Scribble regulates an EMT polarity pathway through modulation of MAPK-ERK signaling to mediate junction formation

Abstract

The 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 and express very little endogenous Crumbs3, to show that inducing expression of the polarity protein Scribble is sufficient to promote tight junction formation. We show this occurs through an epithelial to mesenchymal (EMT) pathway that involves Scribble suppressing ERK phosphorylation, leading to down regulation of the EMT inducer ZEB. Inhibition of ZEB relieves the repression on Crumbs3, resulting in increased expression of this crucial tight junction regulator. The combined effect of this Scribble mediated pathway is the upregulation of 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.

Full text

Journal of Cell Science
Scribble regulates an EMT polarity pathway through
modulation of MAPK-ERK signaling to mediate
junction formation
Imogen A. Elsum
1
, Claire Martin
1
and Patrick O. Humbert
1,2,3,4,
*
1
Cell Cycle and Cancer Genetics, Research Division, Peter MacCallum Cancer Centre, Melbourne, Australia
2
The Sir Peter MacCallum Department of Oncology, Melbourne, Australia
3
Department of Pathology, University of Melbourne, Parkville, Australia
4
Department of Molecular Biology and Biochemistry, University of Melbourne, Parkville, Australia
*Author for correspondence (Patrick.Humbert@petermac.org)
Accepted 23 May 2013
Journal of Cell Science 126, 3990–3999
ß2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.129387
Summary
The 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 and
express very little endogenous Crumbs3, to show that inducing expression of the polarity protein Scribble is sufficient to promote tight
junction 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 in
increased expression of this crucial tight junction regulator. The combined effect of this Scribble-mediated pathway is the upregulation
of 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
Introduction
The 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 (Gardiolet 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

Journal of Cell Science
2012; Gonza
´lez-Mariscal et al., 2008; Steed et al., 2010; Yang
et al., 2012). In turn, components of numerous intracellular
signaling pathways including G-proteins, PLCcand PKC have
been implicated in the regulation and maintenance of tight
junction 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 regulation
is among the plethora of cellular processes the Ras MAPK
(mitogen activated protein kinase)-ERK (extracellular-signal-
regulated kinase) pathway is involved in. The MAPK-ERK
cascade involves a series of phosphorylation events to regulate
machinery controlling physiological processes such as
proliferation, growth, apoptosis and migration (Dhillon et al.,
2007; Shaul and Seger, 2007). The role MAPK-ERK signaling
plays in tight junction integrity appears complex and context
dependent. In intestinal cells, MAPK-ERK activation results in
increased expression of crucial tight junction proteins (Yang
et al., 2005). In Caco2 cells, MAPK-ERK signaling regulates
oxidative-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-1
in a rat epithelial cell line promotes downregulation of the tight
junction proteins Occludin and Claudin1 (Li and Mrsny, 2000).
Although the precise mechanisms remain unclear, these and
numerous other examples illustrate that MAPK-ERK signaling
can influence junction dynamics.
Epithelial-to-mesenchymal transition (EMT) signaling is
among the numerous pathways mediated by MAPK-ERK
signaling. Recently ERK2 was shown to regulate the
expression of ZEB through Fos related antigen-1 (Fra1) (Shin
et al., 2010). The zinc finger transcription factors ZEB1 and
ZEB2 (also known as dEF1 and SIP1 respectively) are classic
examples of EMT-induced transcriptional regulators that are
activated during tumor cell dissemination and invasion
(Schmalhofer et al., 2009; Spaderna et al., 2008). They have
been shown to suppress tight junction proteins including
Occludin, 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 the
AP-1 transcriptional complex, is reported to be involved in tumor
progression and invasion in a variety of different human tumor
cell lines (Young and Colburn, 2006). Furthermore, activation of
the MAPK-ERK pathway is known to increase expression of
Fra1 with ERK-mediated phosphorylation stabilizing Fra1 to
promote 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 a
pathway that encompasses both polarity and signaling molecules
to regulate tight junction formation through regulation of MAPK-
ERK activity. Using MCF10A cells, a mammary epithelial cell
line that despite forming desmosomes and adherens junctions,
lack tight junctions under standard tissue culture conditions
(Underwood et al., 2006), we show that increased expression of
Scribble promotes the formation of functional tight junctions. We
provide evidence for a pathway in which Scribble regulates
MAPK-ERK signaling which in turn mediates the expression of
the EMT inducer ZEB via Fra1. Inhibition of Fra1 leads to a
reduction in ZEB levels and consequently an increase in the
polarity protein Crumbs3, an essential component of tight
junctions. The data presented here show a novel role for
Scribble in positively regulating tight junction assembly
through modulation of an EMT signaling and transcriptional
program.
Results
Scribble promotes tight junction formation
To study the effect of aberrant Scribble expression in MCF10A
cells, stable lines were generated by retroviral transduction in
which human Scribble was knocked down or overexpressed.
Extensive microarray analysis was performed to investigate how
Scribble expression affects the global transcriptional programme
(I.A.E., unpublished data). We found that cells ectopically
expressing Scribble (Scrib
OE
) at levels approximately ten-fold
greater 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 adhesion
molecules’ pathway with a P-value of 7.9610
22
. Examination of
the gene list (supplementary material Table S1) indicated many
proteins associated with desmosomes and tight junctions,
including Occludin and Claudin1, were upregulated by Scribble
overexpression (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 localization
of 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, Scrib
OE
cells displayed smooth, continuous ZO1 staining, typical of intact
tight junctions, and quantification of ZO1 staining showed a
significant 3.3-fold increase in unbroken continuous staining
in Scrib
OE
cells compared to control cells (Fig. 1C).
Immunostaining for other tight junction markers, Occludin and
Claudin1, also showed an increase in continuous staining in
Scrib
OE
cells, supporting the ZO1 staining (Fig. 1C). It is
interesting to note that despite being a stable population with all
cells expressing Scribble, only a subset of the Scrib
OE
cells
localize ZO1, Occludin or Claudin1 to junctions. The patchy
nature of the rescued tight junction protein localization is
puzzling, however we observed this in stable cell lines
expressing Scribble as well as following use of chemical
inhibitors and siRNA (see below). This suggests that additional
extrinsic factors such as high cell confluency may also enhance
ZO1 restoration in Scrib
OE
cells at a population level.
To functionally characterize Scribble-mediated promotion of
adhesion, we carried out a range of assays including transmission
electron 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 in
MCF10A vector and Scrib
OE
cells using TEM revealed an
increase in adhesion and cell junctions in Scrib
OE
cells
(supplementary material Fig. S1A). Additionally, we carried
out TER measurements to determine the ion permeability of
Scrib regulates junctions through EMT signaling 3991

