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RESEARCH ARTICLE

aPKC alters the TGFb response in NSCLC cells through bothSmad-dependent and Smad-independent pathways

Adrian Gunaratne1,*, Eddie Chan1, Tarek H. El-Chabib1,`, David Carter2 and Gianni M. Di Guglielmo1,§

ABSTRACT

Transforming growth factor b (TGFb) signaling controls many cellular

responses including proliferation, epithelial to mesenchymal

transition and apoptosis, through the activation of canonical (Smad)

as well as non-canonical (e.g. Par6) pathways. Previous studies from

our lab have demonstrated that aPKC inhibition regulates TGFb

receptor trafficking and signaling. Here, we report that downstream

TGFb-dependent transcriptional responses in aPKC-silenced

NSCLC cells were reduced compared with those of control cells,

despite a temporal extension of Smad2 phosphorylation. We

assessed SARA–Smad2–Smad4 association and observed that

knockdown of aPKC increased SARA (also known as ZFYVE9)

levels and SARA–Smad2 complex formation, increased cytoplasmic

retention of Smad2 and reduced Smad2–Smad4 complex formation,

which correlated with reduced Smad2 nuclear translocation.

Interestingly, we also detected an increase in p38 MAPK

phosphorylation and apoptosis in aPKC-silenced cells, which were

found to be TRAF6-dependent. Taken together, our results suggest

that aPKC isoforms regulate Smad and non-Smad TGFb pathways

and that aPKC inhibition sensitizes NSCLC cells to undergo TGFb-

dependent apoptosis.

KEY WORDS: Transforming growth factor beta, Atypical protein

kinase C, p38 MAPK, Apoptosis, Nuclear translocation, SARA

INTRODUCTIONTransforming growth factor b (TGFb) signaling regulates many

cellular processes including proliferation, apoptosis, and epithelial

to mesenchymal transition (EMT), and, in addition to being a key

pathway during embryonic development, aberrant TGFb signaling

is a hallmark of several pathological conditions, including cancer

and fibrosis (Derynck and Akhurst, 2007; Derynck et al., 2001;

Elliott and Blobe, 2005; Massague, 1992; Massague and Chen,

2000). The canonical TGFb pathway involves the cell-surface

binding of TGFb ligand to the TGFb type II receptor (TbRII),

which then binds to and phosphorylates the TGFb type I receptor

(TbRI) (Massague, 1998). Phosphorylation of TbRI leads to its

activation and its ability to transduce intracellular signaling through

the phosphorylation of substrate proteins such as the receptor-

regulated Smads (R-Smads) Smad2 and Smad3 (Massague, 1998;

Massague and Chen, 2000). Once phosphorylated, R-Smads

accumulate in the nucleus, where they act as transcription factors

to regulate subsequent TGFb gene response (Attisano and Wrana,

2002; Massague, 1998; Massague and Chen, 2000; Shi and

Massague, 2003; Siegel and Massague, 2003). Entry of R-Smads

into the nucleus is facilitated by direct binding of these proteins to

the nucleopore complex, binding to importins (for Smad3) or by the

binding of the common Smad, Smad 4 (Massague, 2003; Shi and

Massague, 2003)

Importantly, proteins that control the membrane trafficking and

endocytosis of TGFb receptors play a role in regulating the

intensity and duration of TGFb signals. For example, the efficient

regulation of Smad signaling can be facilitated by adaptor proteins

such as the Smad cytosolic cofactor SARA (Smad anchor for

receptor activation, also known as ZFYVE9). SARA contains a

Smad-binding domain, as well as a TGFb receptor complex-

interacting region, and it acts as a bridge, facilitating R-Smad

presentation to the activated receptor complex (Tsukazaki et al.,

1998; Wu et al., 2000). SARA contains a phosphatidylinositol 3-

phosphate (PI3P)-binding FYVE domain, which links it to the

early endosome, implicating receptor endocytosis and trafficking

in efficient Smad signal transduction (Di Guglielmo et al.,

2003; Tsukazaki et al., 1998). Once SARA-bound R-Smads are

phosphorylated, they dissociate from the SARA–receptor complex,

bind to Smad4 and accumulate in the nucleus to regulate

transcription (Xu et al., 2000). Interestingly, although receptor

endocytosis has been reported to be dispensable for the

phosphorylation of R-Smads, it has been reported that

endocytosis is required for the efficient dissociation of R-Smads

from SARA, nuclear accumulation and subsequent transcriptional

response (Runyan et al., 2005). The precise regulation of

transcriptional activity of Smads in the nucleus is important for

the proper execution of embryonic development by controlling

tissue patterning and normal organ development, and it is also

important for controlling cellular growth and apoptotic response in

adult tissues; deviations are associated with various pathologies

(Derynck et al., 2001; Heldin et al., 2009; Pardali and Moustakas,

2007; Shi and Massague, 2003; Siegel and Massague, 2003).

Although it is established that Smads are central regulators of

gene response to TGFb, multiple Smad-independent pathways

are also initiated upon TGFb receptor activation (Derynck and

Zhang, 2003; Heldin et al., 2009; Massague, 2003; Moustakas

and Heldin, 2005; Pardali and Moustakas, 2007). In addition to

Smads, TGFb can also activate the mitogen-associated protein

kinase family (MAPK). There are three principle classes of

MAPK proteins – ERK, JNK, and p38 – each of which has a

complex but apparent role in the development and progression of

cancer (Wagner and Nebreda, 2009). The p38 MAPK pathway

downstream of TGFb has gained considerable interest as a

pathway that regulates apoptosis. Briefly, TGFb receptor

1Department of Physiology and Pharmacology, Western University, London, ONN6A 5C1, Canada. 2London Regional Genomics Centre, Robarts ResearchInstitute, London, ON N6A 5B7, Canada.*Present address: Lunenfeld-Tanenbaum Research Institute, Toronto, ON M5G1X5, Canada. `Present address: Department of Medicine, University of Toronto,Toronto, ON M5S 1A1, Canada.

