Plakoglobin regulates cell motility through Rho- and ... · 2Department of Chemistry, 1110 W....

3576 Research Article

IntroductionEpithelial tissues derive positional and functional cues from theirsurroundings, in part through cell–cell and cell–substrateinteractions involving cadherins and integrins, respectively (Hynes,2002; Wheelock and Johnson, 2003). Adhesion-dependent signalingpathways are critical for normal development and differentiationin adult tissues; defects in both types of adhesive contact contributeto disease pathogenesis, including inherited disease and cancerprogression (Dusek et al., 2007; Janes and Watt, 2006; Jeanes etal., 2008; Watt, 2002).

In addition to their independent roles in tissue morphogenesisand homeostasis, cadherin- and integrin-based cell–substrateadhesive contacts engage in ‘cross-talk’ mechanisms, in which onejunction type affects the expression, assembly, turnover and/orfunction of the other junctions or junction components (Hodivalaand Watt, 1994; Levenberg et al., 1998; Marsden and DeSimone,2003; Monier-Gavelle and Duband, 1997; Ojakian et al., 2001). Avariety of signaling mediators contribute to communication betweencadherins and integrins. These include cell surface adhesionreceptors such as nectins (Ogita and Takai, 2008), receptor-mediatedgrowth-factor-dependent pathways such as TGF/SMAD (Kim etal., 2009), non-receptor kinases and such as Src family members(Arregui et al., 2000; Avizienyte and Frame, 2005), small GTPases(Balzac et al., 2005; Fukuhara et al., 2004; Takai et al., 2008), andmechanotransduction pathways (Chen and Gumbiner, 2006; Tsaiand Kam, 2009).

Desmosomes are a type of cadherin-based intercellular junctioncrucial for structural stability of tissues during embryonic

development and adult tissue morphogenesis (Cheng and Koch,2004; Garrod and Chidgey, 2008; Green and Gaudry, 2000). Defectsin desmosome structure and function, resulting from mutation ofits individual components or inhibition of desmosomal cadherinadhesive functions by bacterial toxins and autoimmune antibodies,lead to skin and/or heart disease (Bazzi and Christiano, 2007;Getsios et al., 2004b; McGrath and Wessagowit, 2005). Moreover,desmosomal molecules are aberrantly expressed in many types ofcancer (Chidgey and Dawson, 2007). In addition to its roles inmaintaining tissue integrity, evidence is emerging that desmosomesalso participate in signal transduction and transcriptional regulation(Garrod and Chidgey, 2008; Green and Simpson, 2007). Despitethe growing body of literature indicating the importance of thesepathways in tissue development and homeostasis, much less isknown about the potential roles of desmosome molecules asmolecular intermediates that regulate cell–substrate adhesion.

Plakoglobin (PG; also known as junction plakoglobin, JUP) isa member of the Armadillo family of proteins, and a close relativeof -catenin. Although it is able to associate with the cytoplasmictails of both classic and desmosomal cadherins (Cowin et al.,1986), PG is thought to be present predominantly in desmosomesof epithelial cells with mature intercellular contacts (Hinck et al.,1994). In desmosomes, PG participates in linking the cytoplasmictail of desmosomal cadherins to intermediate filaments (North etal., 1999; Yin and Green, 2004). Accordingly, PG plays a criticalrole in desmosomal integrity, as demonstrated by reducedexpression and membrane incorporation of other desmosomalproteins (Yin et al., 2005a), as well as impaired cell–cell adhesion

Plakoglobin regulates cell motility through Rho- andfibronectin-dependent Src signalingViktor Todorovic1, Bhushan V. Desai1, Melanie J. Schroeder Patterson2, Evangeline V. Amargo1, Adi D. Dubash1, Taofei Yin1, Jonathan C. R. Jones3 and Kathleen J. Green1,*1Department of Pathology, 303 E. Chicago Avenue, Northwestern University, Chicago, IL 60611, USA2Department of Chemistry, 1110 W. Belden Avenue, DePaul University, Chicago, IL 60614, USA3Department of Cell and Molecular Biology, 303 E. Chicago Avenue, Northwestern University, Chicago, IL 60611, USA*Author for correspondence ([email protected])

Accepted 1 July 2010Journal of Cell Science 123, 3576-3586 © 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.070391

SummaryWe previously showed that the cell–cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contactwith each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PG–/– cells onto ECM deposited by PG+/– cellspartially restored normal cell morphology and inhibited PG–/– cell motility. In over 70 adhesion molecules whose expression wepreviously showed to be altered in PG–/– cells, a substantial decrease in fibronectin (FN) in PG–/– cells stood out. Re-introduction ofPG into PG–/– cells restored FN expression, and keratinocyte motility was reversed by plating PG–/– cells onto FN. Somewhatsurprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not adecrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PG–/– cells exhibited an increasein activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PG–/– cells on ECM deposited by PG+/–

keratinocytes. PG–/– cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoAinhibition attenuated PG–/– keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM–Srcand RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability.

Key words: Armadillo protein, Desmosome, Extracellular matrix, Keratinocyte

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(Caldelari et al., 2001) and adhesive strength (Acehan et al., 2008)in keratinocytes lacking PG. Moreover, PG-null mice and patientswith pathogenic homozygous PG mutations have impaired tissueintegrity associated with skin and heart defects (Aberle et al.,1995; Bierkamp et al., 1996; McKoy et al., 2000; Ruiz et al.,1996). PG is also found in the cytoplasm and nucleus (Green andSimpson, 2007; Schmidt and Koch, 2007), where it is able to actindependently of its function in intercellular adhesion. Its adhesion-independent functions are still not well defined, but the data suggestthat PG can regulate gene expression and protein stability (Aktaryet al., 2010; Hakimelahi et al., 2000; Shimizu et al., 2008) in botha -catenin-dependent and -independent manner (Raurell et al.,2006; Teuliere et al., 2004; Yin and Green, 2004; Zhurinsky et al.,2000).

