Akt isoforms regulate intermediate filament protein levels in epithelial carcinoma cells.
ABSTRACT Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is differentiation- and tissue-specific, and is maintained during tumorigenesis. Vimentin IF is often co-expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in different epithelial cancer cell lines. Over-expression of Akt1 increases K8/18 proteins. Akt2 up-regulates K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results represent the first indication that Akt isoforms regulate IF expression and support the hypothesis that IFs are involved in PI3K/Akt pathway.
- SourceAvailable from: Monique Cadrin
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ABSTRACT: Keratins 8 and 18 (K8/18) are simple epithelial-specific intermediate filament proteins. Keratins are essential for tissues integrity and are involved in intracellular signaling pathways which regulate cell response to injuries, cell growth and death. K8/18 expression are maintained during tumorigenesis, hence their use as diagnostic marker in tumor pathology. In recent years, studies provided evidence that keratins should not be considered only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression play an active role in EMT. We show that K8/18 stable knockdown using shRNA increases collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells show PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increases cisplatin-induced apoptosis. Increased FasR membrane targeting suggest that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and support the hypothesis that keratins play an active role in cancer progression.Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
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ABSTRACT: Human skin produces numerous neurohormones and neuropeptides. Recent evidence has shown that the neuroendocrine regulation of human skin biology also extends to keratins, the major structural components of epithelial cells. For example, thyrotropin-releasing hormone, thyrotropin, opioids, prolactin, and cannabinoid receptor 1-ligands profoundly modulate human keratin gene and protein expression in human epidermis and/or hair follicle epithelium in situ. Since selected keratins are now understood to exert important regulatory functions beyond mechanical stability, we argue that neuroendocrine pathways of keratin regulation are important for maintaining skin and hair follicle homeostasis. This invites innovative neuroendocrine therapeutic interventions for skin disorders characterized by abnormal keratin expression, ranging from psoriasis to genodermatoses, and for promoting skin wound healing and hair growth. This strategy can be probed in simple, but instructive and readily available human skin and hair follicle organ culture assays as ideal models for exploring this new neuroendocrine frontier in translational epithelial biology.BioEssays 05/2014; · 5.42 Impact Factor
Akt isoforms regulate intermediate filament protein levels in epithelial
Anne-Marie Fortier, Céline Van Themsche, Éric Asselin, Monique Cadrin*
Department of Chemistry-Biology, University of Quebec at Trois-Rivieres, Trois-Rivieres, Quebec, Canada G9A 5H7
a r t i c l ei n f o
Received 2 October 2009
Revised 11 December 2009
Accepted 11 January 2010
Available online 28 January 2010
Edited by Veli-Pekka Lehto
a b s t r a c t
Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is dif-
ferentiation- and tissue-specific, and is maintained during tumorigenesis. Vimentin IF is often co-
expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling
pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in
these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in differ-
ent epithelial cancer cell lines. Over-expression of Akt1 increases K8/18 proteins. Akt2 up-regulates
K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results rep-
resent the first indication that Akt isoforms regulate IF expression and support the hypothesis that
IFs are involved in PI3K/Akt pathway.
? ? 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Intermediate filaments (IFs) constitute an extensive cytoskele-
tal network whose protein constituents are encoded by a large
family of genes that are expressed in a tissue- and differentiation
state-specific manner . Keratins are the major IF proteins ex-
pressed in epithelial cells and are associated in obligate hetero-
polymers. The keratins 8 and 18 (K8/18) are typically co-
expressed and constitute the primary keratin pair in simple epithe-
lial cells. As part of the cytoskeleton, IFs are important in providing
mechanical stability and integrity of cells and tissues submitted to
mechanical and toxic stress. Moreover, recent studies have shown
that IFs are involved in signaling pathways which regulate epithe-
lial cell growth, resistance to apoptosis and motility .
K8/18 are expressed in most epithelial tumors (carcinomas) .
