MCPIP1 Regulates Fibroblast Migration in 3-DCollagen Matrices Downstream of MAP Kinases andNF-κBJie Chao1,2, Xiaoniu Dai2, Tiffany Peña1, David A. Doyle1, Timothy M. Guenther1 and Mark A. Carlson1,3,4

The fibroblast-populated three-dimensional (3-D) collagen matrix has been used to model matrix contraction,cell motility, and general fibroblast biology. MCPIP1 (monocyte chemotactic protein–induced protein 1) has beenshown to regulate inflammation, angiogenesis, and cellular motility. In the present study, we demonstratedinduction of MCPIP1 in human fibroblasts embedded in the stress-released 3-D collagen matrix, which occurredthrough activation of mitogen-activated protein kinases, phosphoinositide 3-kinase, and NF-κB. Furthermore,MCPIP1 induction was associated with inhibition of fibroblast migration out of the nested collagen matrix.MCPIP1 induction or ectopic expression also upregulated p53. RNA interference of p53 prevented the inhibitionof migration produced by induction or ectopic expression of MCPIP1. Our findings suggest a new role forMCPIP1 as a molecular switch that regulates fibroblast migration in the nested collagen matrix model.

Journal of Investigative Dermatology advance online publication, 24 September 2015; doi:10.1038/jid.2015.334

INTRODUCTIONRecently, we observed (Chao et al., 2014) that foreskinfibroblasts preconditioned in a rigidly anchored collagenmatrix migrated out of that matrix when it was re-embedded(“nested” Grinnell et al., 2006) in cell-free, anchoredcollagen, whereas fibroblasts preconditioned in a stress-released matrix had relatively poor motility under the sameconditions (5% serum). This observation provided us with anopportunity to study mechanoregulation of fibroblast motilityin the fibroblast-populated three-dimensional (3-D) collagenmatrix (FPCM) model (Grinnell, 1994). A relevant signalingmolecule was MCPIP1 (MCP-1-induced protein 1, alsoknown as ZC3H12A), a 66 kDa protein identified in human

peripheral blood monocytes and cardiomyocytes stimulatedwith MCP-1 (monocyte chemotactic protein 1; Zhou et al.,2006; Liu et al., 2015). The known functions of MCPIP1include the following: downregulation of inflammationthrough induction of apoptosis genes (Zhou et al., 2006;Skalniak et al., 2013); induction of angiogenesis inendothelial cells (human umbilical vein endothelial cells;Niu et al., 2008); inhibition of Toll-like receptor signaling andmacrophage activation (Huang et al., 2012); upregulation ofadipogenesis independent of peroxisome proliferator–activated receptor-γ (Younce et al., 2009); RNAse activityagainst viral DNA (Suzuki et al., 2011; Lin et al., 2013);inhibition of c-Jun N-terminal kinase (JNK) and NF-κB (Lianget al., 2008; Liu et al., 2013); and protection againstlipopolysaccharide-induced shock (Huang et al., 2013).MCPIP1-deficient mice developed a severe inflammatorysyndrome with T-cell activation, increased cytokine produc-tion, and a 50% 8-week mortality (Miao et al., 2013).MCPIP1 also was noted to promote migration in human

umbilical vein endothelial cells (Niu et al., 2008), and MCP-1knock-out mice demonstrated delayed wound re-epithelialization and angiogenesis (Low et al., 2001). Thus, itseemed logical to test whether MCPIP1 participated inmechanoregulation of fibroblast healing functions, such asproliferation, contraction, and migration. Herein we report datademonstrating that MCPIP1 induction after stress release of theFPCM inhibits fibroblast migration, working through a signalingpathway involving the mitogen-activated protein kinases(MAPK), NF-κB, and p53. This brake on fibroblast migrationappears to be a new function for MCPIP1 and implicates thisprotein as a participant in the wound healing process.

