Fluid shear stress-induced JNK activity leads to actin remodeling for cell alignment

Fluid Shear Stress-Induced JNK Activity Leads to ActinRemodeling for Cell Alignment

Meron Mengistu1, Hannah Brotzman1, Samir Ghadiali2,3, and Linda Lowe-Krentz1,3

1Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA18015.2Department of Mechanical Engineering and Mechanics, 19 Memorial Drive West, Bethlehem, PA18015.3Bioengineering Program, Bethlehem, PA 18015.

AbstractFluid shear stress (FSS) exerted on endothelial cell surfaces induces actin cytoskeleton remodelingthrough mechanotransduction. This study was designed to determine whether FSS activates Jun N-terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative tothe actin cytoskeleton in endothelial cells exposed to different FSS conditions, and to evaluate theeffects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higheractivity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSStreatments, JNK activity increased with increasing exposure time, peaking 30 minutes after flowonset with an 8-fold activity increase compared to cells in static culture. FSS-induced phospho-JNK co-localized with actin filaments at cell peripheries, as well as with stress fibers.Pharmacologically blocking JNK activity altered FSS-induced actin structure and distribution as aresponse to FSS. Our results indicate that FSS-induced actin remodeling occurs in three phases,and that JNK plays a role in at least one, suggesting that this kinase activity is involved inmechanotransduction from the apical surface to the actin cytoskeleton in endothelial cells.

KeywordsJNK; Fluid shear stress; Actin remodeling

IntroductionHemodynamic shear stresses have been shown to play a significant role in atheroscleroticplaque localization. Specifically, regions of arteries exposed to relatively lower FSSconditions (less than 4 dyn/cm2) are prone to these plaque formations, while regionsexposed to higher FSS conditions (greater than 15 dyn/cm2) are atheroprotected(Cunningham and Gothlieb, 2005). Endothelial cells (ECs) that form the endothelial liningof blood vessels are constantly exposed to FSS from the blood, which dictates their structureand modulates function. The FSS is primarily steady in some vessels while it is moreoscillatory in others and is even turbulent at specific locations such as bifurcations. WhenECs are exposed to fluid mechanical forces from the blood, they adapt to changes in FSSthrough rapid displacement and deformation of cytoskeletal filaments, where all threecytoskeletal filaments, the actin filaments, microtubules, and intermediate filaments, align in

Corresponding author: Linda J. Lowe-Krentz, Department of Biological Sciences, Lehigh University, 111 Research Drive, B217,Bethlehem, PA 18015, Phone: 610-758-5084, Fax: 610-758-4004, [email protected].

NIH Public AccessAuthor ManuscriptJ Cell Physiol. Author manuscript; available in PMC 2012 March 1.

Published in final edited form as:J Cell Physiol. 2011 January ; 226(1): 110–121. doi:10.1002/jcp.22311.

NIH

-PA Author Manuscript

NIH


NIH


the direction of flow (Franke et al., 1984; Galbraith et al., 1998; Helmke et al., 2001; Malekand Izumo, 1996). These cytoskeletal reorganizations drive changes in EC morphology,where cells elongate and also align their longer axes in the direction of flow, an adaptationthat allows them to decrease spatial stress gradients they experience from the blood flow(Hu et al., 2003).

Both in vivo and in vitro, ECs respond differentially to different FSS magnitudes andexposure times. When exposed to low FSS conditions, ECs adopt a polygonal cell shape andtheir actin filaments are predominately found at cell peripheries with very few and thin actinstress fibers. But in response to higher FSS conditions, ECs elongate and have increasedactin stress fiber formations, which start out being randomly oriented throughout theintracellular space, and then reorganize and align in the direction of flow with increasedexposure times (Birukov et al., 2002; Dewey et al., 1981; Mott and Helmke, 2007). Therealignment of the cytoskeleton is accompanied by the realignment of focal adhesions andeven the underlying fibronectin fibers (Mott and Helmke, 2007).

The fundamental problem addressed in this study is the mechanism by which FSS, which isexerted on EC surfaces, is transmitted to the cytoskeleton to induce actin reorganization,leading to morphological adaptations. We examined mechanotransduction events, which areintracellular biochemical signals that translate FSS, a mechanical stimulus, into actinremodeling, a biological response. The stress activated protein kinase JNK, which belongs tothe family of mitogen-activated protein kinases (MAPKs), is transiently activated by FSS,and has been shown to be involved in remodeling of the actin cytoskeleton (Azuma et al.,2001; Chien, 2007; Yoshizumi et al., 2003).

JNK is an effector kinase found downstream from Rac and Cdc42, which are involved inactin reorganization (Garrington and Johnson, 1999; Hall, 1998; Ip and Davis, 1998). Gβ/γ,phosphatidylinositol-3-kinase-γ, Ras, Src and FAK protein kinases have been shown toactivate JNK and induce actin depolymerization leading to its remodeling (Azuma et al.,2001; Bagrodia et al., 1995; Hoefen and Berk, 2002; Li et al., 1997; Otto et al., 2000).Previously we identified active JNK associated with actin in wounded endothelial layers, butnot in static confluent endothelial layers (Hamel et al., 2006). An earlier study (Otto et al.,2000) identified a downstream target of JNK, p150-Spir, which has been shown to beinvolved in actin reorganization (Bi and Zigmond, 1999; Ramesh et al., 1999). Morerecently it has become clear that JNK activity is required for actin realignment in endothelialmigration (Rush et al., 2007). However, much remains to be determined about the specificrole(s) of JNK in actin rearrangements and how JNK is involved in response to FSS.

In this study, we examined the role of JNK in signaling events that lead to FSS-inducedactin remodeling. We visualized JNK activity in ECs exposed to high and low FSSconditions to determine spatial distribution of JNK and its involvement in actin remodeling.We also blocked JNK activity using pharmacological methods, which altered actinreorganization as a response to the different FSS conditions.

Materials and MethodsCell Culture

Endothelial cells were isolated from bovine aortas (BAECs) as previously described(Dougherty and Lowe-Krentz, 1998), and cultured in minimal essential media (MEM)(Sigma, St, Louis, MO), supplemented with 10% heat inactivated, pre-tested fetal bovineserum (FBS), glutamine, penicillin-streptomycin antibiotics, sodium pyruvate, and MEMnon-essential amino acids, and incubated at 37°C in 5% CO2. BAECs were plated on 0.17-mm thick glass coverslips 30 mm d (Fisher Scientific, Pittsburg, PA) coated with 60 µg/mL

Mengistu et al.

J Cell Physiol. Author manuscript; available in PMC 2012 March 1.

NIH


NIH


NIH


bovine Collagen Type I (BD, San Jose, CA), placed in 6-well plates, and grown for 3–5 daysuntil they formed a confluent monolayer. ECs were then synchronized during a 48-hourincubation in shear media, (MEM containing HEPES to maintain pH and minimize bubbleformation, supplemented with 0.5% FBS). The coverslips were mounted onto a modified-POC mini parallel-plate flow chamber (Yalcin et al., 2007) for exposure to different FSSconditions. Typically, BAECs between 5 and 14 passages were used. For the cycloheximideexperiments, cells were exposed to 1.5 µg/mL cycloheximide for 1 hr prior to FSStreatment. For TNF-α treatment, TNF-α (R&D Systems, Minneapolis MN) was used at 2 ng/ml media for times noted in the text.

