Role of Calcineurin in Thrombin-Mediated

Endothelial Cell Contraction

Bernadett Kolozsvari,1 Zsolt Szıjgyarto,2 Peter Bai,1 Pal Gergely,1,2

Alexander Verin,3 Joe G. N. Garcia,4 Eva Bako1*

� AbstractBarrier function and shape changes of endothelial cells (EC) are regulated by phospho-rylation/dephosphorylation of key signaling and contractile elements. EC contractionresults in intercellular gap formation and loss of the selective vascular barrier to circu-lating macromolecules. EC dysfunction elicited by thrombin was found to correlatewith actin microfilament redistribution. It is known that calcineurin (Cn) is involved inthrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activityand the phosphorylation state of EC myosin light chain is also affected by Cn activity.Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed withGFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endo-thelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin.Cytotoxic effects of transfections or treatments and the efficiency of transfections wereassessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A andFK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibi-tion of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. Wedetected attenuated thrombin-induced stress fiber formation and changes in cell shapeonly when cells were transfected with constitutively active CnA and not with variousCnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treat-ments is negligible. We observed that Cn is involved in the recovery from thrombin-induced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, whileoverexpression of constitutively active CnA resulted in reduced thrombin-inducedstress fiber formation. ' 2009 International Society for Advancement of Cytometry

� Key termsendothelium; barrier function; isoforms of catalytic subunit of calcineurin; stress fibers;immunofluorescence

REVERSIBLE covalent modification of proteins by phosphorylation and dephospho-

rylation plays a dominant role in controlling many cellular processes including cell

contractility. Actin–myosin interaction, stress fiber formation, cell contraction

induced by thrombin are the result of myosin light chain (MLC) phosphorylation

catalyzed by Ca21/calmodulin(CaM)-dependent MLC kinase (1,2). Increased levels

of MLC phosphorylation is followed by actin redistribution, filament formation and

EC barrier dysfunction (2). The direct involvement of type 1 phospho-Ser/Thr speci-

fic protein phosphatase (PP1) in EC MLC dephosphorylation has been demonstrated

(3,4), furthermore, each of the four main classes of protein phosphatases (PP1,

PP2A, PP2B, and PP2C) is able to dephosphorylate MLC in vitro (5). The role of

PP2A in the regulation of EC cytoskeleton structure is to protect EC barrier via de-

phosphorylation of other specific cytoskeletal targets (6). Calcineurin (Cn), which is

also called as PP2B (7,8), associated with a detergent-insoluble actin-enriched cellular

fraction of pulmonary artery EC affected the phosphorylation state of MLC (9).

Inhibition of Cn potentiated the thrombin-induced increase in PKC activity (10). To

elucidate potential substrates of Cn and connection with other enzymes involved in

the regulation of EC cytoskeleton requires further studies.

1Department ofMedical Chemistry,Research Center forMolecularMedicine,Medical and Health Science Center,University of Debrecen, Debrecen, Hungary2Cell Biology and Signaling ResearchGroup of the Hungarian Academy ofSciences, Debrecen, Hungary3Vascular Biology Center, MedicalCollege of Georgia, Augusta, Georgia4Department of Medicine, Pritzker Schoolof Medicine, University of Chicago, Illinois

Received 1 October 2008; RevisionReceived 15 December 2008; Accepted6 January 2009

Grant sponsor: Hungarian ScientificResearch Fund; Grant number: OTKAK60620; Grant sponsor: HungarianScience and Technology (TET) Founda-tion; Grant number: FR-11/2007; Grantsponsor: University of Debrecen, Medicaland Health Science Center, Hungary;Grant number: Mec-1/2008; Grant spon-sor: Bolyai fellowship, Hungarian Acad-emy of Sciences (PB); Grant sponsor:NIH; Grant number: RO1 HL067307.

*Correspondence to: Dr. �Eva Bak�o;Department of Medical Chemistry,Medical and Health Science Center,University of Debrecen, Life ScienceBuilding. Nagyerdei krt 98, H-4032Debrecen, Hungary

Email: bakoeva@dote.hu

Published online 20 February 2009 inWiley InterScience (www.interscience.wiley.com)

DOI: 10.1002/cyto.a.20707

© 2009 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 75A: 405�411, 2009

Vascular endothelium is a critical, semi-selective cellu-

lar barrier to fluid and solute flux across blood vessel

wall. Increased endothelial permeability is a result of inter-

cellular gap formation evoked by bioactive agents such as

the coagulation protease thrombin (11–14). Thrombin

induces a sequence of biochemical events, including Ca21

mobilization, which precedes and initiates Ca21/CaM-

dependent protein phosphatase (PP2B) activation in

endothelium (9).

