a novel anti-inflammatory peptide inhibits endothelial cell cytoskeletal rearrangement, nitric oxide...

JOURNAL OF CELLULAR PHYSIOLOGY 172:171–182 (1997)

A Novel Anti-Inflammatory Peptide InhibitsEndothelial Cell Cytoskeletal Rearrangement,

Nitric Oxide Synthase Translocation, andParacellular Permeability IncreasesQIN WANG,1 WAYNE F. PATTON,1* HERBERT B. HECHTMAN,2

AND DAVID SHEPRO1

1Microvascular Research Laboratory, Biological Science Center, Boston University,Boston, Massachusetts

2Department of Surgery, Harvard Medical School, Boston, Massachusetts

The endothelial cell (EC) membrane-cytoskeletal interface in part maintains plasmamembrane integrity and promotes cell–cell apposition. Nonmuscle filamin (ABP-280),an actin crosslinking protein, promotes orthogonal branching of F-actin and is the majorprotein that links the peripheral actin network to the plasma membrane through its C-terminal glycoprotein binding site. In response to bradykinin, filamin translocates fromthe cell periphery to the cytosol within 1 min. A synthetic peptide, corresponding tofilamin’s C-terminal calcium/calmodulin-dependent protein kinase II phosphorylationsite (CaM peptide), prevents calcium-activated filamin translocation in permeabilizedbovine pulmonary artery EC. The myristoylated permeable form of this peptide inhibitsbradykinin-induced filamin translocation and F-actin rearrangement in cultured intactECs. In addition, bradykinin-induced paracellular gap formation is significantly attenu-ated by CaM peptide, which suggests that the presence of a filamin-based peripheralF-actin network is essential for maintaining EC barrier function. Moreover, CaM peptidereduces wound-induced EC migration rate by 40%, which indicates that F-actin rear-rangement is required for efficient cell motility. The CaM peptide affects other bradyki-nin-induced inflammatory responses. EC nitric oxide synthase (eNOS) translocates fromthe cell membrane to the nuclear fraction within 1–2 min of bradykinin treatment.Pretreatment with CaM peptide inhibits eNOS translocation. However, the peptide hasno effect on bradykinin-induced von Willebrand Factor release. In summary, the CaMpeptide exhibits several anti-inflammatory properties that include maintaining EC junc-tional stability and inhibiting eNOS translocation. J. Cell. Physiol. 172:171–182,1997. q 1997 Wiley-Liss, Inc.

One primary function of microvascular endothelial 1992). Nonmuscle filamin (ABP-280) is a dimeric actincrosslinking protein and provides the major mode forcells (EC) is to provide a selective barrier between bloodattaching the cortical F-actin network to membraneand tissues. Activation of various inflammatory media-glycoprotein GP1ba and FcgR1, in platelets and leuko-tors, such as bradykinin, results in increased vasoper-cytes, respectively (Fox, 1985; Ohta et al., 1991). Asmeability, which is mainly modulated by changes in thelittle as one filamin molecule is capable of crosslinkingdiameters of interendothelial junctional gaps (Shepro,up to 1,000 actin molecules, making filamin the most1994). Other inflammatory responses induced by bra-potent known actin crosslinking protein (Janson et al.,dykinin in vivo include endothelium-dependent vasodi-1991). In a variety of eukaryotic cells, filamin playslation, secretion of von Willebrand factor (vWF), anda key role in stabilizing the membrane–cytoskeletonthe release of arachidonic acid metabolites. In vitro,interface (Hartwig and Kwiatkowski, 1991). Melanomabradykinin increases intracellular Ca2/, activates vari-cells lacking filamin, but not other actin crosslinkingous protein kinases, rearranges peripheral F-actin, re-proteins such as a-actinin, exhibit membrane blebbingleases nitric oxide, and induces paracellular gap forma-

tion in cultured EC (Mombouli and Vanhoutte, 1995).The importance of cytoskeletal proteins in mediatingfunctional EC gap formation in response to inflamma- Contract grant sponsor: NIH; Contract grant number: HL-43875;

Contract grant number: HL-48553; Contract grant number: GM-tory agonists has long been recognized, though the spe-24891; Contract grant number: GM-35141.cific complement of EC cytoskeletal proteins involved*Correspondence to: Wayne F. Patton, Ph.D., Microvascular Re-are only beginning to be elucidated. Proteins at thesearch Laboratory, Biological Science Center, Boston University,membrane–cytoskeleton interface may coordinate a5 Cummington Street, Boston, MA 02215.number of functions essential to cell motility and the

maintenance of EC barrier function (Luna and Hitt, Received 6 January 1997; Accepted 12 March 1997

q 1997 WILEY-LISS, INC.

JCP-0498/ 891E$$0498 06-24-97 14:07:15 wlcpal W Liss: JCP

WANG ET AL.172

and diminished cell motility (Cunningham et al., 1992). duced filamin translocation and F-actin rearrangementin intact EC. In addition, bradykinin-induced gap for-Direct interaction between filamin and b2-integrin sub-

unit CD18 at focal contacts is found at the leading edge mation is significantly attenuated by CaM peptide.Wound-induced EC migration rate is reduced as muchduring leukocyte movement, and this interaction is

thought to be essential for cell locomotion (Sharma et as 40% by CaM peptide as well as by the pharmacologi-cal CaM PKII inhibitor, KN-62. Moreover, CaM peptideal., 1995). Moreover, filamin associates with E-selectin

upon binding of leukocytes to the luminal surface of prevents endothelial cell nitric oxide synthase (eNOS)translocation from the membrane fraction to the nu-EC (Yoshida et al., 1996).