ORIGINAL ARTICLE

1Department of Surgery, University of Nebraska Medical Center, Omaha,Nebraska, USA; 2Department of Physiology, School of Medicine, SoutheastUniversity, Nanjing, Jiangsu, China; 3Department of Surgery, VA Nebraska–Western Iowa Health Care System, Omaha, Nebraska, USA and 4Department ofGenetics, Cell Biology and Anatomy, University of Nebraska Medical Center,Omaha, Nebraska, USA

Correspondence: Mark A. Carlson, Department of Surgery, VA Nebraska–Western Iowa Health Care System, 4101 Woolworth Ave, Surgery 112,Omaha, Nebraska 68105, USA. E-mail: macarlso@unmc.edu

Location where work was done: Omaha, Nebraska, USA, and Nanjing,Jiangsu, China

Received 11 May 2015; revised 27 July 2015; accepted 3 August 2015;accepted article preview online 26 August 2015

Abbreviations: 3-D, three-dimensional; FPCM, fibroblast-populatedcollagen matrix; GFP, green fluorescent protein; HFF, human foreskinfibroblast; HUVEC, human umbilical vein endothelial cell; JNK, c-JunN-terminal kinase; MAPK, mitogen-activated protein kinase; MCPIP1,monocyte chemotactic protein–induced protein 1; TPCK, N-p-tosyl-L-phenylalanine chloromethyl ketone; USP10, ubiquitin specific peptidase 10

© 2015 The Society for Investigative Dermatology www.jidonline.org 1

RESULTSMechanoregulation of migration in the restrained nested matrix;upregulation of MCPIP1 in the stress-released FPCMFibroblast migration was assayed using serum-treatedattached or stress-released collagen matrices populated withgreen fluorescent protein (GFP)-expressing human foreskinfibroblasts (HFFs) (Chao et al., 2014) in the restrained nestedmatrix (Supplementary Figure S1A online; Miron-Mendozaet al., 2010). Fibroblast migration out of the nested stress-released matrix was decreased relative to the nested stressed(attached) matrix (Figure 1a and b and Supplementary FigureS4F online). Immunoblotting demonstrated that the MCPIP1protein was upregulated after matrix release (Figure 1c and d).The MCPIP1 signal reached a maximum at ~ 1 hour andremained elevated for several days. Immunocytochemistry ofMCPIP1 in attached versus released matrices also demon-strated induction of this protein in the released state(Figure 1e). The specificity of the MCPIP1 fluorescence inthe released matrix was particularly impressive while focusingup and down through the 3-D microscopy specimen.

Effect of MCPIP1 RNAi on the mechanoregulation of FPCMcontraction, matrix cell number, and fibroblast migrationIn order to determine whether MCPIP1 induction associatedwith FPCM stress release was biologically relevant, the effect ofMCPIP1 knockdown on FPCM contraction, matrix cell number,and fibroblast migration was determined in attached versusreleased matrices. The efficiency of MCPIP1 RNA interference(RNAi) in the attached versus released FPCM (72 hours aftertransfection, 24 hours after release) was near complete byimmunoblotting (Figure 2a). RNAi of MCPIP1 had minimaleffect on contraction in the floating collagen matrix assay(“dermal equivalent” (Grinnell and Petroll, 2010)); see Figure 2band c. RNAi of MCPIP1 did not affect the decrease in matrix cellnumber (Figure 2d) known to occur after matrix stress release(Carlson and Longaker, 2004). Using attached or stress-releasedmatrices populated with GFP-expressing HFFs nested intorestrained cell-free collagen, it was observed that MCPIP1knockdown disinhibited migration out of the released, nestedmatrix (Figure 2e and f)––i.e., the decrease in fibroblast migration(the inhibition) precipitated by release of the nested matrix wasprevented (disinhibited) if MCPIP1 expression was blocked.

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Figure 1. Effect of fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM) release on fibroblast migration and expression of monocytechemotactic protein–induced protein 1 (MCPIP1). (a) Migration of green fluorescent protein (GFP)-expressing fibroblasts out of nested matrices was decreased24 hours after matrix release. Fibroblast migration shown at the interface between the nested matrix and the restrained cell-free matrix. Left scale bar=200 μm,right scale bar= 80 μm. (b) Plot of migration (three separate experiments from panel a). (c) MCPIP1 induction after FPCM release. Whole-cell lysates fromattached or released matrices immunoblotted for MCPIP1 and β-actin. (d) MCPIP1 densitometry (four separate experiments from panel c). (e) MCPIP1immunocytochemistry in the attached versus released FPCM. Blue=DAPI; green=MCPIP1. Scale bar= 20 μm. Data are mean± SEM.; *Po0.05 versus attached(unpaired t-test).