Recently we demonstrated that PG not only inhibits motility ofkeratinocytes in contact, but also inhibits Src-dependent single cellmotility (Yin et al., 2005b). The observed changes in motility andaltered cell morphology of PG–/– keratinocytes suggested to us thatPG could be regulating cell–substrate interactions by modulatingcomponents of the extracellular matrix (ECM), its integrin receptorsand/or the molecules involved in ECM-triggered motility cues.Using a combination of live cell imaging and cross plating, weshow here that PG expression has a potent impact on theorganization of actin, its associated membrane protrusions, focaladhesions and Src-dependent motility, in large part throughregulation of the expression levels of the underlying ECMcomponents. In particular, the ability of PG to regulate fibronectin(FN; also know as Fn1) mRNA stability was identified as a novelmechanism contributing to PG-dependent suppression ofkeratinocyte motility. Further analysis indicated that PG-dependentalterations in activity of the small GTPases Rac1 and RhoA act inparallel with FN/Src-dependent regulation of cortical actin

3577Plakoglobin regulates motility via ECM

structures to fine tune the motile behavior of keratinocytes.Collectively, these results indicate that a desmosomal molecule,PG, is capable of regulating single cell motility through matrixdeposition in concert with Rho GTPases, independently of its roleas a cell–cell adhesion molecule.

ResultsPG regulates keratinocyte cell polarity and single-cellmotilityTo begin to address the mechanism by which PG regulates singlecell motility, we first carried out live cell imaging analysis toidentify individual components of motility behavior (Fig. 1A;supplementary material Movie 1). Analysis of motility of individualcells revealed that both net displacement and total distance traveledwas approximately twofold greater in PG–/– keratinocytes comparedwith PG+/– cells. The resulting directed migration index (netdisplacement/total distance traveled) was not significantly higherin PG-null cells (Fig. 1B,C) indicating that PG deficiency increasesrandom rather than directional motility. Reconstituting PGexpression by adenoviral transduction reduced the motility of PG–

/– cells, suggesting that single cell keratinocyte motility is controlledby PG (see below). The average cell velocity of PG–/– cells waselevated over twofold (Fig. 1D). However, when velocity wascalculated between each pair of time points analyzed, to create a‘velocity map’, it became evident that PG–/– and PG+/– cellsexhibited distinctively different motility signatures. PG+/– cellshad a slower but more constant motility; by contrast, PG–/– cellsexhibited periods of very rapid movement interrupted by periodsof slower or even no movement (Fig. 1E).

Because keratinocyte motility is largely regulated by the abilityof cells to polarize and form lamellipodia, we next determined thedifferences in cell morphology and number of lamellipodia between

Fig. 1. Plakoglobin regulateskeratinocyte motility by increasing cellvelocity. (A)Representative tracks of tenrandomly chosen PG+/– and PG–/– cellsfrom five 5-hour trials involving aminimum of 50 cells per trial. Theintersection of the x- and y-axes was takenas the starting point of each cell path.Each tick mark on the axes represents 50pixels. (B)Graph showing the netdisplacement (left) and total distancetraveled (right) of PG+/– and PG–/– cells(mean ± s.d.; n≥3 trials, with at least 50cells per trial). (C)Directed migrationindex (ratio of the ‘net displacement’ and‘total distance traveled’) of PG+/– andPG–/– cells. (D)The average velocity of≥50 PG+/– and PG–/– cells, from threeindependent experiments (mean ± s.d.;n≥3 trials, with at least 50 cells per trial).(E)Velocity maps of a representativePG+/– (solid line) and PG–/– (dotted line)keratinocyte. Cells were followed for 5hours, with velocity measured at 5-minuteintervals.

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PG–/– and PG+/– cells. The organization of actin and associatedfocal adhesions was also assessed. Over 70% of PG+/– cellsexhibited an epithelioid morphology characterized by a lack oflamellipodia and prominent cortical actin (Fig. 2A-C). In addition,focal contacts were more numerous and prominent in PG+/– cells,as illustrated by staining for FAK(Tyr397-P) [phospho-tyrosine397 is a focal adhesion kinase (FAK) autophosphorylation sitedependent upon focal adhesion formation; Fig. 2D]. In contrast toPG+/– cells, 80% of PG–/– cells had at least one lamellipodiumpresent (Fig. 2B), consistent with an increased propensity formotile behavior. A third of the PG–/– cells had more than onelamellipodium accompanied by a more elongated, fibroblast-likemorphology (Fig. 2A,B). The presence of more than onelamellipodium has been related to frequent switches in the directionof motion (Sehgal et al., 2006) (supplementary material Movie 1),consistent with an increase in random, rather than directional,motility of PG–/– cells.

PG regulates the expression of molecules involved in cell–substrate interactionsBecause cell–substrate interactions play a crucial role in theregulation of cell motility and actin cytoskeleton remodeling(DeMali et al., 2003; Ridley et al., 2003), we proceeded todetermine whether PG regulates the expression of ECMcomponents, integrins and other adhesion-related molecules.

