Original Article

Fibrillin-1 regulates periostin expression during maintenance of periodontal homeostasis

Yoshikazu Manabea, Momotoshi Shigaa, Kaori Kometani-Gunjigakea, Kayoko NakaoKuroishia, Masahiro Mizuharaa, Takashi Toyonob, Yuji Setab, Tatsuo Kawamotoa*aDivision of Orofacial Functions and Orthodontics, Kyushu Dental University, Kitakyushu,

Japan.
b

Division of Anatomy, Kyushu Dental University, Kitakyushu, Japan.

*Corresponding author:
Division of Orofacial Functions and Orthodontics, Kyushu Dental University, 2-6-1
Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan. Fax: +81-93-582-6000.
E-mail address: r15kawamoto@fa.kyu-dent.ac.jp (T. Kawamoto)
Running title
FBN1 regulates expression of POSTN
Abstract
Background/purpose: Human periodontal ligament consists of elastic system fibers, mainlyfibrillin-1 (FBN1). Periostin (POSTN) maintains periodontal homeostasis. A previous study
showed that the expression of Postn in periodontal ligament cells was decreased in mice
underexpressing Fbn1. However, the relationship between FBN1 and POSTN is not fully
understood in the context of mechanical stress. FBN1 contributes to transforming growth factor
β1 (TGF-β1) activation; TGF-β1 upregulates the expression of POSTN in human periodontal
ligament cells. This study examined whether FBN1 contributed to the maintenance of
periodontal homeostasis in cultured human periodontal ligament cells.

Materials and methods: Human periodontal ligament fibroblasts (HPDLFs) were exposed tomechanical force via centrifugation. The expression of POSTN was examined by quantitative
reverse transcription polymerase chain reaction. The phosphorylation of Smad2 in the TGFβ/Smad signaling pathway was monitored by western blotting.

Results: The expression levels of FBN1 and POSTN were not significantly decreased bycentrifugation. However, the expression of POSTN after centrifugation significantly decreased
upon knockdown of FBN1. The phosphorylation of Smad2 after centrifugation was decreased,regardless of FBN1 knockdown. Supplementation with 0.1 ng/ml recombinant human TGF-β1
rescued POSTN expression after centrifugation in HPDLFs upon knockdown of FBN1.

Conclusion: FBN1 regulates the expression of POSTN to maintain periodontal homeostasis via
TGF-β/Smad signaling during centrifugation.

Key words
Human periodontal ligament fibroblasts, fibrillin-1, periostin, periodontal homeostasis, TGFβ1

Introduction
Periodontal tissue contains the periodontal ligament, which senses mechanical stress (e.g.,
occlusion). Under mechanical stress, human periodontal ligament cells promote collagen fiber
production and remodeling according to the magnitude and direction of strain.1-3 They induce
molecules such as prostaglandin-endoperoxide synthase 2 and respond to mechanical stress.4
The periodontal ligament thus contributes to the maintenance of periodontal homeostasis.
However, the underlying mechanisms have been unknown.
The human periodontal ligament consists of two types of fibers: elastic system andcollagen. Elastic system fibers are present in tissues that require elasticity, such as blood vessels;
they work against external forces.5,6 Elastic system fibers in the periodontal ligament exist
parallel to the long axis of the root.7 Elastic system fibers consist of microfibrils and elastin;8
they are classified into three types according to the relative proportions of microfibrils andelastin. Most elastic system fibers in the periodontal ligament are oxytalan fibers, which include
only microfibrils.9 The major constituent protein of microfibrils is fibrillin-1 (FBN1),10 which
is important for the storage of transforming growth factor β1 (TGF-β1) via latent TGF-βbinding protein 1 (LTBP1);11-13 it also controls TGF-β1 activity in the matrix.14 FBN1 ispresumed to regulate human periodontal ligament cell alignment.15
The periodontal ligament also contains periostin (POSTN).16 POSTN is an important
mediator of the biomechanical properties of fibrous connective tissue.17 In a previous study,

