The Pennsylvania State University
The Graduate School
Department of Chemical Engineering
EVOLUTION OF CELL GENERATED FORCES DURING EPITHELIAL
MYOFIBROBLAST TRANSITION
A Thesis in
Chemical Engineering
by
Sandeep Mouli Nalluri
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
August 2015
The thesis of Sandeep Mouli Nalluri was reviewed and approved* by the following:
Esther W Gomez
Assistant Professor of Chemical Engineering &
Assistant Professor of Bioengineering
Thesis Advisor
Manish Kumar
Assistant Professor of Chemical Engineering
Phillip E. Savage
Head of the Department of Chemical Engineering
Walter L. Robb Family Endowed Chair
*Signatures are on file in the Graduate School
iii
ABSTRACT
Myofibroblasts are cells that aid in wound healing and upon aberrant activation they can promote
diseases including organ fibrosis and cancer. Myofibroblasts can develop from epithelial cells
through an epithelial-mesenchymal transition (EMT), which is characterized by loss of cell-cell
junctions, changes in gene expression and increased cell motility. While many transcriptional
programs that govern EMT are well studied, little is known about how the cytoskeleton
contributes to EMT progression. Here, we find that the expression levels of cytoskeletal
associated proteins are increased during transforming growth factor (TGF)-beta 1 induced EMT
which correlates with cytoskeletal rearrangements and increased exertion of contractile forces by
cells. Furthermore, we find that inhibition of ERK 1/2 and p38 MAPK dramatically impacts the
acquisition of myofibroblast characteristics during EMT. These studies identify key cytoskeletal
associated signaling pathways that regulate the development of myofibroblasts and suggest
approaches to promote myofibroblast development for reparative functions or to abrogate the
activation of myofibroblasts in pathological contexts. Furthermore the regulation of focal
adhesion proteins during EMT in cancer cells with extracellular matrix stiffness was discussed.
iv
TABLE OF CONTENTS
List of Figures .......................................................................................................................... v
List of Tables ........................................................................................................................... vi
Acknowledgements .................................................................................................................. vii
Chapter 1 Background ............................................................................................................. 1
Epithelial-mesenchymal transition ........................................................................... 1 Evolution of cell generated forces and cytoskeletal signaling during EMT............. 3 Role of ERK and p38 MAPK in development of myofibroblasts through EMT ..... 4
Chapter 2 Evolution of cell generated forces ........................................................................... 5
Introduction .............................................................................................................. 5 Materials and Methods ............................................................................................. 5 Results ...................................................................................................................... 10 Discussion ................................................................................................................ 15
Chapter 3 Role of ERK ½ and p38 MAPK in acquisition of myofibroblast characteristics .... 18
Introduction .............................................................................................................. 18 Results ...................................................................................................................... 18 Discussion ................................................................................................................ 28
Chapter 4 Regulation of focal adhesion proteins expression during epithelial
mesenchymal transition of HCC1954 cells with matrix stiffness .................................... 31
Introduction .............................................................................................................. 31 Materials and Methods ............................................................................................. 32 Results ...................................................................................................................... 33 Conclusion ................................................................................................................ 35
Chapter 5 Conclusion and Future ............................................................................................ 37
References ........................................................................................................................ 39
v
LIST OF FIGURES
Figure 1-1 Epithelial mesenchymal transition is characterized by loss of cell-cell
junctions, acquisition of an elongated morphology and formation of stress fibers.
Mesenchymal cells further progress through EMT to form myofibroblasts. ................... 2
Figure 2-1 TGFβ1induces changes in epithelial and mesenchymal proteins. NMuMG
cells treated with control and TGFβ1 are stained for E-cadherin, α-SMA,
tropomyosin, and caldesmon at 24, 48 and 72 h. Scale bar: 25 μm. ................................ 11
Figure 2-2 Western blot of control and TGFβ1 treated cells at 24, 48 and 72 h for E-
cadherin, α-SMA, tropomyosin, p-caldesmon, caldesmon, and α-tubulin, the loading
control. The expression of actin binding proteins caldesmon, p-caldesmon and
tropomyosin is upregulated during TGFβ1 induced EMT and α-SMA is expressed at
72 h following the treatment with TGFβ1. ....................................................................... 12
Figure 2-3 During the progression of EMT the actin cytoskeleton rearranges from a
cortical architecture to prominent stress fibers. Focal adhesion number and area
increase during the progression of EMT. Scale bar: 25 μm. ............................................ 13
Figure 2-4 Cells exhibit increased focal adhesions following induction of EMT. (A)
Immunofluorescence staining for vinculin. Scale bar: 25 μm. Quantification of (B)
focal adhesion number and (C) focal adhesion area. ....................................................... 14
Figure 2-5 TFM analysis of NMuMG cells (A) Maps of traction stresses along the cells.