We used a previously described method of cell ‘de-roofing’(removing cell cytoplasm and nuclei) by mildly basic hypotonicsolution (Langhofer et al., 1993; Sehgal et al., 2006) to obtainsamples enriched in ECM and cell–ECM-adhesion-relatedmolecules. The samples were trypsinized and analyzed by massspectrometry (MS). Details of this novel method for massspectrometry sample preparation and analysis are discussedelsewhere (Todorovic et al., 2010). Differences in protein levelswere observed between integrin receptors, ECM and cytoskeletalmolecules, as well as between membrane glycoproteins and matrixproteolysis regulators (Table 1). Using a microarray specificallytargeting ECM and adhesion molecules, we further expanded thelist of molecules regulated by PG (Table 1) and have shown thatat least some of them (including FN) are regulated at the mRNAlevel. From the mass spectrometry and array data, we selectedseveral well-characterized molecules involved in keratinocyteadhesion and motility that were predicted to be significantly

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differentially expressed in PG+/– and PG–/– cells, and tested theirprotein levels by immunoblotting. Integrin 1 essential for motilityand adhesion to LN-332 and FN (Mizushima et al., 1997; van derFlier and Sonnenberg, 2001), but also found to be inhibitory tokeratinocyte motility on LN-332 during wound re-epithelialization(Margadant et al., 2009), FN and Col IV (provisional matrix andbasement membrane components, respectively) (Clark, 1990; Sadoet al., 1998), were all lower in PG–/– cells, consistent withexpression patterns established by MS and microarray analysis(supplementary material Fig. S1A). Consistent with the enrichmentof basal cell components, de-roofed cells retained FN and integrina6 while losing their nuclei (supplementary material Fig. S1B).Not all ECM molecules relevant for keratinocyte adhesion andmotility are differentially regulated by PG: a major basementmembrane component laminin-332 was not affected (Table 1)(Todorovic et al., 2010).

PG-dependent changes in extracellular matrix regulate theadhesive and motile behavior of mouse keratinocytesIn order to test whether PG controls keratinocyte motility byregulating the deposition of the underlying substrate, we allowedPG+/– and PG–/– cells to deposit their own matrix, and then removedthe cells by ‘de-roofing’ them while retaining the basal cellcomponents (supplementary material Fig. S1B). PG-deficient

Table 1. Protein and RNA expression analysis in PG+/– andPG–/– mouse keratinocytes

Molecule Protein ratio (het/ko) RNA ratio (het/ko)

Integrin 1 2.8 2.4Integrin a6 3.0 ndIntegrin 4 2.8 ndFibronectin 59 100Laminin 3 chain 0.86 ndLaminin a3 chain 0.86 ndLaminin g2 chain 0.86 ndTenascin 30 ndTimp-3 Enriched in het. (LOQ) 30ColIVa1 nd 30ColIVa2 nd 11

Protein ratios calculated by average mass spectrometry spectral counts(4 separate analyses). RNA ratios calculated using Super Array proprietaryRNA expression analysis software. LOQ, limit of quantitation for lowabundance proteins; nd, not detected; N/A, not included on the array.

Fig. 2. Plakoglobin regulates actincytoskeleton organization in mousekeratinocytes. (A)DIC images of PG+/– andPG–/– cells. Scale bar: 20m. (B)Number oflamellipodia per cell (PG+/– white bars; PG–/–

black bars). (C)Alexa-Fluor-350-conjugatedphalloidin-actin staining of PG+/– and PG–/–

cells. Scale bar: 20m. (D)PG+/– and PG–/–

keratinocytes stained for FAK(Tyr397-P)(FAKpY397) to show the focal contacts. Scalebar: 20m.

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keratinocytes were plated onto the pre-deposited matrices and theirmotility was analyzed. In contrast to the behavior on their ownmatrix, on a population basis, PG–/– cells exhibited significantlydiminished motility when plated on matrix deposited by PG+/–

keratinocytes (Fig. 3A,B; supplementary material Movie 2). Thevelocity map revealed some interesting sub-features of PG–/– cellbehavior on PG+/–-deposited matrix. These cells still exhibitedperiods of rapid migration, but they were fewer in number andinterspersed with periods of greatly reduced velocity (Fig. 3C;supplementary material Movie 2). This behavior resulted in anoverall decrease in average velocity of the PG–/– cells (Fig. 3D).

Along with the restoration of more normal motility behavior,the altered morphology and actin-associated structures observed inPG-deficient keratinocytes were also partially restored when platedon the substrate deposited by PG+/– keratinocytes (Fig. 4A,C).These changes included an increase in the number of cells with nolamellipodia from 10% to 40% and an increase in the number ofcuboidal and round cells with more pronounced cortical actin, andpartial normalization of focal contact structures (Fig. 4B–F).

PG-induced FN expression regulates keratinocyte motilityThe ability of PG+/– matrix to attenuate the increased motilityexhibited by PG–/– keratinocytes led us to further examinedifferentially expressed ECM molecules for their ability to regulatePG-dependent cell motility. From the analysis of the expressionpatterns of the ECM components (Table 1; supplementary materialFig. S1A,B), a 50-fold decrease in expression of fibronectin (FN)in PG–/– cells stood out, whereas the level of laminin-332 (LN-332) remained unchanged (Table 1) (Todorovic et al., 2010).

To confirm that PG was responsible for this change in FNexpression, we re-established PG expression in PG-null cells byadenoviral transduction. Ectopically expressed PG effectivelyreduced keratinocyte migration as assessed by live cell imaging


and tracking analysis (Fig. 5A). Furthermore, PG–/– keratinocytesshowed an increase in the overall level of FN upon PG transduction(Fig. 5B). Infected PG-expressing cells that were re-plated to allowfor the de novo deposition of matrix also showed increaseddeposition of FN as compared with adjacent non-transduced, orcontrol GFP-transduced cells (Fig. 5C).

