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Molecular mechanism of transforming growth factor-beta signaling in skin wound healing

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Molecular mechanism of transforming growth factor-beta signaling in skin wound healing

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Content MOLECULAR MECHANISM OF TRANSFORMING GROWTH FACTOR- β
SIGNALING IN SKIN WOUND HEALING

by

Arum Han

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)

December 2011

Copyright 2011 Arum Han
ii

ACKNOWLEDGEMENTS
First and foremost, I heartily thank my advisor Dr. Wei Li. This thesis would not have
been possible without his utmost patience. He has taught me how to understand and
conduct all of my projects and has supported me in numerous ways, including the
countless hours spent working with me making suggestions for my project and the
gracious funding from his lab. Most importantly, his vast knowledge and his continuous
and cheerful enthusiasm have helped me understand how to achieve a research
momentum that has led me to the accomplishment of my thesis.
Next, I owe my complete gratitude to the members of my thesis committee, Dr. Joseph
Landolph, Dr. Bangyan Stiles, and Dr. Agnieszka Kobielak, who gave me incredibly
useful ideas and great advice.
I would also like to thank my lab members for supporting me through many discussions
and our friendships, especially Divya Sahu and Gabrielle Yee. Additionally, I would like
to show my appreciation for Dr. Mei Chen for her continual support, and Dr. Kwang-won
Jung for his help and interest in my project.
Last but not least, I owe many thanks to my loving parents, Sangyong and Sarah, my
sisters Amber and Bolum, my brother Jihun, and the rest of their families for their tireless
encouragement and support through prayer.

iii

TABLE OF CONTENTS

Acknowledgements ii
List of Figures iv
Abstract vi
Chapter 1: Introduction 1
Table 1: TGF- β superfamily and type I/II receptors 4
Chapter 2: T βRI/ALK5-independent T βRII signaling to ERK1/2
in human skin cells according to distinct levels of T βRII
expression 7
Chapter 3: Cross-talk between TGF- β and PKA signaling pathways
inhibits human dermal cell migration 35
Chapter 4: Conclusion 89
Bibliography 96

iv

LIST OF FIGURES
Fig. 1-1: Signaling by TGF- β through serine/threonine kinase
receptors and Smad proteins 3
Fig. 2-1: Opposite effects of TGF- β3 on ERK1/2 in human dermal
versus epidermal cells 10
Fig. 2-2: In vitro and in vivo profiles of T βRII–T βRI subunit in four
human skin types 18
Fig. 2-3: T βRII expression levels determine how TGF- β regulates
ERK1/2 20
Fig. 2-4: T βRI is dispensable for TGF- β signal to ERK1/2 25
Fig. 3-1: Cutaneous wound healing 36
Fig. 3-2: Steps in cell migration 39
Fig. 3-3: T βRI is required for TGF- β-induced inhibition
of dermal cell migration 43
Fig. 3-4: Overexpression of T βRI-TD mutant is sufficient to inhibit
dermal cell migration which is driven by PDGF-BB 46
Fig. 3-5: T βRI is required for TGF β-induced inhibition
of dermal cell migration 50
Fig. 3-6: TGF- β3 inhibits PDGF-BB-induced migration of human
DFs in a Smad-dependent manner 55
Fig. 3-7: TGF- β3 activates CREB by cAMP dependent kinase
(PKA) in a Smad-dependent manner 58
Fig. 3-8: Forskolin inhibits dermal cell migration in a dose-
dependent manner 61
v

Fig. 3-9: Inhibition of PKA abolished TGF- β3’s anti-motility effect 68
Fig. 3-10: Effect of TGF- β3 on the phosphorylation patterns
which are induced by PDGF-BB 70
Fig. 3-11: TGF- β3 induces overexpression of paxillin 74
Fig. 3-12: Over-expression of paxillin by TGF- β3 results in the
disruption of polarization induced by PDGF-BB
in migrating cells 79
Fig. 3-13: Regulation of paxillin expression by TGF- β3 80

vi

ABSTRACT
TGF- β is a secreted cytokine, which plays an important role in cell development,
differentiation, and homeostasis in both physiologic and pathologic conditions, such as
tumorigenesis, abnormal wound healing and skin cancer. TGF- β transmits signal from
the extracellular environment to the intracellular signaling networks via its cell surface
receptor complexes, the TGF- β type II/ I receptor (T βRII/T βRI) heterodimers. TGF- β
ligand binds to T βRII, which in turn recruits and activates T βRI, resulting in the
activation of receptor-regulated Smads (R-Smads) and common Smad4. TGF- β
stimulation is also known to activate R-Smad-independent signaling pathways, such as
the extracellular signal-regulated kinase (ERK1/2) pathway. However, two long-standing
questions remained: 1) why TGF- β activates ERK depending on the cell context and 2)
whether or not T βRII is able to mediate the TGF- β signaling without the participation of
T βRI. In the chapter two, it is shown that TGF- β activates ERK in human dermal
fibroblasts (DFs), but inhibits ERK in human keratinocytes (HKs). While the T βRI
expression remains similar in both cell types, the expression level of T βRII in DFs is
seven-fold higher than that in HKs. Downregulation of T βRII in DFs led to the inhibition
of ERK activation even though TGF- β is present. In contrast, upregulation of T βRII in
HKs resulted in the activation of ERK rather than the deactivation of ERK by TGF- β.
Most intriguingly, the T βRII-mediated TGF- β-stimulated ERK activation or inactivation
in these cells did not require T βRI. Thus, this study illustrates that the expression levels
vii

of T βRII determine how TGF- β regulates ERK in various cell types and provides direct
evidence for the T βRI-independent signaling by T βRII.
In wound healing, TGF- β controls cell proliferation and migration. In the chapter three,
the mechanism of TGF- β’s anti-motility was revealed. Anti-migration is one of the
primary effects of TGF- β on non-transformed cell types. We previously reported that
TGF- β3 inhibits PDGF-BB-induced dermal cell migration during wound healing.
However, it was not clear what the underlying mechanism was. In the chapter three, it is
shown that TGF- β3 activates PKA pathway by R-Smads/Smad4 complex. Activation of
PKA led to over-expression and phosphorylation of paxillin, a focal adhesion molecule.
As a result, cell polarization induced by PDGF-BB was disrupted because of overly-
formed focal adhesions throughout the cell and thereby migration rate was decreased.
This study elucidates the mechanism of TGF- β3 for the inhibition of cell migration.

1

CHAPTER 1: INTRODUCTION
The transforming growth factor (TGF) superfamily include TGF- β, inhibin, activin, bone
morphogenic proteins (BMPs), müllerian inhibiting substance/ anti-müllerian hormone
(MIS/AMH), growth differentiation factor-1 (GDF-1) and nodal. TGF- β superfamily are
secreted cytokines, which play an important role in cell development, differentiation, and
homeostasis. TGF- β is also involved in pathologic conditions such as tumorigenesis,
abnormal wound healing, and cancer (Derynck and Zhang, 2003). To date, three
mammalian TGF- β isoforms has been found: TGF- β1, TGF- β2, and TGF- β3 (Bachman
and Park, 2005). Even though there are high similarities in sequence among the three
TGF- β isoforms, TGF- β3 has a different structure and biological function compared to
TGF- β1 and TGF- β2 (Laverty et al, 2009).
Figure 1-1 shows a brief TGF- β signaling pathway (Miyazono, 2000). TGF- β exists as a
latent 390-amino acid dimeric precursor, bound to latency associated peptide (LAP)
(Wakefield et al., 1988: Miyazono et al., 1988). After dissociation from LAP by cleavage,
active TGF- β binds to TGF- β type I receptor and type II serine/threonine kinase receptors.
Based upon ligand binding, TGF- β type II receptor (T βRII) phosphorylates gly/ser rich
(GS) domain in TGF- β type I receptor (T βRI/ALK). The activated T βRI phosphorylates
receptor regulated (R)-Smad2/3 at their extreme C-terminal serine residues (Shi and
Massagué, 2003). Table 1 shows the TGF- β superfamily and its specific type I and type II
receptors. Based upon a combination of TGF- β type I and type II receptors, TGF- β has
differential downstream effects (Derynck and Feng, 1997: Derynck and Zhang, 2003).
2

The R-Samd2/3 forms a complex with the common Smad4 and translocates to the
nucleus, where the smad complex binds the promoter of target genes. The TGF- β
signaling pathway is negatively regulated by Inhibitory (I)-Smads, such as Smad6 and
Smad7 (Itoh and Dijke, 2007).

The canonical Smad pathway has become the central focus of studies on TGF- β signaling.
However, an increasing amount of evidence indicates that TGF- β activates non-Smad
pathways, such as MAPK pathways, Rho GTPase pathways, JNK pathways, and
phosphatidylinositol-3-kinase (PI3K)/AKT pathways. For example, several studies
unequivocally demonstrated the existence of Smad-independent pathways that mediate
TGF- β signaling to induce programmed cell death. Perlman and colleagues reported that
T βRII interacts with the adaptor protein Daxx, leading to activation of JNK and induction
of apoptosis in epithelial cells and hepatocytes (Perlman et al, 2001). However, it remains
unclear whether the non-Smad signaling pathways could function in the absence of T βRI,
T βRII and/or canonical R-Smad activation. Independent signaling by either T βRI or
T βRII has been reported (Feng et al., 1995, Zhao et al., 2008). Disrupting the T βRII
function impaired the anti-proliferation effect of TGF- β while sustaining plasminogen
activator inhibitor-1 gene activation (Chen et al., 1993). There are also increasing studies
of cross-talk between Smads and the non-Smad signaling molecules (Derynck and Zhang,
2003). R-Smads and co-Smad4 have MH1 and MH2 domains in which there are
phosphorylation target sites of different kinases such as MAPK, ERK, JNK, PKC, and
CamKII. Interactions between the Smad pathway and non-Smad pathways
3

Figure 1-1. Signaling by TGF- β through serine/threonine kinase receptors and Smad
proteins. I-Smads inhibit signaling by R-Smad-Co-Smad complexes. R-I and R-II
represent TGF- β type I and type II receptors, respectively. (Miyazono, 2000)

LAP
TGF- β
4

Table 1. The TGF- β superfamily and type I/II receptors

Ligand Type II receptors Type I receptors
TGF- β TRII TbRI/ALK-5, ALK-1
Inhibin ActR-IIB ALK-4
Actibin ActR-IIB ALK-4
BMP-2, -4, -7/OP-1 BMPR-II, BRK3 ALK-3, ALK-6
BMP-4, GFD-5 ActR-II/ActR-IIB ALK-3, ALK-6
BMP-7/OP-1 ActR-II/ActR-IIB ALK-2
AMH/MIS AMHR-II ALK-2, ALK-3, ALK-6
Nodal ActR-IIB ALK-4
DPP Punt, AtRII Tkv, Sax

5

fine-tune the effect of the TGF- β signaling pathway to modulate different cell events.

Multifunctional roles of TGF- β in wound healing have been suggested since the early
1990’s. Cutaneous wound healing is a highly organized cell event, composed of
inflammation, re-epithelialization, deposition of extra cellular matrix (ECM) molecules,
and tissue remodeling. Dermal fibroblast functions are very critical in wound healing in
terms of migration, proliferation, and ECM deposition. After injury, platelets release
cytokines and growth factors which cause dermal fibroblasts to migrate into the wound
site and deposit ECM molecules for tissue remodeling (Ashcroft and Roberts, 2000).
Abnormal ECM deposition causes keloids and ulcers. TGF- β is a critical factor regulating
the ECM production, cell proliferation, and cell migration during the wound healing
process (Gailit et al, 1994: Massague, 1990: Wang et al., 2006).

