Chapter I Literature Review
1.2 Growth/Differentiation Factor-5 (GDF-5)
1.2.4 The role of GDF-5 in tooth development and differentiation
TGF-β superfamily members play inductive and regulatory roles in tooth
development and repair. The expression of mRNA for TGF-β1, TGF-β2, BMP-2,
BMP-4, BMP-6, BMP-7, and activin, is confirmed by in situ hybridization studies,
RT-PCR, and Northern blot analyses in tooth germ and adult pulp (Roberts et al. 1991,
Vaahtokari et al. 1991, Nakashima et al. 1994, Thesleff et al. 1996).
Until 1997, GDF-5 mRNA is identified for the first time in both dental papilla and
follicle at the root-forming stage of odontogenesis (Oida et al. 1997; Morotome et al.
1998; Sena et al. 2003). In adult human teeth, the expression of GDF-5 and its receptor
mRNA is found in periodontal ligament. In PDL-derived cells, rhGDF-5 stimulates cell
proliferation and glycosaminoglycan synthesis, but inhibits osteoblastic differentiation,
which may be one mechanism by which PDL is maintained around roots (Nakamura et
al. 2003).
More recently, the effect of GDF-5 on the growth and differentiation of porcine
dental papilla- and follicle-derived cells is investigated. The results reveal that GDF-5
regulates differentiation of both dental papilla and follicle during odontogenesis
(up-regulation effect in dental papilla-derived cells in a dose-dependent manner, and
down-regulation effect in dental follicle-derived cells), co-operatively with other growth
factors such as BMP-2 (Sumita et al. 2010). However, the role of GDF-5 in adult dental
pulp remains largely unknown.
1.3 Signaling transduction pathways of GDF-5
1.3.1 Receptors of GDF-5
In most of cells, there are three types of TGF-β receptors, and they are classified as
type I, II, and III (Tai et al. 2008). As a member of TGF-β superfamily, GDF-5
transduces its signals through two types of serine/threonine kinase receptors, termed
type I and type II receptors.
In animal studies, dental pulp tissue express six different type I receptors (termed
activin receptor-like kinases (ALK)-1 to -6) and two different type II receptors (TGF-β
receptor II (TβR-II) and BMP receptor II (BMPR-II)).
GDF-5 share BMP type I receptors with BMP-2, but the affinities to the receptors
differ. GDF-5 binds to the BMP type IB receptor (BMPR-IB, also termed ALK-6) with
high affinity, but binds to BMP type IA receptor (BMPR-IA, also termed ALK-3) with
low affinity (Sebald et al. 2004; Nickel et al. 2005). Moreover, GDF-5 preferentially
binds to ALK-6 in ROB-C26 osteoprogenitor cells (Nishitoh et al. 1996), GDF-5 and
ALK-6 interaction has been shown to be important in chondrogenesis of mouse distal
limb formation (Baur et al. 2000). Both of ALK-6 and ALK-3 can induce
phosphorylation of Smad-1, Smad-5 and Smad-8, meanwhile, ALK-4/5/7 promotes the
phosphorylation of Smad-2, and Smad-3 [Figure 1-1].
1.3.2 Smad pathway
At first, the ligands bind to type II receptor and activate the type II receptor. Then
the GS domain of the type I receptor is phosphorylated. Dimers initiate signaling by
binding to both type I and type II receptors and induced the formation of
heterotetracomplexes. The heterotetracomplexes promotes the phosphorylation of two
Ser residues in the motif Ser-Ser-X-ser of the C-terminal region of an intracellular
protein called receptor- regulated Smad (R-Smad), made up of Smad- 1/2/3/5/8.
Two phosphorylated R-Smad molecules form a heterotrimeric complex with Smad
4, a common-partner Smad (Co-Smad). The R-Smad/Co-Smad complexes then
translocate into the nucleus and regulate transcription of target genes by interaction with
various transcription factors and transcriptional co-activators or co-repressors. The third
class of Smads, inhibitory Smad (I-Smad, including Smad-6/7), negatively regulates
signaling by R-Smad and Co-Smad.