§Author for correspondence (john.diguglielmo@schulich.uwo.ca)

Received 17 April 2014; Accepted 24 November 2014

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 487–498 doi:10.1242/jcs.155440

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activation leads to the recruitment and Lys63-linkedautoubiquitylation and activation of TRAF6 (TNF-receptor-

associated factor 6), an E3 ubiquitin ligase. This stimulates acascade that culminates in the activation of p38 MAPK and,ultimately, apoptosis of various cell types (Edlund et al., 2003;Sorrentino et al., 2008; Yamashita et al., 2008). Interestingly,

atypical protein kinase C (aPKC) isoforms interact with TRAF6to mediate cytokine signaling (Sanz et al., 2000), but less isknown about the involvement of aPKC in modulating the TGFb–

p38 MAPK pathway.We have previously shown that aPKC isoforms can alter TGFb

signaling patterns in non-small cell lung cancer (NSCLC) cells

by altering receptor trafficking, degrading specific receptorcomplexes and by enhancing Par6-dependent phosphorylation(Gunaratne et al., 2012; Gunaratne et al., 2013). However, we had

not examined gene changes on a large scale. Furthermore, we havenot examined whether aPKC isoforms alter TGFb-induced MAPKpathways. The aPKC isoforms, which consist of PKCi and PKCf,are a subset of the protein kinase C family that are Ca2+ and

diacylglycerol (DAG)-independent (Griner and Kazanietz, 2007).Importantly, the aPKCs show altered expression and activities invarious cancers (Huang and Muthuswamy, 2010), and PKCi has

been described as an oncogene (Fields and Regala, 2007; Regalaet al., 2005). Interestingly, aPKC isoforms are known to play a role

in p38-MAPK-induced apoptosis, as inhibition or knockdown ofaPKC sensitizes glioblastoma cells to chemotherapeutic agentsthrough a p38-dependent mechanism (Baldwin et al., 2006).

In this report, we examine gene changes in aPKC-silenced cells

by microarray analyses, and examine how knockdown of aPKCalters Smad and MAPK signaling pathways and stimulates TGFb-dependent apoptosis of NSCLC cells.

RESULTSKnockdown of aPKC isoforms alters TGFb-inducedgene expressionWe reported previously that aPKC gene silencing using smallinterfering RNA (siRNA) temporally extended TGFb-induced

Smad2 phosphorylation (Gunaratne et al., 2012) and inhibitedPar6-dependent EMT (Gunaratne et al., 2013). In order to assessthe effects of aPKC silencing on TGFb-dependent transcription,we silenced PKCi and PKCf in combination (PKCi/f) using

siRNA and conducted microarray analysis. Table 1 summarizesfold change differences between siControl- and siPKCi/f-treatedcells following TGFb induction as determined by microarray

Table 1. aPKC knockdown alters TGFb gene response as shown by microarray analysis

Gene Assignment Gene Symbol siControl (+TGFb) siPKCi/f (+TGFb)

NM_004530 // MMP2 // matrix metallopeptidase 2 MMP2 7.4224 4.82898NM_000575 // IL1A // interleukin 1, alpha IL1A 5.70595 1.99935NM_001845 // COL4A1 // collagen, type IV, alpha 1 COL4A1 4.81464 3.94456NM_002421 // MMP1 // matrix metallopeptidase 1 MMP1 4.5775 2.44365NM_000888 // ITGB6 // integrin, beta 6 ITGB6 3.99775 3.11059NM_003068 // SNAI2 // snail homolog 2 SNAI2 3.85485 4.29951NM_033274 // ADAM19 // ADAM metallopeptidase domain 19 ADAM19 3.34159 2.57803NM_000602 // serpin peptidase inhibitor, (nexin, PAI-1) SERPINE1 3.3258 1.86086NM_002425 // MMP10 // matrix metallopeptidase 10 MMP10 3.30959 2.26383NM_005985 // SNAI1 // snail homolog 1 SNAI1 2.52485 3.1199NM_004994 // MMP9 // matrix metallopeptidase 9 MMP9 2.20114 1.30477NM_001204 // bone morphogenetic protein receptor, type II BMPR2 2.14816 1.54296NM_000641 // IL11 // interleukin 11 IL11 2.08731 1.57918NM_001025366 // vascular endothelial growth factor A VEGFA 1.89192 1.33097NM_005239 // v-ets erythroblastosis virus E26 oncogene homolog 2 ETS2 1.86189 2.17121NM_002229 // JUNB // jun B proto-oncogene JUNB 1.85935 1.73865NM_005414 // SKIL // SKI-like oncogene SKIL (SnoN) 1.85549 1.56457NM_001901 // CTGF // connective tissue growth factor CTGF 1.83944 1.63558NM_001130955 // Rho/Rac guanine nucleotide exchange factor 18 ARHGEF18 1.83241 1.76128NM_015675 // growth arrest and DNA-damage-inducible, beta GADD45B 1.82457 1.69444NM_005860 // FSTL3 // follistatin-like 3 (secreted glycoprotein) FSTL3 1.76007 1.77974NM_002658 // PLAU // plasminogen activator, urokinase PLAU 1.67966 1.26324NM_017449 // EPHB2 // EPH receptor B2 EPHB2 1.67728 1.27402NM_002205 // ITGA5 // integrin, alpha 5 ITGA5 1.67226 1.71087NM_022739 // SMURF2 // SMAD E3 ubiquitin protein ligase 2 SMURF2 1.66886 1.1137NM_002430 // MN1 // meningioma MN1 1.63051 1.41255NM_002228 // JUN // jun oncogene JUN 1.5784 1.51449NM_002659 // plasminogen activator, urokinase receptor PLAUR 1.57811 1.22978NM_005904 // SMAD7 // SMAD family member 7 SMAD7 1.57346 1.49838NM_000214 // JAG1 // jagged 1 (Alagille syndrome) JAG1 1.5104 1.37755NM_006690 // MMP24 matrix metallopeptidase 24 MMP24 21.5844 21.05835NM_002166 // ID2 // inhibitor of DNA binding 2 ID2 21.7217 21.95497NM_004938 // DAPK1 // death-associated protein kinase 1 DAPK1 21.92878 21.8769NM_001202 // BMP4 // bone morphogenetic protein 4 BMP4 22.47374 22.07877NM_004360 // CDH1 // cadherin 1, type 1, E-cadherin (epithelial) CDH1 23.08221 22.04549

A549 cells transfected with control (siControl) or siRNA directed against aPKC isoforms (siPKCi/f) were serum starved and treated with 250 pM TGFb for 1 h,washed and further incubated for 24 h in low-serum medium. Total RNA was then extracted and subjected to gene expression array analysis. Shown is aselected list of gene responses in control and aPKC-silenced cells after 24 h for several genes regulated by TGFb (list adapted from Siegel and Massague,2003). Fold change comparisons are expressed relative to untreated siControl cells (siControl, 2TGFb) and represent the average of three separateexperiments (n53). In bold are genes that exhibited a reduced response in aPKC-knockdown cells.