Moreover, vimentin, a mesenchymal-specific IFs protein, is often
co-expressed with keratins in late-stage epithelial cancer cells pre-
senting a dedifferentiated phenotype [3–5]. To understand the sig-
nificance of IFs protein increase expression in cells, different
researchers have addressed the question as to whether IFs expres-
sion affects tumor fate and behavior. It was reported that K18 over-
expression correlates with reduced invasive/metastatic potential
and tumorigenicity in a human breast cancer [6,7], whereas an en-
hanced migratory and invasive potential is observed in mouse
fibroblasts . The co-expression of keratins with vimentin is asso-
ciated with an increased metastatic potential  and down-regula-
tion of vimentin expression resulted in impaired invasion of colon
and breast cancer cell lines . For this reason, it is generally
thought that the balance between keratins and vimentin expres-
sion in carcinoma cells would dictate cellular invasiveness and
metastatic potential. However, the molecular mechanisms regu-
lated by IFs as well as the regulation of their own expression pat-
tern in a context of normal and cancer cells remain poorly
Similar to IFs, phosphatidylinositol 3-kinase (PI3K)/Akt signal-
ing pathway plays a pivotal role in cell proliferation, survival and
migration. There are three isoforms of Akt. Amplification of Akt1
and/or Akt2 isoforms is frequently found in human cancers 
and, increasing number of studies demonstrate isoforms-specific
functions for Akt kinases in carcinoma cells [12,13]. In breast can-
cer cells for example, it has been shown that over-expression of
Akt1 decreases cellular invasiveness  whereas over-expression
of Akt2, to the contrary, increases cellular invasiveness . Akt3
has a minimal induction effect on these tumors . Downstream
targets of Akt isoforms which mediate their role in cancer progres-
sion, however, are only partially elucidated.
0014-5793/$36.00 ? 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Abbreviations: IF, intermediate filaments; K8/18, keratins 8 and 18; vim,
vimentin; PI3K, phosphatidylinositol 3-kinase; CA-Akt1, constitutively activated
Akt1; TGF-b1, transforming growth factor b1; shRNA, short hairpin RNA; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate buffered saline; CHX,
* Corresponding author. Address: Université du Québec à Trois-Rivières, 3351,
Boul. des Forges, CP 500, Trois-Rivières, PQ, Canada G9A 5H7. Fax: +1 819 376 5057.
E-mail address: email@example.com (M. Cadrin).
FEBS Letters 584 (2010) 984–988
journal homepage: www.FEBSLetters.org
Akt is known to regulate cell proliferation  and there is
increasing evidences that keratins could play a role in cell growth,
protein synthesis and cell cycle by regulating key signaling mole-
cules, like 14-3-3 proteins and proteins of the Akt-mTor pathway
[17,18]. Another function of Akt is to prevent apoptosis in both
intrinsic and extrinsic pathway [13,19]. IFs have been shown to
attenuate pro-apoptotic signals such as TNFa and Fas by scaffold-
ing and organizing death effector proteins [20–23]. Moreover,
Akt is an important modulator of cell motility and invasion 
and IFs are involved in cell migration and wound healing through
their interaction with adhesion proteins [25–27]. All these findings
suggest that IFs could be associated with Akt-regulated cellular
In the present study, we have investigated whether Akt1 and/or
Akt2 regulate the expression and organization of K8/18 and vimen-
tin in different epithelial cancer cell lines.
2. Materials and methods
2.1. Cell lines and reagents
Human endometrial carcinoma KLE cell line, human cervical
carcinoma HeLa cell line and human hepatocellular carcinoma
HepG2 cell line were purchased from ATCC. Cells were maintained
in growth medium supplemented with serum and 50 lg/mL genta-
mycin. Akt1, Akt2, pAkt (Ser-473) and pGSK3b (Ser-9) antibodies
were purchased from Cell Signaling Technology (Beverly, MA).
K8/18 antibodies were a generous gift from Dr. M. Bishr Omary
(University of Michigan, MI). Vimentin and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) antibodies were purchased
from Abcam (Cambridge, MA). LY294002 was from Cell Signaling
Technology. Cycloheximide and b-actin were from Sigma (St. Louis,
MO). Transforming growth factor b1 (TGF-b1) was purchased from
Calbiochem (San Diego, CA).