Of the eight Smad proteins identified in mammals, Smad-1/5/8 can activated by
BMP type I receptors, whereas Smad-2/3 are activated by activin and TGF-β type I
receptors. Smad-1/5/8 are structurally highly similar to each other, and functional
differences between them have not been fully determined. Smad-1/5, but not Smad 8,
are efficiently activated by BMP-6 and BMP-7, whereas all Smad-1/5/8 are activated by
BMP-2 (Aoki et al. 2001). However, the interaction between the Smad pathway and
GDF-5 is still not fully understood. The only clue is in the Smad-1/5/8 phosphorylation
study of Xu et al., rhGDF-5 could not stimulate an obvious Smad-1/5/8 phosphorylation
in the nascent human mesenchymal stem cells (HMSCs) but induced the Smad-1/5/8
phosphorylation in the rhTGFβs treated HMSCs (Xu et al. 2006).
1.3.3 Mitogen activated protein kinase (MAPK) pathway
TGF-β & BMPs are shown to modulate cellular activities via Smad-independent
pathways. Signaling mechanisms including MAPK pathways are recently found to
mediate the biological effects of TGF-β and BMPs (Sowa et al. 2002, Lebrin et al.
2005). The MAPK pathway involves three different cascades: and extracellular
signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38)
[Figure 1-2].
Previous studies have shown that GDF-5 induced the phosphorylation of ERK1/2
and p38 in chondrogenic ATDC5 cell line (Nakumura et al. 1999), activated ERK1/2 in
human umbilical vein smooth muscle cells (Chen et al. 2006), and induced osteogenic
differentiation of human ligamentum flavum cells through activation of ERK1/2 and
p38 MAPK (Zhong et al. 2010). However, the relationship between GDF-5 and MAPK
pathway has not been clarified in dental pulp cells yet.
1.4 The role of cyclin-dependent kinase 1 (CDK1), CDC25C, cyclin B1, p21 in cell proliferation
1.4.1 Cell cycle
The cell cycle or cell-division cycle means the series of events that takes place in a
cell leading to its division and duplication [Figure 2]. The cell cycle consists of four
distinct phases: G1 phase, S (synthesis) phase, G2 phase, and M (mitosis) phase.
Activation of each phase is dependent on the proper progression and completion of the
previous one. When cells have temporarily or reversibly stopped dividing, the state of
quiescence called G0 phase.
1.4.2 The role of cyclins and cyclin-dependent kinases (CDKs)
Cyclin-dependent kinases (CDKs) are a family of protein kinases first discovered
for their role in regulating the cell cycle (Nigg et al. 1995). CDKs are relatively small
proteins, with molecular weights ranging from 34 to 40 kDa, and contain little more
than the kinase domain. Animal cells contain at least nine CDKs, four of which, CDK1,
2, 3, and 4, are directly involved in cell cycle regulation. The known CDKs, their cyclin
partners and their function in human are listed in table 3.
By definition, a CDK binds a regulatory protein called a cyclin. Without cyclin,
CDK has little kinase activity; only the cyclin-CDK complex is an active kinase. The
concentration of cyclins varies in a cyclic fashion during the cell cycle. They are
produced or degraded as needed in order to drive the cell through the different stages of
cell cycle. There are two main groups of cyclins: G1/S cyclins and G2/M cyclins. G1/S
cyclins, including cyclin A, cyclin D, and cyclin E, is essential for the control of the cell
cycle at the G1/S transition. G2/M cyclins is important for the control of the cell cycle at
the G2/M transition. G2/M cyclins accumulate steadily during G2 and are abruptly
destroyed as cells exit from mitosis (at the end of the M-phase). The typical G2/M
cyclins is cyclin B, and it reacts with CDK1 (as known as cell division control protein
2(CDC2) homolog) to regulate progression from G2 to M phase.