JNK Inhibitor TreatmentsJNK activity was inhibited using JNK Inhibitor I from Calbiochem (La Jolla, CA), aninhibitor which competitively binds the activation domain of phospho-JNK and preventsdocking and activation of JNK substrates, or using SP600125 (Calbiochem), a competitiveinhibitor for JNK (Bogoyevitch et al., 2004). ECs that were grown as above were incubatedwith inhibitors at 10 µM in shear media for 1 hour. For comparison, in some experimentscells were exposed to the inhibitor during flow rather than before flow. There were noapparent differences between the two methods of inhibitor treatment. Then the coverslipswere mounted onto the POC mini flow chamber for FSS exposure. Cells were harvested forwestern blots and analyzed as reported previously (Dougherty and Lowe-Krentz, 1998;Hamel et al., 2006). Phospho-MAPKAP-K2 and phospho-c-Jun primary antibodies wereobtained from Cell Signaling Technology (Danvers, MA).

Flow ExperimentsA POC mini chamber from Hemogenix (Colorado Springs, CO) was modified by adding agasket with a rectangular flow channel to create an adjustable-height parallel-plate flowchamber as previously described (Yalcin et al., 2007). For settings in which the width-to-height ratio was greater than 10, the magnitude of fluid shear stress was calculated according

to the equation, , where τw is the wall shear stress, μ is viscosity (0.007 dyn.s/cm2

@ 37°C), Q the flow speed, and W & H the width and height of the gasket, respectively. ECswere exposed to different magnitudes of FSS using the following W, H & Q combinations: 2dyn/cm2: W=1cm, H=0.03cm, & Q=2.57 mL/min; 4 dyn/cm2: W=0.5cm, H=0.03cm, &Q=2.57 mL/min, OR W=0.2cm, H=0.05cm, & Q=2.86mL/min; 10 dyn/cm2: W=0.5cm,H=0.03cm, & Q=6.43 mL/min, OR W=0.2 cm, H=0.03cm, & Q=2.57 mL/min; 15 dyn/cm2:W=0.2cm, H=0.03cm, & Q=3.86mL/min, OR W=1cm, H=0.01cm, & Q=2.14mL/min; 20dyn/cm2: W=1cm, H=0.01cm, & Q=2.86mL/min.

To eliminate responses caused by a static-to-flow transition, we carried out controlexperiments where we pre-exposed ECs to 10 dyn/cm2 FSS for 10 minutes (using W=0.5cm,H=0.03cm, & Q=6.43mL/min for 4 dyn/cm2 experiments or using W=0.2cm, H=0.03cm, &Q=2.86mL/min for 15 dyn/cm2 experiments) before experimental FSS conditions of 4 or 15dyn/cm2. A constant flow of shear media was driven into the POC mini parallel-plate flowchamber using the REGLO Digital continuous flow pump from ISMATEC International(Glattbrugg, Switzerland). The flow experiments were carried out at 37°C.

Immunofluorescence ExperimentsAfter inhibitor (when applicable) and FSS treatments, ECs on coverslips were washed withPBS, fixed and permeabilized with ice-cold methanol for 5 min and washed again with PBS.The coverslips were incubated with primary antibodies against phospho-JNK and actin,either simultaneously or separately, overnight at 4°C. Primary antibodies against phospho-JNK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and actin antibodies

Mengistu et al.


NIH


NIH


NIH


from Sigma. Cells were then incubated with secondary antibodies conjugated to FITC andTRITC from Jackson ImmunoResearch (West Grove, PA) for 2 hours at 37°C. Both primaryand secondary antibodies were used at dilutions recommended by suppliers. Coverslips weremounted in mowoil (Calbiochem) to minimize photobleaching.

Actin was alternatively labeled with Phalloidin conjugated with Tetramethylrhodamine Bisothiocyanate (TRITC) or Fluorescein Isothiocyanate (FITC) from Sigma. In theseexperiments, EC samples were fixed by using 16% formaldehyde (Sigma) andpermeabilized with 0.2% Triton X-100 (Sigma).

Duolink™ Proximity Ligation AssayPhospho-JNK association with actin filaments was further characterized by using theDuolink™ Proximity Ligation Assay (PLA) obtained from Olink Bioscience (Uppsala,Sweden), (Jarvius et al., 2006; Soderberg et al., 2006). Coverslips exposed to FSS wereincubated with primary antibodies against phospho-JNK and actin as described above. ThenPLA probes, which are conjugated with oligonucleotides (oligos), were introduced torecognize the primary antibodies. A solution that promotes hybridization between the PLAoligos was then added, where a hybridization reaction only occurred if the primaryantibodies were in close proximity (40 nm), but not if they were far apart. This reaction wasfollowed by ligation of the oligos and a rolling-circle amplification reaction, where arepeated sequence product was made. This product was then detected using fluorescentlylabeled oligos, where a phospho-JNK – actin association appeared as red dots offluorescently labeled oligos under the microscope.

To observe where the phospho-JNK – actin associations occur relative to the actincytoskeleton, BAECs were incubated with FITC-conjugated phalloidin. After the detectionstep, coverslips were washed with the saline-sodium citrate wash series recommended by themanufacturer, and then incubated with FITC-conjugated phalloidin for 10 minutes at roomtemperature. Slides were mounted onto microscopic slides using MOWOIL mountingmedia, and observed using the Zeiss© Laser Scanning Microscope (LSM) 510 Meta.

Imaging and Image Analysis of JNK Activity LevelsPhospho-JNK and actin were fluorescently labeled to study FSS-induced changes in theiractivity and localization. These fluorescent labels were visualized using the Zeiss© LaserScanning Microscope (LSM) 510 Meta using a 63X oil-immersion lens at room temperature,and distribution of these proteins was tracked in 3D, where X, Y scans of EC monolayerswere taken at multiple regions on the coverslips, and 0.38-µm slices were taken through themonolayers in the Z-direction. The z-slices shown are those with significant actin fibers,typically near the basal surface. To minimize cross-talk between the FITC (Exmax: 495nm,Emmax: 520nm) and TRITC (Exmax: 557nm, Emmax: 576nm) fluorescence labels, the multi-tracking setting of the Zeiss LSM 510 Meta AIM software was used, which sequentiallyilluminated and detected one fluorophore at a time (Anderson et al., 2006).

FSS-induced changes in JNK activity levels were quantified using MetaMorph (MolecularDevices) or the LSM 510 Meta AIM (Zeiss) programs, where phospho-JNK intensity levelsin individual cells and their nuclei were used as indicators for activity levels. Intensity levelsfor all treatments in a given experiment were normalized to control conditions (no flow andno inhibitors), where the settings used to obtain control images were saved and used forimaging of successive inhibitor and FSS-treated samples. JNK activity was determined to beinside the nucleus by simultaneously obtaining the DIC image of BAECs. This DIC imagewas used to select nuclei as regions of interest (ROI), and intensity levels in the ROI wereused as indicators of nuclear activity levels.

Mengistu et al.


NIH


NIH


NIH


ResultsBovine aortic endothelial cells (BAECs) were grown to confluence on collagen-coated glasscoverslips, and exposed to different FSS conditions. The role of JNK MAPK in signalingevents that lead to FSS-induced actin remodeling was examined. The active form of JNK(phospho-JNK), which is dually phosphorylated at Threonine 183 and Tyrosine 185, wasfluorescently labeled to visualize FSS-induced changes in its intracellular distribution andassociation with the actin cytoskeleton. Changes in JNK activity levels in BAECs and theirnuclei were also quantified using intensity levels of the phospho-JNK fluorescence signalsas indicators of activity levels.

FSS-Induced Spatial and Temporal Distribution of Phospho-JNKFluid shear stress-induced changes in JNK activity levels and spatial distribution weretracked by fluorescently labeling active phosphorylated JNK in confluent BAECs exposed to2, 4, 10, 15, and 20 dyn/cm2 FSS conditions for 0, 5, 15, 30, and 60 minutes. The spatialdistribution of phospho-JNK was differentially regulated in BAECs treated with differentFSS conditions (Fig. 1A).