Journal of Cell Science
assayed junctions. Scrib
OE
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-fold
higher 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.cm
2
(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 Scrib
OE
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 Scrib
OE
cells relative to the vector
cells. A significant difference was seen in the diffusion of the
larger 70 kDa molecule yet not the smaller 4 kDa suggesting that
the rudimentary tight junctions formed in cells overexpressing
Scribble are more successful in blocking larger molecules. As
MDCK cells have well-formed tight junctions, they were used as a
positive control and showed values close to zero across both sizes
of dextran.
Inhibition of MAPK-ERK signaling promotes tight
junction assembly
We have previously shown that Scribble overexpression suppresses
MAPK activity in MCF10A cells in the context of oncogenic Ras
(Ras
V12
) (Dow et al., 2008). We carried out biochemical analysis
on Scrib
OE
cells to further look at how altered Scribble expression
alone can influence MAPK signaling. We found that in resting
cells, Scribble overexpression suppresses phosphorylation at
multiple tiers of the MAPK-ERK pathway including MEK and
ERK [Fig. 2A(i)]. Upon EGF stimulation, the phosphorylation
Fig. 1. Scribble promotes adhesion and cell
junctions. (A) MCF10A cells stably expressing
Scribble (Scrib
OE
) 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.cm
2
) 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

Journal of Cell Science
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 Scrib
OE
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
(Scribble
OE
) 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.
Scrib regulates junctions through EMT signaling 3993

Journal of Cell Science
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 vector
controls (supplementary material Fig. S2). Consistent with
previous reports (Fogg et al., 2005), cells overexpressing
Crumbs3 (Crumbs3
OE
) 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 Crumb3
OE
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
Crumb3
OE
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 Crumb3
OE
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 Scrib
OE
cells (Fig. 1E) and once more suggests that the
Crumb3
OE
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 Scrib
OE
and Crumb3
OE
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 Scrib
OE
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 Scrib
OE
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.
Journal of Cell Science 126 (17)3994

