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TGF-β signaling regulates gingival epithelial wound healing and barrier function

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TGF-β signaling regulates gingival epithelial wound healing and barrier function

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TGF-β Signaling Regulates
Gingival Epithelial Wound Healing
and Barrier Function

By
Nicha Ungvijanpunya
May 2018

A Thesis Presented to the
Faculty of the USC Graduate School
University of Southern California
In Partial Fulfillment of the
Requirements for the Degree
Master of Science
(Craniofacial Biology)
2
Acknowledgements
I would like to give special thanks to Dr. Jian Xu for kindly helping me throughout this project
with invaluable guidance and Dr. Stephen Yen for his mentorship.
In addition, I would like to thank Lam Vuong for providing useful data, Tingwei Zhang for
providing mouse tissues, and everyone in Xu’s Lab at CCMB for helping me with experiments.
I would like to thank Dr. Sameshima for his suggestions about statistical analysis.
Finally, I would like to thank my family for all the support and encouragement.

3
Tables of Contents
I. Abstract………………………………………………………………………………..4
II. Introduction
1. Periodontitis……………………………………………………………………….5
2. The biology of gingival epithelium………………………………………………..7
3. Transforming growth factor (TGF-β) signaling………………………………….16
III. Materials and Methods
1. Mouse experiment model of ligature-induced periodontitis……………………..20
2. Cell culture for Western blotting and scratch assay……………..……………….21
3. Tissue preparation and frozen sectioning………………………………………..21
4. Immunostaining………………………………………………………………….22
5. In vitro wound-healing (scratch) assay…………………………………………..23
6. Western blotting………………………………………………………………….24
7. Statistical analysis………………………………………………………………..27
IV. Results
1. TGF-β signaling is activated in gingival epithelium of the diseased animal, but is
impaired at the injury site…………………………………………………….28-30
2. TGF-β promotes gingival epithelial cell wound healing…………………28, 31-40
3. TGF-β is involved in maintaining tissue integrity………………………..28, 41-46
V. Discussion……………………………………………………………………………47
VI. Conclusion and future direction……………………………………………………...52
VII. References……………………………………………………………………………53

4
I. Abstract
The transforming growth factor-β (TGF-β) signaling pathway is involved in the
regulation of cellular growth and differentiation and has multiple roles during craniofacial
development and injury repair. The aim of this study is to assess whether TGF-β signaling is an
underlying mechanism for impaired gingival epithelial tissue repair by examining its role in the
promotion of wound healing and maintenance of epithelial junction integrity.
We used a mouse model of ligature insertion-induced periodontitis and immunostaining
with phospho-Smad3 to measure TGF-β-induced intracellular signaling activity in the vicinity of
the wound site. To assess the role of TGF-β in gingival epithelial wound healing and barrier
function, we incubated human gingival epithelial cell (HGEC) with TGF-β for 48 hours to assess
cell motility using wound healing assay and examine epithelial junction protein expression using
Western blot analysis.
We found that during ligature-induced periodontitis, TGF-β signaling is activated in
gingival epithelium of the diseased animal, but is impaired at the injury site, as determined by the
absence of phospho-Smad3 staining, an indicator of TGF-β signaling activity. Wound healing
assay showed significant higher percentages of wound closure in TGF-β-treated group but lower
in SB431542-treated group. Western blot analysis showed a significant increase of claudin-1 in
TGF-β-treated group but decrease in SB431542-treated group. For ZO-1 detection, there was no
significant difference between control and treatment groups. We concluded that TGF-β promoted
junctional integrity and wound healing in gingival epithelial tissue.

5
II. Introduction
1. Periodontitis
Periodontitis is a chronic inflammatory disease, which occurs when there is an imbalance
of the host immune status, often triggered by microbial dysbiosis, and leads to periodontal tissue
breakdown. Prevalence of periodontitis in the United States was found to be 46% in adults.
Periodontitis can be found at any age of life. However, the prevalence of periodontitis increases
with age (Eke et al, 2015).
Periodontitis has been associated with several systemic diseases including cardiovascular
disease, diabetes, respiratory disease, etc. Periodontitis can also lead to other problems which
decrease patients’ quality of life including bad breath, tooth loss, reduction in masticatory
efficacy, reduced esthetics, decreased self-esteem and decreased social activities (Nagpal et al,
2015). As aging population starts to increase, people especially health care professionals are
more aware of this problem and try to find the way to help understand the disease, improve the
treatment or even prevent the disease from happening.

6

Adapted from Mediators of Inflammation (Nagpal et al, 2015)
Figure 1 Periodontitis is related to multiple systemic diseases/conditions.

Under normal conditions, gingival epithelium serves as a first barrier to defend against
pathogens and toxins. Once there is an immune imbalance, the integrity of the gingival
epithelium will be disturbed. This would enable the invasion of microorganisms and toxins to
induce or sustain inflammation and alter the wound healing response, which initiates the disease
process of periodontitis (Cekici et al, 2014).

Periodontitis
Cardiovascular
disease
Diabetes
Respiratory
disease
Bacteremia
Adverse
pregnancy
outcomes
Nonalcoholic
liver diseases
Rheumatoid
arthitis
Pancreatic/Oral
cancers
7
2. The biology of gingival epithelium
Both chemical and mechanical barrier are involved in this protection. In terms of
chemical barrier, junctional epithelium produce or allow protective substances such as cytokines,
enzymes, antimicrobial peptides and immune cells to translocate from connective tissue to
gingival sulcus and prevent pathogenic microorganisms from invasion (Bartold et al, 2000;
Nakamura, 2017).
For mechanical barrier, epithelial junctions play an important role to help maintain
gingival epithelial integrity. These epithelial junctions comprise of tight junctions, adherens
junctions, and desmosomes (Bosshardt & Lang, 2005; France & Turner, 2017; Schneeberger &
Lynch, 2004).

Adapted from Nature Reviews Molecular Cell Biology (Tsukita et al, 2001)
Figure 2 Epithelial junctions comprise of tight junctions, adherens junctions, and desmosomes.