Under no flow conditions, JNK activity is detected in the nuclei of BAECs (as noted in theMethods, DIC imaging was employed to identify the nuclei as regions of interest (ROI)),while cytoplasmic levels were relatively low (Fig. 1A). The image settings used to acquirethese no-flow images were saved and reused to obtain images of FSS-treated cells in orderto quantify changes in the levels of phospho-JNK caused by these flow treatments.Cytoplasmic intensity levels (Fig. 1B) as well as specifically nuclear levels were quantified(Fig. 1C) to see if FSS-induced changes in JNK activity levels were occurring in the nucleusor the cytoplasm.

Phospho-JNK Activity Levels and Distribution in BAECs Exposed to 2, 4, and 10 dyn/cm2

FSSThe spatial and temporal activity and distribution of phospho-JNK in BAECs exposed to 2and 4 dyn/cm2 FSS conditions were similar to each other and to control cells not exposed toFSS, and are represented by the 4 dyn/cm2 treatment images (Fig. 1A, top row of images).BAECs exposed to 2 and 4 dyn/cm2 FSS did not undergo any significant changes incytosplasmic phospho-JNK levels (Fig. 1B, diagonal & horizontal lines) compared to cellsthat were not exposed to flow. Nuclear activity levels in these BAECs exposed to the lowerFSS conditions decreased somewhat between 5 and 15 minutes of flow exposure, and thenreturned to the no-flow levels by 30 minutes (Fig. 1C, diagonal & horizontal lines).

When exposed to the intermediate FSS of 10 dyn/cm2, BAECs did not undergo significantchanges in active JNK distribution similar to the lower FSS conditions (Fig. 1A, middle rowof images), and exhibited only a modest approximately 4-fold increase in cytoplasmic levelsafter 30 minutes of flow exposure (Fig. 1B, lt. grey). Nuclear JNK activity levels alsoremained similar to the no-flow conditions, except for approximately doubling after 5minutes of flow exposure (Fig. 1C, lt. grey).

Phospho-JNK Activity Levels and Distribution in BAECs Exposed to 15 and 20 dyn/cm2

FSSExposure to 15 and 20 dyn/cm2 FSS induced similar phospho-JNK behavior in BAECs, andresults are represented by images from the 15 dyn/cm2 treatments (Fig. 1A, bottom row ofimages). The higher FSS treatments induced a more pronounced response, with cytoplasmicactivity levels increasing by 5 to 6-fold at the 5- and 15-minute time points, and activitylevels peaking after 30 minutes exposure with an approximate 8-fold increase (Fig. 1B, dark

Mengistu et al.


NIH


NIH


NIH


grey & black). Most of this increase in activity occurred in the cytoplasm. JNK activity inthe nucleus approximately doubled by 5 minutes, and later decreased below the no-flowlevels by 60 minutes (Fig. 1C, dark grey & black). Under these higher FSS conditions, JNKactivity exhibited a fibrous-like pattern in the cytoplasm of BAECs, and a strongfluorescence signal was detected right outside the nucleus of many cells by the 15-minutetime point, and at cell peripheries at the 30-minute time point (Fig. 1A bottom row, arrowsat 15 and 30 min).

To examine whether there was a change in total JNK enzyme location as opposed tolocalized activation, identical experiments were carried out using an antibody against totalJNK 1 rather than the primary antibody against active JNK. There was no obvious change inthe distribution of full length JNK 1 in cells exposed to higher FSS (Fig. 1D). Therefore,dramatic rearrangement of the JNK and changes in JNK levels both appear less likely thanaltered activation of JNK already spatially distributed.

Similar results to those seen in Fig. 1 were found in BAECs that were pre-exposed to 10dyn/cm2 FSS for 10 minutes, then subjected to 4 or 15 dyn/cm2 FSS conditions for 0, 5, 10,15, 30, and 60 minutes (data not shown). Specifically, cells taken from no flow to 15 dyn/cm2 FSS for 5 min had the same JNK staining as cells exposed to 10 dyn/cm2 FSS for 10min followed by 5 min at 15 dyn/cm2. Therefore, the resulting changes in JNK activitylevels and localization were a response to the specific FSS treatments and were not shockresponses due to the static-to-flow transitions. As described in the Methods, differentchamber geometries yielding the shear stress rates noted were used in different trials.Changes in chamber geometry had no additional effect on the cell responses, and data fromdifferent geometries yielding the same FSS rates are included in the results.

Comparison with TNF-α Treatment-Induced Phospho-JNK DistributionActivation of JNK in endothelial cells is typically induced by inflammatory mediators.Endothelial cells were cultured under identical conditions, but exposed to TNF instead ofFSS. Activation of JNK by cell treatment with TNF-α resulted primarily in nuclearlocalization of activated JNK (Fig. 1E) rather than the cytoplasmic localization induced byhigh FSS.

Phospho-JNK Association with F-ActinPhospho-JNK (Fig.2A, green) and actin (Fig.2A, red) were fluorescently labeledsimultaneously, and their localizations relative to each other were visualized in BAECsexposed to 15 dyn/cm2 FSS for 0, 15 and 30 minutes. In the absence of FSS, active JNK ispredominantly in the nucleus and no co-localization with F-actin fibers can be seen (Fig.2A). Exposure to 15 dyn/cm2 FSS for at least 15 min induced a higher level of JNK activity,which exhibited a fibrous-like pattern in the cytoplasm of BAECs that co-localized withstress fibers (Fig. 2 Merge, orange-yellow). In addition to the phospho-JNK – actin fiber co-localization, a region of active JNK right outside the nucleus also co-localized with actinsignals in many cells (Fig. 2 Merge, orange-yellow). This region typically extended throughseveral z stacks. Phospho-JNK and actin were found predominately at cell peripheries inBAECs exposed to 30 minutes of 15 dyn/cm2 FSS, where they also co-localized (Fig. 2A,arrows are shown in the JNK, green, stained panel). Actin and phospho-JNK were alsopresent in the nuclei of BAECs. Similar co-localization and actin pattern changes wereobserved when the actin staining in these experiments was accomplished with phalloidinrather than anti-actin antibodies (data not shown). Because phalloidin staining of methanol-fixed cells is poor, while JNK staining in formaldehyde fixed cells was typically lessintense, the dual-antibody staining was employed for these co-localization studies. Relativeincreases in actin fluorescence signals were also an indication of the presence of more actin

Mengistu et al.


NIH


NIH


NIH


filaments in BAECs exposed to 30 minutes of 15 dyn/cm2 FSS because these experimentswere imaged using identical conditions and image settings between samples andexperiments.

To examine the question of protein synthesis, cells were treated with cycloheximide,subjected to 15 dyn/cm2 flow for 0, 15, or 30 minutes and stained for actin using anti-actinantibody (Fig. 2B). The cycloheximide-treated cells did exhibit some actin reorganization,but the small pool of actin staining near the nucleus was not observed after 15 min of FSSexposure, nor did there appear to be a significant increase in actin fibers over time ofexposure to flow. Rather, total stained actin actually appeared to decrease by 30 min. Theactin reorganization observed in the cycloheximide treated cells appeared in the form ofslightly more cortical actin than in the cells not exposed to flow.

