Scribble regulates an EMT polarity pathway through ......Scrib regulates junctions through EMT...

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Scribble regulates an EMT polarity pathway throughmodulation of MAPK-ERK signaling to mediatejunction formation

Imogen A. Elsum1, Claire Martin1 and Patrick O. Humbert1,2,3,4,*1Cell Cycle and Cancer Genetics, Research Division, Peter MacCallum Cancer Centre, Melbourne, Australia2The Sir Peter MacCallum Department of Oncology, Melbourne, Australia3Department of Pathology, University of Melbourne, Parkville, Australia4Department of Molecular Biology and Biochemistry, University of Melbourne, Parkville, Australia

*Author for correspondence ([email protected])

Accepted 23 May 2013Journal of Cell Science 126, 3990–3999� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.129387

SummaryThe crucial role the Crumbs and Par polarity complexes play in tight junction integrity has long been established, however very few

studies have investigated the role of the Scribble polarity module. Here, we use MCF10A cells, which fail to form tight junctions andexpress very little endogenous Crumbs3, to show that inducing expression of the polarity protein Scribble is sufficient to promote tightjunction formation. We show this occurs through an epithelial-to-mesenchymal (EMT) pathway that involves Scribble suppressing ERK

phosphorylation, leading to downregulation of the EMT inducer ZEB. Inhibition of ZEB relieves the repression on Crumbs3, resulting inincreased expression of this crucial tight junction regulator. The combined effect of this Scribble-mediated pathway is the upregulationof a number of junctional proteins and the formation of functional tight junctions. These data suggests a novel role for Scribble in

positively regulating tight junction assembly through transcriptional regulation of an EMT signaling program.

Key words: Polarity, Scribble, ERK, Tight junctions, EMT, Mammary epithelium

IntroductionThe formation and maintenance of apically located tight junctions

is key for the establishment of correct tissue architecture and

epithelial cell polarity. Major functions attributed to tight junctions

include the regulation of paracellular permeability and a ‘fence’ or

physical barrier function to control the diffusion of lipids and

proteins within the plasma membrane thus contributing to the

organization of the apical and basal domains (Ebnet, 2008; Shin

et al., 2006).

There are three evolutionary conserved polarity complexes; the

Scribble, Par and Crumbs complexes that act as regulators of

apical-basal polarity. Members of each of these complexes have

been reported to play important roles in establishment and

maintenance of tight junctions (Chen and Macara, 2005; Fogg

et al., 2005; Hurd et al., 2003; Ivanov et al., 2010; Joberty et al.,

2000; Lemmers et al., 2004; Michel et al., 2005; Qin et al., 2005;

Shin et al., 2005; Straight et al., 2004; Stucke et al., 2007; Suzuki

et al., 2001; Suzuki et al., 2009). Most of these studies have

focused on the Par and Crumbs complexes, which are intimately

associated with tight junctions however, more recently an

important role for the Scribble complex has also emerged.

Scribble knockdown in MDCK cells results in a delay in tight

junction assembly and impaired recruitment of E-cadherin to the

membrane (Qin et al., 2005), and loss of Scribble in human colon

cells has been shown to impair tight junction assembly (Ivanov

et al., 2010).

Members of the Scribble complex, Scribble, Discs Large (Dlg)

and Lethal Giant Larvae (Lgl), were all identified in Drosophila

melanogaster as neoplastic tumor suppressors, with loss of

function disrupting apical basal polarity and junctional integrity,

and inducing inappropriate proliferation and tissue overgrowth

(Bilder and Perrimon, 2000; Mechler et al., 1985; Woods

and Bryant, 1991). Since then, Scribble has been implicated

in numerous cellular processes including proliferation,

differentiation, apoptosis, stem-cell maintenance, migration and

vesicle trafficking (Elsum et al., 2012; Humbert et al., 2008).

Human Scribble is a functional homologue of Drosophila Scribble

(Dow et al., 2003) and is a target for ubiquitin-mediated

degradation by the human papilloma virus (HPV) E6 proteins

and E6AP protein ligase (Nakagawa and Huibregtse, 2000). HPV

is frequently associated with cervical carcinomas, which often

show decreased Scribble expression (Nakagawa et al., 2004).

Other oncogenic viruses such as human T-cell leukemia virus type

1 (HTLV-1) Tax, a causative agent for adult T-cell leukemia, are

known to target Scribble (Elsum et al., 2012; Javier and Rice,

2011; Okajima et al., 2008). Additionally, mislocalization or

deregulated Scribble expression has been reported in a variety of

epithelial cancers (Gardiol et al., 2006; Kamei et al., 2007; Navarro

et al., 2005; Ouyang et al., 2010; Pearson et al., 2011; Vaira et al.,

2011; Zhan et al., 2008). The mechanism by which Scribble

influences tumorigenesis is unclear, yet may lie in the interactions

with oncogenic signaling cascades such as Ras, Wnt and GPCR

signaling (Humbert et al., 2008).