However, it is still controversial as to whether TGF- β plays a positive or negative role in
the wound healing process. Topical treatment with exogenous TGF- β3, called Avotermin,
on a wound site accelerated wound healing with less scarring by the reduction of
myofibroblasts and deposited ECM molecules (Occleston et al, 2009). As well as TGF-
β3, the application of the exogenous TGF- β1 accelerated the wound healing process in
animal models (Pierce et al., 1989; Mustoe et al, 1987), and the reduction of TGF- β1
impaired the wound healing by impaired matrix formation in chronic ulcers (Jude et al.,
2002). However, Smad3-null mice showed accelerated wound healing with reduced
inflammation, reduced amount of local TGF- β1, and improved re-epithelialization
6

compared to wild-type mice (Ashcroft et al, 1999). Prolonged TGF- β1 delayed wound
healing by augmenting inflammation and TGF- β1 null mice showed accelerated
reepithelialization (Wang et al, 2006: O’Kane and Furguson, 1997: Koch et al., 2000).

The effect of TGF- β on skin cell migration during wound healing is paradoxical. TGF- β
can stimulate or inhibit migration of keratinocytes (O’Kane and Ferguson, 1997:
Andresen and Ehlers, 1998: Seomun et al., 2008: Tsuboi et al., 1992). Fibroblast
migration can be stimulated by TGF- β as well as PDGF-BB (Postlethwaite et al., 1987).
On the other hand, many research groups, including ours, showed that skin fibroblast cell
migration was inhibited by TGF- β (Ellis and Schor, 1996: Bandyopadhyay et al., 2006).
In order to answer this question, the molecular mechanism of TGF- β signaling pathway
needs to be elucidated.

In this thesis, I will examine 1) how TGF- β differently regulates ERK1/2 in dermal
fibroblasts and epidermal keratinocytes, and 2) what signaling pathways are used for the
TGF- β-induced anti-motility signal, and how TGF- β reorganizes focal adhesion to inhibit
cell migration.

7

CHAPTER 2: T βRI/ALK5-independent T βRII
signaling to ERK1/2 in human skin cells according to
distinct levels of TβRII expression

INTRODUCTION

When skin is wounded and the dermal blood vessels in the wound

are damaged, the
resident skin cells are bathed in plasma-converted

serum for the first time. A key factor in
human serum, but not

in plasma, is transforming growth factor- β3 (TGF- β3).

TGF- β3 has
a positive role in wound healing by differentially

regulating the motility of epidermal and
dermal cells, depending

upon on their naturally occurring levels of the TGF- β receptor

II
(T βRII) (Bandyopadhyay et al., 2006). Therefore, we

are particularly interested in the
mechanism of TGF- β3.

TGF- β signals are transmitted via a cell surface receptor

complex, the T βRII and
T βRI/Alk5 heterodimer. TGF- β

binds to T βRII, which in turn recruits,
transphosphorylates

and activates T βRI, thereby achieving cross-membrane signaling

to
the inside of the cell (Derynck and Feng, 1997: Shi and Massagué,

2003). Once the signal
is inside the cell, the post-receptor

signaling events are divided into R-Smad-dependent
and R-Smad-independent

pathways (Derynck and Zhang, 2003). The R-Smad-dependent
signaling

pathway mediates TGF- β signaling to transcriptional activation

of target genes
(Miyazono, 2000). R-Smad-independent signaling

pathways, including TAK1–MEKK1,
the extracellular signal-regulated

kinases 1 and 2 (ERK1/2), Jun N-terminal kinase (JNK),
p38-mitogen-activated

kinase (p38-MAPK), phosphatidylinositol 3-kinase (PI3K) and

8

Rho family GTPases, mediate a range of effects of TGF- β,

whose outcomes are often
dependent upon cell type and cellular

context (Mulder, 2000; Derynck and Zhang, 2003:
Moustaka and

Heldin, 2005). TGF- β-induced Smad2/3 activation occurs in

almost all cell
types and has been thoroughly characterized.

TGF- β-stimulated R-Smad-independent
pathways, however, often

vary in different cell types (Moustakas and Heldin, 2005). In

this chapter, we show that differences in the level of

T βRII expression determines
whether or not TGF- β activates

or inhibits ERK1/2, and that T βRII alone is able to
mediate

TGF- β signaling to ERK1/2 without participation of T βRI/Alk5.
RESULTS
TGF- β3 selectively activates ERK1/2 in dermal cells, but inhibits ERK1/2 in
epidermal cells, yet TGF- β3 universally activates Smad2/3
To investigate why TGF- β-stimulated activation of R-Smad-independent

pathways often
depends upon the cell type, we screened the three

major human skin cell types that are
involved in repair and

regeneration: 1) keratinocytes, 2) dermal fibroblasts and 3)
microvascular

endothelial cells, for TGF- β3-stimulated ERK1/2 activation.

The three cell
types were primary cells isolated from human

neonatal foreskins, and all express
physiological levels of T βRI

and T βRII. In quiescent human dermal fibroblasts (DFs)

and
human dermal microvascular endothelial cells (HDMECs), TGF β3

stimulation induced a
dose-dependent phosphorylation of ERK1/2

(Fig. 2-1Aa and c). In human keratinocytes
(HKs), there was a

basal level of ERK1/2 phosphorylation (Fig. 2-1Ae, lane 1). This

was
probably due to previously reported epidermal growth factor

secretion and autocrine
signaling in these cells (Kansra et

al., 2004). However, in contrast to dermal cells, TGF-
9

β3

stimulation induced a transient and dose-dependent decrease

in ERK1/2
phosphorylation in these cells (Fig. 2-1Ae, lanes 2–5).

Consistently, TGF- β3 induced a
time-dependent increase in

ERK1/2 phosphorylation in DFs (Fig. 2-1Ba) and HDMECs
(Fig. 2-1Bc),

whereas it caused a transient decrease in ERK1/2 phosphorylation

in HKs
(Fig. 2-1Be).

We were curious to determine whether selective activation of ERK1/2 by TGF- β3

in
dermal but not epidermal cells also applied to Smad2/3 phosphorylation.

TGF- β3
stimulation universally induced Smad2/3 phosphorylation

in DFs (Fig. 2-1Ca), HDMECs
(Fig. 2-1Cc) and HKs (Fig. 2-1Ce), which

followed a similar kinetics, with maximum
Smad2/3 phosphorylation

between 45 and 90 minutes. These results indicated that the

cell-type-specific effects of TGF- β3 on ERK1/2 phosphorylation

do not apply to TGF- β3-
induced Smad2/3 phosphorylation in

the same cells. Quantitatively, TGF- β1 is the most
abundant

TGF- β isoforms in skin wounds (Bandyopadhyay et al., 2006). By comparison,
we did not detect any significant differences

in ERK1/2 phosphorylation in dermal cells
in response to stimulation

by TGF- β1 or TGF- β3, although more TGF- β1 than TGF- β3

was required (Fig. 2-1D). We further asked whether the inability

of TGF- β3 to activate
ERK1/2 in epidermal cells was due

to an intrinsic defect in the ERK1/2 pathway in these
cells. We compared stimulation

of ERK1/2 phosphorylation by TGF- β3 with that of TGF
(a

major serum growth factor for HK growth). Although TGF- β3

induced a temporal

10

Figure 2-1. Opposite effects of TGF- β3 on ERK1/2 in human dermal versus
epidermal cells. HKs, DFs and HDMECs in cultures were serum-starved overnight and
subjected to (A) dose-dependent (5 minute stimulation) or (B,C) time course (1.0 ng/ml
stimulation) with recombinant human (rh) TGF- β3. Equalized cell lysates (~40 μg/lane)
were subjected to western blotting analysis with anti-phospho-ERK1/2 and anti-ERK1/2
antibody (A,B) or anti-phopho-Smad2 and anti-Smad2/3 antibodies (C). ECL results
were subjected scanning densitometry to measure the ratio (fold) of phospho-ERK1/2
over total ERK1/2. (D) Comparison of 3 ng/ml TGF- β1-stimulated and 1 ng/ml TGF β3-
stimulated kinetics of ERK1/2 phosphorylation. (E) Serum-starved HKs were treated
without or with TGF- β3 (1.0 ng/ml) or TGF- α (200 ng/ml) and subjected to western
blotting analysis analysis, as indicated. (F) The effect of PD901 on TGF- β3- or PDGF-
BB-stimulated ERK1/2 activation. DFs were pre-treated with PD901 (10 μM) for 30
minutes (and continued presence of PD901) before addition of the growth factors.

11

Continued Fig. 2-1

12

Continued Fig. 2-1

13

Continued Fig. 2-1

14

Continued Fig. 2-1

15

decrease in the basal phosphorylation of

ERK1/2 (Fig. 2-1Ea, lanes 2–4 vs. lane 1), TGF
stimulation

(via binding to EGFR) induced a two- to three- fold increase in

ERK1/2
phosphorylation over the basal level (Fig. 2-1Ea, lanes

6–9 vs. lane 1). These results
demonstrate that there is

no intrinsic defect in the ERK1/2 pathway in epidermal cells.

We next investigated whether TßRII directly activates ERK1/2

or acts via MEK1. We
found that PD901, a specific inhibitor

of MEK1, dramatically inhibited both TGF- β3- and
PDGF-BB

(platelet-derived growth factor-BB)-stimulated ERK1/2 phosphorylation

(Fig.
2-1F). These results suggest that the activated T βRII

also acts via the Ras–Raf–MEK1
cascade to activate

ERK1/2.
TßRII expression determines how TGF- β communicates with ERK1/2
To elucidate the molecular basis for differential regulation

of ERK1/2 by TGF-β3 in
dermal versus epidermal cells, we

focused on the expression levels of T βRII and
T βRI/Alk5

subunits – the first TGF- β-interacting proteins involved

in cross-membrane
signaling. Although variable levels of T βRI

expression were found in HKs, DFs and
HDMECs (Fig. 2-2Aa), there

was no correlation between the differences in T βRI levels

(Fig. 2-2Aa, lanes 1, 3 and 4) and the selective activation of

ERK1/2 in dermal, but not
epidermal, cells in response to TGF- β3.

Neural-crest-originated epidermal melanocytes
(MCs) were also

included as a control (Fig. 2-2Aa, lane 2). By contrast, we found

a strong
correlation between T βRII expression levels and

ERK1/2 activation. DFs and HDMECs
exhibited 7- to 18-fold higher

levels of T βRII expression than HKs (Fig. 2-2Bc, lanes 3

16

Figure 2-2. In vitro and in vivo profiles of T βRII–T βRI subunit in four human skin
types. (A, B) Equalized cell lysates of HKs, DFs and HDMECs and melanocytes (MCs),
were subjected to western blotting analysis with antibodies against T βRI/Alk5 (A) or
T βRII (B). (C) Indirect immunofluorescence staining of normal human skin with
antibodies against T βRII from three independent sources, as indicated. Solid yellow line
outlines the basement membrane. Epi, epidermis; Derm, dermis. Scale bar: 20 μm.

17

Continued Fig. 2-2

Epidermal
Epidermal
Dermal
Dermal
A

B
18

Continued Fig. 2-2

C
19

and 4 vs. lane 1). To confirm these results, we subjected sections

of normal human skin to
immunostaining with three anti-T βRII

antibodies against distinct epitopes and from three
independent

commercial sources. All three anti-T βRII antibodies showed

stronger
staining of T βRII in the dermis rather than in the epidermis

(Fig. 2-2Cb, c and d vs. a). By
contrast, anti-T βRI antibody

showed equal staining of both the dermis and epidermis, as
our laboratory has

previously shown (Bandyopadhyay et al., 2006). It should be

noted that,
unlike epidermis that is >90% composed of HKs,

the sparse staining of T βRII in the
dermis reflects the

normal distribution of DFs in the dermis, where scattered DFs

are
embedded in large areas of connective tissue. Therefore,

a given section could only reveal
a few DFs.