Average (B) area, ##p<0.001 and #p<0.0001, *p<0.001 in comparison to 24 h
TGFβ1 (C) force, # p < 0.0001; *** p < 0.0001 in comparison to 24 h and 48 h
TGFβ1 (D) net contractile moment # p < 0.000001; *** p < 0.01 in comparison to
24 h and 48 h TGFβ1 and (E) traction stress #p<0.5, ##p<0.0001 and *p<0.01 in
comparison to all other samples. ...................................................................................... 16
Figure 2-6 TFM analysis of MDCK cells treated with control or TGFβ1 at 72 h. (A)
Maps of traction stresses along the cells. Average (B) area (C) force (D) net
contractile moment and (E) traction stress. *p<0.0001 in comparison to respective
control samples. ............................................................................................................... 17
Figure 3-1 Western blot showing (A) ERK1/2 phosphorylation at 24, 48 and 72 h in
TGFβ1 and control treated NMuMG cells (B) ERK phosphorylation upon treatment
with TGFβ1 + U0126 and TGFβ1+dmso at 72 h (C) ERK phosphorylation,
expression of α-SMA, tropomyosin, caldesmon and p-caldesmon at 72 h in NMuMG
cells treated with control/TGFβ1 plus DMSO/U0126, also shown are α-SMA,
tropomyosin, caldesmon and p-caldesmon. ..................................................................... 19
Figure 3-2 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus
DMSO or U0126. Transcript levels for (A) E-cadherin, **p<0.001, #p<0.05, (B) α-
SMA, *p<0.01, #p<0.5 compared to TGFβ1 plus DMSO, (C) caldesmon, #p<0.5,
*p<0.01 compared to control plus DMSO, and (D) tropomyosin, *p<0.01, #p<0.5
compared to control plus DMSO. .................................................................................... 20
vi
Figure 3-3 Staining for actin at 72 h in NMuMG cells treated with control or TGFβ1 plus
DMSO or U0126. U0126 did not block TGFβ1 induced stress fibers formation at 72
h. Scale bar ~ 25μm. ........................................................................................................ 21
Figure 3-4 U0126 did not block TGFβ1 induced focal adhesions formation at 72 h. (A)
Immunofluorescence staining for vinculin at 72 h in NMuMG cells treated with
control or TGFβ1 plus DMSO or U0126, and quantification of (B) focal adhesion
area and (C) focal adhesion number. ................................................................................ 22
Figure 3-5 Traction force microscopy at 72 h post the treatment of NMuMG cells treated
with control/TGFβ1 plus DMSO/U0126 was used to generate (A) traction stress
maps, and to calculate (B) cell area, #p<0.0001, *p<0.0001 comparison to TGFβ1 +
DMSO, (C) force exerted by the cells, ##p<0.001, #p<0.0001, *p<0.0001
comparison to TGFβ1 + DMSO, (D) net contractile moment of the cells, #p<0.0001,
*p<0.0001 comparison to TGFβ1 + DMSO, and (E) average traction stress exerted
by the cells, p<0.05 in comparison to control plus DMSO. N=50 cells per condition
and ##p<0.001, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO. ...................... 23
Figure 3-6 Western blot showing expression of p-ERK1/2, ERK1/2, α-SMA, E-cadherin,
and α-tubulin, the loading control, in MDCK cells at 72 h treated with control or
TGFβ1 plus DMSO or U0126. ......................................................................................... 25
Figure 3-7 Traction force microscopy at 72 h post the treatment of NMuMG cells treated
with control or TGFβ1 plus DMSO or SB203580. (A) cell area, (B) force exerted
by the cells, (C) net-contractile moment of the cells, and (D) average traction stress
exerted by the cells; #p<0.05 and *p<0.0001. ................................................................. 26
Figure 3-8 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus
DMSO or SB203580. Transcript levels for (A) E-cadherin, *p<0.001, (B) α-SMA,
(C) caldesmon, and (D) tropomyosin. .............................................................................. 27
Figure 3-9 Traction force microscopy at 72 h on MDCK cells treated with control or
TGFβ1 plus DMSO or U0126. (A) traction maps showing traction stress across the
cells, (B) cell Area, #p<0.001 and *p<0.001 compared to TGFβ1 plus DMSO, (C)
forces exerted by the cells, #p<0.001 and *p<0.001 compared to TGFβ1 plus
DMSO, (D) net contractile moment of the cells, #p<0.001 and ##p<0.05, and (E)
average traction stress exerted by the cells, #p<0.001 and ##p<0.05. Minimum of 50
cells were used per condition over three different experiments. ...................................... 29
Figure 4-1 Western blot showing expression of vinculin, LPP, and GAPDH, the loading
control, in HCC1954 cells at 96 h treated with control or TGFβ1 in (A) low serum
medium and (B) high serum medium. .............................................................................. 34
Figure 4-2 Immunofluorescence staining for vinculin in HCC1954 cells at 96 h post the
treatment of cells with control or TGFβ1 on 130 Pa, 1800 Pa and 6000 Pa gels. ............ 34
Figure 4-3 Traction force microscopy analysis of HCC1954 cells treated with control or
TGFβ1 at 96 h. (A) Traction maps of HCC1954 cells, (B) Cell area, (C) total force
exerted, (D) traction stress, and (E) net contractile moment of HCC1954 cell. .............. 36
vii
ACKNOWLEDGEMENTS
I owe my deepest gratitude to my family for giving me the opportunity to pursue higher
studies at The Pennsylvania State University. They have helped and supported me in every way
possible towards achieving my career goals.
I’m very grateful to my research advisor, Dr. Esther W Gomez, for her guidance and
support for pursuing my thesis work and following my career dreams. She has given me lots of
opportunities to develop my scientific knowledge and improve my technical communication
skills. I definitely improved myself a lot and became a better researcher because of her. I couldn’t
have imaged a better adviser and mentor.
I would like to thank Dr. Manish Kumar and Dr. Phillip E. Savage for agreeing to be in
my defense committee. They were supportive and encouraging which helped to the successful
completion of my thesis.
I would like to thank my lab members, Joseph W. O’Connor, Joelle Khouri, Natalie
Morrissey and Todd Thompson for their help and advice for performing my experiments. They
were very helpful in finishing my experiments on time.
I would like to express my sincere gratitude to Dr. Wayne Cutis and Dr. Thomas Wood
for letting me use their instruments for my research
I would also like to thank my other lab members Matt Bierowski, Akanksha Gupta, Dan Ye,
Joseph Wokpetah and Steve Pohler for making the lab environment more lively and joyful to
work. Thanks are also due to my friends who helped me at every time to realize my dream come
true.
1
Chapter 1
Background
Myofibroblasts are cells found in the body that help in wound healing but when they are
activated in pathological contexts they can contribute to the formation of fibrosis and lead to
cancer progression [1-9]. During wound healing they exert large contractile forces on the
extracellular matrix (ECM) and secrete ECM components that help in the closure of the wound
[1, 2]. Similarly, in pathological context these factors can lead to fibrosis and ultimately to the
failure of organs [3-6]. When present near the tumor front, myofibroblasts secrete certain
cytokines that promote the metastasis of tumor cells [7-9]. Myofibroblasts can also attack the
implanted biomaterials leading to their failure [10]. Myofibroblasts can be formed through
fibroblasts differentiation or they can also be formed through epithelial mesenchymal transition
(EMT) process [1, 11, 12]. Here, we study the mechanisms underlying myofibroblast formation
through EMT.
Epithelial-mesenchymal transition
Myofibroblasts can develop from lung [13-16], kidney [17-19], and breast [20-22]
epithelial cells through EMT. Transcriptional programs governing EMT are well documented;
there is down-regulation of epithelial markers such as E-cadherin (E-cadherin) and cytokeratins,
and up-regulation of various mesenchymal markers such as N-cadherin (N-cad) and vimentin [23,
24]. In EMT cells loose intercellular contacts, acquire an elongated morphology, and display
increased motility (Figure 1-1) [25-29]. During this process there is a dramatic remodeling of the
cytoskeleton and formation of stress fibers [23, 24, 30]. Cells can further progress through EMT
2
to express alpha-smooth muscle actin (α-SMA) protein, which is a hallmark of myofibroblast
formation [31].
Figure 1-1 Epithelial mesenchymal transition is characterized by loss of cell-cell junctions,
acquisition of an elongated morphology and formation of stress fibers. Mesenchymal cells further
progress through EMT to form myofibroblasts.
Transforming growth factor (TGF)-β1 is an important cytokine that is present near
wound, cancer and fibrosis sites, which is known to induced EMT in epithelial cells [32-34].
