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Expression and localization of cystic fibrosis transmembrane conductance regulator in human gingiva
Louis C Ajonuma1, Qian Lu, Becky PK Cheung, W. Keung Leung, Lakshman P Samaranayake and Lijian Jin1
Faculty of Dentistry, University of Hong Kong, Hong Kong SAR, People's Republic of China
CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-activated chloride channel that regulates electrolyte and water transport. The present study investigated the expression and localization of CFTR in human gingiva and explored the possible association of CFTR with periodontal conditions. CFTR expression in gingival biopsies from five periodontally healthy subjects and ten subjects with chronic periodontitis and in the RHGE (reconstituted human gingival epithelia) was detected by immunohistochemistry, whereas its expression in gingival biopsies was analysed by immunofluorescence staining. CFTR mRNA was analysed by reverse transcription-PCR. CFTR mRNA was detected in human gingival epithelia and RHGE. CFTR protein was detected in gingival biopsies from both healthy subjects and individuals with periodontitis and in RHGE. In healthy subjects, CFTR expression was mainly confined to the granular and spinous layers of epithelia and localized on the cell membrane. In patients with periodontitis, CFTR was detected in all layers of epithelia and the underlying connective tissues. The mean CFTR expression levels in periodontitis patients were significantly higher than those in healthy subjects. The present study for the first time showed the expression and localization of CFTR in human gingival epithelia. Elevated CFTR expression in periodontitis subjects implies the possible involvement of CFTR in periodontal disease pathogenesis. Further study is warranted to confirm the present findings.
Key words: CFTR, gene expression, gingival epithelium, periodontal disease
Abbreviations: AL, attachment loss, BOP, bleeding on probing, CF, cystic fibrosis, CFTR, cystic fibrosis transmembrane conductance regulator, DAB, 3-3-diaminobenzidine, ENaC, epithelial sodium channel, GADPH, glyceraldehyde-3-phosphate dehydrogenase, GCF, gingival crevicular fluid, ORCC, outwardly rectifying chloride channel, PD, probing depth, RHGE, reconstituted human gingival epithelia, ROMK2, inwardly rectifying potassium channel
1Correspondence may be addressed to either of these authors (email Lcajonuma@graduate.hku.hk or firstname.lastname@example.org).
CFTR (cystic fibrosis transmembrane conductance regulator), a unique member of the ABC (ATP binding cassette) family (Higgins, 1992), is a single polypeptide chain of 1480 amino acids that is located at chromosome 7, spans ∼250 kb of genomic DNA and contains 27 exons (Zielenski et al., 1991). More than 1000 CFTR gene mutations have been identified so far and they are responsible for the hallmarks of CF (cystic fibrosis), the most common autosomal recessive disorder in Caucasians (Collins, 1992). It is a cAMP-activated chloride channel that regulates electrolyte and water transport across epithelia (Stutts et al., 1995; Sheppard and Welsh, 1999) and interacts with other epithelial ion channels and transporters such as aquaporin water channels, calcium activated chloride channels and sodium hydrogen exchangers (Ajonuma et al., 2002). It is expressed in a number of epithelia in humans, including those lining the lumen of the gastrointestinal tract (Kälin et al., 1999), where it is involved in transepithelial transport of fluid and electrolytes. There are no previous reports on CFTR expression in human gingiva as well as in other parts of the oral cavity and pharynx.
Human gingival epithelium is the first barrier of periodontal tissues and it plays very important protective and defence roles by performing both physical and biological barrier functions (Dale, 2002; Lu et al., 2004, 2005). Gingival epithelial cells are not passive bystanders in the periodontal tissues, but rather are metabolically active and capable of reacting to external stimuli by synthesizing a number of cytokines, adhesion molecules, growth factors and enzymes (Bartold et al., 2000). While the exact mechanism involved in the regulation of transepithelial transport of GCF (gingival crevicular fluid) remains unknown, the functions of its cellular components and the various effector molecules concerned are unclear. Production of GCF is a result of an increase in the permeability of the vessels underlying sulcular and junctional epithelia in response to various stimuli and during infections (Alfano et al., 1976; Griffiths, 2003; Emingil et al., 2006). However, its exact origin, formation, flow and transportation across the gingival epithelium are still not well understood. Previous studies have provided molecular and electrophysiological evidence of CFTR involvement in transepithelial fluid transport in general (Chan et al., 1999, 2002; Ajonuma et al., 2005b). Hence, CFTR may be expressed in the human gingiva, and plays an important role in the regulation of fluid balance and maintenance of periodontal homoeostasis. The present study for the first time investigated the expression and localization of CFTR in human gingiva, and explored the possible association of CFTR with periodontal conditions.