We then studied whether the differences in T βRII levels

indeed determine how TGF- β3
regulates ERK1/2 using two approaches:

(1) downregulation of T βRII in DFs and (2)
upregulation

of T βRII in HKs. We used the lentiviral system FG-12 to

deliver shRNA to
knock down T βRII. FG-12 offered a >99%

transduction efficiency in DFs (Fig. 2-3A)
and the shRNA dramatically

downregulated the endogenous T βRII (Fig. 2-3Ca, lane 3),

in comparison to infections with vector alone or vector carrying

a non-specific shRNA
(Fig. 2-3Ca, lanes 1 and 2). As expected,

the shRNA did not affect T βRI/Alk5 in the
same cells (Fig. 2-3Cb). In the control cells, TGF- β3 stimulation induced a

time-
dependent phosphorylation of both Smad2/3 (Fig. 2-3Cd) and

ERK1/2 (Fig. 2-3Ch).
However, in the DFs with downregulated T βRII,

TGF- β3-stimulated phosphorylation of
both Smad2/3 (Fig. 2-3f) and ERK1/2 (Fig. 2-3Cj) was completely blocked. These results

indicate that T βRII is crucial for mediation of the TGF- β3

signaling to both Smad2/3 and
20

Figure 2-3. T βRII expression levels determine how TGF- β regulates ERK1/2. (A)
Lentiviral gene transduction efficiency, indicated by GFP expression, in DFs. (B)
Lentiviral gene transduction efficiency in HKs. (C) Downregulation of endogenous
T βRII (a, lane 3 vs. lanes 1 and 2) but not T βRI (b, lane 3). Equalized lysates of TGF- β3
stimulated DFs were subjected to anti-phospho-Smad2 (d and f), anti-Smad2/3 protein (e
and g) antibodies, anti-phospho-ERK1/2 (phospho-ERK1/2) (h and j) or anti-ERK
protein (ERK1/2) (i and k) antibody immunoblotting analyses. (D) Overexpression of the
wt or DN mutant of T βRII in HKs (a, lanes 3 and 4 vs. lane 2), with DF lysates as a
comparison (lane 1). Western blotting analysis analyses with anti-phospho-Smad2 (c,e,g),
anti-Smad2/3 protein (d,f,h), anti-phospho-ERK1/2 (i,k,m) or anti-ERK1/2 protein (j,l,n)
antibodies.

21

Continued Fig. 2-3

B
A
22

Continued Fig. 2-3

C
23

Continued Fig. 2-3

D
24

ERK1/2 pathways in DFs.
To further address the importance of T βRII levels in ERK1/2

activation, we carried out
the reverse experiments. We reasoned

that if the failure of TGF- β3 to activate ERK1/2 in
HKs

was due to their lower level of T βRII expression, then

raising the T βRII level to that
of DFs should switch the

inhibition of ERK1/2 to activation of ERK1/2 in these cells.

To
overexpress a gene of interest, we used the lentiviral vector,

pRRLsin.MCS-Deco, which
also offered a >97% gene transduction

efficiency in HKs (Fig. 2-3B). We overexpressed
the wild type

(WT) or a kinase-defective (DN) T βRII in HKs from the endogenous

low
level (Fig. 2-3D, lane 2) to a level that was similar to that

in DFs (Fig. 2-3D, lanes 3 and
4 vs. lane 1). We found that, in

the T βRII-WT-overexpressing HKs, TGF- β3 induced a
rapid

and time-dependent ERK1/2 activation (Fig. 2-3Dk), similarly to

DFs. TGF- β3 still
induced a decrease in ERK1/2 phosphorylation

in vector control HKs (Fig. 2-3Di).
Overexpression of the T βRII-DN

mutant inhibited TGF- β3-stimulated phosphorylation of
Smad2/3

(Fig. 2-3Dg), as expected. Furthermore, unlike T βRII-WT,

the T βRII-DN failed
to mediate ERK1/2 activation (Fig. 2-3Dm). Instead, it caused a slight increase in the
basal ERK1/2

phosphorylation and delayed TGF- β3-induced inhibition of

ERK1/2 (Fig.
2-3Dm), which is the exact opposite of the results when

T βRII-WT is overexpressed.
These results therefore indicate that

the T βRII expression levels and kinase activity
determine

whether or not TGF- β3 activates or inhibits ERK1/2.

25

Figure 2-4. T βRI is dispensable for TGF- β signal to ERK1/2. (A) Downregulation of
the endogenous T βRI (b ′, lanes 1 and 3 vs. lane 2), but not T βRII (a ′), by two shRNAs to
knock down T βRI. TGF- β3-stimulated phospho-Smad2 (d ′ and f ′), Smad2 protein (e ′ and
g′), phospho-ERK1/2 (h ′ and j ′) and ERK protein (i ′ and k ′) were analyzed. (B) In HKs,
overexpression of the WT or TD mutant of T βRI over the endogenous T βRI (a ′, lanes 2
and 3 vs. lane 1). Lysates of TGF- β3-stimulated cells were subjected to phospho-Smad2
(c ′,e ′,g ′) or phospho-ERK1/2 blotting analysis (i ′,k′,m ′), as previously described. (B) In
DFs, rhTGF- β3 (1.0 ng/ml, 45 minutes) stimulated, in the absence or presence of various
concentrations of SB431542, phospho-Smad2 (a ′) or phospho-ERK1/2 (b ′). To use the
same membrane for all antibodies, anti-AKT blotting analysis was used. (D) A schematic
representation of the main findings in this study. The question mark indicates an
unknown linker. The ‘n’ in x=n was not defined by a specific number, but low in HKs
and higher in DFs and HDMECs.

26

Continued Fig. 2-4

A
27

Continued Fig. 2-4

B
28

Continued Fig. 2-4

C
D
29

T βRI is not required for TGF- β signaling to ERK1/2
The widely accepted dogma for TGF- β signaling is the sequential

events of TGF- β–
T βRII–T βRI–intracellular

signaling pathways, as previously mentioned. However, we
found

little correlation between T βRI expression levels and the

differential effects of
TGF- β3 on ERK1/2. We questioned,

therefore, whether T βRI had any role in mediating
TGF- β3

signaling to ERK1/2. First, to answer this question in DFs,

we used two
independent approaches: (1) downregulation of endogenous

T βRI to an undetectable level,
which became technically

feasible with the lentiviral RNAi system, FG-12 (Qin et al.,

2003), and (2) inhibition of T βRI kinase. Two independent

shRNAs (RNAi1 and RNAi2)
either dramatically or completely downregulated

the endogenous T βRI/Alk5 (Fig. 2-4Ab',
lanes 1 and 3 vs. lane

2), but did not affect T βRII in the same cells (Fig. 2-4Aa',

lanes 1
and 3 vs. lane 2). We chose to use the RNAi2-infected

DFs, where the T βRI was reduced
to an undetectable level

(Fig. 2-4Ab', lanes 1). As expected, TGF- β3 was no longer

able
to induce any detectable Smad2/3 phosphorylation (Fig. 2-4Af') in these cells, in
comparison with the control cells (Fig. 2-4Ad'). However, the TGF- β3-induced ERK1/2
phosphorylation

remained unchanged in these cells (Fig. 2-4Aj'), in comparison

with the
control cells (Fig. 2-4Ah').
To confirm this surprising

finding, we used a T βRI kinase-specific inhibitor, SB431542
(inhibiting Alk4, Alk5 and Alk7) (DaCosta Byfield et al., 2004). Treatment of DFs with
increasing concentrations of SB431542 led to a complete blockade of TGF- β3-stimulated
Smad2/3

phosphorylation (Fig. 2-4Ca', lanes 3–5 vs. lane 2). However,

SB431542
30

showed little inhibition of TGF- β3-stimulated ERK1/2

activation at any of the
concentrations used in the same cells

(Fig. 2-4Cb', lanes 3–5 vs. lane 2).

Second, to answer the same question in HKs, we took an advantage

of the constitutively
activated T βRI mutant, T βRI-TD,

which would initiate TGF- β signaling in the absence of
upstream

activators. The idea was that, if TGF- β3 signaling to ERK1/2

goes through
T βRI, similarly to Smad2/3, the T βRI-TD

mutant should induce a constitutive activation
of both Smad2/3

and ERK1/2 even in the absence of TGF- β. There was a four-

to six fold
increase in T βRI-WT and T βRI-TD mutant

expression over the endogenous T βRI (Fig. 2-
4Ba', lanes 2

and 3 vs. lane 1). As expected, in T βRI-TD-overexpressing

HKs, Smad2/3
phosphorylation became constitutive even in the

absence of TGF β3 stimulation (Fig. 2-
4Bg', lanes 2–4

vs. lane 1). Therefore, the T βRI-TD mutant worked as expected.

However,
in the same cells, T βRI-TD was unable to either

cause ERK1/2 activation over its basal
level or rescue the TGF- β3-induced

inhibition of ERK1/2 (Fig. 2-4Bm' vs. i' and k'), in
sharp contrast

to the effect of overexpressed WT T βRII (Fig. 2-3B).

In addition, the
negative results with the constitutively activated

T βRI-TD kinase on ERK1/2 also ruled
out any possible involvement

of other Alk isoforms that might be present in HKs.

Taken
together, the above findings provide direct evidence for

T βRI-independent signaling by
T βRII to an R-Smad-independent

pathway, as schematically represented in Fig. 2-4D.

31

DISCUSSION

Since the initial reports that TGF- β activates ERK1/2 in

epithelial cells and breast cancer
cells (Hartsough and Mulder,

1995), ERK1/2 activation has been linked to a number of
TGF- β-regulated

cellular events, including CKIs p21
Cip1
and p27
Kip1
gene expression

and
growth arrest (Hartsough et al., 1996: Frey and Mulder,

1997), the epithelial-
mesenchymal transition (EMT) (Zavadil

et al., 2001) and breast cancer cell motility
(Dumont et al.,

2003). An important question was how the T βRI–T βRII

complex activates
ERK1/2. Lee and colleagues reported that,

following T βRII activation, T βRI in the T βRI–
T βRII

complex recruits and phosphorylates ShcA, an SH2 adapter protein.

Tyrosine-
phosphorylated ShcA in turn recruits the Grb2/Sos complex

that activates the Ras–Raf–
Mek1–ERK1/2 cascade

(Lee et al., 2007). By contrast, Imamichi and co-workers showed

that the T βRI inhibitor SB431542 could not fully block

TGF- β-induced ERK1/2
activation, suggesting that T βRI

is not involved in ERK1/2 activation (Imamichi et al.,
2005).

However, none of these studies specifically addressed the roles

of T βRII and T βRI
in TGF- β signaling to ERK1/2.

Using FG12-mediated complete knockdown of the
endogenous T βRs

and pRRLsin-mediated overexpression of the constitutively activated

T βRI-TD mutant, the current study provides direct evidence

that (1) the differences in
T βRII expression levels determine

ERK1/2 activation or inhibition and (2) T βRII is
crucial

and T βRI/Alk5 is dispensable for TGF- β signaling to

ERK1/2.

This finding appeared to be at odds with a previous notion that

T βRII is constitutively
activated, even in the absence of

TGF- β. If this is true, how can ERK1/2 activation still

32

remain sensitive to TGF- β stimulation? It should be pointed

out that previous studies
were based on exogenously overexpressed

TGF- β receptors and/or in vitro biochemical
kinase assays. Under similar

conditions, even tyrosine kinase receptors such as HER2 in
breast

cancer cells and EGFR in the carcinoma cell line A431 become

constitutively
active. Recently, Chen's group has shown that

T βRII forms dimers

in response to TGF- β
stimulation under physiological expression levels (Zhang et al., 2009). Thus,

the first
likely explanation is that the ‘excess’

of T βRII (which do not have T βRI molecules to
form

heterodimers at the cell surface) in DFs and HDMECs form homodimers

among
themselves. This homo-dimerization leads to the activation

of T βRII but not T βRI, and
activation of ERK1/2. Second,

the excess T βII receptors might form a complex with a
new

gene product. For instance, Qiu and colleagues have recently

reported that T βRII can
form a complex and directly phosphorylate

the parathyroid hormone type I receptor
(PTH1R), which is important

for bone production and absorption (Qiu et al., 2010).