TGFβ1 activates various Smad and non-Smad pathways; Smad pathway induces transcription
factors that include, dEF1/ZEB1, SIP1/ZEB2, and Snail/SNAI1, and non-Smad pathways include
ERK, p38 MAPK, RhoA, and Rac1, regulate cell fate during EMT [35]. In this study we have
induced EMT in normal murine gland epithelial cells (NMuMG) and Madin-Darby canine kidney
(MDCK) epithelial cells using TGFβ1, and we examined the roles of ERK and p38 in the
regulation of EMT.
3
Evolution of cell generated forces and cytoskeletal signaling during EMT
Transcriptional programs governing during EMT have been studied extensively,
However, less is known about how actin cytoskeletal remodeling and cell generated forces evolve
during EMT progression [36-40] . Cytoskeletal remodeling during EMT involves loss of thin
cortical actin filaments and formation of thick parallel stress fibers made up of actin filaments
[41, 42]. Factors regulating, cytoskeletal rearrangements and the impact of cytoskeletal signaling
on EMT progression is poorly understood. Furthermore, the evolution of cell generated forces
during myofibroblast formation through EMT has not been studied. In this thesis, we examine the
evolution of cell generated forces during myofibroblast formation through EMT using the traction
force microscopy (TFM) technique. In addition, we examine the role of key signaling molecules
in the regulation of cytoskeletal remodeling and force generation.
EMT involves upregulation of a variety of cytoskeletal associated proteins such as α-
SMA [31], tropomyosin isoforms [43, 44] and caldesmon [44]. Incorporation of α-SMA into
stress fibers plays an important role in increased contractility of myofibroblasts formed through
fibroblasts[45]. Tropomyosin, upon binding to actin, activate actomyosin ATPase and promote
stress fibers formation [46]. Several studies have shown that an increase in caldesmon expression
stabilizes stress fibers [47]; however, others have shown that overexpression of caldesmon can
disrupt stress fibers [48]. It has also been shown that phosphorylation of caldesmon at ser527
releases the inhibitory effect of caldesmon on actomyosin ATPase activity leading to stabilization
of stress fiber formation [49-51] . Here, for the first time we correlate the expression of these
actin binding proteins (α-SMA, caldesmon, phosphorylated caldesmon at ser527 (p-caldesmon)
and tropomyosin) with cytoskeletal rearrangement and cell generated forces during myofibroblast
formation through EMT.
4
Role of ERK and p38 MAPK in development of myofibroblasts through EMT
Activation of various pathways such as MAPK pathways, RhoA, and Rac1 are needed for
stress fiber formation during EMT [36-40, 52] . The role of ERK1/2 MAPK in regulation of EMT
and associated stress fiber formation varies with cell types. In some cell types, blocking ERK1/2
activation using pharmacological inhibitors blocked EMT, α-SMA expression, and stress fibers
associated with EMT, whereas, in other cell types the pharmacological inhibitors did not block
EMT and associated stress fibers formation [40, 53-55]. Here, we examine the impact of the ERK
signaling pathway on cytoskeletal rearrangement and force generation in NMuMG and MDCK
cells. We find that inhibition of ERK increased the expression of α-SMA and cell-generated
forces in NMuMG cells during TGFβ1 induced EMT. However, in MDCK cells, inhibition of
ERK decreased the expression of α-SMA and associated cell generated forces.
The p38 MAPK plays an important role during induction of EMT and associated
stress fibers formation [26, 56-58]. The p38 MAPK is a part of MKK3/6-p38MAPK-ATF2
pathway which is activated during TGFβ1 induced EMT [56]. The p38 MAPK inhibitors such as
SB203580 and SB202190 (specific p38MAPK inhibitors) block the stress fibers formation and
change in cell morphology during EMT [56]. In NMuMG cells blocking p38 MAPK activation
has been shown to block the EMT characteristics such as loss of E-cadherin, acquisition of a
spindle-shaped fibroblast morphology, and formation of actin stress fibers [26]. In this
study we show the effect of p38 MAPK inhibitor, SB203580, on cell generated forces
and expression of actin binding proteins at transcript levels.
5
Chapter 2
Evolution of cell generated forces
Introduction
Evolution of cell generated forces during TGFβ1 induced EMT at 24, 48 and 72h were
studied on 1.8 KPa gels, which is comparable to the stiffness of an average breast tumor [59]. 1.8
KPa gels were also necessary because optimal beads movement was observed on this stiffness
during TFM technique which was used to measure the forces exerted by the cells. Hence, all the
experiments used in this study were conducted on 1.8 KPa gels. The total number of focal
adhesions and total focal adhesions area per cell was calculated at these time points. Phalloidin
staining of actin filaments was used to show stress fibers formation during myofibroblasts
formation through EMT. The expression of actin binding proteins caldesmon, p-caldesmon,
tropomyosin and α-SMA were also studied at these time points and correlated with cell generated
forces.
Materials and Methods
Cell culture
Normal murine mammary gland (NMuMG) epithelial cells were obtained from American Type
Culture Collection (ATCC CRL-1636) and were cultured in DMEM supplemented with 10% fetal
bovine serum (FBS; Atlanta Biologicals), 10 μg/ml insulin (Sigma), and 50 μg/ml gentamicin
(Life Technologies). Madin-Darby canine kidney (MDCK) epithelial cells were obtained from
Celeste Nelson (Princeton University) and were cultured in Eagle’s Minimum Essential Medium
6
supplemented with 10% FBS and 50 µg/ml gentamicin. Cells were grown in their culture media
in a 37°C humidified incubator with 5% CO2. For all experiments, cells were serum starved
overnight (2% serum in culture medium) and then treated with 10 ng/ml recombinant human
TGFβ1 (R&D Systems) or carrier vehicle (control) (1mg/ml BSA and 4mM HCl in H2O). For
inhibitor studies, cells were treated either with MEK1/2 inhibitor U0126 (10 μM; VWR) or with
p38 MAPK inhibitor SB203580 (10 μM; VWR) diluted in dimethyl sulfoxide (DMSO).
Preparation of substrates
Polyacrylamide (PA) gels with a modulus comparable to that of a mammary tumor (1.8
kPa) were fabricated following a modified protocol [60, 61]. Glass slides were incubated in 0.1N
sodium hydroxide (NaOH) for 15 minutes, thoroughly rinsed with water, and dried in air. The
slides were then incubated in 2% (v/v) aminopropyl trimethoxysilane (APTMS) diluted in
acetone for 30 minutes and were then rinsed with acetone and dried in a fume hood. Next, the
treated slides were incubated in 0.5% glutaraldehyde diluted in 1× phosphate buffered saline
(PBS) for 30 minutes followed by thorough washing with deionized (DI) water before drying in
air. A solution of 5% acrylamide, 0.06% bis-acrylamide, and DI water was degassed for 30
minutes before adding 0.5% of ammonium persulphate (10% w/v) and 0.05% of
tetramethylethylenediamine (TEMED) to initiate polymerization. This mixture was pipetted onto
the treated glass slide followed by placing a cover slip treated with Rain-X on the top of the
solution. Polymerization occurred over a 30 minute time frame.