8
2.1 Tight Junction
One of the important intracellular structures that maintain epithelial barrier integrity and
homeostasis is tight junction. It seals the adjacent cells together and has mechanisms which
allow only specific particles to travel between cells, being a selectively permeable barrier which
depends on charge and size of the particles (Anderson & Van Itallie, 2009). Tight junctions are
mainly composed of transmembrane proteins such as claudins, occludin, and junctional adhesion
molecule (JAM) with cytosolic proteins such as zonula occludens (ZO) (Fanning et al, 1999;
Giepmans & van Ijzendoorn, 2009; Howe et al, 2005).
Claudins are 20-27 kDa in size. They form two extracellular loops with cytosolic C- and
N- terminals. These two loops varies in composition among different types of claudins
(Schneeberger & Lynch, 2004). Claudin-1 was found to be highly expressed in barrier
epithelium of the airway (Coyne et al, 2002), intestine (Saeedi et al, 2015), and gingiva (Fujita et
al, 2010). It was also found to be essential for tight junctional integrity of intestinal epithelial
cells (Saeedi et al, 2015)
Interaction of claudin-1 with ZO-1 may be essential for claudin-1 as a component of tight
junction (Schneeberger & Lynch, 2004). There are 3 ZO proteins (ZO-1, -2, and -3) which are
involved in tight junction (Schneeberger & Lynch, 2004). ZO-1 was the first one that was found.
It is a dominant ZO in tight junction with 225 kDa in size. ZO-1 was found to play important role
in leak pathway of the tight junction and control adherens junction which is important for
formation of tight junction (France & Turner, 2017; Schneeberger & Lynch, 2004). ZO-1 was
also found to be important in regulating tight junction in epithelial cells(Shen, 2012).

9
Occludin is another tetraspan protein forming two extracellular loops with both C- and N-
terminals in the cytosol similar to claudin. Occludin is 60 kDa in size (Schneeberger & Lynch,
2004). It also interacts with ZO-1 (Mitic & Anderson, 1998). There was a study with occludin
knockout mice. However, no significant change in epithelial barrier was observed. Its function is
still not clearly explained (Anderson & Van Itallie, 2009).
Junctional adhesion molecule (JAM) is a member of immunoglobulin (Ig) superfamily
with about 40 kDa in size. JAM was found to be associated with transportation of immune cells
(Chiba et al, 2008; Fanning et al, 1999; Schneeberger & Lynch, 2004; Tsukita et al, 2001).

Adapted from Biochimica et Biophysica Acta (Chiba et al, 2008)
Figure 3 Main components of tight junction are claudins, occludin, JAMs, and cytosolic proteins
such as ZO-1.

10

Adapted from Journal of the American Society of Nephrology (Fanning et al, 1999)
Figure 4 Structures of transmembrane proteins of tight junction were shown: occludin, claudin,
and JAM. Occludin has similar structure as claudin but is bigger.

2.2 Adherens Junctions
Adherens junctions (zonula adherens) locate slightly below tight junctions. They are
found to be important for the stability of tight junctions. E-Cadherin is the main component of
adherens junction (France & Turner, 2017). Decrease in E-Cadherin in epithelial tissue was
found to be detrimental for tissue integrity (Fujita et al, 2010).

11

Adapted from International Review of Cell and Molecular Biology (Ivanov & Naydenov, 2013)
Figure 5 Adherens junctions comprise of multiple proteins with E-Cadherin as a main
component.

2.3 Desmosomes
Desmosomes (macula adherens) locate at the most inferior area of epithelial tissues
among the three types of cell junctions. They are important as stations for intermediate filaments
in order to form tensile strength. Cadherin proteins are also important components in desmosome
(Alberts B, 2002; Garrod & Chidgey, 2008)

12

Adapted from Biochimica et Biophysica Acta (Garrod & Chidgey, 2008)
Figure 6 Main components of desmosomes were shown. Deficiency in each protein leads to
different diseases.

13
2.4 Special features of gingival epithelium
Gingival epithelium comprises 3 types of epithelium: oral epithelium, sulcular
epithelium, and junctional epithelium (Bartold et al, 2000). Oral epithelium is a keratinized
epithelium with four layers of cells. Rete ridges define separation of oral epithelium and
underlying connective tissue layer. Sulcular epithelium, unlike oral epithelium, is a non-
keratinized epithelium. It lines the gingival sulcus and is continuous with junctional epithelium.
Junctional epithelium is a very unique tissue. It is a non-keratinized epithelium that is attached to
the tooth surface. Compared to oral epithelium and sulcular epithelium, junctional epithelium has
wider intercellular space and lower differentiation (Jiang et al, 2014; Schroeder & Listgarten,
2003). It is more permeable than the oral or sulcular epithelium, and therefore serves as the
preferred route of passage for bacterial products and fluid. It was found to be an interesting tissue
that may play an important role as a first line barrier in periodontal disease (Shimono et al,
2003). Accumulation of numerous protective agents and cells was found in this tissue (Bartold et
al, 2000). It becomes more interesting structure for the researcher nowadays. There were several
studies claiming that tight junction was not found in human junctional epithelium (Hatakeyama
et al, 2006). However, claudin-1 which is one of 3 components of tight junction was highly
expressed in rat junctional epithelium and localizes to the cell-cell boundary. Further study is
therefore needed to determine the formation and function of tight junctions in oral and junctional
epithelium (Damek-Poprawa et al, 2013; Fujita et al, 2010; Fujita et al, 2017).
Oral epithelium is a keratinized tissue. Therefore, it has keratinized layer as a first barrier
from bacterial and toxin invasion. However, sulcular and junctional epithelium do not have
keratinized layer as oral epithelium does, epithelial junctions therefore play important role in
protective mechanism in these non-keratinized tissues.
14

Adapated from Journal of Periodontology (Bartold et al, 2000)
Figure 7 There are 3 types of gingival epithelium: oral epithelium, sulcular epithelium, and
junctional epithelium.

Adapted from Journal of Electron Microscopy (Tokyo) (Shimono et al, 2003)
Figure 8 Three components of gingival epithelium under light microscope were shown: oral
epithelium (OE), sulcular epithelium (SE), and junctional epithelium (JE) (D= Dentin, E =
Enamel)
15

Adapted from Journal of Periodontology (Bartold et al, 2000)
Figure 9 H&E staining showing 4 layers of oral epithelium with low-power view (A) and high-
power view (B): stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS), and
stratum basale (SB)