Postn-null mice showed irregular periodontal ligament, alveolar bone resorption, and externalroot resorption because of occlusal loading.18 Those findings suggested that POSTN maintains
periodontal tissue structure and contributes to periodontal ligament homeostasis. Mechanicalforce causes the release of signaling molecules (e.g., cytokines and colony-stimulating factors)
via blood vessels.19 Although some studies have described homeostasis of the extracellularmatrix, the underlying mechanisms have been unknown. Here, we focused on FBN1 and
POSTN. A previous study reported that the expression of Postn in periodontal ligament cells
was decreased in mice underexpressing Fbn1;20 however, it has been unclear whether FBN1
regulates the expression of POSTN under mechanical force. Thus, we investigated whether
FBN1 maintained the expression of POSTN in human periodontal ligament fibroblasts(HPDLFs) under mechanical force.

Materials and methods
Cell isolation and culture
HPDLFs were purchased from Lonza Japan, Inc. (Tokyo, Japan). The research protocol wasapproved by the Research Ethics Committee of Kyushu Dental University (permission number:
17-37). HPDLFs were cultured in alpha-minimum essential medium (Gibco BRL, Grand Island,
NY, USA), supplemented with 10% fetal bovine serum (Biosera, Nuaillé, France), 100 U/mL
penicillin G (Gibco BRL), 100 µg/mL streptomycin (Gibco BRL) and 100 µg/mL amphotericin
B (Gibco BRL); they were incubated at 37°C with 5% CO2. Cells from passages four throughsix were used for all experiments.

Mechanical force application
To simulate the compression side during orthodontic tooth movement, we applied mechanicalforce to HPDLFs by centrifugation using a previously published protocol.21 HPDLFs were
cultured at 33.6 × 103 cells/well in six-well plates until they reached sub-confluence; themedium was subsequently exchanged for Dulbecco’s modified Eagle medium (Gibco BRL)
supplemented with 90 µL/well HCl (Nacalai Tesque Inc., Kyoto, Japan) to stabilize pH andcells were incubated at 37°C for 24 hours (Yamato Scientific Co., Ltd., Tokyo, Japan). Finally,a centrifuge (Kubota Co., Tokyo, Japan) in the incubator was used to apply mechanical forcesof 40, 90, and 135 g for 12 and 24 hours.

Cell damage assay
Lactate dehydrogenase release from cells was quantified to evaluate cell damage caused bymechanical force. After centrifugation treatment, we measured the absorbance (optical density[OD] value) of formazan dye at 490 nm with a spectrophotometer (iMark™ Microplate
Absorbance Reader, Bio-Rad Laboratories, Inc., Hercules, CA, USA), using the Cytotoxicity
Lactate Dehydrogenase Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan). The
supernatant from cells not subjected to mechanical force was used as a low control; thesupernatant with lysis buffer added 30 minutes before collection was used as a high control.
After the background control OD value was subtracted from the OD value of each well,cytotoxicity was calculated using the following formula: cytotoxicity (%) = (OD value ofexperimental group − OD value of low control) / (OD value of high control − OD value of low
control) × 100%.

HPDLF treatments
For gene silencing experiments, small interfering RNA targeting human FBN1 (5′ACCGGTTTACCCGTTGATATT-3′) was purchased from Qiagen (Qiagen, Venlo, Limburg,
the Netherlands); the expression of FBN1 was suppressed using Lipofectamine™ RNAiMAX
Transfection Reagent (Invitrogen Co., Carlsbad, CA, USA) in accordance with themanufacturer’s instructions. The All Stars Negative control (Qiagen) was used as a scrambled
control for gene silencing experiments. For stimulatory experiments, 0.001, 0.01, 0.1, 1.0, and10 ng/ml recombinant human TGF-β1 (PeproTech, Cranbury, NJ, USA) were used.

