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의학 석사 학위 논문
Roles of transglutaminases
in the regulation
of dendritic cell function
수지상세포 기능의 조절에 있어서
트랜스글루타미네이즈의 역할
2012 년 8월
서울대학교 대학원
의과대학 의과학과
노 지 아
수지상세포 기능의 조절에 있어서
트랜스글루타미네이즈의 역할
지도교수 김 인 규
이 논문을 의학석사 학위논문으로 제출함
2012 년 8월
서울대학교 대학원
의과대학 의과학과 전공
노 지 아
노지아의 의학석사 학위논문을 인준함
2012 년 8월
위 원 장 (인)
부위원장 (인)
위 원 원 (인)
Roles of transglutminases
in the regulation
of dendritic cell function
by
Jiah Noh
A Thesis Submitted in Partial Fulfillment
of the Requirements for the Degree of
Master of Science in Medicine
(Biomedical Sciences)
at the Seoul National University College of Medicine
October 2012
Approved by thesis committee:
Professor Chairman
Professor Vice Chairman
Professor
i
Abstract
Jiah Noh
Major in Biomedical Sciences
Department of Biomedical Sciences
Seoul National University Graduate School
Dendritic cell (DC) is professional antigen presenting cell, regulating
innate immunity and adaptive immunity. It has been reported that
treatment of DC with LPS up-regulates the expression of TGase 2, a
member of calcium-dependent enzyme family that catalyzes the
post-translational modification of protein by forming polyaminated
proteins or cross-linked proteins. In this study, role(s) of LPS-
induced TGase 2 in DC was investigated using TGase 2-deficient DC.
When bone marrow cells isolated from wild-type and TGase 2-
deficient mice were compared, there was no difference between
wild-type and TGase 2-deficient cells in the ability of differentiation
and maturation. Moreover, TGase 2-deficient DC showed no
difference in the level of secreted cytokine, phagocytosis, migration,
and apoptosis compared with those of wild-type DC. However, wild-
type DC showed higher level of ROS than TGase 2-deficient DC. In
addition, Real time RT-PCR revealed that increase of TGase activity
by LPS treatment is due to expression of TGase 4. These results
vi
indicate that TGase activity might be involved in the generation of
ROS and in the process of DC maturation.
Key word : Transglutaminase, Dendritic cell, Differentiation,
Maturation
vii
Contents
Abstracts ------------------------------------------- ⅰ
Contents ------------------------------------------ ⅲ
List of Tables ------------------------------------- ⅳ
List of Figures ------------------------------------ ⅳ
Introduction ---------------------------------------- 1
Purpose ------------------------------------------ 28
Materials and Methods --------------------------- 29
Results ------------------------------------------- 36
Discussion ----------------------------------------- 61
Reference ---------------------------------------- 63
Abstract (in Korea) ------------------------------ 69
viii
List of Tables
Table 1. Summary of characteristics of nine transglutaminases-----6
Table 2. Known inducers of TGase 2 expression---------------14
Table 3. NOX isoenzymes --------------------------------23
Table 4. Primers for TGases ------------------------------45
List of Figures
Figure1. Schematic representation of enzymatic reaction of
transglutaminase ---------------------------------------- 5
Figure 2. The schematic representation of TGase 2 structure ----- 8
Figure 3. Structure of TGase 2 ----------------------------- 9
Figure 4. Correlation between the development of myeloid and
lymphoid dendritic cells (DCs) ---------------------------- 19
Figure 5. Domain structure of regulatory proteins for NOX enzymes --
---------------------------------------------------- 26
Figure 6. Activation of reactive oxygen species (ROS) generation by
assembly of Phox regulatory proteins ---------------------- 27
Figure 7. A process of mDCs differentiation and maturation ------ 31
Figure 8. TGase 2 is not involved in DC differentiation ------- 37
ix
Figure 9. TGase 2 is not involved in LPS-induced DC maturation - 39
Figure 10. Cystamine inhibits DC maturation via TGase 2-
independent pathway ----------------------------------- 41
Figure 11. KCC009 also inhibits DC maturation via TGase 2-
independent pathway ------------------------------------ 42
Figure 12. TGase 4 is a strong candidate that is involved in DC
maturation -------------------------------------------- 46
Figure 13. TGase 4 is increased along with differentiation of DC -- 47
Figure 14. TGase 4 is involved in maturation of DC ------------- 48
Figure 15. TGase 2 is not involved in function of matured DCs ---- 50
Figure 16. TGase 2 is not involved in phagocytosis of DCs ------- 52
Figure 17. TGase 2 is not involved in migration of maturated DC -- 54
Figure 18. TGase 2 is not involved in expression of CCR7 ------- 55
Figure 19. TGase 2 is not involved in apoptosis of maturated DC -- 57
Figure 20. TGase 2 is not involved in ROS release of maturated DC --
---------------------------------------------------- 59
Figure 21. TGase 2 is involved in mRNA level of NOX2 components --
--------------------------------------------------- 60
1
Introduction
1. Transglutaminase
The transglutaminases (TGases) catalyze Ca2+-dependent acyl
transfer reactions between proteins or protein and polyamines
(Lorand and Graham, 2003). TGase-mediated modification of
glutamine residue can be made by three ways: polyamine
incorporation, deamidation, and crosslinking reaction. This reaction
involves a glutamine-containing protein or peptides as acyl acceptor
substrate. TGase forms a covalently linked γ-glutamythioester, the
acylezyme intermediate, at the active site cysteine with release of
ammonia (Iismaa, Mearns et al. 2009). This initial acylation step is
followed by a direct attack by small molecule amine (polyamine
incorporation), water (deamidation), and amino group of protein-
bound lysine residues (crosslinking) (Figure 1).
To date, nine TGase isoforms have been identified in human
(Esposito and Caputo, 2005). TGase 1 (encoded by the gene TGM1
on human chromosome 14q11.2) is expressed in almost all kinds of
epithelial cells and associated with plasma membrane in lung, kidney,
and liver (Hiiragi T and M, 1999). It is also expressed at low level in
proliferating keratinocytes differentiation (Steinert PM, 1996). It
functions mainly in the formation of cornified cell envelope or
terminally differentiated epidermal keratinocyte (Chakravarty R,
2
1989). It has been reported that mutation in the TGase 1 gene were
found in lamellar ichthyosis, autosomal recessive sin disease. This
finding indicated that TGase 1 has esterase enzyme activity in
addition to transamidation activity for lipid and protein barrier
formation.