We reported that in cultured EC, filamin responds to clear fraction in response to bradykinin. However, bra-dykinin-induced vWF secretion, though a calcium/cal-various stimuli, including Ca2/ ionophore, bradykinin,

and H2O2, by translocating from the cell periphery modulin-dependent inflammatory response, is not in-hibited by CaM peptide. Thus, this filamin-basedto the cytosol, as demonstrated by immunofluores-

cence microscopy and subcellular fractionation studies peptide exhibits several anti-inflammatory propertiesthat include maintaining EC junctional stability and(Wang et al., 1996; Hastie et al., 1997). Bradykinin-

induced filamin translocation correlates with disrup- inhibiting eNOS translocation, but does not appear toinfluence the inflammatory secretion response.tion of the F-actin dense peripheral band (unpublished

observations). Whereas H2O2-induced filamin translo-MATERIALS AND METHODScation is Ca2/ independent, intracellular Ca2/ increases

Materialsare essential for bradykinin-induced filamin transloca-tion (Wang et al., 1996; Hastie et al., 1997). The kinetics KN-62 is obtained from Calbiochem (La Jolla, CA).

Bradykinin and des-Arg9-bradykinin are obtained fromof bradykinin-induced filamin translocation coincidewith intracellular Ca2/ increases, and can be blocked Sigma (St. Louis, MO).by pharmacological inhibitors of calcium/calmodulin-

Endothelial cell isolation and cultivationdependent protein kinase II (CaM-PKII). Human ECfilamin contains nine potential CaM-PKII phosphoryla- Bovine pulmonary artery ECs are isolated as pre-

viously described (Shepro et al., 1974; Mineau-tion sites (Gorlin et al., 1990). In addition, in vitro stud-ies demonstrate that chicken gizzard filamin can be Hanschke et al., 1990). ECs are removed by gentle

scraping with forceps of the exposed lumen of dissectedphosphorylated by CaM-PKII, which reduces filaminaffinity for actin filaments (Ohta and Hartwig, 1995). vessels. After incubation in 0.1% collagenase in phos-

phate buffered saline (PBS) for 15 min at 377C, the cellsFilamin phosphorylation by cAMP-dependent proteinkinase and p90 ribosomal protein S6 kinase have also are centrifuged at 2000 rpm for 4 min, resuspended in

Dulbecco’s modified Eagle’s medium (DMEM) con-been reported (Chen and Stracher, 1989; Ohta andHartwig, 1996). taining 20% bovine calf serum (BCS), penicillin (100

U/ml), streptomycin (100 ug/ml), L-arginine (1 mM), L-To characterize filamin translocation functionallyand its regulation by CaM-PKII, a peptide (CaM pep- glutamine (2 mM), and seeded onto 6-well plates at

approximately 7.51 104 cells per plate. One week later,tide) that corresponds to one of filamin’s C-terminalCaM-PKII phosphorylation sites is synthesized. In per- the cells are refed with 7% BCS. Cells are subsequently

re-fed every 2–3 days and subpassaged by trypsiniza-meabilized EC, filamin translocation can be induced bythe addition of Ca2/, ATP, and calmodulin. The omis- tion as necessary. All experiments are performed with

passage 4–10 cells.sion of any of the three cofactors reduces filamin trans-location significantly. Pretreatment with CaM peptide

Preparation of cell lysates and subcellularprevents filamin translocation induced by Ca2/, ATP,fractionsand calmodulin. The myristoylated form of CaM pep-

tide, which is cell permeable, prevents bradykinin-in- ECs seeded onto 100 mm culture dishes are rinsedtwice in PBS (Ca2//Mg2/ free), and lysed by adding1007C sample buffer I containing 2.5% sodium dodecylsulfate (SDS), 200 mM DTT in 10 mM Tris, pH 8.0,and protease inhibitors (5 mM ethylene glycol-bis (b-amino-ethyl ether) N,N,N*N*-tetraacetic acid (EGTA),1 ug/ml leupeptin, 1 mM phenylmethylsulfonyl fluorideAbbreviations(PMSF), and 0.11 IU aprotinin). The lysate is allowed

cAMP adenosine 3*-5*-cyclic monophosphate to cool on ice. Chilled sample buffer II containing 1 mg/PLC phospholipase Cml DNase I and 0.25 mg/ml RNase A is added to thePLD phospholipase D

IP3 inositol 1,4,5-triphosphate culture dish and the lysate is incubated on ice for anCa2/ calcium additional 5 min. The cell lysate is collected using anDAG sn-1,2-diacyl glycerol automated pipette, precipitated in ice-cold acetone (fi-CaM-PK II calcium/calmodulin dependent protein kinase II

nal concentration 80%), and redissolved in sampleMLCK myosin light chain kinasePKC protein kinase C buffer I.PMA phorbol myristate acetate ECs are fractionated into cytosol, membrane/organ-EC endothelial cell elle, nucleus, and cytoskeleton fractions, as previouslyEGTA ethylene glycol-bis (b-amino-ethyl ether) N,N,N*N*-

described (Ramsby et al., 1994; Wang et al., 1996).tetra acetic acidPMSF phenylmethylsulfonyl fluoride Briefly, ECs seeded onto 100-mm culture dishes areDDF differential detergent fractionation rinsed twice with PBS (Ca2//Mg2/ free) and extractedCaM-peptide a synthetic peptide corresponding to one of the fi- in ice-cold digitonin buffer (0.01% digitonin, 10 mMlamin’s C-terminal CAM-PK II phosphorylation

PIPES, pH 6.8, 300 mM sucrose, 100 mM NaCl, 3 mMsitesPIPES 1,4-Piperazine bis-(ethanesulfonic acid) MgCl2, 10 ug/ml phalloidin and protease inhibitors).