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Figure 2. Effect of monocyte chemotactic protein–induced protein 1 (MCPIP1) RNAi or ectopic expression on matrix contraction, matrix cell number, andfibroblast migration. (a) Immunoblots of lysates from fibroblast-populated three-dimensional (3-D) collagen matrix (FPCMs) expressing siRNA (MCPIP1 versusnonsense). (b and c) Effect of MCPIP1 RNAi on FPCM contraction (well diameter=19mm); plot= three experiments. (d) Effect of MCPIP1 RNAi on FPCM cellnumber, 1-day post release. (e and f) Effect of MCPIP1 RNAi on migration out of the released, nested FPCM (scale bar=80 μm); plot= three experiments.(g) Immunoblots of lysates from FPCMs expressing MCPIP1-Flag versus Flag. (h and i) Effect of MCPIP1-Flag expression on FPCM contraction; plot= threeexperiments. (j) Effect of MCPIP1-Flag expression on FPCM cell number, 1-day post release. (k and l) Effect of MCPIP1-Flag expression on migration out of theattached, nested FPCM (scale bar= 80 μm); plot= three experiments. *Po0.05, unpaired t-test.

J Chao et al.MCPIP1 Regulates Fibroblast Migration

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Effect of MCPIP1 ectopic expression on the mechanoregulationof FPCM contraction, matrix cell number, and fibroblastmigrationIn order to corroborate the findings in Figure 2, an analogousset of experiments was performed using plasmid-expressedFlag-tagged MCPIP1 (Figure 2g and l). Confirmation ofMCPIP1-Flag expression after plasmid transfection in theFPCM is shown in Figure 2g and Supplementary Figure S1Bonline. Addition of the MAT-Tag-Flag sequence (see Materialsand Methods section) added 15 amino acids to the 599 aminoacid sequence of MCPIP1, but no shift was seen on theimmunoblots of MCPIP1 versus MCPIP1-Flag. Subsequentectopic expression of MCPIP1-Flag had no effect on contrac-tion in the floating collagen matrix assay (Figure 2h and i).Expression of MCPIP1-Flag also did not affect the decrease inmatrix cell number, which occurred after matrix stress release(Figure 2j). In experiments analogous to those in Figure 2eand f, expression of MCPIP1-Flag inhibited GFP-HFF migra-tion out of the attached matrix nested into restrained, cell-freecollagen (Figure 2k and l). Ectopic expression of MCPIP1-Flagin monolayer fibroblasts actually increased migration in ascratch assay (Supplementary Figure 5A and B online)––i.e.,the opposite effect to that observed in the 3-D culture model.

Effect of FPCM release on phosphorylation of MAPK and AktPrevious reports have indicated that activation of MAPK andthe phosphoinositide 3-kinase (PI3K)/Akt pathway bothstimulate fibroblast migration (Li et al., 2004; Clement et al.,2013). In order to see whether there was a link between thesekinase pathways and MCPIP1-associated inhibition of cellularmigration, phosphorylation of these kinases in the attachedversus released FPCM was evaluated first (Figure 3). Within5minutes of matrix stress release, there was increasedphosphorylation of Erk, which tapered off by 6 hours (Figure3a and c). Within 5–10minutes of release, p38 demonstratedincreased phosphorylation, reaching a peak around 30–60minutes and then tapering off (Figure 3a and b). JNK alsodemonstrated a burst of activation from 5 to 30minutes afterrelease (Figure 3d and e). The data of Figure 3 were consistentwith previous work of other investigators, who demonstratedErk and p38 activation after stress release of the FPCM undersimilar but not identical conditions (Lee et al., 2000).Akt also demonstrated a burst of activity 1 hour after

release, with tapering thereafter (Figure 3d and f). Of note,previously published reports demonstrated that Akt wasdephosphorylated during a longer time course (46 hours) ofFPCM stress release (Tian et al., 2002; Carlson et al., 2004;Xia et al., 2004). A long time course of Akt activity after matrix

release was repeated for corroboration purposes in Supple-mentary Figure S2A and B online, which again demonstratedrapid and transient Akt phosphorylation, followed by gradualdephosphorlyation evident at 6–12 hours (consistent withpreviously published data).