Adapted from BMC Oral Health (Jiang et al, 2014)
Figure 10 (A) showing oral epithelium (OGE), sulcular epithelium (SE), and junctional
epithelium (JE). The arrows in (B) indicated a boundary between junctional epithelium and
sulcular epithelium. Junctional epithelium and sulcular epithelium are different in cell shape and
the size of intercellular space.
16
3. Transforming growth factor (TGF-β) signaling
Since gingival tissues are constantly challenged by microbes that are normally present in
the oral cavity, they secrete a variety of anti-inflammatory cytokines to maintain tissue
homeostasis, one of which is the TGF-β family of cytokines. TGF-β proteins act as disulfide-
linked dimers and activate cellular responses through binding to heteromeric cell surface receptor
complexes (Xin-Hua Feng, 2016). There are two types of TGF-β receptors, types I and II, both
with serine/threonine kinase activity. Type II receptors are considered constitutively active.
When ligands bind to cell surface type I and type II receptors, the complex induce the
phosphorylation and activation of type I receptors. Activated type I receptors then phosphorylate
downstream cytoplasmic transcription factors called receptor-activated Smads (R-Smads) with
help from the protein Smad anchor for receptor activation (SARA) (Gou et al, 2015). Smad1,
Smad5, and Smad8 are activated by BMP receptors, while Smad2 and Smad3 are directly
activated by TGF-β receptors (Massague & Wotton, 2000). Type I receptor-mediated
phosphorylation induces R-Smads to accumulate in the nucleus and control transcription (Shi &
Massague, 2003).
Increase or decrease in Smad signaling corresponds with alterations in TGF-β signaling.
TGF-β signaling regulates several processes such as cell proliferation, cell differentiation,
morphogenesis, tissue homeostasis, and regeneration. Alteration or disruption in TGF-β
pathways, therefore, leads to abnormal functions in the aforementioned processes and diseases
(Massague, 2012). One of its primary functions is to drive scar formation and attenuate the
inflammation that accompanies wound healing (Kumar, 2005).
17

Adapted from Journal of Periodontology (Schroeder & Listgarten, 1997)

Figure 11 A schematic diagram showing TGF-β signaling from cell membrane to the nucleus.
Orange arrows indicate ligand and receptor activation. Gray arrows indicate Smad and receptor
inactivation. Green arrows indicate Smad activation and formation of a transcriptional complex.
Blue arrows indicate Smad nucleocytoplasmic shuttling.

18
SB431542 is a TGF-β receptor kinase inhibitor. It was proved to be a selective (does not
have a direct effect on ERK, JNK, or p38 MAP kinase pathways) and potent TGF-β inhibitor.
SB431542 blocks TGF-β receptor kinase-induced Smad2/3 activation, and through this
mechanism, inhibits the downstream complex formation between Smad2/3 and Smad4, prevents
translocation of Smad2 and Smad3 to the nucleus, and inhibits transcriptional responses.
Therefore, this inhibitor is a useful tool to help understand the TGF-β signaling process (Halder
et al, 2005; Inman et al, 2002).

Numerous studies support the role of TGF-β in promoting tissue integrity. For example,
TGF-β induced signaling enhanced epithelial barrier function (Howe et al, 2005). TGF-β also
restored intercellular junction strength of intestinal epithelial cells after exposure to the toxin that
disrupted the epithelial junctions (Roche et al, 2000). TGF-β has been documented to regulate
tight junction (Capaldo & Nusrat, 2009). In endothelial cells, TGF-β-induced phosphorylation of
Smad2 and Smad3 led to the production of tight junction protein such as ZO-1 (Jo et al, 2013). In
renal epithelial cells (MDCK-2), TGF-β treatment significantly increased the expression of
claudin-1 and ZO-2 (Feldman et al, 2007). ZO-1 expression level was found to be increased in
TGF-β-stimulated A549 lung cancer cells via Smad-dependent pathway and contributed to
enhanced motility (Lee et al, 2015).

However, several studies showed the opposite results. For example, TGF-β was found to
disrupt ZO-1 pattern in tight junction in mouse mammary epithelial cells (Woo et al, 1996). An
increase in TGF-β correlated with a decrease in ZO-1 and claudin-1 in intestinal epithelial layers
of weaned pigs (Xiao et al, 2014). In human renal proximal tubular epithelial cells, they found
that TGF-β downregulated occludin and ZO-1 expression (Zhang et al, 2013). A study in
19
epithelial tracheal cells found that TGF-β reduced cell-cell contacts by affecting adherens
junction without affecting tight junction since ZO-1 expression level did not change(Boland et al,
1996).

Tight junctions locate at the most superficial layer of the gingival epithelium, it is
therefore considered essential for mechanical barrier. ZO-1 and claudin-1 are key components of
the tight junctions and highly expressed in gingival epithelium. Therefore, we focused on
changes in these 2 proteins with modulation of TGF-β. We aim to assess whether repression of
TGF-β signaling is an underlying mechanism for impaired gingival epithelial tissue repair by
examining its role in the promotion of wound healing and maintenance of tissue integrity.

20
III. Materials and Methods
1. Mouse experiment model of ligature-induced periodontitis
To induce periodontitis in mouse, 5-0 silk ligature was used to allow bacterial
accumulation that induces periodontitis (Graves et al, 2008). Silk ligature was placed between
maxillary left first and second molar and secured in place by knots tied on both ends. This
ligature-inserted side served as the experimental (suture) side. The contralateral side without
ligature insertion served as control. A group of mice with no ligature insertion on either side was
also included as a negative control group. After 10 days of ligature placement, mice were
sacrificed. Maxillae were harvested and prepared for immunostaining.
Adapted from Journal of Dental Research (Zhang et al, 2018)
Figure 12 A silk ligature was placed between maxillary first and second molar to induce
periodontitis (A). Micro CT showed significant bone loss as a sign of periodontitis on suture side
(C) more than contralateral side without ligature insertion (B). A group of mice with no ligature
insertion was also included in the experiment (D,E).
21
2. Cell culture for Western Blotting and scratch Assay
Human primary gingival epithelial cells (HGEC) were purchased from Cell Biologics
Inc. (Catalog No. H-6202). Cells were plated on 10-cm plate in epithelial cell medium (basal
medium containing essential and non-essential amino acids, vitamins, organic and inorganic
compounds, hormones, growth factors, and trace minerals) with supplement (Catalog No.
M6621-kit, containing Insulin-Transferrin-Selenium (ITS), epithelial growth factor (EGF), L-
Glutamine, antibiotic-antimycotic solution, and fetal bovine serum (FBS)).

3. Tissue preparation and frozen sectioning
Maxilla were harvested and fixed in 4% paraformaldehyde (PFA) in PBS overnight, then
they were decalcified in 10% Ethylenediaminetetraacetic acid (EDTA) in PBS on a shaker at
37°C. At least 1 month later, the tissues were washed with 1XPBS overnight, then they were
perfused in 30% sucrose solution in 1XPBS overnight, 60% sucrose solution in 1XPBS
overnight, and finally half 60% sucrose solution in 1XPBS half OCT compound (Tissue-Tek,
Catalog No. 25608-930) overnight. After perfusion, the tissues were embedded in OCT on dry
ice. Tissues were oriented so that the buccal side of molars were parallel to the base of the block.
The samples were kept in -80°C freezer for at least 1 hour before sectioning. The maxilla
samples were longitudinally sectioned (all molars were presented in each section) with a cryostat
5µm in thickness. The tissue sections were transferred to positive-charged slides (Denville
Scientific Inc., Catalog No. M1021). The slides were dried on the warm plate overnight and then
kept in -80°C freezer before staining.

