Modulation of protease-activated receptor expression by Porphyromonas gingivalis in human gingival epithelial cells

Gingival epithelial cell culture

Primary human GECs were isolated from healthy human gingival tissue samples from patients
undergoing third molar extraction at the Dental Department, Sir Run Run Shaw Hospital,
School of Medicine, Zhejiang University, China. Written informed consent was obtained
from all individuals participating in this study. The study were evaluated and approved
by the Ethics Committee of the Affiliated Sir Run Run Show Hospital of Zhejiang University
School of Medicine (20131120). Fresh gum tissue was placed into D-Hanks containing
300 U/ml penicillin G and 300 ?g/ml streptomycin and incubated at 4 °C. Within 1 h,
tissue was prepared to obtain epithelial cells. Briefly, the tissue was cut into small
pieces (1 mm?×?1 mm), treated with a solution of 25 % dispase II (Sigma–Aldrich, St
Louis, MO, USA) and incubated for 18 h at 4 °C. After incubation, the epidermal layer
of human keratinocytes was lifted from the dermis and placed into a 15 ml sterile
centrifuge tube containing 2 ml trypsin–EDTA. The tissue was incubated at 37 °C for
approximately 10 min. Subsequently, isolated GECs were seeded into T-75 flasks (BD
Biosciences) at a cell density of approximately 3?×?10
⁶
cells per flask in 10–15 ml serum-free keratinocyte medium (keratinocyte-SFM) to which
supplements were added according to the manufacturer’s instructions (Gibco BRL, Life
Technologies, Rockville, MD, USA). Fluids in the flasks were exchanged for fresh complete
medium and gassed with 5 % CO
₂
every 2–3 days. Cells were passaged when 75–80 % confluence was reached.

Reverse transcription-PCR (RT-PCR) analysis for the determination of PARs expression

To examine the expression of PARs mRNA, total RNA was isolated from GECs (grown to
70 % confluence) using TRIzol® reagent (Gibco BRL, Life Technologies, Rockville, MD,
USA) according to the manufacturer’s suggested protocol. The synthesis of the first
strand cDNA and RT-PCR were performed using a PromeScript® RT-PCR Kit (Takara Biotechnology
Co., Ltd, Dalian, China). The primers for PAR-1, PAR-2, PAR-3, PAR-4 and ?-actin were
synthesized by Sangon Biotech Co., Ltd (Shanghai, China; Table 1). For amplification of PAR-1, PAR-2 and ?-actin products, PCR was performed for 30 cycles.
The first cycle included a denaturation step of 5 min at 94 °C. Cycles 2–30 had a
denaturation step of 30 s at 94 °C, 30 s of annealing at 60 °C and 45 s of elongation
at 72 °C. The last cycle included an elongation step of 10 min at 72 °C. For PAR-3
and PAR-4 amplification, PCR was performed for 30 cycles. The first cycle included
a denaturation step of 5 min at 94 °C. Cycles 2–30 had a denaturation step of 30 s
at 94 °C, 30 s of annealing at 65 °C and 45 s of elongation at 72 °C. The last cycle
included an elongation step of 10 min at 72 °C. DNA products and molecular weight
marker DL1,000â„¢ DNA Marker (Takara Biotechnology Co., Ltd, Dalian, China) were separated
in 1.5 % agarose gel, after which the gels were stained with GelRed™ and visualized
under UV light.