Effect of pharmacological inhibition of MAPK or Akt on MCPIP1induction and fibroblast migration after FPCM releasePharmacological inhibition of the above kinases was used todetermine whether the pathways of interest (JNK, ERK, p38,and PI3K/Akt) regulated (i) the expression of MCPIP1 and (ii)fibroblast migration out of the nested matrix (Figure 3g and l).Justification for the 30-minute point was the relatively largeincrease in MCPIP1 expression in the released matrix presentat this time; the 24-hour time point was chosen because bothMCPIP1 upregulation and migration inhibition were evident24 hours after release (Figure 1). Treatment of FPCMs withcommercially available small molecules U0126 (MEK inhi-bitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), orLY294002 (PI3K inhibitor) at the manufacturer-recommendeddose decreased the stress-release-induced phosphorylation ofthe respective target kinase (see Supplementary Figure S2Cand F online).Inhibitor pre-treatment for 2 hours prior to FPCM stress

release diminished the induction of MCPIP1 at both 30minutes and 24 hours after stress release (Figure 3g and j). Atthe 30-minute time point, the MCPIP1 signal still appearedto be increased in the stress-released matrix in the presenceof each kinase inhibitor, but this reached significance onlywith the p38 inhibitor (SB203580). At the 24-hour time point,induction of MCPIP1 expression after stress release was stillblunted by each kinase inhibitor, but effects were lesspronounced compared with the 30-minute time point(Figure 3j).Analysis of variance and unpaired t-testing performed on

the MCPIP1/actin expression ratios for the attached matrix inFigure 3j (i.e., attached vehicle versus attached SP600125versus attached U0126 versus attached SB203580 versusattached LY294002) revealed that the attached LY294002ratio (indicated with an asterisk over that bar) was differentfrom the attached vehicle ratio. Thus, although kinaseinhibition may have decreased MCPIP1 expression in theattached FPCM, our assay detected this only for the PI3 Kinhibitor. This observation might decrease the relevance ofthe decrease in MCPIP1 induction observed in the LY294002-treated released matrix; the absolute effect of the PI3Kinhibitor in the released matrix, however, was still largecompared with the effect in the attached matrix.

Figure 3. Kinase activity, inhibition, monocyte chemotactic protein–induced protein 1 (MCPIP1) expression, and migration in the fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM). (a–f) p38, Erk1/2, c-Jun N-terminal kinase (JNK), and Akt phosphorylation after FPCM release (immunoblots of wholelysates), with densitometry of n=4 experiments. (g–j) Effect of kinase inhibitors (JNK= SP600125, MEK=U0126, p38= SB203580, PI3K= LY294002) on FPCMMCPIP1, 30 minutes and 24 hours post release (whole-lysate immunoblots), with densitometry (n=4 experiments each). *Po0.05 versus vehicle-attached(unpaired t-test); #Po0.05 versus vehicle-released (unpaired t-test). (k and l) Effect of kinase inhibitors on migration out of the released, nested matrix (upper scalebar=200 μm, lower scale bar= 80 μm); plot represents n= 3 experiments/condition. *Po0.05, unpaired t-test.

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The effect of pharmacologic inhibition of kinase activity onfibroblast migration from stress-released matrices nested intorestrained cell-free matrices then was evaluated (Figure 3kand l). Pre-treatment of the FPCM for 2 hours prior to stressrelease with the MEK, p38, or JNK inhibitor enhancedfibroblast migration out of the nested released matrix (i.e.,resulted in disinhibition of fibroblast migration). Pre-treatmentof matrices with the PI3K inhibitor LY294002, however, didnot have an effect––that is, fibroblast migration out of thenested released matrix was similar to vehicle treatment,meaning barely detectable. There was no significant effect ofany of these inhibitors on fibroblast migration out of thenested attached matrix (Supplementary Figure S3A and Bonline). The data of Figure 3 suggested that MAPK activationwas upstream of MCPIP1 induction in a putative pathwaythat inhibited fibroblast migration after stress release ofthe FPCM.