Tight junctions are recognized to have important roles in

acting as signaling platforms in a variety of cellular processes

(Balda and Matter, 2008; Balda and Matter, 2009; Farkas et al.,

3990 Research Article

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2012; González-Mariscal et al., 2008; Steed et al., 2010; Yanget al., 2012). In turn, components of numerous intracellular

signaling pathways including G-proteins, PLCc and PKC havebeen implicated in the regulation and maintenance of tightjunction integrity (Nunbhakdi-Craig et al., 2002; Nusrat et al.,1995; Stuart and Nigam, 1995; Terry et al., 2011; Walsh et al.,

2001; Ward et al., 2002). Of note, tight junction regulationis among the plethora of cellular processes the Ras MAPK(mitogen activated protein kinase)-ERK (extracellular-signal-

regulated kinase) pathway is involved in. The MAPK-ERKcascade involves a series of phosphorylation events to regulatemachinery controlling physiological processes such as

proliferation, growth, apoptosis and migration (Dhillon et al.,2007; Shaul and Seger, 2007). The role MAPK-ERK signalingplays in tight junction integrity appears complex and contextdependent. In intestinal cells, MAPK-ERK activation results in

increased expression of crucial tight junction proteins (Yanget al., 2005). In Caco2 cells, MAPK-ERK signaling regulatesoxidative-stress-induced disruption of tight junctions in a manner

that is dependent on their differentiation state (Aggarwal et al.,2011; Basuroy et al., 2006). In Ras transformed MDCK cells,inhibition of the MAPK-ERK pathway restores tight junctions

(Chen et al., 2000) and expression of a constitutively active Raf-1in a rat epithelial cell line promotes downregulation of the tightjunction proteins Occludin and Claudin1 (Li and Mrsny, 2000).

Although the precise mechanisms remain unclear, these andnumerous other examples illustrate that MAPK-ERK signalingcan influence junction dynamics.

Epithelial-to-mesenchymal transition (EMT) signaling is

among the numerous pathways mediated by MAPK-ERKsignaling. Recently ERK2 was shown to regulate theexpression of ZEB through Fos related antigen-1 (Fra1) (Shin

et al., 2010). The zinc finger transcription factors ZEB1 andZEB2 (also known as dEF1 and SIP1 respectively) are classicexamples of EMT-induced transcriptional regulators that are

activated during tumor cell dissemination and invasion(Schmalhofer et al., 2009; Spaderna et al., 2008). They havebeen shown to suppress tight junction proteins includingOccludin, Claudins, Tricellulin, JAM and ZO3 (Aigner et al.,

2007; Comijn et al., 2001; Peinado et al., 2007; Vandewalle et al.,2005), as well as several polarity proteins, such as Crumbs3,PATJ and Lgl2, through direct binding to E-boxes in the

promoter regions (Aigner et al., 2007; Spaderna et al., 2008).Fra1, a member of the Fos family and a key component of theAP-1 transcriptional complex, is reported to be involved in tumor

progression and invasion in a variety of different human tumorcell lines (Young and Colburn, 2006). Furthermore, activation ofthe MAPK-ERK pathway is known to increase expression of

Fra1 with ERK-mediated phosphorylation stabilizing Fra1 topromote efficient activity (Hoffmann et al., 2005; Treinies et al.,1999; Young et al., 2002).

We have previously shown that Scribble mediates MAPK-

ERK signaling both in vitro and in vivo (Dow et al., 2008;Pearson et al., 2011). Here we demonstrate the existence of apathway that encompasses both polarity and signaling molecules

to regulate tight junction formation through regulation of MAPK-ERK activity. Using MCF10A cells, a mammary epithelial cellline that despite forming desmosomes and adherens junctions,

lack tight junctions under standard tissue culture conditions(Underwood et al., 2006), we show that increased expression ofScribble promotes the formation of functional tight junctions. We

provide evidence for a pathway in which Scribble regulatesMAPK-ERK signaling which in turn mediates the expression of

the EMT inducer ZEB via Fra1. Inhibition of Fra1 leads to areduction in ZEB levels and consequently an increase in thepolarity protein Crumbs3, an essential component of tight

junctions. The data presented here show a novel role forScribble in positively regulating tight junction assemblythrough modulation of an EMT signaling and transcriptionalprogram.

ResultsScribble promotes tight junction formation

To study the effect of aberrant Scribble expression in MCF10Acells, stable lines were generated by retroviral transduction in

which human Scribble was knocked down or overexpressed.Extensive microarray analysis was performed to investigate howScribble expression affects the global transcriptional programme

(I.A.E., unpublished data). We found that cells ectopicallyexpressing Scribble (ScribOE) at levels approximately ten-foldgreater than vector controls (Fig. 1B) had a strong cell adhesion

signature. DAVID bioinformatics analysis (http://david.abcc.ncifcrf.gov/) showed enrichment in the KEGG ‘cell adhesionmolecules’ pathway with a P-value of 7.961022. Examination ofthe gene list (supplementary material Table S1) indicated manyproteins associated with desmosomes and tight junctions,including Occludin and Claudin1, were upregulated by Scribbleoverexpression (Fig. 1A). Western blot analysis confirmed this

upregulation occurred at a protein level (Fig. 1B).

ZO1, a scaffolding protein associated with tight junctions, can

be used as a marker of junction integrity. We found localizationof ZO1 to be dramatically altered by Scribble overexpression(Fig. 1C) yet total expression levels remained unchanged(Fig. 1B). Consistent with previous reports (Fogg et al., 2005),

vector control MCF10A cells displayed a diffuse pattern of ZO1,with punctate staining around the membrane. In contrast, ScribOE

cells displayed smooth, continuous ZO1 staining, typical of intact

tight junctions, and quantification of ZO1 staining showed asignificant 3.3-fold increase in unbroken continuous stainingin ScribOE cells compared to control cells (Fig. 1C).

Immunostaining for other tight junction markers, Occludin andClaudin1, also showed an increase in continuous staining inScribOE cells, supporting the ZO1 staining (Fig. 1C). It isinteresting to note that despite being a stable population with all

cells expressing Scribble, only a subset of the ScribOE cellslocalize ZO1, Occludin or Claudin1 to junctions. The patchynature of the rescued tight junction protein localization is

puzzling, however we observed this in stable cell linesexpressing Scribble as well as following use of chemicalinhibitors and siRNA (see below). This suggests that additional

extrinsic factors such as high cell confluency may also enhanceZO1 restoration in ScribOE cells at a population level.