Quantitative reverse transcription-polymerase chain reaction
Total RNA was isolated from HPDLFs and purified using the RNAqueous Total RNA Isolation
Kit (Ambion, Inc., Austin, TX, USA), in accordance with the manufacturer’s instructions. Total
RNA was treated with DNase (Thermo Fisher Scientific, Waltham, MA, USA); 0.5 µg RNA
was reverse transcribed into cDNA using SuperScript VILO Master Mix (Invitrogen Co.). The
expression levels were evaluated by quantitative reverse transcription polymerase chain
reaction using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA)and QuantStudio 3 Real-Time PCR System (Applied Biosystems). Primers for the following
gene transcripts were used: FBN1, POSTN, bone morphogenetic protein 2 (BMP2), connective
tissue growth factor (CTGF), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Table1). Polymerase chain reaction amplification consisted of 40 cycles of denaturation at 95°C for
15 seconds, followed by annealing and extension at 60°C for 1 minute. The specificity of thepolymerase chain reaction product was confirmed by melt curve analysis. GAPDH was used asa reference gene. Relative expression was quantified using the ΔΔCt method.

Western blotting
Cells were lysed in sample buffer supplemented with protease and phosphatase inhibitor
cocktails (both from Nacalai Tesque). Protein concentrations were determined using protein
assay reagent (Cytoskeleton, Denver, CO, USA); for each sample, 20 µg protein were separated
by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred topolyvinylidene difluoride membranes. Membranes were incubated for 12 hours at room
temperature with primary antibodies against Smad2/3 (1:2000, Cat. No.: 8685; Cell Signaling
Technology, Beverly, MA, USA) and phospho-Smad2 (1:1000, Cat. No.: 3108; Cell Signaling
Technology); they were incubated for 1 hour at room temperature with a primary antibody
against β-actin (1:5000, Cat. No.: sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Membranes were then washed and incubated at room temperature for 1 hour with horseradish
peroxidase-conjugated secondary antibody (1:25000, Cat. No.: 715035150; Jackson
ImmunoResearch, West Grove, PA, USA). Protein bands were visualized using Western
Chemiluminescent HRP Substrate (Millipore, Boston, MA, USA). Images were quantified by
ImageJ software (version 1.53e, National Institutes of Health, Bethesda, MD, USA). The
densities of protein bands were normalized to β-actin.

Statistical analysis
Data are shown as means ± standard errors. Two-way analysis of variance was used to analyzecytotoxicity after exposure to mechanical force. One-way analysis of variance was used toanalyze changes in the expression levels of genes after exposure to mechanical force and FBN1
knockdown; it was also used to analyze changes in phospho-Smad2 levels. To identify
statistically significant differences between groups, Tukey's test was used for post hoc multiple
comparisons. Values of p < 0.05 were considered statistically significant.

Results
Evaluation of cytotoxicity after exposure to mechanical force
To evaluate the effect of mechanical force on HPDLFs, cytotoxicity was investigated. Afterapplication of 135 g for 12 or 24 hours, cytotoxicity was significantly increased compared withapplication of 40 g for 12 or 24 hours (p < 0.05). However, there were no significant differencesin cytotoxicity between 40 and 90 g for 12 or 24 hours (Fig. 1). The mechanical forces createdby centrifugation at 40 g and 90 g did not cause different degrees of cytotoxicity, consistent
with previous findings.22 Thus, subsequent experiments were conducted using 90 g for 24 hours.

Changes in expression levels of FBN1 and POSTN in HPDLFs after exposure to mechanicalforce
To evaluate the effects of mechanical force on HPDLFs, expression levels of FBN1 and POSTN
were examined. There were no significant differences in the expression of FBN1 (Fig. 2A) or
POSTN (Fig. 2B).

Mechanical force-induced changes in expression levels of POSTN in HPDLFs upon knockdown
of FBN1
To clarify the involvement of FBN1 in POSTN expression under mechanical force, FBN1 was
knocked down and changes in POSTN gene expression were examined. First, FBN1 knockdown

was confirmed (Fig. 3A). POSTN expression was significantly decreased upon suppression of
FBN1 expression. This reduction of POSTN expression was greater after exposure tomechanical force (Fig. 3B).

