Multi-drug Resistance Protein 4 (MRP4)-mediated Regulationof Fibroblast Cell Migration Reflects a Dichotomous Role ofIntracellular Cyclic Nucleotides*□S

Received for publication, November 12, 2012, and in revised form, December 16, 2012 Published, JBC Papers in Press, December 22, 2012, DOI 10.1074/jbc.M112.435925

Chandrima Sinha‡, Aixia Ren‡, Kavisha Arora‡, Chang-Suk Moon‡, Sunitha Yarlagadda‡, Weiqiang Zhang‡,Satish B. Cheepala§, John D. Schuetz§, and Anjaparavanda P. Naren‡1

From the ‡Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee 38163 and the§Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

Background:MRP4 is an endogenous transporter of cyclic nucleotides that can regulate cell migration. The role of MRP4in fibroblast migration is unknown.Results:MRP4-deficient fibroblasts migrate faster and have a moderately higher level of intracellular cyclic nucleotides.Conclusion: Inhibition of MRP4 increases fibroblast migration via alteration of intracellular cyclic nucleotide levels.Significance: Inhibition of MRP4 facilitates wound repair.

It has long been known that cyclic nucleotides and cyclic nucle-otide-dependent signalingmolecules control cellmigration. How-ever, the concept that it is not just the absence or presence of cyclicnucleotides, but a highly coordinated balance between these mol-ecules that regulates cell migration, is new and revolutionary. Inthis study, we used multidrug resistance protein 4 (MRP4)-ex-pressingcell linesandMRP4knock-outmiceasmodel systemsandwound healing assays as the experimental system to explore thisuniqueandemergingconcept.MRP4,amemberofa large familyofATP binding cassette transporter proteins, localizes to the plasmamembrane and functions as a nucleotide efflux transporter andthus plays a role in the regulation of intracellular cyclic nucleotidelevels. Here, we demonstrate that mouse embryonic fibroblasts(MEFs) isolated from Mrp4�/� mice have higher intracellularcyclic nucleotide levels and migrate faster compared with MEFsfromMrp4�/�mice.UsingFRET-basedcAMPandcGMPsensors,we show that inhibition of MRP4 with MK571 increases bothcAMP and cGMP levels, which results in increased migration. Incontrast to these moderate increases in cAMP and cGMP levelsseen in the absence ofMRP4, a robust increase in cAMP levels wasobserved following treatment with forskolin and isobutylmethylx-anthine, which decreases fibroblast migration. In response toexternally added cell-permeant cyclic nucleotides (cpt-cAMP andcpt-cGMP),MEFmigrationappears tobebiphasic.Altogether, ourstudies provide the first experimental evidence supporting thenovel concept thatbalancebetweencyclicnucleotides is critical forcell migration.

Multidrug resistance protein 4 (MRP4/ABCC4)2 is a mem-brane protein belonging to the C subfamily of ATP-binding

cassette transporters. MRP4 is identified as an endogenoustransporter of cyclic nucleotides and utilizes the energy of ATPhydrolysis to translocate the substrates across the membrane(1, 2). As an endogenous efflux transporter of cyclic nucleo-tides, MRP4 can regulate the intracellular cAMP and cGMPconcentrations together with the different isoforms of phos-phodiesterase. Thus, MRP4 can control various cyclic nucle-otide-dependent signaling pathways. The role of cyclic nucleo-tides and their downstream signalingmolecules in regulation ofdifferent types of cellmigration is well established (3, 4). Earlier,some studies showed the two nucleotides had an oppositeeffect; and, according to the “Yin-Yang” hypothesis (4), cAMPinhibits, whereas cGMP stimulates, cell migration. But theactual role of cAMP and cGMP in cell migration is still contro-versial. Some recent studies have revealed the importance ofcAMP and cAMP-dependent protein kinase A (PKA) for local-ized cell protrusion and efficient cell migration (5, 6). The lead-ing edge of a migrating cell accumulates more PKA comparedwith the rest of the cell body, and this compartmentalized acti-vation of PKA is an important early step in directional cellmigration. PKA has been found to both facilitate and inhibitactin cytoskeleton dynamics and cell migration because of thediversity of the substrate for PKA. But inhibition of PKA activ-ity as well as interference with PKA anchoring leads to a defec-tive pseudopod formation followed by an attenuated cellmigra-tion in a Rac GTPase-dependent fashion (7). Paradoxically,elevation of cAMP inhibits lamellipodia formation in mouseembryonic fibroblast cells (MEFs) andmouse breast tumor cellsby acting downstream of small GTPase Rac (8). In contrast,cGMP-dependent protein kinase (PKG) activation decreasesphosphorylation of the actin-severing protein cofilin (Ser-3)and hence helps in generating actin monomer for polymeriza-tion of new filaments in the cell protrusions. Conversely, inhypoxic conditions, adhesion andmigration of vascular smoothmuscle cells increase due to down-regulation of PKG (9). Cells

* This work was supported, in whole or in part, by National Institutes of HealthGrants RO1-DK080834, RO1-DK093045 (to A. P. N.), and RO1-GM60904 (to J. D. S).

□S This article contains supplemental Movie 1.1 To whom correspondence should be addressed: Dept. of Physiology, Uni-

versity of Tennessee Health Science Center, 894 Union Ave., 426 NashResearch Bldg., Memphis, TN 38163. Tel.:1-901-448-3137; Fax: 1-901-448-7126; E-mail:anaren@uthsc.edu.