MATERIALS AND METHODS
Cells and antibodies
Primary human neonatal human keratinocytes (HKs), melanocytes

(MCs), dermal
fibroblasts (DFs), and human dermal vascular endothelial

cells (HDMECs) were
purchased from Clonetics (San Diego, CA)

and cultured as previously described
(Bandyopadhyay et al.,

2006). Recombinant human TGF- β1, TGF- β2 and TGF- β3

were
purchased from the R&D Systems (Minneapolis, MN). Anti-ERK1/2 antibody

(03-6600)
33

was from Zymed Laboratories (South San Francisco,

CA). Anti-ERK1/2-P antibody
(V803A) was from Promega (Madison,

WI). The sources of human T βRI and T βRII,
including T βRI-TD,

T βRII-KD cDNAs, antibodies against TβRI/Alk5 and T βRII

(Santa
Cruz, SC-400, Upstate, 06-277 and Cell Signaling, #3713),

anti-Smad2-P (Ser465/467)
antibody, anti-Smad2/3 antibody, anti-Akt

antibody, anti-MEK1 antibody and anti- β actin
antibody

were used as previously described (Bandyopadhyay et al., 2006). Anti-Smad3-P
antibody (#52903) was purchased from Abcam (Cambridge,

MA). PD0325901 (PD901)
was purchased from Calbiochem (San Diego, CA).
Lentiviral gene-overexpressing and gene down-regulating systems
The lentivirus-derived vectors, FG-12 and pRRLsinh-CMV, were used

as described (Qin
et al., 2003; Bandyopadhyay et al.,

2006).

293T cells were plated the day before
transfection. On the day of transfection, 293T cell media were changed with fresh growth
media. DNA and CaCl2 were mixed in water and added dropwise to 2X HBS solution.
After 30 minutes of incubation, the DNA-CaCl2 solution was added to the cells. 24 hours
after transfection, the 293T cells were incubated with fresh growth media containing
10mM Sodium Butyrate for 8 hours. The culture medium was changed again with fresh
growth media. The following day, the culture medium was collected for infecting human
dermal fibroblasts or epidermal keratinocytes. Protein expression of infected genes was
detected and quantified using Western assays.
Staining human skin tissue with anti-T βRII antibodies
The protocols for staining frozen human skin sections with the

indicated antibodies were
34

described previously (Woodley et al., 2004). The tissue specimen on slides was fixed
with acetone for 5 min, washed with PBS, and blocked with 10% normal goat serum for
60 min. The slides were incubated with anti-T βRII antibodies for 2 hr. After three times
of wash with PBS, FITC-conjugated secondary antibody was added onto the tissue area.
The results were analyzed under a microscope (TE-2000U Eclipse; Nikon).

35

CHAPTER 3: MOLECULAR MECHANISM OF
TGF- β FOR THE INHIBITION OF HUMAN
DERMAL CELL MIGRATION

INTRODUCTION

Wound healing is a highly orchestrated cell event, composed of inflammation,
granulation, re-epithelialization, and tissue remodeling (Singer and Clark, 1999). When
normal wound healing fails, it causes ulcers, hypertrophic scars, and keloids. Following
skin injury, platelets form platelet plugs by which many growth factors and cytokines are
released such as platelet-derived growth factor (PDGF), transforming growth factor- β,
and vascular endothelial growth factor (VEGF) (Fig. 3-1). The growth factors and
cytokines are involved in cell migration and proliferation, angiogenesis, extracellular
matrix synthesis, and tissue remodeling. After injury, epidermal cells proliferate and
migrate laterally at the margin of the wound, degrading extracellular matrix and fibrin
clots by producing collagenases such as matrix metalloproteinases (MMPs). Three to four
days after injury, granulation tissue appears, and dermal fibroblasts migrate into the
wound site. Fibroblasts produce new ECM molecules based upon stimulation primarily
by PDGF and TGF- β1. In about two weeks, granulation tissue converts to a scarring, and
the wound contracts. After completion of angiogenesis, ECM deposition, and tissue
remodeling, cells undergo apoptosis due to an unknown mechanism. Complete tissue
remodeling takes up to two years.

36

Figure 3-1. Cutaneous wound healing (A) Three days after injury. Growth factors and
cytokines, fibroblasts, and inflammatory cells are shown. (B) Five days after injury.
(Singer and Clark, 1999).

A
37

Continued Fig. 3-1

B
38

Skin cell migration is a highly coordinated process, which is critical in reepithelialization
during wound healing process. After injury, keratinocytes migrate into the wound site to
form an epithelial barrier, and dermal fibroblasts move in later to deposit ECM molecules
to remodel the tissue. Impairment of epidermal and dermal cell migration can results in
severe pathologic conditions such as chronic non-healing wounds due to the failure of
epidermal repair. For example, the failure of keratinocytes to migrate was found in
chronic venous ulcers (Sivamani et al., 2007, Adair et al., 1977, Andriessen et al., 1995).
Dermal fibroblasts from aged human patients showed impaired motility and wound
healing (Reed M. J. et al., 2001).

Ridley and colleagues reviewed cell migration in detail (Ridley et al, 2003). The multiple
steps of the migration and regulation of proteins are shown in Figure 2. The migration
process is a cyclic event of cell polarization, protrusion, adhesion to the ECM, and rear
detachment and retraction. Maintaining polarization is very important for migrating cells,
and this is mediated by Rho GTPases such as Rac and Cdc42, phospho-inositide 3-
kinases (PI3K), PTEN, integrins, and microtubules. At the leading edge, focal complexes
form by binding integrins to ECM. When Paxillin enters into the focal complex, it
becomes a more stable focal adhesion compared to a newly-formed adhesion (Webb et al.,
2002). In the rear of migrating cells, adhesions generate tension to break integrin-actin
cytoskeleton. Focal adhesions are known to inhibit cell migration (Galbraith and Sheetz,
1998). Over the last decade, there have been increasing studies on microtubule dynamics
and regulating molecules in cell migration; however, it remains unclear as to how cells
39

Figure 3-2. Steps in cell migration. (A) Cdc42, along with Par proteins and aPKC, are
involved in the generation of polarity. Several additional proteins are implicated in
polarity, which results in directed vesicle trafficking toward the leading edge,
organization of microtubules, and the localization of the MTOC and Golgi apparatus in
front of the nucleus. In the presence of a chemotactic agent, PIP
3
is produced at the
leading edge through the localized action of PI3K, which resides at the leading edge, and
PTEN, a PIP
3
phosphatase that resides at the cell margins and rear. PTEN and myosin II
are implicated in restricting protrusions to the cell front. The migration cycle begins with
the formation of a protrusion (B) WASP/WAVE proteins are targets of Rac and Cdc42
and other signaling pathways and regulate the formation of actin branches on existing
actin filaments by their action on the Arp2/3 complex. Actin polymerization, in turn, is
regulated by proteins that control the availability of activated actin monomers (profilin)
and debranching and depolymerizing proteins (ADF/cofilin), as well as capping and
severing proteins. Protrusions are stabilized by the formation of adhesions. This process
requires integrin activation, clustering, and the recruitment of structural and signaling
components to nascent adhesions. Integrins are activated by talin binding and through
PKC-, Rap1-, and PI3K-mediated pathways. Integrin clustering results from binding to
multivalent ligands and is regulated by Rac. At the cell rear, adhesions disassemble as the
rear retracts (C). This process is mediated by several possibly related signaling pathways
that include Src/FAK/ERK, Rho, myosin II, calcium, calcineurin, calpain, and the
40

delivery of components by microtubules. Many of these molecules may also regulate the
disassembly of adhesions at the cell front, behind the leading edge. (Ridley et al, 2003)
Continued Fig. 3-2

41

maintain polarity by those regulators. In addition, the mechanism is unknown by which
pro-motility factors and anti-motility factors control the regulators in order to alter the
migration process.

Major pro-motility or anti-motility factors are growth factors and cytokines. Many
growth factors are involved in epidermal and dermal cell migration, including epidermal
growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), keratinocyte
growth factor (KGF), platelet-derived growth factor (PDGF), transforming growth factor
(TGF)- α, and TGF- β (Singer and Clark, 1999). TGF- β1 is released by granulated
platelets, monocytes, keratinocytes, and fibroblasts (Ashcroft and Roberts, 2000). It was
reported that many signaling pathways are involved in cell migration, but it is unclear
how TGF- β activates different downstream effectors to regulate migration.

In this study, we show that TGF- β3 activates the cAMP-dependent kinase pathway via
TGF- β receptor I/II complexes. Inhibition of PKA abolished the anti-migration effect of
TGF- β3 on PDGF-induced migration. PKA activation by Smads activates gene
expression and phosphorylation of paxillin. Over-expression and phosphorylation of
paxillin disrupted polarization that was induced by PDGF-BB, resulting in the inhibition
of dermal cell migration.

42

RESULTS
T βRI is required for the anti-migratory signal of TGF-β3 on PDGF-induced
migration of human dermal fibroblasts
We previously reported that TGF- β3 inhibits the PDGF-induced migration of human
dermal fibroblasts and that high levels of T βRII expression are required for the anti-
motility effect (Bandyopadhyay et al., 2006). We questioned whether T βRI is required
for the anti-motility effect of TGF- β3 because recent studies showed that T βRII can
activate signal molecules such as PTH receptor and ERK, independent of T βRI (Derynck
and Zhang, 2003: Qiu et al., 2010: Bandyopadhyay et al., 2011). In order to answer the
question, endogenous T βRI levels in DFs were downregulated by lentiviral shRNA
against T βRI without altering the levels of T βRII (Fig. 3-3A). Then, we subjected these
T βRI-null cells to colloidal gold migration assay in the presence of TGF- β3 and PDGF-
BB, along with control cells which were infected with an empty-lentiviral vector. In the
control DFs, we got similar result as we previously reported (Bandyopadhyay et al.,
2006): PDGF-BB stimulated migration around four times more than that of serum-free
(SF) and TGF- β3 blocked the stimulatory effect (Fig. 3-3B). However, when T βRI was
completely depleted in the cells, the anti-motility effect of TGF- β3 on PDGF-induced
migration was completely abolished (Fig. 3-3B). This data signifies that T βRI is required
for the anti-migration signal of TGF- β3.

To confirm that solely T βRI is mediating the anti-migration signal from TGF- β ligand
without diverging to other pathways, we overexpressed constitutively-active T βRI
43

Figure 3-3. T βRI is required for TGF- β-induced inhibition of dermal cell migration.
DFs were infected with different lentivirus carrying shRNA against T βRI (A-B), or
carrying pRRLsin-CMV-T βRI-TD which is a constitutively active mutant (C-E). An
empty vector was used as control lentivirus. (A) 48 h after infection, whole cell lysates
were extracted for checking downregulation of the levels of endogenous T βRI.
Endogenous protein levels of T βRI were almost abolished after shRNA infection
compared to the control while not affecting on the levels of T βRII. (B) T βRI-null DFs
were serum starved overnight and migration assays were performed in the presence of
PDGF-BB and/or TGF- β3.

44

Continued Fig. 3-3

β-actin
Alk5/T βRI
shT βRI Control
A
B
45

in DFs (Fig. 3-4A). As expected, phosphorylation of Smads was saturated in the T βRI-
TD cells in comparison to the control cells (Fig. 3-4B). Control DFs and T βRI-TD DFs
were subjected to a colloidal gold migration assay to see whether the constitutive
activation of T βRI can drive the anti-migration signal in the absence of TGF- β3 ligand
binding or not. Surprisingly, PDGF-BB did not drive cell migration at all in the T βRI-TD
cells (Fig. 3-4C). No migration is due to the fact that the anti-migration signaling is
constitutively on in the T βRI-TD cells, so the initiation of migration was blocked. This
result ruled out the possibility that anti-migration signal of TGF- β3 can be a T βRII-
independent signaling pathway.