To test how FN deposition would influence motility of PG–/–

cells, we plated cells on pre-deposited recombinant FN andrecorded their motility with live cell imaging. PG–/– keratinocytesshow significantly reduced motility on pre-deposited FN ascompared with their own matrix whereas pre-deposited matrixenriched in LN-332 did not appear to have a dramatic inhibitoryeffect (Fig. 5D). These results suggest that FN is one of thematrix molecules specifically involved in PG-dependent inhibitionof cell motility. The data demonstrate that PG can control themotility of mouse keratinocytes through regulation of FNexpression and deposition.

PG regulates FN mRNA stabilityThe RNA array results (Table 1) suggested that the decrease inFN protein expression correlates with a decrease in FN mRNA in PG–/– cells. To test this directly, we conducted quantitative reversetranscriptase PCR (qRT-PCR) and confirmed that PG–/– cellscontained at least fivefold less FN mRNA than the control cells(Fig. 6A). Because both PG and its closest family member -catenin are well established as regulators of transcription (Maedaet al., 2004; McCrea et al., 2009), and because we observedupregulation of both FN protein and mRNA, we next addressedwhether FN promoter activity depends on PG. Surprisingly, usinga luciferase reporter downstream of a 1.2 kb FN promotersequence (Michaelson et al., 2002) we showed that FN promoteractivity was increased, not reduced as might have been predictedbased on the observed reduction in FN mRNA levels (Fig. 6B).

Fig. 3. Plakoglobin-dependent ECM deposition regulates the motile behavior of mouse keratinocytes. (A)Representative tracks showing motile behavior of10 randomly chosen PG–/– cells on the ECM deposited by either PG–/– or PG+/– keratinocytes. (B)Graph showing the distribution of total distances traveled forindividual PG+/– and PG–/– keratinocytes on pre-deposited matrices (n110, horizontal black bars indicate the median value). (C)Velocity maps of a representativePG–/– keratinocyte on PG–/– ECM (solid line) and PG+/– ECM (dotted line). (D)Average velocity of PG–/– cells on PG–/–- and PG+/–-deposited ECMs (n≥50; errorbars represent ± s.d.).

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As FN levels can be controlled through stabilization of its mRNA(Mimura et al., 2004), we assessed the stability of FN mRNA inPG+/– and PG–/– cells by inhibiting de novo RNA synthesis withactinomycin D and measuring the relative levels of mRNA byqRT-PCR. Whereas the level of FN mRNA was relatively stablein PG+/– cells until the later 6-hour time point, a rapid decline inFN mRNA occurred over time, dropping to ~60% of its initialvalue as early as 15 minutes after actinomycin D treatment (Fig.6C). These results suggest that PG regulates FN levels at least inpart through maintenance of mRNA stability in mousekeratinocytes.

PG regulates Src activity in a matrix-dependent mannerPreviously we demonstrated that the increase in motility of PG-deficient cells is dependent upon the activity of Src kinase (Yin et al.,2005b), leading us to hypothesize that PG can regulate Src activity.In order to test this, we compared the levels of activated Src in PG+/–

and PG–/– cells by immunoblotting using an antibody recognizing theactivated form of Src [Src(Tyr416-P)]. The results indicate an increasein the levels of activated Src in PG–/– cells (Fig. 7A).

As Src activity is regulated by ECM interactions with integrins(Playford and Schaller, 2004), we investigated whether the PG-dependent changes in ECM composition were responsible for theobserved difference in Src activity between PG–/– and PG+/– cells.When plated on the matrix deposited by PG+/– cells, Src activityin PG–/– cells was significantly reduced (Fig. 7A,B). This finding


supports the idea that PG modulates Src activity at least in partthrough the regulation of ECM expression and deposition.

One of the major motility pathways regulated by cell–ECMinteractions is mediated through Src-dependent phosphorylation ofFAK and subsequent changes in focal adhesions. Changes in theinteraction of the cells with the underlying matrix leading to Src-dependent translocation of FAK from robust focal adhesions tosmaller focal complexes have been correlated with increased cellmotility (Beningo et al., 2001; Beningo and Wang, 2002).Consistent with this, less motile PG+/– keratinocytes exhibitedmore prominent FAK staining corresponding to zyxin-containingmature focal adhesions (Fig. 2D and not shown). However, PG–/–

cells exhibited more punctate and immature focal complexes.To test whether the formation of robust focal adhesions in PG+/–

keratinocytes was dependent upon decreased Src activity in thesecells (Fig. 7A), we used a chemical inhibitor of Src kinase (PP2)to treat PG–/– cells and assessed its ability to rescue focal adhesionformation. Treatment of PG–/– cells with PP2 led to a significantincrease in robust focal adhesions, as demonstrated by FAK(Tyr397-P) staining (Fig. 7C), corresponding to decreased motility of thetreated cells (Fig. 8A) (Yin et al., 2005b).

Taken together, these results suggest that PG inhibits cell motilityby regulating the expression levels of ECM molecules (includingFN). These PG-dependent changes in ECM composition in turninhibit motility through the downregulation of Src activation andincrease in mature focal adhesion formation.