2. Materials and methods
2.1. Subjects and collection of samples
Ten Chinese adults with a mean age of 41.4±9.6 years were recruited for the study. They had untreated advanced chronic periodontitis, with ≥5.0 mm of PD (probing depth), ≥3.0 mm of clinical AL (attachment loss) and radiographic evidence of alveolar bone loss on at least two teeth per quadrant. After basic periodontal treatment, all subjects exhibited unresolved periodontitis in need of periodontal surgery. Gingival biopsies were collected during periodontal surgery in unresolved periodontitis sites with PD ≥6 mm, AL ≥5 mm and significant loss of alveolar bone on radiographs. Five gingival samples, as controls, were obtained from five periodontally healthy subjects with a mean age of 21.6±5.4 years who attended Prince Philips Dental Hospital and required tooth extraction for orthodontic treatment purposes or crown lengthening procedures. Inclusion criteria were: (i) systemically healthy condition; (ii) no sites with PD >4 mm or AL >1 mm in the whole dentition; (iii) no radiographic evidence of periodontal bone loss after a full-mouth radiographic examination; (iv) a full-mouth score of BOP (bleeding on probing) <15%; (v) no antibiotics or anti-inflammatory drugs taken within the preceding 6 months. These sites sampled met the following criteria: (i) PD not exceeding 3 mm; (ii) absence of BOP; (iii) AL not exceeding 1 mm; and (iv) no radiographic evidence of alveolar bone loss. The general health of all subjects was good and none received antibiotics within the preceding 6 months. None reported receiving any prior immunosuppressive therapy. All subjects were non-smokers. The purposes and procedures of the study were explained to participants and informed consent was obtained from all subjects. The study protocol was approved by the ethics committee of the University of Hong Kong.
2.2. Reconstituted human gingival epithelia model
We reconstituted 0.5 cm2 RHGE (reconstituted human gingival epithelia) (Skinethic Laboratories) cultures of normal human gingival keratinocytes (second passage) for 5 days in serum-free and chemically defined medium on inert polycarbonate filters. This model features a stratified epithelium with a thin stratum corneum, few granular layer cells and mostly spinous layer cells, histologically resembling the outer cell layers of human gingiva and functionality equivalent to the gingival epithelia of humans in vivo. Upon arrival, RHGE were placed in an incubator at 37°C, 5% CO2 in air, and saturated humidity in a medium containing insulin (5 μg/ml), calcium chloride (1.5 mM), gentamycin (25 μg/ml) and hydrocortisone (0.4 μg/ml) for 24 h.
2.3. RT-PCR (reverse transcriptase-PCR)
Total RNA was extracted from the homogenized gingival biopsies and RHGE using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. From each sample, 1 μg of total RNA was reverse-transcribed into cDNA in a final volume of 20 μl using the SuperScript™ First-Strand Synthesis System (Invitrogen). CFTR mRNA expression was detected by PCR. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was amplified together with the target gene as an internal control. The specific oligonucleotide primers for human GAPDH mRNA expression were as follows: 5′-GTGGGGCGCCCCAGGCACCA-3′ (sense) and 5′-CTCCTTAATGTCACGCACGATTTC-3′ (antisense), with expected cDNA of 515 bp. The specific oligonucleotide primers for human CFTR were as follows: 5′-AGCTGGACCAGACCAATTTTGAGGAAA-3′ (sense) and 5′-CCACACGAAATGTGCCAATGCAAGTCC-3′ (antisense), with expected cDNA of 554 bp. The PCR conditions were as follows: denaturation at 94°C for 45 s, annealing at 53°C, 58°C for 60 s, extension at 72°C for 60 s, and subsequently 30 and 33 cycles for GAPDH and CFTR respectively (Ajonuma et al., 2005a). Optimal amplification cycles were determined based on the linear relationship between the amount of PCR product detected and the number of amplification cycles. The PCR products were analysed using 2% agarose gel electrophoresis stained with ethidium bromide and visualized in an Uvidoc gel documentation system (Bio-Rad). The band intensities of CFTR gene were normalized to those of GAPDH, which were amplified simultaneously. Experiments in the absence of CFTR primers were run as negative controls.