Following the polymerization, PA gel surfaces were treated with 0.5 mM N-
Sulfosuccinimidyl-6-(4'-azido-2'nitrophenylamino) hexanoate (sulfo-SANPAH; Pierce) diluted in
50 mM HEPES buffer (pH 8.5) and the gel surfaces were exposed to ultraviolet light (CL-1000
Ultraviolet Crosslinker; UVP) for 10 minutes. The activation step was then repeated. After
7
activation, the PA gels were washed three times in 50 mM HEPES before incubation with10
μg/ml of human plasma fibronectin (BD Biosciences) for 2 hours at room temperature. The gels
were then washed three times with 50 mM HEPES buffer to remove excess fibronectin solution.
The PA gels were finally incubated with DMEM supplemented with 2% FBS for 1 hour in an
incubator prior to plating cells for experiments.
Western Blotting
Total protein was extracted from cells cultured on PA gels in the presence of TGFβ1 or
control vehicle for 24, 48, or 72 hours. Cells were washed twice with ice cold 1× PBS and then
lysed with ice cold RIPA buffer (Pierce) supplemented with protease and phosphatase inhibitors
(Pierce). The lysate was then centrifuged at 14000 rpm for 15 minutes at 4 ºC. The supernatant
was collected and the protein concentration was measured using a BCA Protein Assay Kit
(Pierce). Equal amounts of lysate were loaded and separated on a NuPAGE Novex 4-12% Bis-
Tris Protein Gels (Life Technologies) and transferred to a PVDF membrane. Membranes were
blocked with 5% non-fat dry milk and analysis was performed with primary antibodies against E-
cadherin (1:1000, Cell Signaling), αSMA (1:2500; Sigma), p-caldesmon (1:700; Abcam),
caldesmon (1:10000; Abcam), tropomyosin (1:700; Sigma), α-tubulin (1:1000, Sigma), ERK 1/2
(1:1000, Cell Signaling), and phosphorylated ERK 1/2 (1:1000, Cell Signaling) . Membranes
were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies
(1:2000; Cell Signaling) and SuperSignal West Pico Chemiluminescent Substrate (Pierce) for
detection. The blots were then imaged using FluorChem FC2 system (Cell Biosciences).
8
Traction force microscopy
Double-layer PA gels for traction force microscopy were fabricated as described
previously [62]. Briefly, a thin polyacrylamide gel (1800 Pa) without fluorescent beads was
attached to a gluteraldehyde treated coverslip as described above. Then, a second layer of PA gel
containing fluorescent beads (0.2 µm diameter; Life Technologies) was polymerized on top of the
first PA gel layer. Fibronectin was then cross-linked to the PA gel surface through the use of
sulfo-SANPAH chemistry.
NMuMG cells were seeded at 4000 cells/cm2 on PA gels embedded with fluorescent
beads. Experiments were conducted on an inverted epifluorescence microscope (Nikon Eclipse
TiE) equipped with a LiveCell environmental control chamber (Pathology Devices Inc.). Phase
contrast images of cells and fluorescent images of beads embedded in the gel before and after
removing the cells from the PA gel surface were collected with a 20× air objective. Bead
registration of the two fluorescent images was carried out by aligning the images with ImageJ
[63] and the TurboReg plug-in [64]. The displacements of the beads were determined and
traction stress maps were generated using LIBTRC 2.4 software (generously provided by
Professor Micah Dembo at Boston University) [65]. LIBTRC was also used to compute cell area,
total force |F|, average traction stress |T|, and net contractile moment.
Immunofluorescence staining
Samples were fixed at various time points with 4% paraformaldehyde in 1x PBS at room
temperature for 15 min, except for α-SMA which is fixed with 1:1 methanol/acetone at -20 °C for
10 min. After fixing, the cells were washed three times with 1x PBS, permeabilized with 0.1%
Triton X-100, blocked with 10% goat serum (Sigma) and were incubated with primary antibodies
9
for 1 h: α-SMA (1A4, Sigma), tropomyosin (TM311, Sigma), caldesmon (E89, Abcam) and E-
cadherin (Cell Signaling). Cells were then washed with 1x PBS 3 times before incubating them
with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Following rinsing cells
were washed with 1x PBS 3 times and nuclei were counterstained with Hoechst 33342 (Life
Technologies).
For vinculin and actin staining, cells were fixed with 4% paraformaldehyde in 1x PBS,
cell were washed twice with wash buffer (0.05% Tween-20 in 1x PBS) and permeabilized with
0.1% Triton X-100 in 1x PBS for 5 min at room temperature. Cells were then washed twice with
wash buffer followed by blocking with 1% BSA solution in 1x PBS for 30 min at room
temperature. Anti-vinculin (V9131, Sigma) was diluted (1:100) to a working concentration in the
blocking solution and cells were incubated with antibody for 1 h at room temperature followed by
washing with wash buffer three times. Cells were then incubated with Alexa Fluor-conjugated
secondary antibodies (1:100, Life Technologies) in 1x PBS for 60 min at room temperature. To
stain for actin, cells were incubated with Alexa Fluor 594 phalloidin (1:250, Life Technologies)
simultaneously with the secondary antibody for 60 min at room temperature. Samples were then
washed three times with wash buffer. Following washing, nuclei were counterstained with
Hoechst 33342 (Life Technologies) at room temperature for 2 min, followed by washing three
times with wash buffer. All samples were then mounted on cover glass slide with Fluormount-G
(Electron Microscopy Sciences).
Microscopy and Focal Adhesion Analysis
Samples were imaged using a 20x or 40 x air objectives on a Nikon Eclipse Ti-E inverted
fluorescence microscope equipped with a Photometrics CoolSNAP HQ2 CCD camera. To
determine focal adhesion properties, stack containing vinculin images of single cells were
10
uploaded to ‘The Focal Adhesion Analysis Server’ (FAAS) [66]. Briefly, free hand tool in
ImageJ software is used to draw a boundary along a single cell per image to isolate it from group
of cells present in the vinculin immunofluorescence images taken at 40x by using clear outside
command in the edit menu. After all the single cells isolated from images were saved, they were
stacked using ImageJ and saved in ‘Tiff’ format which is uploaded to the FAAS. For the analysis
in the FAAS, detection threshold is set as 2 and minimum focal adhesions size is set as 10 pixels2,
which is equivalent to 0.256 µm2. Also, ‘Only Calculate Static Properties’ option is selected since
these are the static images. The FAAS gives the number of focal adhesions per cell and area of
each focal adhesion. This data was then used to plot histograms of the number of focal adhesions
per cell and the total area of all focal adhesions per cell using different treatment conditions.
Minimum of 50 cells were analyzed per sample condition over three different experiments.