NOVEL ANTI-INFLAMMATORY PEPTIDE 173

The cells are incubated on ice with gentle agitation for Synthetic peptide10 min. The supernatant (cytosolic fraction) is collected A synthetic peptide corresponding to one of filamin’sand the residual cell components are sequentially ex- C-terminal CaM-PKII phosphorylation sites (CaM pep-tracted in Triton X-100, Tween-40/deoxycholate, and tide) is synthesized by Boston University’s peptide syn-then SDS. Triton X-100 extraction is performed for 30 thesis facility and uses the deduced amino acid se-min in 0.5% Triton X-100, 10 mM PIPES, pH 7.4, 300 quence of human umbilical vein endothelial cell filaminmM sucrose, 100 mM NaCl, 3 mM MgCl2, 3 mM EGTA, (Gorlin et al., 1990). The peptide contains amino acidprotease inhibitors, and 1002 mg/ml phalloidin. After residues 2517–2529 of filamin: NH2-Thr-Gly-Pro-Arg-the supernatant (membrane/organelle fraction) is col- Leu-Val-Ser-Asn-His-Ser-Leu-His-Glu-COOH. Peptidelected, the residual material is extracted on ice for 10 purity is confirmed by reverse phase HPLC and lasermin with a Tween-40/deoxycholate buffer, containing desorption mass spectrometry. A single species with m/1% Tween 40, 0.5% deoxycholate, 10 mM PIPES, pH z ratio of 1467.75 is observed with mass spectrometry.7.4, and protease inhibitors. The supernatant (nuclear A control peptide, corresponding to amino acid residuesfraction) is collected, and hot (1007C) SDS buffer con- 564–579 of filamin, is also synthesized by the facility.taining 2.5% SDS in 10 mM tris-HCl, pH 8, 200 mM It has the amino acid sequence of NH2-Ser-Pro-Phe-DTT and protease inhibitors, is added to solubilize the Glu-Val-Lys-Val-Gly-Thr-Glu-Cys-Gly-Asn-Gln-Lys-cytoskeleton fraction. The culture dish is scraped and COOH and corresponds to a structural b-sheet regionchilled on ice. 50 ul sample buffer II (as described in of filamin. The m/z ratio of this peptide is found towhole cell lysate preparation) is added and the dish is be 1664.88. The myristoylated form of CaM peptide isincubated on ice for 5 min. The cytoskeletal fraction is synthesized by Quality Controlled Biochemicals, Inc.collected and heated at 1007C for 5 min. All subcellular (Hopkinton, MA). Peptide purity is confirmed by re-fractions are then acetone-precipitated and redissolved verse phase HPLC and laser desorption mass spectrom-in equal volumes of sample buffer I (as described for etry. A single species with m/z ratio of 1664.1 is ob-the cell lysate extraction). served with mass spectrometry.

Peptide phosphorylation by CaM-PKIIElectrophoresis, immunoblotting, and proteinquantification Phosphorylation of the synthetic peptide (CaM pep-

tide) and a commercial CaM-PKII peptide substrateCell extracts, dissolved in sample buffer, are sub- with the sequence of NH2-Lys-Arg-Gln-Gln-Ser-Phe-jected to SDS-polyacrylamide gel electrophoresis using Asp-Leu-Phe-COOH (New England Biolabs, Beverly,4–15% gradient gels (Pharmacia, Piscataway, NJ) ac- MA) is carried out at 307C for 3 min and 5 min, in acording to manufacturer’s protocol. Equal volumes or reaction mixture containing 450 mM peptide substrate,equal amounts of protein are loaded in each lane, de- 2.4 mM calmodulin, 2 mM CaCl2, 100 mM ATP, 10 mMpending upon the experiment. Protein is quantified us- MgCl2, 0.5 mM DTT, and 0.1 mM EDTA in 20 mMing the BCA protein assay (Pierce, Rockford, IL). After Tris-HCl, PH 7.5. The reaction is started by addingelectrophoresis, proteins are electroblotted for 1 h to 1,000 units of purified CaM-PKII (New England Bio-0.4 mm pore size nitrocellulose membrane (BioRad, labs, Beverly, MA) and terminated at 3 min and 5 min,Hercules, CA) using the Pharmacia Phast system (Tow- by adding 5 mM EGTA and heating at 857C. The reac-bin et al., 1979). Alternatively, subcellular fractions are tion mixture is then dot-blotted onto nitrocelluloseapplied directly to nitrocellulose using a Bio-Dot SF membrane in duplicates. After drying, the membranevacuum apparatus (Bio-Rad Laboratories, Hercules, is incubated in blocking solution (as described in immu-CA) according to the manufacturer’s instructions. After noblotting section) for 10 min, followed by incubationelectroblotting or slot-blotting, the membrane is incu- in 1:500 dilution of mouse antiphosphoserine IgM (Cap-bated in blocking buffer containing 5% BSA, 0.1% pel, Durham, NC) for 1 h. Antiphosphoserine antibodyTween-20 for at least 1 h, followed by incubation in is used to avoid detection of CaM-PKII autophosphory-1:250 dilution of mouse antihuman nonmuscle filamin lation at the threonine residue (Thr-286/Thr-287). Theantibody (Chemicon, Temecula, CA) overnight. Alter- membrane is then incubated in 1:2000 dilution ofnatively, for eNOS detection, the membrane is incu- horseradish peroxidase-conjugated goat antimousebated in 1:500 dilution of mouse antihuman eNOS anti- IgM for 1 h, followed by visualization with a diamino-body (Transduction laboratory, Lexington, KY) over- benzidine, nickel chloride (Sigma, St. Louis, MO), andnight. The membrane is then incubated in alkaline H2O2 (Fisher Scientific, Pittsburgh, PA) chromogenphosphatase-conjugated goat antimouse IgG (Cappel, system.Durham, NC) and filamin bands are visualized using