Involvement of NF-κB in the expression of MCPIP1 andinhibition of fibroblast migration after FPCM releaseNF-κB activation has been documented in the contractileFPCM (Xu and Clark, 1997; Xu et al., 1998; Carlson et al.,2013), and NF-κB signaling has been implicated in theinduction of both MCP-1 and MCPIP1 in endothelial cells (Qiet al., 2010; Yao et al., 2010). In addition, NF-κB activationcan occur secondary to MAPK activation (Troppmair et al.,1998; Dhawan and Richmond, 2002; Dong et al., 2012).Hence, it was hypothesized that MCPIP1 induction in thestress-released FPCM occurred as a consequence of MAPK–mediated NF-κB activation. During the 5–30 -minute intervalafter matrix release, there was an increase in the level of thephosphorylated p65 (p-p65) subunit of NF-κB in the nuclearfraction of the HFFs (Figure 4a and b), with a concomitant lossof this subunit in the cytoplasm (Supplementary Figure S4Aonline), consistent with pilot data (Carlson et al., 2013). This

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increase in p-p65 after matrix release appeared to beabrogated if the matrices were pre-treated with pharmacolo-gic inhibitors of Erk, p38, or JNK; pre-treatment with the PI3Kinhibitor had less effect (Figure 4c and d). These resultssuggested that MAPK activation after FPCM release resulted inNF-κB activation.The MCP-1 promoter has an NF-κB binding site (Yao et al.,

2010); a chromatin immunoprecipitation assay was utilized todetermine whether a similar site was present on the MCPIP1promoter (Figure 4e). Chromatin was cross-linked to proteinin attached versus 20-minute stress-released FPCMs, andsonicated extracts were processed with a chromatin immuno-precipitation kit (EMD Millipore, Billerica, MA). Subsequentagarose gel electrophoresis of the amplified, immunoprecipi-tated DNA revealed much greater NF-κB binding in extractsfrom the released matrix compared with the attached(Figure 4f). This was consistent with NF-κB binding to theMCPIP1 promoter in extracts from the stress-released FPCM.In order to determine whether NF-κB activation after FPCM

release regulated MCPIP1 induction, matrices were pre-treatedwith pharmacologic inhibitors of NF-κB activation (Figure 4gand i, Supplementary Figure S1C and D, and S4D and F online),including TPCK (N-p-tosyl-L-phenylalanine chloromethylketone) and SC-514 (4-Amino-[2,3"]bithiophenyl-5-carboxylicacid amide). TPCK, a serine protease inhibitor, will block NF-κBactivation by preventing the proteolysis of IκB-α SC-514, anATP-competitive inhibitor selective for IκB kinase-2 (IKK-2), will

block NF-κB activation by preventing phosphorylation of IκB-α(Henkel et al., 1993; Kishore et al., 2003; Karin et al., 2004; Haet al., 2009). Compared with vehicle-treated matrices, MCPIP1induction after release in matrices pre-treated with SC-514 orTPCK or was not present at 24 hours (Figure 4g and hand Supplementary Figure S4D and E online, respectively).Furthermore, FPCM pre-treatment with either SC-514 orTPCK disinhibited HFF migration in the released matrix thatwas nested into a restrained, cell-free matrix (Figure 4i,Supplementary Figure S1C and D, and S4F online). In otherwords, pharmacologic inhibition of NF-κB allowed fibroblaststo migrate out of the collagen matrix under conditions in whichthey normally would not. The results thus far suggested thatinhibition of fibroblast migration associated with FPCM releaseinvolved a pathway with sequential upregulation of MAPK,NF-κB, and MCPIP1.

Interaction of MCPIP1 with p53 after release of the FPCMIt has been shown that p53 is upregulated after release ofthe FPCM (Carlson et al., 2004, 2009, 2013). In order todetermine whether there was co-localization of MCPIP1and p53 after release of the FPCM, double-immunocyto-chemistry for these two antigens was performed (Figure 5aand Supplementary Figure S3E online). The orange-yellowcoloring in the merged images indicated that there was someco-localization of MCPIP1 and p53 in the 1 day-releasedmatrix. Immunoprecipitation of either MCPIP1 or p53

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Figure 5. Monocyte chemotactic protein–induced protein 1 (MCPIP1) and p53 association after fibroblast-populated three-dimensional (3-D) collagenmatrix (FPCM) release. (a) MCPIP1 and p53 immunocytochemistry in attached versus 1 day-released FPCM. Blue=DAPI; green=MCPIP1; red=p53; scalebar=20 μm. (b and c) MCPIP1 and p53 co-localization after FPCM release (whole lysates from attached versus 1 day-released FPCMs immunoprecipitated forMCPIP1 or p53 and then immunoblotted for the other; IgG used as immunoprecipitate control). (d–f) Effect of RNAi of MCPIP1 or p53 on p53 or MCPIP1 in1 day-released FPCM (whole-lysate immunoblots); n= 4 experiments for densitometry. *Po0.05 versus attached (unpaired t-test).