22
4. Immunostaining
Tissue samples in frozen sections were prepared from negative control group (mice with
no ligature insertion on either sides), the control contralateral side, and the experimental ligature-
inserted side.
Tissue slides were washed in 1XPBS for 10 – 15 minutes. ImmEdge Hydrophobic Barrier
PAP Pen (Vector Laboratoties, Catalog No. H-4000) was used to draw the border for each
section. Tissues were rinsed briefly with 1XPBS 3 times. Afterwards, tissues were incubated
with 0.5% Triton X-100 in PBS for 15-20 minutes. Tissues were rinsed with 1XPBS 3 times with
5 minutes each before being incubated with blocking buffer, which was 10% goat serum (Life
Technology) for 1 hour. After that, the blocking buffer was suctioned from all the samples
except the negative control. All tissues except the negative control group were then incubated
with phospho-Smad3 Rabbit mAb (Cell Signaling, #9520) as a primary antibody at concentration
of 1:100 in 10% goat serum overnight in 4°C fridge. The next morning, all tissue sections were
washed with 1XPBS 2-3 minutes per time for 3 times before being incubated with fluoro-
conjugated secondary antibody (anti-rabbit, Life Technologies) at concentration of 1:300 and
DAPI at concentration of 1:1000 (Sigma Aldrich) for counterstaining for 1 hour. Then the tissues
were washed with 1XPBS briefly once followed by two more washes of 5 minutes each. 1XPBS
was suctioned from the tissue and 1 drop of ProLong Gold Antifade Mountant (Thermo Fisher
Scientific, Catalog No. P36930) was added on the tissues before covering with cover slips. The
cover slips were sealed to the slides with clear nail polish lining all the edges.
Photos were taken with confocal microscopy at 20X and 60X to compare phospho-
Smad3 signaling in gingival epithelium between experimental and control groups.

23
5. In Vitro Wound-Healing (Scratch) Assay
To assess the role of TGF-β in gingival epithelial wound healing response, HGEC were
used as an in vitro model. The cells were split into 6-cm plates and incubated with 2 ng/ml TGF-
β (Humanzyme), or 5 µM of SB431542 (Sigma Aldrich), or no intervention for 24 hours.
Afterwards, the 6-well tissue culture plate was prepared by drawing 4 squares at the bottom of
each well with a Sharpie. The cells were seeded into 24-well tissue culture plate at a density that
after 24-hour of growth, they should reach approximately 70-80% confluence as a monolayer.
Afterwards, TGF-β, or SB431542, or nothing was added to each plate. A line was scraped
through the cells with a 200 µl pipette tip to create a cell-free area on the confluent cell layer
across the center of the well. The long-axial of the tip was perpendicular to the bottom of the
well. The resulting gap distance therefore equaled to the outer diameter of the end of the pipette
tip. After scratching, the wells were washed with 1XPBS to remove the detached cells. The wells
were replenished with fresh epithelial cell medium and were added with TGF-β, or SB431542, or
nothing. The speed of wound closure as an indicator of cell migration was quantified by taking
snapshot photos at specific landmarks under the phase contrast light microscope at time intervals
(30 minutes – for T0, 6 hours, 12 hours, and 24 hours) following the scratch. The gap distance
was quantitatively evaluated using the ImageJ software. To reduce variability in results, we
documented multiple views of each well and repeated multiple times for each experimental
group.

24
The percentages of wound closure were calculated from each time point (6h, 12h, and
24h) by using wound closure % formula (Figure 13)

Adapted from Journal of Biomolecular Screening (Yue et al, 2010)
Figure 13 The formula that is used to calculate percentages of wound closure was shown above.

6. Western Blotting
After cells were divided in 3 plates, we wait until 30-40% of area surface of the plate was
covered by cells. Afterwards, 1 plate was used as a control group, another one was treated with
0.4 µl of TGF-β and the other one was treated with 10 µl of SB431542. All 3 plates were
incubated for another 48 hours and the cells were harvested.

Cell Harvest
Plates were washed 1 time with 1XPBS. Cells were lysed in 200 µl lysis buffer (150 mM
NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5, 50 mM NaF) on ice. A scraper was used to scrape
cells while the plate was tilted to let the cells accumulate at one edge of the plate. The cells in

𝑊𝑜𝑢𝑛𝑑 𝐶𝑙𝑜𝑠𝑢𝑟𝑒 (%) = 0
𝑊
1234
−𝑊
12∆4
𝑊
1234
7 × 100%

𝑊
1234
is the gap distance of the wound measured 30
minutes after scratching
𝑊
12∆4
is the gap distance of the wound measured h
hours after the scratch is performed

25
lysis buffer were transferred to 1.5 ml microcentrifuge tubes. The tubes were rotated on rotator
for 15 minutes at 4°C. Afterwards, the tubes were centrifuged at 13,500 rpm for 10 minutes at
4°C. The supernatants were transferred to new 1.5 ml microcentrifuge tubes and the pellets at the
bottom of the tubes were discarded.

Determination of Protein Concentration
Protein concentration was determined by Bio-Rad Protein Assay Dye Reagent
Concentrate (Bio-Rad, Catalog No. 5000006). 120 µl of the reagent was mixed with 480 µl of
deionized water and put in each well of 9 wells in 48-well plate. 6 dilutions of BSA (Sigma
Aldrich were prepared in the first 6 wells (0, 0.125, 0.25, 0.5, 1, and 2 µg/µl). 2 µl of each
sample was added to the other 3 wells and mixed thoroughly. Optical Density (OD) values of
solution in 9 wells were measured. Standard curve of protein assay was obtained from the OD
values of BSA dilutions. The protein concentration of each samples was determined from this
standard curve.