TGF- β3 inhibits PDGF-BB-induced migration of human DFs in a Smad-
dependent manner

Since R-Smads are primary targets of activated T βRI, we examined whether Smads are
required in the anti-motility signal of TGF- β3 by down-regulating R-Smad2/3 and co-
Smad4 with the lentiviral system. Endogenous Smad2, Smad3 and Smad4 were
dramatically down-regulated in DFs when the cells were infected with shRNA targeting
each Smad (Fig. 3-5A). A non-specific shRNA (shLacZ) was used as control. Smad-
downregulated DFs were subjected to migration assay in the presence or absence of
PDGF-BB and TGF- β3. Down-regulation of Smad2, Smad3 and Smad4 all resulted in
the abolition of the anti-migration effect of TGF- β3 on the PDGF-BB-induced migration
stimulation (Fig. 3-5B, C).

46

Figure 3-4. Overexpression of T βRI-TD mutant is sufficient to inhibit dermal cell
migration which is driven by PDGF-BB. DFs were infected with different lentivirus or
carrying pRRLsin-CMV-T βRI-TD which is a constitutively active mutant (A-C). (A) 48
h after infection, overexpression of the wild-type and constitutively active T βRI-TD was
confirmed by western blotting analysis with the anti-T βRI antibody. The levels of
overexpressed WT- and T βRI-TD were at least 3-4 times higher than that of control DFs.
(B) DFs infected with vector, or T βRI-TD were serum starved overnight and treated with
TGF- β3 for the indicated time periods. The whole cell lysates were extracted and
subjected to western blotting analysis with anti-Smad3 or anti-Smad3-p antibodies. (C)
DFs, infected with the vector or TβRI-TD, were subjected to migration assay with
PDGF-BB or a combination of PDGF-BB and TGF- β3.

47

Continued Fig. 3-4

T βRI/Alk5
β-actin
Control wt TD
T βRI
p-Smad3
Smad3
p-Smad3
Smad3
Control

TGF β3: 0 5’ 15’ 45’
T βRI-TD

A
B
48

Continued Fig. 3-4

C
49

Interestingly, both Smad2 and Smad3 were required for the anti-migration signal of TGF-
β3. It has been debated what the combination of R-Smads binding to a co-Smad is. Both
Smad2 and Smad3 are receptor-regulated Smads. Two R-Smad molecules bind to one co-
Smad4 forming a trimeric Smad complex which then translocates into the nucleus
(Derynck and Zhang, 2003: Chacko et al., 2004: Feng and Derynck, 2005). Smad2 and
Smad3 were suggested for their distinct functions in the signaling pathway (Kretschmer
et al., 2003). However, it is also believed that Smad2 and Smad3 are interchangeable and
redundant. In addition to the redundancy of their functional roles, Feng and colleagues
reported that TGF- β-induced p15inkB gene expression require all Smad2, Smad3 and
Smad4 functions and that all three Smads interact with the gene promoter (Feng et al.,
2000). Our data also shows that the inhibition of any one of Smad2, Smad3 and Smad4
abolished TGF- β-induced anti-motility effect, suggesting that Smad2, Smad3 and Smad4
might exist in the same complex.

TGF- β3 induces phosphorylation of CREB on Ser133 in DFs, but not in KCs
In our previous report, we showed that the differential expressions of endogenous T βRII
in DFs and KCs resulted in differential responsiveness to the TGF- β3 in terms of anti-
migration (Bandyopadhyay et al., 2006). It seems that there is a threshold in the levels of
T βRII for mediating the anti-migration signaling. In figure 3-3, we demonstrated that
Smad activation via T βRI/ T βRII complex is required for TGF- β3’s anti-motility
signaling. However, in those two skin cell types, the kinetics of phosphorylation of
Smad2/3 by TGF- β3 were similar (Bandyopadhyay et al., 2006). In addition, constitutive
50

Figure 3-5. TGF- β3 inhibits PDGF-BB-induced migration of human DFs in a Smad-
dependent manner (A) DFs were infected with different lentivirus carrying shRNA
against Smad2, Smad3 and Smad4. shLacZ was used as control virus. 48 h after infection,
whole cell lysates were extracted and subjected to western for downregulation of each
Smads. (B) Representative images of the colloidal gold migration assay in (C). (C) Smad-
downregulated DFs were subjected to colloidal gold migration assay with PDGF-BB or
combination of PDGF-BB and TGF- β3.

51

Continued Fig. 3-5

A
52

Continued Fig. 3-5

a
SF
PDGF PDGF+ TGF β
Control
ShSmad2

ShSmad3
ShSmad4

b c
f e d
g h i
l k j
B
53

Continued Fig. 3-5

C
54

activation of T βRI was sufficient to drive anti-migration without activation of T βRII.
Thus, we hypothesized that there should be another signaling mediator involved in the
anti-migration signaling along with Smad signaling pathway. Therefore, we performed a
human phospho-kinase array to see if there were any activated phospho-molecules after
TGF- β3 treatment in DFs, showing a different phosphorylation pattern than HKs. This
array detects relative levels of phosphorylation on approximately 50 phosphorylation
sites of human kinases.

When we determined the phosphorylation status in DFs and HKs, we found that
phosphorylation of ERK, cAMP response element binding protein (CREB), and Src were
upregulated only in DFs (Fig. 3-6A). Phosphorylation of β-catenin was upregulated only
in HKs. Because both ERK and Src are part of migration signaling of PDGF-BB, we
decided to investigate CREB. CREB is a second messenger being activated when it is
phosphorylated on Ser133. We confirmed the human phospho-kinase array result by
Western blotting analysis using anti-phospho-CREB antibody from the same company
(Fig. 3-6B). As expected, phosphorylation of CREB was dramatically induced by TGF-
β3 in DFs, but there was no increase in the level of phosphorylated CREB in HKs. This
raises the possibility that lack of activation of CREB by TGF- β3 in HKs might be
important for the failure of TGF- β’s anti-motility effect.

55

Figure 3-6. TGF- β3 induces phosphorylation of CREB on Ser133 in HDF, but not in
HKC in a Smad-dependent manner (A) DFs and KCs were serum starved overnight
and treated with TGF- β3 for 10 min. The whole cell lysates were extracted and subjected
to human phospho-kinase array based on manufacturer’s instructions. (B) DFs and KCs
were serum starved overnight and treated with TGF- β3 for indicated time periods. After
treatment, western blotting analysis was performed using the whole cell lysates with anti-
phospho-CREB antibody.

56

Continued Fig. 3-6

A
B
HDF
SF
TGF β3
HKC
•phospho-CREB
•phopho-ERK1/2
p-CREB
p38
TGF β3: - + - +
HDF HKC
57

TGF- β3 activates CREB by cyclic AMP dependent kinase (PKA) in Smad-
dependent manner

CREB is phosphorylated in response to cyclic AMP (cAMP) by PKA and also in a
cAMP-independent manner by different kinases, such as Calcium-calmodulin-dependent
kinase II/IV, AKT, PP90, MSK-1, protein kinase B, and protein kinase C (Gonzalez and
Montminy, 1989: Mayr and Montminy, 2001). In 1991, Kramer et al. reported that TGF-
β phosphorylated CREB in ML-CCI64 but the underlying mechanism how TGF- β
activates CREB was unknown (Kramer et al., 1991). It is possible that TGF- β activates a
protein kinase directly through the interaction with TGF- β receptor complexes or through
the cross-talk with Smad complexes.

PKA is the primary kinase for CREB phosphorylation. In order to know whether TGF- β3
phosphorylates CREB via PKA kinase, we checked the phosphorylation of CREB after
treating DFs with TGF- β3 and/or H89. H89 is a PKA-selective small molecule inhibitor
which does not affect cAMP level. H89 itself did not show any toxicity without changing
any cell morphology and it did not affect TGF- β’s Smad phosphorylation (Fig3-7A). H89
completely abolished TGF- β3-induced phosphorylation of CREB in DFs. This result
means that PKA activity is required for the phosphorylation of CREB by TGF- β3. We
further questioned whether the PKA activation by TGF- β3 is Smad-dependent. We
downregulated Smad4 by using shRNA in DFs and subjected these cells to Western
blotting assay to check the phosphorylation status of CREB. As shown in Figure 3-7B,
Smad4-downregulated DFs no longer activated CREB in response to TGF- β3 while DFs

58

Figure 3-7. TGF- β3 activates CREB by cAMP dependent kinase (PKA) in Smad-
dependent manner (A) DFs were serum starved overnight and treated with TGF- β3
combination with H89 for 10 min. Extracted whole cell lysates were subjected to Western
blotting analysis with anti-phospho-CREB and anti-phospho-Smad3. (B) DFs were
infected with lentivirus carrying shRNA against Smad4. shLacZ was used as a control
virus. 48 h after infection, cells were serum starved and treated with TGF- β3 for 10 min.
Extracted whole cell lysates were subjected to Western blotting analysis with anti-
phospho-CREB.

59

Continued Fig. 3-7

A
B
TGF β3:
p-CREB
p-CREB
p-Smad3
p38
p38
60

with control shRNA showed a robust increase in the levels of phospho-CREB. Our data
suggests that TGF- β3 activates PKA pathway by a Smad-dependent manner. In the same
notion, a recent study supports our data that TGF- β activated PKA by direct interaction
between PKA and a Smad3/Smad4 complex (Zhang et al., 2004).

Forskolin inhibits dermal, but not epidermal cell migration in a dose-
dependent manner

TGF- β has many biological functions in DFs, including inhibition of proliferation and
migration, and stimulation of ECM production. Thus, we tested whether the activation of
PKA pathway by TGF- β3 is important for inhibiting dermal cell migration or not. PKA
was artificially activated in DFs by treatment with forskolin, followed by migration assay.
Forskolin is a diterpene which activates adenylate cyclase, resulting in increased levels of
intracellular cAMP which leads to the activation of PKA. DFs and HKs showed the
phosphorylation of CREB after treatment with 10uM of forskolin (Fig.3-8A). This result
confirms that PKA pathway can be activated in both cell types by forskolin. DFs and
HKs were subjected to a migration assay with PDGF-BB or TGF- α in combination with
forskolin to see whether or not forskolin affects cell migration induced by the motility
factors. In DFs, increasing amounts of forskolin correlated with decreased migration
indexes in a dose-dependent manner (Fig. 3-8B, C). However, in HKs, forskolin
treatment failed to block TGF- α-induced migration although forskolin treatment
abolished TGF- α-induced migration in DFs (Fig. 3-8D, E). This data implies that the
anti-migration effect of TGF- β3 was not growth factor-specific. The reason why forskolin

61

Figure 3-8. Forskolin inhibits dermal cell migration in a dose-dependent manner (A)
DFs and KCs were serum starved overnight and treated with 10µM forskolin for 30 min.
Whole cell lysates were extracted and subjected to western blotting analysis with anti-
phospho-CREB. (B) Representative images of the colloidal gold migration assay in (C).
(C) DFs were serum starved overnight and subjected to migration assay in the absence or
presence of PDGF-BB and PDGF-BB plus increasing amount of forskolin as indicated.
(D) Representative images of the colloidal gold migration assay in (E). (E) DFs and KCs
were serum starved overnight and subjected to migration assay in the absence or presence
of TGF- α, TGF- β3, and forskolin. 10 ng/ml of TGF- α was used.

