaling functions during angiogenesis. In the embryo, ECM is equally critical for angiogenesis and vessel stabilization, although there are likely important distinctions from the adult because of differences in composition and abundance of specific ECM components.     

Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells

Vascular endothelial cells (ECs) seeded sparsely on extracellular matrix (ECM) will proliferate in the absence of exogenous basic fibroblast growth factor (bFGF). This ECM will also stimulate neurite outgrowth in PC12 cells in the absence of exogenous growth factors. We have previously shown that bFGF is found in subendothelial ECM (Vlodavsky, I., J. Folkman, R. Sullivan, R. Fridman, R. Ishai-Michaeli, J. Sasse, and M. Klagsburn. 1987. Proc. Natl. Acad. Sci. USA. 84:2292-2296) and in basement membranes (Folkman, J., M. Klagsburn, J. Sasse, M. Wadzinski, D. Ingber, and I. Vlodavsky. 1988. Am. J. Pathol. 130:393-400). The actual requirement of ECM-associated bFGF for the growth of ECs and differentiation of PC12 cells was shown in two ways. First, polyclonal anti-bFGF antibodies added to subendothelial ECM inhibited both EC proliferation and PC12 neurite outgrowth. Secondly, PF-HR-9 cells, which do not synthesize bFGF and which produce an ECM not permissive for EC proliferation and PC12 neurite outgrowth, were transfected with bFGF cDNA. PF-HR-9 cells transfected with bFGF, but not with the dominant selectable marker SV2-neomycin, were found to express bFGF and to produce an ECM which did support both EC proliferation and PC12 differentiation. The ECM-mediated stimulatory effects were inhibited by anti-bFGF antibodies but not by anti-nerve growth factor antibodies or nonimmune rabbit IgG. These results indicate that bFGF associated with ECM is a required ECM component for ECM-mediated cell proliferation and differentiation.     

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.     

Lymphocyte adhesion-dependent calcium signaling in human endothelial cells

Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3- labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino- phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion- dependent EC signals. mAb studies indicate that the beta 2 integrin- intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1- mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion.     

Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport

The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin alpha5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin alpha5, whereas Rad associates with importins alpha3, alpha5 and beta through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex 'in situ' as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function.     

Incomplete and transitory decrease of glycolysis

During vessel sprouting, a migratory endothelial tip cell guides the sprout, while proliferating stalk cells elongate the branch. Tip and stalk cell phenotypes are not genetically predetermined fates, but are dynamically interchangeable to ensure that the fittest endothelial cell (EC) leads the vessel sprout. ECs increase glycolysis when forming new blood vessels. Genetic deficiency of the glycolytic activator PFKFB3 in ECs reduces vascular sprouting by impairing migration of tip cells and proliferation of stalk cells. PFKFB3-driven glycolysis promotes the tip cell phenotype during vessel sprouting, since PFKFB3 overexpression overrules the pro-stalk activity of Notch signaling. Furthermore, PFKFB3-deficient ECs cannot compete with wild-type neighbors to form new blood vessels in chimeric mosaic mice. In addition, pharmacological PFKFB3 blockade reduces pathological angiogenesis with modest systemic effects, likely because it decreases glycolysis only partially and transiently.     

Adhesion of T and B lymphocytes to extracellular matrix and endothelial cells can be regulated through the beta subunit of VLA