Cell permeabilizationa nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate p-toluidine chromogen system (Sigma, St. Confluent EC monolayers are washed in cytoskeletalLouis, MO). stabilization buffer (50 mM KCl, 10 mM imidazole, 1

Electroblots are subsequently scanned at 300 dots mM EGTA, 1 mM MgSO4, 0.5 mM DTT, 5 ug/ml aproti-per inch (dpi) resolution with 256 gray-scale levels us- nin, 5 ug/ml leupeptin, 0.1 mM PMSF, 20 mM PIPES,ing a flat-bed scanner interfaced to an Apple Macintosh 2 M Glycerol, PH 6.5) twice and permeabilized with 25Power PC microcomputer. Proteins are quantified us- ug/ml saponin in cytoskeletal stabilization buffer foring NIH Image, a public domain graphics program de- 5*. EC permeabilization is monitored microscopicallyveloped by Dr. W. Rasband, Research Services Branch, with 0.1% trypan-blue. The monolayers are washedNIMH ([email protected]). This assay is linear over with cytoskeletal stabilization buffer twice and incu-a 24-fold range (R Å 0.96), which easily encompasses bated in cytoskeletal stabilization buffer with or with-

out 100 mM synthetic peptide for 20* at 377C, in a hu-the working range of the translocation experiments.


WANG ET AL.174

midified 5% CO2 atmosphere. The cultures are then Statisticsincubated in cytoskeletal stabilization buffer with or Results are expressed as means { SD. Differenceswithout 10 mM Ca2/, 2 mM ATP, 5 ug/ml calmodulin in two groups are considered statistically significantfor 30*, during which time 100 mM of the corresponding when P õ 0.05 using the Welch’s t-test.synthetic peptides are also included for peptide-pre-treated monolayers. RESULTS

CaM peptide prevents filamin translocation inF-actin visualization using permeabilized bovine pulmonary artery EC

rhodamine-phalloidinEC monolayers are fractionated into cytosol, mem-

brane, nuclear, and cytoskeleton fractions using a se-ECs are seeded onto glass coverslips, treated withagonists as indicated in the figure legends, and fixed quential detergent fractionation technique (Ramsby et

al., 1994; Wang et al., 1996). Cell permeabilization byin a fixative solution, 1G4F, containing 99 ml formalinstock solution (0.9% NaCl in 10% buffered formalin saponin is monitored by trypan blue exclusion. Saponin

treatment for 5 min does not release filamin into thesolution) and 1 ml 25% aqueous glutaraldehyde for 10min at 377C. The cells are then permeabilized with extracellular space (data not shown), and in unstimu-

lated control cells, the percentage of filamin in the cyto-0.2% triton X-100 in PBS for 10 min at room tempera-ture, followed by incubation in PBS, containing 1 mM sol and membrane fraction is 48% and 52%, respec-

tively (Fig. 1). The addition of Ca2/, ATP, and calmodu-rhodamine phalloidin for 1 h. The coverslips aremounted on slides in glycerol:H2O (9:1) and sealed with lin to permeabilized EC for 30 min induces significant

filamin translocation to the cytosol (Fig. 1), with 80%nail varnish. The slides are monitored under an in-verted IM-35 Zeiss fluorescent microscope. in the cytosol and 20% in the membrane fraction. Ca2/

alone or Ca2/ and calmodulin alone does not cause fi-lamin translocation (data not shown). To understandQuantification of agonist-induced EC gapthe regulatory role of CaM-PK II activation in filaminformationsubcellular distribution, a peptide corresponding to one