2.2. Transfection with short hairpin RNAs (shRNAs) and plasmids
Cells were transfected as described before  with Akt1,
Akt2 or control (scrambled) shRNAs (SABiosciences, Frederick,
MD), or constitutively active Akt1 vector (CA-Akt1), constitu-
tively active Akt2 vector (CA-Akt2) or empty pcDNA3.1 vector
2.3. Western blots and qRT-PCR
Total cellular proteins and mRNA were extracted as described
. Quantitative real-time PCR was performed with LightCycler?
480 (Roche, Laval, Canada) using K8 sense primer 50-agggctgacc-
gacgagat-30and antisense 50-caccacagatgtgtccgaga-30; K18 sense
aaagtgtggctgccaagaac-30and antisense 50-agcctcagagaggtcagcaa-30
(Institute for Research in Immunology and Cancer, Montreal, Can-
ada). Samples were analyzed in triplicates from three independent
experiments. Relative quantification of expression was calculated
by the comparative 2?DDCTmethod  with GAPDH and TBP as
Cells were grown on glass coverslips, fixed with 4% paraformal-
dehyde for 10 min, washed twice in phosphate buffered saline
(PBS) and permeabilized for 10 min in citrate solution (0.1% so-
dium citrate, 0.1% Triton X-100 in PBS) at room temperature. Triple
immunofluorescence staining was performed as described before
2.5. Statistical analyses
The data were subjected to one-way ANOVA analysis of vari-
ance (PRISM software version 3.03; GraphPad, San Diego, CA). Dif-
ferences between experimental groups were determined by the
Tukey’s test. Statistical significance was accepted when P < 0.05.
3.1. Akt1 and Akt2 isoforms differentially-regulates IF proteins levels in
epithelial carcinoma cells
To determine whether Akt isoforms can affect IFs expression
in epithelial carcinoma cells, we used three epithelial carcinoma
cell lines whose various differentiation state and p-Akt levels
constitute an interesting experimental model: two p-Akt-nega-
tive cell lines, HepG2 and HeLa cells, obtained, respectively,
from well and moderately-differentiated epithelial carcinoma,
and one p-Akt-positive and poorly-differentiated cell line, KLE
We have investigated the role of Akt1 and Akt2 isoforms in
regulating the expression of IFs proteins in p-Akt-negative HeLa
and HepG2 cells. Cells were transfected with constitutively
active Akt (CA-Akt) constructs inducing the expression of myri-
stoylated Akt proteins, which are constitutively phosphorylated/
activated in the cells . Increased phosphorylation of Akt pro-
teins as well as one of their substrate GSK3b (Fig. 1A and B) con-
firms that CA-Akt proteins are active in our experimental
Our results show that over-expression of CA-Akt1 isoform in p-
Akt-negative HeLa and HepG2 cells increases K8 and 18 protein
levels in the two cell lines. Vimentin, which is only detectable in
HeLa cells, is not significantly affected (Fig. 1A). On the other hand,
expression of CA-Akt2 isoform up-regulates K18 protein level in
the two cell lines, while K8 is not affected. Interestingly, unlike
CA-Akt1, over-expression of CA-Akt2 isoform significantly up-reg-
ulates vimentin protein levels (Fig. 1B). These results suggest that
activation of Akt1 and Akt2 regulates IFs protein expression and
that the regulation is Akt isoform-specific.
We also examined whether increase in K8/18 was regulated at
transcriptional or posttranscriptional level. We found that trans-
fection of HepG2 and HeLa cells with CA-Akt1 and CA-Akt2 had
no effect on K8, K18 or vimentin mRNAs (Fig. 1C). Thus, the regu-
lation of IFs protein levels by Akt1 and Akt2 activation occurs at the
In the opposite manner, we have analyzed the role of Akt1 and
Akt2 isoforms in regulating the expression of IFs in p-Akt-positive
KLE cells using shRNA. Knockdown of Akt1 decreases K8 protein le-
vel, whereas K18 and vimentin expression were not affected
(Fig. 1D). This could be explained by the presence of active Akt2
isoform in KLE cells, which could maintain K18 and vimentin at a
high level. For instance, Akt2 knockdown significantly decreases
K18 as well as K8 protein levels (Fig. 1E). Knockdown of Akt1 or
Akt2 individually in KLE cells was not sufficient to decreased
vimentin protein levels (Fig. 1D). Altogether, these results reveal
that Akt regulates IFs protein levels in carcinoma cells, in an iso-
To determine whether the increase in IF proteins following Akt
over-expression requires new protein, the effect of cycloheximide,
a protein synthesis inhibitor, on Akt2-induced IFs expression was
studied in HeLa cells. The induction of IFs proteins by Akt2 was re-
duced following cycloheximide treatment (Fig. 2), suggesting that
new protein synthesis is required for the Akt2-induced IFs up-reg-
ulation. This suggests that Akt2 could modulates the stability of IFs
mRNA and leads to the increased IFs expression.