2 The abbreviations used are: MRP4, multidrug resistance protein 4; MEF,mouse embryonic fibroblast; IBMX, isobutylmethylxanthine; PKA, cAMP-

dependent protein kinase A; PKG, cGMP-dependent protein kinase; RWD,relative wound density.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 6, pp. 3786 –3794, February 8, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

3786 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 6 • FEBRUARY 8, 2013

that overexpress vasodilator-stimulated phosphoprotein, amajor substrate of PKG, have a higher rate of protrusion forma-tion but have a slower cell migration rate. PKG-dependentphosphorylation of vasodilator-stimulated phosphoprotein canrelieve this overall negative effect on cellmigration (10, 11). TheRac GTPase and p21-activated kinase-dependent feedbackmechanism is essential for repetitive behavior of cell migration,and cGMP positively regulates the activity of Rac and p21-acti-vated kinase and thus stimulates the process of cell migration.PKA and PKG can also exert differential effects on contractilityand tension transmission inmigrating cells via phosphorylationand activation of both myosin light chain kinase and myosinphosphatase (10). Thus, cyclic nucleotides and cyclic nucle-otide-dependent kinases are involved in and regulate differentsignaling events that compose cell migration. Highly regulatedPKA and PKG activities and therefore a balanced intracellularcyclic nucleotide concentration are essential for proper cellmovement.In our studies, we hypothesize that it is not just the overall

increase or decrease in intracellular cyclic nucleotide level butrather a tight balance between cAMP and cGMP that is essen-tial for the process of cell migration. To test our hypothesis andto understand the dichotomous role of cyclic nucleotides in cellmigration, we used MEFs from Mrp4�/� and Mrp4�/� miceand NIH 3T3 cells for migration assays. We found that (a)MRP4 is an intracellular cAMP/cGMP transporter in thesefibroblasts and has a profound effect on intracellular cyclicnucleotide dynamics; (b) cyclic nucleotides have a biphasiceffect on fibroblast migration; and (c) a highly regulated intra-cellular cAMP/cGMP concentration is important for fine-tuned fibroblast migration.

EXPERIMENTAL PROCEDURES

Reagents—MRP4 inhibitor MK571 was obtained from Cay-man Chemical (Ann Arbor, MI). Forskolin was obtained fromTocris (Ellisville, MO). IBMX, cpt-cAMP, and cpt-cGMP werepurchased from Sigma-Aldrich. Zaprinast was purchased fromEnzo Life Sciences (Farmingdale, NY).Cell Cultures—MEF cells andNIH 3T3 cells were cultured in

DMEM (Invitrogen)media containing 10% FBS and 1% penicil-lin/streptomycin andmaintained in 5%CO2 incubator at 37 °C.Lipofectamine LTX reagent was used to transfect theMrp4�/�

and Mrp4�/� primary MEFs with 1 �g of total SV40 genomicDNA. One day post transfection the cells were serially diluted(1000 to 10 cells per well) and plated in flat bottom 96-wellplates. SV40 immortalized clones were picked from the lowerdilutions wells where the media turned into yellow. Lipo-fectamine 2000 (Invitrogen) was used for all transient transfec-tions, and MRP4-overexpressing cell lines were generated byusing lentivirus vector and selected by puromycin (2 �g/ml).Animals—Mrp4�/� andMrp4�/� (C57BL/6)mice (12) were

provided by Dr. John Schuetz (St. Jude Children’s ResearchHospital, Memphis, TN).Skin Explant Outgrowth Assay—Full thickness dorsal skin

biopsies (5 mm) were excised from 8 weeks old MRP4�/� andwild-type mice after removing the hair and were fixed infibronectin-coated six-well plates, placing the dermal sidedown. The skin explants were cultured in 2ml of DMEMmedia

containing 10% FBS and 1% penicillin/streptomycin and main-tained in a 5% CO2 incubator at 37 °C for 5 days (13, 14). Rep-resentative images of 100� magnification of the cellular out-growth from each explant were taken, and the proximaldistance of cellular migration was measured by NIH Image Jsoftware.High Content Microscopy—Cells were grown for 24 h in a

fibronectin-coated 96-well Essen BioScience ImageLockmicroplate in a standard CO2 incubator. Wounds were madeprecisely by the 96-pin Wound-Maker provided with theIncuCyteTM (15). After washing thoroughly with PBS toremove the detached cells, the cells were placed in theIncuCyteTM with appropriate media. The wound images wereautomatically acquired by IncuCyteTM (Essen Bioscience) fromthe incubator at 1-h intervals. The kinetics of the relativewound density (RWD) was analyzed by IncuCyteTM software.Wound Healing Assay—Cell migration was measured

according to the previously describedmethod (16). In brief, theconfluent monolayers of fibroblast cells were wounded byscraping with a pipette tip across the monolayer. Cells werewashed with PBS and incubated with appropriate media.Images of 100� magnification were taken at the initial time ofwounding and then at 6 h or 10 h post-wounding using a cooledelectron microscope (EM)-CCD camera (Hamamatsu; Bridge-water; Denver, CO). The cell migration was analyzed by ImageJ software and represented as a percentage of initial woundlength.Immunoblotting—For detection of MRP4 in fibroblast cells

using Western blotting, the cells were lysed in lysis buffer (1�PBS, containing 0.2% Triton X-100 and protease inhibitors: 1mM phenylmethylsulfonyl fluoride, 1 �g/ml pepstatin A, 1�g/ml leupeptin, and 1 �g/ml aprotinin). The lysate was cen-trifuged at 16,000 � g for 10 min at 4 °C, and the clear superna-tant was mixed with 5� Laemmli sample buffer (containing2.5% �-mercaptoethanol), denatured, subjected to SDS-PAGEon 4–15% gel (Bio-Rad), transferred to a PVDFmembrane, andthe membranes were then incubated with specific antibodies.We used anti-MRP4 antibody (rabbit polyclonal antibody) (12)and anti-�-actin antibody (mouse) that was purchased fromSigma-Aldrich. The protein bands were detected by chemi-luminescence using ECLTM Western blotting detectionreagents from GE Healthcare. We loaded 50 �g of total pro-tein per lane.Fluorescence Resonance Energy Transfer Microscopy—CFP-