Western Blotting
After protein concentration of samples was determined, 25 µg of each samples was added
into the running gel. Bio-Rad Precision Plus Protein Dual Color Standards (Bio-Rad, Catalog
No. 1610374) was added for a ladder. The gel was ran at 85V for 30 minutes and changed to
100V until the lowest bands almost reached the bottom of the gel. The proteins were transferred
to a membrane at 100V at 4°C for 3 hours. Afterwards, the membrane was washed with 1X Tris-
Buffered Saline (TBS) (50mM Tris-Cl, pH 7.6; 150 mM NaCl) for 5 minutes and cut as needed
for different primary antibody blocking. Membranes were blocked in 10 ml of 5% milk in TBST
26
(Tris-Buffered Saline, 0.1% Tween 20) for 1 hour on rotator at room temperature. Afterwards,
membranes were washed with TBST 3 times (5 minutes each). Membranes were then incubated
with primary antibody in 3% BSA and 0.1% Sodiam Azide at 4°C on rotator overnight. Primary
antibodies were anti-ZO-1 (Cell Signaling, Catalog No. D7D12) at concentration of 1:1,000,
anti-Claudin-1 (Cell Signaling, Catalog No. 4933) at concentration of 1:1,000, and anti-Tubulin
(Santa Cruz, sc-8035) as a loading control at concentration of 1:5,000. The next morning,
membranes were washed 3 times with 5 minutes each with TBST. Afterwards, membranes were
incubated with 10 ml of 5% milk solution with secondary antibody that corresponded to each
primary antibody for 1 hour. Secondary antibodies are Anti-Rabbit H+L (Jackson
ImmunoResearch, Catalog No.111-035-003) for ZO-1 and Claudin-1 at concentration of
1:10,000 and Anti-Mouse H+L (Jackson ImmunoResearch, Catalog No.115-035-003) for
Tubulin at concentration of 1:10,000. After blocking with secondary antibodies, membranes
were washed 3 times with 5 minutes each with TBST before protein detection.
Proteins were detected with Amersham ECL Western Blotting Detection Reagent (GE
Healthcare). The images were developed on HyBlot CL Autoradiography films (Denville
Scientific Inc., Catalog No. E3018) in the dark room. The protein bands were quantified with
NIH- ImageJ software.

Quantification with ImageJ
The photos of protein bands were imported to ImageJ. The photos were then converted to
8-bit. The background was subtracted and changed to light background. The photos’ colors were
inverted. Integrated density of each protein bands was measured by surrounding the margin of
each band with selection tools.
27
7. Statistical analysis
In Vitro Wound-Healing (Scratch) Assay
The data (wound closure %) from TGF-β-treated and SB431542-treated groups were
compare with control group using appropriate statistical analysis. Shapiro-Wilk test was used to
check normality of the data. Kruskal-Wallis test was used to compare among 3 groups at each
time point since the data were not normal distribution. The significant level was p ≤ 0.05

Western blotting
Quantification of protein bands of claudin-1 and ZO-1 from TGF-β-treated and
SB431542-treated groups were compared with control group. Average measurements from a set
of the biological data was used. Shapiro-Wilk test was used to check normality of the data. If the
data had normal distribution, independent T-test would be used. If the data did not have normal
distribution, Mann-Whitney test would be used. The significant level was p ≤ 0.05

28
IV. Results
1. TGF-β signaling is activated in gingival epithelium of the diseased animal, but is
impaired at the injury site
Immunostaining showed phospho-Smad3 staining in the cytosol of both control and
experimental group. When compared with the control group, experimental group at the
contralateral (no suture) side showed an increase in phospho-Smad3 signal. At the suture-treated
side, however, there was a significant decrease in phospho-Smad3 signal (Figure 14 and 15).

2. TGF-β promotes gingival epithelial cell wound healing
The TGF-β-treated group showed significant acceleration in wound closure of HGECs
when compared with the control group at 6h, but not at 12h and 24h when the wound was almost
sealed, while the use of a TGF-β signaling inhibitor (SB431542) significant decreased
percentages of wound closure at 6h and 12h but not at 24h. This result indicated that TGF-β
promotes gingival epithelial cell wound healing. Without TGF- β, the wound healing is impaired
(Figure 16-17, Table 1-17).

3. TGF-β is involved in maintaining tissue integrity
We found that the expression level of tight junction protein, claudin-1, significantly
increased in TGF-β-treated group and significantly decreased in SB431542-treated group when
compared with control group. The expression level of ZO-1 showed a mild increase in the TGF-
β-treated group and a mild decrease in SB431542-treated group when compared with control
group, but the changes were statistically insignificant. The experiment was repeated 3 times. The
results that were shown were the representative of 3 assays (Figure 18-20, Table 18-25).
29

Figure 14 Immunostaining of gingival epithelium were shown. They were negative controls
with no primary antibody. From left to right: no suture control, contralateral no-suture side with
periodontitis, and suture side with periodontitis.
Control –
No suture
Experiment –
No Suture
Experiment –
Suture
DAPI
Negative
Overlay
30

Figure 15 Immunostaining of phospho-Smad3 in gingival epithelium were shown. From left to
right: no suture control, contralateral no-suture side with periodontitis, and suture side with
periodontitis. Significantly higher signal level of phospho-Smad3 in cytosol was seen in no-
suture experimental group but lower in suture experimental group when compared with control.
DAPI
Control –
No suture
Experiment –
No suture
Experiment –
Suture
p-Smad3
Overlay
31

Figure 16 TGF-β promotes wound healing in HGEC. Light microscope images of wound
closure at the different time points following injury.

Control TGF-β SB431542
6h 28.3075 42.335 18.7925
12h 88.525 100 46.94
24h 100 100 82.5

Table 1 Average wound closure (%) of control, TGF-β, and SB431541 groups at different time
points

32

Figure 17 TGF-β-treated group showed higher percentages of wound closure when compare to
control group at 6h and 12h. SB431542-treated group showed lower percentages of wound
closure when compare to control group at all 3 time points.

0
10
20
30
40
50
60
70
80
90
100
110
6h 12h 24h
% Wound Closure
Time point
Control
TGF-β
SB431542
33

Time point
0h 6h 12h 24h
Control
group1 6.75 4.82 1.61 0
group2 7.5 5.31 0 0
group3 7.03 5.11 1.55 0
group4 7.5 5.39 0 0
TGF-β
group1 6.09 3.8 0 0
group2 6.12 3.62 0 0
group3 6.13 3.45 0 0
group4 6.72 3.55 0 0
SB431542
group1 12.66 9.86 6.59 2.33
group2 13.29 11.22 7.79 4.49
group3 11.35 9.13 5.44 0
group4 11.67 9.58 6.26 2.08

Table 2 Wound size (gap distance) from each group

Control TGF-β SB431542
group1 28.59 37.6 22.12
group2 29.2 40.85 15.58
group3 27.31 43.72 19.56
group4 28.31 47.17 17.91

Table 3 Wound closure (%) at 6h time point
34
Control TGF-β SB431542
W 0.971988 0.99783 1.000092
p-value 0.853752 0.993024 No value
alpha 0.05 0.05 0.05
normal yes yes no value

Table 4 Shapiro-Wilk Test to test normality of data at 6h time point

Control TGF-β SB431542
median 28.45 42.285 18.735
rank sum 26 42 10
count 4 4 4 12
r^2/n 169 441 25 635
H