The native form of Cn is a heterodimer of two tightly

bound subunits: calcineurin A (CnA), a 58–64 kDa catalytic

and CaM-binding subunit, and calcineurin B (CnB), a 19 kDa

Ca21-binding regulatory subunit. The two-subunit structure

is essential for Cn activity (15). Cn in different tissues is a

widely but not evenly distributed protein phosphatase. CnA is

represented by three isoforms (a, b, and c), which are pro-

ducts of different genes, the highly conserved CnB is encoded

by a single gene in all tissues except testis (16,17). CnA genes

encode for polypeptides with variable N- and C-terminal tails

and consisting highly conserved amino acid sequences of the

catalytic and the regulatory domains (18). The regulatory

domain contains subdomains as the CnB-binding helix, the

CaM-binding and autoinhibitory (AI) subdomain. The enzy-

matic activity of Cn is repressed in the native protein, but it

becomes fully active when the CaM-binding and AI-domains

are cleaved by proteases (15).

Pharmacological agents, cyclosporin A (CsA), and FK506,

inhibit Cn in the presence of their respective cytoplasmic

immunophillin proteins, cyclophillin, and FK506-binding

proteins (19). Cn has much narrower in vitro substrate speci-

ficity than the other two major Ser/Thr phosphatases, PP1 or

PP2A (20–22) . Two of its best known substrates in vitro and

in vivo include inhibitor-1 and DARPP-32 (23). When phos-

phorylated, they are strong and specific inhibitors of PP1, thus

Cn also controls PP1 activity (24).

It appears likely that Cn functions as a key enzyme in a

complex cascade system, which controls the activity of other

enzymes with much broader substrate specificity. Tight asso-

ciation of Cn with the nonmuscle cytoskeleton (25,26) has

raised the possibility that Cn may be involved in regulating

contractility of specific cell types and Cn also could regulate

cytoskeletal dynamics (27,28).

The role of Cn in endothelium has been elucidated by

Verin et al. (9). It has been found that beside the constitu-

tively active myosin-associated PP1, thrombin-inducible

Ca21/CaM-dependent protein phosphatase, Cn may be also

involved in agonist-mediated EC activation. To further

characterize the role of Cn in endothelium, we investigated

the specific roles of CnA isoforms by transfection of EC

with pEGFP constructs and we demonstrated the effect of

Cn inhibitors on thrombin-induced stress fiber formation

in the present study.

Although the various isoforms of CnA had no effect

on the cytoskeleton structure of thrombin-induced

endothelial cells, the constitutively active truncated form of

Cn catalytic subunit (DCnA) attenuated the stress fiber

formation.

MATERIALS AND METHODS

Reagents

Thrombin, cyclosporin A, and FK506 monohydrate were

purchased from Sigma-Aldrich (St Louis, MO, USA). Texas

Red-phalloidin and ProLong Gold Antifade medium were

from Molecular Probes (Eugene, OR).

Cell Culture

Bovine pulmonary artery endothelial cells (BPAEC) (cul-

ture line-CCL 209) were obtained frozen at passage 16 (Amer-

ican Type Tissue Culture Collection, Rockville, MD), and were

utilized at passages 17–22 as previously described (29). Cells

were maintained in MEM (Gibco-BRL, Chagrin Falls, OH)

supplemented with 20% (v/v) colostrum-free bovine serum

(Irvine Scientific, Santa Ana, CA), 15 mg/ml EC growth

supplement (Collaborative Research, Bedford, MA), 1% anti-

biotic, and antimycotic solution (penicillin, 10,000 U/ml;

streptomycin, 10 mg/ml; and amphotericin B, 25 mg/ml; K.C.

Biologicals, Lenexa, KA), and 0.1 mM nonessential amino

acids (Gibco-BRL, Chagrin Falls, OH). All cells were main-

tained at 378C in a humidified atmosphere of 5% CO2 and

95% air.

Plasmids

The entire coding sequences of different CnA isoforms

were amplified by PCR and were subcloned to pEGFP-C1 vector

from Clontech (Palo Alto, CA). The truncated a isoform, DCnAwas subcloned to pEGFP-C1 vector from pcDL-SRa296 (30).

The specific primers with the restriction sites were as follows.

CnAa: Hind III 50-GCTCAAGCTTCTATGTCCGAGCC-30 andBamH I 50-CCGGCGGATCCTCACTGAATATT-30;

CnAb: Sal I 50-CTACAGTCGACATGGCCGCCCCGGAG-30 andBamH I 50-GCACGGGATCCTCACTGGGCAGTATGG-30;

CnAc: Sal I 50-CTGCAGTCGACATGTCCGGGAGG-30 and BamH

I 50-GCCCGGGATCCTCATGAATGGGC-30,

respectively. All constructs and their open reading frames were

analyzed by sequencing using vector and insert specific

primers.

Transfection

Cells were grown to 70–80% confluency and transfected

(31) using Fugene HD transfection reagent (Roche, Indiana-

polis, IN) according to the manufacturer’s instructions. After

24–48 h of incubation, cells were washed three times with PBS

and used for further experiments.

Immunofluorescence

After specific treatments, BPAEC were plated onto glass

coverslips and grown to confluence. The cells were washed

once with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM

Na2HPO4, 1.47 mM KH2PO4, pH 7.4) and fixed in 3.7% para-

formaldehyde (PFA) in PBS for 10 min at room temperature.

Between each step, the cells were rinsed three times with PBS.

The cells were permeabilized with PBS 1 0.25% Triton-X 100

ORIGINAL ARTICLE

406 Endothelial Dysfunction and Calcineurin

(TBST) at room temperature for 30 min, blocked with 2% BSA

in TBST (for 1 h at room temperature) (32). Then, the cells were

incubated with the primary antibody diluted in blocking solution

for 1 h at room temperature. Actin filaments were stained with

Texas Red-phalloidin. Cover slips were rinsed and mounted in

ProLong Gold Antifade (Molecular Probes, Eugene, OR) and

observed on a Nikon Eclipse TE300 microscope. Images were

processed using PhotoShop Imaging software.

Flow Cytometry

The possible cytotoxic effects of the transfections or treat-

ments and the efficiency of the transfections were assessed by

flow cytometry analysis. Cells were plated in six-well plates in

triplicates and were either treated with 50 nM thrombin for 30

min or with 0.1 lM FK506 for 4 h or with 2 lM CsA for 4 h.

Treatments of another set of samples were as follows 50 nM

thrombin for 30 min after that with 0.1 lM FK506 or 2 lMCsA for 4 h.

H2O2 (1 mM, 4 h) treated endothelial cells were used as

a positive control. Cells were stained with propidium iodide

(5 lg/ml, 30 min) then cells were washed twice with PBS and

then trypsinized for 5 min to obtain cell suspension. There

was no significant cell loss during treatments, i.e. cell counts

before and after the procedures were the same (�5%). Cells

were collected by centrifugation at 1200g for 10 min, resus-

pended in 1 ml ice-cold PBS and rate of cells death was deter-

mined by flow cytometry analysis using a FACS Calibur

instrument (Becton-Dickinson, San Jose, CA) counting 20,000

cells. To determine transfection efficiency, cells were plated

into Petri dishes; triplicate samples were transfected as

described earlier. Forty-eight hours later, cells were trypsinized

and subjected to flow cytometry analysis. The efficiency of

transfection was low and to acquire statistically relevant num-

ber of double positive cells 1.1 million of cells were counted.

Statistical Analysis

For statistical analysis Student’s t-test was applied and the

P\ 0.05 was considered as significant.

RESULTS

Inhibition of Cn Prolongs the Thrombin-Induced

Stress Fiber Formation

The focus of this study was to determine the role of Cn in

EC cytoskeleton regulation and identify its cytoskeletal protein

targets involved in the endothelial barrier function using

pharmacological inhibitors (CsA and FK506) and thrombin

Figure 1. Inhibition of calcineurin prolongs the thrombin-induced stress fiber formation. Monolayers of BPAEC were treated as follows.

(A) Untreated control; (B) 50 nM thrombin for 30 min; (C) 50 nM thrombin for 30 min followed by serum free medium for 4 h; (D) 2 lM CsA

for 4 h; (E) 50 nM thrombin for 30 min followed by 2 lM CsA for 4 h; (F) 0.1 lM FK506 for 4 h; (G) 50 nM thrombin for 30 min followed by

0.1 lM FK506 for 4 h. Typical patterns of Texas Red-phalloidin staining are shown from three independent experiments. Scale bars: 20 lm.[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE

Cytometry Part A � 75A: 405�411, 2009 407

(Fig. 1). Actin microfilaments were stained with Texas Red-

phalloidin. The fluorescent pictures show the stress fiber for-

mation in the actin cytoskeleton after different treatments of

the cells. First, we treated BPAEC with 50 nM thrombin for 30

min, this agent evoked stress fiber formation and barrier com-

promise. Immunostaining of BPAEC for F-actin demonstrates

that treatment of EC with thrombin increased stress fiber for-

mation (Fig. 1B) as it has been demonstrated by earlier obser-

vations (1,10,33,34). Recovery of cytoskeleton structure of

BPAEC could be detected; attenuated stress fiber formation

was obtained in the endothelial cytoskeleton if thrombin treat-

ment was followed by serum free medium for 4 h, (Fig. 1C).

When Cn activity was inhibited with CsA (2 lM, 4 h), no dif-

ference in the actin cytoskeleton could be detected when com-

pared with the control cells (Fig. 1D). When pharmacological

inhibitor, CsA (2 lM, 4 h) of Cn, was added to BPAEC after

pretreatment with thrombin (50 nM, 30 min) augmented

actin filaments stained. The inhibition of Cn caused prolonged

contraction of the cells, the established stress fibers were

exhibited in the presence of CsA even after 4 h (Fig. 1E). The

cytoskeleton structure was not altered by FK506 alone, which

is another specific Cn inhibitor (Fig. 1F). In contrast, induc-

tion of the cells with thrombin (50 nM, 30 min) followed by

FK506 treatment, increased thrombin-induced formation of

stress fibers (Fig. 1G) confirming possible involvement of Cn

activity in EC barrier regulation.

Effect of Overexpression of CnA Isoforms on

Thrombin-Induced Stress Fibers Formation

To further clarify the role of the isoforms of CnA in the

regulation of EC cytoskeleton structure, we cloned the three

major isoforms of CnA (a, b, and c). We also used a constitu-

Figure 2. Effect of overexpression of CnA isoforms on thrombin induced stress fiber formation. Immunofluorescent detection of the cyto-

skeleton structure by Texas red-phalloidin staining in PFA fixed BPAE cells. Untreated EC (left panel) and thrombin-induced (50 nM for 30

min) EC (right panel). Overexpression of GFP (A, B), CnAa-pEGFP (C, D), CnAb-pEGFP (E, F), CnAc-pEGFP (G, H), DCnA-pEGFP (I, J). Typicalpatterns are shown from three independent experiments. Arrows indicate the cells transfected by pEGFP or Cn-pEGFP isoforms. Scale

bars: 20 lm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE

408 Endothelial Dysfunction and Calcineurin

tive active, truncated a isoform (DCnA) missing CaM-binding

and AI domains of the catalytic subunit of Cn.

The overexpressed proteins inserted into pEGFP expres-

sion plasmids were investigated for the F-actin cytoskeleton

structure by immunofluorescent imaging (Fig. 2). The overex-

pressed catalytic subunit isoforms had no effect on cytoskele-

ton structure of BPAEC either in untreated or thrombin-sti-

mulated cells as revealed by Texas Red-phalloidin staining. On

the other hand, transfection of BPAEC by the constitutive

active, truncated form of Cn catalytic subunit (DCnA-pEGFP)attenuated the stress fiber formation in the presence of

thrombin.

Efficiency of Transfection and Viability of Cells as

Revealed by FACS Analysis

The cell viability was not affected by treatments of throm-

bin, CsA, or FK506 as revealed by FACS analysis (data not

shown). The cell viability after transfection was detected by

FACS as described in Materials and Methods section and

shown in Figure 3. In the upper panel (Fig. 3A), region R2

represents nontransfected viable, R3 the propidium-iodide

positive nontransfected, R4 the GFP positive transfected cells,

while R5 represents the double positive cells. Efficiency of

transfection of endothelial cells with these mammalian expres-

sion constructs was 5–10%. Transfection with pEGFP, CnAa-pEGFP, CnAb-pEGFP, CnAc-pEGFP, or DCnA-pEGFP had no

significant effect on cell survival. As a positive control, H2O2

(1 mM, 4 h) was used (Fig. 3B).

DISCUSSION

Cn, also known as a key regulator of T-lymphocyte acti-

vation via dephosphorylation and consequent nuclear translo-

cation of the transcription factor Nuclear Factor of Activated

T-lymphocytes (NFAT) (21). Cn inhibitors, CsA, and FK506

(tacrolimus) are used as immunosuppressive drugs in clinical

transplantations. The side effect of Cn inhibitors is that they

induce endothelial dysfunction in renal transplant recipients

(35). Disruption of barrier function of EC occurs also during

inflammatory disease such as acute lung injury (11). The

major contributor in the inflammation-induced barrier dys-

Figure 3. Efficiency of transfection (A) and viability of cells (B) as revealed by FACS analysis. (A) Histograms show the transfection of

BPAEC with pEGFP and various plasmids of CnA catalytic subunit. (B) Cell viability for the transfected cells was expressed in percent as

(R4/R4 1 R5) 3 100, for control and H2O2-treated cells the viability was calculated as (R2/R2 1 R3) 3 100, where R2, R3, R4, R5 represent

the number of cells in the appropriate regions. Transfection by various CnA isoforms does not impair cellular viability; asterisk indicates

significant difference when compared with control (P\0.001).

ORIGINAL ARTICLE

Cytometry Part A � 75A: 405�411, 2009 409

function is the paracellular transport. Effect of inflammatory

mediators such as alpha thrombin and histamine results in

increased endothelial permeability accompanied by cell round-

ing and interendothelial gap formation (36,37). The normal

function of endothelium is highly dependent on the endothe-

lial cytoskeleton. Changes in the F-actin cytoskeletal structure

of renal microvascular endothelial cells after ischemia/reperfu-

sion caused capillary hyperpermeability and endothelial cell

detachment (38). Regulation of paracellular transport in endo-

thelial cells is associated with modulation of actin-based

systems which anchor the cell to its neighbor or extracellular

matrix maintaining endothelial integrity (37–39).

It is well-established that thrombin-induced EC dysfunc-

tion is critically dependent on increased levels of MLC

phosphorylation, which results in stress fiber formation and

increased contraction (12). Little is known regarding the

events that reverse inflammatory responses reducing the con-

tractile response and initiating relaxation. In this study, we

have provided evidence for the involvement of Cn activity in

the recovery from thrombin-induced EC dysfunction using an

approach, which primarily utilizes pharmacologic inhibitors

and overexpression of constitutively active form of Cn cataly-

tic subunit. The fluorescent pictures show the stress fiber for-

mation in the actin cytoskeleton after treatment with 50 nM

thrombin for 30 min which attenuated after 4 h, marking the

recovery of the cytoskeleton structure. EC dysfunction

induced by thrombin was significantly prolonged by the addi-

tion of Cn inhibitors (CsA or FK506). Differences in morpho-

logical and biochemical effects of these immunosuppressive

agents on endothelial function were reported in a capillary

tube assay in vitro (40). In our experiments with bovine

pulmonary artery endothelial cell monolayers, we could not

detect any significant difference between the effect of CsA and

FK506 (Fig. 1). On the other hand, overexpression of constitu-

tively active form of Cn catalytic subunit attenuated stress

fiber formation caused by thrombin treatment (Fig. 2) indicat-

ing the role of Cn in cytoskeletal rearrangement.

In the next set of experiments we intended to clarify iso-

forms-specific effect of Cn. The Cn isoforms may be differen-

tially expressed and/or the activity of each may be differen-

tially regulated, leading to tissue specific functions. Cn is a

heterodimer composed by CnA catalytic and CnB regulatory

subunits. Information is limited about the functional differ-

ences between CnA isoforms. The three isoforms of mamma-

lian CnA differs mostly at the variable N- and C-terminal tails,

whose functions are not known. Sequence differences may be

potentially important for function and regulation of Cn

(17,41). The three isoforms (a, b, c) of CnA could be detected

by Northern and Western blotting methods almost in equal

amount in EC (unpublished observation). Overexpression of

different isoforms of the catalytic subunit of Cn in BPAEC had

no effect on cytoskeleton structure either of untreated or

thrombin-stimulated cells. The reason could be that heterodi-

meric (CnA and CnB) structure is essential for the catalytic

function of Cn (15). Therefore, the transfection of BPAEC by

the constitutive active, truncated form (DCnA-pEGFP) atte-

nuated stress fiber formation in the presence of thrombin

indicating the role of Cn activity in the recovery from EC dys-

function. The shape of cells were also different when com-

pared with the control cells or the cells transfected by CnA a,b, and c isoforms. Further studies are required to investigate

functional significance of the presence of different CnA iso-

forms in endothelium and their role in the barrier function.

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