Different combinations of the cyclin and CDK determine the different downstream
proteins. The mechanism of cyclin-CDK interaction is shown in Figure 3. Once the cell
receives a pro-mitotic extracellular signal, G1 cyclin-CDK complexes become active to
prepare the cell for S phase. It promotes the expression of transcription factors that
induce the expression S-phase cyclins and enzymes necessary to DNA replication.
Active S-phase cyclin-CDK complexes phosphorylated proteins that make up the
pre-replication complexes assembled during G1 phase on DNA replication origins.
Mitotic cyclin-CDK complexes are synthesized but inactivated during S phase and G1
phase. The complex promotes the initiation of mitosis by stimulating downstream
proteins involved in chromosome condensation and mitotic spindle assembly.
1.4.3 CDC25C
CDC25C activates cyclin dependent kinase by the removal of phosphates from
residues in the CDK active site. M-CDK complex activates CDC25C, which is the
activator of CDK1. The switch-like behavior forces entry into mitosis is irreversible.
CDC25A to C are known to control the transitions from G1 to S phase and G2 to M
phase.
1.4.4 p21
p21 also known as CDK inhibitor 1 or CDK-interacting protein 1 is a potent CDK
inhibitor. There are two families of genes that prevent the progression of the cell cycle:
the cip/kip family and the INK4a/ARF(Inhibitor of Kinase 4a/ Alternative Reading
Frame). The cip/kip family consists of the genes of p21, p27, and p57. They stop the
cell cycle in G1 phase by binding to and inactivating the cyclin-CDK complexes.
The p21 protein inhibits the activity of cyclin E/CDK2 and cyclin D/CDK4
complexes and functions as a regulator of G1 phase. The expression of this gene is
tightly controlled by the tumor suppressor protein p53, through which this protein
mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress
stimuli. p21 can also interact with proliferating cell nuclear antigen (PCNA), a DNA
polymerase accessory factor, and plays a regulatory role in S phase DNA replication
and DNA damage repair.
1.5 Alkaline phosphatase (ALP) and its role in tissue mineralization
Alkaline phosphatases (ALPs) are a glycosylated, membrane bound enzymes that
are present in nearly all living organisms. ALPs are cell surface glycoproteins that
hydrolyze a variety of monophosphate esters. In humans, there are at least four types of
genetically different iso-enzymes, namely, tissue-nonspecific alkaline phosphatase
(TNAP), intestinal alkaline phosphatase, placental alkaline phosphatase, and
placentallike alkaline phosphatase (Goseki-Sone et al. 2002). Intestinal alkaline
phosphatase, and placental alkaline phosphatase are tissue-specific and relatively
thermostable. TNAP are thermolabile iso-enzyme, characteristic for liver/bone/kidney
and present in most cell types. Placental-like phosphateses are present at low levels in
adult germ cells (Chang et al. 1994).
The bone isoenzyme has long been thought to play a role in mineralization of bone
and cartilage (Robinson 1923; Beertsen and van den Bos 1992). In dental follicale, this
activity is also used as an early marker of cementoblast/osteoblast differentiation (Zhao
et al. 2002; Morsczeck 2006). Likewise, as a marker of odontoblast differentiation,
Goggins and Fullmer (1967) reported that ALP activity is greatest in the
pre-odontoblastic cell-rich zone near the lateral and occlusal walls of the rat molar pulp
chamber. Furthermore, ALP is likely a prerequisite for differentiation and specialization
of pulp cells in vivo (Miller et al. 1976). They suggest this is because ALP activity is
greater in the pre-odontoblastic layer than in the mature odontoblastic layer and greater
in pulp cells just prior to differentiation into odontoblasts. ALP is an enzyme which can
be used in detection of mineralized tissue formation in cells. ALP as well as osteopontin
(OPN), osteocalcin (OCN), osteonectin, and other specific markers of dentin
differention are protein markers presented at early stage of pulpal cell differentation
(Narayanan et al. 2001).