Journal of Cell Science
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
Scrib
OE
and Crumb3
OE
cells (supplementary material Fig. S5). There
was a greater difference in the diffusion of both the small and large
dextrans in the Crumb3
OE
cells than in the Scrib
OE
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 Scrib
OE
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 Scrib
OE
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 Scrib
OE
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 Scrib
OE
,
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).
Discussion
Active 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, Scrib
OE
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-ERK
signaling, 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).
Scrib regulates junctions through EMT signaling 3995

Journal of Cell Science
loss and junction dissolution (Godde et al., 2010; Moreno-Bueno
et al., 2008; Peinado et al., 2007). In this study we build on this
notion and have identified the polarity protein Scribble as a novel
regulator of an EMT signaling and transcriptional pathway
involved in the establishment of tight junctions. We have shown
that Scribble-mediated suppression of MAPK-ERK signaling
results in downregulation of the ZEB proteins through changes to
the expression of the gene product Fra1. Consequently, the
polarity protein Crumbs3 is transcriptionally upregulated leading
to the formation of functional tight junctions. The data presented
here also indicates that Scribble is likely to act on additional
pathways that regulate tight junction integrity to that described
above as overexpression of Scribble also results in an increase in
Occludin and Claudin1 expression (Fig. 1A), this effect is not
seen upon Crumbs3 overxpression (Fogg et al., 2005). It is
possible that the upregulation of Occludin and Claudin1 and the
upregulation of Crumbs3 are processes that lie parallel and
separately 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 and
colleagues demonstrated that ERK2, but not ERK1, specifically
mediates upregulation of ZEB and induction of an EMT
phenotype. Although we see no difference in efficacy of tight
junction formation with siERK1 vs siERK2 (supplementary
material Fig. S1D), we see a greater increase in Crumbs3
expression and a greater decrease in Fra1 and Zeb1 expression
with ERK2 depletion than ERK1 (data not shown). These results
are therefore consistent with Shin et al. (Shin et al., 2010) and
suggest that ERK2 is playing a greater role than ERK1 in
controlling the EMT phenotype, however this does not correlate
to changes in tight junction integrity and suggests that other
pathways than EMT signaling contribute to aspects of tight
junction integrity.
What other Fra1 and Zeb targets may be involved in tight
junction regulation? Fra1 is part of the AP-1 transcriptional
complex that interacts with several proteins including
retinoblastoma, 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, TGFb
and also of note, NFkB signaling (Chua et al., 2007; Dillner and
Sanders, 2004; Dohadwala et al., 2006; Peinado et al., 2007;
Shirakihara et al., 2007). As overexpression of the NFkB subunit
p65 in MCF10A cells results in increased expression of ZEB1 and
ZEB2 (Chua et al., 2007), these data suggest that ZEB and Fra1
could mediate part of their effects through regulation of NFkB
pathway. Nevertheless, we found that depletion of various
components of the NFkB pathway in MCF10A cells had no
discernible effect on tight junction formation (supplementary
material Fig. S6). This data strengthens our hypothesis for a
specific role of the ERKRFra1RZEBRCrumbs3 pathway in tight
junction assembly (Fig. 5).
Our laboratory has previously shown that Scribble suppresses
Ras dependent transformation through inhibition of MAPK
activity (Dow et al., 2008). It is likely that in Scrib
OE
MCF10A
cells, Scribble-mediated suppression of MAPK-ERK signaling
prevents stabilization of Fra1 and results in the decreased ZEB
expression reported here. This raises the question of how Scribble
is suppressing MAPK-ERK signaling. Recently a direct protein
interaction between Scribble and ERK was reported (Nagasaka
et al., 2010b). In this study the authors suggest that ERK
activation enhances the interaction between Scribble and ERK,
the formation of such a Scribble–ERK complex then acts to
inhibit MAPK activity. Varying phosphorylation sites within
Scribble have been proposed to alter its ability to bind ERK and
the 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 of
signaling and/or a scaffold. Scribble may act to differentially
regulate several cellular processes in a manner that is dependent
on the level of activity of particular signaling pathway. When
Scribble is ectopically expressed, as reported here, the system
may become saturated so that Scribble binds ERK, forming a
complex to alter downstream signaling events including
inhibition of EMT programs while additionally functioning at
other 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 as
ZO2, bPix and b-catenin (Audebert et al., 2004; Me
´tais et al.,
2005; Sun et al., 2009). Recently Scribble was shown to recruit
the phosphatase PHLPP1 to the cell membrane to negatively
regulate AKT signaling (Li et al., 2011). We and others have
previously shown that Scribble regulates the phosphorylation
status 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 influence
signaling and junction dynamics is a question that needs to be
further addressed.
A key downstream consequence of the Scribble-mediated
suppression of MAPK signaling demonstrated here is the
increased expression of the apical polarity gene Crumbs3.
Several studies performed in cell lines with either
microdeletions or low levels of endogenous Crumbs3 have
shown that reconstituting with ectopic Crumbs3 is sufficient to
promote the formation of tight junctions (Fogg et al., 2005; Karp
et al., 2008; Rothenberg et al., 2010). Furthermore,
downregulation of Crumbs3 by EMT inducers such as Snail
results in loss of tight junctions (Whiteman et al., 2008). The
functional role Crumbs3 plays in tight junctions is unclear yet it
is 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. We
have 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 broader
implications regarding how Scribble and other polarity proteins
may feed into the MAPK pathway and influence tumor
progression through modulation of other processes where EMT
has been implicated such as invasion and metastasis. Notably, the
present study is among the first to describe how one polarity
protein can transcriptionally regulate another. This is important in
understanding how interactions and feedback within the polarity
network occur. This study and some of the examples outlined
above, illustrate that a finely tuned balance between different
polarity proteins is required for the correct establishment and
maintenance of tight junctions.
Materials and Methods
Reagents
Immunostaining and western blotting were performed with the following
antibodies: 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),
Journal of Cell Science 126 (17)3996