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followed by immunoblotting for the opposite antigen demon-strated an association of MCPIP1 and p53 in whole-celllysates from the released (but not attached) FPCM (Figure 5band c). Control immunoprecipitation experiments that usedRNAi to knockdown either MCPIP1 or p53 prior to pull-downconfirmed the specificity of the MCPIP1-p53 association(Supplementary Figure S3C and D online. The results fromFigure 5a and c suggested that there was a physical inter-action between MCPIP1 and p53 after FPCM stress release.In order to determine whether one of these proteins

regulated the concentration of the other, RNAi of MCPIP1or p53 was performed, and protein levels were determinedwith immunoblotting (Figure 5d and f). Fibroblasts transfected

with siRNA against MCPIP1 did not have p53 upregulation24 hours after matrix release (Figure 5f), even though theknockdown of MCPIP1 did not appear complete (Figure 5e).Treatment with siRNA against p53 prevented most of the post-release increase in p53 (Figure 5d and f). Dissimilar to thesituation with MCPIP1 RNAi, however, partial knockdown ofp53 did not affect the induction of MCPIP1 in the releasedmatrix (Figure 5d and e). These data suggested that MCPIP1regulated p53 induction after FPCM release––i.e., MCPIP1was upstream of p53 in a putative pathway.Further exploration of an MCPIP1-p53 relationship was

carried out using ectopic expression of MCPIP1-Flag com-bined with p53 RNAi (Figure 6). Expression of MCPIP1-Flag

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Figure 6. Monocyte chemotactic protein–induced protein 1 (MCPIP1) regulates cell migration through p53. (a–c) Effect of MCPIP1-Flag expression and/or p53RNAi on MCPIP1 and p53 in attached versus 1 day-released fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM; whole-lysate immunoblots);n= 4 experiments for densitometry. (d and e) Effect of MCPIP1-Flag expression and/or p53 RNAi on migration out of attached versus 1 day-released FPCMs (scalebar=80 μm); plot represents n=3 experiments/condition. *Po0.05 versus control-attached (unpaired t-test); #Po0.05 versus control-released (unpaired t-test).(f and g) The Putative pathway of the MCPIP1-centric pathway regulating migration in the FPCM. Stress release of the FPCM activates PI3K, ERK, p38, and c-JunN-terminal kinase (JNK). Activated NK-κB induces expression of MCPIP1, which upregulates p53, which then inhibits fibroblast migration through unknownmechanisms. A pointed arrowhead head= stimulatory effect; flat arrowhead= inhibition.

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upregulated p53 in the attached FPCM, with no significanteffect in the released matrix (Figure 6a and c); as expected,MCPIP1-Flag inhibited cell migration out of the nestedattached FPCM (Figure 6d and e). Consistent with Figure 5,RNAi of p53 did not affect induction of MCPIP1 associatedwith matrix release (Figure 6a and b) but did result indisinhibition of cell migration that occurred in the nestedreleased FPCM (Figure 6d and e). Expression of MCPIP1-Flagcould not overcome the effect of p53 RNAi on cell migration(Figure 6d and e). In other words, MCPIP1 could inhibitthe ability of fibroblasts to migrate out of the attached matrixthat was nested into a restrained, cell-free matrix if p53 wasnot inhibited. If p53 was knocked down, however, thenthe fibroblasts could migrate out of either an attached or areleased matrix, regardless of the state of MCPIP1.MCPIP1 associated with USP10 (ubiquitin specific pepti-