Duolink™ Proximity Ligation Assay to Assess Phospho-JNK Association with ActinPhospho-JNK co-localized with stress fibers and actin networks at EC peripheries in theimmunofluorescence experiments. Although images where taken with a confocalmicroscope, which allowed us to visualize fluorescent signals that are occurring on the sameplane, we were still limited by a resolution of ~ 1 µm in the Z direction of the microscopeand the 0.38-µm slice size for image acquisition. The small flow chambers did not result insufficient cells for physical isolation of the actin/phospho-JNK complexes, a problemcompounded by the difficulty of separating the cells exposed to flow from the other cells onthe coverslips. Therefore, the PLA technique was employed to facilitate identification ofassociated proteins. For a better resolution of the association between phospho-JNK andactin, we carried out a proximity ligation assay (PLA), where a fluorescent signal wouldonly occur if a phospho-JNK and actin association occurred. This assay identifies proteinsthat are within 40 nm of each other.

A Duolink™ PLA was carried out in BAECs exposed to a 15 dyn/cm2 FSS for 0, 5, 15, 30and 60 minutes (Fig. 3) as described in the Materials and Methods section, where phospho-JNK – actin associations are visualized as dots of fluorescent oligos bound to amplifiedproduct. The DIC images of BAECs were used to trace their contours and the location oftheir nuclei (Fig. 3A, white lines). In some experiments these cells were simultaneouslylabeled with FITC-phalloidin (Fig. 3B, single cells stained green) in order to observe wherethe FSS-induced phospho-JNK – actin associations (red dots) occur relative to the actincytoskeleton.

Under static conditions, very few phospho-JNK – actin associations were seen in BAECs.This result is consistent with our immunofluorescence data and provides a negative control(no protein association) where faint background staining, without larger red dots, isobserved. Interestingly, despite apparent nuclear presence of both anti-actin and anti-phospho-JNK staining in Figure 2, there was no PLA signal in the nuclei in Figure 3indicating no close association. When exposed to 15 dyn/cm2 FSS for 5 minutes,associations between the active phosphorylated JNK and actin occurred in the cytoplasmwhere PLA signals co-localized with actin fibers (Fig. 3B, yellow dots and dots partly redand partly yellow), while PLA fluorescent signals were significant at cell peripheries inBAECs subjected to flow for 30 minutes (e.g. arrows in A and B). Phospho-JNK – Actinassociation near the nucleus after 15 minutes of exposure to 15 dyn/cm2 FSS condition wasalso seen using the PLA, where some fluorescent signals for PLA (Fig. 3B,) can be seen.The phalloidin staining (Fig. 3B) does not appear to co-localize with PLA near the nucleus,nor is there any evidence of phalloidin staining in the nuclei where both anti-actin and anti-active JNK antibodies stain as in Fig. 2. Thus, the PLA staining only identifies where theactin and active JNK are very close and probably associated, and not all places where themerged yellow color in standard dual double immunofluorescent experiments suggests they

Mengistu et al.


NIH


NIH


NIH


are in the same location. Exact co-localization of the PLA staining and the actin filaments isyellow, but because the red color is product produced in co-localized regions, theamplification product could extend beyond the stained actin filaments. In addition, it oftenappears that actin fibers actually associated with the PLA detection system stain less wellwith phalloidin than adjacent fibers, probably due to steric hindrance.

Effect of JNK Activity Inhibition on FSS-Induced Actin RemodelingIn response to FSS, phospho-JNK associated with stress fibers and actin networks at ECperipheries in a time-dependent manner. We examined whether inhibitors of JNK activitywould alter the JNK association with actin fibers. Neither JNK inhibitor I, a peptideinhibitor which competitively binds the active site of phospho-JNK and prevents bindingand subsequent activation of its substrates, or SP600125, which binds in the ATP bindingsite of phospho-JNK and inhibits its activity, altered the low level of resting JNK activitythat is associated with the nucleus (Fig. 4, panel A). When inhibitor-treated cells weresubjected to flow at 15 dyn/cm2 there was no change in staining for active JNK (Fig. 4,panel B) implying that either JNK must be activated to associate with the fibers or JNK mustbe activated after fiber association in response to the flow treatment. There did not appear tobe any increase in the levels of phospho-JNK after FSS exposure with either inhibitortreatment (Fig. 4 panel B). These results verify that flow-induced active JNK did notincrease in the presence of the inhibitors.

JNK Inhibitor I and SP600125 Inhibition of JNK Activity and SpecificityTo confirm that JNK activity is inhibited by JNK inhibitor I and SP600125 treatments, itwas necessary to identify a JNK substrate to monitor. Because the JNK substrate(s) in flowtreated cells is unknown, we altered the JNK activation conditions so that we could examinethe known JNK substrate, c-Jun. Cells grown as for FSS experiments were conditioned withflow media, treated with inhibitor for one hour, and then changed to fresh flow mediacontaining TNF-α or hydrogen peroxide. Phospho-JNK and JNK activity (based onphosphorylation of the substrate c-Jun) were determined over a one hour TNF-α or hydrogenperoxide treatment. Both treatments resulted in a short time course of activation with lowlevels of JNK activity in control cells (e.g. see Fig. 1E). There was no increase in phospho-Jun during TNF-α exposure in cells pre-treated with SP600125 (Fig. 4C). However,phospho-c-Jun did increase in cells treated with JNK inhibitor I similar to the increase incells not treated with inhibitor, indicating that the JNK inhibitor I did not block c-Jun-phosphorylation activity of JNK in the nucleus under these conditions. Similar results wereobtained in cells activated with hydrogen peroxide (not shown).

To examine whether these JNK inhibitors were specific to the JNK pathway, we evaluatedthe phosphorylation of MAPKAP-K2, a kinase substrate of the MAPK p38 with effects onthe cytoskeleton. Figure 4, panel D illustrates the phosphorylation of MAPKAP-K2 inresponse to TNF-α treatment of endothelial cells conditioned with flow media. In thepresence of either JNK inhibitor I or SP600125, there is no difference in phosphorylation ofMAPKAP-K2 compared to cells not treated with a JNK inhibitor, although in someexperiments as shown in Fig. 4D, at the longest treatment point with SP600125 there is adecrease in MAPKAP-K2. In cells treated with the specific p38 MAPK pathway inhibitor,SB203580, there is no phosphorylation of the p38 substrate MAPKAP-K2 (Figure 4D).

Effect of JNK Inhibitor I and SP600125 on FSS-Induced Actin DistributionWithout any inhibitor treatments, FSS induced actin remodeling to form a cortical ring after30 minutes of exposure to 15 dyn/cm2 FSS (Fig. 5A). These actin patterns are similar to theanti-actin antibody staining of filaments illustrated in Figure 2. Phalloidin staining is shownto emphasize the stained stress fibers. This FSS-induced organization of actin at cell

Mengistu et al.


NIH


NIH


NIH


peripheries induced morphological changes in BAECs where they first became rounded inshape. With extended exposure to this high FSS treatment, BAECs began to align in thedirection of flow as the thick cortical actin reorganized into stress fibers which also alignedin the direction of flow (Fig. 5A). Both JNK inhibitor I & SP600125 induced changes in thisprocess of actin adaptation to flow.

BAECs were treated with 10-µM SP600125 for one hour, exposed to 15 dyn/cm2 FSS for 0,30, 60, and 120 minutes, and changes in FSS-induced actin remodeling were observed usingfluorescence labeling of the actin cytoskeleton with TRITC-conjugated phalloidin.SP600125, which inhibited ATP-dependent JNK activity, caused disruptions in actinstructure (Fig. 5B). Even without exposure to FSS, this inhibitor treatment slightly alteredactin structure at cell peripheries, where cortical actin at cell-cell junction sites was affectedthe most (Fig. 5B). After a 30-minute exposure to 15 dyn/cm2 FSS, the thick cortical actinseen in inhibitor-free BAECs occurred only to a limited extent in SP600125-treated cells,and cells appeared to lose connections to their neighbors as treatment progressed. Increasedexposure to FSS further weakened cell-cell associations, and increasingly fewer actinfilaments could be seen in BAECs. By 120 minutes, the lack of actin filaments and cell-cellcontacts compromised the mechanical integrity of BAECs, making them susceptible tobeing washed off by the flow treatment. Inclusion of SP600125 in the flow medium ratherthan pre-treating the cells resulted in similar defects in actin remodeling (data not shown).