Mechanical force-induced changes in expression levels of BMP2 and CTGF in HPDLFs upon
knockdown of FBN1
BMP2 and CTGF function to upregulate the expression of POSTN. Therefore, to determine theinvolvement of POSTN in expression of other genes, expression levels of BMP2 and CTGF
were examined. Centrifugation caused a significant increase in BMP2 expression; however,there were no significant differences in the expression levels of BMP2 upon knockdown of
FBN1 (Fig. 4A). Additionally, there was no significant difference in the expression of CTGF
under mechanical force, regardless of FBN1 knockdown (Fig. 4B).

Mechanical force-induced changes in Smad2 phosphorylation in HPDLFs upon knockdown of
FBN1
TGF-β1 binds to FBN1 via LTBP1; it is released from LTBP1 to activate and upregulate POSTN.
Therefore, to elucidate the involvement of FBN1 in POSTN expression, we examined thephosphorylation of Smad2, which functions downstream of the TGF-β/Smad signaling pathway.
Knockdown of FBN1 caused a significant decrease in the phosphorylation of Smad2.

Surprisingly, centrifugation enhanced the decrease in Smad2 phosphorylation (Fig. 5A, B).

Effects of TGF-β1 supplementation in HPDLFs upon knockdown of FBN1 during exposure tomechanical force
To explore the possibility that TGF-β1 affects the expression of POSTN, the medium wastreated with 0.001, 0.01, 0.1, 1.0, or 10 ng/ml TGF-β1. The expression of POSTN was
significantly increased upon supplementation with ≥ 0.1 ng/ml TGF-β1 (p < 0.01) (Fig. 6A).
Importantly, the mechanical force-induced reduction of POSTN expression upon knockdown of
FBN1 in HPDLFs was rescued by supplementation with 0.1 ng/ml TGF-β1 (p < 0.01) (Fig. 6B).

Discussion
To our knowledge, this is the first study to show that FBN1 maintains the expression of POSTN
under mechanical force. Our study examined the interrelationship between FBN1 and POSTN
in HPDLFs after exposure to mechanical force. The expression levels of POSTN in HPDLFs
did not significantly decrease under mechanical force. However, the expression of POSTN
significantly decreased upon knockdown of FBN1. These findings suggest that FBN1 is
important for maintaining periodontal tissue homeostasis under mechanical force.
Regarding FBN1 expression under mechanical force, a previous study showed that
FBN1 expression did not change when cyclic stretching was applied to human periodontal
ligament cells.23 Similarly, the present study showed that FBN1 expression did not change after
centrifugation (Fig. 2A). These results suggest that FBN1 expression does not change undermechanical force. To our knowledge, our report is one of few concerning FBN1 expression
under mechanical force.
Regarding POSTN expression under mechanical force, Panchamanon et al. subjected
human periodontal ligament cells to compression force using weighted coins; they found that
POSTN expression increased with compression force of 1.0 g/cm2 and decreased withcompression force of 2.0 g/cm2.24 The magnitude of the force used in the previous study was
equivalent to a centrifugal force of 1.9 g to 3.8 g, which considerably differed from thecentrifugal force of 90 g used in our study. Because we observed no changes in the expression

levels of FBN1 and POSTN under mechanical force, we presume that FBN1 contributes to themaintenance of POSTN expression.
Factors involved in the expression of POSTN include BMP2,25 CTGF,26 and TGF-β1.27
To evaluate these factors, we examined changes in their expression levels upon knockdown of
FBN1. The expression of BMP2 was significantly increased after exposure to mechanical force,
regardless of FBN1 knockdown (Fig. 4A). However, there were no significant differences inthe expression levels of CTGF (Fig. 4B). BMP2 induces new bone formation;28 it also activates
osteoclasts.29 The increased expression of BMP2—in response to tension force in the previous
study30 and compressive force in the present study—suggests increased bone metabolism. In
the present study, upon knockdown of FBN1, the expression of BMP2 was increased, whereas
the expression of POSTN was decreased. These findings implied that BMP2 was not involvedin the interaction between FBN1 and POSTN. Previous studies have shown that Ctgf is highly
expressed in rat osteoblasts during bone formation.31 CTGF may be important during boneformation. However, upon knockdown of FBN1, we found that the expression of CTGF did notchange, whereas the expression of POSTN was decreased (Fig. 4B). Our findings indicate that
CTGF is not involved in the interaction between FBN1 and POSTN.
The phosphorylation of Smad2, which functions downstream of the TGF-β1 signaling
pathway, was decreased after exposure to mechanical force, regardless of FBN1 knockdown(Fig. 5). A previous study showed that TGF-β1 was induced by intermittent compressive force