Taken together, ALP activity is an appropriate early indicator of
cementoblast/osteoblast and odontoblast differentiation (Nuki and Bonting 1961;
Yoshiki and Kurahashi 1971; Tonomura et al. 2007). ALP activity was gradually
increased, reached the peak on day 14 and then declined during the differentiation of
both human and rat dental pulp cells.
Chapter II The Purpose of the Study
The purpose of our study is to investigate whether GDF-5 influences the
morphological changes, cell proliferation and viability, cell differentiation, and collagen
formation of human dental pulp cells in vitro. In addition, this study also tests the
signaling transduction pathways in GDF-5-induced cell proliferation and differentiation.
Chapter III Materials and Methods
3.1 Materials
3-(4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT), alkaline
phosphatase (ALP) staining assay reagents, dimethylsulfoxide (DMSO) and
dorsomorphin are purchased from Sigma (Sigma Chemical Company, St. Louis, MO,
USA). Noggin and Recombinant GDF-5 are obtained from PeproTech Inc. (NJ, USA).
U0126 is purchased from Promega (Promega Corporation, Madison, Wisconsin, USA).
SB431542 and SB203580 are from Tocris (Tocris bioscience, St. Louis, MO, USA).
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS),
penicillin/streptomycin are from Gibco (Life Technologies, Grand Island, NY, USA).
The SuperScript TM III First-Strand DNA synthesis system for RT-PCR is purchased
from Invitrogen (Invitrogen Corporation, Carlsbad, CA, USA). RNA isolation kit, and
NucleoSpin RNAII are purchased from Macherey-Nagel (Macherey-Nagel Inc, Easton,
PA, USA). Tris Base is from Amresco (Amresco Inc, Solon, Ohio, USA). Sircol
collagen assay kits are from Biocolor (Belfast, N. Ireland, UK)
3.2 Culture of human dental pulp cells
Human dental pulp cells were cultured by an explant technique and characterized
as previous studies (Chan et al. 2005, Tai et al. 2008). Briefly, human dental pulp cell
were derived from the extracted human third molars or premolars of patients under 25
y/o of age with proper informed consent before providing samples. Teeth were split
with a hammer to get vital pulp tissue. Pulp tissues were cut into 1 x 1 x 1 mm3 small
pieces by a surgical knife and cultured in DMEM containing 10% FBS and 1X
penicillin/streptomycin in culture dishes. The medium were changed every 2 or 3 days.
Pulp cells were cultured at 37°C, in a humidified atmosphere with 95% air and 5% CO2.
When pulp cells were growing from the explants reached near confluence, they were
trypsinized and subcultured at a ratio of 1:3. The cell passage numbers from 3 to 8 were
used for all experiments in this study.
3.3 Morphology of human dental pulp cells
Briefly, 5 x 103 pulp cells were incubated onto 24-well culture plate in DMEM
containing 10% FBS. Twenty-four hours later, the culture medium was changed by
serum-free DMEM and various concentrations of GDF-5 were added, depending on the
experimental conditions. The concentrations of GDF-5 were ranged from 0, 1, 10, 50,
100, 250 ng/ml. 5 days after GDF-5 treatment, the morphological changes of human
dental pulp cells were observed and photographed under a phase contrast microscope
(Olympus IX71, Olympus America Inc.).