9.846154
df

2
p-value

0.007277
alpha

0.05
sig

yes

Table 5 Kruskal-Wallis Test to compare among 3 groups at 6h time point

35
TUKEY'S HSD / TUKEY-KRAMER

Alpha 0.05
Groups c mean n ss c^2/n c*mean
Control 1 28.3525 4 1.863275 0.25 28.3525
TGF-β -1 42.335 4 49.9209 0.25 -42.335
SB431542 18.7925 4 22.76028 0 0
12 74.54445 0.5 -13.9825
Q TEST

std err q-stat df q-crit lower upper sig
1.438985 -9.71692 9 3.948 -19.6636 -8.30139 yes

Table 6 Post-hoc test to identify the group that is significantly different from each other among 3
groups: control and TGF-β groups showed significance difference from each other at 6h time
point

TUKEY'S HSD / TUKEY-KRAMER

Alpha 0.05
Groups c mean n ss c^2/n c*mean
Control 1 28.3525 4 1.863275 0.25 28.3525
TGF-β 42.335 4 49.9209 0 0
SB431542 -1 18.7925 4 22.76028 0.25 -18.7925
12 74.54445 0.5 9.56
Q TEST

std err q-stat df q-crit lower upper sig
1.438985 6.64357 9 3.948 3.878885 15.24111 yes

Table 7 Post-hoc test to identify the group that is significantly different from each other among 3
groups: control and SB431542 groups showed significance difference from each other at 6h time
point

36
TUKEY'S HSD / TUKEY-KRAMER

Alpha 0.05
Groups c mean n ss c^2/n c*mean
Control 28.3525 4 1.863275 0 0
TGF-β 1 42.335 4 49.9209 0.25 42.335
SB431542 -1 18.7925 4 22.76028 0.25 -18.7925
12 74.54445 0.5 23.5425
Q TEST

std err q-stat df q-crit lower upper sig
1.438985 16.36048 9 3.948 17.86139 29.22361 yes

Table 8 Post-hoc test to identify the group that is significantly different from each other among 3
groups: TGF-β and SB431542 groups showed significance difference from each other at 6h time
point

Control TGF-β SB431542
group1 76.15 100 47.95
group2 100 100 41.38
group3 77.95 100 52.07
group4 100 100 46.36

Table 9 Wound closure (%) at 12h time point

37
Control TGF-β SB431542
W 0.763753 No value 0.989211
p-value 0.051516 No value 0.95339
alpha 0.05 0.05 0.05
normal yes No value yes

Table 10 Shapiro-Wilk Test to test normality of data at 12h time point

Control TGF-β SB431542
median 88.975 100 47.155
rank sum 30 38 10
count 4 4 4 12
r^2/n 225 361 25 611
H

8
df

2
p-value

0.018316
alpha

0.05
sig

yes

Table 11 Kruskal-Wallis Test to compare among 3 groups at 12h time point

38
TUKEY'S HSD / TUKEY-KRAMER

Alpha 0.05
Groups c mean n ss c^2/n c*mean
Control 1 88.525 4 528.3225 0.25 88.525
TGF-β -1 100 4 0 0.25 -100
SB431542 46.94 4 58.587 0 0
12 586.9095 0.5 -11.475
Q TEST

std err q-stat df q-crit lower upper sig
4.037703 -2.84196 9 3.948 -27.4158 4.46585 no

Table 12 Post-hoc test to identify the group that is significantly different from each other among
3 groups: control and TGF-β groups showed no significance difference from each other at 12h
time point

TUKEY'S HSD / TUKEY-KRAMER

Alpha 0.05
Groups c mean n ss c^2/n c*mean
Control 1 88.525 4 528.3225 0.25 88.525
TGF-β 100 4 0 0 0
SB431542 -1 46.94 4 58.587 0.25 -46.94
12 586.9095 0.5 41.585
Q TEST

std err q-stat df q-crit lower upper sig
4.037703 10.29917 9 3.948 25.64415 57.52585 yes

Table 13 Post-hoc test to identify the group that is significantly different from each other among
3 groups: control and SB431542 groups showed significance difference from each other at 12h
time point

39
TUKEY'S HSD / TUKEY-KRAMER

Alpha 0.05
Groups c mean n ss c^2/n c*mean
Control 88.525 4 528.3225 0 0
TGF-β 1 100 4 0 0.25 100
SB431542 -1 46.94 4 58.587 0.25 -46.94
12 586.9095 0.5 53.06
Q TEST

std err q-stat df q-crit lower upper sig
4.037703 13.14114 9 3.948 37.11915 69.00085 yes

Table 14 Post-hoc test to identify the group that is significantly different from each other among
3 groups: TGF-β and SB431542 groups showed significance difference from each other at 12h
time point

Control TGF-β SB431542
group1 100 100 81.6
group2 100 100 66.22
group3 100 100 100
group4 100 100 82.18

Table 15 Wound closure (%) at 24h time point

40
Control TGF-β SB431542
W No value No value 0.949662
p-value No value No value 0.714004
alpha 0.05 0.05 0.05
normal No value No value yes

Table 16 Shapiro-Wilk Test to test normality of data at 24h time point

Control TGF-β SB431542
median 100 100 81.89
rank sum 32 32 14
count 4 4 4 12
r^2/n 256 256 49 561
H

4.153846
df

2
p-value

0.125315
alpha

0.05
sig

no

Table 17 Kruskal-Wallis Test to compare among 3 groups at 24h time point. There was no
difference among 3 groups. Therefore, post-hoc test was not needed.

41

Figure 18 TGF-β is involved in maintaining tissue integrity. The expression level of claudin-1
significantly increased in TGF-β-treated group and significantly decreased in SB431542-treated
group when compared with control group. However, the expression level of ZO-1 insignificantly
changed.