Statistical Analysis
A minimum of three independent trials were performed for all experiments. A student’s t-test or
analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test for multiple comparisons
were used for sample comparisons using Kaleidagraph 4.1 software. Differences were considered
significant for p < 0.05.
Results
To examine how TGFβ1 regulates EMT and actin binding proteins expression,
immunofluorescence staining and western blot were performed
Immunofluorescence staining at 24, 48 and 72 h on NMuMG cells treated with control
and TGFβ1 was performed for E-cadherin, α-SMA, caldesmon, and tropomyosin. Staining for
11
the epithelial marker E-cadherin showed that there is reduced expression of E-cadherin at cell-cell
junctions in TGFβ1 treated samples compared to controls at 24 , 48 and 72 h. α-SMA was found
to be expressed at 72 h post the treatment of cells with TGFβ1 but not in control treated cells,
which suggests that myofibroblasts form at 72 h. Caldesmon and tropomyosin were upregulated
in TGFβ1 treated cells at 24 , 48 and 72 h compared to control cells, also, these proteins seemed
to localize to stress fibers as seen in the figure Figure 2-1.
Figure 2-1 TGFβ1induces changes in epithelial and mesenchymal proteins. NMuMG cells treated
with control and TGFβ1 are stained for E-cadherin, α-SMA, tropomyosin, and caldesmon at 24,
48 and 72 h. Scale bar: 25 μm.
Western blot showed that the expression levels of caldesmon, p-caldesmon, and
topomyosin are increased in NMuMG cells at 24 h after the treatment with TGFβ1 compared to
control vehicle treated cells. The expression levels of these actin binding proteins remained
12
elevated at 48 h and 72 h in TGFβ1 treated cells. The expression of α-SMA was observed at 72 h
post treatment with TGFβ1, which suggests the formation of myofibroblasts at 72 h.
Figure 2-2 Western blot of control and TGFβ1 treated cells at 24, 48 and 72 h for E-cadherin, α-
SMA, tropomyosin, p-caldesmon, caldesmon, and α-tubulin, the loading control. The expression
of actin binding proteins caldesmon, p-caldesmon and tropomyosin is upregulated during TGFβ1
induced EMT and α-SMA is expressed at 72 h following the treatment with TGFβ1.
Immunofluorescence staining for vinculin and phalloidin staining for filamentous actin
was performed on cells treated with control vehicle or TGFβ1 at 24, 48 and 72 h. Thick, parallel
actin filaments formed in TGFβ1 treated cells at all the time points, whereas control treated cells
showed cortical actin around the cell peripheries (Figure 2-3). Focal adhesion analysis on cells
stained with vinculin showed that there is an increase in the total number of focal adhesions and
in the total focal adhesion area per cell from 24 h to 72 h after the treatment of NMuMG cells
with TGFβ1 (Figure 2-4). Also, TGFβ1 treated samples showed higher total focal adhesion area
13
and more focal adhesions per cell when compared to corresponding control treated cells at all the
time points. As seen from the immunofluorescence images there is an increase in the size of focal
adhesions at 72 h time point in TGFβ1 treated samples, which correlates with increased α-SMA
expression.
Figure 2-3 During the progression of EMT the actin cytoskeleton rearranges from a cortical
architecture to prominent stress fibers. Focal adhesion number and area increase during the
progression of EMT. Scale bar: 25 μm.
Traction maps generated from TFM analysis on NMuMG cells show that there is increase
in traction stresses across the cells as they undergo TGFβ1 induced EMT (Figure 2-3. A). TFM
analysis also showed that there is increase in area, total forces, and net-contractile moment
(measure of over-all contractility of the cell) in the cells from 24h to 72 h in TGFβ1 treated
samples (Figure 2-3 B, C & D). The increased expression of forces in TGFβ1 treated cells at 72 h
correlates compared to 24 h and 48 h correlate with expression of α-SMA and increase in number
and total area of focal adhesions, which was observed in immunofluorescence staining and
western blots. Also at each time point control treated cells have shown less area, total forces, and
net-contractile moment compared to TGFβ1 treated cells (Figure 2-3 B, C & D).
14
Figure 2-4 Cells exhibit increased focal adhesions following induction of EMT. (A)
Immunofluorescence staining for vinculin. Scale bar: 25 μm. Quantification of (B) focal adhesion
number and (C) focal adhesion area.
15
Discussion
NMuMG cells cultured on 1.8 kPa gels show increased expression of caldesmon, p-
caldesmon, and tropomyosin upon treatment with TGFβ1 as cells undergo EMT. After initial
upregulation of these actin binding proteins, expression of α-SMA is observed at 72 h. Previous
studies showed that an increase in cellular tension is necessary for the induction of αSMA
expression during formation of myofibroblast through fibroblast differentiation [67]; however,
this has not yet been demonstrated during myofibroblast formation through EMT. Here, using
TFM, we show that the tension in cells is increased before expression of α-SMA and there is
increase in focal adhesions size in TGFβ1 induced EMT. Also, it is known that, during
myofibroblast formation through fibroblasts the expression of α-SMA actin increases the size of
focal adhesions and cell generated forces [45, 68]. Even in our experiments we show that the
size of focal adhesions and traction forces increase at 72 h time point correlating with α-SMA
expression.
Traction force microscopy was also performed on MDCK cells at 72 h post the treatment
with control and TGFβ1 treatment with 1.8 kPa gels. Traction maps show traction stresses
increase along the cell during TGFβ1 induced EMT compared to control treated cells (Figure 2-6
A). Average area, total force exerted, net contractile moment and traction stress, per cell,
increased with TGFβ1 treatment compared to control treated cells (Figure 2-6 B, C, D, & E).
16
Figure 2-5 TFM analysis of NMuMG cells (A) Maps of traction stresses along the cells. Average
(B) area, ##p<0.001 and #p<0.0001, *p<0.001 in comparison to 24 h TGFβ1 (C) force, # p <
0.0001; *** p < 0.0001 in comparison to 24 h and 48 h TGFβ1 (D) net contractile moment # p <
17
0.000001; *** p < 0.01 in comparison to 24 h and 48 h TGFβ1 and (E) traction stress #p<0.5,
##p<0.0001 and *p<0.01 in comparison to all other samples.
Figure 2-6 TFM analysis of MDCK cells treated with control or TGFβ1 at 72 h. (A) Maps of
traction stresses along the cells. Average (B) area (C) force (D) net contractile moment and (E)
traction stress. *p<0.0001 in comparison to respective control samples.