Journal of Cell Science
anti-total MEK1/2 all purchased from Cell Signaling, Danvers, MA. Rabbit anti-
Claudin1 (51-9000 Zymed, San Francisco, CA), rabbit anti-occludin (71-1500
Zymed), 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 Santa
Cruz, Santa Cruz, CA), mouse anti a-tubulin (B512) (T5168 Sigma, St Louis, MO)
and mouse anti-ZO1 (339100, Invitrogen, Grand Island, NY). Mouse monoclonal
anti-Scribble (7C6-D10) has been previously described (Dow et al., 2003). Inhibitor
experiments were performed using the MEK1 inhibitor PD98059 (20 mM) (P125
Sigma Aldrich, St Louis, MO), the PI3K inhibitor LY294002 (20 mM) (440202,
Merck, Germany) and the JNK inhibitor SP600125 (20 mM) (420119 Merck).
Cell culture
MCF10A cells were maintained in DMEM:F12 (Dulbecco’s Modified Eagle
Medium: F12) supplemented with 5% donor horse serum (Gibco, NY), 10 mg/ml
insulin (Novo Pharmaceuticals, UK), 0.5 mg/ml hydrocortisone (Sigma Aldrich),
20 ng/ml EGF (Cytolab Ltd, Switzerland), 100 ng/ml cholera toxin (Sigma
Aldrich), penicillin (100 U/ml) and streptomycin (100 U/ml) as previously
described (Debnath et al., 2003). 293T cells were maintained in DMEM
supplemented with 10% foetal bovine serum, penicillin (100 U/ml) and
streptomycin (100 U/ml). All cultures were maintained at 37˚
Cin5%CO
2
.
Retroviral constructs and infection
Full length human Scribble was cloned into MSCV-IRES-GFP as previously
described (Dow et al., 2003). The coding sequence of Crumbs3 had previously
been PCR amplified and cloned into MSCV-IRES-GFP vector using EcoRI sites.
All constructs were verified by sequencing. Virus was generated by transfecting
293T cells by calcium phosphate precipitation using the amphotrophic packaging
vector RD114 and appropriate DNA constructs. Stable MCF10A cell lines were
generated essentially as described previously (Debnath et al., 2003). Cultured
MCF10A cells were infected with virus 30, 42 and 54 hours post transfection and
allowed to recover for 24 hours before selection. To generate polyclonal stable
populations, cells were either selected for one week in 2 mg/ml puromycin and/or
sorted for GFP
+
cells on a FACStar flow cytometer (Becton Dickinson, Franklin
Lakes, NJ). All stable cell lines were monitored and regularly checked for
expression and re-selected if necessary.
EGF stimulation
MCF10A cells were plated out at 7.5610
4
cells in 6-well dishes. 24 h later culture
media was replaced with EGF-depleted media. Cells were cultured in EGF-
depleted media for 48 h before replacing with pre-warmed media containing
20 ng/ml EGF. Cells were harvested for protein analysis at designated timepoints.
Quantification was performed by densiometry of immunoblot bands using ImageJ
software (ImageJ, NIH USA).
JNK kinase assay
MCF10A cells were grown in 10 cm dishes till they reached 80% confluency. Cells
were UV irradiated (40 J/m
2
), washed in PBS and fresh media applied for 30 min
before harvesting. JNK activity was assessed using a non-radioactive kinase assay
kit (9810, Cell Signaling) according to the manufacturer’s instructions.
Western blotting
Protein concentrations from prepared whole cell lysates were determined using a
Lowry Protein Assay (BioRad, Hercules, CA) and resolved on pre-cast gradient
gels (Invitrogen). Samples were transferred overnight at 4˚C and probed with the
relevant antibodies. Proteins were visualised using LumiLight ECL reagents
(Roche, Germany) according to the manufacturer’s instructions.
RNA isolation, cDNA synthesis and qRT-PCR
RNA was harvested using TRizol reagent (Invitrogen) and purified using
chloroform extraction and ethanol precipitation. RNA quality and concentration
was measured using a Nanodrop ND-1000 spectrophotometer (ThermoScientific,
Vic, Aust). cDNA was made from 2 mg of RNA using standard methods (Dow
et al., 2008). Samples were run in triplicate on a StepOnePlus Real-Time PCR
System (Applied Biosystems, Carlsbad, CA). All samples were normalized to
GAPDH control (or L32 when using siGAPDH) and fold change between samples
was calculated using the comparative C(T) method. Primers used for qRT-PCR are
listed in supplementary material Table S2. qRT-PCR experiments were performed
on multiple independently derived cell lines and at least 3 independent siRNA
transfections. Statistics were calculated on results from 3–6 runs using GraphPad
Prism data software (GraphPad version 5.0b).
siRNA transfections
All 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. For
downstream protein or RNA applications MCF10A cells were plated out in 1.6 ml
of antibiotic-free media at 1.1610
5
cells per well in a 6-well dish ,16 hours prior
to transfection. Prior to transfection, siRNAs were diluted to a concentration of