Control TGF-β SB431542
group 1 1944 2522 1258
group 2 1960 2222 1558
group 3 2163 2675 1720
group 4 2438 2808 1780
group 5 1940 2222 1360
group 6 2200 2600 1548
group 7 2079 2875 1806
group 8 2520 3210 1680

Table 18 The quantification values from western blot analysis of claudin-1 protein

Control TGF-β SB431542
Claudin-1
ZO-1
Tubulin
42

Figure 19 TGF-β-treated group showed higher claudin-1 expression level when compared to
control group

Control TGF- β SB431542
W 0.8769062 0.9519466 0.91879223
p-value 0.17588558 0.73083429 0.42014016
alpha 0.05 0.05 0.05
normal yes yes yes

Table 19 Shapiro-Wilk Test to test normality of data from claudin-1 expression level

0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control TGF-β SB431542
Arbitrary Units (FoldS)
43
SUMMARY

Hyp Mean Diff 0
Groups Count Mean Variance Cohen d
Control 8 2155.5 49992.5714
TGF-β 8 2641.75 110645.929
Pooled 80319.25 1.71573335

Table 20 T-Test to compare between control and TGF-β group for claudin-1 expression level

SUMMARY

Hyp Mean Diff 0
Groups Count Mean Variance Cohen d
Control 8 2155.5 49992.5714
SB431542 8 1588.75 39093.6429
Pooled 44543.1071 2.68535233

Table 21 T-Test to compare between control and SB431542 group for claudin-1 expression level

T TEST: Unequal Variances

Alpha 0.05
std err t-stat df p-value t-crit lower upper sig effect r
One Tail 141.703255 3.43146669 12.253141 0.00248609 1.78228756

yes 0.70003544
Two Tail 141.703255 3.43146669 12.253141 0.00497218 2.17881283 -794.99487 -177.50513 yes 0.70003544
T TEST: Unequal Variances

Alpha 0.05
std err t-stat df p-value t-crit lower upper sig effect r
One Tail 105.52619 5.37070465 13.7935464 4.9346E-05 1.76131014

yes 0.82249408
Two Tail 105.52619 5.37070465 13.7935464 9.8692E-05 2.14478669 340.418832 793.081168 yes 0.82249408
44
Control TGF-β SB431542
group 1 2009 2616 2140
group 2 1512 2197 1787
group 3 1707 2594 1712
group 4 2105 2717 1670
group 5 2830 3576 2527
group 6 3366 4352 2870
group 7 4083 4908 3284
group 8 4459 5811 3962

Table 22 The quantification values from western blot analysis of ZO-1 protein

Figure 20 TGF-β-treated group showed higher ZO-1 expression level when compared to control
group

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control TGF-β SB431542
Arbitrary Units (Folds)
45
Control TGF-β SB431542
W 0.91299883 0.89965404 0.90779794
p-value 0.37566524 0.28689299 0.33881754
alpha 0.05 0.05 0.05
normal yes yes yes

Table 23 Shapiro-Wilk Test to test normality of data from ZO-1 expression level

SUMMARY

Hyp Mean Diff 0
Groups Count Mean Variance Cohen d
Control 8 2758.875 1238342.13
TGF- β 8 3596.375 1699107.13
Pooled 1468724.63 0.6910582

T TEST: Unequal Variances

Alpha 0.05
std err t-stat df p-value t-crit lower upper sig effect r
One Tail 605.954748 1.38211641 13.663806 0.09429549 1.76131014

no 0.35022232
Two Tail 605.954748 1.38211641 13.663806 0.18859097 2.14478669 -2137.1437 462.143676 no 0.35022232

Table 24 T-Test to compare between control and TGF-β group for ZO-1 expression level

46
SUMMARY

Hyp Mean Diff 0
Groups Count Mean Variance Cohen d
Control 8 2758.875 1238342.13
SB431542 8 2494 691036.286
Pooled 964689.205 0.26967908

T TEST: Unequal Variances

Alpha 0.05
std err t-stat df p-value t-crit lower upper sig effect r
One Tail 491.092966 0.53935816 12.9573434 0.29937821 1.7709334

no 0.14818287
Two Tail 491.092966 0.53935816 12.9573434 0.59875642 2.16036866 -796.06685 1325.81685 no 0.14818287

Table 25 T-Test to compare between control and SB431542 group for ZO-1 expression level