2.4. Immunohistochemistry and image analysis
Serial paraffin sections of gingival tissue biopsies and RHGE were cut at 4 μm and mounted onto slides for immunohistochemistry analysis. Briefly, the slides were deparaffinized in xylene, rehydrated in graded ethanol and washed in distilled water. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide incubation for 30 min. The slides were placed in a cooling jar with boiled sodium citrate buffer at pH 6.0 and boiled for 5 min for antigen retrieval. After cooling, the slides were rinsed in PBS and incubated in normal blocking serum (Vectastain Elite ABC kit, Vector Laboratories) for 30 min and washed with PBS. The slides were incubated with the primary antibody (CFTR 01:500 v/v, Alexis Biochemicals) and controls in normal blocking serum at 4°C overnight. Tissue sections were then washed with PBS and immunostaining was performed using a biotinylated secondary antibody, a horseradish peroxidase-H conjugate and a substrate–chromogen mixture (Vector Laboratories). Finally, the slides were incubated for 5–15 min with the chromogen DAB (3-3-diaminobenzidine) substrate (International Laboratory), rinsed in water and counterstained with haematoxylin (Dako). After the tissue sections were dehydrated in graded ethanol (75%, 95% and 100%) and xylene, slides were permanently mounted. CFTR expression was depicted by positive DAB staining which was analysed by a true-colour computerized image system with a digital camera (Leica DC 300 V2.0, Leica) and software (Qwin V2.6, Leica). The experiment was repeated three times and negative controls were applied by using normal serum without primary antibody. CFTR immunostaining intensity values of healthy and periodontitis tissues were analysed using Image-Pro Plus version 6.3 (Media Cybernetics).
2.5. Immunofluorescence assay
Serial paraffin sections (4 μm thick) of gingival tissue biopsies were cut and mounted onto slides with standard procedures. The slides were deparaffinized in xylene, rehydrated in graded ethanol and washed in distilled water. Endogenous peroxidase activity was quenched by incubating slides in a solution of 0.3% of hydrogen peroxide in water for 30 min. The slides were washed in water and incubated in normal blocking serum (Vector Laboratories) for another 30 min to block non-specific sites. Excess serum was removed from the slides by blotting and incubated overnight in primary antibody (CFTR 1: 500 v/v; Alexis Biochemicals Corp.) and controls in normal blocking serum at 4°C. Tissue sections were washed three times for 5 min with PBS and incubated for 30 min in a diluted biotinylated secondary antibody solution (Vector Laboratories). They were again washed three times for 5 min in PBS and incubated for 20–30 min in FITC-conjugated anti-mouse antibody (Vector Laboratories), washed again in PBS, mounted with glycerol, and observed under an epifluorescence microscope (Nikon) fitted with SPORT image acquisition system and software (Diagnostic Instruments). The cultured cells on cover slips were observed under a Confocal Laser Scanning Microscope (Olympus FluoView FV1000) fitted with Olympus DP Controller DP71 software (Olympus).
2.6. Statistical analysis
Differences in staining intensities of CFTR immunoreactivity between the groups were assessed using analyses of variance and Newman–Keuls multiple comparison test. Statistical analysis was done using GraphPad Prism version 5.01 (GraphPad Software). A P value of ≤0.05 was considered statistically significant.
mRNA expression of CFTR in gingival tissues from periodontally healthy subjects, patients with periodontitis and RHGE is shown in Figure 1. CFTR mRNA was detected in the gingival biopsies from periodontally healthy subjects (lane 1) and patients with periodontitis (lane 3) as well as in RHGE (lane 2).
The expression of CFTR protein was detected by immunostainning in the gingival biopsies from periodontally healthy subjects, patients with periodontitis and RHGE. In the gingival biopsies from periodontally healthy subjects, immunoreactive CFTR was strongly detected on the granular and spinous layers of gingival epithelia (Figure 2A). Higher magnification showed dense CFTR expression on the epithelial cell membrane in normal gingival biopsies (Figure 2B). In the gingival biopsies from patients with chronic periodontitis, CFTR was detected in all layers of the gingival epithelium including the basal layers and underlying connective tissues (Figure 2C). The staining intensity of the biopsies from patients with periodontitis was significantly higher than those from periodontally healthy subjects (P<0.05, Figure 3). CFTR was also sparsely expressed on the superficial layers of RHGE (Figure 4). Immunofluorescence assay confirmed the CFTR expression profile in human gingival biopsies (Figure 5).