on human periodontal ligament cells and the expression of POSTN was induced by TGF-β1,27suggesting enhancement of Smad2 phosphorylation. Similar to our results regarding theexpression of POSTN upon knockdown of FBN1, the expression of POSTN may depend on the
TGF-β1 signaling pathway. Furthermore, in a study by Panchamanon et al., TGF-β1 and
POSTN expression levels increased when human periodontal ligament cells were subjected to
a compression force of 1.0 g/cm.24 These results indicate an important role for FBN1 in theexpression of POSTN under mechanical force via Smad2 phosphorylation and TGF-β1
signaling.
To further clarify the involvement of TGF-β1 in POSTN expression, we evaluatedchanges in POSTN expression upon TGF-β1 supplementation to HPDLFs. We found that theexpression of POSTN was rescued (Fig. 6B), implying that the expression of POSTN undermechanical force depends on TGF-β/Smad signaling. A previous study reported that FBN1
binds to TGF-β1 via latency-associated propeptide and LTBP1; TGF-β1 is activated upon
release from LTBP1.12,14,32 FBN1 is important for the storage of TGF-β1. The present study
demonstrated that the expression of POSTN was decreased upon knockdown of FBN1. The
underlying mechanism is that the expression of POSTN depends on active TGF-β1 release fromlatency-associated propeptide. FBN1 presumably binds to integrins present on the cell
membrane and participates in cell signaling;33 the integrin expression levels are increased with
24 hours of cyclic extension.34 These findings suggest that integrin expression decreases during

compression; TGF-β1 cell signaling was suppressed by reducing the expression of FBN1 forintegrin binding. A previous study showed that shear stress significantly reduced the expression
levels of the two major TGF-β receptors (types I and II) in mouse embryo mesenchymal
progenitor cells.35 The suppression of Smad2 phosphorylation in this study may be related todecreased TGF-β receptor expression. Additionally, a previous study showed that angiotensin
II was involved in the TGF-β1-mediated alteration of Postn expression.36 Another study
reported that mechanical force to HPDLFs led to enhanced secretion of angiotensin II.37 Further
studies are needed to elucidate the detailed mechanism underlying POSTN expression in
response to mechanical force.
Regarding the clinical significance of our findings, a previous study revealed fewer
oxytalan fibers in the central region of mouse periodontal ligament7; periodontal tissue
homeostasis might be maintained by controlling the application of orthodontic force. In
conclusion, we demonstrated that FBN1 contributes to the maintenance of POSTN expressionin HPDLFs after centrifugation treatment. Our results may help elucidate the mechanisms
involved in the maintenance of periodontal tissue homeostasis.

Declaration of competing interest
The authors declare that they have no conflicts of interest.

Acknowledgments
We thank Ryan Chastain-Gross, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draftof this manuscript.

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Figure Captions
Fig. 1 Cytotoxicity under mechanical force. Cytotoxicity was significantly increased by theapplication of 135 g for 12 and 24 hours compared with 40 g. However, it was not significantly
increased by the application of 90 g for 12 and 24 hours compared with 40 g (n = 3). For
subsequent experiments, 90 g mechanical force was applied for 24 hours. *p < 0.05, 40 g versus
90 g versus 135 g.

Fig. 2 Changes in the expression levels of FBN1 and POSTN under mechanical force in
HPDLFs, evaluated by quantitative reverse transcription-polymerase chain reaction (n = 3). The
relative expression levels are shown with the 0 g group regarded as 1. There were no significant
differences in the expression of FBN1 (A) and POSTN (B) upon exposure to 90 g for 24 hours.
FBN1: fibrillin-1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, HPDLFs: humanperiodontal ligament fibroblasts, POSTN: periostin.