EPAC-YFPwas a generous gift fromDr. Kees Jalink (Division ofCell Biology, The Netherlands Cancer Institute, Amsterdam,The Netherlands), and Cygnet 2.1 was a generous gift from Dr.Wolfgang RDostmann (Department of Pharmacology, Univer-sity of Vermont, Burlington, VT). Cells were transiently trans-fectedwith CFP-EPAC-YFP (cAMP sensor) (17) or Cygnet 2.1(cGMP sensor) (18) and were grown in 35-mm glass-bot-tomed dishes (MatTek) for 24–48 h; cells were then washedwith Hanks Balanced Salt Solution and mounted on anOlympus microscopy system for FRET imaging. Images wererecorded with a cooled CCD camera Hamamatsu ORCA285(Hamamatsu, Japan) mounted on the Olympus microscopeIX51 (U-Plan Fluorite 60 � 1.25 NA oil-immersion objective),and the system was controlled by SlideBook software (version

MRP4 Regulates Fibroblast Migration

FEBRUARY 8, 2013 • VOLUME 288 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 3787

4.1, Intelligent Imaging Innovations; Denver, CO) with ratioand FRETmodules used to obtain and analyze the images. Exci-tation light was provided by a 300-watt Xenon lamp and atten-uated with a Neutral Density filter with 50% light transmission.Images were captured using a JP4 CFP/YFP filter set (Chroma;Brattleboro, VT), including a 430/25-nm excitation filter, adouble dichroic beam splitter, and two emission filters (470/30-nm for CFP and 535/30-nm for FRET) alternated by a filter-changer Lambda 10-3 (Sutter Instruments; Novato, CA). Time-lapse images were acquired with 100 ms of exposure time, and1-min intervals. Multiple regions of interest on the cell wereselected after background subtraction for quantitative dataanalysis (4–6 cells per condition were averaged). The emissionratio images (CFP/FRET)were obtained at different time pointsas described previously (19). The representative pseudocolorcell images of 600� magnification were shown to highlight thechanges in the ratio of CFP/FRET fluorescence intensity.Cyclic AMP and cGMP Measurement—cAMP and cGMP

weremeasured by specific competitive immunoassay accordingto the manufacturer’s instructions (Enzo Life Sciences; com-plete ELISA kit for cAMP and cGMP). The Fluostar Omega(BMG Labtech) microplate reader was used to measure theoptical density at 405 nm.Statistical Analysis—Student’s t test (two-tailed) was per-

formed to compare the mean values of different groups; p val-ues � 0.05 were considered to be significant. All of the resultsare represented as mean � S.E., with n equaling the number ofexperiments.

RESULTS

MRP4-deficient Mice Fibroblast Cells Migrate Rapidly andHealWounds Faster—Cyclic nucleotides and cyclic nucleotide-dependent kinases are important for different hallmark steps ofcell migration (5, 10). Among the endogenous substrates,MRP4 has been reported to have high affinity for cAMP andcGMP (10, 20). Hence, MRP4 can regulate the intracellularcyclic nucleotide level; and MRP4-deficient cells have alteredcyclic nucleotides levels, leading to modified cAMP/cGMP-mediated signaling pathways, including cell migration. Here,we study the role of MRP4 in cell migration using the ex vivoskin explant outgrowth assay. We isolated the skin fromMrp4�/� and Mrp4�/� mice and then made biopsy patches.They were grown in fibronectin-coated wells for 4 days asdescribed under “Experimental Procedures.” The fibroblastcells migrated out from the skin patches after 2 days. The rep-resentative images of cellular outgrowth from skin explants atday 4 post-culture are shown in Fig. 1A. Coating with fibronec-tin (10 �g/ml) helped the fibroblasts to migrate properly. Anextensive halo of cells appeared aroundMrp4�/� and wild typemice skin explants. ButMrp4�/� cells migrated 40%more rap-idly than the wild type (Fig. 1B).For in vitro wound healing assays, we used two most com-

monly used fibroblast cell types: 1) MEFs and 2) NIH 3T3. Wegenerated MEFs from wild type andMrp4�/� mice (21). Usinga high-content microscopic technique, we investigated the reg-ulation of fibroblast migration by MRP4 in vitro. The precisewounds were made in the confluent monolayer of MEFs iso-

FIGURE 1. MRP4-deficient fibroblast cells can migrate faster. Biopsy patches (5 mm) isolated from skin of Mrp4�/� mice or wild type mice were cultured withappropriate media in fibronectin-coated (10 �g/ml PBS) six-well dishes. A, the halo of fibroblast cells came out from the skin explant after 2 days, and the imageswith 100� magnification represent the 4th day post-seeding. B, the proximal distance of the cellular outgrowth from the explant is represented in the bargraph. C, the MEFs isolated from Mrp4�/� or Mrp4�/� mice were grown on fibronectin-coated 96-well Essen BioScience ImageLock dishes, and the woundswere made precisely using the 96-pin Wound-Maker provided with the IncuCyteTM. The kinetics of the RWD was analyzed using IncuCyteTM software. D, theconventional wound healing assay was performed on the Mrp4�/� or Mrp4�/�or MK571-treated Mrp4�/� MEFs, and images were taken with 100� magnifi-cation at the 0 and 6th hour. E, the initial and final wound lengths were calculated, and cell migrations as a percentage of initial wound length are representedin the bar graph. F, the immune blots data show the expression of MRP4 in Mrp4�/�, Mrp4�/�, and MK571-treated Mrp4�/� MEFs. All data represent at leastthree independent experiments (n � 3). *, p � 0.05.