To functionally characterize Scribble-mediated promotion of

adhesion, we carried out a range of assays including transmissionelectron microscopy (TEM), transepithelial resistance (TER)and size-selective assessment of tight junction paracellular

permeability using fluorescently labelled dextrans (PPFD).Analysis of the microstructure of the epithelial sheet inMCF10A vector and ScribOE cells using TEM revealed an

increase in adhesion and cell junctions in ScribOE cells(supplementary material Fig. S1A). Additionally, we carriedout TER measurements to determine the ion permeability of

Scrib regulates junctions through EMT signaling 3991

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assayed junctions. ScribOE cells were found to have a modest yet

significant increase in TER when grown for 13 days on transwell

filters (Fig. 1D). As vector control and wild-type MCF10A cells

lack tight junctions, readings were similar to the blank wells.

However, Scribble overexpressing cells recorded values ,1.5-foldhigher than vector cells. MDCK cells, which form complete, intact

tight junctions, were used as a positive control and produced TER

readings close to 500 ohms.cm2 (data not shown). Tight junctions

regulate diffusion of ions and molecules both by charge and size

and so, to effectively measure tight junction functionality, both

must be measured. TER assays determined the diffusion of ions

and to assay size diffusion we performed size-selective assessment

of tight junction paracellular permeability using fluorescently

labelled dextrans (PPFD) (Matter and Balda, 2003). Vector control

and ScribOE cells were grown for 13 days on transwell filters prior

to addition of fluorescently labeled 70 kDa Rhodamine and 4 kDa

FitC dextran molecules (Fig. 1E). As the experimental values in a

sample group varied despite the trends between sample groups

remaining consistent, the data has been shown here as the change

in fluorescent diffusion in the ScribOE cells relative to the vectorcells. A significant difference was seen in the diffusion of thelarger 70 kDa molecule yet not the smaller 4 kDa suggesting that

the rudimentary tight junctions formed in cells overexpressingScribble are more successful in blocking larger molecules. AsMDCK cells have well-formed tight junctions, they were used as apositive control and showed values close to zero across both sizes

of dextran.

Inhibition of MAPK-ERK signaling promotes tightjunction assembly

We have previously shown that Scribble overexpression suppressesMAPK activity in MCF10A cells in the context of oncogenic Ras

(RasV12) (Dow et al., 2008). We carried out biochemical analysison ScribOE cells to further look at how altered Scribble expressionalone can influence MAPK signaling. We found that in resting

cells, Scribble overexpression suppresses phosphorylation atmultiple tiers of the MAPK-ERK pathway including MEK andERK [Fig. 2A(i)]. Upon EGF stimulation, the phosphorylation

Fig. 1. Scribble promotes adhesion and cell

junctions. (A) MCF10A cells stably expressing

Scribble (ScribOE) or vector control were analysed by

microarray as described in the Materials and Methods.

Ectopic Scribble expression resulted in upregulation of

numerous tight junction and desmosome proteins.

(B) Cell lysates from MCF10A cells stably expressing

Scribble or vector control analysed by western blotting

and probed for the indicated antibodies including tight

junction markers. (C) MCF10A stable cell lines were

grown to confluency and (i) stained for the tight

junction markers ZO1, Occludin and Claudin1.

(ii) Quantification of ZO1 staining was carried out as

described in Materials and Methods. Error bars

represent s.d., n54. P-values were calculated using a

paired Student’s t-test. Scale bar: 20 mm.(D) Measurements of transepithelial resistance

(ohms.cm2) on cells stably expressing Scribble or vector

control, grown for 13 days on transwell filters. Error

bars represent s.d., n56, P-values were calculated using

a paired Student’s t-test. (E) Size-selective assessment

of tight junction paracellular permeability on cells

stably expressing Scribble or vector control using

fluorescently labelled dextrans. Cells were grown for 13

days on transwell filters prior to addition of

fluorescently labeled 70 kDa-dextran–Rhodamine

(pink) and 4 kDa-dextran–FitC (green) as described in

the Materials and Methods. Error bars represent s.d.,

n53 with experiments performed in duplicate using two

independent cell lines. P-values were calculated using a

paired Student’s t-test.

Journal of Cell Science 126 (17)3992

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response of MEK was also muted [Fig. 2A(ii)]. Given this, we were

interested in looking at the role of MAPK signaling in tight junction

formation in MCF10A cells. By using specific inhibitors against

MEK1 (PD98059), PI3K (LY294002) and JNK (SP600125)

(supplementary material Fig. S1B) we were able to assess the

individual contributions of each of these pathways to tight junction

formation. Of the tested pathways, only inhibition of the MEK arm

was sufficient to promote tight junction formation (Fig. 2B).

In agreement with these results, siRNA-mediated knockdown

of ERK1 and ERK2 was found to promote the formation of

tight junctions (Fig. 2C). Quantification of staining revealed a

significant 3.8-fold increase in continuous ZO1 staining in

siERK1/2 cells compared to the siControl [Fig. 2C(ii)]. No

detectable differences were seen in ZO1 staining between cells

individually transfected with siERK1 and siERK2 or combined

transfections (supplementary material Fig. S1D). Further

confirmation of tight junction formation was seen by staining

for Occludin and Claudin1 in cells where both ERK1 and ERK2

had been depleted (Fig. 4B).