47
V. Discussion
The purpose of this study is to investigate the role of TGF-β in gingival epithelial wound
healing and tissue integrity.
We first studied with immunostaining of tissues from periodontitis-induced mice. Our
result showed that the signal of phospho-Smad3, which is directly activated by TGF-β, was
increased in experimental group at no ligature-inserted sides. This is consistent with previous
reports that TGF-β expression is enhanced during periodontitis (Gurkan et al, 2006; Howe et al,
2005). However, the ligature-inserted side showed decrease in TGF-β signal. This suggests a
possibility that the decrease in TGF-β signal may correlate with gingival tissue damage. Our
hypothesis is that TGF-β maintains gingival epithelium tissue integrity (homeostasis, via the
regulation of tight junction) and promotes tissue repair.
In non-ligature insertion side of periodontitis model, there was elevated inflammation
from periodontitis-induced from the other side but tissue was largely undamaged. There may still
have adequate reparative function. That may explain why there was an increase in TGF-β signal
since the need for repair increases.
In ligature insertion side of periodontitis model, there was not only inflammation but also
extensive damage of the gingival tissue. This may impair reparative function and TGF-β signal
decreased since the TGF-β pathway was also disrupted.
According to our immunostaining result, the signal of phospho-Smad3 increased in
cytosol in periodontitis model at no-suture side. This may imply that during gingival
inflammation, Smad3 is activated by TGF-β and may contribute to tissue repair. However, the
activated phospho-Smad3 did not undergo nuclear translocation to induce transcriptional
changes, suggesting distinct cytosolic functions. Smad3 might be involved in junctional protein
48
regulation. The possibility of the mechanisms involving in this regulation may include a decrease
in protein degradation, an increase in protein recycling, a facilitation in subcellular localization,
etc. However, further study is needed to investigate the mechanism(s) involved.
In the current study, we found that TGF-β has important roles in tissue integrity.
According to study by P Ye, TGF-β3 was found to be involved in regulating of tight junction
formation (Ye, 2012). Moreover, according to study by Howe KL et al., epithelial cells with
TGF-β exposure showed an increase in claudin-1 which is consistent to our result (Howe et al,
2005). They also found that neither occludin nor ZO-1 expression was affected by TGF-β
exposure which supports our result. There were studies that supported tight junction role in terms
of prevention of bacteria invasion. It was found that some periodontal pathogenic bacteria
damaged proteins which are important in tight junction integrity, such as E-cadherin, occludin,
and ZO-1. As a result, bacteria were able to invade the tissue and cause periodontal tissue
breakdown (Ji et al, 2015).
Another factor that might affect the expression level of junctional proteins upon TGF-β
exposure is cell density. It was found that with the same concentration of TGF-β, different cell
density modulates signal transduction in different ways (Zi et al, 2012). Therefore, the number of
cells in each experiment should be controlled in order to obtain consistent and reliable result.
The observation time possibly affects the expression level of junctional proteins upon
TGF-β treatment. It was shown that TGF-β concentration significantly changes with time
especially for low concentration (Zi et al, 2012). Cells harvested at 24 hours after TGF-β
treatment may give different result in Western blot analysis when compared to the ones that were
harvested at 48 hours. Further investigation will be needed to confirm this assumption.
49
Different doses of TGF-β exert different effects to the cells. The study of epithelial tight
junction integrity in chronic sinusitis with nasal polyps showed that the treatment with 10ng/ml
of TGF-β1 for 72 hours led to a significant decrease and disruption in immunostaining patterns
of both occludin and ZO-1. However, the treatment with 1ng/ml of TGF-β1 did not disrupt the
expression pattern of these tight junction proteins (Jiao et al, 2017). Another study tried to induce
EMT by treating NMuMG cells with 2ng/ml of TGF-β, the same dose as we used in wound
healing assay. This treatment induced the complete disassembly of ZO-1 positive junctions
(Conway et al, 2017).
The type of cells also affects the type of cellular response upon TGF-β treatment (Sporn,
2006). This might be according to involvement in different signaling pathway for each cell types.
However even in the same cell type, TGF-β may lead to different effects depending on the
presence of other growth factor (Sporn, 2006). This finding explains the conflict in results from
several studies on the roles of TGF-β in maintaining tissue integrity which were mentioned in the
introduction part.
Wound healing and repair are important processes that appear to be deregulated in the
disease state of periodontitis. In this study we used in vitro scratch assay which is simple to
perform and closely mimic the cell migration/healing in vivo (Liang et al, 2007). Our findings
suggested that TGF-β signaling promotes wound closure in HGEC.
Significant difference in percentages of wound closure of TGF-β-treated cells when
compared with control group was observed at 6h time point. This difference decreased with time
because the wound was close to sealed at 12h and 24h time points. Another factor that comes
into play at these later time points are cell proliferation, which started to influence the wound
closure over the effect of TGF-β in cell migration. Therefore, it is better to compare wound
50
closure (%) at early stage of the experiment. We noticed that the initial gap distances of wounds
created in SB431542-treated group were larger than the other 2 groups. However, we repeated
the experiment at least 3 times and each time we performed 4 points of assay in each group to
minimize variability in results. This might be according to impaired ability of SB431542-treated
cells to attach to the plate, thus causing contraction of the wound edge. Coated plate might
improve the attachment of the cells to the plate.
In a review article published in 2014, TGF-β was shown to be an inducer of epithelial-
mesenchymal transition (EMT). This occurs through SMAD-mediated and non-SMAD signaling
to shift the expression pattern of target genes in epithelial cells to become more “mesenchymal”
(Lamouille et al, 2014). As a result, cells acquire changes in their cytoskeleton and junctional
proteins to allow for the formation of actin stress fibers and front-rear polarity. The
reorganization of actin architecture that is consistent with mesenchymal markers enables cells to
acquire motility and invasive capacities. These findings support our results in terms of TGF-β’s
role in wound healing, since our assay measured the migration of HGECs following injury.
However, it does not appear to support our current data regarding the barrier function of TGF-β,
since junctional proteins should be repressed in cells during EMT. TGF-β-induced Smad2/3
activates or represses target genes to achieve the repression of junctional proteins. Our results
demonstrated cytosolic localization of phospho-Smad3, which suggests distinct mechanisms that
may be independent of transcription. This possibility needs to be further examined.
The result for TGF-β as a regulator of wound healing is further supported when the host
immune response after injury is examined. In a paper published in 2017, TGF-β was determined
to have regulatory functions on virtually every innate and adaptive immune cell, including
dendritic cells, B cells, NK cells, innate lymphoid cells, and granulocytes (Kelly et al, 2017).
51
Following the acute stage of inflammation, TGF-β curbs the immune response mainly by
inhibiting the differentiation and proliferation of T and B lymphocytes and by promoting
differentiation of Tregs. Resident macrophages and epithelial cells release TGF-β, which signals
these cells to reorganize the extracellular matrix and migrate to close the wound. Our results
suggest that TGF-β has a role in promoting wound healing; however this function may be
impaired at the site of periodontal injury. One possible explanation may be that, because
periodontitis is a chronic condition, the host immune system at the immediate site of injury may
not be conducive for TGF-β to terminate the immune response and initiate the processes of
wound healing.

52
VI. Conclusion and Future direction
TGF-β promoted epithelial integrity and wound healing. Our future studies aim to
determine whether restoring TGF-β signaling promotes epithelial integrity and wound healing in
vivo. We will use the ligature-induced periodontitis mice model and TGF-β supplementation to
test our hypothesis.
Further studies focusing on specific periodontal pathogenic bacteria and proteins,
important in maintaining tissue integrity, will provide interesting information about how TGF-β
regulates tissue integrity and repair.
In order to support the results that TGF-β is involved in epithelial junction integrity,
functional assay will be needed. Transepithelial electrical resistance (TEER) is widely used to
measure the integrity of epithelial cells, especially tight junction. It is found to be reliable,
sensitive, and non-invasive (Srinivasan et al, 2015).

53
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Abstract (if available)

Abstract The transforming growth factor-β (TGF-β) signaling pathway is involved in the regulation of cellular growth and differentiation and has multiple roles during craniofacial development and injury repair. The aim of this study is to assess whether TGF-β signaling is an underlying mechanism for impaired gingival epithelial tissue repair by examining its role in the promotion of wound healing and maintenance of epithelial junction integrity. ❧ We used a mouse model of ligature insertion-induced periodontitis and immunostaining with phospho-Smad3 to measure TGF-β-induced intracellular signaling activity in the vicinity of the wound site. To assess the role of TGF-β in gingival epithelial wound healing and barrier function, we incubated human gingival epithelial cell (HGEC) with TGF-β for 48 hours to assess cell motility using wound healing assay and examine epithelial junction protein expression using Western blot analysis. ❧ We found that during ligature-induced periodontitis, TGF-β signaling is activated in gingival epithelium of the diseased animal, but is impaired at the injury site, as determined by the absence of phospho-Smad3 staining, an indicator of TGF-β signaling activity. Wound healing assay showed significant higher percentages of wound closure in TGF-β-treated group but lower in SB431542-treated group. Western blot analysis showed a significant increase of claudin-1 in TGF-β-treated group but decrease in SB431542-treated group. For ZO-1 detection, there was no significant difference between control and treatment groups. We concluded that TGF-β promoted junctional integrity and wound healing in gingival epithelial tissue.

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Creator Ungvijanpunya, Nicha (author)

Core Title TGF-β signaling regulates gingival epithelial wound healing and barrier function

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 03/12/2018

Defense Date 03/02/2018

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

Tag barrier function,claudin-1,gingival epithelial,OAI-PMH Harvest,SB431542,Smad3,TGF-beta,TGF-β,tight junction,tissue integrity,wound healing,ZO-1

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Xu, Jian (committee chair), Paine, Michael (committee member), Sameshima, Glenn T. (committee member)

Creator Email dr.nichaung@gmail.com,ungvijan@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-485044

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