MRP4 Regulates Fibroblast Migration

3788 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 6 • FEBRUARY 8, 2013

lated from eitherMrp4�/� orMrp4�/� mice, and images weretaken at 1-h intervals for 18 h using the IncuCyteTM live cellimaging system.TheRWD, the spatial cell density in thewoundarea relative to the spatial cell density outside the wound area,wasmeasured at every time point. The RWDparameter has theadvantage of being self-normalized for changes in cell densitydue to cell proliferation outside the wound area. The kinetics ofthe RWD is depicted in Fig. 1C. The MRP4-deficient MEFswere found to have faster RWD kinetics than Mrp4�/� MEFs.These data were supported by the conventional in vitro woundhealing assays in which cell migration was represented as a per-centage of initial wound length. Cell migration was measuredfor Mrp4�/�, Mrp4�/�, and MRP4 inhibitor MK571-treatedMrp4�/� MEFs. MK571-treated MEFs had a similar migrationrate as Mrp4�/� MEFs, and both migrated significantly fasterthan the wild typeMEFs (Fig. 1,D and 1E). MK571 did not alterthe MRP4 expression in MEFs (Fig. 1F) but antagonized thefunction ofMRP4. All of these data indicated thatMRP4 has aninhibitory effect on MEF migration. To further investigate theeffect ofMRP4 on fibroblast cell migration, we used the in vitrowound healing assay with control and MRP4-overexpressingNIH 3T3 cell lines. The MRP4-overexpressing stable cell linehad a higher level of functionally active MRP4 compared withthe control NIH 3T3 cell line (Fig. 2A). The high-contentmicroscopy studies revealed slower RWD kinetics for NIH 3T3

cells overexpressing MRP4 compared with the control cells(Fig. 2B). In conventional wound healing experiments, theMRP4 overexpression also showed an inhibitory effect on cellmigration (Fig. 2,C andD). These data suggest thatMRP4 playsa key role in fibroblastmigration.Given thatwe and others havealready shown that MRP4 has an effect on intracellular cyclicnucleotide dynamics (19, 22), and cAMP and cGMP areinvolved in many hallmark steps of cell migration (6, 10), werationalized that theMRP4mediated regulation of intracellularcyclic nucleotides levels is important in this aspect.Inhibition of MRP4 Augments the Intracellular Cyclic Nucle-

otide Levels in Fibroblast Cells—It has been reported thatMRP4 is an endogenous efflux transporter of cyclic nucleotidesand thereby regulates the intracellular cyclic nucleotidedynamics (1). To investigate the effect of MRP4 inhibition onintracellular cAMP and cGMP dynamics in live fibroblast cells,we used FRET microscopy. We transiently transfected theMRP4-overexpressing NIH 3T3 cells either with cAMP sensorCFP-EPAC-YFP (23) or with cGMP sensor, Cygnet 2.1 (18).Using these unimolecular FRET-based sensors, we were able tomonitor the intracellular cAMP and cGMP dynamics with avery high temporal and spatial resolution. Application of 20�M

(19) of the MRP4 inhibitor MK571 induced an increase in theintracellular cAMP level represented by the CFP/FRET emis-sion ratio (Fig. 3, A and B) in fibroblast cells. Similarly, thecGMP level was also increased by the addition of 20�MMK571(Fig. 3, C and D). For both of the cases, the effect was ratherrapid (within 30 s). These data confirmed that inhibition ofMRP4 is responsible for the increases in both intracellularcAMP and cGMP levels. The effect was more profound, how-ever, on the cAMP level compared with the cGMP level, possi-bly indicating a higher affinity of MRP4 for cAMP.IncreasedAccumulation of IntracellularCyclicNucleotides in

Mrp4�/� MEF Cells—MRP4 as an efflux transporter of cyclicnucleotides is capable of regulating the intracellular cAMP/cGMP level. In the absence of MRP4, there should be a dimin-ished cyclic nucleotides extrusion rate and increased intracel-lular cAMP/cGMP levels. We used a highly sensitive FRET-based cAMP sensor (CFP-EPAC-YFP) and cGMP sensor(Cygnet 2.1) for monitoring the intracellular cyclic nucleotidelevels inMrp4�/� andMrp4�/�MEFs. In the absence ofMRP4,theMEFs showed a higher basal level of intracellular cAMP andcGMP (Fig. 4, A and C) compared with the MEFs that endoge-nously express MRP4. When we treated the cAMP sensor-transfected cells with 10 �M forskolin, a global elevator ofcAMP, and 100 �M IBMX, a nonspecific phosphodiesteraseinhibitor, the rise in cAMP level was higher inMrp4�/� MEFscompared with the Mrp4�/� MEFs. We also noticed that theeffect of forskolin and IBMX on cAMP level was more promi-nent compared with the absence of MRP4 (Fig. 4, A and B).After addition of zaprinast, a specific phosphodiesterase 5inhibitor, to the cGMP sensor-transfected cells, intracellularcGMP level increased more inMrp4�/� cells than inMrp4�/�

MEFs (Fig. 4,C andD). Therewas, however, a transient increasein cGMP level after addition of zaprinast, whereas a sustainedincrease in cAMP level was obtained after addition of forskolinand IBMX. Using specific competitive immunoassays forcAMP and cGMP, we further found the higher intracellular

FIGURE 2. MRP4 overexpression decreases fibroblast cells migration. A,MRP4-overexpressing stable NIH 3T3 cells had a higher expression of MRP4compared with the NIH 3T3 parental cells. B, the MRP4-overexpressing andparental NIH 3T3 cells were grown on fibronectin-coated 96-well Essen Bio-Science ImageLock dishes, and the wounds were made precisely using the96-pin Wound-Maker provided with the IncuCyteTM. The kinetics of the RWDwas analyzed using IncuCyteTM software. C, the conventional wound healingassay was performed on the MRP4-overexpressing or parental NIH 3T3 cells,and images were taken with 100� magnification at the 0 and 10th hour. D,the initial and final wound lengths were calculated, and cell migrations as apercentage of initial wound length are represented in the bar graph. All datarepresent at least three independent experiments (n � 3). *, p � 0.05.

MRP4 Regulates Fibroblast Migration

FEBRUARY 8, 2013 • VOLUME 288 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 3789

cAMP and cGMP levels in MRP4-deficient MEFs (Fig. 4, E andF). When we treated the Mrp4�/� MEFs with 10 �M forskolinand 100 �M IBMX for 10 min, the intracellular cAMP levelincreased to a higher extent compared with the Mrp4�/�

MEFs. The data suggest that the intracellular cAMP level can bemoderately regulated by MRP4, whereas forskolin and IBMXcan strongly elevate the cAMP level inside the cells.Migration of MEFs Is Attenuated in the Presence of Excessive

cAMP—We observed that the inhibition of the cyclic nucleo-tide transporter MRP4 led to accumulation of more cAMPinside the cells and resulted in an increased migration rate. Asthe importance of cAMP in directional migration is well estab-lished (5), the global cAMP-inducing agent forskolin and thenonspecific phosphodiesterase inhibitor IBMX should regulatecell migration. The strong elevation with forskolin and reduceddegradationwith IBMX lead to a robust increase in intracellularcAMP level. So, we next tried to find the effect of forskolin andIBMX treatment onMEF migration. The high content micros-copy studies revealed that the RWDkinetics is faster for controlMEFs compared with the forskolin- and IBMX-treated MEFs(Fig. 5A). A similar result was found with the conventionalwound healing experiments, which showed significantly slowercellmigration after forskolin and IBMX treatment (Fig. 5,B andC). However, using the CFP-PKA regulatory subunit and YFP-PKA catalytic subunit as FRET-based cAMP sensor, we foundinhibition of MRP4 increased the cAMP level near the leadingedge of amigrating fibroblast, whereas forskolin or IBMX treat-ment caused a robust increase in the cAMP level throughoutthe cell (supplemental Movie S1). Thus, forskolin and IBMX,which strongly elevate the intracellular cAMP level, had aninhibitory effect on MEF cell migration; whereas inhibition ofMRP4, which moderately increased the cAMP level inside thecell, stimulated cell migration.

Cyclic Nucleotides Show a Biphasic Effect on MEF CellMigration—The effect ofMRP4 inhibition on fibroblast migra-tion was found to be opposite to the effect of forskolin andIBMX treatment. This paradoxical effect of cAMP on cellmigration compelled us to investigate whether cyclic nucleo-tides regulate cell migration in a concentration-dependentmanner. We treated the MEFs with different concentrations ofcell-permeable cpt-cAMP and cpt-cGMP (0, 10, 20, 50, 100,and 200 �M). With conventional in vitrowound healing assays,we found both cAMP and cGMP had a biphasic effect on cellmigration. Up to 20 �M cAMP can stimulate cell migration in adose-dependent fashion; but after that, it began to decrease themigration rate (Fig. 6A). We further noticed that the intracel-lular concentration of cAMP for Mrp4�/� MEFs was � 7 �M

and forMrp4�/�MEFs� 10�M, whereas prolonged treatmentwith 10 �M forskolin and 100 �M IBMX (�30 min) stronglyincreased the concentration up to� 42�M (Fig. 6A).Where theabsence of MRP4 increased the intracellular cAMP concentra-tion to a level at which cAMP exerted its stimulating effect onmigration, extended treatment with forskolin or IBMX furtherraised the concentration; and, as a consequence, cell migrationwas inhibited. cGMP had a similar biphasic effect, but the con-centration range for stimulationwas broader, and the effectwasmilder than with cAMP. It positively regulated cell migrationup to 100 �M and then exerted the inhibitory effect (Fig. 6B).Altogether, these data suggest that a moderate elevation ofcyclic nucleotides stimulates cell migration but that a furtherincrease leads to inhibition.

DISCUSSION

Our study demonstrates that inhibition ofMRP4moderatelyincreases the intracellular cyclic nucleotide level and stimulatesfibroblast cell migration. MRP4 was first identified as an efflux

FIGURE 3. Inhibition of MRP4 augments intracellular cAMP and cGMP levels. MRP4-overexpressing NIH 3T3 cells were transfected with a FRET-based cAMPsensor CFP-EPAC-YFP or cGMP sensor Cygnet 2.1. A, the pseudocolor images of CFP/FRET with 600� magnification before and 5 min after addition of MK571(20 �M) for cells expressing cAMP sensor are shown. B, the line graph indicates the intracellular cAMP dynamics before and after addition of MK571 as a changein CFP/FRET ratio. C, the pseudocolor images of CFP/FRET with 600� magnification before and 5 min after addition of MK571 (20 �M) for cells expressing cGMPsensor are shown. D, the line graph indicates intracellular cGMP dynamics before and after addition of MK571 as a change in CFP/FRET ratio. Images in eachpanel were captured from the same field of view. The colored bar shows the magnitude of the emission ratio. All data represent at least three independentexperiments (n � 3). *, p � 0.05.

MRP4 Regulates Fibroblast Migration

3790 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 6 • FEBRUARY 8, 2013

transporter of a nucleoside-based antiviral drug in a humanT-lymphoid cell line (12). Besides having a role in conferringdrug resistance, MRP4 was also found to regulate quite diversephysiological phenomena via alteration of the intracellular con-centration of its different endogenous substrates (24). How-ever, among the endogenous substrates, MRP4 has beenreported to have the high affinity for cAMP (Km � 45 �M) andcGMP (Km � 10 �M), and these two cyclic nucleotides are veryimportant signaling molecules involved in many physiologicaland pathological events. These elegant but simple secondarymessengers regulate different complex cellular phenomena,mostly via the activation of their corresponding kinases (25, 26).

Cell migration is a highly integrated multistep process thatplays a central role in embryonic development, tissue repair,cancer, and many other physiological and pathological events(27, 28). These secondary messengers play important roles inthe signaling pathways at various stages of the cell migrationprocess, either directly or by activating their correspondingkinases. The PKA activity increases at the leading edge of amigrating cell in a Rac GTPase-dependent manner and isimportant for maintaining cell polarity and pseudopod forma-tion (6). Conversely, cAMP has been reported to inhibit Rac-induced lamellipodia formation (8). Similarly, PKG has beenshown to be down regulated during vascular smooth musclecell migration (9), whereas PKG is also important for maintain-ing the repetitive behavior of migration by phosphorylatingvasodilator-stimulated phosphoprotein (10). The complex andequivocal effects of cAMP and cGMP on cell migration ensurea tightly regulated and balanced intracellular cyclic nucleotidelevel for fine-tuned cell migration.Our ex vivo skin explant outgrowth study as well as the high-

content microscopy and conventional wound healing datashowed significantly increased cell migration for MRP4-defi-cient fibroblast cells, suggesting an important role of MRP4 inthe regulation of fibroblast migration. In similar experiments,inhibition of MRP4 significantly increased cell migration,whereas overexpression of MRP4 reduced fibroblast motilitysuggesting a negative effect of MRP4 on fibroblast cell migra-tion. To understand the mechanism, we focused on differentendogenous substrates for MRP4 that are important in thisaspect. As MRP4 has high affinity for cAMP and cGMP andthese 2-s messengers were already reported to be involved indifferent hallmark steps in cell migration, we tried to monitorthe effect of MRP4 inhibition on intracellular cyclic nucleotidelevels. Using FRET-based sensors for cAMP and cGMP, wefound that MRP4 inhibition by MK571 increased both cAMPand cGMP levels inside the cells and that the effectwas strongerfor cAMP compared with cGMP, indicating the higher affinityof MRP4 for cAMP. Using the same sensor, we found thatMrp4�/� MEFs had higher basal levels of cAMP and cGMP.The intracellular cyclic nucleotide level is controlled by phos-phodiesterase-mediated hydrolysis and by the process involv-ing active efflux transport from the cell. The phosphodiesterase5 inhibitor zaprinast prevents breakdown of cGMP and thusincreases the intracellular cGMP level. In the absence ofMRP4,the extrusion of cGMP from the cell was also reduced, and therewas more accumulation of intracellular cGMP; therefore, thezaprinast-induced elevation of cGMP is greater in Mrp4�/�

MEFs. Forskolin, which elevates the cAMP level by stimulatingadenylate cyclase, and the nonspecific phosphodiesteraseinhibitor IBMX cause a huge and prolonged increase in theintracellular cAMP level. The MRP4-deficient cells accumu-lated more cAMP than Mrp4�/� MEFs upon treatment withforskolin and IBMX because of less efflux transportation ofcAMP. We found that the sustained and robust increase incAMP level evoked by forskolin and IBMX inhibited fibroblastmigration, whereas a stimulatory effect was shown for a mod-erate increase in cAMP level, mostly near the leading edge of amigrating fibroblast, by the inhibition of MRP4. Thus, MRP4inhibition may facilitate fibroblast cell polarization, which is a

FIGURE 4. MRP4-deficient MEFs have moderately higher levels of intra-cellular nucleotides. Mrp4�/� MEFs or Mrp4�/� MEFs were transfected witha FRET-based cAMP sensor CFP-EPAC-YFP or cGMP sensor Cygnet 2.1. A, thepseudocolor images of CFP/FRET with 600� magnification before and 5 minafter addition of forskolin (10 �M) and IBMX (100 �M) for cells expressingcAMP sensor are shown. B, the bar graphs represent the CFP/FRET ratio inbasal condition and after treatment in MEFs from Mrp4�/� (black bars) orMrp4�/� (gray bars). C, the pseudocolor images of CFP/FRET with 600� mag-nification before and 5 min after addition of zaprinast (200 �M) for cellsexpressing cGMP sensor are shown. D, the bar graphs represent the CFP/FRETratio in basal condition and after treatment in MEFs from Mrp4�/� (black bars)or Mrp4�/� (gray bars). Images in each panel were captured from the samefield of view. The colored bar shows the magnitude of the emission ratio. E, theintracellular cAMP levels in Mrp4�/�, Mrp4�/�, and forskolin (10 �M) and IBMX(100 �M) treated Mrp4�/� MEFs were measured using competitive immuno-assay. F, in similar experiments, cGMP levels were also measured for Mrp4�/�

and Mrp4�/� MEFs. All data represent at least three independent experi-ments (n � 3). *, p � 0.05.

MRP4 Regulates Fibroblast Migration

FEBRUARY 8, 2013 • VOLUME 288 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 3791

hallmark step for directionalmigration. In wound healing stud-ies with cell-permeable cyclic nucleotides, cpt-cAMP in con-centrations up to 20 �M enhanced the cell migration in a con-centration-dependent manner; after that, the effect wasinhibitory. The intracellular cAMP level ofMrp4�/� MEFs wasmoderately higher than that ofMrp4�/� MEFs (1.5 fold), andit fell within the stimulatory range (1–20 �M). Prolonged treat-ment (�30 min) with forskolin and IBMX, however, increasedthe level further (6.5 fold), at which point the effect of cAMPwas no longer stimulatory, but instead inhibitory to cell migra-tion. This explains the opposing effects of forskolin, IBMXtreatment, and MRP4 inhibition on cell migration. A similarbut milder biphasic effect was also shown for cpt-cGMP, whichstimulated cell migration when used in concentrations up to100 �M. Hence, balanced and highly controlled intracellularcyclic nucleotides levels are implicated as important in fibro-blast cell migration.Similar to our findings, the knockdown of MRP4 has already

been reported to enhance human retinal vascular endothelial

cell migration (29). However, some other studies with dendriticcells (24) and arterial smooth muscle cells (22) showed a stim-ulatory effect of MRP4 on cell migration. This dichotomy ofMRP4 as an effector may be related to the ambiguous role ofcyclic nucleotides in cell migration and can be explained by thetissue-specific expression level of MRP4 and its interactome.Variation inMRP4-dependent regulation of intracellular cyclicnucleotide levels and differential effects of cyclic nucleotides ondifferent types of cell migration could be the major issue. Theother endogenous substrates of MRP4 may also be involved inthis process of regulation (30). Taken together, our findingssuggest thatMRP4 can negatively regulate fibroblast cellmigra-tion, at least in part, in a cyclic nucleotide-dependent manner.Our observation that inhibition of MRP4 increases fibroblastcell migration warrants the use of MRP4 inhibitor as a new andpotent wound healing agent. During the use of MRP4 inhibitorto reduce chemotherapy resistance, the indirect effect on can-cer metastasis should be taken into consideration. Furtherdetailed studies are required to examine the role of MRP4 in

FIGURE 5. Forskolin and IBMX inhibit MEF migration by strongly elevating intracellular cAMP levels. A, dimethyl sulfoxide (DMSO)-treated or forskolin (10�M) and IBMX (100 �M) treated Mrp4�/� MEFs were grown on fibronectin-coated 96-well Essen BioScience ImageLock dishes, and the wounds were madeprecisely using the 96-pin Wound-Maker provided with the IncuCyteTM. The kinetics of the RWD was analyzed by IncuCyteTM software. B, the conventionalwound healing assay was done with dimethyl sulfoxide- or forskolin (10 �M) and IBMX (100 �M) treated Mrp4�/� MEFs, and images were taken with 100�magnification at the 0th and 6th hour. C, the initial and final wound lengths were calculated, and cell migrations as a percentage of initial wound length arerepresented in the bar graph. All data represent at least three independent experiments (n � 3). *, p � 0.05.

MRP4 Regulates Fibroblast Migration

3792 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 6 • FEBRUARY 8, 2013

different types of cancer metastasis. However, together withenhanced wound repair, augmentation of intracellular cAMPby inhibition of MRP4 can be a good strategy to reduce fibro-blast to myofibroblast transformation (31, 32) for preventingunwanted tissue fibrosis.

REFERENCES1. Russel, F. G., Koenderink, J. B., and Masereeuw, R. (2008) Multidrug re-

sistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugsand signaling molecules. Trends Pharmacol. Sci. 29, 200–207

2. Schinkel, A. H., and Jonker, J. W. (2003) Mammalian drug efflux trans-porters of the ATP binding cassette (ABC) family: an overview.Adv. DrugDeliv. Rev. 55, 3–29

3. Elferink, J. G., and de Koster, B. M. (1993) The effect of cyclic GMP andcyclic AMP on migration by electroporated human neutrophils. Eur.J. Pharmacol. 246, 157–161

4. Elferink, J. G., andVanUffelen, B. E. (1996)The role of cyclic nucleotides inneutrophil migration. Gen. Pharmacol. 27, 387–393

5. Howe, A. K. (2004) Regulation of actin-based cell migration by cAMP/PKA. Biochim. Biophys. Acta 1692, 159–174

6. Lim, C. J., Kain, K.H., Tkachenko, E., Goldfinger, L. E., Gutierrez, E., Allen,M. D., Groisman, A., Zhang, J., and Ginsberg, M. H. (2008) Integrin-mediated protein kinaseA activation at the leading edge ofmigrating cells.Mol. Biol. Cell 19, 4930–4941

7. Howe, A. K., Baldor, L. C., andHogan, B. P. (2005) Spatial regulation of thecAMP-dependent protein kinase during chemotactic cell migration. Proc.Natl. Acad. Sci. U.S.A. 102, 14320–14325

8. Chen, L., Zhang, J. J., andHuang, X. Y. (2008) cAMP inhibits cellmigrationby interfering with Rac-induced lamellipodium formation. J. Biol. Chem.283, 13799–13805

9. Negash, S., Narasimhan, S. R., Zhou,W., Liu, J.,Wei, F. L., Tian, J., and Raj,J. U. (2009) Role of cGMP-dependent protein kinase in regulation of pul-monary vascular smooth muscle cell adhesion and migration: effect ofhypoxia. Am. J. Physiol. Heart Circ. Physiol. 297, H304–312

10. Guo, D., Tan, Y. C., Wang, D., Madhusoodanan, K. S., Zheng, Y., Maack,T., Zhang, J. J., and Huang, X. Y. (2007) A Rac-cGMP signaling pathway.Cell 128, 341–355

11. Krause, M., Dent, E. W., Bear, J. E., Loureiro, J. J., and Gertler, F. B. (2003)Ena/VASP proteins: regulators of the actin cytoskeleton and cell migra-tion. Annu. Rev. Cell Dev. Biol. 19, 541–564

12. Schuetz, J. D., Connelly, M. C., Sun, D., Paibir, S. G., Flynn, P. M., Srinivas,R. V., Kumar, A., and Fridland, A. (1999)MRP4: A previously unidentifiedfactor in resistance to nucleoside-based antiviral drugs. Nat. Med. 5,1048–1051

13. Kopecki, Z., Arkell, R., Powell, B. C., and Cowin, A. J. (2009) Flightless Iregulates hemidesmosome formation and integrin-mediated cellular ad-hesion and migration during wound repair. J. Invest. Dermatol. 129,2031–2045

14. Mazzalupo, S., Wawersik, M. J., and Coulombe, P. A. (2002) An ex vivoassay to assess the potential of skin keratinocytes for wound epithelializa-tion. J. Invest. Dermatol. 118, 866–870

15. Liu, L., Wang, Y. D., Wu, J., Cui, J., and Chen, T. (2012) Carnitine palmi-toyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR andmediates PAX3-FKHR-dependent motility in alveolar rhabdomyosar-coma cells. BMC Cancer 12, 154

16. Ghosh,M. C.,Makena, P. S., Gorantla, V., Sinclair, S. E., andWaters, C.M.(2012) CXCR4 regulatesmigration of lung alveolar epithelial cells throughactivation of Rac1 and matrix metalloproteinase-2. Am. J. Physiol. LungCell Mol. Physiol. 302, L846–856