3.4 MTT assay
MTT assay is a rapid colorimetric assay for cellular growth and survival. It can be
applicated for evaluation of cell proliferation and cytotoxicity. In short, human pulp
cells were incubated onto 24-well culture plate in DMEM with 10% FBS. (In condition
I, 5 x 103 human pulp cells per well is indicated; in condition II, the cell number alters
to 1 x 104 human pulp cells per well.) After twenty-four hours for attachment of cells on
the plate, the culture medium was replaced by serum-free DMEM and various
concentrations of GDF-5 were added, depending on the experimental conditions. (I) The
concentration of GDF-5 was ranged from 0, 1, 10, 50, 100, 250 ng/ml. (II) Cells were
individually pretreated with 1 or 5μM of U0126 (ERK inhibitor), 0.5or 1μM of
SB431542 (ALK-4/5/7 specific inhibitor), or 500 or 1000 ng/ml of Noggin (BMP
antagonist) for 30 minutes. Then 250 ng/ml of GDF-5 was added for co-incubation.
Cells were cultured under the same condition for another 5 days. Then, 20ul of
MTT reagent was added into each well and incubated at 37℃ for 2 hours. After 2-hour
incubation, the cultured medium was decanted and the produced formazan was
dissolved in 150 ul of DMSO. Then, 100ul of dissolved formazan solution from each
well was transferred to 96-well plate. The amount of dissolved formazan was monitored
by the readings against blank sample (DMSO) at OD 540 nm using a Dynatech
Microwell plate reader (Dynatech Microwell, Dynatech, Alexandria, Virginia).
Cell viability = (sample value of OD540nm-blank) / (negative control value of
OD540nm- blank) X 100%
3.5 Alkaline phosphatase (ALP) staining
ALP activity of pulp cells was determined. Briefly 1 x 105 human pulp cells were
inculated onto 24-well culture plate in DMEM containing 10% FBS. Twenty-four hours
later, medium was changed and according to experimental conditions various
concentrations of GDF-5 were added. (I) The concentration of GDF-5 was ranged from
0, 10, 50, 100, 250, 500 ng/ml. (II) Cells were individually pretreated with 1 or 5 μM of
U0126 (ERK inhibitor), 0.5 or 1 μM of SB431542 (ALK-4/5/7 specific inhibitor), 1 or 5 μM of SB203580 (p38 inhibitor), 1 or 5 μM of Dorsomorphin (ALK-2/3/6 specific
inhibitor; AMPK inhibitor) for 30 minutes. Then 250 ng/ml of GDF-5 was added for
co-incubation.
Cells were cultured at 37℃, in a humidified atmosphere with 95％ air and 5％
CO2 for another 5 days or 10 days. (In condition I, the 5-day and 10-day experiments
were both done, and the cultured medium was changed in 5th day. In condition II, only
the 5-day experiment was done.) Then, the medium was collected and cells were
washed with PBS three times. ALP activity of human dental pulp cells was evaluated by
histochemical staining using azo-dye coupling method as described before (Chan et al.,
2005; Tai et al., 2008). After removal of PBS, cells were fixed by 2% of
paraformaldehyde/PBS under room temperature for 20 minutes. Fresh stock substrate
solution was prepared [Figure 4]. The fixation medium was removed and cells were
rinsed with PBS containing Mg2+ and Ca2+ ions [Figure 5]. Then cells were flooded with 1000μl of stock substrate. At least, 1000ul/ well of incubation solution [Figure 6]
were added. Pulp cells were incubated under room temperature for 15 min in the dark.
The ALP staining of cells were observed and photographed.
3.6 Quantitative assay of Alkaline phosphatase activity
3.6.1 Cell lysate collection
1 x 105 human pulp cells were seeded onto 24-well culture plate in DMEM
containing 10% FBS. After twenty-four hours for attachment of cells on the plate, the
medium was changed and according to experimental conditions various concentrations
of GDF-5 were added. (I) The concentration of GDF-5 was ranged from 0, 10, 50, 100,
250, 500 ng/ml. (II) Cells were individually pretreated with 1 or 5 μM of U0126 (ERK
inhibitor), 0.5 or 1 μM of SB431542 (ALK-4/5/7 specific inhibitor), 1 or 5 μM of
SB203580 (p38 inhibitor), 1 or 5 μM of Dorsomorphin (ALK-2/3/6 specific inhibitor;
AMPK inhibitor) for 30 minutes. Then 250 ng/ml of GDF-5 was added and
co-incubated.