ECs seeded onto glass coverslips are treated with of filamin’s C-terminal CaM-PKII phosphorylationagonists, as indicated in the figure legends, and fixed sites (CaM peptide) and a control peptide correspond-in 1G4F for 10 min at 377C. The cells are then stained ing to amino acid residues 564–579 of filamin are eval-with rhodamine phalloidin as described in the previous uated in permeabilized bovine pulmonary artery ECsection, or with Coomasie Blue stain. Micrographs of monolayers. CaM peptide, as well as a commercialat least five different fields for each time point are ana- CaM-PKII substrate, are readily phosphorylated onlyzed for gap formation. Briefly, intercellular gaps are their respective serine residues by CaM-PKII in an intraced onto paper, and scanned at 72 dots per inch (dpi) vitro CaM-PKII assay (data not shown). Incubation ofresolution with 256 gray-scale levels using a flat-bed permeabilized monolayers with 100 mM CaM peptidescanner interfaced to an Apple Macintosh Power PC or the control peptide for 20 min in cytoskeleton stabili-microcomputer. The number of gaps in each field and zation buffer does not alter filamin subcellular distribu-the average gap area are quantified using NIH image. tion (data not shown); however, preincubation of per-

meabilized cells with 100 mM CaM peptide blocksvon Willebrand Factor assay filamin translocation induced by Ca2/, ATP, and cal-

modulin (Fig. 1). The control peptide does not affectThe concentration of von Willebrand Factor (vWF) in translocation when Ca2/, ATP, and calmodulin arethe supernatant of cultured primary EC is measured added.using an ELISA kit (Asserachrom, American Biopro-ducts, Parsippany, NJ). Primary cultured ECs are Myristoylated CaM peptide preventsgrown to confluence in 6-well culture plates, and bradykinin-induced filamin translocation andwashed twice with Krebs Ringer bicarbonate buffer F-actin rearrangement(KRBB), containing 120 mM NaCl, 4.75 mM KCl, 1.2

Exposing confluent bovine pulmonary artery ECs tomM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM10 nM bradykinin results in filamin translocation fromNaHCO3, 5 mM glucose, 25 mM Hepes, PH 7.4, supple-the cell periphery to the cytosol fraction (Fig. 2A). Inmented with 2 mM L-Glutamine, 100 units/ml pen/postconfluent EC, filamin is mainly found in the mem-strep, 2% BSA. The cells are then incubated with KRBBbrane and cytoskeleton fractions, with less in the cyto-alone or with indicated chemicals diluted in KRBB forsol fraction. In response to 10 nM bradykinin treat-the indicated period of time at 377C. The supernatantsment, filamin translocates from the cell periphery intoare collected and assayed for vWF concentration by thethe cytosol fraction with filamin levels in the nuclearmanufacturer’s instructions.and cytoskeleton fractions remaining fairly constant.Filamin translocation occurs within as little as 1 min ofWound-healing assaysbradykinin treatment, and remains at maximal levelsthrough 2 min. Filamin starts to return to the mem-ECs are grown to confluence in 24-well culture plates

and the monolayers are scraped with a 1000 ul pipet brane fraction after 5 min bradykinin treatment. Thereversible nature of filamin translocation, as well astip. The width of the wounds generated in the mono-

layer is about 4–5 mm. The cells are incubated in me- the inability of a cell permeable calpain inhibitor (cal-peptin) to block translocation indicates that filamin re-dia alone or in media containing the indicated chemi-

cals. The migration distance of EC from the wound edge distribution is not due to proteolysis (Wang et al.,1996).is examined and quantified under the microscope every

24 h for up to 96 h. An N-terminal myristoylated derivative of CaM pep-



Fig. 1. Inhibition of filamin translocation by a synthetic peptide cor- ment. Lanes 3, 4: Permeabilized monolayer exposed to cytoskeletonresponding to one of filamin’s C-terminal CaM-PKII phosphorylation stabilization buffer with 10mM Ca2/, 2mM ATP, 5ug/ml calmodulinsites (CaM peptide). Permeabilized EC are preincubated with 100mM for 5 min. Lanes 5, 6: Permeabilized cells pretreated with the controlCaM peptide, the control peptide, or cytoskeleton stabilization buffer peptide. Lanes 7, 8: Permeabilized cells pretreated with CaM peptide.for 20 min, followed by incubation in buffer with or without 10mM Lanes 1, 3, 5, 7: Cytosol fractions; lanes 2, 4, 6, 8: Membrane fractions.Ca2/, 2mM ATP, 5ug/ml calmodulin for 30 min. The cytosol and mem- B: Inhibition of filamin translocation by CaM peptide pretreatment.brane fractions are isolated by DDF. A: Electroblotted cytosol and Open bars, cytosol fraction; filled bars, membrane fraction. Error barsmembrane fractions from CaM peptide and control peptide treated are means { standard deviation (n Å 3).cells. Lanes 1, 2: Permeabilized control cells with no further treat-

tide is synthesized to facilitate incorporation into live with 10 mM myristoylated CaM peptide for 30 minsignificantly attenuates bradykinin-induced filamincells. This peptide, like the nonmyristoylated CaM pep-

tide, is readily phosphorylated by CaM PKII in an in translocation in live EC monolayers (Figure 2B),whereas 10 mM nonmyristoylated CaM peptide pre-vitro CaM PKII assay (data not shown). Pretreatment


WANG ET AL.176

treatment has no effect on bradykinin-induced filamintranslocation (Fig. 2C).