TGase 2 (encoded by TGM2 on human chromosome 20q11-12), a
74-to 80-kDa protein, is ubiquitously expressed, and found in all
mammalian tissues (Begg, Carrington et al., 2006). It is localized in
cytosol, nuclear, membrane, cell surface, and extracellular. It is not
present in the mitochondria (Krasnikov, Kim et al., 2005). TGase 2
functions both as transamidating enzyme and GTP binding and
hydrolyzing enzyme. The transamidase activity plays an extracellular
role in matrix stabilization, and it essential for angiogenesis, wound
healing, and bone remodeling. And it also plays an intracellular role
in the cross-linking of proteins during apoptosis.
TGase 3 (encoded by TGM3 on human chromosome 20q11-12) is
also expressed in epithelial cells and hair follicle (Kim HC, 1990).
The predicted protein contains 692 amino acids and is 75% identical
with that of mouse. It has been reported that while TGase 3 is of
unique functional importance in hair development, loss of TGase 3 in
the epidermis could be compensated by other TGase family members.
Hair lacking TGase 3 is thinner, and shows major alterations in the
cuticle cells and markedly decreased cross-linking of hair protein
(John, Thiebach et al., 2012).
3
TGase 4 (encoded by TGM4 on human chromosome 3q21-22) is
unique in that it is espressed in the prostate gland (Willian-Ashman,
1972). Recently, it has been shown that TGase 4 may be involved in
prostate cancer cell growth, migration and invasiveness, tumor–
endothelial interaction and epithelial–mesenchymal transition.
Moreover, TGase 4 has a wide-range of interaction with other
molecular complexes, implicating that TGase 4 could be used as a
possible biomarker of aggressive cancer as well as a therapeutic
factor (Jiang and Ablin, 2011).
TGase 5 (encoded by TGM5 on human chromosome 15q15.2) is
very similar to that of TGase 1 in aspect of tissue distribution and
functions. TGase 5 is widely expressed in the epidermis and involved
in protein cross-linking. It is thought to be required for structural
integrity of the outermost epidermal layers (Cassidy, van Steensel et
al., 2005), Recently it is reported that TGM5 mutations were found
in acral peeling skin syndrome (Pigors, Kiritsi et al., 2012).
TGase 6 (encoded by TGM6 on human chromosome 20q11) is
expressed in a human carcinoma cell line with neuronal
characteristics and in mouse brain. Biochemical data show that
TGase 6 is allosterically regulated by Ca2+ and guanine nucleotides
(Thomas, Beck et al., 2011). TGase 7 is encoded by TGM7 on human
chromosome 15q15.2. Enzymatic properties and biological function of
TGase 6 and 7 are not fully characterized yet.
Factor XⅢa (encoded by F13A1 on human chromosome 6p24-25)
is expressed in blood. It is zymogen that is activated by thrombin
4
cleavage. The activated Factor XⅢa crosslinks fibrin monomer to
stabilize the blood clot (McDonagh and McDonagh, 1975). Factor
XⅢa consists of 731-amino acids with a molecular mass of 83kDa
(Takahashi, Takahashi et al., 1986).
Lastly, band4.2 (encoded by EPB42 on human chromosome
15q15.2) is a catalytically inactive member of the TGase family due
to absence of cysteine at active site of enzyme. It contributes to
membrane shape and stability and associates with the Rhesus protein
complex in RBC membranes (Mouro-Chanteloup, Delaunay et al.,
2003). Characteristics of TGases are summarized in Table 1.
5
Figure 1. Schematic representation of enzymatic reaction of
transglutaminase
Transglutaminases catalyze Ca2+-dependent protein-modification of
glutamine residue: polyamine incorporation, deamidation, and
crosslinking reaction.
6
Table 1. Summary of characteristics of nine transglutaminases
(Lorand and Graham, 2003)
7
2. Transglutaminase 2
2.1 Cloning and genomic structure
TGase 2 may be the most interesting member of TGases. It is
first isoenzyme identified among TGase family. TGase 2 was purified
from guinea pig liver homogenate (Sarkar et al., 1957), and then
cDNA clone was isolated from mouse macrophage and human
endothelial cell. By fluorescence in situ hybridization (FISH), TGase
2 maps on chromosome 20q11.2-12 (Genitil V, 1991). Analysis of
genomic structure showed that TGase 2 gene is 32.5 kilobases (Fraij
and Gonzales, 1996). There are two alternatively splied isoform of
TGase 2. One is TGase-H (TGase-homologue) or TGase-S (TGase-
short) that is 1.9 kilobases transcript and coded 584 amino acids
protein. The other is TGase-H2 that is 2.4 kilobases transcript and
coded 349 amino acids protein.
TGase 2 is comprised of a dimer form in complex and has four
distinct domains, N-terminal β-sandwich, catalytic core domain and
two C-terminal β-barrel domains (Figure 2). In the absence of
calcium, TGase 2 shows a compact conformation. When TGase 2 is
activated, there is a remarkably large conformational change from
the compact form to extended form, and then TGase active site is
exposed. The catalytic triad of enzyme consists of a cysteine,
histidine, and aspartate residue, and a conserved tryptophan
stabilizes the transition state. These are all essential for catalytic
activity (Iismaa, Mearns et al., 2009) (Figure 3).
8
Figure 2. The schematic representation of TGase 2 structure
Transglutaminase 2 consists of four domains; β-sandwich, catalytic
core, β-barrel 1, and β-barrel 2 (Fesus L 2002).
9
(A)
(B)
Figure 3. Structure of TGase 2
(A) Compact, inactive TGase 2 conformation (left) and extented
inhibitor-bound TGase 2 conformation (right) (B) TGase 2 is
comprised of four domains and catalytic core domain consists of a
cysteine, histidine, aspartate, and tryptophan residue.
10
2.2 Functions
2.2.1 The function of TGase 2 in cells
Seeing as the in vivo expression pattern of the enzyme, TGase 2
may have multiple roles. The cell type that expresses TGase 2 can
be divided into two groups. There are cell type such as smooth
muscle and endothelial cells that constitutively express TGase 2 at
high levels. In the other group, TGase 2 is induced by signalling
pathways.
At the membrane, TGase 2 is involved in transmitting signals from
seven-transmembrane helix receptor to phospholipase C (Iismaa, Wu
et al., 2000). TGase 2 also has important roles in diverse cellular
processes like apoptosis, cell adhesion, cell cycle and receptor-
mediated endocytosis. TGase 2 is thought to be responsible for the
formation of intracellular cross-linked protein polymers that
constitute the major component in an apoptotic envelope and it
stabilizes the apoptotic body (Melino G, 1998). TGase 2 activated by
Ca2+ interacts with and modifies key components of the cytoskeleton.