A.-M. Fortier et al./FEBS Letters 584 (2010) 984–988
3.2. Akt2 isoforms induces a reorganization of cytoplasmic IFs network
in epithelial carcinoma cells
Epifluorescence microscopy using triple staining for pAkt, kera-
tins/vimentin and nuclei shows that transfection of CA-Akt2 in-
duces modifications of keratins and vimentin networks. In CA-
Akt2-transfected HeLa cells, pAkt shows a diffuse cytoplasmic
staining with a prominent localization at the cell membrane
(Fig. 3). In these cells, keratins and vimentin form a dense juxtanu-
clear cluster with little fibrillar extension which is not observed in
non-transfected cells. These results indicate that IFs network orga-
nization is modulated by Akt2 activation. Akt is known to remodel
cytoskeleton by its interaction with actin in a cell migration con-
text . We performed a rhodamin–phalloidin staining to deter-
mine if Akt2-induced IFs reorganization could induce actin
network remodelling. We observed that over-expression of Akt2
in HeLa cells induced actin cytoskeleton cortical remodelling,
which is characterized by a denser actin network at the cell periph-
ery. Taken together these results reveal that Akt2 regulates IFs and
actin networks organization in carcinoma cells.
3.3. IFs are up-regulated by TGF-b1 and insulin in PI3K-dependent
manner in epithelial carcinoma cells
To examine the regulation of IFs protein expression under a
more physiological condition, we treated p-Akt-positive KLE cells
with TGF-b1 or insulin, which are known to induce Akt activation
[31,32]. Exposure of the cells to TGF-b1 (Fig. 4A) or insulin (Fig. 4B)
increased the levels of Akt phosphorylation but did not modulate
the levels of K8/18. However, vimentin protein level is significantly
up-regulated (Fig. 4A). Treatment with PI3K inhibitor LY294002
blocked the TGF-b1-induced up-regulation of vimentin. These find-
ings show that activation of PI3K/Akt pathway following treatment
with physiological concentration of TGF-b1 is associated with
upregulation of vimentin, in agreement with our findings that
over-expression of constitutively active Akt proteins (Akt2 in this
case) increases vimentin protein levels (Fig. 1B).
IFs protein expression pattern is commonly used as diagnostic
marker in tumor pathology . In fact, epithelial tumors maintain
the keratin expression patterns of their respective cell type of ori-
gin and co-expression of vimentin is considered to be a marker of
de-differentiation (epithelial to mesenchymal transition) and inva-
siveness. In recent years, different studies have shown that IFs
should not be considered only as markers proteins but also as reg-
ulators of differentiation and that they might play an active role in
In this work, we investigated whether Akt isoforms could regu-
lates IFs expression in epithelial carcinoma cells. Our results show
that Akt1 up-regulates both K8 and K18 protein levels, whereas
Akt2 increases K18 and vimentin expression, while K8 expression
+- + -
shRNA CTL + -
shRNA Akt1 -
shRNA CTL + -
shRNA Akt2 -
Fig. 1. Intermediate filaments are differentially regulated by Akt1 and Akt2
isoforms in epithelial carcinoma cells. Protein levels of Akt1 or Akt2, pAkt, pGSK3,
K8/18 and vimentin were analyzed by Western blot in HeLa and HepG2 cells
transfected with control (empty) vector or with CA-Akt1 vector (A) or CA-Akt2
vector (B). mRNA levels of K8, K18 and vimentin were analyzed by quantitative
real-time RT-PCR in HeLa and HepG2 cells transfected with empty vector or with
CA-Akt1 vector (C) or CA-Akt2 vector (D). Protein levels of Akt1, Akt2, pAkt, K8/18
and vimentin were analyzed by Western blot in KLE cells transfected with control
(scrambled) shRNA or with Akt1 shRNA (E) or Akt2 shRNA (F). GAPDH or b-actin
was used as a loading control. H indicates a P-value of <0.05.