18
Chapter 3
Role of ERK ½ and p38 MAPK in acquisition of myofibroblast characteristics
Introduction
TGFβ1 activates various MAPK pathways that include ERK1/2 and p38 MAPK
pathways [26, 40, 53-58]. ERK1/2 MAPK is a part of Ras/Raf/MEK/ERK pathway, whereas p38
MAPK is a part of MKK3/6-p38MAPK-ATF2 pathway [56, 69]. Studies show P38 MAPK is
necessary for TGFβ1 induced EMT, whereas role of ERK in EMT varies with cell types [26, 56,
70]. How these MAPK pathways regulate myofibroblast characteristics during EMT is not
studied. We used U0126, a specific pharmacological inhibitor for MEK1/2 which blocks the
activation of MEK1/2 which in turn abolishes the phosphorylation of ERK1/2, to study the effect
of the ERK1/2 pathway in acquisition of myofibroblast characteristics. Also for blocking the
activation of p38 MAPK we have used SB203580 inhibitor to study traction forces and transcript
levels of actin binding proteins during TGFβ1 induced EMT.
Results
Phosphorylation of ERK1/2 was studied at 24 , 48 and 72 h following the treatment of
cells with TGF𝛽1 or control vehicle using western blot, which showed that there’s no notable
upregulation of ERK1/2 phosphorylation during myofibroblasts formation through EMT in
NMuMG cells at 48 and 72 h (Figure 3-1. A). At 24 h the ERK1/2 phosphorylation seems
upregulated in TGFβ1 treated samples compared to control treated samples.
19
Figure 3-1 Western blot showing (A) ERK1/2 phosphorylation at 24, 48 and 72 h in TGFβ1 and
control treated NMuMG cells (B) ERK phosphorylation upon treatment with TGFβ1 + U0126
and TGFβ1+dmso at 72 h (C) ERK phosphorylation, expression of α-SMA, tropomyosin,
caldesmon and p-caldesmon at 72 h in NMuMG cells treated with control/TGFβ1 plus
DMSO/U0126, also shown are α-SMA, tropomyosin, caldesmon and p-caldesmon.
Cells were treated with MEK1/2 specific inhibitor, U0126, which blocked
phosphorylation of ERK1/2 (Figure 3-2. B&C). Treatment of U0126 increased the expression
of α-SMA in TGFβ1 treated samples and there is no expression of α-SMA in control plus
DMSO or U0126 treated samples (Figure 3-1 B & C). Western blot was performed in
samples treated with TGFβ1 plus DMSO or U0126 treated samples at 24, 48 and 72 h on
NMuMG cells. ERK1/2 phosphorylation was blocked at all the time points in TGFβ1
plus U0126 treated samples. In TGFβ1 plus U0126 treated samples, initially, there is
reduced expression of caldesmon, p-caldesmon, and tropomyosin at 24 h time point
20
compared to TGFβ1 plus DMSO treated samples, but at 48 h and 72 h, the expression
levels of these proteins was similar in both TGFβ1 plus DMSO and U0126 treated
samples (Figure 3 1. B).
Figure 3-2 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus DMSO
or U0126. Transcript levels for (A) E-cadherin, **p<0.001, #p<0.05, (B) α-SMA, *p<0.01,
#p<0.5 compared to TGFβ1 plus DMSO, (C) caldesmon, #p<0.5, *p<0.01 compared to control
plus DMSO, and (D) tropomyosin, *p<0.01, #p<0.5 compared to control plus DMSO.
The expression levels of caldesmon, p-caldesmon, and tropomyosin remained
upregulated in TGFβ1 plus DMSO or U0126 treated samples compared to control plus DMSO
21
treated sample. Interestingly, control samples treated with U0126 also showed expression up-
regulation of caldesmon, p-caldesmon, and tropomyosin compared to control plus DMSO treated
samples at 72 h (Figure 3 1. C).
Real time- PCR shows that transcript levels of EMT marker E-cadherin is downregulated
in both TGFβ1 plus DMSO or U0126 samples compared to control plus DMSO or U0126 (Figure
3-2 A). α-SMA, caldesmon, and tropomyosin transcript levels are upregulated with U0126
treatment in TGFβ1 treated samples at 72 h in NMuMG cells (Figure 3-2 B, C & D). The
transcript levels of α-SMA are more in TGFβ1 plus U0126 treated samples compared to TGFβ1
plus DMSO treated samples (Figure 3-2 B). The transcript levels of caldesmon and tropomyosin
are more in TGFβ1 plus DMSO or U0126 samples compared to control plus DMSO or U0126
treated samples (Figure 3-2 C &D).
Figure 3-3 Staining for actin at 72 h in NMuMG cells treated with control or TGFβ1 plus DMSO
or U0126. U0126 did not block TGFβ1 induced stress fibers formation at 72 h. Scale bar ~ 25μm.
F-actin and vinculin staining was performed on NMuMG cells treated with control
vehicle or TGFβ1 plus DMSO or U0126 at 72 h. TGFβ1 plus DMSO or U0126 treated samples
showed actin stress fibers indicating that U0126 did not block TGFβ1 induced stress fibers
formation at 72 h (Figure 3-2). The total focal adhesion area and total focal adhesion number per
22
cell increased in TGFβ1 plus U0126 treated cells when compared to TGFβ1 plus DMSO treated
cells and control plus DMSO or U0126 treated cells (Figure 3 3).
Figure 3-4 U0126 did not block TGFβ1 induced focal adhesions formation at 72 h. (A)
Immunofluorescence staining for vinculin at 72 h in NMuMG cells treated with control or TGFβ1
plus DMSO or U0126, and quantification of (B) focal adhesion area and (C) focal adhesion
number.
Traction force microscopy revealed a significant increase in cell area, forces exerted, and
net contractile moment by the TGFβ1 plus U0126 treated cells compared with TGFβ1 plus
23
Figure 3-5 Traction force microscopy at 72 h post the treatment of NMuMG cells treated with
control/TGFβ1 plus DMSO/U0126 was used to generate (A) traction stress maps, and to calculate
24
(B) cell area, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO, (C) force exerted by the
cells, ##p<0.001, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO, (D) net contractile
moment of the cells, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO, and (E) average
traction stress exerted by the cells, p<0.05 in comparison to control plus DMSO. N=50 cells per
condition and ##p<0.001, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO.
DMSO treated cells (Figure 3-4 A, B, C, & D). Also, TGFβ1 plus DMSO or U0126 treated cells
have a larger area, exert more forces and have more net contractile moment compared to control
plus DMSO or U0126 treated cells, respectively. This increased in forces exerted by TGFβ1 plus
U0126 treated cells correlates with increased expression of α-SMA, and an increase in total focal
adhesion area and total focal adhesion number per cell compared to TGFβ1 plus DMSO treated
cells (Figure 3-1 and Figure 3-3).