Filamin translocation is accompanied by F-actin re-arrangement. In postconfluent cultured EC, F-actin isorganized predominantly into dense peripheral bandsat the cell periphery (Fig. 3A). Exposing EC to 10 nMbradykinin for as little as 1 min leads to disruption ofthe actin dense peripheral band and aggregation of ac-tin in the cytoplasm (Fig. 3B), with a few gaps observedbetween the cells. This actin rearrangement persiststhrough 10 min bradykinin treatment (Fig. 3C). With10 mM myristoylated CaM peptide pretreatment for 30min, bradykinin-induced actin rearrangement is sub-stantially attenuated. The disruption of the actin denseperipheral band does not occur until after 10 min bra-dykinin treatment (Fig. 3D–3F).

Myristoylated CaM peptide attenuatesbradykinin-induced gap formation

Bradykinin-induced EC cytoskeletal protein changesare accompanied by the formation of intercellular gaps(Fig. 4A). Exposing postconfluent EC to 10 nM bradyki-nin for as little as 1 min leads to increases in gap num-ber as well as average gap area. Maximal gap formationis observed after 10 min bradykinin treatment. Subse-quently, gap number and average gap area return grad-ually to baseline levels. A similar time course of gapformation is observed with 1 mM des-Arg9-bradykinin-treated cells (data not shown). Pretreatment with 10mM myristoylated CaM peptide for 30 min attenuatesbradykinin-induced gap formation (Fig. 4B). Comparedto bradykinin-stimulated controls, increases in averagegap area are substantially inhibited, and increases ingap number are not observed until after 5 min bradyki-nin treatment. Gap number after 10 min bradykinintreatment is reduced by 58% in the presence of CaMpeptide.

Myristoylated CaM peptide reduces migrationof EC in a wound healing model

EC cultures are scraped once with a 1000 ul pipettip that produces a 4–5 mm-wide furrow across themonolayer. The re-endothelialization process is moni-tored using an inverted microscope. ECs begin to mi-grate from wound edges to the denuded region of theculture plastic approximately 30 min after injury. Overa 96-hr observation period, EC are found to migrate ata roughly constant rate of 800 um/24 hr. Incubation ofmonolayers in 5 mM KN-62, a pharmacological CaM-PKII inhibitor, or 10 mM CaM peptide, substantiallyreduces EC migration rate (Fig. 5). A statistically sig-nificant inhibition in migration rate in injured mono-layers exposed to CaM peptide or KN-62 when com-pared to injured monolayers not exposed to the agents

Fig. 2. Myristoylated, but not nonmyristoylated, CaM peptide pre-vents bradykinin-induced filamin translocation. Cells are preincu-bated with culture media, 10 mM myristoylated or nonmyristoylatedCaM peptide for 30 min, followed by treatment with 10 nM bradykininfor the indicated times. The cytosolic and membrane fractions areisolated by DDF. Filamin in each fraction is quantified by densitome-try. A: Bradykinin-stimulated control. B: Pretreatment with myristoy-lated CaM peptide. C: Pretreatment with nonmyristoylated CaM pep-tide. Data are presented as the percentage of filamin in the cytosol andmembrane fraction. Open bars, cytosol fraction; filled bars, membranefraction. Data are expressed as means { standard deviation (n Å 3).



Fig. 3. Myristoylated CaM peptide prevents bradykinin-induced ac- B,C: Cells treated with bradykinin alone for 1 min and 10 min. D:tin rearrangement. F-actin is detected by rhodamine phalloidin stain- Cells treated with the myristoylated CaM peptide for 30 min. E,F:ing. Cells are incubated in media alone or with 10 mM myristoylated Cells treated with the peptide for 30 min, followed by bradykininCaM peptide for 30 min, followed by stimulation with 10 nM bradyki- treatment for 1 min and 10 min. Scale bar: 10mM.nin for the indicated times. A: Control cells without any treatment.

is observed after 48 h (P Å 0.0011). Dose response ex- inhibits EC migration by 35–50% while KN-62 inhibitsEC migration by 30–40% during this time period.periments using myristoylated CaM peptide indicate

that the peptide is effective over a concentration rangeMyristoylated CaM peptide preventsof 0.1–100 mM. The percentage inhibition observed for

bradykinin-induced eNOS translocation10 mM myristoylated CaM peptide and 5 mM KN-62 arefairly constant from 48 to 96 hr after their application Bradykinin is known to stimulate eNOS activity and

modulate its subcellular distribution (Michel et al.,to injured monolayers. The myristoylated CaM peptide


WANG ET AL.178

Fig. 4. Myristoylated CaM peptide attenuates bradykinin-induced lated CaM peptide. Circles, gap number/mm2; squares, average gapgap formation. Cells are incubated with media alone or 10 mM peptide area (mm2). Data are expressed as means { standard deviation of atfor 30 min, followed by 10 nM bradykinin treatment for the indicated least five fields from one experiment, which is representative of attimes. Gap formation is quantified as stated in materials and methods. least two experiments.A: Bradykinin-stimulated control. B: Pretreatment with myristoy

1993). Exposing postconfluent EC to 10 nM bradykinin eNOS starts to return to the membrane fraction after 5min bradykinin treatment and full recovery is observedresults in rapid subcellular translocation of eNOS from

the cell membrane fraction to the nuclear fraction (Fig. within 10–20 min. Minimal amounts of eNOS are re-covered in the cytosol and cytoskeleton fractions before6A,B). Eighty percent of total cellular eNOS is recov-

ered in the membrane fraction in untreated cells, with or after bradykinin treatment. Pretreatment of ECwith 10 mM myristoylated CaM peptide does not changethe other 20% recovered in the nuclear fraction. In re-

sponse to 10 nM bradykinin, eNOS translocates from the basal subcellular distribution of eNOS; however,it prevents bradykinin-induced eNOS translocationthe membrane fraction to the nuclear fraction within