Similarly, BAECs were treated with 10-µM JNK inhibitor I for one hour, exposed to 15 dyn/cm2 FSS for 0, 30, 60, and 120 minutes, and images were taken to examine if FSS-inducedactin remodeling is regulated through a kinase-substrate relationship (Fig. 5C). JNKinhibitor I induced slight increase in actin fiber formation in the absence of flow comparedto untreated BAECs, where more filaments can be seen in the treated cells. This inhibitor didnot prevent cortical actin formation after 30 minutes of 15 dyn/cm2 FSS exposure, but rathercells treated with JNK inhibitor I exhibited denser cortical actin staining. However, thecortical actin persisted with increased exposure to FSS of up to 120 minutes, instead ofremodeling into stress fibers.

DiscussionThe spatial and temporal actin distribution in BAECs exhibited very high sensitivity to thedifferent flow treatments in our studies. FSS conditions that were 4 dyn/cm2 or lower did notinduce significant change in JNK activity or actin distribution in BAECs exposed to over 60minutes of flow. At the higher FSS conditions (15 & 20 dyn/cm2), most of the change inJNK activity occurred in the cytoplasm of these cells. Unlike results with chemical stressors,there was little increase in nuclear JNK activity induced by FSS. Altered nuclear levels ofphospho-JNK, the presence of which could have resulted in transcription changes due toJNK activation of transcription factors, were limited, but a role for such small changescannot be dismissed. FSS has been shown to not only protect vessels from atherosclerosis,but recently it has also been documented that FSS protects endothelial cells from TNF-αinduced increases in JNK activity (Li et al., 2008).

Previous work in our lab, (Hamel et al., 2006), identified associations of activephosphorylated JNK with stress fibers in BAECs that were in sub-confluent cultures, as wellas with cortical actin during cell spreading. Since that report, Yang et al. (2007) alsoreported JNK association with stress fibers. The authors suggest that JNK activation andassociation with actin depends on α–actinin, and that phospho-JNK appeared to activatetranscription (Yang et al., 2007). Therefore, we hypothesized that the fibrous phospho-JNKpattern was due to the association of this kinase with the actin cytoskeleton. Here, we showin BAECs exposed to high FSS conditions, the co-localization between phospho-JNK and

Mengistu et al.


NIH


NIH


NIH


actin fluorescent signals (Fig. 2), which represents associations between the signalingprotein and the cytoskeleton. This co-localization occurred in the form of (i) stress fibers,(ii) a pool outside the nucleus, and (iii) cortical actin. This association was confirmed inflow treated cells using the PLA experiments, indicating that phospho-JNK and actin wereoften within 40 nm of each other. There was essentially no co-localization of these proteinsin confluent cells not subjected to flow, providing a negative control. The co-localization ofphospho-JNK with the actin pool outside the nucleus first, and later with cortical actin at cellperipheries could imply a role for JNK in the transport of actin to form cortical actin. Furtherexperiments are needed to examine the reason for phospho-JNK association with this actinpool.

A recent proteomics report indicates that mice deficient in JNK/stress-activated proteinkinase-associated protein 1 (JSAP1) are not only deficient in JNK activation, but their cellscontain altered levels of four different β–actin forms as well as decreased levels of theHsp27 actin chaperone protein (Ha et al., 2008). The phospho-JNK/actin association weidentify (Figs. 1, 2, and 3) might be related to one, or more, of these actin pools as suggested(Ha et al., 2008). Consistent with our studies are reports of a “tent-like” actin structure inendothelial cells exposed to equi-biaxial stretching which induced cytoskeletal remodeling(Wang et al., 2001), and an actin collection near the nucleus in cells treated by parallel plateFSS to induce remodeling (Noria et al., 2004).

We also found that both actin and phospho-JNK were present in the nucleus of BAECs.Evidence suggests some of this actin could be polymeric despite the lack of phalloidinbinding (Pederson, 2008; Vartiainen, 2008). An intriguing suggestion is that some nuclearactin might be involved in mechanical coupling through the cytoskeleton to the extracellularmatrix (Wang et al., 2009). Our data do not indicate that this nuclear actin is associated withphospho-JNK.

The results of the present study, summarized in Fig. 6 suggest JNK activity was required forsome aspects of FSS-induced actin remodeling, which occurred in 3 phases:

1. Nucleation of actin fibers within 5 minutes of exposure to high FSS

2. Cortical actin ring formation, which is most pronounced around 30 minutes of highFSS exposure, inducing morphological changes where BAECs become moreregular and rounded in shape

3. Some cortical actin remodeling into stress fibers that align in the direction of flow,aligning BAECs along with it.

Treatment with inhibitors of JNK activity, JNK inhibitor I and SP600125, did not preventthe formation of stress fibers (phase 1) and cortical actin (phase 2), suggesting that theseevents did not depend on JNK phosphorylation of substrate(s), and can be initiated withoutJNK activity. However, some loss of fibrous actin in SP600125 treated cells did occur priorto flow, and may contribute to the changes in rearrangement observed with this inhibitor.The fact that an inhibitor of JNK added to resting cells causes a decrease in actin fibers isalso consistent with a role for JNK in actin fiber rearrangement and/or maintenance. JNKappears to be playing a role in the actin realignment, specifically through its association withthe cortical region. JNK inhibitor I-treated BAECs appeared to be arrested in anintermediate state where cortical actin could not be further remodeled into aligned stressfibers. Since JNK activity in the nucleus did not appear to be blocked by inhibitor I, thiseffect is not likely to be the result of changes in gene expression. This situation could beexplained by the complex that has been suggested to form between JSAP1, JNK, FAK, andp130Cas, (Takino et al., 2005) because JSAP1 is involved in a phosphorylation-dependentcooperation with FAK to alter JNK activity and adhesion-triggered actin cytoskeleton

Mengistu et al.


NIH


NIH


NIH


reorganization (Chae et al., 2006). The dense cortical actin ring helps maintain cell-celladhesions between ECs and gives them the capability to contract and regulate endothelialpermeability (Dudek and Garcia, 2001). With increased exposure to FSS, SP600125-treatedcells lost more cell-to-cell associations and had thinner actin filaments, which compromisedtheir mechanical integrity and allowed them to be washed off by flow. Loss of cell-cellcontacts in these cells suggests that either JNK activity or JNK induced protein synthesismight be required for the maintenance of the contractile cortical ring. The involvement ofJNK in maintaining cell-cell attachment during remodeling has recently been shown instudies of epithelial sheet migration (Liense and Martin-Blanco, 2008; Melani et al., 2008).This MAPK has also been shown to modulate substrate interactions facilitating coordinatedmigration, at least partly through paxillin (Liense and Martin-Blanco, 2008). Thiscoordinated control could explain the decreased cell contacts between our endothelial cellstreated with SP600125. Another reason for the difference between JNK inhibitor 1 andSP600125 effects on JNK remodeling could be the fact that the SP600125 inhibitor clearlyis blocking JNK activity in the nucleus, while JNK inhibitor I did not effectively blocknuclear JNK activity in our TNFα-activated cells (Figure 4).