Pulp cells were cultured at 37℃, in a humidified atmosphere with 95％ air and 5
％ CO2 for another 5 days or 10 days. (In condition I, the 5-day and 10-day
experiments were both done, and the cultured medium was changed in 5th day. In
condition II, only the 5-day experiment was done.) Then, the medium was collected and
cells were washed with PBS twice.
First, 0.5％ Triton X-100 solution (extraction buffer) was prepared: 250 ul Triton
and 80 ul 1M MgCl2 were mixed in 40 ml ddH2O. After aspirating the PBS to dry, 250
ul of extraction buffer was added immediately to each well. Then, place the culture plate
on ice in the shake for 30 minutes. After 30-minute incubation, the cell lysate was
collected for protein measurement.
3.6.2 Alkaline phosphatase activity measurement
p-Nitrophenol standard solution was prepared in 1.5 ml eppendorfs into various
concentrations. Add each standard solution with 2.5 ul in volume to standard well of
96-well plate. 50 ul stock substrate (phosphatase substrate) was added into blank and
sample wells; 50 ul water was added into standard wells. Then, 50 ul alkaline buffer
was added to each well. The culture plate was covered with Parafilm® to avoid the
reaction of CO2 and incubated at 4℃ for 30 minutes. After 30-minute incubation, 12.5
ul of 0.5％ triton was added into blank and standard wells; 12.5 ul cell lysate was
added into sample wells. Finally, the culture plate was incubated at 37℃ for 45 minutes.
The reaction was quenched by adding 1 N NaOH and the absorbance at 405 nm was
read on the plate reader.
3.7 Collagen content assay
SircolTM Collagen Assay kit. (Biocolor Ltd., Newtownabbey, Northern Ireland) is a
quantitative dye-binding method that has been designed for analysis of acid-soluble
collagens extracted from mammalian tissues and collagens released into culture medium
by mammalian cells during in vivo culture. Synthesis of collagen by pulp cells in this
experiment was measured by Sircol Collagen Assay kit.
1 x 105 human pulp cells were seeded onto 24-well culture plate in DMEM
containing 10% FBS. Twenty-four hours later, according to experimental conditions the
medium was changed and various concentrations of GDF-5 were added. (I) In condition
I, we used DMEM containing 10%FBS as changing medium, and GDF-5 with various
concentrations (0, 10, 50, 100, 250, 500 ng/ml) were added. (II) In condition II,
serum-free DMEM was used as changing medium, and 0-500 ng/ml GDF-5 were added;
meanwhile, serum-containing DMEM without GDF-5 was used as positive control
group.
Pulp cells were cultured at 37℃, in a humidified atmosphere with 95％ air and 5
％ CO2 for another 5 days or 10 days. (In condition I, the 5-day and 10-day
experiments were both done, and the cultured medium was changed in 5th day and the
various concentrations of GDF-5 were added. In condition II, only the 5-day experiment
was done.)
Then, culture medium was collected and the pulp cells were washed with PBS
twice. 50 ul of 0.5 M acetic acid was added into each well for cell dissolving. Then,
pulp cells were stained by 200 ul of Sircol dye reagent (Sirius Red) in a slight shaking
motion for 30 minutes. Sirius Red is an anionic dye with sulphonic acid chain groups.
These groups may react with the side chain groups of the basic amino acids present in
collagen. After 30 minutes, the dye reagent was decanted. The complex of Sirius Red
binding to collagen was extracted with 200 ul of alkaline reagent for 10 minutes.
Alkaline reagent contained 0.5N sodium hydroxide, which released Sircol dye form the
collagen-dye complex. 100 ul of the reagent solution from each well was taken into
collagen-dye complex. 100 ul of the reagent solution from each well was taken into