62

Continued Fig.3-8

A
63

Continued Fig.3-8

SF PDGF PDGF+TGF β
PDGF+
forskolin 0.12 μM
PDGF+
forskolin 0.37 μM
PDGF+
forskolin 1.1 μM
PDGF+
forskolin 3.3 μM
PDGF+
forskolin 10 μM

a b
h
g
f e
d c
B
64

Continued Fig. 3-8
C
65

Continued Fig.3-8

e
a
SF TGF α+ TGF β TGF α+ forskolin
HK
DF

h
g
f e
D
TGF α
66

Continued Fig.3-8

E
67

failed to block migration in HKs is due to the fact that increased cAMPs affect on the
keratinocyte migration. That is supported by the previous report that exogenous addition
of dibutyryl cAMP (DBcAMP) significantly induced the migration of human
keratinocytes at the concentrations between 10
-5
µM and 10
-6
µM (Iwasaki et al., 1994).

Inactivation of PKA pathway abolishes anti-motility effect by TGF- β3
We showed the activation of PKA pathway either by TGF- β3 or forskolin only blocks
dermal cell migration, not in epidermal keratinocytes above. We confirmed that the
activation of PKA is required for anti-migration signaling by inhibiting PKA in the
presence of TGF- β3 in DFs. Dermal cell migration was analyzed in the presence or
absence of PDGF-BB, TGF- β3 and H89. H89 is a cell-permeable PKA-selective inhibitor
(Fig. 3-9A). Inhibition of PKA by H89 resulted in the loss of the anti-motility effect of
TGF- β3 (Fig. 3-9A, B). This data provides direct evidence that the activation of PKA
pathway by TGF- β3 is required for the anti-motility effect.

TGF- β3 does not affect on migration signaling induced by PDGF-BB
We questioned whether activated PKA by TGF- β3 targets the migration signaling
pathway of PDGF-BB. DFs were serum starved overnight and treated with PDGF-BB in
combination with TGF- β3 and forskolin for 10 minutes. PDGF-BB was shown to
stimulate migration by activating ERK1/2, p38, AKT, FAK, Src, paxillin and so on (Li et
al., 2004: Patsenker et al., 2007). We subjected the treated DFs to Western blotting assay
to check the levels of phosphorylated ERK1/2, AKT, p38, and paxillin. Neither TGF-
68

Figure 3-9. Inhibition of PKA abolished TGF- β3’s anti-motility effect. (A)
Representative images of the colloidal gold migration assay in (B). (B) DFs were serum
starved and subjected to migration assay in the absence or presence of PDGF, TGF- β3
and 1 μM H89. H89 is a selective small molecule inhibitor of PKA.

SF PDGF+ TGF β PDGF+ TGF β
+H89
PDGF

a b d c
A
B
69

β3 nor forskolin had an affect on PDGF-BB-induced phosphorylation status of ERK1/2,
AKT, p38, and paxillin (Fig. 3-10A). This result implies that the anti-motility effects of
TGF- β3 and forskolin on migration are not by blocking the signaling transduction,
induced by PDGF-BB. This data also suggests that the activation of PKA by TGF- β3 and
forskolin might affect gene expressions for migration inhibition.

Surprisingly, the basal level of phosphorylated paxillin was high under serum-free
condition in DFs and the phospho-paxillin level was dramatically decreased after PDGF-
BB treatment (Fig. 3-10A). This result was interesting because PDGF-BB was known to
activate the phosphorylation of paxillin in order to form focal complexes at the leading
edge in migrating cells (Abedi and Zachary, 1997). On the other hand, by a different
research group, it was reported that PDGF-BB disrupts the phosphorylation of paxillin at
a high concentration (30 ng/ml), but not at low concentrations (1-5 ng/ml) in Swiss 3T3
cells (Rankin and Rosengurt, 1994). Therefore, we treated DFs and Swiss 3T3 cells with
increasing concentrations of PDGF-BB for 10 min and investigated the phosphorylation
levels of paxillin using two different antibodies detecting the phosphorylation at Tyrosine
31 and Tyrosine 118. In DFs, the levels of phospho-paxillin were dramatically decreased
in a dose dependent manner in respond to PDGF-BB (Fig. 3-10B). However, in Swiss
3T3 cells, the phosphorylation of paxillin was robustly increased by PDGF-BB in a dose
dependent manner (Fig. 3-10B). This data implies that PDGF-BB might reorganizes focal
adhesion molecules differently in various cells upon the onset of pro-motility stimulation.

70

Figure 3-10. Effect of TGF- β3 on the phosphorylation of paxillin which are induced
by PDGF-BB (A) DFs were serum starved overnight and treated in combination with
PDGF-BB, TGF- β3 and forskolin for 10 min. The whole lysates were extracted and
subjected to western blotting analysis with anti-phospho-ERK, anti-phospho-AKT, anti-
phospho-Paxillin, anti-Paxillin, anti-phospho-p38, and anti-p38 antibodies. (B) DFs and
Swiss 3T3 cells were serum starved overnight and treated with different dosage of
PDGF-BB as indicated for 10 min. The whole lysates were extracted and subjected to
western blotting analysis with anti-phospho-paxillin and anti-paxillin antibodies.

71

Continued Fig. 3-10

p-ERK
p-AKT
p-paxillin (T118)
p-p38
paxillin
PDGF:
TGF β:
-
+
-
- -
-
+ +
+
+
-
-
p-paxillin (T31)
p38
Forskolin:
A
72

Continued Fig. 3-10

Swiss 3T3:
PDGF (ng/ml)
-
5 1 15 30 50
p-paxillin (T31)
p38
p-paxillin (T118)
paxillin
PDGF (ng/ml)
p-paxillin (T31)
p38
HDF:
-
5 1 15 30 50
p-paxillin (T118)
paxillin
B
73

TGF- β3 induces overexpression of paxillin via PKA pathway
Activation of PKA results in various end products such as gene expression. We
speculated the PKA activation by TGF- β3 might change gene expression profile since
TGF- β3 did not affect on the migration signaling pathways of PDGF-BB (Fig. 3-11A).
Thus, we identified gene expression profiles that are known to be involved in cell
migration by quantitative reverse transcription PCR (RT-PCR): TGF- β3 upregulated the
transcription of PTEN, actin, and paxillin, but downregulated the transcription of ROCK2
and KRT4 (Keratin 4) (Fig. 3-11A). There was no effect on the gene transcriptions of Fli-
I (flightless I homolog), ITGA2 (Integrin, α 2), IGFBP1 (insulin-like growth factor
binding protein 1), CSCL12 (chemokine ligand 12), PKIB (protein kinase inhibitor β I),
and MGP (matrix Gla protein) (data not shown).

Among these up- or downregulated genes by TGF- β3 treatment, only paxillin
transcription was significantly abolished by H89 co-treatment (Fig. 3-11A). This means
that paxillin overexpression by TGF- β3 depends on the PKA function. We speculated
whether the upregulation of paxillin changes the phosphorylation levels of paxillin which
indicate the amount of focal adhesions. Thus, we detected the levels of phospho-paxillin
and total paxillin by Western blotting assay using dermal cell lysates collected 24 hours
after the treatment with PDGF and/or TGF- β3 in DFs (Fig. 3-11B). As we expected, the
levels of total paxillin were upregulated by TGF- β3 treatment. The phosphorylation level
of paxillin was maintained low after 24 hours in the presence of PDGF-BB. Surprisingly,
treatment of dermal cells with TGF- β3 alone resulted in the remarkable increase of
74

Figure 3-11. TGF-β3 induces overexpression of paxillin. (A) DFs were serum starved
overnight, and total mRNA was isolated 8 h after treatment with TGF- β3 alone or
combination with H89, followed by reverse transcription. Quantitative PCR was
performed with primers against PTEN, ROCK2, KRT4, paxillin and Actin. Relative
expression levels were normalized to GAPDH mRNA levels. (B) DFs were serum
starved overnight and treated with PDGF-BB and/or TGF- β3 for 24 h. The whole cell
lysates were extracted and subjected to western blotting analysis with anti-FAK, anti-
phospho-paxillin, anti-paxillin, or anti-p38 antibodies. (C) DFs and KCs were serum
starved overnight and treated with PDGF-BB or TGF- α in the combination with TGF- β3
and forskolin for 24 h. The whole cell lysates were extracted and subjected to western
blotting analysis with anti-phospho-paxillin, anti-paxillin, or anti-p38 antibodies.

75

Continued Fig. 3-11
A
76

Continued Fig. 3-11

TGF β:
FAK
paxillin
p38
PDGF:
-
+
-
+
-
+ +
-
p-paxillin (T118)
p-paxillin (T31)
B
77

Continued Fig. 3-11

TGF β:
Forskolin:
-
- -
- - +
-
+ + +
+
-
p-paxillin (T118)
p38
paxillin

p-paxillin (T31)
PDGF:
TGF β:
Forskolin:
-
- -
- - +
TGF α:
-
+ + +
+
-
p-paxillin (T118)
p38
paxillin
p-paxillin (T31)
C
HDF:
HKC:
78

Figure 3-12. Overexpression of paxillin by TGF- β3 results in the disruption of
polarization induced by PDGF-BB in migrating cells. DFs were grown on coverslips
for 24 hrs and followed by serum starvation overnight. Subsequently, DFs were treated
with PDGF-BB and/or TGF- β3 for 24hr. After fixation, cells were stained with anti-
phospho-paxillin (Tyr118) antibody overnight. Arrows indicate membrane ruffles.

a b c
SF PDGF PDGF+ TGF β
79

phosphorylation levels of paxillin. Increased phospho-paxillin is likely due to the increase
of total paxillin. Surprisingly, TGF- β3 maintained the levels of phospho-paxillin high in
the presence of PDGF-BB. Forskolin treatment showed the same results as TGF- β3 in
DFs (Fig. 3-11C). In keratinocytes, TGF- β3 did not increase the levels of phospho-
paxillin and total paxillin (Fig. 3-11C). This data suggests the possible mechanism of
TGF- β3 by which migration is inhibited by overexpressing paxillin.

Overexpression of paxillin by TGF- β3 results in the disruption of polarization
induced by PDGF-BB in migrating cells.

The hallmark of cell migration is the presence of membrane ruffling. The first step of a
migration cycle is the polarization of focal complexes along with actin and microtubule
rearrangement. In order to know how the upregulations of phospho- and total paxillin
affect on the polarization of paxillin, DFs were immunostained with phospho-paxillin
antibody to visualize the polarization pattern. Under serum-free condition, phospho-
paxillin was distributed evenly inside of the cells indicating no migration (Fig. 3-12a).
When cells were stimulated by PDGF-BB overnight, phospho-paxillin was localized near
membrane ruffles at the leading edge (Fig. 3-12b). However, TGF- β3 increased the
intensity of the phospho-paxillin staining with even distribution inside the cell that
represents disrupted polarization of paxillin (Fig. 3-12c). This data means that the
overexpression of paxillin by TGF- β3 is the key mechanism of inhibiting cell migration
by which cell polarization is disrupted. This data also signifies that maintaining
polarization of paxillin by PDGF-BB is critical for cell migration.

80

Figure 3-13. Regulation of paxillin expression by TGF- β3.

Smad3
Smad2
Smad4
PKA
P
CREB
CREB
P
P
paxillin
paxillin
paxillin
paxillin
TGF β
Focal
adhesion
Gene expression
P
81

DISCUSSION
TGF- β plays multiple roles in the steps of wound healing in almost all skin cells. One of
the important roles of TGF- β3 is regulating cell migration. Most of the studies about
TGF- β in skin cell migration were conducted in keratinocytes. However, the importance
of its role in modulating migration of dermal fibroblasts has been underestimated
although DFs play a critical role in reepithelialization in wound healing. After injury,
dermal fibroblasts migrate into a wound site 3-4 days after keratinocyte migration. The
sequential migration of keratinocytes and dermal fibroblasts is important for ensuring the
completion of epithelial barrier by keratinocytes. TGF- β3 controls the sequential
migrations by selectively inhibiting the migration of dermal fibroblasts (Bandyopadhyay
et al., 2006).