50 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 the
siGFP or siGAPDH control. The lipid and siRNA mix werecombined and allowed to
duplex for 20 min before adding to cells. Cells were washed and fresh media applied
after 24 hours. For RNA analysis, cells were harvested 48 hours following
transfection and for protein analysis, cells were harvested 72 hours post
transfection. For downstream immunofluorescent staining MCF10A cells were
plated out in 400 ml of antibiotic-free culture media at 3.6610
4
cells per well onto
sterile poly-L-lysine-coated coverslips (#354085, Becton Dickinson) in a 48-well
dish ,20 hours prior to transfection. As above Dharmafect3 lipid
(ThermoScientific) was used and complexed with the appropriate siRNAs at
50 nM. Fresh media was applied after 24 hours. Cells were used for subsequent
immunofluorescent staining 72 hours post transfection.
Immunostaining and quantification
MCF10A cells were grown on sterile coverslips prior to fixing in 100% ice-cold
methanol for 20 mins for ZO1 staining or 4% paraformaldehyde for 15 min at
room temperature followed by a 10 min permeabilization step in 1% SDS for
Occludin and Claudin1 staining. Coverslips were washed in PBS before blocking
in 2% BSA/PBS for 1 hour at room temperature. Samples were incubated with
mouse anti-ZO1 (339100, Invitrogen), rabbit anti-occludin (71-1500, Zymed) or
rabbit anti-claudin1 (51-9000, Zymed) overnight at 4˚
C. Coverslips were washed
with PBST before probing with secondary anti-mouse antibodies conjugated to
Alexa 488 flurochromes (Molecular Probes, Grand Island, NY)). ProLong Gold
Antifade containing DAPI (Invitrogen) was used to visualize nuclei. Images were
acquired on a FV1000 BX51 scanning confocal microscope (Olympus Corp,
Japan) using a 406oil objective. To quantify tight junctions, continuous ZO1
staining was assessed using MetaMorph 6.3 software (Molecular Devices Corp.,
Downington, PA). Slides stained for ZO1 were viewed under a confocal
microscope and multiple images taken randomly across the entire sample.
Images were analyzed with MetaMorph software using the ‘Angiogenesis Tube
Formation’ application. From the analysis, the value describing the ‘tube length/
set’ was used as a measure of continuous ZO1 staining. This value was divided by
the number of cells in the image (determined by DAPI staining). For each
experiment at least 1000 cells were analyzed across multiple images. Identical
analysis was carried out for controls and values expressed as fold change relative
to control. Data was plotted and analyzed using GraphPad Prism data software
(GraphPad version 5.0b).
Transepithelial electrical resistance measurements
Cells were seeded at 6610
5
per well onto polyester transwell filters (Sigma
Aldrich, CLS3450) and readings recorded every second day using a Millicell-ERS
(MERS00001 Millipore, Billerica, MA) according to the manufacturer’s
instructions. Resistance was calculated as resistance of a unit area5resistance
(V)6effective membrane area (cm
2
). Sample readings were measured in triplicate
for each reading. Media was replaced following each reading.
Size-selective assessment of tight junction paracellular permeability
Cells were seeded at 6610
5
or 2610
5
cells per well on polyester transwell filters
(Sigma Aldrich, CLS3450) for 13 or 3 days. Media was replaced every 2 days and
on the day indicated TER measurements were recorded and the media was
replaced with 1 ml in the bottom well and 250 ml in the top well. Fluorescently
labeled 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 at
37˚C for approx 3 hours. Media from the bottom plate was aliquoted to 3 wells in a
black-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-Stack
automated plate reader (BioTak, Millennium Science, Australia) (Matter and
Balda, 2003). The average of the readings were taken as technical replicates and
the 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, then
rinsed in PBS containing 5% sucrose. Post-fixation was carried out using 2%
osmium tetroxide in PBS followed by dehydration through a graded series of
alcohols, two acetone rinses and embedding in Spurrs resin. Sections ,80 nm
thick were cut with a diamond knife (Diatome, Switzerland) on an Ultracut-S
ultramicrotome (Leica) and contrasted with uranyl acetate and lead citrate. Images
were captured with a Megaview II cooled CCD camera (Olympus) on a JEOL
1011 transmission electron microscope.
Microarray experiments and analysis
RNA integrity was assessed using a bioanalyzer (Agilent Technologies, USA) and
RNA with a RNA integrity number (RIN) of 10 were used for microarray
Scrib regulates junctions through EMT signaling 3997

Journal of Cell Science
experiments. Microarray experiments were carried out using the GeneChip Human
Gene 1.0 ST Array kit (Affymetrix, Santa Clara, CA) according to the
manufacturer’s instructions. Quality control metrics were carried out using
Expression Console software (Affymetrix) and Partek software (Partek
Incorporated, USA). Gene lists were generated using Tukey’s biweight 1 step
summarisation and batch effect was applied to passage number and cell line.
Pathway and gene ontology analysis was applied using the online DAVID
bioinformatics database (http://david.abcc.ncifcrf.gov).
Acknowledgements
We thank members of our laboratory for helpful discussions. Special
thanks to the Peter MacCallum Cancer Centre Microscopy Core, in
particular Stephen Asquith for preparation of the TEM samples. We
also thank the Peter MacCallum Cancer Centre Microarray Core in
particular Dr Jason Li for help and advice with analysis.
Author contributions
I.A.E. designed, planned and carried out the experiments, analyzed
and interpreted the data and wrote the manuscript; C.M. assisted with
experimental work and approach, interpretation of data and edited
the manuscript; P.O.H. designed the experiment, sourced funding,
provided reagents and mentorship, edited the manuscript.
Funding
This study was supported by a project grant from the National Health
and Medical Research Council of Australia [grant number
APP1004434 to P.O.H.]. I.A.E was supported by a Cancer Council
Victoria Postgraduate Cancer Research Scholarship and P.O.H by a
Biomedical Career Development Award from the National Health
and 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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