Fig. 4. Plakoglobin-dependent ECM depositionregulates actin cytoskeleton organization.(A)DIC images of PG–/– on PG–/–- and PG+/–-deposited ECM. Scale bar: 20m. (B)Number oflamellipodia per PG–/– cell (PG–/– ECM, whitebars; PG+/– ECM, black bars). (C)Alexa-Fluor-350-conjugated phalloidin-actin staining of PG–/–

keratinocytes on PG+/– ECM and PG–/– ECM.(D)Diagram showing the percentage of PG+/–-and PG–/– cells (n≥50) on PG+/–- and PG–/–-deposited matrices with cortical actin stainingabsent (no ring), or present over one third (onethird), two thirds (two thirds) or whole cell border(complete ring). (E)Bar and whisker graphrepresenting the radial symmetry of PG+/– andPG–/– cells on predeposited matrices (n>100; y-axis represents the ratio between the twoperpendicular cell diameters, with higher valuealways being used as a numerator; the closer theratio is to 1, the more round or cuboidal the cell;black diamonds indicate the mean value; white baris 50th percentile; gray box delineates 25th and75th percentile, whiskers represent minimum andmaximum values). (F)PG–/– keratinocytes stainedfor FAK(Tyr397-P) to show the focal contacts onPG+/– and PG–/– ECM. Scale bars: 20 m.

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PG regulates RhoGTPase-dependent motility in a Src-independent mannerFN-rich PG+/– matrix was largely effective at restoring PG–/– cellmorphology and motile behavior. However, in live cell imagingexperiments it appeared that a proportion of cells with fibroblasticmorphology continued to move rapidly, if they were able to breakaway from the matrix. This behavior may have led to the incompletereversion of the velocity maps (Fig. 3C). Moreover, the restorationof PG–/– cell morphology and cortical actin symmetry on PG+/–

deposited ECM was incomplete as well (Fig. 4A-E). To exploreadditional factors that might contribute to PG-dependent regulationof motility, we carried out analysis of the Rho small GTPases,known to be important regulators of the actin cytoskeleton andmotility, often downstream from Src kinase (Arthur and Burridge,2001; Arthur et al., 2000). To assess whether Rho activity wasrequired for the increased motility of PG–/– cells, we treated themwith the cell-penetrating C3 transferase, a potent and specific inhibitorof RhoA. The inhibition of RhoA led to a significant reduction inPG–/– motility (Fig. 8A). Rho G-LISA assays revealed that bothRhoA and Rac1 activity were elevated in PG–/– cells (Fig. 8B).Surprisingly, the Src inhibitor PP2 was unable to reduce the activityof either RhoA or Rac1 (Fig. 8B). Likewise, the Rho inhibitor C3failed to significantly lower the level of activated Src in PG–/– cells(Fig. 8C), suggesting that PG regulates keratinocyte motility throughat least two independent signaling branches, ECM–Src on the onehand and RhoGTPase on the other. In addition, whereas the inhibition


of Src leads to significant decrease in the number of lamellipodia,the selective inhibition of RhoA has an opposite effect, with cellsextending many protrusions but not able to move (Fig. 8D,E).

DiscussionThe desmosomal armadillo protein plakoglobin links desmosomalcadherins, desmocollins and desmogleins, to the plakin family ofmolecules and the associated intermediate filament cytoskeleton(Cowin, 1994). Compromising these linkages by ablation of PGresults in severe loss of tissue integrity in vivo (Ruiz et al., 1996;Bierkamp et al., 1996) and cell–cell adhesion strength in vitro(Yin et al., 2005a). However, recently PG has also been shownto be involved in cell–cell adhesion-independent activities,including inhibition of motility of cells with disrupted cell–cellcontacts (Yin et al., 2005b), and regulation of transcription (Greenand Simpson, 2007).

Here we demonstrate that PG regulates cell–cell adhesion-independent motility through modulating expression and depositionof the extracellular matrix. Using a novel approach for preparationand mass spectrometric analysis of cell-adhesion-related molecules(Todorovic et al., 2010), we show that PG affects the expressionlevels of basal cell components involved in cell–ECM interactions,which in turn leads to changes in cell–substrate adhesion, actincytoskeleton organization, focal adhesion maturation and cellpolarity. Moreover, PG regulates Src activity in an ECM-dependentmanner, as well as RhoGTPase activity, both of which are

Fig. 5. Fibronectin contributes to plakoglobin-dependentinhibition of cell motility. (A)Representative tracks (n10) showingthe decrease in motility of PG–/– cells upon introduction of PG byadenovirus as compared to GFP-only control. (B)Western blot analysisof FN expression in the whole cell lysates of PG+/–, control (GFP) andPG-transduced PG–/– cells. (C)Immunofluorescence staining showingrestoration of FN expression and deposition in PG–/– cells transducedwith adenovirus expressing PG (green) compared with GFP-expressing control cells. Scale bar: 20 m. (D)Representative tracks(n10) showing the motile behavior of PG–/– keratinocytes plated ontoplastic- (no predeposited matrix), fibronectin (FN)- and laminin-332(LN-332)-coated dishes.

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instrumental in its regulation of keratinocyte motility. Thesefindings establish a regulatory role for a desmosomal molecule incontrolling the expression of, and cellular interactions with, ECMcomponents.

Among ECM molecules a role for both FN and LN-332 in theregulation of keratinocyte motility has been well established.Whereas LN-332 is the main component of skin basementmembrane and is required for both activation and inhibition ofkeratinocyte motility through distinct pathways (Litjens et al.,2006; Mercurio and Rabinovitz, 2001; Rabinovitz and Mercurio,1997), FN is important in regulating cell migration over theprovisional matrix in skin wound healing, disappearing after woundclosure (Santoro and Gaudino, 2005). Here, we show that althoughLN-332 is not affected by PG expression, FN mRNA levels aresignificantly reduced in PG–/– cells, with protein levels being by50-fold higher in control.