In the chapter three, we showed that the anti-migration effect by TGF- β3 is R-Smad-
dependent (Fig.3-4). As expected, the downregulation of T βRI abolished Smad-
dependent TGF- β3 signaling that leads to anti-migration in dermal cells (Fig.3-3).
Interestingly, even though co-Smad4 was not completely down-regulated by lenti viral
shRNA, the anti-migration effect induced by TGF- β3 was completely abolished.
Furthermore, the downregulation of co-Smad4 also completely blocked the activation of
PKA/CREB pathway, which is required for downstream gene regulation in anti-migration.
This signifies the role of co-Smad4 for anti-migration.

PKA pathway has been known to play an important role in cell migration. It was reported
82

that the PKA pathway is involved in an integrin-mediated migration signal. Either
overactivation or inactivation of the PKA pathway can alter normal cell migration.
Although the individual roles of PKA and TGF- β in regulating cell migration were
reported, it has not been studied whether the cross-talk between TGF- β and PKA
pathways plays a role in migration or anti-migration. However, the effects of TGF- β on
PKA pathways were reported in other studies. In early 1990s, Kramer and colleagues
showed that the phosphorylation of CREB was induced by TGF- β1 without changing
cAMP levels (Kramer et al., 1991). They suggested that CREB protein binds to a TGF- β
responsive element (TRE) in a different manner of its CRE binding. However, it was not
clear how TGF- β1 activates CREB. About a decade later, Zhang and colleagues reported
that TGF- β activates PKA pathway through direct interaction between PKA kinase and
Smad3/Smad4 complexes (Zhang et al., 2004). Here we show that Smad4 is required in
the activation of PKA pathway by TGF- β3 (Fig. 3-6).

Regulation of cell migration is very complex in which many regulatory molecules are
involved. Paxillin is one of cytoskeletal proteins regulating the assembly of focal
adhesion and its turnover. The alteration of paxillin levels results in either promotion or
inhibition of migration in different cells (Petit et al., 2000: Schaller and Schaefer, 2001:
Panetti, 2002: Yano et al., 2000). Reorganization of focal complexes and actin
cytoskeleton is an initial step of migration. However, the initiation and maintenance of
cell polarization by a motility factor are unclear. Interestingly, we showed here that
PDGF-BB reduced the levels of phospho-paxillin in order to initiate and maintain
83

migration (Fig. 3-9). The rapid reduction of phospho-paxillin by PDGF-BB within 5
minutes indicates the possible involvement of phosphatases. For example, protein
tyrosine phosphatase-PEST (PTP-PEST) was reported to disassemble focal adhesions
through targeting p130CAS (Turner C.E, 2000).

TGF- β3 induced overexpression of paxillin as well as increase of its phosphorylation,
overriding PDGF’s effect on paxillin mentioned above (Fig. 3-10B). We hypothesized
that the increase of paxillin by TGF- β3 led to the incorporation of the excess paxillin into
focal adhesions (Fig. 3-10B, C). Thereby, increased focal adhesions resulted in
disbalance in organized paxillin, leading to the disruption of cell polarity (Fig. 3-11). It is
possible that increased paxillin levels cause a stronger attachment to actin cytoskeletons
that further ensure inhibition of migration. In the same notion, it was reported that TGF-
β1 up-regulated paxillin in malignant astrocytomas, enhancing cytoskeletal attachment
(Han et al., 2001). Our hypothesis is further supported by data from Tian and Phillips
(Tian and Phillips, 2003). They reported that addition of TGF- β1 to HK-2 cells decreased
migration rate by increase in the levels of total paxillin and vinculin that is another focal
adhesion molecule. They also showed that there was increased assembly with actin
cytoskeletons as well as increased β-integrin expressions. However, it was unclear what
the mechanism of TGF- β is in inducing overexpression of paxillin and vinculin.

TGF- β3 also regulated expressions of other genes such as PTEN, ROCK2, KLT4, and
actin in a PKA-independent manner (Fig. 3-10). It is also possible that TGF- β3 regulates
84

those genes simultaneously with paxillin in order to inhibit migration or the genes are
involved in other cell events which are regulated by TGF- β. PTEN is known as a tumor
suppressor but it can modulate migration in various cells (Raftopoulou et al., 2004).
Overexpression of PTEN inhibited cell migration by reducing focal adhesions and
altering actin cytoskeleton (Tamura et al., 1998). ROCK2 is a Rho-associated, coiled-coil
containing protein kinase. Overexpression of ROCK2 enhanced cell motility and knock-
down of ROCK2 reduced cell migration in hepatocellular carcinoma cells (Wong et al.,
2009).

To date, PDGF-BB is the only FDA-approved wound healing agent, but the PDGF-BB
treatment is not efficient. Exogenous TGF- β treatment of chronic wounds was also not
successful. PDGF-BB is a secreted growth factor upon injury which derives cell
migration of DFs and stimulates cells to proliferate. On the other hand, TGF- β3 inhibits
cell migration of DFs, overriding PDGF-BB’s pro-motility effect (Qui et al., 2004:
Bandyopadhyay et al., 2006). Thus, understanding the mechanism of TGF- β in wound
healing is important in order to get high efficacy in wound treatment with PDGF-BB. We
hypothesized previously that TGF- β inhibits PDGF-BB-induced cell migration in
different cell context. Here this thesis suggests that TGF- β inhibits cell migration by
regulating the levels of paxillin which result in altering focal adhesion and disrupting
polarization via Smad/PKA pathway (Fig. 3-13).

85

MATERIALS AND METHODS
Antibodies and reagents
Recombinant humanTGF- β3 and

Platelet-derived Growth Factor (PDGF)-BB were
purchased from the R&D Systems (Minneapolis, MN). H89, dihydrochloride was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Forskolin was purchased
from Sigma-Aldrich (Saint Louis, MO). Anti-phospho-CREB and anti-Smad4 antibodies
were from the R&D Systems (Minneapolis, MN). Anti-ERK1/2 antibody

was from
Zymed Laboratories (South San Francisco,

CA). Anti-phospho-ERK1/2 antibody was
from Promega (Madison,

WI). Anti-T βRI/Alk5, anti-phospho-AKT, anti-phospho-p38,
anti-phospho-paxillin, anti-Smad2, and anti-Smad3 antibodies were from cell signaling
(Danvers, MA). Anti-paxillin antibody was from BD Biosciences (San Jose, CA). Anti-
T βRII antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-
Smad3 antibody was from Abcam (Cambridge,

MA).
Primary cell culture
Primary human neonatal keratinocytes (HKs) and dermal fibroblasts (DFs) were
purchased from Clonetics (San Diego, CA).

HKs were cultured in EpiLife medium with
supplemented HKGS (Cascade Biologics, Protland, Oregon) and DFs were maintained in
DMEM with 10% FBS.

86

Lentiviral shRNA-mediated gene down-regulation
The lentivirus-derived vector, FG-12, was used

as previously described (Qin et al., 2003;
Bandyopadhyay et al.,

2006). The sense oligonucleotide shRNA sequence for T βRI,
Smad2, Smad3, Smad4, and PKA catalytic α subunits were as follows: Smad2, 5’-
GGACTGAGTACACCAAATA-3’ and 5’-GCAGAACTATCTCCTACTA-3’, Smad3,
5’-GCCTGGTCAAGAAACTCAA-3’ and 5’-GGTGCTCCATCTCCTACTA-3’, Smad4,
5’-GGTGGAGAGAGTGAAACAT-3’ and 5’-GAATCCATATCACTACGAA-3’. PKA
C α, 5’-GGGTCTCCATCAATGAGAA-3’ and 5’-CCATCCAGATCTATGAGAA-3’.
Colloidal gold migration assay
The colloidal gold migration assay was previously described by Albrecht-Buehler (1997)
and modified by Woodley et al. (1988) and Li et al. (2004). Coverslips were coated with
1% bovine serum albumin (BSA). Gold solution was prepared by mixing 9% gold salt
with 30% Na2CO3. After boiling the gold solution, 0.1% formaldehyde was added and
aliquoted onto BSA-coated coverslips in 8-well plates. After rinsing with HBSS, the
coverslips were incubated in 40ug/ml of type I Collagen for 2 h. Cells were trypsinized,
seeded onto the coverslips, and incubated overnight. The following day, medium was
removed and coverslips were fixed with 0.1% formaldehyde for 10 min. Migration was
examined under dark field optics and photographed. Twenty fields were randomly
selected and analyzed with an attached CCD camera (Model KP-MIU, Hitachi-Denshi)
and a computer using the NIH Image 1.6 program. Migration indexes represent the
percentage of individual cell migration area over the total field area viewed by
microscope that is covered by cell migration tracks.
87

Human phospho-kinase array
Subconfluent DFs and HKs were serum-starved overnight, and treated with TGF- β3 for
10 min. Cells were lysed using 1X10
7
cells/ml of lysis buffer, supplied by the
manufacturer, at 4°C for 30 min. 250µg of the whole cell lysates were applied to the
Proteome Profiler Human Phospho-Kinase Array Kit (Catalog # ARY003: R&D Systems,
Minneapolis, MN) according to the manufacturer’s instructions. Signal was detected to
film by applying ECL (Amersham, Piscataway, NS).
Real-time quantitative reverse transcription-PCR
Real-time quantitative reverse transcription-PCR analysis was performed according to
previously described protocols (Jeong et al., 2009). Total RNA was isolated from DFs
with Trizol (Invitrogen) after treatment with TGF- β3 alone or combination with H89 as
indicated, and subjected to reverse transcription by the iScript cDNA synthesis kit
(BioRad). 2 μl of 10-fold diluted reverse transcription product was used for quantitative
PCR analysis with the following primers; β-actin, 5’-
ACCCCATCGAGCACGGCATCG-3’ (forward) and 5’-
GTCACCGGAGTCCATCACGATG-3’ (reverse) ; GAPDH, 5’-
TCTGGTAAAGTGGATATTGTTG-3’ (forward) and 5’-GATGGTGATGGGATTTCC-
3’ (reverse) ; PTEN, 5’-CGAACTGGTGTAATGATATGT-3’ (forward) and 5’-
CATGAACTTGTCTTCCCGT-3’ (reverse) ; ROCK2, 5’-
TCCCCCATCAACGTGGAGAGCT-3’ (forward) and 5’-
TGCCTTGTGACGAACCAACTGCA-3’ (reverse) ; KRT4, 5’-
CTCCAGCAAAAACCTTGAGC-3’ (forward) and 5’-AAGTCATTCTCGGCTGCTGT-
88

3’ (reverse) ; paxillin, 5’-TCTTCGAGCGGGATGGAC-3’ (forward) and 5’-
GCCGGATGAGGAACTCACAT-3’ (reverse). Relative expression levels were
normalized to GAPDH mRNA levels.