Inhibition of Fra1 and ZEB1/2 promotes tight junction

assembly

ERK2 has been reported to regulate expression of the EMT

inducer ZEB, through Fra1 (Shin et al., 2010). We investigated

whether Scribble expression influences ZEB expression through

such a pathway.

Overexpression of Scribble resulted in a decrease in Fra1 at

both the protein and mRNA level and a decrease in Fra1 was also

seen upon knockdown of ERK1/2 (Fig. 3A). As ZEB has been

shown to lie downstream of Fra1 (Shin et al., 2010), we looked at

whether changes in upstream components such as Scribble or

ERK could alter ZEB expression and consequently tight junction

formation. Forced Scribble expression, siERK1/2 or siFra1 all

resulted in downregulation of ZEB1 and ZEB2 (Fig. 3B). To test

the functional consequences of these findings, MCF10A cells

were depleted of Fra1 or ZEB1/2 using siRNAs (supplementary

material Fig. S2) and stained for ZO1, Occludin and Claudin1

(Fig. 3C,D). In all cases knockdown modulated tight junction

formation and quantification of continuous ZO1 staining showed

an approximate 1.5- and 3-fold increase for Fra1 and ZEB

respectively compared to the controls.

Given that EMT inducers are known to influence tight junction

integrity (Aigner et al., 2007; Ikenouchi et al., 2003; Vandewalle

et al., 2005) we were interested in investigating whether enforced

Scribble expression resulted in altered expression of EMT

inducers other than ZEB. We found that similarly to the

changes in ZEB expression, Snail and Slug mRNA levels were

reduced in ScribOE cells. However, unlike ZEB, depletion of

Fig. 2. MAPK-ERK signaling regulates tight

junction assembly in MCF10A cells.

(A) (i) Western blot on lysates harvested from

resting vector- and Scribble-expressing

(ScribbleOE) stable MCF10A cell lines and probed

for the indicated proteins. (ii) Representative

western blot on lysates harvested from EGF

stimulations of starved vector- and Scribble-

expressing MCF10A cells probed for

phosphorylated MEK (pMEK1/2) and total MEK.

Quantitative densitometry analysis of pMEK

relative to total MEK was calculated using ImageJ

software and is shown on right. (B) Wild-type

MCF10A cells treated for 48 hours with DMSO

(control) or inhibitors against MEK1 (PD98059),

PI3K (LY294002) or JNK (SP600125) and

stained for ZO1. Scale bar: 20 mm.(C) (i) Immunofluorescence staining for ZO1,

Occludin and Claudin1 in wild-type MCF10A

cells 72 hours following transfection with

siControl or siERK1/2. (ii) Quantification of ZO1

staining was carried out as described in the

Materials and Methods. Error bars represent s.d.,

n54. P-values were calculated using a paired

Student’s t-test. Scale bar: 50 mm.


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either Snail or Slug was insufficient to promote the formation of

tight junctions (supplementary material Fig. S3). Together these

data suggest a specific, functional role for the ZEB proteins in

tight junction assembly in MCF10A cells.

Crumbs3 is required for Scribble and MAPK-dependent

tight junction formation

Exogenous expression of Crumbs3 in MCF10A cells is sufficient

to induce tight junction structures (Fogg et al., 2005). As several

studies have identified Crumbs3 as a ZEB target (Aigner et al.,

2007; Spaderna et al., 2008), we investigated the possibility of a

ZEB dependent involvement for Crumbs3 in Scribble-mediated

tight junction formation. We first confirmed that ectopic

Crumbs3 expression induced tight junction structures. Stable

MCF10A cell lines were generated using retroviral transduction

to express Crumbs3 at levels ,15–20 times greater than vectorcontrols (supplementary material Fig. S2). Consistent with

previous reports (Fogg et al., 2005), cells overexpressing

Crumbs3 (Crumbs3OE) had smooth continuous ZO1 and

Occludin staining compared to vector controls, indicative of the

formation of tight junctions (supplementary material Fig. S4A).

Additionally, TEM analysis demonstrated the presence of tight

junctions in Crumb3OE cells yet only desmosomes were seen in

vector control cells (supplementary material Fig. S4B). TER

measurements showed an increase of more than 4-fold in

Crumb3OE cells compared to control cells (supplementary

material Fig. S4C). Size diffusion was assayed using PPFD and

the percentage of molecules that had diffused through the tight

junctions in the Crumb3OE cells were compared to the vector

control cells. A larger and more significant difference was seen

with the diffusion of the larger 70 kDa dextran molecule rather

than the smaller 4 kDa molecule (supplementary material Fig.

S4D). This is a similar trend as to what was observed in

the ScribOE cells (Fig. 1E) and once more suggests that the

Crumb3OE cells partially block larger molecules and the smaller

dextran molecules to a lesser extent. We also looked at an earlier

time point to address if there was a difference in the kinetics of

tight junction formation in the ScribOE and Crumb3OE cells. We

Fig. 3. Scribble and ERK1/2 regulate Fra1

and ZEB1/2 transcription to mediate tight

junction assembly. (A) (i) Western blot of

MCF10A cells stably expressing Scribble or

vector control probed for Fra1 and a-tubulin.

Band density was calculated and normalised to

a-tubulin using ImageJ software.

Quantification is shown from four western

blots using three independent cell lines.