For NMuMG cells, treatment with U0126 enhances the acquisition of myofibroblast
characteristics during EMT, including increased levels of α-SMA and increased cell generated
traction forces. Next, we used MDCK cells to examine the role of ERK1/2 signaling in increasing
myofibroblast characteristics is cell type specific. Western blot analysis showed that ERK1/2
phosphorylation was blocked in all TGFβ1 and control plus DMSO and U0126 treated samples at
the 72 h time point (Figure 3-5). Interestingly, ERK 1/2 phosphorylation was increased in TGFβ1
plus DMSO treated cells compared to control plus DMSO treated cells. TGFβ1 treated cells
showed reduced expression of α-SMA when treated with U0126 compared to DMSO treated cells
(Figure 3-5). Furthermore, TGFβ1 induced high contractile forces at 72 h in MDCK cells
compared to control treated cells (Figure 2-6). TFM analysis was also performed at 72 h with
control or TGFβ1 plus DMSO or U0126 to study the effect of ERK1/2 signaling on cells
generated forces. Inhibition of EKR1/2 phosphorylation reduced cell generated traction forces
(Figure 3-6). These results show that U0126 inhibition of ERK1/2 activation results in reduced
myofibroblast characteristics in MDCK cells.
25
Figure 3-6 Western blot showing expression of p-ERK1/2, ERK1/2, α-SMA, E-cadherin, and α-
tubulin, the loading control, in MDCK cells at 72 h treated with control or TGFβ1 plus DMSO or
U0126.
Traction force microscopy was performed with NMuMG cells using p38 MAPK inhibitor
SB203580 during TGFβ1 induced EMT (Figure 3-7). The area of cells treated with TGF β1 plus
SB203580 increased compared to that of control plus SB203580, but when compared with TGF
β1 plus DMSO treated cells, the TGF β1 plus SB203580 cells showed less cell area (Figure 3-7
A). Total forces in cells treated with TGF β1 plus SB203580 appeared to be same when compared
with TGF β1 plus DMSO (Figure 3-7 B). Net contractile moment of TGF β1 plus SB203580
decreased when compared to TGF β1 plus DMSO treated cells (Figure 3-7 C). There were no
significant differences in average traction stresses exerted by the cells when treated with TGF β1
plus SB203580 or DMSO (Figure 3-7 D).
26
Figure 3-7 Traction force microscopy at 72 h post the treatment of NMuMG cells treated with
control or TGFβ1 plus DMSO or SB203580. (A) cell area, (B) force exerted by the cells, (C)
net-contractile moment of the cells, and (D) average traction stress exerted by the cells; #p<0.05
and *p<0.0001.
Real time-PCR was also performed with inhibitor SB203580 on NMuMG cells during
TGFβ1 induced EMT (Figure 3-8). E-cadherin transcript levels in TGF β1 plus SB203580
decreased compared to control plus DMSO treated samples (Figure 3-8 A). Transcript levels of α-
27
SMA, caldesmon and tropomyosin appeared same in TGF β1 plus SB203580 treated samples
when compared with TGF β1 plus DMSO treated samples (Figure 3-8 B, C, and D).
Figure 3-8 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus DMSO
or SB203580. Transcript levels for (A) E-cadherin, *p<0.001, (B) α-SMA, (C) caldesmon, and
(D) tropomyosin.
28
Discussion
Blocking of ERK phosphorylation has been shown to block EMT in some cell types and
does not have any effect in other cell types [26, 56]. In renal tubular cells it was shown that
treatment with a MEK inhibitor blocks actin fiber formation and α-SMA synthesis [54].
Here, we show that blocking ERK phosphorylation increases the number and total area of
focal adhesions, the α-SMA expression, and cell generated forces in NMuMG cells.
However blocking ERK activation in MDCK cells led to downregulation of α-SMA and
cell generated forces. Hence, the results here show that ERK might play different role in
acquisition of myofibroblast characteristics in different cell types. Inhibition of ERK1/2
signaling could be a useful strategy in abrogating myofibroblast activation in pathological
contexts or for activating them in wound healing contexts, but how the ERK pathway
behaves in different epithelial cells types in guiding them towards myofibroblast should
be further studied.
29
Figure 3-9 Traction force microscopy at 72 h on MDCK cells treated with control or TGFβ1 plus
DMSO or U0126. (A) traction maps showing traction stress across the cells, (B) cell Area,
30
#p<0.001 and *p<0.001 compared to TGFβ1 plus DMSO, (C) forces exerted by the cells,
#p<0.001 and *p<0.001 compared to TGFβ1 plus DMSO, (D) net contractile moment of the cells,
#p<0.001 and ##p<0.05, and (E) average traction stress exerted by the cells, #p<0.001 and
##p<0.05. Minimum of 50 cells were used per condition over three different experiments.
31
Chapter 4
Regulation of focal adhesion proteins expression during epithelial
mesenchymal transition of HCC1954 cells with matrix stiffness
Introduction
Lipoma-preferred partner (LPP) and vinculin are proteins that are recruited to focal
adhesions during TGFβ1 induced EMT [71, 72]. This chapter discusses the focal adhesion
properties and forces exerted by the cells during TGFβ1 induced EMT in Human mammary gland
epithelial cells (HCC1954). The expression levels of focal adhesion proteins including vinculin
and LPP are studied on gels of varying stiffness during TGFβ1 induced EMT in HCC1954 cells.
LPP is known to localize to focal adhesions during TGFβ1 induced EMT and help in the
promotion of carcinoma invasion [73]. LPP is shown to regulate migration and invasion of breast
cancer cells during TGFβ1 induced EMT; TGFβ1 re-localizes LPP to focal adhesion complexes,
which is critical for TGFβ1 mediated focal adhesion turn over [71]. During EMT LPP also acts
as a sensor for extracellular signals promoting invasiveness of tumor cells [74]. Vinculin is a
protein that is recruited from cytoplasm to focal adhesion during TGFβ1 induced EMT [72].
Actin filaments converge into the focal adhesion complexes where they associate to FN receptors,
α5β1 integrin and bind to vinculin [75]. Here we showed that cancer cells acquired matured focal
adhesions on treatment with TGFβ1, and since matured focal adhesions are known to increase
cell generated forces, traction force microscopy analysis was performed on HCC1954 cells during
TGFβ1 induced EMT to check for modulation of cell generated forces.
32
Materials and Methods
Human mammary gland epithelial cells (HCC1954) were purchased from American Type
Culture Collection (ATCC) and maintained in RPMI 1640 media supplemented with 10% fetal
bovine serum (FBS; Atlanta Biologicals) and 50 µg/ml gentamicin (Life Technologies). Cells
were grown in the culture media in a 37°C humidified incubator with 5% CO2. For all
experiments, cells were serum starved overnight (2% serum in culture medium), and then treated
with 2.5 ng/ml recombinant human TGFβ1 (R&D Systems) or carrier vehicle (control) (1mg/ml
BSA and 4mM HCl in H2O) for 96 h.