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analysis. This list was selected from a set of genes commonlyknown to be regulated by TGFb (Siegel and Massague, 2003).

The full microarray dataset can be accessed online at the NCBIGene Expression Omnibus website (GEO; GSE26241). Weobserved that several classical TGFb-regulated genes hadsimilar expression patterns between control and aPKC-silenced

cells, including BMP4, SNAI1 and SNAI2. However, there wereseveral genes that displayed a reduced TGFb-dependent responsein aPKC-silenced cells compared to that of control cells,

including IL1A, SMURF2, MMP2 and MMP9 (shown in bold).In order to assess gene changes in further detail, we investigatedseveral transcripts using real-time PCR (qPCR).

Knockdown of aPKC isoforms alters TGFb-induced geneexpression in qPCR analysisWe first assessed the expression of PKCi, PKCf and PKCa toconfirm that our siRNA approach was efficient and specific, andwe observed that siRNA treatment was successful in reducing thegene expression of the atypical PKCs (aPKCi and f) but not the

classical PKCa (Fig. 1A). We next assessed the transcription ofknown TGFb-dependent genes in response to TGFb in controland aPKC-knockdown cells at 4 and 24 h after stimulation with

TGFb. Several genes showed a significantly reduced induction byTGFb in aPKC-silenced cells at 4 and 24 h after ligand-mediated

stimulation. These included SMURF2 (Fig. 1B) in A549 cells (butnot in H1299 cells; supplementary material Fig. S1A), SERPINE1

(also known as PAI-1; Fig. 1C; supplementary material Fig. S1B)and MMP9 (Fig. 1D; supplementary material Fig. S1C). We alsoobserved cases in which the TGFb-dependent response was eitherpartial (SNAI1; Fig. 1E; supplementary material Fig. S1D) or was

similar in both aPKC-knockdown and control cells (CDH1; Fig. 1F).Finally, KLF10 (also known as TIEG1) was unresponsive to TGFbin both treatment groups (data not shown). Taken together, our

microarray and qPCR data suggest that TGFb-dependenttranscription is muted in aPKC-knockdown cells, despite thefact that these cells exhibited extended Smad2 phosphorylation

in response to TGFb induction (Gunaratne et al., 2012;supplementary material Fig. S2).

Knockdown of aPKC increases cytosolic retention of Smad2by SARAActivated TGFb receptors phosphorylate receptor-regulatedSmads (Smad2 and Smad3) on a C-terminal SSXS motif, which

facilitates their dissociation from SARA, association with Smad4and accumulation in the nucleus to modulate transcription (Shiand Massague, 2003). Therefore, the reduced TGFb-dependent

transcriptional response that we observed in aPKC-silenced cellsmight be due to a reduced nuclear translocation of Smad2. The

Fig. 1. aPKC silencing alters TGFb-dependentgene induction. Real-time PCR analysis ofTGFb-induced mRNA levels in A549 controlsiRNA cells versus cells with knockdown of bothPKCi and PKCf (siPKCi/f). RNA extracts wereisolated from cells treated for 1 h with TGFbfollowed by 4-h or 24-h incubation in the absenceof ligand. Two-way ANOVA analysis followed bypost-hoc Bonferonni’s tests were used todetermine statistical significance of geneexpression changes of PKCi, PKCf and PKCa(A), SMURF2 (B), SERPINE1 (PAI-1; C), MMP-9(D), SNAI1 (Snail; E) and CDH1 (E-cadherin; F).All data show the mean6s.e.m.; *P#0.05;**P#0.01.

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subcellular localization of R-Smads can be controlled by abalance between binding factors that retain them in the cytoplasm

versus transcription factors that retain them in the nucleus. Onesuch cytoplasmic retention factor is SARA, a protein enriched onearly endosomes (Itoh et al., 2002; Tsukazaki et al., 1998).

SARA preferentially binds to the non-C-terminally phosphorylated

form of Smad2, and it is thought that the activated receptorcomplex formed at the plasma membrane is captured by SARA inthe early endosome, which then presents the bound R-Smad to the

receptor for phosphorylation (Massague and Chen, 2000). Smad2then dissociates from SARA and associates with Smad4 prior tonuclear translocation and the initiation of transcription (Massague

and Chen, 2000). Furthermore, although phosphorylated R-Smadscan activate transcription alone, a full TGFb response requirescomplex formation by Smad2 and Smad4 (Levy and Hill, 2005;

Wrana, 2009). To test this, we immunoprecipitated Smad2 fromcontrol and aPKC-silenced cells treated with TGFb to examinewhether SARA would dissociate from Smad2 upon TGFb addition.TGFb addition reduced SARA association with Smad2 and

resulted in a concomitant increase in binding to Smad4(Fig. 2A). Interestingly, in aPKC-silenced cells, TGFb additionreduced SARA–Smad2 dissociation as well as the binding of

Smad2 to Smad4, suggesting a deficit in this exchange. Thisindicated that SARA might be retaining Smad2 in the cytoplasm toa greater degree in aPKC-silenced cells, and might reduce nuclear

translocation of Smad2. Because we have previously reportedalterations in the membrane trafficking of TGFb receptors uponPKC inhibition (Gunaratne et al., 2012), we next examined

whether SARA levels were altered in aPKC-silenced cells.Interestingly, aPKC-silenced cells showed significantly increasedtotal protein levels of SARA compared with control cells, althoughno appreciable changes were observed with TGFb addition