1–2 min, with 43% recovered in the nuclear fraction. (Fig. 6A,C).



strate for CaM PKII. Utilizing synthetic peptides tomodulate intracellular signal transduction pathwayshas been reported in a number of studies. Camstatins,peptide antagonists of calmodulin, bind to calmodulinin a Ca2/-independent manner and inhibit calmodulin-dependent enzymes such as neuronal nitric oxide syn-thase (nNOS) (Slemmon et al., 1996). Not only doesCaM peptide inhibit filamin translocation, but it alsoreduces bradykinin-induced F-actin rearrangement,possibly by inhibiting the effects of CaM PKII on vari-ous actin binding proteins including filamin.

More importantly, bradykinin-induced EC gap for-mation is substantially attenuated by CaM peptide. In-creased interendothelial cell junctional diameters arethought to result by a combination of EC retractionand contraction (Garcia and Schaphorst, 1995). EC aretethered to each other and the underlying substratethrough adhesion molecules, which are linked to theF-actin cytoskeleton. Changes in the tethering forcesresult in retraction of EC leading to intercellular gapFig. 5. Myristoylated CaM peptide as well as a CaM-PKII inhibitor,

KN-62, reduce migration of endothelial cells in a wound-healing formation. In addition, actin stress fiber interactionsmodel. Cell monolayers are wounded with pipet tips and are incubated with myosin, which are in part regulated by the activa-with culture media, 5 mM KN-62 or 10 mM myristoylated CaM peptide tion of myosin light chain kinase (MLCK), generatefor the indicated times. Cell migration distance is quantified from

contractile forces that mediate EC contraction, and pos-micrographs. Open circles, control cells; filled circles, cells treatedwith myristoylated filamin CaM peptide; filled squares, cells treated sibly gap widening. Two actin/myosin-based syntheticwith 5 mM KN-62. Data are presented as means { standard deviation peptides, SM-1 and myosin peptide, prevent EC con-(n ¢ 3). traction as well as changes in cytoskeleton and cell

morphology (Wysolmerski and Lagunoff, 1991; Sims etal., 1992), which illustrates the importance of acto–

Myristoylated CaM peptide has no effect on myosin based contraction in EC motility. SM-1 is abradykinin-induced von Willebrand factor pseudosubstrate peptide of MLCK, and inhibits MLCK

(vWF) secretion phosphorylation of myosin. As a result, SM-1 inhibitsEC contraction and F-actin distribution induced by theThough myristoylated CaM peptide is effective in in-

hibiting EC motility, it does not appear to affect brady- addition of exogenous Ca2/ and ATP in permeabilizedEC monolayers. SM-1 also binds Ca2/-calmodulin andkinin-induced vWF secretion. Constitutively released

by EC under normal conditions, vWF secretion can also it is conceivable that it inhibits EC contraction byblocking Ca2/-calmodulin activation of MLCK ratherbe stimulated in a Ca2/-dependent manner by a num-

ber of vasoactive agonists, including bradykinin. In re- than blocking the active site of MLCK. On the otherhand, myosin peptide competes for the binding of myo-sponse to 10 nM bradykinin treatment for 1 h, vWF

secretion increases approximately 80% over control lev- sin to F-actin and inhibits cell morphological andcytoskeletal changes. In an analogous manner to theels. Pretreatment with 10 mM myristoylated CaM pep-

tide does not inhibit this bradykinin-induced vWF se- myosin-based peptides, CaM peptide, which can bephosphorylated by CaM-PKII, prevents cytoskeletal re-cretion (Fig. 7).arrangement and EC gap formation. In addition, CaM

DISCUSSION peptide, as well as the CaM PKII inhibitor, KN-62,reduces EC migration rate by up to 40%. This is consis-This study demonstrates that, in permeabilized bo-

vine pulmonary artery EC, a synthetic peptide that tent with the notion that F-actin gelation and solationat the leading lamellapodia are essential for cell motil-corresponds to one of filamin’s C-terminal CaM-PKII

phosphorylation sites (CaM peptide), prevents filamin ity (Stossel, 1994).To demonstrate further that CaM peptide inhibitstranslocation from the membrane fraction to the cytosol

fraction in a manner dependent upon exogenous Ca2/, calcium-calmodulin dependent signaling pathways,eNOS is studied. eNOS is a typical calmodulin-depen-ATP, and calmodulin. The omission of any of the three

cofactors results in minimal filamin translocation. Pre- dent enzyme that is associated with the cell mem-brane. Bradykinin induced eNOS activation is rapid,treatment with 100 mM CaM peptide, but not the con-

trol peptide, prevents filamin translocation. These re- transient, and tightly coupled to intracellular Ca2/

increases (Buckley et al., 1995). eNOS may also besults confirm our previous observations that activationof CaM PKII plays important regulatory roles in fi- regulated by other factors, since eNOS phosphoryla-

tion and subcellular translocation from the cell mem-lamin subcellular localization (Wang et al., 1996). Inaddition, the myristoylated CaM peptide prevents bra- brane to the cytosol have been observed in EC stimu-

lated with Ca2/-mobilizing agonists (Michel et al.,dykinin-induced filamin translocation in intact ECs.The myristoylated CaM peptide has no effect on brady- 1993). We observe that, upon stimulation with brady-

kinin, eNOS translocates from the membrane frac-kinin-induced intracellular Ca2/ increases, and in vitrophosphorylation assays demonstrate that CaM peptide tion to the nuclear fraction within a minute. CaM

peptide pretreatment prevents eNOS translocation.is readily phosphorylated by CaM PKII (data notshown). Thus, CaM peptide may interfere with the eNOS translocation from the membrane to the cy-

toskeleton in bradykinin-stimulated cultured bovineCaM PKII pathway by acting as a competitive sub-