Fig. 3 Changes in the expression levels of FBN1 and POSTN under mechanical force in
HPDLFs upon knockdown of FBN1, evaluated by quantitative reverse transcriptionpolymerase chain reaction. (A) Expression of FBN1 in HPDLFs. The relative expression levels
are shown with Scr regarded as 1. Expression of FBN1 was decreased compared with the Scrgroup (p < 0.01) (n = 3). This result confirmed reduced expression of FBN1. (B) Expression of

POSTN under mechanical force of 90 g in HPDLFs upon knockdown of FBN1. The relative
expression levels are shown with Scr-0 g regarded as 1. Expression of POSTN in Si-0 g was
decreased compared with Scr-0 g (p < 0.01) (n = 3). Similarly, the expression of POSTN in Si90 g was significantly decreased compared with Si-0 g (p < 0.05) (n = 3). (A) *p < 0.05, Scrversus Si. (B) * p < 0.05 or ** p < 0.01, Scr-0 g versus Scr-90 g versus Si-0 g versus Si-90 g.
FBN1: fibrillin-1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, HPDLFs: humanperiodontal ligament fibroblasts, POSTN: periostin, Scr: scrambled siRNA, Si: siRNA against
FBN1.

Fig. 4 Changes in the expression levels of BMP2 and CTGF under mechanical force in HPDLFs
upon knockdown of FBN1, evaluated by quantitative reverse transcription-polymerase chain
reaction. The relative expression levels are shown with Scr-0 g regarded as 1. (A) Expressionof BMP2 was significantly increased under mechanical force of 90 g, regardless of FBN1
knockdown (p < 0.01) (n = 3). (B) There was no change in the expression of CTGF undermechanical force of 90 g, regardless of FBN1 knockdown (n = 3). **p < 0.01 versus Scr-0 g.
BMP2: bone morphogenetic protein 2, CTGF: connective tissue growth factor, FBN1: fibrillin1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, HPDLFs: human periodontal
ligament fibroblasts, POSTN: periostin, Scr: scrambled siRNA, Si: siRNA against FBN1.

Fig. 5 Changes in the phosphorylation of Smad2 under mechanical force in HPDLFs upon
knockdown of FBN1, evaluated by western blotting. β-actin was used as the internal loading
control. (A) FBN1 knockdown caused a decrease in the phosphorylation of Smad2. Furthermore,
centrifugation caused a decrease in the phosphorylation of Smad2. (B) The phosphorylation
rate of Smad2 shown in (A) was quantified by ImageJ software. The relative expression levels
are shown with Scr-0 g regarded as 1. The phosphorylation of Smad2 was significantly
decreased under mechanical force of 90 g, regardless of FBN1 knockdown (p < 0.05) (n = 3).
*p < 0.05 **p < 0.01, Scr-0 g versus Scr-90 g versus Si-0 g versus Si-90 g. FBN1: fibrillin-1,
HPDLFs: human periodontal ligament fibroblasts, Scr: scrambled siRNA, Si: siRNA against
FBN1.

Fig. 6 Changes in the expression of POSTN upon treatment with TGF-β1, evaluated byquantitative reverse transcription-polymerase chain reaction. (A) Expression of POSTN was
increased by supplementation with 0.1, 1.0, or 10 ng/ml TGF-β1 (p < 0.01) (n = 3). (B)Expression of POSTN in Si-90 g was rescued by supplementation with 0.1 ng/ml TGF-β1 (p <0.01) (n = 3). (A) **p < 0.01, versus control. (B) **p < 0.01, versus Scr-0 g and Si-0 g (without
TGF-β1 supplementation). FBN1: fibrillin-1, GAPDH: glyceraldehyde 3-phosphate
dehydrogenase, HPDLFs: human periodontal ligament fibroblasts, POSTN: periostin, Scr:
scrambled siRNA, Si: siRNA against FBN1.

Table 1. Primers used for quantitative reverse transcription-polymerase chain reaction. BMP2:
bone morphogenetic protein 2, CTGF: connective tissue growth factor, FBN1: fibrillin-1,
GAPDH: glyceraldehyde 3-phosphate dehydrogenase, POSTN: periostin.

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

Table.1