Table 1. Oligonucleotide sequences used for RT-PCR

Bacteria culture and supernatants collection

P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA,
USA) and anaerobically cultured (80 % N
₂
, 10 % H
₂
and 10 % CO
₂
) in a brain–heart infusion (BHI; Oxoid) agar plate containing 5 % defibrinated sheep
blood enriched with 5 g/l yeast extract, 5 mg/l hemin and 10 mg/l menadione (Sigma–Aldrich,
Dorset, UK) at 37 °C for up to 5 weeks. Liquid cultures were prepared by inoculation
of bacterial colonies (3–4 days old) from blood agar plates into 10 ml BHI broth supplemented
with 5 g/l yeast extract, 5 mg/l hemin and 10 mg/l menadione and incubated for 24 h.
Ten percent inoculum was transferred to 90 ml of the same medium and incubated for
6 days. After this culture period, bacteria were harvested by centrifugation at 10,000?g for 15 min at 4 °C and supernatants were collected, filter-sterilized over a 0.2 mm
filter and stored at ?80 °C until use. Before treatment, aliquots of the supernatant
were used for pre-incubation (10 min) with 1 mmol/l of the serine and cysteine protease
inhibitor tosyl-L-lysine chloromethylketone (TLCK; Sigma–Aldrich, St Louis, MO, USA), which inhibits
gingipains 15], 21].

Characterization of bacterial culture supernatants and treatment

P. gingivalis supernatants were diluted in cell culture medium and their concentration expressed
as the total bacterial protein (mg/ml) present in the cell cultures. The protein concentration
was determined with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Absorbance
was measured at 562 nm on a SpectraMax® Plus plate reader. Protease activity was measured
with a Protease Assay™ Kit (G-Biosciences, St Louis, MO, USA) 22]. The absorbance of the dye-labeled peptide was measured at 570 nm for determination
of the protease activity. Chemically stabilized trypsin (MSG-Trypsinâ„¢) was supplied
with the kit as a general protease standard. GECs were grown to 80 % confluence and
stimulated with either 50 ?g/ml culture supernatant protein from P. gingivalis supernatants or TLCK-preincubated supernatants, for 6 h 22]. Unstimulated GEC medium served as a control for the stimulation experiments. Each
stimulation experiment was performed in triplicate and cells from two to five different
donors were tested.

Quantitative real-time RT-PCR (QRT-PCR)

After stimulation, total RNA was extracted using an RNeasy Kit (Qiagen, Valencia,
CA, USA) and reverse-transcribed using a SuperScript® RT-PCR Kit (Takara, Tokyo, Japan).
Quantitative real-time RT-PCR was performed using the Applied Biosystems 7500 PCR
machine and SYBR Premix Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. PCRs were carried out in 96-well plates in a total volume of 20 ?l,
including 1 ?l cDNA and 0.8 ?l primers (10 ?M; Table 2). Sample expression was normalized against that of the housekeeping gene ?-actin,
which was included in each QPCR run. PCR controls were performed using water instead
of cDNA. All reactions were carried out in duplicate. At each time point, the expressions
of the selected mRNAs in cells incubated with P. gingivalis supernatants or TLCK-preincubated supernatants were calculated relative to the housekeeping
gene ?-actin (?C_t
) for each sample and then expressed relative to untreated cells at the same time
point using the 2
^-??Ct
method 23].

Table 2. Oligonucleotide sequences used for QRT-PCR

Flow cytometry

Cells were washed in phosphate-buffered saline (PBS), incubated for 30 min at 4 °C
with PBS containing 20 % heat-inactivated normal human serum, washed again and then
incubated for 30 min at 4 °C with 15 nM of specific monoclonal antibodies (mAb) to
human PAR-1–4 (PE-conjugated; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
Flow cytometry analyses were performed on a FACScan (BD Biosciences, San Jose, CA,
USA). Control cells incubated with PE-conjugated nonspecific antibodies obtained from
the same manufacturers were used to set the threshold for the fluorescence parameter,
such that the fraction of cells with positive fluorescence was 2.5 % of the total
cells. The percentage of PAR-1–4 positive cells was determined from the fraction of
cells in the sample incubated with specific antibodies that exceeded the threshold
for the fluorescence signal intensity obtained with the control sample.

Statistical analyses

All data are shown as the mean?±?the standard deviations (SD). QRT-PCR data are expressed
as C_t
(cycle threshold), ?C_t
(C_t
PAR mRNA – C_t
?-actin mRNA) and relative quantification (RQ; expressed as fold change). The fold
changes of PARs mRNA expression were calculated using the 2
^-??Ct
method 23]. Student’s t-test was used for comparison between two groups. SPSS version 19.0 (SPSS Inc., Chicago,
IL, USA) was used for statistical analysis and P???0.05 was considered statistically significant.