17. Ponsioen, B., Zhao, J., Riedl, J., Zwartkruis, F., van der Krogt, G., Zaccolo,M., Moolenaar, W. H., Bos, J. L., and Jalink, K. (2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epacas a novel cAMP indicator. EMBO Reports 5, 1176–1180

18. Honda, A., Adams, S. R., Sawyer, C. L., Lev-Ram, V., Tsien, R. Y., andDostmann, W. R. (2001) Spatiotemporal dynamics of guanosine 3,5-cyclic monophosphate revealed by a genetically encoded, fluorescent in-dicator. Proc. Natl. Acad. Sci. U.S.A. 98, 2437–2442

19. Li, C., Krishnamurthy, P. C., Penmatsa, H., Marrs, K. L., Wang, X. Q.,Zaccolo,M., Jalink, K., Li, M., Nelson, D. J., Schuetz, J. D., andNaren, A. P.(2007) Spatiotemporal coupling of cAMP transporter to CFTR chloridechannel function in the gut epithelia. Cell 131, 940–951

20. Sellers, Z. M., Naren, A. P., Xiang, Y., and Best, P. M. (2012) MRP4 andCFTR in the regulation of cAMP and �-adrenergic contraction in cardiacmyocytes. Eur. J. Pharmacol. 681, 80–87

21. Suetsugu, S., Yamazaki, D., Kurisu, S., and Takenawa, T. (2003) Differen-tial roles ofWAVE1 andWAVE2 in dorsal and peripheral ruffle formationfor fibroblast cell migration. Dev. Cell 5, 595–609

22. Hara, Y., Sassi, Y., Guibert, C., Gambaryan,N., Dorfmüller, P., Eddahibi, S.,Lompré, A. M., Humbert, M., and Hulot, J. S. (2011) Inhibition of MRP4prevents and reverses pulmonary hypertension inmice. J. Clin. Invest.121,2888–2897

23. Penmatsa, H., Zhang, W., Yarlagadda, S., Li, C., Conoley, V. G., Yue, J.,Bahouth, S. W., Buddington, R. K., Zhang, G., Nelson, D. J., Sonecha,M. D., Manganiello, V., Wine, J. J., and Naren, A. P. (2010) Compartmen-talized cyclic adenosine 3,5-monophosphate at the plasma membraneclusters PDE3A and cystic fibrosis transmembrane conductance regulatorinto microdomains.Mol. Biol. Cell 21, 1097–1110

24. van de Ven, R., Scheffer, G. L., Reurs, A. W., Lindenberg, J. J., Oerlemans,R., Jansen, G., Gillet, J. P., Glasgow, J. N., Pereboev, A., Curiel, D. T.,Scheper, R. J., and de Gruijl, T. D. (2008) A role for multidrug resistanceprotein 4 (MRP4; ABCC4) in human dendritic cell migration. Blood 112,2353–2359

FIGURE 6. cAMP and cGMP have a biphasic effect on fibroblast cell migra-tion. The conventional wound healing assay was done with Mrp4�/� MEFsusing different concentrations (0, 10, 20, 50, 100, and 200 �M) of cell-perme-able cpt-cAMP (A) or cpt-cGMP (B). Images were taken with 100� magnifica-tion at the 0 and 6th hour. The initial and final wound lengths were calculated,and the fold increases in cell migration rates were represented. A, the intra-cellular cAMP concentration were measured by competitive immunoassayfor Mrp4�/�, Mrp4�/�, and forskolin (10 �M) and IBMX (100 �M) treatedMrp4�/� MEFs. The intracellular cAMP levels corresponding to those cells areindicated by arrows. All data represent at least three independent experi-ments (n � 3).

MRP4 Regulates Fibroblast Migration

FEBRUARY 8, 2013 • VOLUME 288 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 3793

25. Scott, J. D. (1991) Cyclic nucleotide-dependent protein kinases. Pharma-col. Ther. 50, 123–145

26. Copsel, S.,Garcia,C.,Diez, F.,Vermeulem,M., Baldi,A., Bianciotti, L.G., Russel,F. G., Shayo, C., and Davio, C. (2011) Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMPcellular levels and controls human leukemia cell prolif-eration and differentiation. J. Biol. Chem.286, 6979–6988

27. Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H.,Borisy, G., Parsons, J. T., and Horwitz, A. R. (2003) Cell migration: inte-grating signals from front to back. Science 302, 1704–1709

28. Lauffenburger, D. A., andHorwitz, A. F. (1996) Cellmigration: a physicallyintegrated molecular process. Cell 84, 359–369

29. Tagami, M., Kusuhara, S., Imai, H., Uemura, A., Honda, S., Tsukahara, Y.,and Negi, A. (2010) MRP4 knockdown enhances migration, suppresses

apoptosis, and produces aggregated morphology in human retinal vascu-lar endothelial cells. Biochem. Biophys. Res. Commun. 400, 593–598

30. Lin, Z. P., Zhu, Y. L., Johnson, D. R., Rice, K. P., Nottoli, T., Hains, B. C.,McGrath, J., Waxman, S. G., and Sartorelli, A. C. (2008) Disruption ofcAMP and prostaglandin E2 transport by multidrug resistance protein 4deficiency alters cAMP-mediated signaling and nociceptive response.Mol. Pharmacol. 73, 243–251

31. Swaney, J. S., Roth, D. M., Olson, E. R., Naugle, J. E., Meszaros, J. G., andInsel, P. A. (2005) Inhibition of cardiac myofibroblast formation and col-lagen synthesis by activation and overexpression of adenylyl cyclase. Proc.Natl. Acad. Sci. U.S.A. 102, 437–442

32. Camelliti, P., Borg, T. K., and Kohl, P. (2005) Structural and functionalcharacterisation of cardiac fibroblasts. Cardiovasc. Res. 65, 40–51

MRP4 Regulates Fibroblast Migration

3794 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 6 • FEBRUARY 8, 2013