(ii) qRT-PCR for Fra1 mRNA in ScribOE cells

and cells transfected with siERK1/2. Values

are expressed relative to the appropriate vector

or siControl cells. Error bars represent s.d.,

n53. P-values were calculated using a paired

Student’s t-test. (B) qRT-PCR for ZEB1 and

ZEB2 mRNA in ScribOE cells, or cells

transfected with siERK1/2 or siFra1. Values

are expressed relative to the appropriate vector

or siControl cells. Error bars represent s.d. P-

values were calculated using a paired Student’s

t-test. (C,D) Wild-type MCF10A cells were

transfected with siControl and (C) siFra1 and

(D) siZEB1/2, then 72 hours later stained for

ZO1, Occludin and Claudin1. A quantification

of continuous ZO1 staining, shown in upper-

right panel of both C and D, was carried out as

described in Materials and Methods. Scale bar:

50 mm.


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performed PPFD 3 days after plating onto transwell filters. Again

there was a significant difference in the diffusion of the large 70 kDa

dextran molecule but not the smaller 4 kDa molecule in both the

ScribOE and Crumb3OE cells (supplementary material Fig. S5). There

was a greater difference in the diffusion of both the small and large

dextrans in the Crumb3OE cells than in the ScribOE cells suggesting

that the former have more intrinsically intact tight junctions.

To determine if Scribble expression or MAPK-ERK signaling

influences Crumbs3 transcription via a Fra1/ZEB pathway we

measured Crumbs3 mRNA in ScribOE cells, cells treated with

siERK1/2, siFra1 or siZEB1/2. All conditions resulted in modest

yet significant increases in Crumbs3 (Fig. 4A). Despite extensive

efforts, we were unfortunately unable to measure the protein levels

and localization of Crumbs3 in ScribOE cells, cells treated with

siERK1/2, siFra1 or siZEB1/2 as the available commercial

antibodies did not reliably detect endogenous Crumbs3. To test

the requirement of Crumbs3 in the tight junction phenotype

observed in ScribOE cells, siERK1/2, siFra1 and siZEB1/2 cells,

we made use of siRNAs targeted against Crumbs3 (supplementary

material Fig. S2). Co-transfection of siCrumbs3 in ScribOE,

siERK1/2, siFra1 or siZEB1/2 cells abrogated the formation of

tight junctions (Fig. 4B) indicative of an essential role for

Crumbs3 in tight junction assembly in MCF10A cells. Of note,

Scribble expression or localization was not altered in the presence

of siCrumbs3 (data not shown). Taken together, our results support

a model in which Scribble acts to regulate tight junction formation

in MCF10A cells through a pathway involving MAPK-ERK

signaling, expression of ZEB, Fra1 and Crumbs3 (Fig. 5).

DiscussionActive EMT programs are known to deregulate the expression of

many essential polarity and junction proteins, resulting in polarity

Fig. 4. Crumbs3 is required for tight junction

assembly downstream of ERK1/2, Fra1 and

ZEB1/2. (A) qRT-PCR for Crumbs3 mRNA in

MCF10A cells stably expressing Scribble or vector

control transfected with siERK1/2, siFra1 or

siZEB1/2. Values are expressed relative to the

relevant control. Error bars represent s.d., n54–6.

P-values were calculated using a paired Student’s t-

test. (B) siControl cells, ScribOE cells, siERK1/2,

siFra1 or siZEB1/2 cells were each co-transfected

with siControl (top panel) or with siCrumbs3

(bottom panel) and stained for ZO1 72 hours

following transfection. Scale bar: 20 mm.

Fig. 5. Scribble regulates tight junctions through a MAPK-

ERKRFra1RZEBRCrumbs3 axis. Scribble suppresses MAPK-ERKsignaling, possibly through direct protein interactions, resulting in decreased

ZEB expression through regulation of Fra1. Decreased ZEB expression leads

to an increase in expression of the polarity protein Crumbs3, a crucial

component of tight junctions (see text for further details).


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loss and junction dissolution (Godde et al., 2010; Moreno-Buenoet al., 2008; Peinado et al., 2007). In this study we build on this

notion and have identified the polarity protein Scribble as a novelregulator of an EMT signaling and transcriptional pathwayinvolved in the establishment of tight junctions. We have shownthat Scribble-mediated suppression of MAPK-ERK signaling

results in downregulation of the ZEB proteins through changes tothe expression of the gene product Fra1. Consequently, thepolarity protein Crumbs3 is transcriptionally upregulated leading

to the formation of functional tight junctions. The data presentedhere also indicates that Scribble is likely to act on additionalpathways that regulate tight junction integrity to that described

above as overexpression of Scribble also results in an increase inOccludin and Claudin1 expression (Fig. 1A), this effect is notseen upon Crumbs3 overxpression (Fogg et al., 2005). It ispossible that the upregulation of Occludin and Claudin1 and the

upregulation of Crumbs3 are processes that lie parallel andseparately contribute to tight junction formation.

As mentioned previously, ERK2 has been shown to regulate

the expression of ZEB through Fra1 (Shin et al., 2010). Shin andcolleagues demonstrated that ERK2, but not ERK1, specificallymediates upregulation of ZEB and induction of an EMT

phenotype. Although we see no difference in efficacy of tightjunction formation with siERK1 vs siERK2 (supplementarymaterial Fig. S1D), we see a greater increase in Crumbs3

expression and a greater decrease in Fra1 and Zeb1 expressionwith ERK2 depletion than ERK1 (data not shown). These resultsare therefore consistent with Shin et al. (Shin et al., 2010) andsuggest that ERK2 is playing a greater role than ERK1 in

controlling the EMT phenotype, however this does not correlateto changes in tight junction integrity and suggests that otherpathways than EMT signaling contribute to aspects of tight

junction integrity.