JNK inhibitor I-treated BAECs could not remodel their actin cytoskeleton into aligned stressfibers, and did not lose their integrity. Probably the extensive build-up of the cortical actin inJNK inhibitor 1-treated cells reflected the overlap of cortical actin accumulation in phase 2and stress fiber alignment in phase 3, which is blocked by the inhibitor.

The multiple stages of actin rearrangement could reflect the need to first enhance barrierfunction, an important role for cortical actin in endothelial cells (Prasain and Stevens, 2009).Stress fiber production without alignment actually decreases barrier function (Prasain andStevens, 2009), suggesting that the formation of cortical actin may facilitate neededimprovement in barrier function and correct alignment of the stress fibers during theirformation. JNK activity appears to be required to initiate phase 3 remodeling of corticalactin into stress fibers. As noted above, JNK inhibitor I might alter signaling complexformation (Takino et al., 2005), thereby protecting cell-cell interactions, but blocking furtheractin remodeling. SP600125 might also be affecting the cells by inhibiting JNK and alsoother kinases, causing a more dramatic response than that caused by JNK inhibitor 1.

A recent review of JNK inhibitors (Bogoyevitch and Arthur, 2008) notes that bothinhibitors, JNK Inhibitor I and SP600125, have been used extensively to block JNK activity,and have sometimes been reported to have different results. We employed 10 µM inhibitorconcentrations, a level often used to obtain complete JNK inhibition in cell culture situations(Amagasaki et al., 2006; Yang et al., 2007). Because the sensitivity of JNK is at least 300fold greater than other MAPKs (Bogoyevitch and Arthur, 2008; Bogoyevitch et al., 2004), itis likely that the effects caused by SP600125 are due to blocking the JNK activity.Evaluation of p38 MAPK activity in the presence of SP600125 indicates that p38 was notinhibited in our conditions, but it is impossible to completely rule out effects on otherkinases by this inhibitor, which might then account for the different effects of the twoinhibitors. Previous studies have identified a requirement for p38 MAPK in endothelialrealignment in response to shear stress (Azuma et al., 2001; Kadohama et al., 2006). Clearly,multiple MAPK enzymes play roles in shear stress induced endothelial realignment. Furtherstudies of the inhibitors will be needed to determine for certain the explanation for thedifferent responses.

It is not surprising that JNK is playing a role in actin remodeling in response to FSS.Numerous published studies identify a role for JNK in cell migration-directed actinremodeling, and recently these reports have included roles for JNK in endothelial migration(David et al., 2007; Rush et al., 2007). A significant variety of JNK substrates have been

Mengistu et al.


NIH


NIH


NIH


identified (Bogoyevitch and Kobe, 2006). Of those, the focal adhesion adaptor proteinpaxillin is one that appears likely to be important (Amagasaki et al., 2006; Huang et al.,2004). Huang et al. (Huang et al., 2004) proposed a model of how FAK induced JNKphosphorylation of paxillin could result in adhesion site turnover and allow rapid migration.Whether this same target is important in actin reorganization in the FSS system here remainsto be determined. Further support for the idea that JNK involvement in actin remodelingresults in JNK activation comes from a study which uses transgenic over-expression ofprofilin 1 (Moustafa-Bayoumi et al., 2007). In this study, over-expression of profilin 1 invascular smooth muscle cells resulted in the increase in actin dynamics (stress fiberformation and membrane ruffles), which was accompanied by increased levels of activeJNK. Since the profilin 1 caused increased changes in actin dynamics, this process mostlikely induced the increased JNK activity, but may have been modulated by it as well.

In summary, the data reported here provide evidence for the role of JNK in the realignmentof actin in response to atheroprotective FSS. Active JNK is associated with fibrous actinfrom as early as 5 minutes of FSS exposure, then with cortical actin and pools of actinduring remodeling, and with stress fibers during realignment. In addition, two separate JNKinhibitors interfere with one or more aspects of actin remodeling during the realignmentprocess. Additional research will be needed to identify the actual target(s) of JNK activityand to determine how JNK activity is integrated with other signaling systems that play rolesin endothelial cell mechanotransduction in response to FSS.

AcknowledgmentsThis work was supported in part by the Biosystems Dynamics Summer Institute, funded through an HHMIundergraduate education grant to Lehigh University and NIH award HL54269 to LJL-K. The authors thank JoshuaSlee and Raymond Pugh for help with inhibitor specificity experiments and Maria Brace for help with illustrations.

Abbreviation list

BAEC bovine aortic endothelial cell

ECs endothelial cells

FSS fluid shear stress

JNK Jun N-terminal kinase

LSM laser scanning microscope

MAPK mitogen activated protein kinase

PLA proximity ligation assay

ROI regions of interest

Reference ListAmagasaki K, Kaneto H, Heldin C-H, Lennartsson J. c-Jun N-terminal kinase is necessary for platelet-

derived growth factor-mediated chemotaxis in primary fibroblasts. Journal of Biological Chemistry.2006; 281:22173–22179. [PubMed: 16760468]

Anderson K, Sanderson J, Gerwig S, Peychl J. A new configuration of the Zeiss LSM 510 forsimulltaneous optical separation of green and red fluorescent protein pairs. Cytometry. 2006;69:920–929. [PubMed: 16969813]

Azuma N, Akasaka N, Kito H, Ikeda M, Gahtan V, Sasajima T, Sumpio B. Role of p38 MAP kinase inendothelial cell alignment induced by fluid shear stress. American Journal of Physiology - Heartand Circulatory Physiology. 2001; 280:H189–H197. [PubMed: 11123233]

Mengistu et al.


NIH


NIH


NIH


Bagrodia S, Derijard B, Davies RJ, A CR. Cdc42 and PAK-mediated signaling leads to Jun kinase andp38 mitogen-activated protein kinase activation. Journal of Biological Chemistry. 1995; 270:27995–27998. [PubMed: 7499279]

Bi E, Zigmond SH. Where the WASP stings. Current Biology. 1999; 9:160–163.Birukov KG, Birukova AA, Dudek SM, Verin AD, Crow MT, Zhan X, DePaola N, Garcia JGN. Shear

stress-mediated cytoskeletal remodelling and cortactin translocation in pulmonary endothelial cells.American Journal of Respiratory Cell and Molecular Biology. 2002; 26:453–464. [PubMed:11919082]

Bogoyevitch MA, Arthur P. Inhibitors of c-Jun N-teminal kinases - JuNK no more? BiochimicaBiophys Acta. 2008; 1784:76–93.

Bogoyevitch MA, Boehm I, Oakley A, Ketterman AJ, Barr R. Targeting the JNK MAPK cascade forinhibition: basic science and therapeutic potential. Biochimica et Biophysica Acta. 2004; 1697:89–101. [PubMed: 15023353]

Bogoyevitch MA, Kobe B. Uses for JNK: the many and varied substrates of the c-Jun N-terminalkinases. Microbiological and Molecular Biological Reviews. 2006; 70:1061–1095.