dase 10) after genotoxic stress, enhancing the latter’sdeubiquitinase activity, thereby producing deubiquitinationof NEMO (IKK-γ), which resulted in inhibition of NF-κBactivation (Niu et al., 2013). Genotoxic stress also produced aTANK-MCPIP1-USP10 complex that effected/enhanced deu-biquitination of both TRAF6 (E3 Ubiquitin Protein Ligase) andNEMO, which in turn inhibited NF-κB activation (Wang et al.,2015). Therefore, in the setting of genotoxic stress, MCPIP1appears to function as a facilitator of USP10 deubiquitinase,thereby acting as a “brake” (negative feedback mechanism)on NF-κB activation. In order to determine whether MCPIP1facilitation of deubiquitination was relevant for MCPIP1-induced upregulation of the p53 protein, we performed FPCMexperiments with fibroblasts transfected with MCPIP1(ΔZF),an MCPIP1 mutant that does not promote deubiquitination(Liang et al., 2010). Our data suggested that MCPIP1-facili-tated deubiquitination is important for induction of p53by MCPIP1 (Supplementary Figure 4B and C online),which suggested that the increase in the p53 protein afterFPCM stress release was mediated at least in part by adecrease in p53 ubiquitination. Whether MCPIP1 affects theubiquitination status of p53 through USP10 will need furtherinvestigation.The data of Figure 6 were consistent with a pathway in

which MCPIP1, acting upstream and through p53, effectedinhibition of cell migration out of the nested released FPCM.Stress release of the FPCM also has been shown both toincrease apoptosis and inhibit the cell cycle in the residentfibroblasts (Fluck et al., 1998; Grinnell et al., 1999; Tian et al.,2002; Carlson and Longaker, 2004; Hadjipanayi et al., 2009;Carlson et al., 2013), which likely is an effect of p53upregulation. Although cell survival and proliferation werenot the focus of this study, we did demonstrate that theBax protein increased after FPCM stress release and that thisinduction was abrogated by p53 knockdown (SupplementaryFigure 5C online). This finding would be consistent with ap53-modulated increase in apoptosis associated with FPCMstress release.

DISCUSSIONThe data suggested the existence of an MCPIP1-centricnetwork that regulated fibroblast migration in the nested

FPCM model (Figure 6f and g). In this putative network,MCPIP1 in the nested attached FPCM was undetectable,allowing fibroblasts to migrate out of the FPCM and into theacellular neomatrix. If the matrix was released, however,phosphorylation of MAPK (Erk, p38, and JNK) ensued andproduced NF-κB activation. The p65 subunit of NF-κB thenbound to the promoter region of the MCPIP1 gene, followedby increased MCPIP1 expression. This induction of MCPIP1upregulated p53, possibly through binding events betweenMCPIP1 and p53 (and perhaps some unspecified scaffoldingproteins). Upregulation of p53 produced, through unidentifiedadditional steps, inhibition of fibroblast migration out of thenested released matrix.Although we have implied that MCPIP1 induction after

FPCM release was secondary to increased transcription andtranslation, we have not ruled out other protein turnovermechanisms, such as transcript stability or protein degrada-tion. Our assumption of increased MCPIP1 transcription wasbased on two observations: (i) p65 NF-κB bound to theMCPIP1 promoter after FPCM release; and (ii) inhibition ofNF-κB activation abrogated both the induction of MCPIP1and the inhibition of migration that occurred after matrixrelease (Figure 6).It is conceivable that other signaling events (e.g., involving

the Rho GTPases (Raftopoulou and Hall, 2004)) may havecontributed to the inhibition of migration associated withFPCM stress release. Nevertheless, various “molecularswitches” have been described in which a change in theprotein level and/or activity produced a large downstreamevent (Milburn et al., 1990; Murphy et al., 2004; Drees et al.,2005); hence, it is not inconceivable that increased MCPIP1protein contributed to the inhibition of fibroblast migration.The effect of MAPK inhibition on MCPIP1 induction inFigure 6 was partial, suggesting that the MAPK inhibitors mayhave had produced other downstream effects (e.g., on myosinlight chain kinase (Huang et al., 2004)) that resulted indisinhibition of migration after matrix stress release.Previous reports have suggested that activation of onco-