Investigating the regulation of very late antigen (VLA)-mediated functions, we found that TS2/16, a mAb directed against the beta chain of the VLA group of integrins, can induce binding of resting peripheral blood lymphocytes, cloned T lymphocytes, and Epstein Barr virus-transformed B cells to extracellular matrix components, fibronectin, laminin, and collagen, but not to fibrinogen. The antibody stimulates VLA-4-, VLA-5-, and VLA-6-mediated binding. Furthermore, it induces VLA-4-mediated binding to vascular cell adhesion molecule-1 expressed by rTNF-alpha-stimulated endothelial cells, but it does not stimulate homotypic aggregation of cells as described for a number of anti-VLA-4 alpha antibodies (Bednarczyk, J.L., and B. W. McIntyre. 1990. J. Immunol. 144: 777-784; Campanero, M. R., R. Pulido, M. A. Ursa, M. Rodríguez-Moya, M. O. de Landázuri, and F. Sánchez-Madrid. 1990. J. Cell Biol. 110:2157-2165). Therefore, the stimulating activity of this anti-beta 1 antibody clearly contrasts with that of the anti-VLA-4 alpha antibodies, which induce homotypic cell aggregation, but not binding of cells to extracellular matrix components or endothelial cells, indicating that TS2/16 may generate different signals. The observation that also F(ab')2 or Fab fragments of this anti-beta 1 antibody stimulate binding to extracellular matrix components and endothelial cells excludes the possibility that binding requires receptor crosslinking, or is Fc receptor mediated. Induction of this adhesion is cation and energy dependent and requires an intact cytoskeleton. Although changes in the conformation of VLA integrins induced by this antibody may regulate their functional activity, the dependence on metabolic energy indicates that intracellular processes may also play a role.     

Functional dissection of Rab GTPases involved in primary cilium formation

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125-148; Singla, V., and J.F. Reiter. 2006. Science. 313:629-633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517-524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.     

Hyaluronan and Its Receptors as Regulatory Molecules of the Endothelial Interface

On the surface of endothelial cells (ECs) lies the glycocalyx, a barrier of polysaccharides that isolates the ECs from the blood. The role of the glycocalyx is dynamic and complex, thanks to not only its structure, but its vast number of components, one being hyaluronan (HA). HA is a critical component of the glycocalyx, having been found to have a wide variety of functions depending on its molecular weight, its modification, and receptor–ligand interactions. As HA and viscous blood are in constant contact, HA can transmit mechanosensory information directly to the cytoskeleton of the ECs. The degradation and synthesis of HA directly alters the permeability of the EC barrier; HA modulation not only alters the physical barrier but also can signal the initiation of other pathways. EC proliferation and angiogenesis are in part regulated by HA fragmentation, HA-dependent receptor binding, and downstream signals. The interaction between the CD44 receptor and HA is a driving force behind leukocyte recruitment, but each class of leukocyte still interacts with HA in unique ways during inflammation. HA regulates a diverse repertoire of EC functions.     

Mechanotransduction in endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.     

The non-transmembrane form of Delta1, but not of Jagged1, induces normal migratory behavior accompanied by fibroblast growth factor receptor 1-dependent transformation

The interactions between Notch (N) receptors and their transmembrane ligands, Jagged1 (JI) and Delta1 (Dl1), mediate signaling events between neighboring cells that are crucial during embryonal development and in adults. Since the non-transmembrane extracellular form of J1 acts as an antagonist of N activation in NIH 3T3 mouse fibroblast cells and induces fibroblast growth factor 1 (FGF1)-dependent transformation (Small, D., Kovalenko, D., Soldi, R., Mandinova, A., Kolev, V., Trifonova, R., Bagala, C., Kacer, D., Battelli, C., Liaw, L., Prudovsky, I., and Maciag, T. (2003) J. Biol. Chem. 278, 16405-16413), we examined the potential redundant functions of the two subfamilies of Notch ligands and report that while the soluble (s) forms of both Dl1 and J1 act as N signaling antagonists in NIH 3T3 cells, they do display disparate functions. While sJ1 induced an attenuation of cell motility which is accompanied by a decrease in actin stress fibers and an increase in adherence junctions, sDl1 does not. However, sJ1, like sDl1, induces a NIH 3T3 cell tranformed phenotype mediated by FGF signaling. Because the inhibition of N signaling by sJ1 and sDl1 is rescued by dominant-negative Src expression, we suggest that there may be cooperation between the Notch and Src signaling pathways.     