For the following methods refer to chapter 2, materials and methods section for detailed
protocol. Cells were cultured on polyacrylamide gels of various stiffness’s including 130 Pa,
1800Pa, 5000 Pa and 6000 Pa. Gels were activated with sulfo-SANPAH, and for western blot
experiments 300,000 cells were added to the gels, for immunofluorescence experiments 150,000
cells and for TFM experiments 50,000 cells were added per sample. For performing TFM
analysis 5 kPa polyacrylamide gels were used. For western blot experiments and
immunofluorescence experiments cells were cultured on 130 Pa, 1800Pa, 5000 Pa and 6000 Pa
polyacrylamide gels.
Immunofluorescence staining for vinculin staining was performed using the same
protocol that was followed in chapter 2. For LPP staining cell signaling staining protocol is
followed, briefly, 4% paraformaldehyde fixed samples were washed with 1x PBS followed by
blocking in blocking buffer (1x PBS / 5% normal serum / 0.3% Triton™ X-100) for 60 min. LPP
(Cell Signaling) primary antibody was diluted (1:100) in antibody dilution buffer (1x PBS / 1%
BSA / 0.3% Triton™ X-100) and added to the samples after aspirating the blocking solution.
Samples were incubated overnight at 4°C and rinsed three times in 1x PBS for 5 min each.
Specimen were incubated in fluorochrome-conjugated secondary antibody (goat anti mouse)
33
diluted in antibody dilution buffer for 1 h at room temperature in the dark. Following washing,
nuclei were counterstained with Hoechst 33342 (Life Technologies) at room temperature for 2
min, followed by washing three times with wash buffer. All samples were then mounted on cover
glass slide with fluormount-G (Electron Microscopy Sciences).
Method for performing western blot used is same as that mentioned in chapter 2. For
western blot vinculin is used at dilution of 1:500 and LPP is used at dilution of 1:1000. GAPDH
is used as loading control at dilution of 1:1000.
Results
Western blot analysis shows that stiffness of extracellular matrix regulates the expression
of vinculin and LPP in low serum medium (2% FBS). Both vinculin and LPP are upregulated at
higher stiffness of 6000 Pa compared to 130 Pa or 1800 Pa gels in both control and TGFβ1
treated samples at 96 h (Figure 4-1 A). But, in high serum medium (10% FBS) there was no
difference of levels of vinculin and LPP expression on 130 Pa and 6000 Pa gels (Figure 4-1 B).
34
Figure 4-1 Western blot showing expression of vinculin, LPP, and GAPDH, the loading control,
in HCC1954 cells at 96 h treated with control or TGFβ1 in (A) low serum medium and (B) high
serum medium.
Figure 4-2 Immunofluorescence staining for vinculin in HCC1954 cells at 96 h post the treatment
of cells with control or TGFβ1 on 130 Pa, 1800 Pa and 6000 Pa gels.
35
Immunofluorescence staining for vinculin protein also shows that TGFβ1 increases the
induction of vinculin protein to focal adhesions on all the gels of 130 Pa, 1800 Pa and 6000 Pa
stiffness (Figure 4-2). Also, the cells grown on higher stiffness of 6000 Pa appeared to have the
higher number of focal adhesions compared to cells grown on lower stiffness of 130 Pa and 1800
Pa in TGFβ1 Treated samples.
Traction force microscopy analysis at 96 h on control and TGFβ1 treated samples show
that the traction stress in the cells increase with TGF β1 treatment compared with control treated
samples (Figure 4-2 A). Cell area of HCC1954 cells, and total force exerted, traction stress and
net contractile moment of HCC1954 cells increased with TGF β1 treatment compared to control
treated cells on 5000 Pa gels (Figure 4-2 B, C, D & E).
Conclusion
During TGFβ1 induced EMT the upregulation of vinculin and LPP were found to be
related to the stiffness of extracellular matrix on which the cells were growing in low serum
medium but not in high serum medium. The expression of these focal adhesion proteins increased
in 6000 Pa gels compared to 1800 Pa and 130 Pa gels. Traction force microscopy was performed
with HCC1954 showed that cell area, total forces exerted by the cells, net contractile moment and
traction stress exerted by the cells increased during TGF β1 induced EMT at 96 h correlating with
increased expression of focal adhesion proteins.
36
Figure 4-3 Traction force microscopy analysis of HCC1954 cells treated with control or TGFβ1
at 96 h. (A) Traction maps of HCC1954 cells, (B) Cell area, (C) total force exerted, (D) traction
stress, and (E) net contractile moment of HCC1954 cells. Minimum of 50 cells over 3 different
experiments were used per condition, #p < 0.01, *p < 0.0001 when compared to control treated
samples.
37
Chapter 5
Conclusion and Future
TFM was used to show the evolution of cell generated forces during EMT for the first
time. In this study we have shown that cell generated forces increase before expression of α-SMA
by NMuMG cells. The expression of α-SMA further correlates with increased contractile activity
of the cells, and total number and area of focal adhesion per cell. Previous it was known that
expression of α-SMA in myofibroblasts formation through α-SMA negative fibroblasts cells
required certain amount of cellular tension, and expression of α-SMA further increases the size of
focal adhesions, but this was not show in myofibroblasts formation through EMT [45, 55]. The
results obtained in this study show that this similar pattern is observed in myofibroblast formation
through EMT as seen in myofibroblast formation through fibroblast. However, blocking α-SMA
protein expression and seeing how it affects the cell generated forces, and focal adhesions size
and number, will further strengthen this theory.
ERK is known to have no effect on TGFβ1 induced EMT and stress fibers formation in
some cell types and blocks EMT in other cell types [26, 56]. In renal tubular cells it is shown that
TGFβ1 induced α-SMA and EMT is blocked during EMT [54]. However, how inhibiting the
ERK phosphorylation controls the expression of α-SMA and corresponding cell generated forces
have not been studied in detail. Here we have shown that inhibiting ERK in NMuMG cell
increases the myofibroblast characteristics, which are increased expression of α-SMA and cell
generated forces. We have also show that this increase in α-SMA is correlated with increase in
total focal adhesion area and number per cell. In contract, inhibiting ERK phosphorylation in
MDCK cell has downregulated the expression of α-SMA and cell generated forces. These results
38
show that ERK regulation of myofibroblasts development through EMT varies with different cell
types. This ERK regulation can be helpful in abrogating the myofibroblast in pathological
contexts or to activate the myofibroblast formation in reparative conditions.
Experiments with HCC1954 cells showed that cells upregulated vinculin and LPP with
stiffness in low serum medium but not in high serum medium. Traction force microscopy analysis
showed that cell area, total forces exerted by the cells, net contractile moment and traction stress
exerted by the cells increased during TGF β1 induced EMT. Further studies needed to be done to
know the area and number of focal adhesions per cell during TGFβ1 induced EMT to correlate
with cell generated forces.
39
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