What other Fra1 and Zeb targets may be involved in tightjunction regulation? Fra1 is part of the AP-1 transcriptional

complex that interacts with several proteins includingretinoblastoma, SMAD3, SMAD4 and the p65 subunit of NFkB(Young and Colburn, 2006). In addition to MAPK signaling,regulation of ZEB expression has been linked to hormones, TGFband also of note, NFkB signaling (Chua et al., 2007; Dillner andSanders, 2004; Dohadwala et al., 2006; Peinado et al., 2007;Shirakihara et al., 2007). As overexpression of the NFkB subunitp65 in MCF10A cells results in increased expression of ZEB1 andZEB2 (Chua et al., 2007), these data suggest that ZEB and Fra1could mediate part of their effects through regulation of NFkBpathway. Nevertheless, we found that depletion of variouscomponents of the NFkB pathway in MCF10A cells had nodiscernible effect on tight junction formation (supplementary

material Fig. S6). This data strengthens our hypothesis for aspecific role of the ERKRFra1RZEBRCrumbs3 pathway in tightjunction assembly (Fig. 5).

Our laboratory has previously shown that Scribble suppresses

Ras dependent transformation through inhibition of MAPKactivity (Dow et al., 2008). It is likely that in ScribOE MCF10Acells, Scribble-mediated suppression of MAPK-ERK signaling

prevents stabilization of Fra1 and results in the decreased ZEBexpression reported here. This raises the question of how Scribbleis suppressing MAPK-ERK signaling. Recently a direct protein

interaction between Scribble and ERK was reported (Nagasakaet al., 2010b). In this study the authors suggest that ERKactivation enhances the interaction between Scribble and ERK,

the formation of such a Scribble–ERK complex then acts toinhibit MAPK activity. Varying phosphorylation sites within

Scribble have been proposed to alter its ability to bind ERK andthe cellular localization (Nagasaka et al., 2010a; Yoshihara et al.,2011). Phosphorylation events within Scribble may therefore

alter its function thus allowing it to function as a modulator ofsignaling and/or a scaffold. Scribble may act to differentiallyregulate several cellular processes in a manner that is dependenton the level of activity of particular signaling pathway. When

Scribble is ectopically expressed, as reported here, the systemmay become saturated so that Scribble binds ERK, forming acomplex to alter downstream signaling events including

inhibition of EMT programs while additionally functioning atother cellular levels to promote the formation of tight junctions.Such functions may include acting as a dynamic scaffolding

molecule by interacting with known binding partners such asZO2, bPix and b-catenin (Audebert et al., 2004; Métais et al.,2005; Sun et al., 2009). Recently Scribble was shown to recruitthe phosphatase PHLPP1 to the cell membrane to negatively

regulate AKT signaling (Li et al., 2011). We and others havepreviously shown that Scribble regulates the phosphorylationstatus of several components of MAPK signaling (Dow et al.,

2008; Elsum and Humbert, 2013; Nagasaka et al., 2010b).Precisely how Scribble interacts with phosphatases to influencesignaling and junction dynamics is a question that needs to be

further addressed.

A key downstream consequence of the Scribble-mediatedsuppression of MAPK signaling demonstrated here is the

increased expression of the apical polarity gene Crumbs3.Several studies performed in cell lines with eithermicrodeletions or low levels of endogenous Crumbs3 have

shown that reconstituting with ectopic Crumbs3 is sufficient topromote the formation of tight junctions (Fogg et al., 2005; Karpet al., 2008; Rothenberg et al., 2010). Furthermore,

downregulation of Crumbs3 by EMT inducers such as Snailresults in loss of tight junctions (Whiteman et al., 2008). Thefunctional role Crumbs3 plays in tight junctions is unclear yet itis likely to be involved in recruiting or stabilizing other protein

complexes critical for tight junction formation.

In summary, we have found that expression of Scribble in

MCF10A cells results in the formation of tight junctions. Wehave provided evidence that Scribble acts via a MAPK-ERKRFra1RZEBRCrumbs3 axis to regulate tight junctions.Although this study has focused on tight junctions as a

physiological read out, these findings have far broaderimplications regarding how Scribble and other polarity proteinsmay feed into the MAPK pathway and influence tumor

progression through modulation of other processes where EMThas been implicated such as invasion and metastasis. Notably, thepresent study is among the first to describe how one polarity

protein can transcriptionally regulate another. This is important inunderstanding how interactions and feedback within the polaritynetwork occur. This study and some of the examples outlined

above, illustrate that a finely tuned balance between differentpolarity proteins is required for the correct establishment andmaintenance of tight junctions.

Materials and MethodsReagents

Immunostaining and western blotting were performed with the followingantibodies: rabbit anti-pAKT (ser473), anti-total AKT, anti-total ERK1/2, anti-total JNK, anti-pMEK1/2 (41G9) and mouse anti-pERK1/2 (Thr202/Tyr204),


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anti-total MEK1/2 all purchased from Cell Signaling, Danvers, MA. Rabbit anti-Claudin1 (51-9000 Zymed, San Francisco, CA), rabbit anti-occludin (71-1500Zymed), rabbit H-Ras (MC57) (05-775 Upstate, Billerica, MA), rabbit anti-Fra1 (R-20) (sc605) and goat anti-Crumbs3 (C-15) (sc27904) (both purchased from SantaCruz, Santa Cruz, CA), mouse anti a-tubulin (B512) (T5168 Sigma, St Louis, MO)and mouse anti-ZO1 (339100, Invitrogen, Grand Island, NY). Mouse monoclonalanti-Scribble (7C6-D10) has been previously described (Dow et al., 2003). Inhibitorexperiments were performed using the MEK1 inhibitor PD98059 (20 mM) (P125Sigma Aldrich, St Louis, MO), the PI3K inhibitor LY294002 (20 mM) (440202,Merck, Germany) and the JNK inhibitor SP600125 (20 mM) (420119 Merck).