Chae H-J, Ha H-Y, Im J-Y, Song J-Y, Park S, Han P-L. JASP1 is required for the cell adhesion andspreading of mouse embryonic fibroblasts. Biochemical and Biophysical ResearchCommunications. 2006; 345:809–816. [PubMed: 16707108]

Chien S. Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. AmericanJournal of Physiology - Heart and Circulatory Physiology. 2007; 292:H1209–H1224. [PubMed:17098825]

Cunningham K, Gothlieb A. The role of shear stress in the pathogenesis of atherosclerosis. LaboratoryInvestigation. 2005; 85:9–23. [PubMed: 15568038]

David L, Mallet C, Vailhe B, Lamouille S, Feige J-J, Bailly S. Activin receptor-like kinase 1 inhibitshuman microvascular endothelial cell migration: potential roles for JNK and ERK. Journal ofCellular Physiology. 2007; 213:484–489. [PubMed: 17620321]

Dewey C, Bussolari S, Gimbrone M, Davies P. The dynamic response of vascular endothelial cells tofluid shear stress. Journal of Biomechanical Engineering. 1981; 103:177–185. [PubMed: 7278196]

Dougherty CS, Lowe-Krentz L. Heparin increases protein S levels in cultured endothelial cells bycausing a block in degradation. Journal of Vascular Research. 1998; 35:437–448. [PubMed:9858869]

Dudek SM, Garcia JGN. Cytoskeletal regulation of pulmonary vascular permeability. Journal ofApplied Physiology. 2001; 91:1487–1500. [PubMed: 11568129]

Franke R, Graffe M, Schnittler H, Seiffge D, Mittermayer C. Induction of human vascular endothelialstress fibers by fluid shear stress. Nature. 1984; 307:648–649. [PubMed: 6537993]

Galbraith CG, Skalak R, Chien S. Shear stress induces spatial reorganization of the endothelial cellcytoskeleton. Cell Motility and the Cytoskeleton. 1998; 40:317–330. [PubMed: 9712262]

Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinasepathways. Current Opinion in Cell Biology. 1999; 11:211–218. [PubMed: 10209154]

Ha H-Y, J-B K, Cho I-H, Kim K-S, Lee K-W, Sunwoo H, Im J-Y, Lee J-K, Hong J-H, Han P-L.Morphogenetic lungg defects of JSAP1-deficient embryos proceeds via the disruption of thenormal expressions of cytoskeletal and chaperone proteins. Proteomics. 2008; 8:1071–1080.[PubMed: 18324732]

Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998; 279:509–514. [PubMed: 9438836]Hamel M, Kanyi D, Cipolle M, Lowe-Krentz L. Active stress kinases in proliferating endothelial cells

associated with cytoskeletal structures. Endothelium. 2006; 13:157–170. [PubMed: 16840172]Helmke BP, Thakker DB, D GR, F DP. Spatiotemporal analysis of flow-induced intermediate filament

displacement in living endothelial cells. Biophysical Journal. 2001; 80:184–194. [PubMed:11159394]

Hoefen R, Berk B. The role of MAP kinases in endothelial activation. Vascular Pharmacology. 2002;38:271–273. [PubMed: 12487031]

Hu S, Chen J, Fabry B, Numaguichi Y, Gouldstone A, Ingber D, Fredberg J, Butler J, Wang N.Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of

Mengistu et al.


NIH


NIH


NIH


living cells. American Journal of Physiology - Cell Physiology. 2003; 285:C1082–C1090.[PubMed: 12839836]

Huang C, Jacobson K, Schaller MD. A role for JNK-paxillin signaling in cell migration. Cell Cycle.2004; 3:4–6. [PubMed: 14657652]

Ip TY, Davis RJ. Signal transduction by the c-jun N-terminal kinase (JNK)-from inflammation todevelopment. Current Opinion in Cell Biology. 1998; 10:205–219. [PubMed: 9561845]

Jarvius J, Melin J, Goransson J, Stenberg J, Fredriksson S, Gonzalez-Rey C, Bertilsson S, Nilsson M.Digital quantification using amplified single-molecule detection. Nature Methods. 2006; 3:725–727. [PubMed: 16929318]

Kadohama T, Akasaka N, Nishimura K, Hoshino Y, Sasajima T, Sumpio B. p38 mitogen-activatedprotein kinase activation in endothelial cell is implicated in cell alignment and elongation inducedby fluid shear stress. Endothelium. 2006; 13:43–50. [PubMed: 16885066]

Li L, Tatake R, Natarajan K, Taba Y, Garin G, Tai C, Leung E, Surapisitchat J, Yoshizumi M, Yan C,Abe J-i, Berk B. Fluid shear stress inhibits TNF-mediated JNK activation via MEK5-BMK1 inendothelial cells. Biochemical and Biophysical Research Communications. 2008; 370(1):159–163.[PubMed: 18358237]

Li S, Kim M, Hu Y, Jalali S, Schlaepfer DD, Hunter T, Chien S, Shyy JY. Fluid shear stress activationof focal adhesion kinase. Linking ot mitogen activated protein kinases. Journal of BiologicalChemistry. 1997; 272:30455–30462. [PubMed: 9374537]

Liense F, Martin-Blanco E. JNK signaling controls border cell cluster integrity and collective cellmigration. Current Biology. 2008; 18:538–544. [PubMed: 18394890]

Malek A, Izumo S. Mechanism of endothelial cell shape change and cytoskeletal remodeling inresponse to fluid shear stress. Journal of Cell Science. 1996; 109:713–726. [PubMed: 8718663]

Melani M, Simpson KJ, Brugge JS, Montell D. Regulation of cell adhesion and collective cellmigration by hindsight and its human homolog RREB1. Current Biology. 2008; 18:532–537.[PubMed: 18394891]

Mott RE, Helmke BP. Mapping the dynamics of shear stress-induced structural changes in endothelialcells. American Journal of Physiology - Cell Physiology. 2007; 293:C1616–C1626. [PubMed:17855768]

Moustafa-Bayoumi M, Alhaj MA, El-Sayed O, Wisel S, Chotani MA, Abouelnaga ZA, HassonaMDH, Rigatto K, Morris M, Nuovo G, Zweier JL, Goldschmidt-Clermont P, H H. Vascularhypertrophy and hypertension caused by transgenic overexpression of profilin 1. Journal ofBiological Chemistry. 2007; 282:37632–37639. [PubMed: 17942408]

Noria S, Xu F, McCue S, Jobes M, Gotlieb AI, Langille BL. Assembly and reorientation of stressfibers drives morphological changes to endothelial cells exposed to shear stress. American Journalof Pathology. 2004; 164:1211–1223. [PubMed: 15039210]

Otto IM, Raabe T, Rennefahrt UE, Bork P, Rapp UR, Kerkhoff E. The p150-Spir protein provides alink between c-jun N-terminal kinase function and actin reorganization. Current Biology. 2000;10:345–348. [PubMed: 10744979]

Pederson T. As functional nuclear actin comes into view, is it globular filamentous, or both? J CellBiology. 2008; 180:1061–1064.

Prasain N, Stevens T. The actin cytoskeleton in endothelial phenotypes. Microvascular Research.2009; 77:53–63. [PubMed: 19028505]

Ramesh N, Anton IM, Marinez-Quiles N, Geha RS. Waltzing with WASP. Trends in Cell Biology.1999; 9:15–19. [PubMed: 10087612]

Rush S, Khan G, Bamisaive A, Bidwell P, Leaver HA, Rizzo MT. C-jun amino-terminal kinase andmitogen activated protein kinase 1/2 mediate hepatocyte growth factor-induced migration of brainendothelial cells. Experimental Cell Research. 2007; 313:121–132. [PubMed: 17055484]

Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Webster K, HydbringP, Bahram F, Larsson LG, Landegren U. Direct observation of individual endogenous proteincomplexes in situ by proximity ligation. Nature Methods. 2006; 12:995–1000. [PubMed:17072308]

Mengistu et al.


NIH


NIH


NIH


Takino T, Nakada M, Miyamori H, Watanabe Y, Sato T, Gantulga D, Yoshioka K, Yamada KM, SatoH. JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cellmigration. Journal of Biological Chemistry. 2005; 280:37772–37781. [PubMed: 16141199]

Vartiainen M. Nuclear actin dynamics - From form to function. FEBS Letters. 2008; 582:2033–2040.[PubMed: 18423404]

Wang J-C, Goldschmidt-Clermont P, Wille J, Yin F-P. Specificity of endothelial cell reorientation inresponse to cyclic mechanical stretching. J Biomechanics. 2001; 34:1563–1572.