In recent years PG has been established as a transcriptionalregulator both independently and through Wnt/-catenin pathwayregulation (Maeda et al., 2004; McCrea et al., 2009). Therefore, weexpected to see PG directly regulating transcription of FN mRNAespecially because -catenin has already been shown to regulateFN expression in Xenopus fibroblasts (Gradl et al., 1999).Surprisingly, we show here that PG regulates FN mRNA level byenhancing its stability, rather than enhancing its promoter activity


(Fig. 6). Whereas the increase in FN promoter activity might beexplained as a compensatory attempt of the cells to synthesize themissing protein, the mechanism by which PG regulates the mRNAstability of fibronectin is an unexplored area of Armadillo proteinbiology. Recent data show the ability of plakophilins to bind to andregulate mRNA transcription (Borrmann et al., 2006; Wolf et al.,2010). Moreover, the Wnt/-catenin pathway has been implicatedrecently in regulating the turnover of labile mRNAs throughadenylate and/or uridylate-rich elements and their binding proteins(Briata et al., 2003). It remains to be seen whether PG stabilizesFN mRNAs directly or indirectly; however, this finding brings tolight a new subset of PG cell–cell adhesion-independent activities.

A PG-dependent increase in FN expression was associated withattenuated migration of single keratinocytes in vitro. That FNdecreased, rather than increased, motility might be explained byprevious observations that the process of FN deposition andpolymerization requires tight control to properly titrate cellbehavior. Indeed, in certain cases, cells have been shown to respondin a biphasic manner to increasing concentrations of FN, and athigh concentrations, FN has been shown to decrease the rate ofmigration, as in the case of airway epithelial cells (Hocking andChang, 2003); or decrease protrusiveness, as in the case of CHOcells (Cox et al., 2001).

In tissue culture mouse keratinocytes produce large amounts ofFN and LN-332, whereas human keratinocytes predominantlyproduce LN-332 (J.C.R.J. unpublished observations). In addition,human keratinocytes are more motile than mouse keratinocytes inculture. Thus, it seems possible that PG relieves a FN-dependentinhibition of mouse keratinocyte motility, allowing them to movefreely on the underlying LN-332 (Hamill et al., 2009; Sehgal et al.,2006). Although the mechanism of such inhibition is unclear, it istempting to speculate that FN and LN-332 might be competing forthe integrin receptors required for motility on LN-332. Furthermore,the decrease in FN observed in PG–/– cells is accompanied by a

Fig. 6. Plakoglobin increases fibronectin RNA levels through increasing itsstability. (A)qRT-PCR analysis of the FN RNA, which is significantlydecreased in PG–/– cells as compared with controls (experiment done intriplicate with two sets of primers and normalized to Gapdh; error barsrepresent ± s.d.). (B)Luciferase activity analysis shows an increase in FNpromoter activity in PG–/– cells, suggesting that PG does not increase FNlevels through promoter activation (Firefly luciferase activity normalized toRenilla activity used as a transfection efficiency control; experiment done inquadruplicate, error bars represent ± s.d.). (C)FN RNA stability analysis inPG+/– and PG–/– cells treated with actinomycin D (2g/ml) for the specifiedtimes. Samples from treated cells were normalized to their matching PG+/– andPG–/– controls (time 0), both of which were given a relative value of 1(experiment done in triplicate with two different sets of primers; error barsrepresent ± s.d.).

Fig. 7. Plakoglobin regulates Src activity through ECM deposition.(A)Western blot analysis of the levels of activated Src [Src(Tyr416-P); Scr-p416] in PG+/– and PG–/– keratinocytes in steady state. (B)Western blotshowing a decrease in activated Src in PG–/– cells on the ECM deposited byPG+/– cells 5 hours after replating. (C)Immunofluorescence showing morerobust focal adhesions in PG–/– cells treated with the Src inhibitor PP2. Scalebar: 20 m.

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decrease in 1 integrin expression (supplementary material Fig.S1; Table 1) (Todorovic et al., 2010), which may be due to the lossof FN as a primary a51 integrin ligand. Thus, becausea31integrin has been recently implicated in the inhibition ofkeratinocyte motility on newly deposited LN-332 (Margadant etal., 2009), the decrease of 1 integrin could lead to attenuation ofthis inhibitory effect. Mass spectrometry also revealed an increasein Syndecan-1 in the PG–/– keratinocytes (M.J.S.P. and V.T.unpublished observations). Because Syndecan-1 enhanceskeratinocyte motility through regulation of ECM deposition andintegrin surface expression (Stepp et al., 2007) it is possible thatthis glycoprotein contributes to PG-dependent regulation ofkeratinocyte motility.