Cell cultureMCF10A cells were maintained in DMEM:F12 (Dulbecco’s Modified EagleMedium: F12) supplemented with 5% donor horse serum (Gibco, NY), 10 mg/mlinsulin (Novo Pharmaceuticals, UK), 0.5 mg/ml hydrocortisone (Sigma Aldrich),20 ng/ml EGF (Cytolab Ltd, Switzerland), 100 ng/ml cholera toxin (SigmaAldrich), penicillin (100 U/ml) and streptomycin (100 U/ml) as previouslydescribed (Debnath et al., 2003). 293T cells were maintained in DMEMsupplemented with 10% foetal bovine serum, penicillin (100 U/ml) andstreptomycin (100 U/ml). All cultures were maintained at 37 C̊ in 5% CO2.

Retroviral constructs and infectionFull length human Scribble was cloned into MSCV-IRES-GFP as previouslydescribed (Dow et al., 2003). The coding sequence of Crumbs3 had previouslybeen PCR amplified and cloned into MSCV-IRES-GFP vector using EcoRI sites.All constructs were verified by sequencing. Virus was generated by transfecting293T cells by calcium phosphate precipitation using the amphotrophic packagingvector RD114 and appropriate DNA constructs. Stable MCF10A cell lines weregenerated essentially as described previously (Debnath et al., 2003). CulturedMCF10A cells were infected with virus 30, 42 and 54 hours post transfection andallowed to recover for 24 hours before selection. To generate polyclonal stablepopulations, cells were either selected for one week in 2 mg/ml puromycin and/orsorted for GFP+ cells on a FACStar flow cytometer (Becton Dickinson, FranklinLakes, NJ). All stable cell lines were monitored and regularly checked forexpression and re-selected if necessary.

EGF stimulationMCF10A cells were plated out at 7.56104 cells in 6-well dishes. 24 h later culturemedia was replaced with EGF-depleted media. Cells were cultured in EGF-depleted media for 48 h before replacing with pre-warmed media containing20 ng/ml EGF. Cells were harvested for protein analysis at designated timepoints.Quantification was performed by densiometry of immunoblot bands using ImageJsoftware (ImageJ, NIH USA).

JNK kinase assayMCF10A cells were grown in 10 cm dishes till they reached 80% confluency. Cellswere UV irradiated (40 J/m2), washed in PBS and fresh media applied for 30 minbefore harvesting. JNK activity was assessed using a non-radioactive kinase assaykit (9810, Cell Signaling) according to the manufacturer’s instructions.

Western blottingProtein concentrations from prepared whole cell lysates were determined using aLowry Protein Assay (BioRad, Hercules, CA) and resolved on pre-cast gradientgels (Invitrogen). Samples were transferred overnight at 4 C̊ and probed with therelevant antibodies. Proteins were visualised using LumiLight ECL reagents(Roche, Germany) according to the manufacturer’s instructions.

RNA isolation, cDNA synthesis and qRT-PCRRNA was harvested using TRizol reagent (Invitrogen) and purified usingchloroform extraction and ethanol precipitation. RNA quality and concentrationwas measured using a Nanodrop ND-1000 spectrophotometer (ThermoScientific,Vic, Aust). cDNA was made from 2 mg of RNA using standard methods (Dowet al., 2008). Samples were run in triplicate on a StepOnePlus Real-Time PCRSystem (Applied Biosystems, Carlsbad, CA). All samples were normalized toGAPDH control (or L32 when using siGAPDH) and fold change between sampleswas calculated using the comparative C(T) method. Primers used for qRT-PCR arelisted in supplementary material Table S2. qRT-PCR experiments were performedon multiple independently derived cell lines and at least 3 independent siRNAtransfections. Statistics were calculated on results from 3–6 runs using GraphPadPrism data software (GraphPad version 5.0b).

siRNA transfectionsAll siRNAs used were purchased from Dharmafect SMARTpool(ThermoScientific, Rockford, IL) and siGFP or siGAPDH were used as controls.Each siRNA pool contained four oligos targeting separate mRNA regions. Fordownstream protein or RNA applications MCF10A cells were plated out in 1.6 mlof antibiotic-free media at 1.16105 cells per well in a 6-well dish ,16 hours prior

to transfection. Prior to transfection, siRNAs were diluted to a concentration of50 nM in 200 ml OpitiMEM and a lipid mix made using 3 ml of Dharmafect3 lipid(ThermoScientific) and 197 ml OptiMEM. For experiments using multiple siRNAs,the highest combined concentration was used to determine the concentration of thesiGFP or siGAPDH control. The lipid and siRNA mix were combined and allowed toduplex for 20 min before adding to cells. Cells were washed and fresh media appliedafter 24 hours. For RNA analysis, cells were harvested 48 hours followingtransfection and for protein analysis, cells were harvested 72 hours posttransfection. For downstream immunofluorescent staining MCF10A cells wereplated out in 400 ml of antibiotic-free culture media at 3.66104 cells per well ontosterile poly-L-lysine-coated coverslips (#354085, Becton Dickinson) in a 48-welldish ,20 hours prior to transfection. As above Dharmafect3 lipid(ThermoScientific) was used and complexed with the appropriate siRNAs at50 nM. Fresh media was applied after 24 hours. Cells were used for subsequentimmunofluorescent staining 72 hours post transfection.