Wang N, Tytell J, Ingber D. Mechanotransduction at a distance: mechanically coupling theextracellular matrix with the nucleus. Nature Reviews Molecular Cell Biology. 2009; 10:75–82.

Yalcin HC, Perry SF, Ghadiali SN. Influence of Airway Diameter and Cell Confluence on EpithelialCell Injury in an In-Vitro Model of Airway Reopening. Journal of Applied Physiology. 2007;103:1796–1807. [PubMed: 17673567]

Yang C, Patel K, Harding P, Sorokin A, Glass WF II. Regulation of TGF-β1/MAPK-mediated PAI-1gene expression by the actin cytoskeleton in human mesangial cells. Experimental Cell Research.2007; 313:1240–1250. [PubMed: 17328891]

Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses: atheroprotectiveeffect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated proteinkinases. Journal of Pharmacological Sciences. 2003; 91:172–176. [PubMed: 12686737]

Mengistu et al.


NIH


NIH


NIH


Mengistu et al.


NIH


NIH


NIH


Figure 1.FSS-induced changes in JNK activity and spatial distributions in BAECs. Confluentmonolayers of BAECs were exposed to 2, 4, 10, 15 and 20 dyn/cm2 FSS for 0, 5, 15, 30, and60 minutes. Active JNK was fluorescently labeled to visualize changes in intracellulardistribution (A). The same image acquisition settings were used to obtain these images inorder to quantify relative changes in phospho-JNK intensity levels, which were used asindicators of activity levels (B - cytoplasm & C - nuclei). Activity levels in BAECs treatedwith 2 (diagonal lines), 4 (horizontal lines), 10 (light grey), 15 (dark gray), and 20 (black)dyn/cm2 FSS, for 0, 5, 15, 30, and 60 minutes (x-axis), were quantified. Error bars representstandard deviations in intensity levels from 20 cells taken from multiple regions within thearea(s) exposed to FSS. For cell analysis illustrated in the graphs, each trial was done at leastthree times and cells for analysis were chosen from the different trials. The images in A aresimilarly representative and those illustrated for a single FSS value all came from the sameexperiment. At 4 and 10 dyn/cm2, these were representative of at least two experiments, andat 15 dyn/cm2 the images were representative of four or more experiments. Staining with aprimary antibody to total JNK1 is illustrated in D (no flow, 5, 15, and 30 min) at 15 dyn/cm2. The pictures in panel D are from representative regions in a single trial each. BAECswere cultured on cover slips as for FSS experiments, but were then treated with TNF-α(Panel E) for 0, 5, 15 or 30 min and stained for phospho-JNK as in A. These data are from asingle experiment which produced identical staining to more than five similar experimentswithout transfer to flow media. Scale bars = 20-µm for all images.

Mengistu et al.


NIH


NIH


NIH


Figure 2.Fluid shear stress-induced phospho-JNK association with actin. In panel A, BAECs exposedto 15 dyn/cm2 FSS conditions for 15 and 30 minutes were co-labeled with antibodies againstphospho-JNK (green) and actin (red), where their relative spatial distribution was examinedand compared to cells not exposed to flow. Co-localization between phospho-JNK and actincan be seen by a yellow signal, which result from overlapping the green and red signals(merge). Image acquisition settings designed to eliminate stain overlap were obtained withBAECs that were not treated with flow. These settings were saved and reused for imaging ofFSS-treated BAECs in order to visualize FSS-induced changes in phospho-JNK and Actinintensity. Images shown are representative from at least two experiments at each condition.

Mengistu et al.


NIH


NIH


NIH


In panel B an identical experiment carried out in the presence of cycloheximide to blocknew protein synthesis is shown (actin staining only). Scale bars = 20-µm.

Mengistu et al.


NIH


NIH


NIH


Figure 3.Fluid shear stress-induced phospho-JNK association with actin examined with PLA.Phospho-JNK – actin associations were recognized with the Duolink™ PLA as described inMethods, and the position of these associations in BAECs was observed by tracing the celland nucleus contours (white lines) using the DIC images of the cells. Scale bars = 10-µm(A). Panel B illustrates single cells where simultaneous labeling of the actin cytoskeletonwith FITC-conjugated phalloidin (green) is shown with the red PLA. Scale bars = 5-µm.

Mengistu et al.


NIH


NIH


NIH


Figure 4. JNK inhibitors specifically block further JNK activation and JNK activityCells exposed to JNK inhibitor I or SP600125 at 10 µM for 60 min were stained forphospho-JNK as in Methods (A). Cells treated with drugs or left untreated were thenexposed to 15 dyn/cm2 FSS for 30 min and stained for phospho-JNK (B). Panels A and Bare from different experiments. Scale bars = 10 µm. Panel C illustrates western blotting forthe JNK substrate, phospho-c-Jun, in confluent endothelial cells prepared as for flowincluding incubation with JNK Inhibitor I or SP600125 as shown. TNF-α-induced activationwas employed in place of flow. Blots are representative of three separate experiments. PanelD illustrates a similar experiment to assess the specificity of these inhibitors. MAPKAP-K2,a substrate for the MAPK p38, was evaluated by western blotting after preparation of cells

Mengistu et al.


NIH


NIH


NIH


for flow and JNK inhibitors as noted. The blots are representative of three separateexperiments. Inhibition of p38 activity with the p38 inhibitor, SB203580, from an identicalexperiment is also shown as a control.

Mengistu et al.


NIH


NIH


NIH


Figure 5.Affects of JNK inhibitors I and SP600125 on fluid shear stress-induced actin remodeling.Flow-induced actin remodeling was observed by fluorescence labeling of the actincytoskeleton with TRITC-conjugated phalloidin in BAECs treated with 15 dyn/cm2 FSS for0, 30, 60, and 120 minutes (A). The role of JNK in FSS-induced actin remodeling wasstudied by observing changes in actin responses to flow in BAECs treated with 10 µM ofSP600125 (B), or JNK inhibitor I (C), for 1hour. Intensity levels of the fluorescence signalswere not normalized, and therefore, are not necessarily reflective of actin levels. Range bar= 20-µm.

Mengistu et al.


NIH


NIH


NIH


Figure 6.Model for the role of JNK in FSS-induced actin remodeling. BAECs in static culture haverandomly oriented actin cytoskeletons. Upon exposure to 15 dyn/cm2 FSS, these cellsundergo actin remodeling in 3 phases: (1) Nucleation of actin fibers within 5 minutes; (2)Cortical actin ring formation by 30 minutes; (3) Cortical actin remodeling into stress fibersthat align in the direction of flow. The role of JNK in this FSS-induced remodeling wasassessed by inhibiting JNK activity using JNK inhibitor I and SP600125. BAECs treatedwith both inhibitors were able to transition to phase 1 similar to untreated cells. During thesecond phase, SP600125-treated BAECs did not form a thin cortical actin ring ( ),which weakened cell-cell associations and made it easier for them to be washed off by theflow treatment. JNK inhibitor I treatment induced thicker cortical actin formation ( ) thatpersisted over the 120-minute flow treatment. In these cells, cortical actin remodeling intoaligned stress fibers (phase 3) was blocked.

Mengistu et al.


NIH


NIH


NIH


Fluid shear stress-induced JNK activity leads to actin remodeling for cell alignment

Documents

Transcript of Fluid shear stress-induced JNK activity leads to actin remodeling for cell alignment