Increased activity of Src and its phosphorylation of FAK havebeen shown to accompany weakening of cell–cell adhesions andde-regulation of E-cadherin (Avizienyte et al., 2002; Frame, 2004).This Src/FAK signaling axis causes a switch in adhesion typepredominance in epithelial cells from cadherin based to integrinbased adhesions, promoting a more motile phenotype (Avizienyteand Frame, 2005). Here we demonstrate that elevated motility inPG–/– keratinocytes relies on alterations to the underlying matrixand maturation state of cell–substrate adhesive structures or focalcontacts. However, the observed elevation of RhoA and Rac1activity were not sensitive to the inhibition of Src and vice versa,


suggesting the existence of multiple parallel pathways for PG incontrolling keratinocyte motility behavior. Supporting this idea,the morphological responses of the PG–/– cells treated with Src andRhoA inhibitors were divergent. On the one hand, the inhibition ofSrc led to formation of strong focal adhesions (Fig. 7C),accompanied by a lack of lamellipodial protrusions (Fig. 8E). Onthe other hand, selective inhibition of RhoA inhibited cell motility(Fig. 8A) (Ridley et al., 2003) but left the cells with manyprotrusions (Fig. 8D,E), most probably as a result of active Rac1(Fig. 8B) (Rottner et al., 1999), which is responsible forlamellipodia formation. This is further supported by the observationthat PG+/– cells, whose Rac1 activation levels are substantiallylower (Fig. 8B), show less prominent protrusions upon Rhoinhibition (Fig. 8D).

Taken together with previous findings, our data are consistentwith a model in which desmosomal cadherins use PG as a molecular‘switch’ to modulate cell–cell adhesion-dependent and -independentfunctions, through regulation of its subcellular localization. Innormal cells, partial or complete detachment from cell–cell contactswould lead to a rise in the amount of free cytoplasmic and nuclearPG, which would inhibit motility and survival mechanisms,preventing the inappropriate spread of cells. Under conditionswhere physiological tissue remodeling and cell migration occur,this regulatory mechanism may be attenuated by recruitment of PG

Fig. 8. Plakoglobin regulates motilitythrough independent regulation ofRhoGTPase and Src activity.(A)Representative tracks (n10) showingthe effects of inhibition of Rho (C3transferase, 2g/ml) and Src (PP2, 10M)on PG–/– keratinocyte motility. (B)Rho andRac1 G-LISA showing that increase intheir activity in PG–/– cells is independentfrom Src activity. (C)Western blotdemonstrating that the increased Srcactivity in PG–/– cells is independent fromRho activity. (D)DIC images of PG+/– andPG–/– keratinocytes, either untreated ortreated with the Rho and Src inhibitors.Scale bar: 20 m. (E)Number oflamellipodia in PG–/– cells (control, whitebars; Src-inhibitor-treated, gray bars; Rho-inhibitor-treated, black bars).Jo

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into intercellular junctions present in the coherent migrating sheetof cells, thus allowing controlled migration in activated cells at theleading edge of a wound. In tumors, this regulatory mechanismmight be completely lost leading to uncontrolled migration andincreased survival of single tumor cells lacking PG. In addition toproviding insight into mechanisms of epithelial remodeling inwound healing and cancer, further investigation of the cell–celladhesion-independent role of PG in regulating matrix depositionand cell–matrix interactions may ultimately shed light on how PGdysfunction contributes to inherited diseases of the skin and heart.

Materials and MethodsCell cultureKeratinocyte cultures established from PG knockout (PG–/–) or heterozygous control(PG+/–) mouse skin (Caldelari et al., 2001) were cultured in defined keratinocyteserum-free medium (Invitrogen), supplemented with 10 ng/ml EGF and 10–10 Mcholeratoxin. Calcium level was adjusted to 0.07 mM using 1 M CaCl2.

ECM preparationKeratinocyte matrix was prepared as described previously (Langhofer et al., 1993).Briefly, cells were plated on tissue culture dishes or glass coverslips and allowed toreach confluency. The culture medium was removed and the cells were washed insterile phosphate-buffered saline (PBS). The cells were ruptured by treating them for5 minutes in sterile 20 mM NH4OH, followed by three rapid washes in steriledistilled water. The remaining ECM and cell membranes were then washed severaltimes in sterile PBS, followed by water, and then immediately analyzed or used forcell plating. Laminin-332-enriched matrix was prepared from 804G cells as describedbefore (Langhofer et al., 1993). Recombinant FN was purchased from Sigma.

Western blottingWhole and ‘de-roofed’ cell samples were prepared using urea sample buffer. Theamount of protein in whole cell lysates was measured using the Amido Black Assay(Sheffield et al., 1987). Equal amounts of protein were fractionated on a 7.5% SDS-polyacrylamide gel, and immunoblotting was performed as described previously(Kowalczyk et al., 1997). Primary antibodies against FN (Sigma), PG (mouse mAbclone 11E4), a-tubulin (12G10), Src(Tyr416-P) and total Src (Cell SignalingTechnology) were used. The following secondary antibodies were used: HRP-conjugated goat anti-mouse and anti-rabbit (Kirkegaard and Perry Laboratories,Gaithersburg, MD).

Indirect immunofluorescencePG+/– and PG–/– mouse keratinocytes were fixed with 4% paraformaldehyde andstained for integrin a6 and fibronectin, using GoH3 (Chemicon) and anti-FN (Sigma)antibody, respectively. Alexa Fluor 488 goat anti-rat and Alexa Fluor 568 goat anti-rabbit (Molecular Probes, Eugene, OR; 1:400 dilution) were used as secondaryantibodies. For visualization of the actin cytoskeleton and focal adhesions, cells werefixed by formal saline (3.7% formaldehyde in PBS) followed by 2 minutes in ice-cold acetone. Cells were then stained using a monoclonal anti-FAK(Tyr397-P)antibody (BD Transduction Labs) together with Alexa Fluor 488 goat anti-mouseantibody and Rhodamine-conjugated phalloidin for F-actin staining.