Immunostaining and quantification

MCF10A cells were grown on sterile coverslips prior to fixing in 100% ice-coldmethanol for 20 mins for ZO1 staining or 4% paraformaldehyde for 15 min atroom temperature followed by a 10 min permeabilization step in 1% SDS forOccludin and Claudin1 staining. Coverslips were washed in PBS before blockingin 2% BSA/PBS for 1 hour at room temperature. Samples were incubated withmouse anti-ZO1 (339100, Invitrogen), rabbit anti-occludin (71-1500, Zymed) orrabbit anti-claudin1 (51-9000, Zymed) overnight at 4 C̊. Coverslips were washedwith PBST before probing with secondary anti-mouse antibodies conjugated toAlexa 488 flurochromes (Molecular Probes, Grand Island, NY)). ProLong GoldAntifade containing DAPI (Invitrogen) was used to visualize nuclei. Images wereacquired on a FV1000 BX51 scanning confocal microscope (Olympus Corp,Japan) using a 406oil objective. To quantify tight junctions, continuous ZO1staining was assessed using MetaMorph 6.3 software (Molecular Devices Corp.,Downington, PA). Slides stained for ZO1 were viewed under a confocalmicroscope and multiple images taken randomly across the entire sample.Images were analyzed with MetaMorph software using the ‘Angiogenesis TubeFormation’ application. From the analysis, the value describing the ‘tube length/set’ was used as a measure of continuous ZO1 staining. This value was divided bythe number of cells in the image (determined by DAPI staining). For eachexperiment at least 1000 cells were analyzed across multiple images. Identicalanalysis was carried out for controls and values expressed as fold change relativeto control. Data was plotted and analyzed using GraphPad Prism data software(GraphPad version 5.0b).

Transepithelial electrical resistance measurements

Cells were seeded at 66105 per well onto polyester transwell filters (SigmaAldrich, CLS3450) and readings recorded every second day using a Millicell-ERS(MERS00001 Millipore, Billerica, MA) according to the manufacturer’sinstructions. Resistance was calculated as resistance of a unit area5resistance(V)6effective membrane area (cm2). Sample readings were measured in triplicatefor each reading. Media was replaced following each reading.

Size-selective assessment of tight junction paracellular permeability

Cells were seeded at 66105 or 26105 cells per well on polyester transwell filters(Sigma Aldrich, CLS3450) for 13 or 3 days. Media was replaced every 2 days andon the day indicated TER measurements were recorded and the media wasreplaced with 1 ml in the bottom well and 250 ml in the top well. Fluorescentlylabeled dextrans were added to the top well, 25 ml of 4 kDa FitC-Dextran (Sigma,FD-4) and 25 ml of 70 kDa Rhodamine-Dextran (Sigma, R-9379) and incubated at37 C̊ for approx 3 hours. Media from the bottom plate was aliquoted to 3 wells in ablack-walled 96-well plate and the following wavelengths; FitC (Exc: 485 nm,Em: 544 nm) and Rhodamine (Exc: 520 nm, Em: 590 nm) on a Bio-Stackautomated plate reader (BioTak, Millennium Science, Australia) (Matter andBalda, 2003). The average of the readings were taken as technical replicates andthe experiments were performed in biological repeats as indicated.

Transmission electron microscopy

Cells were grown to confluency before being fixed in 2% paraformaldehyde, 2.5%glutaraldehyde in 0.08 M Sorensen’s phosphate buffer/PBS for 30 mins, thenrinsed in PBS containing 5% sucrose. Post-fixation was carried out using 2%osmium tetroxide in PBS followed by dehydration through a graded series ofalcohols, two acetone rinses and embedding in Spurrs resin. Sections ,80 nmthick were cut with a diamond knife (Diatome, Switzerland) on an Ultracut-Sultramicrotome (Leica) and contrasted with uranyl acetate and lead citrate. Imageswere captured with a Megaview II cooled CCD camera (Olympus) on a JEOL1011 transmission electron microscope.

Microarray experiments and analysis

RNA integrity was assessed using a bioanalyzer (Agilent Technologies, USA) andRNA with a RNA integrity number (RIN) of 10 were used for microarray


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experiments. Microarray experiments were carried out using the GeneChip HumanGene 1.0 ST Array kit (Affymetrix, Santa Clara, CA) according to themanufacturer’s instructions. Quality control metrics were carried out usingExpression Console software (Affymetrix) and Partek software (PartekIncorporated, USA). Gene lists were generated using Tukey’s biweight 1 stepsummarisation and batch effect was applied to passage number and cell line.Pathway and gene ontology analysis was applied using the online DAVIDbioinformatics database (http://david.abcc.ncifcrf.gov).

AcknowledgementsWe thank members of our laboratory for helpful discussions. Specialthanks to the Peter MacCallum Cancer Centre Microscopy Core, inparticular Stephen Asquith for preparation of the TEM samples. Wealso thank the Peter MacCallum Cancer Centre Microarray Core inparticular Dr Jason Li for help and advice with analysis.

Author contributionsI.A.E. designed, planned and carried out the experiments, analyzedand interpreted the data and wrote the manuscript; C.M. assisted withexperimental work and approach, interpretation of data and editedthe manuscript; P.O.H. designed the experiment, sourced funding,provided reagents and mentorship, edited the manuscript.

FundingThis study was supported by a project grant from the National Healthand Medical Research Council of Australia [grant numberAPP1004434 to P.O.H.]. I.A.E was supported by a Cancer CouncilVictoria Postgraduate Cancer Research Scholarship and P.O.H by aBiomedical Career Development Award from the National Healthand Medical Research Council of Australia.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.129387/-/DC1

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