In response to RA treatment, TGase 2-dependant transamidation of
RhoA results in the increased binding of RhoA GTPase to ROCK-2
protein kinase, autophosphorylation of ROCK-2 and phosphorylation
of vimentin (Singh, Kunar et al., 2001) which can lead to the
formation of stress fibers and increased cell adhesion. In the cells
under the apoptosis in vivo, TGase 2 is induced. Recently it is
reported that TGase 2 sensitizes cells during apoptosis by interacting
11
with mitochondria, and it alters redox status (Mauro Piacentini et al.,
2002).
2.2.2 The function of TGase 2 on the cell surface and in the
extracellular matrix
On the cell surface, integrin-bound TGase 2 offers a binding site
for fibronectin. The TGase 2 binds to the sequences outside the
integrin ligand-binding pocket. On the surface of many different cell
type, some integrins could be complexed with TGase 2 (Akimov SS,
2001). In addition to fibronectin, other proteins were reported to be
substrates for TGase 2. TGase 2 may play an important role in
stabilizing the ECM by making the matrix resistant to mechanical and
proteolytic degradation. Such a stabilization of the ECM has been
proposed to play a role in tumor cell invasiveness as a result of
altered cell-matrix interactions. TGase 2 is also involved in
angiogenesis and wound healing (Haroon, Hettasch et al., 1999).
2.3 Pathogenic role of TGase 2
It has been reported that TGase 2 is involved in many pathogenic
processes such as celiac disease, cataract, bleomycine-induced lung
fibrosis, and neurodegenerative diseases.
In the case of celiac disease, it is a malabsorbtion syndrome
characterized by almost total atrophy of villi in the jejunum on
exposure to dietary glutens. TGase 2 is involved in generating gluten
peptides which stimulates T cell through deamidation of specific
12
glutamines (Anderson, Degano et al., 2000; Vader, de Ru et al., 2002).
The TGase 2-formed disease-triggering epitopes stimulate a
pathological immune response that destroys the jejunal epithelium.
Cataract is a clouding that develops in the crystalline lens of the
eye or in its envelope, varying in degree from slight to complete
opacity by obstructing the passage of light. Age-related cataracts
are characterized by aggregation of lens proteins with subsequent
lens clouding. TGase 2-mediated cross-linking of the β-crystallins,
αB-crystallins, and the intermediate filament protein vimentin is
likely to be involved in cataracogenesis (Lorand, Hsu et al., 1981;
Mulders, Hoekman et al., 1987; Velasco and Lorand 1987).
TGase 2 is activated by oxidative stress in human lens epithelial
cells. Oxidative stress induces the release of transforming growth
factor (TGF)-2 which in turn activates Smad3 signaling pathway.
Modulation of gene expression by Smad signalling pathway results in
activation of TGase 2, leading to crosslinking and aggregation of lens
proteins (Shin, Jeon et al., 2008).
Huntington disease (HD) is a neurodegenerative genetic disorder
that affects muscle coordination and leads to cognitive decline and
psychiatric problems. HD is caused by the expansion of CAG
trinucleotide repeats in the gene encoding huntingtin (htt).
Accumulation of ubiquitinated htt aggregate in the nucleus, and
progressive loss of neuronal cells are observed. One of the proposed
mechanisms of htt aggregation is based on the action of TGase 2
because expanded polyglutamine repeats are excellent glutaminyl-
13
donor substrates for TGase2-catalyzed cross-linking (Lesort,
Tucholski et al., 2000). In addition, TGase 2 is highly expressed in
several cancerous tissues including glioblastoma, malignant
melanoma, and pancreatic ductal adenocarcinoma (Siegel and Khosla,
2007). The cancer cells resistant to chemothrapeutic drug or
obtained from metastatic cancer tissue also showed elevated
expression of TGase 2. Elevated TGase 2 induced constitutive
activation of transcription factor of NF-κB (nuclear factor κB),
important regulator in cell proliferation and survival (Mann et al.,
2006), which is probably associated with TGase 2 up-regulation in
drug resistant and metastatic cells. Furthermore, treatment of
irreversible TGase 2 inhibitors results in apoptosis of cancer cells
and sensitization to chemotherapy in glioblastoma (Yuan, Choi et al.,
2005).
14
Table 2. Known inducers of TGase 2 expression
15
3. Dendritic cell
Dendritic cells (DCs) are immune cells participating in the
mammalian immune system. Although DCs are scanty, they are
broadly distributed in many tissues (Shortman and Naik, 2007). DCs
are at the center of immune control, linking innate immunity and
adaptive immunity. DCs are one of the professional antigen
presenting cells (APC) derived from hemopoietic cells and
specialized for T cell-mediated immunity and tolerance. Major
histocompatibility complex (MHC) of DC surface interacts with T cell
receptor (TCR). DCs present peptide epitope of antigen to T cells,
and associates with TCR, activating naive T cell to effector T cells.
The interaction between DCs and naive T cells initiate immune
response (Shortman and Naik, 2007).
Peripheral tolerance to self-antigens is maintained by immature
DCs. Activation of DCs is essential to induce immunity. DCs derived
from progenitor cells go through maturation step upon encountering
antigen. Then, DCs migrate to lymphoid tissues, possessing ability to
induce T cells. During last several years, it is identified that DCs
have many distinct subtype. Each subtype can be classified according
to many aspects such as location, phenotype and functions (Shortman
and Liu, 2002). Recently, therapeutic DC vaccine has been proposed
as a novel approach for treatment of cancer and chronic infections
(Freire, Kadaria et al., 2010), suggesting potential roles for DCs
other than immune induction (Chauvin and Josien 2008).
16
3.1 History of DCs
In the 19th century, Dendritic cells were first explained by Ralph M.
Steinman and Zanvil A. Cohn, described as a large stellate cells with
distinct properties from the former cell types (Steinman RM 2007). In
1998, Jacques Banchereau and Ralph M. Steinman explain that B and
T lymphocytes are the mediators of immunity, but their function is
under the control of dendritic cell. Thus, dendritic cell is thought to
be as a powerful tool for manipulating the immune system. For this
finding, Steinman was awarded the Nobel Prize in Physiology and
Medicine in 2011. Almost 30 years on, dendritic cells are one of the
most well studied cells in immunology.
3.2 Development of DCs
The key advance in DC biology for the past few years have been
facilitated by the ability to propagate large numbers of DCs in vitro
with defined growth factors. One of the most important finding is DCs
are not a single cell type, but a system of cells that arise from
distinct, bone morrow-derived, hematopoietic lineages. The earliest
DC progenitors are normally present in the bone morrow, and DC
progenitors/precursors are released from the bone morrow and
patrol through the blood and lymphoid organs that are ready to
receive differentiation signals.