Modification of proteins secreted by endothelial cells during modeled low gravity exposure

The exposure of the human body to microgravity, conditions that occurs during space flights, causes significant changes in the cardiovascular system. Many cell types have been involved in these changes, and the endothelium seems to play a major role. In endothelial cells (EC), it has been shown that modeled low gravity impairs nitric oxide synthesis, cell adhesion, extracellular matrix composition, cytoskeleton organization, cytokines, and growth factors secretion. Nevertheless, detailed analysis of EC physiological changes induced by microgravity exposure is still lacking. Secretome analysis is one of the most promising approaches for the identification of biomarkers directly related to the physiopathological cellular state. In this study, we analyzed in details the modifications of EC secretome by using umbilical vein endothelial (HUVE) cells exposed to modeled low gravity conditions. By adopting a two‐dimensional (2‐D) proteomic approach, in conjunction with a technique for the compression of the dynamic range of proteins, we observed that modeled low gravity exposure of HUVE cells affected the secretion of proteins involved in the regulation of cytoskeleton assembly. Moreover, by using Luminex® suspension array systems, we found that the low gravity condition decreased in ECs the secretion of some key pro‐inflammatory cytokines, including IL‐1α and IL‐8, and of the pro‐angiogenic factor bFGF. On the contrary, microgravity increase the secretion of two chemokines (Rantes and Eotaxin), involved in leukocytes recruitment. J. Cell. Biochem. 112: 265–272, 2011. © 2010 Wiley‐Liss, Inc.     

Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).     

Dissociation of ERK and Akt signaling in endothelial cell angiogenic responses to beta-amyloid

Cerebrovascular deposits of beta-amyloid (Abeta) peptides are found in Alzheimer's disease and cerebral amyloid angiopathy with stroke or dementia. Dysregulations of angiogenesis, the blood-brain barrier and other critical endothelial cell (EC) functions have been implicated in aggravating chronic hypoperfusion in AD brain. We have used cultured ECs to model the effects of beta-amyloid on the activated phosphorylation states of multifunctional serine/threonine kinases since these are differentially involved in the survival, proliferation and migration aspects of angiogenesis. Serum-starved EC cultures containing amyloid-beta peptides underwent a 2- to 3-fold increase in nuclear pyknosis. Under growth conditions with sublethal doses of beta-amyloid, loss of cell membrane integrity and inhibition of cell proliferation were observed. By contrast, cell migration was the most sensitive to Abeta since inhibition was significant already at 1 muM (P = 0.01, migration vs. proliferation). In previous work, intracellular Abeta accumulation was shown toxic to ECs and Akt function. Here, extracellular Abeta peptides do not alter Akt activation, resulting instead in proportionate decreases in the phosphorylations of the MAPKs: ERK1/2 and p38 (starting at 1 microM). This inhibitory action occurs proximal to MEK1/2 activation, possibly through interference with growth factor receptor coupling. Levels of phospho-JNK remained unchanged. Addition of PD98059, but not LY294002, resulted in a similar decrease in activated ERK1/2 levels and inhibition of EC migration. Transfection of ERK1/2 into Abeta-poisoned ECs functionally rescued migration. The marked effect of extracellular Abeta on the migration component of angiogenesis is associated with inhibition of MAPK signaling, while Akt-dependent cell survival appears more affected by cellular Abeta.     

EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution

During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.     

Vascular smooth muscle cells promote endothelial cell adhesion via microtubule dynamics and activation of paxillin and the extracellular signal-regulated kinase (ERK) pathway in a co-culture system

Interaction between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays an important role in vascular biology. Cell adhesion to the extracellular matrix provides critical environmental information necessary for cell migration, proliferation, differentiation and survival. In this study, the role of VSMCs in EC adhesion was demonstrated by using a co-culture system. It was shown that the co-cultured VSMCs significantly increased the number of adherent ECs, and induced an increase of total focal adhesion area in ECs. These changes were associated with a low microtubule-to-tubulin ratio, and activation of extracellular signal-regulated kinase (ERK) and paxillin. Both the EC adhesion state and activation of the ERK/paxillin pathway by the co-cultured VSMCs could be inhibited by trichostatin A (TSA). As an inhibitor of histone deacetylase, TSA acts by modulating microtubule polymerization state. Taken together, these data suggest that the co-cultured VSMCs promote EC adhesion by modulating the microtubule cytoskeleton polymerization state, which in turn activates the ERK pathway and up-regulates phosphorylated paxillin expression to accelerate focal adhesion formation.     

Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.