Mass spectrometry and data analysisProtein sample preparation, mass spectrometry and data analysis were conducted aspreviously described (Todorovic et al., 2010). Even though mouse keratinocyte cellculture medium does not contain serum, proteins present in the medium wereidentified using control samples (glass surfaces without cells). The contaminatingproteins could then be deleted from the protein lists generated for the de-roofed cells,leaving only monolayer-isolated proteins for comparison of unique spectra as themetric for relative quantification.

Live cell imagingKeratinocytes were plated at high density on four-chamber borosilicate glasscoverslips overnight to deposit their own matrix. The next day cells were de-roofedand fresh PG–/– cells were plated at 10–20% density. After cells had attached andspread (2 hours), medium was changed to eliminate debris, and live-cell imagingwas conducted using a Leica DMI 6000 microscope (�20, DIC), and a Hamamatsudigital camera. Cells were imaged for 5 hours in 5-minute intervals. Images wereprocessed using Simple PCI (Hamamatsu), and individual cell movement wasanalyzed with Metamorph (Molecular Devices). Motility assays were performed aminimum of three times, with at least 50 cells analyzed in each trial.

Adenoviral constructs and transductionThe pAdEasy adenovirus packaging system kindly provided by Warren G. Tourtellote(Northwestern University Feinberg School of Medicine) was used to generatepreviously characterized myc-tagged, full-length human PG (Palka and Green, 1997)as described previously (Yin et al., 2005a). Infection rates were monitored using

GFP expressed in tandem with PG; at least 30% of replated cells still retained GFPexpression. In order to allow the PG and GFP control transduced cells to depositfresh extracellular matrix, the cells were re-plated for matrix deposition 24 hoursafter infection and left overnight. The next day cells were either fixed and stainedfor fibronectin deposition or de-roofed as described above. Control GFP-transducedcells were plated upon the pre-deposited matrices and live-cell images were taken asdescribed.

RNA arrayThe levels of expression of ECM and membrane related molecules in PG+/– and PG–/– cells was determined by using Oligo GEArray Mouse Extracellular Matrix andAdhesion Molecules MicroArray (SABiosciences, Frederick, MD) according to themanufacturer’s directions. The data was analyzed using GEArray Expression AnalysisSuite.

G-LISAThe levels of active RhoA and Rac1 were determined using RhoA- and Rac1-specific colorimetric activation assays (G-LISA; Cytoskeleton) according to themanufacturer’s directions. A specific Rho inhibitor, cell permeable C3 transferase,was obtained from Cytoskeleton. The absorbances were read at 490 nm on aSynergy2 plate reader (BioTek).

Quantitative RT-PCRRNA was isolated and purified from PG+/– and PG–/– keratinocytes and was treated asdescribed in the text using an RNeasy mini kit (Qiagen) according to manufacturer’srecommendations. First strand synthesis was performed on normalized RNA samplesusing a SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen).qRT-PCR was performed using SBYR Green PCR Master Mix (Applied Biosystems)with the following primers: 5�-CTTTGTCAAGCTCATTTCCTGG-3� (mouse Gadph– forward 1), 5�-TCTTGCTCAGTGTCCTTGC-3� (mouse Gadph – reverse 1), 5�-CCTCTGCGCCCTTGAGCTAGGA-3� (mouse Gadph – forward 2), 5�-CACAA-GAAGATGCGGCCGTCTC-3� (mouse Gadph – reverse 2), 5�-CTTTGGCAGTG-GTCATTTCAG-3� (mouse FN – forward 1), 5�-ATTCTCCCTTTCCATTCCCG-3�(mouse FN – reverse 1), 5�-TGCCTTCAACTTCTCCTGTG-3� (mouse FN – forward2), 5�-CACTAACCACGTACTCCACAG-3� (mouse FN – reverse 2).

FN promoter luciferase assayPG+/– and PG–/– keratinocytes were transiently transfected with a mixture of 0.1 gpRL-TK transfection efficiency control vector (Promega) and 2 g of pGL3 (emptyvector) or pFN(1.2Kb)Luc (generous gift from Jesse Roman) reporter vectors usingthe polyethylenimine method as previously described (Stiehl et al., 2006). Transfectionefficiency was established at 20–40% using pLZRS-pBMN-EGFP (Getsios et al.,2004a) and determining the proportion of green cells. Cell lysates were prepared andluciferase assays conducted using Dual Luciferase Reporter Assay System (Promega)according to manufacturer’s recommendations. Luminescence was measured usinga Synergy2 plate reader luminescence module (BioTek).

The authors thank Eliane Mueller for the PG+/– and PG–/–

keratinocytes, and colleagues for helpful discussions. We also thankJesse Roman for the pFN(1.2KB)Luc and control plasmids, as well asJeffrey D. Ritzenthaler for providing the protocols on how to use theconstructs. In addition, we thank Kimberly Smith for help with settingup the luciferase assays. This work was supported by R01AR43380,R01AR041836-17S1 (American Recovery and Rehabilitation Act),R01CA122151 and the J. L. Mayberry endowment (to K.J.G.) andNIH training grant T32 CA070085 and F32 AR055444 to V.T. V.T.and B.V.D. performed most of the work. T.Y. performed the initialstudy. A.D.D. conducted RhoA and Rac1 G-LISA assays, as well asFN qPCR analysis. M.J.S.P. analyzed differential protein expressionby mass spectrometry. E.A. performed live cell imaging. V.T., K.J.G.,J.C.R.J. wrote the manuscript. K.J.G. and J.C.R.J. provided guidancefor the project. The authors declare that they have no conflict ofinterest. Deposited in PMC for release after 12 months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/123/20/3576/DC1

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