17
3.2.1 Lymphoid-derived DCs
Lymphoid DC is a distinct DC subtype that is closely linked to the
lymphocyte lineage. Lymphoid DCs may be differentiated from
progenitors that also have the potential to mature into T and natural
killer (NK) cells (Marquez, Trigueros et al., 1998). Such progenitors
are distributed in the thymus and in the T cell areas of secondary
lymphoid tissue (Grouard, Rissoan et al., 1997). Lymphoid DCs also
may be developed from thymic progenitors by stimulation with
interleukin 3 (IL-3), but not granulocyte-macrophage colony-
stimulating factor (GM-CSF), and from lymphoid precursors in human
tonsil by treatment with CD40 ligand (CD40L).
A various functions of lymphoid DCs have been reported (Mott,
UnderHill et al., 2008). Above all, they promote negative selection in
the thymus and are costimulators of CD4+ and CD8+T cells.
Lymphoid DCs primarily mediate regulatory rather than stimulatory
immune effector functions. In human, early progenitors of the
lymphoid related DC pathway express CD34, CD10 and CD38;
intermediate precursors have been described as CD25+, CD86+, IL-
3R+, CD4+, MHC class Ⅰ, and MHC class Ⅱ positive.
3.2.2 Myeloid-derived DCs
CD34+ progenitor and PB mononuclear cells (MNC) have described
two other DC pathways, both associated with the myeloid lineage
(Cavanagh LL, 1998). One pathway is derived from a CD14+
precursor and is represented by a CD14+CD1a+ intermediate; the
18
other is derived from a CD14-CD1a+ precursor. Both produce mature
DCs capable of stimulating strong naive T cell responses. Mature
DCs of both path way express high level of MHC class I, MHC class
Ⅱ molecules and CD86. Tumor necrosis factor (TNF), GM-CSF, and
IL-4 induce the maturation of CD14-derived DCs from CD34+
progenitors or CD14+PB monocytes (Rosenzwajg, Camus et al.,
1998). For CD1a-derived DCs, transforming growth factor β (TGF-
β) appears to be a principal differentiation factor. CD14-derived DCs
most likely correspond to interstitial and/or circulating blood DCs,
whereas CD1a-derived DCs are related to epidermal DCs (Strobl et
al., 1998).
19
Figure 4. Correlation between the development of myeloid and
lymphoid dendritic cells (DCs) (Kumar and Jack 2006)
20
3.3 Maturation and activation of DCs
Life cycle of DCs can be divided into two stages: immature and
mature stage based on the expression of surface markers. While
peripheral tolerance to self-antigens is maintained by immature DCs,
activation (maturation) of DC is essential to induce immunity.
Immature DCs have capacity to capture and internalize antigens due
to surface expression of receptors that enables the recognition and
endocytosis of potential antigens. These receptors include members
of pattern recognition receptors (PRRs) family such as Toll-like
receptors, C-type lectin receptors (CLRs), intracytoplasmic
nucleotide oligomerization domain (NOD)-like receptors, and so on.
Immature DCs act as immunological sensors that receive stimuli from
environment to alert the immune system. However, immature DCs
are poor stimulators of T cells and must undergo a maturation
process.
Upon antigen capture y immature DCs, a complex process of
maturation is initiated based on the nature of the stimuli. There are
stimuli that control DC maturation such as cytokines (TNF, IL-1,
IFNs, and TSLP), microbial products (LPS, CpG, dsRNA and MDP),
danger signals (Urates, HSPs), NK cells (INFγ), and T helper cells
(CD40L). And it is showed the series of phenotypic changes that
enables DCs to initiate immunity as professional APCs (Villadangos
and Schnorrer, 2007), which include (1) loss of phagocytic receptor;
(2) morphological changes; (3) secretion of cytokines and
chemokines such as IL-1β, IL-10, IL-12, TNF, CCL19, and CCL22;
21
(4) up-regulation of costimulatory and adhesion molecules such as
CD80, CD86, CD40, CD54 and CD58; (5) translocation of MHC class
Ⅰ,Ⅱ compartents to the cell surface; and (6) migration of mature
DCs into regional/draining lymphoid tissue. As a result, antigen
acquired in peripheral sites is retained, processed and presented to
T cells in lymph nodes.
22
4. NADPH oxidase
Reactive oxygen species (ROS) are a heterogeneous group of highly
reactive molecules that oxidize targets in a biologic system. ROS are
widely recognized as important mediators of cell growth, adhesion,
differentiation, senescence, and apoptosis. During steady-state
conditions, ROS are constantly produced in the electron-transport
chain during cellular respiration and by various constitutively active
oxidases. ROS production can also be induced by activation of
NADPH oxidase family in a process generally referred to as an
oxidative burst. NADPH oxidase is a superoxide (O2−)-generating
enzyme first identified in phagocytes such as neutrophils that shows
bactericidal activities. After that it has also been reported that O2−
is released in an NADPH dependent manner in non-phagocytes. But
studies on NADPH oxidases have been performed mainly in
phagocytes because mutations in the gene encoding the phagocyte
NADPH oxidase were found in patients with chronic granulomatous
disease (CGD) (Royerpokora, Kunkel et al., 1986)
4.1. NADPH oxidase iosenzymes
NADPH oxidases are composed of five homologs that are
distinguished by catalytic subunit from NOX1 to NOX5, and two
related DUOX1, 2.
23
Table 3. NOX isoenzymes
24
4.2 Structure and subunits of NOX2
NOX2/NADPH oxidase is composed of NOX2 (gp91phox), p22phox,
p67phox, p47phox, p40phox, and the small GTP-binding protein Rac.
gp91phox and p22phox are located at the plasma membrane and p22phox
forms a mutually stabilizing complex with gp91phox. The complex of
gp91phox and p22phox is referred to as flavocytochrome b558. p67phox,
p47phox, and p40phox are located at cytosol and referred to as
cytosolic subunits. Under resting conditions, the SH3 (SRC-
homology3) domains of p47phox bind intramolecularly to the
autoinhibitory region (AIR) in the middle of C-terminal, preventing its
binding to p22phox. The phagocyte oxidase (PX) domain in N-terminal
of p47phox is known for role of binding to membrane lipids. Other
cytosolic subunit p40phox binds to PB1 (Phox and Bem1p proteins)
domain of p67phox. Similarly, SH3 domain of p67phox binds to PR
(prolline rich) domain of p47phox. p40phox, p47phox, and p67phox are
assembled into complex in cytosol. A small GTP-binding protein Rac
is interacted with inhibitory protein RhoGDP-dissociation inhibitor
(RhoGDI).
4.3 Activation mechanisms of NOX2
Activation of the NOX2 system can be divided into at least three
signaling triggers; lipid metabolism, phosphorylation, and Guanine-
nucleotide exchange. First, after any stimulation phosphatidylinositol
3-kinase (PI3K) and phospholipase D produce 3-phosphorylated
phosphatidylintositols (PtdInsP) and phosphatidic acid, respectively,
25
providing lipids to which the p47phox and p40phox PX domains bind.
Second, when it comes to complex of cytosolic subunits (p40phox,
p47phox, and p67phox) they translocate to membrane and assemble with
flavocytochrome b558. To translocate from cytosol to membrane,
protein kinases are required.
Protein kinases like AKT catalyse AIR domain of p47phox. It allows
transformation of p47phox, which makes p47phox to bind to p22phox. It
also relieves inhibition of the PX domain of p47phox, allowing binding
to lipids. Last, activation allows GTP binding. The conformational
change of RAC-GDP reduces interaction RAC-GDP with Rho GDI.
And it makes for RAC-GTP to bind to membrane. p67phox subunit has
activation domain that activates electron transfer. It produces
superoxide.
26
Figure 5. Domain structure of regulatory proteins for NOX enzymes
27
Figure 6. Activation of reactive oxygen species (ROS) generation by
assembly of Phox regulatory proteins
28
Purpose
TGase 2 is involved in some inflammatory disease such as celiac
disease and bleomycine-induced lung fibrosis. Moreover, the role of
DCs in septic shock has been highlighted. It has been reported that
TGase 2 is a possible player in molecular and cellular mechanisms
leading to improper regulation of inflammatory response involved in
sepsis (Falasca L, 2008). Moreover, treatment with KCC009, inhibitor
for TGase 2, did not alter monocyte differentiation to phenotypically
immature DCs, but influence the ability of DCs to mature when
stimulated with LPS (Matic, Sacchi et al., 2010). The aim of this
study is to confirm the role of TGase 2 in differentiation and
maturation of DC using TGase 2 knock-out mice. Interestingly,
results of our research with TGase 2 knock-out mice show that
TGase 2 is not involved in DCs maturation and phenotype.
29
Material and Method
1. Mice
C57BL/6J mice were obtained from The Jackson Laboratory. TG2-
null mice were backcrossed to C57BL/6J mice for 10 generations
(N10). Mice were kept in essentially specific pathogen-free
environment at the animal facility of Seoul National University
College of Medicine. All animal experiments were carried out with
the approval of the Institutional Animal Care and Use Committee at
Seoul National University.
2. Cell culture
Bone marrow (BM) cells were isolated by flushing femur and tibia.
The cells were centrifuged and then resuspended in culture medium
consisting of Isocove’s Modified Dulbecco’s Media (Gibco, #12240)
supplemented with 10% heat-inactivated fetal bovine serum (Gibco,
#26140), 0.1 % 2-mercaptoethanol (Gibco, #21985-023), 0.1 %
gentamicine (Gibco, #15750-060), 1% Penicillin-Streptomycin
glutamine (Gibco, #10378), gentamicine (Gibco, #15750-060), 3
ng/ml IL-4, and GM-CSF (peprotech, #214-14 and #315-3). BM
cells were cultured at 1x106 cell/ml in 24well plates in culture
medium. The cells were incubated at 37 ℃ in a humidified atosphere
containing 5% CO2 for 6 days as shown in Figure 7. Media has to be
30
changed per 2 days for 6 days. DCs were maturated for 20 hr by the
addition of 1 μg/ml LPS (sigma, #L6529).
31
Figure 7. A process of mDCs differentiation and maturation
32
3. Flow cytometry
Cells were harvested by vigorous pipetting and removal of
nonadherent cells. Before incubatation with mAbs, the cells were
blocked at 4 ℃ for 20 min in mixture FACS buffer (1 % BSA, 1mM
EDTA, and 0.02% NaN3 in PBS), super block 1:1. Cells were incubated
with directly conjugated mAbs at 4 ℃ for 30 min.
The following mAbs were used: PE Rat anti-mouse CD40 (BD Ph
armingenTM), PE/Cy7 anti-mouse, C86 (BioLegend), FITC Mouse
anti-mousel-A[b](BDPharmingenTM), APC conjugated anti-mouse
CD80(B7-1) (eBioscience.com), APC/Cy7 anti-mouse CD11c
(BioLegend), and PE rat anti-mouse CD11b (BDPharmingenTM). After
washing two times with FACs buffer, cells conjugated mAbs were
measured by flow cytometry.
4. Real-time quantitative PCR
Quantitative assessment of mRNA levels of IL-10, IL-12, CCR7, and
NOX2 subunits was performed by real-time quantitative PCR using a
CFX96TM Real-Time system (Bio-Rad). The cDNA templates from
each cell were amplified using Maxime RT PreMix Kit (iNtRON).
KAPA SYBR® FAST qPCR Master Mix (KAPA Biosystems)
isusedtoreal-timePCR.
33
5. Intracellular TG activity assay
Cells were incubated with 100mM biotinylated pentylamine (BP,
Pierce) for 1 hr at 37 ℃ before harvesting. Cell extracts were
prepared by disrupting the cells in lysis buffer (50 mM Tris-Cl, pH
7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) followed by
centrifugation (12,000 g) for 10 min at 4 ℃ and were subjected to
SDS-PAGE using 10 % gel and then transferred to nitrocellulose
membrane. The proteins that crosslink with BP were visualized by
probing with HRP-conjugated strept-avidin, followed by enhanced
chemiluminescence reagents (Pierce).
6. Western blot analysis
Cells were suspended with 1 % Tritronx100 buffer and sonicated (2
sec pulse and 2 sec pulse, 5 times at 14 % amplitude). Cell lysate
were centrifuged at 12000 g for 10min at 4 ℃ and supernatant was
transferred to e-tube. Samples were boiled in sample loading buffer
at 100 ℃ for 10 min, seperated on 10 % SDS-polyacrylamide gel,
and transferred to a nitrocellulose membranes. The membrane was
incubated with 5 % skim milk in TBS-T (50 mM Tris, 200 mM NaCl,
0.05 % Tween 20, PH7.5) for 1 hr to block nonspecific protein
binding. After washing for 5 min with 0.1 % TBS-T, the membranes
were then incubated with anti-TGase 4 antibody (1:1000), anti-
TGase 2 antibody (1:1000), or anti-gp91phox antibody (1:1000) in 5 %
skim milk in TBST. Immunoreative antibodies were further incubated
34
with appropriate secondary antibodies conjugated to horseradish
peroxidase in 5 % skim milk in TBST for 1 hr. After washing three
times with 0.1 % TBS-T, the membranes were detected by
SuperSignal West Substrate (Pierce, #34080) and exposed to X-ray
film.
7. Phagocytosis assay
BM-drived DCs (105/sample) were suspended with culture medium.
Fluorescein isothiocyanate-dextran particles (sigma, Korea) were
added to each sample at a final concentration of 1 mg/ml and
incubation was continued for 45 min. Cells were rinsed twice with
FACS buffer containing 1 % BSA, 1 mM EDTA, and 0.02 % NaN3 in
PBS and analysed using flow cytometry. First, cells were gated out
according to CD11c+, CD11b+ differentiated DCs population and then
phagocytosing BM-drived DCs were identified on a forward scatter-
side scatter plot and the fluorescence of the cells was measured. Ten
thousands of DCs were counted in each sample.
8. In vitro 3D chemotaxis assays
Mixture of 50ml collagen (1.5 mg/ml) and 50 ml of a cell suspension
containing 5 × 105 BM-drived DCs was placed in 1 μ-Slide Ⅵ flat
ibiTreat microscopy chamber and incubated for 30 min. 30 ml culture
medium containing chemokines (CCL21) was inserted in lower
chamber and 30 ml culture medium in upper chamber. Cells migated
35
from the lower and upper chamber, and were recorded onto confocal
microscope per 2 min for 4 hr.
9. In vitro survival assay
BM-drived DCs were seeded in 96-well micro plates at a
concentration of 1x105/ml in 100ml of culture medium in the
presence or absence of LPS (1 μg/ml) and/or thapsigargin (50 nM).
For each time cells were added 20 μl/well Cell Titer-Blue Reagent
(Promega, #G8080). And then record fluorescence 560/590 nm.
10. Measurement of ROS production
DCs (3x106 cells) from wild type mice and TGase 2 knock-out mice
were treated with PMA or LPS. ROS generation in cultured medium
was measured by luminal-enhanced chemiluminescence. That is, DCs
were collected and resuspended in 200l of CO2-independent
medium containing luminol (12.5 M) and horseradish peroxidase
(6.25 unit). Cell suspensions were preheated to 37℃ in the
thermostated chamber of the luminometer (Berthold-Biolumat LB937)
and allowed to stabilize for 5min. Then, they were stimulated with
50l medium containing 5 g/ml LPS, 2.5 g/ml PMA or 50 M DPI.
Changes in chemiluminiscence were measured over the indicated
times, every 1 min for 120 min.
36
Results
TGase 2 is not involved in differentiation of DC
To investigate the role of TGase 2 in differentiation of DCs, we
examined the protein level of TGase 2 and transamidation activity
during BMCs is induced to differentiate into DCs. Streptavidin-HRP
that recognizes proteins incorporated with biotinylated-pentylamine
(BP) was used to measure the transamidation activity. When BMCs
were differentiated into DCs, the protein level of TGase 2 and
transamidation activity were gradually increased. Moreover, the
protein level of TGase 2 and transamidation activity were further
increased with treatment DCs with LPS at day 6. However, when
compared the percentage of DC population by flow cytometry
measuring the expression of CD11b and CD11c, we found that there
is no difference in differentiated DCs population between BMCs from
wild-type and TGase 2-deficient mice (Figure 8). These results
indicate that TGase 2 is not involved in DC differentiation in contrast
to previous reports (Matic et al., 2010).
37
Figure 8. TGase 2 is not involved in DC differentiation.
We differentiated mouse bone marrow cells (BMCs) into DCs by
treatment of GM-CSF and IL-4 for 6 days. (A) BM-derived DCs are
incubated with 500uM of biotinylated pentylamine for 1hr before it is
collected. After 6 day DCs are treated 1 μg/ml LPS. (B) DCs not
treated LPS are collected and then expression of CD11c, CD11b is
measured by flow cytometry.
38
TGase 2 is not involved in maturation of DC
There was report that TGase 2 plays an important role in
maturation of DCs by LPS (Matic et al., 2010). However, they didn't
show this effect in DCs from TGase 2-deficient mice. So, we tested
whether DCs obtained from TGase 2-deficient mice are abnormally
maturated by LPS or not. After 6 day DCs differentiated from wild-
type BMCs and TGase 2-deficient BMCs were treated with different
concentration of LPS for 20 hr. And then, CD80+, CD86+, CD40+, and
MHCⅡ+ populations that are markers of maturated DCs were
measured. Unlike previous reports, there was no difference between
DCs obtained from wild type mice and TGase 2-deficient mice
(Figure 9). DCs obtained from TGase 2-deficient mice were normally
maturated by LPS dose dependently. Thus, TGase 2 is not involved
in maturation of DCs.
39
Figure 9. TGase 2 is not involved in LPS-induced DC maturation.
Cells were treated with different concentration of LPS for 18 hr.
The expression of CD80+CD86+(A), CD40+ MHC2+(B) that indicate
mature DCs is tested by flow cytometry. The expression level is
increased by LPS dose dependant. But there is no difference
between wile type mice and TGase 2 knock-out mice.
40
Cystamine and KCC009 inhibit maturation of DC via TGase 2-
independent pathway
Although our data showed that TGase 2 is not involved in
maturation of DCs, we decided to test whether DCs maturation is
decreased by TGase 2 inhibitor as previous study showed that TGase
inhibitor suppresses the process of DC maturation. At day 6 of
differentiation, DCs were treated with TGase 2 inhibitor, cystamine,
and LPS. Interestingly, DCs treated with cystamine showed
decreased expression of surface markers in the both DCs obtained
from wild-type and TGase 2-deficient mice. Likewise, TGase
activity was decreased with cystamine treatment in a dose dependent
manner in the both DCs from wild type and TGase 2-deficient mice
(Figure 10). To confirm these results, we treated DC with another
TGase 2 inhibitor, KCC009. The expression of surface markers was
similar with those treated with cystamine (Figure 11). These results
indicate that cystamine and KCC009 inhibit maturation of DCs via
TGase 2-independent pathway, suggesting that other TGase
isoenzyme may play a role in the process of DC maturation.
41
Figure 10. Cystamine inhibits DC maturation via TGase 2-
independent pathway.
BM-drived DCs were collected after 6 days of culture with GM-CSF
and IL-4 and in the presence of the LPS stimulation and the different
concentrations of cystamine, TGase 2 specific inhibior. (A) After LPS
stimulati0n, CD80, CD86, CD40, and MHC2 molecules of each DC
treated cystamine were analyzed by flow cytometry. (B) Maturated
DCs from wild-type and TGase 2-deficient BMCs were lysed and the
lasates were immunoblatted with horseradish peroxidase conjugated
strep-avidin to visualize intracellular TG activity or antibody for
actin.
42
Figure 11. KCC009 also inhibits DC maturation via TGase 2-
independent pathway.
Another TGase 2 specific inhibitor, KCC009, also displays the same
tendency with result from cystamine. (A) flow cytometry analysis (B)
Western blot for TGase activity and actin
43
Expression of TGase 4 is increased during DC maturation
To investigate which TGase isoenzyme(s) is involved in maturation
of DC, we first screened mRNA expression level of from TGase 1 to
FⅩⅢa. DCs were differentiated for 6 day and treated with LPS for 3
hr, 6 hr, and 18 hr. Cells were then collected, and total RNA was
extracted using TRIzol reagent (Invitrogen). Primer pairs used for
RT-PCR were summerized in Table 4. Compared with control DCs,
DCs treated with LPS showed comparable increase of TGase 1,
TGase 2, TGase 3, and TGase 5 mRNA level after 3hr LPS treat, and
decrease of TGase 7 and FⅩⅢa mRNA level. By contrast, the level
of TGase 4 mRNA was increased about 100 folds after 3hr LPS
treatment (Figure 12). To confirm these results in protein level,
BMCs were differentiated to DCs for 1 day, 3 day, and 6 day, and the
treated with LPS for 20 hr. Cells were collected and subjected to
Western blot analysis using TGase 4 specific antibody. Western blot
analysis showed that TGase 4 protein level was increased during
differentiation from BMCs to DCs, and protein level of TGase 4 was
further increased with LPS treatment (Figure 13).
To further confirm the involvement of TGase 4 in DC maturation,
we examined the knock-down effect of TGase 4 on DC maturation.
BMCs were differentiated to DCs for 6 day, and cells were
transfected with siRNA for GFP and TGase 4. GFPi was used as
positive control. Surface markers of maturated DCs were measured
by FACS analysis. DCs transfected with siRNA for TGase 4 showed
significantly decreased expression of CD80, CD86 compared to
44
transfected with siRNA for GFP (Figure 14). These results indicate
that TGase 4 is involved in the maturation of DC.
45
Table 4. Primers of TGases
46
Figure 12. TGase 4 is a strong candidate that is involved in DC
maturation.
After 6 days DCs were treated LPS (1 μg/ml) for 3, 6, and 18 hr.
And then DCs were collected and total RNA was extracted from
cultured DCs using TRIzol reagent (Invitrogen). The RNA was then
employed in the first-strand cDNA synthesis reations using Maxime
RT PreMix Kit (iNtRON). The PCR products were amplfied using
each primer.
47
Figure 13. TGase 4 is increased along with differentiation of DC.
40 mg of concentrated BM-drived DCs for each time point after
differentiation and in the presence of the LPS stimulation were
loaded and immunoblot was performed with anti-TGase 4 monoclonal
antibody.
48
Figure 14. TGase 4 is involved in maturation of DC
We transfect siRNA for TGase 4 and GFP after 6 day of DC culture
And then DCs were treated with LPS or not for 20 hr. Maturation markers
were analyzed by FACS.
49
Functional analysis of TGase 2 deficient DCs: cytokine secretion
Figure 10 and 11 showed significantly higher TGase activity of
wild-type DCs than TGase 2 deficient DCs. To investigate the role of
TGase 2 in DC function, we examined cytokine level secreted by DCs
upon LPS treatment. Under proper stimulation, DCs are able to
secrete IL-10 and IL-12 which play a central role in the regulation of
the immune response (Matic, Sacchi et al., 2010). We measured IL-
10 and IL-12 released from DCs after LPS stimulation. We found no
difference between wild-type and TGase 2-deficient DCs, indicating
that TGase 2 is not involved in cytokine secretion of DC (Figure 15).
50
Figure 15. TGase 2 is not involved in function of matured DCs.
The quantification of mouse IL-10(A), 12(B) mRNA in DCs from wild
-type BMCs as well as TGase 2-deficient BMCs treated with LPS for
20 hr or not by using real-time quantitative PCR.
51
Functional analysis of TGase 2 deficient DCs: antigen uptake
Previous reports showed that TGase 2 play a role in phagocytosis
through phagocytosis-associate crosstalk between macrophages and
apoptotic cells. Moreover, TGase 2 also contributes to phagocytotic
ability of neutrophil (Balajthy et al., 2006). To test whether TGase 2
also play a role in phagocytosis of DC, BMCs were differentiated to
DCs for 6 day, and cells were incubated with FITC labeled dextran
for 15 min, 30 min, and 45 min. Flow cytometric analysis revealed
that there is no difference in percentage of FITC positive DC
populations between wild-type and TGase 2-deficient DCs,
indicating that TGase 2 is not involved in phagocytosis of DC (Figure
16).
52
Figure 16. TGase 2 is not involved in phagocytosis of DCs
DCs differentiated from TGase 2-deficient BMCs and wild-type
BMCs for 6 days were incubated with FITC-labeled E coli at 37 ℃
for each designated times. FITC positive cells out of total BM-drived
DCs were counted by FACS analysis. Each experimental group
included 2 to 6 mice, and each individual experiment was performed
2 or 3 timed in triplicates.
53
Functional analysis of TGase 2 deficient DCs: migration
In regulating immune response, DCs have a unique ability to migrate
peripheral lymph node and then to stimulate naive T lymphocytes.
We tested whether TGase 2 affects migration ability of DCs. When
DCs are activated, DCs express receptors to their surface that
enable to migrate to peripheral lymph node. One of major receptors
participating migration is CCR7. To investigate migration of DCs we
use specially designed microscopy chamber. It has two sides. DCs
were placed on one side and ligand of CCR7, CCL21, were placed on
the other side. And then DCs moving toward CCL21 were measured
by confocal microscopy every 2 min for 4 hr. Four sets were
measured independently; wild type DC, wild-type DC treated with
LPS, TGase 2 deficient DC and TGase 2 deficient DC treated with
LPS. Data obtained from confocal microscopy were analyzed at the
three factors which are accumulated distance, euclidean distance,
and velocity. In all factors, no difference was observed (Figure 17).
Moreover, when measured mRNA and protein level of CCR7, major
factor affecting this assay, both wild-type and TGase 2-deficient
DCs showed the similar expression level of CCT7 mRNA and protein
(Figure 18), indicating that TGase 2 is not involved in migration of
DCs.
54
Figure 17. TGase 2 is not involved in migration of maturated DC.
Accumulated distances, Euclidean distances, and Velocities of
chemotaxing BM-derived single DCs (dot;line,mean) towards CCL21
in the presence or absence of LPS stimulation (1 μg/ml) in 3D
collagen matrices.
55
Figure 18. TGase 2 is not involved in expression of CCR7.
(A) After LPS stimulation, CCR7 positive DCs positive were
analyzed by flow cytometry analysis. (B) The quantification of mouse
CCR7 mRNA in DCs from wild-type BMCs as well as TGase 2-
deficient BMCs treated with LPS for 20 hr or not by using real-time
quantative PCR.
56
Functional analysis of TGase 2 deficient DCs: apoptosis
It has been considered that apoptosis of DC is critical for immune
suppression. Therefore, defect in apoptosis of DC can induce
autoimmune diseases (Chen, Wang et al., 2006). Moreover, TGase 2
is involved in apoptosis. We investigated whether TGase 2 is
involved in DC apoptosis. DCs differentiated from wild type and
TGase 2-deficient BMCs for 6 days were activated with LPS or LPS
and thapsigargin. The percentages of living cells were measured at
various time points. No difference between wild-type and TGase 2
deficient DC was observed (Figure 19), indicating that TGase 2 is not
involved in apoptosis of DCs.
57
Figure 19. TGase 2 is not involved in apoptosis of maturated DC.
BM-drived DCs from wild-type mice as well as TGase 2-deficient
mice survival after incubation with LPS (1 μg/ml) or thapsigargin (50
nM). Survival of unstimulated cells kept in culture medium.
58
Functional analysis of TGase 2 deficient DCs: ROS production
Previous reports showed that gp91phox, one of major components of
NOX2, is down-regulated in TGase 2-deficient neutrophil
granulocyte (Balajthy et al., 2006). In human dendritic cells, NADPH
oxidase is regulated by Toll receptor. Oxygen radicals generated by
NADPH oxidase is not associated with DC differentiation, maturation,
cytokine production, and induction of T cell proliferation (Vulcano et
al., 2004), but is required for DC killing of intracellular Escherichia
coli. Therefore, we tested whether TGase 2 is involved in ROS
generation through modulation of NOX2 expression. When DCs were
treated with LPS, wild-type DC showed significant increase of
p67phox and gp91phox expression, whereas TGase 2-deficient DCs
showed no change in the expression of NOX2 components (Figure
21). Thus, TGase 2 is involved in the regulation NOX2 expression of
DC.
59
Figure 20. TGase 2 is not involved in ROS release of maturated DC.
Release of ROS from BM-drived DCs from wild-type mice and TGase2
-deficient mice left unstimulated or stimulated with LPS (1 g/ml)
(a,b) and with PMA (0.5 g/ml) or PMA and DPI (10 M)
60
Figure 21. TGase 2 is involved in mRNA level of NOX2 components
After 6 days DCs were treated LPS (1 μg/ml) for 3, 6, and 18 hr.
And then DCs were collected and total RNA was extracted from
cultured DCs using TRIzol reagent (Invitrogen). The RNA was then
employed in the first-strand cDNA synthesis reactions using Maxime
RT PreMix Kit (iNtRON). The PCR products were amplified using
each primer.
61
Discussion
Regulating innate immunity and adaptive immunity by DC is one of
the most important immune responses in immune system. It has
capacity to respond directly to an invading pathogen. So, DC is
considered as great therapeutic target. Recently, it has been
reported that treatment of DC with LPS up-regulates the expression
of TGase 2 that catalyzes the post-translational modification of
protein by forming polyaminated proteins or cross-linked proteins.
However, role(s) of TGase 2 in DC has been unclear. In this study,
role(s) of LPS-induced TGase 2 in DC was investigated using TGase
2-deficient DC.
Although DC treated with LPS up-regulates the expression of
TGase 2 as well as transamidation activity, differentiated DC
population and matured DC markers didn’t show difference between
DCs from wild-type and TGase 2-deficient mice. However, like
previous study that showed TGase inhibitor suppresses the process
of DC maturation, our data also showed the same results. That is
other TGase isoenzyme may play a role in the process of DC
maturation. And we found that increase of TGase activity by LPS
treatment is due to expression of TGase 4.
Our data also showed significantly higher TGase activity of wild-
type DCs than TGase 2-deficient DCs and it suggested that TGase 2
may play the important role in DC function. Seeing our data, TGase
62
2-deficient DC showed no difference in the level of secreted
cytokine, phagocytosis, migration, and apoptosis compared with
those of wild-type DC. However, wild-type DC showed higher level
of ROS than TGase 2-deficient DC.
Taken together, our results indicated that role of TGase 2 in DC
function might be involved in the generation of ROS. To be more
definite, we need to more studies by which mechanism(s) TGase 2 is
involved in the generation of ROS.
63
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요약 (국문 초록)
수지상세포는 항원을 present하는 세포로서, innate 와 adaptive 면역의
조절에 중요한 역할을 한다. 트랜스글루타미네이즈 2 (TG2)는 단백질의
글루타민 잔기와 라이신잔기 사이의 교결합 형성하거나 또는 단백질에
폴리아민을 결합시키는 칼슘 의존성 효소이다. 따라서 TG2 는 이러한
수식을 촉매함으로서 단백질 기능을 조절하는 역할을 한다. 또한 TG2는
수지상세포에서도 발현되는데, LPS 로 처리하면 발현이 증가한다. 본
연구는 수지상세포에서 LPS 에 의하여 증가된 TG2 의 기능을 규명하기
위하여 시행되었다. 이를 위하여 wild-type 과 TG2-/-mice 골수에서
채취한 세포를 이용하여, 수지상세포 분화와 성숙 marker 의 발현을
비교하였다. 그 결과, TG2 가 없는 수지상세포는 wild-type 과
비교하여 분화와 성숙능력에서 차이가 없었다. 뿐만 아니라, 분화된
수지상세포의 기능인 사이토카인 분비, 세포이동, 포식작용 그리고
세포사멸에서 두 종류의 수지상세포간에 차이가 없었다. 반면, wild-
type 세포는 활성산소를 많이 생산하였다. 또한 LPS 자극에 의한
TG 활성의 증가는 TG4 발현증가에 기인함을 밝혔다. 이 결과는
수지상세포에서 TG2 가 분화나 성숙과정에는 관여하지 않으나,
활성산소 생산에 중요한 역할을 하며, TG4 가 성숙과정에 중요한
역할을 할 가능성을 보여준다.
주요어 : 트랜스글루타미네이즈, 수지상세포, 분화, 성숙
학 번 : 2010-23740