genic Ras, MAPK, and PI3K all may work to enhancefibroblast migration (Li et al., 2004; Menezes et al., 2008;Clement et al., 2013), in apparent contradiction to our report.These earlier reports all used one or more of the followingconditions: (i) transformed cell lines with forced/sustainedexpression of various signaling proteins; (ii) monolayer cultureconditions; and (iii) variable amounts of soluble growthfactors. In contrast, our experimental system (i) did not utilizeforced expression to make primary observations (Figures 1and 4), (ii) did utilize a 3-D culture system, and (iii)maintained a constant culture medium. These differences inexperimental design might explain the discrepancy betweenthe previous studies and our data. For example, we observedtransient MAPK activation, the effect of which likely isdifferent from activation secondary to forced expression(Marshall, 1995; Sabbagh et al., 2001).Our intent herein was to study fibroblast migration in a

tractable 3-D model that has morphologic and physiologicsimilarities to dermal wounds (Carlson and Longaker, 2004);clearly, these results may not perfectly translate to in vivo

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processes. It is interesting to note that forced expression ofMCPIP1 in a monolayer assay of migration actually appearedto promote fibroblast motility––i.e., the opposite effect to whatwas observed in the 3-D collagen matrix. This discordance ofexperimental results from monolayer versus 3-D culturesystems suggests that it may be better to utilize 3-D culturesystems when studying cells whose natural environment isthree-dimensional not planar (Cukierman et al., 2001).The FPCM model has been in continual use since the 1970s

to study various phenomena related to wound healing, suchas cellular migration, cell-mediated matrix contraction,synthesis and secretion of matrix proteins, cell death,and cellular proliferation. Derivatives of the FPCM modelalso have been used to study tissue engineering (Carlson andLongaker, 2004; Grinnell and Petroll, 2010; Harunaga andYamada, 2011; Kim et al., 2011). Continued progress inwound healing science likely will benefit from the use of (i)culture systems such as the FPCM and (ii) in vivo models.

MATERIALS AND METHODSCell cultureThe use of primary dermal fibroblasts derived from discarded humanneonatal circumcision specimens was approved by the Research andDevelopment Committee of the Omaha VA Medical Center and bythe Institutional Review Board of the University of Nebraska MedicalCenter. Fibroblasts were cultured from explants of human neonatalforeskins, as previously described (Carlson et al., 2009). The collagenmatrix model was utilized, as previously described (Grinnell et al.,1999; Carlson et al., 2009, 2013). Lentiviral-transduced HFFs withstable expression of GFP was described in a separate report (Chaoet al., 2014).

Nested matrix model and cell migrationThe nested collagen matrix model was utilized as previously described(Grinnell et al., 2006; Miron-Mendoza et al., 2010; Chao et al., 2014;Liu et al., 2015) with some modifications; refer to Supplementary FigureS1A and protocol in the Supplementary Information online. For thenested attached matrix, a standard FPCM was incubated in the attachedstate for 72 hours with 5% fetal bovine serum in DMEM; the FPCM thenwas removed from the culture well and placed onto a 60-μl aliquot offresh acellular collagen matrix solution (neomatrix solution) that wascentered inside a 12mm-diameter score on the bottom of a new culturewell. A 140-μl aliquot of neomatrix solution then was used to cover thenewly transferred FPCM. The neomatrix was allowed to polymerize for1 hour at 37 °C and 5% CO2, and then 2ml of DMEM with 5% fetalbovine serum was added to the well. The same procedure was followedfor the nested released matrix, except that the initial incubation of theFPCMwas 48 hours in the attached stated, followed by detachment, andthen 24-hour incubation in the released state (see Supplementary FigureS1A online). Cell migration out of the nested FPCM and into the acellularneomatrix was quantified 24 hours after nesting with fluorescentmicroscopy, as described in the Supplementary Information online. Cellnumber per matrix was determined using a Scepter Cell Counter (EMDMillipore, Billerica, MA), as previously described (Chao et al., 2014).

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSThis study is the result of work supported in part with resources and the use offacilities at the VA Nebraska–Western Iowa Health Care System. The Flag-MCPIP plasmids were kindly provided by Shilpa Buch of the University ofNebraska Medical Center. We express our appreciation for the helpfuldiscussions with Amarnath Natarajan. We acknowledge the technicalassistance of Chris Hansen and Dean Heimann. MAC was supported bygrants from the State of Nebraska, the United States Department of Defense,and the National Institutes of Health. JC was supported by grants of TheNational Natural Science Foundation of China (81473263) and The NaturalScience Foundation of Jiangsu Province, China (No. BK20141347 and No.BK20141497).

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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