(Fig. 2B). We next examined whether increased SARAexpression might disrupt its localization to the early endosome,which, in turn, might disrupt normal Smad signaling in aPKC-

silenced cells. Using immunofluorescence microscopy, we did notdetect any appreciable alteration of SARA colocalization withEEA-1 (an early endosome marker), suggesting that SARA still

accessed the early endosome in aPKC-silenced cells (Fig. 2C). Wenext analyzed whether the increased SARA levels in aPKC-depleted cells could be retaining Smad2 in the cytoplasm.

aPKC knockdown reduces TGFb-induced Smad2nuclear accumulationTo examine TGFb-dependent nuclear translocation of Smad2 in

control and aPKC-silenced cells, we carried out immunofluorescencemicroscopy analysis (Fig. 3A). As expected, in cells transfectedwith control siRNA, TGFb induced an increase in Smad2 nuclear

staining, suggesting nuclear accumulation of Smad2 (Fig. 3A).Consistent with the microarray and qPCR data, aPKC-silencedcells showed reduced nuclear accumulation of Smad2 in response

to TGFb (Fig. 3A). The reduction in Smad nuclear translocation inaPKC-silenced cells was also observed for another TGFb receptorR-Smad, Smad3, and also occurred in another NSCLC cell line,H1299 NSCLC cells (supplementary material Fig. S3A,B).

Because we observed an increase in SARA expression andassociation with Smad2 in aPKC-silenced cells (Fig. 2), we nextassessed whether reducing SARA levels would reverse the

cytoplasmic retention of Smad2 in aPKC-silenced cells. Toinvestigate this possibility, we first silenced SARA using siRNAand observed no differences in Smad2 phosphorylation

(supplementary material Fig. S2C) or nuclear accumulation in

A549 cells (Fig. 3A). Interestingly, concomitant siRNA targetingof aPKC isoforms and SARA partially restored the nuclear

accumulation of Smad2 that was absent in cells with knockdownof aPKC only (Fig. 3A).

To verify and quantify the observation of reduced nuclearSmad accumulation, we also conducted subcellular fractionation

studies and immunoblotting of cellular cytosolic and nuclearfractions when cells were treated in the presence or absence ofTGFb (Fig. 3B). Consistent with our immunofluorescence

microscopy analysis, TGFb treatment stimulated an increase innuclear Smad2 levels in cells transfected with control siRNA. Incontrast, aPKC-knockdown cells contained significantly reduced

nuclear Smad2 levels upon TGFb stimulation (Fig. 3B). Theamount of TGFb-dependent nuclear accumulation of Smad2 inSARA-silenced cells was similar to that of control cells (Fig. 3B).

However, consistent with the immunofluorescence analysis, thesilencing of aPKC isoforms and SARA partially restored theaccumulation of Smad2 in aPKC-silenced cells (Fig. 3B).

Taken together, these results suggest that an increase in SARA

protein in the cytoplasm of aPKC-silenced cells might beinhibiting Smad2 from translocating to the nucleus after TGFbstimulation, and possibly altering transcriptional activity.

However, the results do not exclude the possibility that othersignaling pathways might be altering Smad function upon aPKCknockdown. We therefore examined whether aPKC knockdown

would affect MAPK pathways, as these pathways have beenshown to crosstalk with the Smad pathway and have beenreported to alter R-Smad nuclear targeting.

Knockdown of aPKC enhances phosphorylated p38MAPK levelsSmads shuttle to and from the nucleus, and their subcellular

localization is primarily controlled through phosphorylationevents. MAPK pathways are known to crosstalk with Smadsthrough the phosphorylation of the Smad linker region, which can

alter Smad localization and function, including nuclear exclusion(Massague, 2003; Kretzschmar et al., 1999). Given our observationof reduced nuclear accumulation of Smad2/3 in aPKC-silenced

cells, we next assessed whether MAPK pathways were altered inaPKC-silenced cells. We analyzed the levels of the three activatedMAPK pathways in response to TGFb in control and aPKC-silenced cells (Fig. 4). Interestingly, aPKC knockdown increased

basal and TGFb-induced levels of phosphorylated p38 MAPK atboth 1 h and 24 h time-points, whereas no appreciable differenceswere observed for phosphorylated ERK or phosphorylated JNK

(Fig. 4A).We then assessed shorter timecourses of p38 MAPK activation

in control and aPKC-knockdown cells and further observed that

TGFb-induced p38 MAPK phosphorylation was increased andextended in aPKC-silenced cells at 0.5, 1.5 and 4.5 h after TGFbtreatment (Fig. 4B). Because MAPK crosstalk has been reported

to alter Smad nuclear–cytoplasmic shuttling dynamics (Burchet al., 2010; Kamato et al., 2013; Massague, 2003), we reasonedthat increased p38 MAPK activity might be reducing Smad2nuclear import in aPKC-silenced cells. When we tested this

hypothesis, we observed that p38 MAPK inhibition did notrescue Smad2 nuclear accumulation in aPKC-silenced cells(supplementary material Fig. S3C). This result suggested that

p38 MAPK activity might not be responsible for the reducedSmad2 nuclear accumulation observed in aPKC-silenced cells,and that the Smad2 and p38 MAPK pathways might be

independent of each other.

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Knockdown of aPKC increases TGFb-induced apoptoticresponse through p38 MAPKTGFb receptors can activate the p38 MAPK pathway to stimulate

apoptosis (Derynck and Zhang, 2003; Yu et al., 2002). Wetherefore examined whether the increased p38 MAPK signalingobserved in aPKC-silenced cells sensitized cells to TGFb-inducedapoptotic response. To measure apoptosis, control and aPKC-

silenced cells were treated with or without TGFb for 48 h, and thelevel of apoptosis was measured through the assessment ofnuclear morphology after Hoescht staining (Fig. 5A). Cells

treated with control siRNA showed a modest apoptotic

response to TGFb. This was in contrast to aPKC-silenced cells,which exhibited a significant increase in cell death when treatedwith TGFb (Fig. 5A). Importantly, aPKC-silenced cells treated

with a p38 MAPK inhibitor showed a reduced number ofapoptotic nuclei, indicating that the apoptotic response observedwas downstream of p38 MAPK signaling (Fig. 5A). We observedsimilar results using another NSCLC cell line, H1299 cells

(supplementary material Fig. S4A,B). TGFb-induced cleaved-Parp levels (c-Parp), a marker of apoptosis, were significantlyhigher in aPKC-silenced cells than in control cells, and c-Parp

levels were reduced with the p38 inhibitor (Fig. 5B), although the

Fig. 2. aPKC knockdown alters TGFb-inducedSARA–Smad2–Smad4 interactions. (A) A549cells transfected with control siRNA (siControl) orsiRNA directed against the aPKC isoforms (PKCi/f)were serum starved and treated with or without250 pM TGFb for 1 h prior to lysis. Cell lysateswere then immunoprecipitated (IP) using anti (a)-Smad2 antibodies and subjected to SDS-PAGEand immunoblotting using anti-SARA, anti-Smad4and anti-Smad2 antibodies. IgG heavy chain isindicated. Cell lysates were included to showrelative endogenous protein expression.Densitometric analysis of Smad2-associated SARAor Smad4 levels from three independent replicateexperiments are shown. Data show themean6s.e.m (n53); *P#0.05; **P#0.01 (two-wayANOVA). (B) A549 cells transfected with controlsiRNA or siRNA targeting aPKC isoforms werelysed and immunoblotted using antibodies againstSARA and actin as indicated on the right of thepanels. Densitometric analysis of steady-stateSARA levels from three independent experimentsis shown graphically to the right of therepresentative immunoblots. Data show themean6s.e.m. (n53); *P#0.05 (two-way ANOVA).(C) aPKC knockdown does not inhibit thelocalization of SARA in the early endosome. A549cells were transfected as described in A andprocessed for immunofluorescence microscopy tovisualize EEA1 (green) and SARA (red). DAPI wasused to visualize DNA (blue). The boxed areasare shown at higher magnification in the lowerimages. Representative images from at least threeindependent experiments are shown. Scale bars:10 mm.

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p38 inhibitor had no effect on Smad2 phosphorylation levels(supplementary material Fig. S4C). These results further highlightthe importance of p38 signaling in this apoptotic response.

aPKC knockdown stabilizes TGFb-induced p38 MAPKsignaling through TRAF6TGFb-stimulated apoptosis through p38 MAPK has been

previously reported to occur through the recruitment andactivation of the E3 ubiquitin ligase TRAF6 (Sorrentino et al.,2008; Yamashita et al., 2008). Briefly, upon TGFb activation,

TRAF6 is recruited to TbRI of the TGFb receptor complex. Thiscauses TRAF6 to become auto-ubiquitylated, which activatesTAK1 (also known as MAP3K7; a MAP3K), which triggers the

MAPK cascade to p38 activation (Sorrentino et al., 2008;Yamashita et al., 2008). We have shown previously that aPKC

expression can alter the binding and degradation patterns of TbRIand its substrates (Gunaratne et al., 2012; Gunaratne et al., 2013).We suspected that the increased TGFb–p38 MAPK signals

that we observed in aPKC-silenced cells might be due toincreased levels of TRAF6 and TGFb receptor complexes whenaPKC was depleted. We tested this by immunoprecipitatingendogenous TRAF6 from control and aPKC-silenced cells,

followed by immunoblotting for TbRI (Fig. 6A). Interestingly,TbRI associated to a greater degree with TRAF6 in the absence ofaPKC expression (Fig. 6A). This finding suggested that TbRI–

TRAF6 complexes were more stable in aPKC-knockdown cells.We reasoned that this increase in TbRI–TRAF6 complexes wasresponsible for the increased TGFb-induced p38 MAPK signals

in aPKC-silenced cells. To test this, we next examined whethersiRNA-mediated TRAF6 knockdown could abrogate p38 MAPK

Fig. 3. aPKC silencing reduces TGFb-induced Smad2 nuclear accumulation. (A) A549 cells were transfected with the indicated siRNA, serum starved andtreated with 250 pM TGFb for 1 h. The cells were processed for immunofluorescence microscopy with antibodies against Smad2. DAPI was used to visualizeDNA. Representative images from at least three independent replicate experiments are shown. Scale bars: 10 mm. (B) A549 cells were transfected and treatedwith TGFb as described in A. The cells were then subjected to subcellular fractionation to isolate cytoplasmic and nuclear fractions. The fractions were subjectedto SDS-PAGE and immunoblotted using anti (a)-Smad2, anti-tubulin and anti-histone H3 antibodies to determine the subcellular distribution of Smad2. HistoneH3 and tubulin antibodies were used as loading controls for the nuclear and cytoplasmic fractions, respectively. Average nuclear Smad2 levels from threeindependent replicate experiments were quantified and the data are shown below the representative immunoblots. Data show the mean6s.e.m. (n53); *P#0.05(two-way ANOVA).

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signaling in aPKC-silenced cells. We used siRNA to knock downthe aPKCs (siPKCi/f), TRAF6 alone (siTRAF6) or aPKC andTRAF6 together (siPKCi/f+TRAF6) (Fig. 6B). As we had

observed before, knockdown of aPKC enhanced TGFb-inducedphosphorylated p38 MAPK levels (Fig. 6B, lanes 3 and 4).Interestingly, in cells where aPKC and TRAF6 were knocked down

simultaneously, TGFb-induced p38 MAPK phosphorylation wasabrogated (Fig. 6B, lanes 7 and 8). Furthermore, the p38 signalingpatterns were reflective of the level of apoptotic stimulation, asTRAF6 siRNA also reduced the TGFb-induced apoptotic response

seen in aPKC-silenced cells (Fig. 6C). These results suggested thatthe enhanced p38 MAPK signaling and apoptosis that we hadobserved in aPKC-silenced cells were TRAF6 dependent.

TRAF6 silencing partially inhibits TGFb-dependentgene transcriptionFinally, given that aPKC knockdown increased TRAF6-dependentp38 MAPK activity and decreased TGFb-dependent genetranscription, we assessed whether the two observations werelinked, i.e. whether TRAF6 modulation would alter TGFb-

dependent gene transcription. To do this, we performed qPCRanalysis of SMURF2, SERPINE1, MMP9, CDH-1 and SNAI-1mRNA levels in control and TRAF6-silenced cells following TGFbstimulation for 4 and 24 h (Fig. 7). Unlike our previous resultsregarding SMURF2 mRNA levels in control versus aPKC-silencedcells (Fig. 1B), we observed no differences in SMURF2 transcript

levels between control and siTRAF6-treated cells (Fig. 7A).However, consistent with the results observed with aPKCsilencing (Fig. 1), TRAF6 silencing decreased TGFb-dependent

SERPINE1, MMP9 and SNAI1 levels (Fig. 7A). Interestingly, thereduction of gene transcription in TRAF6-silenced cells wasindependent of Smad2 phosphorylation or nuclear accumulation

(Fig. 7B,C). Taken together, our results suggest that aPKC isoformsnot only regulate TGFb-dependent gene regulation but also directNSCLC cells towards EMT versus apoptotic responses using

multiple signaling pathways.

DISCUSSIONTGFb pathways regulate many developmental and homeostatic

processes, and aberrant signaling is associated with variouspathologies such as fibrosis and cancer. Here, we have found thataPKC knockdown alters both Smad-dependent and the Smad-

independent p38 MAPK signaling pathways and regulateswhether NSCLC cells undergo TGFb-dependent apoptosis.

In this report, we examined the transcriptional changes

associated with TGFb signaling in an aPKC-silencedbackground. In this context, we found that several TGFb-stimulated genes showed reduced transcriptional activity andthis was a result of reduced Smad2 nuclear accumulation.

Furthermore, we found that the knockdown of aPKC increasedthe basal protein levels of SARA. This is an important finding, asit has been reported previously that increased SARA expression

is associated with reduced TGFb receptor degradation (DiGuglielmo et al., 2003), consistent with our previous findingsthat aPKC knockdown reduces TGFb receptor degradation

(Gunaratne et al., 2012). Moreover, increased SARA expressionis associated with the maintenance of epithelial cell phenotype(Runyan et al., 2009), consistent with our findings that aPKC

Fig. 4. aPKC knockdown increases and temporally extendsphosphorylated p38 MAPK levels in response to TGFb. (A) A549cells transfected with control siRNA (siControl) or siRNA directedagainst the aPKC isoforms (PKCi/f) were treated with or without250 pM TGFb for 1 or 24 h prior to lysis. Samples were thenprocessed for SDS-PAGE and immunoblotted with anti (a)-phospho-specific antibodies directed against phosphorylated (P-) forms of ERK,p38 and JNK as indicated on the right of the panels. Shown arerepresentative immunoblots from at least three independent replicateexperiments. Immunoblotting for actin was performed as a loadingcontrol. (B) A549 cells transfected with the indicated siRNA wereserum starved and treated with 250 pM TGFb for 30 min, washed andfurther incubated for 1 or 4 h prior to lysis. Lysates were thenprocessed for SDS-PAGE and immunoblotted with the anti-phospho-specific p38 and total p38 MAPK antibodies as indicated on the right ofthe panels. Phosphorylated p38 MAPK levels from three independentreplicate experiments were quantified, and the data are shown belowthe representative immunoblots. Data show the mean6s.e.m. (n53);*P#0.05; **P#0.01 (two-way ANOVA).

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knockdown reduces TGFb-induced EMT (Gunaratne et al.,2013). Given our previous finding that aPKC alters membranetrafficking of the TGFb receptors (Gunaratne et al., 2012), itwould be interesting to explore whether aPKC alters the function

or localization of receptor-bound SARA to control the contextunder which Smads are signaling. Interestingly, Runyan andcolleagues showed that although inhibiting TGFb receptor

internalization from the membrane only slightly alteredphosphorylated Smad2 levels, it did significantly impact on theability of Smad2 to dissociate from SARA (Runyan et al., 2005).

Moreover, Smad2–Smad4 complex formation has also beenreported to occur in the early endosome (Chen et al., 2007). This

suggests that the coordinated function and subcellular localizationof SARA and associated Smads are important for mediatingTGFb-dependent transcription properly.

A recent report has implicated SARA in general endocytic

processes through classical ESCRT complex machinery (Kostaraset al., 2013). More specifically, the correct subcellular traffickingof the EGFR from the early endosome to late endosomes to

regulate EGFR degradation was dependent on SARA, implicatingSARA with a more general role in endocytic trafficking than wasappreciated previously (Kostaras et al., 2013). This might have

important implications with respect to our findings that aPKCknockdown reduces TGFb receptor degradation and stabilizes

Fig. 5. aPKC knockdown enhances TGFb-induced apoptoticresponse. (A) A549 cells transfected with control siRNA (siControl) orsiRNA directed against the aPKC isoforms (PKCi/f) were serumdeprived and treated with or without 250 pM TGFb for 48 h in thepresence or absence of a p38 MAPK inhibitor. Hoescht 33342 wasused to stain the nuclei of cells prior to image acquisition and cellcounting. Scale bars: 10 mm. Quantification of apoptotic nuclei (yellowarrowheads) from four independent experiments is expressedgraphically below the representative images. Data show themean6s.e.m. (n54); *P#0.05 (two-way ANOVA). (B) A549 cells weretreated as in A and then lysed. Cell lysates were processed for SDS-PAGE and immunoblotted with anti (a)-cleaved (c-)PARP and anti-actinantibodies. Densitometric analysis from four independent replicateexperiments is shown graphically below the representative immunoblot.Data show the mean6s.e.m. (n54); *P#0.05 (two-way ANOVA).

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particular TGFb receptor–protein complexes (Gunaratne et al.,

2012; Gunaratne et al., 2013). One possibility is that depletion ofaPKC leads to an accumulation of SARA and TGFb receptorcomplexes. Indeed, aPKC has been reported previously to be

involved in the trafficking of membrane proteins, as well as beinginvolved in the passage of EGFR to lysosome-targetedendosomes through the anchoring protein p62 (also known as

SQSTM1) (Sanchez et al., 1998). Whether the knockdown ofaPKC in our model is causing a reduced passage of receptors tolysosomes is an important area for future study.

Here, we made the novel finding that the knockdown of aPKC

increases and extends TGFb-induced p38 MAPK activation,

which sensitizes NSCLC cells to undergo apoptosis. We found

that knockdown of aPKC stabilized TbRI–TRAF6 complexes,and that knockdown of TRAF6 in aPKC-silenced cells returnedp38 MAPK activation back to control levels. In line with our

results, increases in p38 MAPK activity have been reportedbefore upon aPKC silencing (Baldwin et al., 2006), indicatingthat aPKC might be attenuating p38 MAPK signaling in multiple

tumor cell types. Interestingly, when aPKC is knocked down, p38MAPK is able to elicit an apoptotic response, indicating that insome situations aPKC might be a viable therapeutic target.However, the role of p38 MAPK in cancer is also complex, and

context dependent, and in addition to sensitizing cells to a death

Fig. 6. aPKC knockdown enhances TGFb-induced p38 MAPK signaling and apoptoticresponse through TRAF6. (A) HEK 293T cellstransfected with control siRNA (siControl) or siRNAtargeting aPKC isoforms (PKCi/f) were co-transfected with cDNA encoding Flag-tagged TGFbtype 1 receptor (FlagTbRI) as indicated. Cells werethen lysed, and endogenous TRAF6 wasimmunoprecipitated (IP) using anti-TRAF6antibodies. The immunoprecipitates were processedfor SDS-PAGE and immunoblotted with anti (a)-Flagand anti-TRAF6 antibodies to visualizeimmunoprecipitated Flag-tagged TbRI and TRAF6(upper panel). Cell lysates were immunoblotted withanti-PKCi, anti-PKCf, anti-Flag and anti-TRAF6antibodies to visualize endogenous aPKC andTRAF6 levels as well as expressed Flag-taggedTbRI (lower panel). Representative immunoblotsfrom at least from three independent replicateexperiments are shown. (B) A549 cells transfectedwith control siRNA or with siRNA directed againstaPKC (siPKCi/f), TRAF6 (siTRAF6) or both aPKCand TRAF6 (siPKCi/f+TRAF6) were serum starvedand treated with 250 pM TGFb for 1 h prior to lysis.Lysates were then processed for SDS-PAGE andimmunoblotted with the anti-phospho-specific p38and total p38 MAPK antibodies as indicated on theright of the panels. Immunoblotting using anti-TRAF6, anti-PKCi and anti-PKCf antibodies wasused to determine knockdown levels.Phosphorylated p38 MAPK levels from threeindependent replicate experiments were quantifiedby densitometric analysis and data are shown belowthe representative immunoblots. Data show themean6s.e.m. (n53); **P#0.01 (two-way ANOVA).(C) A549 cells transfected as in B were serumdeprived and treated with or without 250 pM TGFbfor 48 h. Hoescht 33342 was then used to stain thenuclei of cells prior to image acquisition and cellcounting. Scale bar: 10 mm. Quantification ofapoptotic nuclei (yellow arrowheads) from threeindependent experiments is shown below therepresentative images. Data show the mean6s.e.m.(n53); **P#0.01 (two-way ANOVA).

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response, p38 activity is also associated with cancer cell survivaland both the stimulation and suppression of EMT (Bakin et al.,

2002; Strippoli et al., 2010; Wagner and Nebreda, 2009). We alsoreport here that the enhanced p38 MAPK activation we observedin aPKC-silenced cells was not responsible for the reduction in

Smad2 nuclear accumulation. The role of Smad2 linkerphosphorylation by MAPK members in TGFb signaling hasyielded mixed results. The original reports show that linkerphosphorylation by MAPK blocked Smad2 nuclear accumulation

(Grimm and Gurdon, 2002; Kretzschmar et al., 1999); however,nuclear stabilization of Smad2 by linker phosphorylation has alsobeen reported (Alarcon et al., 2009; Burch et al., 2010),

suggesting that Smad linker phosphorylation is more complexthan originally thought, and requires further examination.

In conclusion, we have found that aPKC plays multiple roles in

TGFb signaling, and the localization and expression patterns of

aPKC might dictate how a cell responds to TGFb. This isespecially important given that aPKC isoforms have recently

been implicated in cancer progression (Huang and Muthuswamy,2010; Kojima et al., 2008) and aPKCi has been classified as ahuman oncogene (Fields and Regala, 2007; Murray et al., 2011).

Whether aPKC might be a viable therapeutic target in TGFb-driven tumor progression remains to be examined.

MATERIALS AND METHODSAntibodies and ReagentsPrimary antibodies were as follows: anti-b-actin (Sigma, A2668), anti-

PKCi (BD Transduction Laboratories, 610175), anti-PKCf (Cell

Signaling Technology, 9372), anti-phospho-Smad2 (Cell Signaling

Technology, 3101), anti-Smad2/3 (BD Transduction Laboratories,

610842), anti-tubulin (Sigma, T4026), anti-H3-histone (Millipore, 05-

499), anti- phospho-p38 (Cell Signaling Technology, 9211), anti-p38

(Cell Signaling Technology, 9212), anti phospho-ERK (Cell Signaling

Fig. 7. TRAF6 silencing alters TGFb-dependent gene induction but notphosphorylation or nuclear accumulation ofSmad2. (A) Real-time PCR analysis of TGFb-induced mRNA levels in A549 control siRNA cells(siControl) versus TRAF6-silenced cells(siTRAF6). RNA was isolated from cells treatedfor 1 h with TGFb followed by 4 or 24 h ofincubation in the absence of ligand. Two-wayANOVA analysis followed by post-hocBonferonni’s test was used to determine statisticalsignificance of gene expression changes ofSMURF2, SERPINE1 (PAI-1), MMP-9, SNAI1(Snail) and CDH1 (E-cadherin). Data show themean6s.e.m. *P#0.05, **P#0.01. (B) A549 cellstransfected with the indicated siRNA were serumstarved and treated with 250 pM TGFb for 1 h.Lysates were then processed for SDS-PAGE andimmunoblotted with anti (a)-phosphorylation (P-)specific Smad2, Smad2/3 or TRAF6 antibodies.(C) A549 cells transfected with the indicatedsiRNA were serum starved and treated with250 pM TGFb for 1 h. The cells were thensubjected to subcellular fractionation to isolatecytoplasmic and nuclear fractions. The fractionswere subjected to SDS-PAGE and immunoblottedusing anti-Smad2, anti-tubulin and anti-histone H3antibodies to determine the subcellulardistribution of Smad2. Histone H3 and tubulinantibodies were used as loading controls for thenuclear and cytoplasmic fractions, respectively.Representative blots from three experimentsare shown.

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Technology, 4370), anti-phospho-JNK (Cell Signaling Technology,

9255s), anti-Smad4 (Abcam, AB40759), anti-SARA (Santa Cruz

Biotechnology, sc-9135), anti-Flag (Sigma Aldrich, F3165), anti-Traf6

(Cell Signaling Technology, 8028s), anti-EEA1 (BD Transduction

Laboratories, 610457). Horseradish-peroxidase-conjugated secondary

goat anti-rabbit-IgG (Thermo Scientific, 31460) and goat anti-mouse-

IgG (Thermo Scientific, 31430) were used for immunoblot analysis.

Fluorescently conjugated donkey anti-mouse-IgG (Life Technologies,

A21206) and donkey anti-rabbit-IgG (Life Technologies, A31572) were

used for immunofluorescence studies. Human siRNA constructs were

purchased from Life Technologies (siPKCf, siPKCi and siControl

catalog numbers were 10620319-HSS183348, 10620319-HSS183318

and 4390844, respectively). TRAF6 siRNA was purchased from Life

Technologies (product number s14389- 4390824). p38 MAPK inhibitor

was purchased from Calbiochem (506126).

Cell culture and transfectionsA549 and H1299 NSCLC cell lines were maintained in F12K and RPMI-

1640 medium, respectively, supplemented with 10% fetal bovine serum.

Cells were kept in a humidified tissue culture incubator at 37 C under 5%

CO2. siRNA and DNA transfections were conducted using Lipofectamine

RNAi max and Lipofectamine LTX (Life Technologies) according to the

manufacturer’s protocol. TGFb treatments (250 pM) were conducted in

low-serum medium (0.2% FBS) for the indicated times after cells were

serum deprived overnight.

Immunoblotting and immunoprecipitationCells were lysed (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA,

0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride and a mixture

of protease inhibitors) and centrifuged at 20,000 g at 4 C for 10 min.

Aliquots of supernatants were collected for analysis of total protein

concentration. Protein concentrations were determined using the Lowry

method (Fisher). Immunoprecipitations and immunoblotting were

conducted as described previously (Gunaratne et al., 2012; Gunaratne

et al., 2013).

Cellular fractionationCytoplasmic and nuclear cellular fractions were isolated using the

Thermo Scientific Kit NE-PERH kit (78833) according to the

manufacturer’s protocols.

Immunofluorescence microscopyCells were fixed with 4% paraformaldehyde, permeabilized with 0.25%

Triton X-100, and incubated with primary antibodies at 4 C overnight.

Following incubation with the appropriate fluorescent-probe-conjugated

secondary antibodies, the probes were visualized by immunofluorescence

microscopy using an inverted IX81 Microscope (Olympus, Canada).

RNA quality assessment, probe preparation andGeneChip hybridizationTo conduct the microarray analyses, siRNA-treated cells (siControl and

siPKCi/f double knockdown) were treated with TGFb for 1 h, followed

by washout and further incubation of cells for 24 h in low-serum

medium. Total RNA was extracted and processed for microarray analysis

as described below.

RNA and GeneChips were processed at the London Regional Genomics

Centre (Robarts Research Institute, London, Ontario, Canada; http://www.

lrgc.ca) as described previously (Guo et al., 2011). Using Partek, any batch

effect due to scan date was removed and an ANOVA (Yijk5m+Condition

6Timeij+eijk) using Method of Moments (Eisenhart, 1947) was run to

determine gene-level P-values. Fold change comparisons are expressed

relative to untreated siControl cells and represent the average of three

separate experiments (three separate GeneChips per condition). A fold

change of 61.6 was considered as the cutoff for induction.

Reverse transcription and qPCRRNA extraction, reverse transcription and real-time PCR were conducted

as described previously (Gunaratne et al., 2013). Primer sequences are

shown in supplementary material Table S1. Gene expression in each

treatment is expressed relative to the control (siControl, no TGFb) and is

an average of three to six independent experimental trials.

Cell death assaysA549 and H1299 cells transfected with the appropriate siRNA

constructs were serum deprived (0.2% FBS) and then incubated with

or without TGFb in low-serum medium in the presence or absence of a

p38 MAPK (1 mM) inhibitor for 48 h. After 48 h, apoptosis of A549

and H1299 cells was analyzed by examining nuclear morphology after

Hoechst 33342 staining. Hoescht stain (1 mg/ml) was added directly to

the medium and samples were incubated for 30 min at 37 C. The cells

were then visualized using a fluorescent microscope (Olympus IX71),

and ten random images were acquired per condition. Normal and

apoptotic nuclei were counted and the apoptotic nuclei (characterized by

condensed chromatin) were scored as a proportion of normal healthy

cells.

Statistical analysisOne-way or two-way ANOVA analyses followed by post-hoc

Bonferonni’s test were used to evaluate the significance of the results.

Statistical analyses were performed using GraphPad PrismH Software 5.0

and P-values of ,0.05 were considered to be statistically significant.

AcknowledgementsThe authors would like to thank the members of the Di Guglielmo laboratory foradvice and support.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsA.G. conceived and carried out the majority of the work presented in themanuscript. E.C. performed and analyzed the nuclear accumulation of Smad2and D.C. carried out the microarray analysis. T.H.E.-C. assisted in theacquisition of the phosphorylated p38 and apoptosis data. G.M.D.G. supervisedthe studies, helped design the overall experimental approach and prepared thefinal manuscript.

FundingThe work carried out in this study was supported by the Canadian Institutes ofHealth Research [grant number MOP-93625 to G.M.D.G.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.155440/-/DC1

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