+ - +
Fig. 2. Akt2-induced intermediate filaments up-regulation requires new protein
synthesis. HeLa cells were transfected with control (empty) vector or CA-Akt2
vector and treated with cycloheximide (CHX) (20 lg/mL) or vehicle for 24 h. Protein
levels of Akt2, K8, K18 and vimentin were analyzed by Western blot. GAPDH was
used as a loading control.
A.-M. Fortier et al./FEBS Letters 584 (2010) 984–988
is not significantly modulated. These results suggest that IFs regu-
lation mediated by Akt is isoform-specific. Although our results
show that the increase in IF proteins does not result from increased
transcription, it required new protein synthesis. Indeed, cyclohex-
imide treatment inhibits Akt2-induced IFs up-regulation. We sug-
gest that Akt could modulates mRNA stability and leads to the up-
regulation of IFs proteins. Some studies have already shown that
keratins protein synthesis could be regulated at a posttranscrip-
tional level, through a stabilization of mRNA transcripts [33–35].
It has been proposed that some physiological block in translation
prevents proteins synthesis until required during specific cell
states and we can extrapolate this hypothesis to our model in that
Akt activation could reverse the blocking of IFs translation in epi-
thelial carcinoma cells [36–38].
As obligate heteropolymers, K8/18 are known to be associated
and expressed in 1:1 ratio . However, imbalance in K8/18 ratio
has been shown to occur in different pathological conditions. For
instance, K8 over-expression is associated with IFs aggregation
and Mallory–Denk body formation in mice hepatocytes . In
vitro K18 over-expression has also been shown to induce IFs
aggregation . However, the functional significance of modifi-
cations in K8/18 ratio in cells is not known. Our results show that
Akt2 induces an increase of K18 protein level, while K8 were not
significantly modulated. The imbalance of K8/18 expression in
Fig. 3. Intermediate filaments cytoskeletal network is reorganized by Akt2 isoform in HeLa cells. HeLa cells were transfected with CA-Akt2 vector and triple
immunofluorescence staining of Akt2 (green), keratins/vimentin/actin (red) and nuclei (blue) was performed.
- + +
Fig. 4. TGF-b1- and insulin-stimulated Akt modulates intermediate filaments
expression via PI3K. KLE cells were treated for 24 h with TGF-b1 (10 nM) (A) or
insulin (200 nM) (B) in presence or not of PI3K inhibitor pre-treatment (LY294002
30 lM for 1 h). Protein levels of pAkt, K8, K18 and vimentin were analyzed by
Western blot. GAPDH was used as a loading control. H indicates a P-value of <0.05.
A.-M. Fortier et al./FEBS Letters 584 (2010) 984–988
Akt2-transfected cells could be related to vimentin, which are
also up-regulated by Akt2 in HeLa cells. It was already shown that
K18 could possibly interact with vimentin to form mixed fila-
ments . Another attractive explanation is that K18 could be
unpolymerized either in insoluble small aggregates  or in fil-
amentous soluble pool, which could be undegraded due to their
association with other proteins such as trichoplein and desmopla-
kin . All these possibilities need further investigation to be
In conclusion, these results represent the first indication that
Akt could regulate IF protein levels in epithelial cancer cells. The
physiological significance of differentially IFs up-regulation by
Akt1 and Akt2 isoforms is not elucidated yet but this work suggest
that IFs proteins are directly involved in PI3K/Akt signaling
The authors want to thank Julie Girouard for performing shR-
NAs tranfections. M.C. and E.A. are supported by a grant from Nat-
ural Sciences and Engineering Research Council of Canada and E.A.
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