WANG ET AL.180

Fig. 6. Myristoylated CaM peptide prevents bradykinin-induced cytosol; lane 2, membrane; lane 3, nuclear fraction). The blot is repre-eNOS subcellular translocation. Cells are preincubated in media sentative of at least three independent experiments. B: Bradykinin-alone, or with 10 mM myristoylated CaM peptide for 30 min, followed induced eNOS translocation. C: Modulation of bradykinin-inducedby 10 nM bradykinin treatment for the indicated times. The cytosolic, eNOS translocation by myristoylated CaM peptide. Hatched bars, cy-membrane, and nuclear fractions are collected by DDF. eNOS in tosol; filled bars, membrane; open bars, nuclear fraction. The data areeach fraction is quantified by densitometry. A: Immunoblots of eNOS expressed as means { standard deviation from three independentin subcellular fractions with or without peptide pretreatment (lane 1, experiments.

aortic EC has recently been reported (Venema et al., cance of eNOS translocation remains to be clarified,this study does provide evidence that CaM peptide1996). However, in that study, EC are only fraction-

ated into triton-soluble (membrane) and triton-insol- modulates EC responses, possibly by interfering withcalcium-calmodulin pathways and/or by maintaininguble (cytoskeleton) fractions instead of the four frac-

tions used in our study. It is unclear whether activa- the integrity of the cortical F-actin network.vWF is either constitutively secreted by EC or storedtion of eNOS by vasoactive agonists or other stimuli

such as mechanical stretch requires participation of along with P-selectin in the Weibel-Palade bodies(WPB). Exocytosis of WPB in inflammation plays anfilamin and F-actin. While the physiological signifi-



are complex and interrelated. Besides activation of cal-cium/calmodulin-dependent protein kinases, bradyki-nin also activates protein kinase C (PKC) (Murray etal., 1991) and phospholipase D (PLD) (Natarajan andGarcia, 1993) in cultured endothelial cells. PKC activa-tion depends upon intracellular Ca2/ increases andgeneration of diacylglycerol (DAG). Inhibition of PKCattenuates bradykinin-induced increases in vasoper-meability (Murray et al., 1991). PKC is known to phos-phorylate several cytoskeletal proteins, including vi-mentin and caldesmon (Stasek et al., 1992). PLD acti-vation by bradykinin involves PKC activation and isdependent upon increases in intracellular Ca2/ (Nata-rajan and Garcia, 1993). Although the exact functionsof PLD in modulating EC paracellular permeability areunclear, PLD activation may lead to increases in ECpermeability by producing DAG, which, in turn, acti-vates PKC. Pretreatment with CaM peptide does notalter bradykinin-induced intracellular Ca2/ increases(data not shown); thus, other signal transduction path-ways that are calmodulin-independent may still be ac-Fig. 7. Myristoylated CaM peptide does not affect bradykinin-in-

duced vWF secretion. Primary culture of EC monolayers are preincu- tivated. Thus, it is not surprising that CaM peptidebated in KRBB buffer alone or with 10 mM myristoylated CaM peptide only partially inhibits the endothelial cell inflamma-diluted in buffer for 1 hr, followed by incubation in buffer alone or 10 tory response.nM bradykinin prepared in buffer for 1 hr. Data are expressed as

In conclusion, we report a filamin-based syntheticpercentage over the control and are presented as means { standarddeviation (n Å 5). peptide that exhibits several anti-inflammatory proper-

ties. This peptide may act as a CAM-PKII substratein activated EC, and thus inhibit various calmodulin-dependent enzymes. As a consequence, CaM peptideimportant role in mediating leukocyte rolling and

platelet adhesion to subendothelium, due to the surface inhibits filamin translocation, cytoskeletal rearrange-ments, EC gap formation, and eNOS translocation.expression of P-selectin and increased release of vWF,

respectively. vWF secretion in response to Ca2/ iono-ACKNOWLEDGMENTSphore, thrombin, and histamine has been demonstrated

(de Groot et al., 1984; Hamilton and Sims, 1987), and The authors thank Laurie Hastie, Nancy Chung-Ca2//calmodulin is necessary for thrombin-induced Welch, Mary F. Lopez, and Michael Lu for useful dis-vWF secretion (Birch et al., 1992). We observe an 80% cussions and careful reading of this manuscript. Thisincrease in vWF secretion in response to bradykinin project was supported by NIH grants HL-43875 andtreatment. However, CaM peptide does not inhibit this -48553, and GM-24891 and -35141.secretory response. This suggests that exocytosis of

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a novel anti-inflammatory peptide inhibits endothelial cell cytoskeletal rearrangement, nitric oxide...

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