89

CHAPTER 4: CONCLUSION
TGF- β is one of the most studied cytokines, and it affects almost all cell events in
physiological conditions such as differentiation, proliferation, apoptosis, and migration.
TGF- β also involves in pathologic conditions such as autoimmune diseases,
neurodegeneration, fibrosis, tumorigenesis and abnormal wound healing. Increasing
studies about the molecular mechanism of TGF- β have been reported, but the mechanism
remains unclear because of its contradictory roles in a wide variety of cell events. The
contradictory role of the TGF- β pathways is due to the diversity and complexity of the
signaling pathway. Diversity and complexity come from differential expression and the
combinatorial T βRI/ T βRII receptor complex formation on cell surface. Intracellulary, a
variety of Smads and differential modulation of its activation give further specificity and
selectivity to the TGF- β signaling in different cell events. Involvement of the non-Smad
pathway has been focused on recently, especially cross-talking between the Smad
pathway and non-Smad pathways.
Non-Smad signaling pathways could be the independent T βRII signaling from T βRI. The
importance of T βRII in TGF- β signaling has been reported more than a decade ago.
T βRII has a different affinity towards three TGF- β isoforms during mammalian
development, suggesting that T βRII plays a role in modulating TGF- β signaling (Lawler
et al., 1994). However, there was no direct evidence that T βRII can regulate the TGF- β
signaling pathway in the absence of T βRI. Here we show two important aspects of T βRII.
First, expression levels of T βRII determine the sensitivity to TGF- β signaling pathway.
90

The strength of T βRII determined ERK activation, but not Smad activation (Fig. 2-1).
This might be because canonical Smad activation requires a lower threshold of activated
T βRII. Furthermore, we previously reported that the differential levels of T βRII
determines anti-migration signal, but not anti-proliferation signal (Bandyopadhay et al.,
2006). This explains how TGF- β selectively activates a signaling pathway in the same
cells, and how it has differential effects in various cells.
The second important notion is that T βRII can activate non-Smad signaling molecules in
the absence of T βRI. Knock-down of endogenous T βRI did not affect ERK activation by
TGF- β3 in the presence of functional endogenous T βRII in vitro (Fig. 2-4). This
represents a novel mechanism of independent signaling of T βRII. It has been believed
that heterodimerization between T βRI and T βRII is required for all TGF- β signaling
pathways. The independent role of T βRII is more supported by T βRI knockout mice
studies. T βRI knockout mice showed prenatal lethality with severe defects including
abnormal angiogenesis. However, surprisingly, hematopoietic potential was normal in the
TGF- β1 knockout mice (Larsson et al., 2001). In addition, Imamich and colleagues
reported that a T βRI chemical inhibitor did not affect ERK activation by TGF- β
(Imamich et al., 2005).
Recently, cross-talk between TGF- β signaling pathways and other signaling pathways
also has been focused. Cross-talk between different signaling pathways generates
diversity and complexity. It has been reported that R-Smads and Smad4 can interact with
other kinases. R-Smads and Smad4 have phosphorylation sites that can be targeted by
91

MAPK, JNK, ERK, PKC, and CamKII kinases (Derynck and Zhang, 2003).
Phosphorylation in the linker region of R-Smads by ERK modulates the activity of Smad,
subsequently inhibiting TGF- β-induced anti-proliferation effect (Kretzschmar et al., 1997,
1999). As well as being the target of different kinases, Smads can activate different
kinases. In this thesis, it was shown that Smads can activate PKA kinase in response to
TGF- β3 (Fig. 3-6).
The activation of the PKA pathway by TGF- β was reported about two decades ago, but
Zhang and colleagues first suggested the actual mechanism only recently (Zhang et al.,
2004). Zhang and colleagues reported that TGF- β inhibits cell proliferation by direct
binding of Smad4 to the PKA that leads to the activation of the PKA. Our data also
clearly shows that PKA activation by TGF- β3 is Smad-dependent. Furthermore, we show
that the activation of PKA by TGF- β3 is critical for the inhibition of dermal cell
migration. However, this cross-talk between TGF- β and PKA pathways depends on cell
context. In epidermal keratinocytes, we did not detect CREB activation after TGF- β3
treatment although phospho-CREB was dramatically induced by TGF- β3 in dermal
fibroblasts. This explains how TGF- β modulates migration differently in various cells as
an inhibitor or as a stimulator. These differential effects of TGF- β are very important,
because of the sequential migration of keratinocytes and dermal fibroblasts. During
reepithelialization, the most important role of keratinocytes is to migrate and generate an
epithelial barrier. In the mean time, dermal fibroblasts halt migration about 3-4 days after
injury.
92

We show here that TGF- β requires gene expression via cross-talk with the PKA pathway
in order to regulate focal adhesion. Migration is a cyclic process in which focal adhesions
assemble and disassemble along with the rearrangement of actin cytoskeleton. Migration
can be inhibited by the impairment of migration dynamics. Although the molecular
mechanism of anti-motility is unclear, it was suggested that the stabilization of focal
adhesions and impaired disassembly by impeding adhesion turnover can result in
migration inhibition. Adhesion turnover is modulated by molecular regulators such as
paxillin, focal adhesion kinase (FAK), Src, and tyrosine phosphatases. The inhibition of
the regulators decreases motility (Lawson and Maxfield, 1995: Klinghoffer et al., 1999).
Our data supports that aberrant overexpression and increased phosphorylation of paxillin
by TGF- β3 might impair the turnover of focal adhesion, leading to disrupted cell polarity
and decreased cell motility. However, it still needs to be solved the involvement of CREB
as transcription activator for the paxillin overexpression or PKA is interacting with other
transcription activators. If CREB is involved as a transcription factor, it needs to be
further investigated whether CREB is transactivating paxillin gene by interacting with the
TGF- β response element (TRE) or the cAMP response element (CRE).
Human recombinant TGF- β3 is already in a clinical trial under the name of Avotermin
(Occleston et al., 2009). In addition to migration, TGF- β plays multiple roles in cell
events such as proliferation, angiogenesis, and ECM production in wound healing. Thus,
it is unclear whether the TGF- β treatment itself will or will not benefit the wound healing
process, because TGF- β has been reported as positive and negative regulators of wound
healing. We showed that TGF- β3 negatively regulated dermal cell migration. Therefore,
93

in theory, the addition of exogenous TGF- β3 might have a negative effect on wound
healing if dermal cells stop migration. PDGF-BB is the only FDA-approved wound
healing reagent. PDGF-BB stimulates dermal cells for migration and ECM deposition.
The treatment of wounds with PDGF-BB was not successful. This could be because
TGF- β3 in serum has an inhibitory effect on PDGF-BB for dermal cell migration.
Theoretically, the inhibition of TGF- β3 can help accelerate wound healing and increase
the efficacy of PDGF-BB in terms of cell migration. In the same notion, Smad3-null mice
showed reduced inflammation and accelerated reepithelialization (Ashcroft et al., 1999).
However, wound healing process is a very complex cell event involving migration,
proliferation, gene regulation, angiogenesis, and apoptosis. TGF- β has an anti-
proliferation effect on both dermal and epidermal cells. Inhibition of keratinocyte
proliferation by TGF- β delays the wound healing process (Zamburuno et al., 1995). In
contrast, reduction of fibroblast causes less ECM deposition, and thereby accelerates re-
epithelialization and scar maturation (Occleston et al., 2009). Reduced inflammation and
ECM deposition correlate with accelerated wound healing. Excess deposition of ECM
with fibroblast proliferation is the hallmark of abnormal wound healing such as keloids
and hypertrophic scars (Singer and Clark, 1999). Although there are opposite reports
about positive and negative roles of TGF- β, Furguson and colleagues suggested the
differential effect of three different isoforms, TGF- β1, TGF- β2 and TGF- β3. Increased
TGF- β1 stimulates ECM deposition that correlates with fibrogenesis, but exogenous
TGF- β3 reduces ECM deposition (Occleston et al., 2009). In the same notion, the
94

neutralization of TGF- β1 or TGF- β2 with anti-TGF- β antibodies led to less inflammation,
less ECM deposition, and reduced scarring (Shah et al., 1995).
Because of the opposite effects of TGF- β in various steps of wound healing, it is
important to understand the underlying mechanism of normal wound healing in order to
apply TGF- β3 to wound healing treatment. More extensive pre-clinical and clinical
studies need to be done, as well as further research for an overall understanding about the
molecular mechanism of TGF- β pathways.
Revealing the mechanism of TGF- β3 in cell migration, proliferation, and angiogenesis
can be applied to cancer progression/invasion in skin. TGF- β plays a role as a tumor
suppressor in normal tissues by maintaining homeostasis and preventing tumorigenesis.
However, TGF- β can have opposite roles in the later stage of tumorigenesis as a tumor
promoter (Wakefield et al., 2002: Akhurst et al., 2001: Laverty et al., 2009). TGF- β3 is
the most abundant in human epidermis, compared to TGF- β1 and TGF- β2 (Schimid et al.,
1993). Expression of TGF- β3 correlates with keratinocyte tumor progression and
melanoma carcinogenesis (Gold et al., 2000: Van Belle et al., 1996). However, the
causative role of TGF- β3 in the tumor progression was unclear (Rodeck et al., 1999).
In this study, we revealed new mechanisms of non-Smad signaling pathway from T βRII
to ERK independent of T βRI in Chapter 2. This gives direct evidence of independent
signaling of T βRII that breaks the central dogma that TGF- β transmits a signal to T βRII-
T βRI complex. In Chapter 3, we revealed the molecular mechanism of anti-motility
signaling by TGF- β3 in dermal fibroblasts. TGF- β3 modulates cell migration through
95

cross-talk with PKA and thereby leads to overexpression of paxillin. The current study
provides the molecular mechanisms for regulation of TGF- β3 in cell events in skin
wound healing.

96

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Abstract (if available)

Abstract TGF-beta is a secreted cytokine, which plays an important role in cell development, differentiation, and homeostasis in both physiologic and pathologic conditions, such as tumorigenesis, abnormal wound healing and skin cancer. TGF-beta transmit signal from the extracellular environment to intracellular signaling networks via its cell surface receptor complex, the TGF-beta type II/ I receptor (TbetaRII/TbetaRI) heterodimer. TGF-beta ligand binds to TbetaRII, which in turn recruits and activates TbetaRI, resulting in activation of downstream signaling complex, receptor Smads (R-Smads) and common Smad4. TGF-beta stimulation is also known to activate R-Smad-independent signaling pathways, such as the extracellular signal-regulated kinase (ERK1/2) pathway. However, two long-standing questions remained: 1) why TGF-beta activates ERK depends on the cell context and 2) whether or not TbetaRII is able to mediate the TGF-beta signaling without the participation of TbetaRI. In the chapter two, it is shown that TGF-beta activates ERK in human dermal fibroblasts (DFs) and inhibits ERK in human keratinocytes (HKs). While the TbetaRI expression remains similar in both cell types, the expression level of TbetaRII in DFs is more than seven fold higher than that in HKs. Down-regulation of TbetaRII in DFs blocked TGF-beta-stimulated ERK activation. In contrast, up-regulation of TbetaRII in HKs to the similar level in DFs resulted in activation of ERK rather than inhibition by TGF-beta. Most intriguingly, the TbetaRII-mediated TGF-beta-stimulated ERK activation or inactivation in these cells did not require any participation of TbetaRI. Thus, this study illustrates that the vii expression levels of TbetaRII determine how TGF-beta regulates ERK in various cell types and provides direct evidence for the TbetaRI-independent signaling by TbetaRII. ❧ In wound healing, TGF-beta controls cell proliferation and migration. In the chapter three, the mechanism of TGF-beta's anti-motility was revealed. Anti-migration is one of the primary effects of TGF-beta on non-transformed cell types. We previously reported that TGF-beta3 inhibits PDGF-BB-induced dermal cell migration during wound healing. However, it was not clear what the underlying mechanism was. In this study, it is shown that TGF-beta3 activates PKA pathway through R-Smads/Smad4 complex. Activation of PKA led to over-expression and phosphorylation of paxillin, a focal adhesion molecule. As a result, polarization by PDGF-bb was disrupted and migration rate was decreased. This study elucidates the new mechanism of TGF-beta3 for inhibition of cell migration.

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Asset Metadata

Creator Han, Arum (author)

Core Title Molecular mechanism of transforming growth factor-beta signaling in skin wound healing

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 11/24/2011

Defense Date 06/27/2011

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag gene expression,migration,OAI-PMH Harvest,signal transduction,skin,TGF-beta,wound healing

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Landolph, Joseph R., Jr. (committee chair), Kobielak, Agnieszka (committee member), Li, Wei (committee member), Stiles, Bangyan L. (committee member)

Creator Email arumhan@usc.edu,arumhn@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-212271

Unique identifier UC11291557

Identifier usctheses-c3-212271 (legacy record id)

Legacy Identifier etd-HanArum-435-1.pdf

Dmrecord 212271

Document Type Dissertation

Rights Han, Arum

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA