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J Periodont Res 2017 2017 John Wiley & Sons A/S.

All rights reserved Published by John Wiley & Sons Ltd

JOURNAL OF PERIODONTAL RESEARCH


doi:10.1111/jre.12445

W. Li1,2,3,*, W. Zhu1,*, J. Hou1,


Vitamin D-binding protein H. Meng1
1
Department of Periodontology, Peking

expression in healthy tooth


University School and Hospital of Stomatology,
Haidian District, Beijing, China, 2Department of
Periodontology, The Affiliated Hospital of
Qingdao University, Qingdao, Shandong

and periodontium: an Province, China and 3Key Laboratory of Oral


Clinical Medicine, College of Stomatology,
Qingdao University, Shinan District, Qingdao,

experimental study both in Shandong Province, China

monkeys in vivo and in


humans in vitro
Li W, Zhu W, Hou J, Meng H. Vitamin D-binding protein expression in healthy
tooth and periodontium: an experimental study both in monkeys in vivo and in
humans in vitro. J Periodont Res 2017; doi: 10.1111/jre.12445. 2017 John Wiley
& Sons A/S. Published by John Wiley & Sons Ltd

Background and Objective: Vitamin D-binding protein (DBP) is a highly


expressed plasma protein with many important functions, including transport of
vitamin D metabolites, sequestration of actin, control of bone metabolism and
modulation of immune and inammatory responses. Previous results of our
study indicated an association between DBP and periodontitis. We hypothesized
that periodontium might be another source of DBP in gingival crevicular uid
other than serum.

Material and Methods: DBP expression was examined in dental and periodontal
tissues of monkeys by immunohistochemistry, and in primary cells isolated from
human dental and periodontal tissues by reverse transcription plus the poly-
merase chain reaction and immunocytochemistry. Huanxin Meng, PhD, Department of
Periodontology, Peking University School and
Results: DBP was constitutively expressed and widely distributed in dental and Hospital of Stomatology, 22 Zhongguancun
periodontal tissues of primates. Their immunoreaction was evident in gingival Nandajie, Haidian District, Beijing 100081,
China
epithelium, particularly in junctional epithelium, and in mineralizing areas of Tel:+86 82195368
the dental pulp, periodontal ligament and bone marrow. Correspondingly, Fax: +86 62173402
mRNA and protein expression were detected in primary human gingival epithe- e-mail: kqhxmeng@bjmu.edu.cn
*Both authors have contributed equally to the
lial cells, dental pulp cells and periodontal ligament cells. work.

Conclusion: DBP is highly expressed and widely distributed in dental and peri- Key words: dental and periodontal tissue;
human; immunochemistry; monkey; vitamin D-
odontal tissues, which may take an active part in local host defense and hard
binding protein
tissue metabolism.
Accepted for publication December 9, 2016

Vitamin D-binding protein (DBP) is a (1). As its name suggests, it was ini- binding sites were occupied (3). Such
multifunctional and highly expressed tially recognized as the major carrier an excess came up with the possibility
plasma protein. It is synthesized pre- of vitamin D and its metabolites that the primary role of DBP
dominantly by hepatic parenchymal throughout the body (2). Soon after- extended far beyond acting solely as a
cells and at lower levels, by tissues wards, it was found that at any one transporter. Thereafter, multiple addi-
such as kidney, testis and placenta time, only 12% of the vitamin D- tional functions have been described,
2 Li et al.

such as extracellular actin scavenging, Laboratory Animal Center of Acad- PDLCs and GFs were maintained in
complement component 5a (C5a)- emy of Military Medical Sciences, Dulbeccos modied Eagles medium
mediated leukocyte chemotaxis, Beijing, China), were rst xed in (Gibco, Grand Island, NY, USA) sup-
macrophage activation and control of 10% buered formalin and then plemented with 10% (v/v) fetal bovine
bone metabolism (4). decalcied in 10% EDTA, dehydrated serum (Gibco) at 37C in a humidied
Periodontitis is an inammatory and embedded in paran. Micro- atmosphere of 5% CO2, while gingival
disorder aecting the supportive tis- tome serial mesio-distal sections epithelial cells replaced with dened
sues of teeth, resulting in attachment (5 lm) were cut and mounted on to keratinocyte serum free medium
loss, formation of periodontal pockets, adhesive slides. All sections were (Gibco). After the cells were 80% con-
resorption of the alveolar bone and examined by hematoxylin and eosin uent, primary cells were digested with
ultimately tooth loss. Although initi- staining. 0.25% (w/v) trypsin and 0.02% (w/v)
ated by bacteria, it is the host immune EDTA and subcultured. Cells at pas-
response that determines the destruc- sages 24 were used for the following
Vitamin D-binding protein
tive process of the disease, in which experiments. The study protocol was
immunohistochemistry
cytokines play important roles. The reviewed and approved by the ethical
aforementioned functions of DBP After deparanization and rehydra- board of Peking University School and
might happen to be implicated in peri- tion, selected sections were soaked in Hospital of Stomatology (PKUSSIRB-
odontitis. As hypothesized, previous 3% hydrogen peroxide for 10 min at 2011007). Human dental pulp cells
studies from our group pointed to an room temperature to inhibit endoge- (DPCs) were kindly donated by Dr.
association between DBP and peri- nous peroxidase and digested by 1 mg/ Sainan Wang (Department of Odon-
odontitis. Patients with generalized mL trypsin for 10 min at 37C for anti- tology, Peking University School and
aggressive periodontitis had signi- gen retrieval. After blocking with 10% Hospital of Stomatology).
cantly higher DBP concentration in normal goat serum at room tempera-
plasma while lower in gingival crevicu- ture for 10 min, sections were incu-
Detection of vitamin D-binding
lar uid compared with healthy con- bated with rabbit anti-recombinant
protein transcription in human
trols (5,6). Further analysis showed human DBP polyclonal antibody
primary cells
that in healthy conditions, the levels of (1 : 1000; Abcam, Cambridge, MA,
DBP in gingival crevicular uid were USA) or normal rabbit IgG (Santa Total RNA was isolated from the
even higher than those in plasma. This Cruz, Santa Cruz, CA, USA) at 4C above cells using TRIzol reagent
indicated that DBP might play a role overnight. The staining of DBP was (Invitrogen, Carlsbad, CA, USA) fol-
in periodontal health and that peri- performed via a polymer/HPR and lowing the manufacturers instructions
odontal tissues might be another DAB chromagen system (Zhongshan and was quantied by spectrophoto-
source of gingival crevicular uid DBP Golden Bridge Biotechnology, Beijing, metric absorbance at 260 nm. Approx-
other than serum (6). Furthermore, we China). Finally, the sections were imate 2 lg total RNA was reverse
carried out this study with the use of counterstained with hematoxylin. All transcribed to cDNA via a RevertAid
dental and periodontal tissues of mon- sections were examined under a digital First Strand cDNA Synthesis kit
keys and tissue cells isolated from microscopic system (Olympus BX51/ (Thermo Fisher Scientic, Waltham,
human dental and periodontal tissues, DP72, Tokyo, Japan). MA, USA). Polymerase chain reac-
to examine systematically the DBP tion (PCR) was then performed with
expression and distribution and to the use of a Taq PCR MasterMix
Culture of human primary cells
infer its possible roles in health condi- (Solarbio Science & Technology, Bei-
tions. For conrmation of the results from jing, China). The primers used were as
monkeys, primary human periodontal follows: GAPDH, forward: CGA-
ligament cells (PDLCs), gingival CAGTCAGCCGCATCTT, reverse:
Material and methods
broblasts (GFs) and gingival epithe- CCAATACGACCAAATCCG
lial cells were cultured with tissue TTG; DBP, forward: TGGCTACCA
Tissue sampling
explant methods generally as described CTTTTACATGGTC, reverse: TCAA
With the approval of the Experimen- previously (911). Briey, PDLCs were ACGTGCCACTGGGAAA. The prod-
tal Animal Welfare Ethical Branch of obtained by explants dissected from ucts were analyzed by agarose gel elec-
Peking University Biomedical Ethics the mid-root of premolars extracted trophoresis and nucleotide sequencing.
Committee (LA2008-006), dental and from donors under orthodontic treat-
periodontal specimens were collected ment with a sharp scalpel and then
Detection of vitamin D-binding
from monkeys (Macaca fascicularis) minced into small pieces. GFs and gin-
protein expression in human
from our earlier experiments (7,8). gival epithelial cells were derived from
primary cells
Excised mandibular premolars and healthy gingival tissues under crown-
molars with their periodontal tissues, lengthening surgeries. The gingival Protein of DBP in the above cells was
from three adult male monkeys (5.5 epithelium and connective tissues were detected by immunocytochemistry.
6.0 years old, weighing 5.15.5 kg; dissected and minced separately. After seeding on glass slides for 8 h,
DBP expression in periodontium 3

cells were xed in 95% ethanol for inhibit endogenous peroxidase for serum for 10 min, cells were incu-
30 min at room temperature. Then, 10 min at room temperature. After bated with the same antibody used
3% hydrogen peroxide was used to blocking with 10% normal goat above or normal rabbit IgG at 4C

B C

D E

F G

H I

Fig. 1. (A) Immunohistochemistry of vitamin D-binding protein in dental and periodontal tissue; (B, D, F, H) magnication of the boxed
areas in gingiva, pulp, periodontal ligament and alveolar bone of (A), respectively; (C, E, G, I) high magnication of the boxed areas in
(B, D, F, H), respectively. AB, alveolar bone; D, dentin; G, gingiva; P, pulp; PDL, periodontal ligament. Solid arrows, osteoblasts in peri-
odontal ligament lying next to the alveolar bone; solid triangles cementoblasts in periodontal ligament lying next to the cementum; aster-
isks, fat cells in alveolar bone marrow; hollow arrows, osteoblasts in alveolar bone marrow lining the marrow cavities. Scale bars: (A)
1 mm; (B, D, F, H) 200 lm; (C, E, G, I) 50 lm.
4 Li et al.

overnight. The staining of DBP was negative for DBP (Fig. 1D). However, Generally, the results were consistent
performed in the same way and odontoblasts lying next to the dentin with those observed in dental and
hematoxylin was used for counter- were signicantly positive, some of periodontal tissues of the monkeys
staining. whose cytoplasmic processes extending in vivo.
into the dentinal tubules even showed
DBP immunoreactivity (Fig. 1E). Peri-
Results Discussion
odontal ligament was diusely positive
for DBP (Fig. 1F), with osteoblasts DBP is characterized by widespread
Vitamin D-binding protein
(Fig. 1G solid arrows) lying next to distribution into the tissues and can
expression in dental and
the alveolar bone and cementoblasts be determined most abundantly in
periodontal tissues of monkeys
(Fig. 1G solid triangles) lying next to plasma and less in cerebrospinal uid,
in situ
the cementum were evenly positive. In seminal uid, saliva and breast milk
DBP was detected in gingival epithe- alveolar bone marrow (Fig. 1H), fat (12). As far as we know, this is the
lium, dental pulp, periodontal liga- cells (Fig. 1I asterisks) and osteoblasts rst study to identify systematically
ment and bone marrow (Fig. 1). (Fig. 1I hollow arrows) lining the bone the expression and distribution of
Specically, in gingival epithelium, marrow cavities were DBP positive as DBP in dental and periodontal tissues
DBP was strongly positive in all layers well. Results of the negative controls both in monkeys in vivo and in
of the junctional epithelium (Fig. 1B conrmed the specicity of the humans in vitro. Signicantly, gingival
and 1C), evenly positive from stratum immunoreaction (data not shown). epithelial cells, DPCs and PDLCs,
basale to stratum granulosum of the these unique cells from dental and
oral and sulcular epithelium (from periodontal tissues, all expressed DBP
Vitamin D-binding protein
other samples, Fig. 2), while much to some extent. Combined with our
expression in human cells isolated
stronger in the supercial hydropic previous results that in healthy condi-
from dental and periodontal tissues
degenerated cells of the sulcular epithe- tions the levels of DBP in gingival
in vitro
lium (Fig. 2C solid triangles) and crevicular uid were higher than those
much weaker in stratum corneum of Reverse transcription plus the PCR in plasma (6), it is reasonable to
the oral epithelium (Fig. 2B). More- revealed the transcription of DBP
over, the staining seemed to become mRNA in human PDLCs, GFs,
weaker from junctional epithelium and DPCs and gingival epithelial cells
sulcular epithelium to oral epithelium. (Fig. 3). Relatively, DBP was primar-
In gingival connective tissue, most cells ily expressed in gingival epithelial
were DBP-negative (Fig. 2A), with cells, less in DPCs and the least in
only some GFs near the inammatory PDLCs. The transcripts of DBP
zone weakly stained (Fig. 2C hollow mRNA in GFs could not be detected Fig. 3. Messenger RNA expression of
triangles). In addition, some polymor- in this condition. Immunocytochem- DBP in primarily cultured human gingival
phonuclear leukocytes (Fig. 2C hollow istry conrmed the results of reverse epithelial cells (lane 1), gingival broblasts
arrow) and mononuclear cells (Fig. 2C transcription-PCR. DBP staining was (lane 2), dental pulp cells (lane 3) and peri-
solid arrow) beneath the sulcular most prominent in gingival epithelial odontal ligament cells (lane 4). GAPDH
epithelium also expressed DBP. In the cells, followed by DPCs and PDLCs, was used as an internal control. DBP, vita-
pulp, cells in the pulp core were and negative in GFs (Fig. 4). min D-binding protein.

A B C

Fig. 2. (A) Immunohistochemistry of vitamin D-binding protein in gingiva; (B, C) high magnication of the boxed areas in oral epithe-
lium (left) and sulcular epithelium (right), respectively. Hollow arrows, polymorphonuclear leukocytes; solid arrows, mononuclear cells;
hollow triangles, weakly positive gingival broblasts; solid triangles, supercial hydropic degenerated cells of the sulcular epithelium. Scale
bar: (A) 200 lm; (B, C) 50 lm.
DBP expression in periodontium 5

A B C D

Fig. 4. Protein expression of vitamin D-binding protein in primarily cultured human human gingival epithelial cells (A), gingival brob-
lasts (B), dental pulp cells (C) and periodontal ligament cells (D). Scale bar: 50 lm.

presume periodontal tissues to be DBP was suggested to be an impor- DBP might have a more generalized
another source of gingival crevicular tant scavenger of endotoxin (19). And regulating role in bone formation and
uid DBP other than serum. the concentrations of DBP were ever remodeling. As alveolar bone is the
DBP is known for its role as a used to predict organ failure in peri- most dynamic in the skeletal system,
transporter of vitamin D. Vitamin D tonitis (20). DBP was also reported to the abundance of DBP in periodontal
has a multilevel role in maintaining be associated with the surface of a ligament and bone marrow might be
periodontal health, including strength- large number of cells, such as neu- critical for mineralization homeostasis
ening epithelial barrier, stimulating trophils, broblasts, B and T cells, in healthy tissues.
antimicrobial peptides, enhancing etc. (21), which was also shown in our In conclusion, our study for the rst
macrophage activities and reducing results (Fig. 2C). When binding to time systematically determined the
bone loss (13). However, binding to C5a, DBP can enhance the C5a- expression and distribution of DBP in
DBP is a requisite for vitamin D to mediated neutrophil and monocyte dental and periodontal tissues. Tissue
be transported within the organism, chemotaxis (22,23). This can act as cells, including gingival epithelial cells,
facilitating access to tissues and cells, the rst defense in the periodontium DPCs and PDLCs, were all able to
as well as regulating the amount of and determine the switch for initiation synthesize DBP, which may be respon-
vitamin D available (14). Previous or resolution of periodontitis particu- sible for local host defense and hard
results from our group demonstrated larly at the initial stage. During tissue metabolism. Further research on
that in vitro human GFs and PDLCs inammation, activated T and B cells DBP expression during periodontal
could uptake 25-hydroxyvitamin D3 are able rapidly to transform the DBP inammation and its function in peri-
to synthesize 1,25-dihydroxyvitamin into a potent macrophage-activating odontal tissues should be carried out
D3 so as to make it possible for vita- factor (MAF) (24). Thereafter, this for understanding its roles in peri-
min D to act in an autocrine/para- DBP-MAF can activate macrophages odontitis.
crine manner in these cells (15). Our for the benet of the host defense
nding of DBP expression in peri- (25). However, if the inammation Acknowledgements
odontal tissues helps to make these overreacts, DBP-MAF can induce
further convincing in the periodontal apoptosis in activated macrophages The authors thank Ms. Mingjie Wei
tissues in vivo. via caspase induction as well (26). (Department of Oral Pathology, Pek-
In addition to vitamin D, DBP also Therefore, the expression of DBP in ing University School and Hospital of
acts as binding protein of actin (14). the periodontium, particularly in the Stomatology) for her expert assistance
Upon cell lysis, actin is released into junctional epithelium, may represent with the preparation of histologic
the extracellular environment, where an important mechanism for local specimens. This study was supported
it goes on to nucleation and polymer- protection in periodontal health and by National Natural Science Founda-
ization and has the potential to block inammation. tion of China 81271149.
and damage the microvasculature Furthermore, DBP-MAF is also
(16). Periodontitis has been shown to involved in bone metabolism. In cases Competing interests
be associated with endothelial and of inammation, the production of
microvascular dysfunctions (17). DBP-MAF could contribute to bone The authors declare no conicts of
Thus, the presence of DBP in peri- resorption through its eects on mod- interest related to this study.
odontal tissues, which binds actin ulating osteoclast calcium sensing
with high anity (18), must have con- (27). In an in vitro assay, DBP-MAF References
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