CFTR transgenic mice (CF mice) are associated with abnormal enamel mineralization, ion concentrations and altered pH regulation during enamel development of teeth (Wright et al., 1996a, 1996b; Arquitt et al., 2002; Sui et al., 2003), which suggests that CFTR may play an important role in the oral cavity. This is the first time that the CFTR gene and protein expression in human gingiva has been detected. RT-PCR results suggest that the expression of CFTR mRNA in human gingiva is similar to that in other epithelial lining, such as gastrointestinal tract (Trezise and Buchwald, 1991; Trezise et al., 1993a) and female reproductive tract (Ajonuma et al., 2005a, 2005b; Trezise et al., 1993b). The presence of CFTR protein in human gingiva was confirmed by immunolocalization of CFTR immunoreactivity by using a specific monoclonal antibody, showing that CFTR protein is densely expressed in human gingival tissues. CFTR expression was seen mostly in the granular and spinous layers of gingival epithelia in human gingival biopsies, and was mainly confined to the epithelial cell membrane.
In terms of the overall expression profile of CFTR, its expression was mainly observed in the granular and spinous layers of gingival epithelia in periodontally healthy tissues, while CFTR was detected in all layers of the gingival epithelium and the underlying connective tissues in the gingival biopsies from periodontitis patients. As CFTR expression was elevated in tissues from periodontal patients, it is tempting to speculate that CFTR expression may be involved in periodontal pathogenesis.
Currently, the mechanisms that entail formation and transport of GCF across gingival epithelium are not well understood. Fluid movements across epithelia are secondary to ion movements. Ions are not actively transported but move in response to osmotic gradients largely established by ion transport across epithelia, particularly chloride ions, through ion channels such as CFTR. The latter is known to regulate a variety of cellular functions both directly and indirectly. Primarily, CFTR has been shown to regulate transepithelial ion transport by acting as an epithelial ion channel (Schwiebert et al., 1998) and influencing the activities of other ion channels such as the ENaC (epithelial sodium channel) (Stutts et al., 1995; Ismailov et al., 1996), ORCC (outwardly rectifying chloride channel) (Schwiebert et al., 1998), ROMK2 (inwardly rectifying potassium channel) (McNicholas et al., 1997), aquaporin water channels, calcium-activated chloride channels and sodium–hydrogen exchangers (Ajonuma et al., 2002). The proposed mechanisms for CFTR-dependent regulation of these channels include direct interactions between CFTR and ENaC (Stutts et al., 1995), the nucleotide binding domain-1 of CFTR and ROMK2 (McNicholas et al., 1997) and the facilitation of ATP-dependent stimulation of ORCC activity by CFTR-mediated ATP release (Schwiebert et al., 1998). Therefore, the localization of CFTR in gingival epithelia of humans observed in this study suggests the possible involvement of CFTR in transepithelial fluid transport across gingival epithelia and maintenance of tissue homoeostasis.
CFTR is involved in the regulation of cytokines that modulate mucosal immunity (Schweibert et al., 1999). It is likely that CFTR in human gingiva is also involved in the regulation of local mucosal immunity directly, since it is involved in the expression of several relevant genes, such as nitric oxide synthases (Steagall et al., 2000) and various cytokines.
Periodontal diseases are considered the most common form of infections in humans, due to the unique anatomic juxtaposition of hard dental tissues and soft gingival tissues. Additionally, pathogenic plaque bacteria that constitute a biofilm permanently colonize this particular niche. The latter, in turn, induces an infective state termed ‘periodontitis’, which is characterized by bacteria-induced inflammatory destruction of tooth-supporting structures and the alveolar bone (Jin, 2008). The increased expression of CFTR in infections as observed in gingival tissues from the present study has also been reported in other tissues (Ajonuma et al., 2005a). Earlier studies have shown that some bacteria, such as Pseudomonas aeruginosa (Pier et al., 1996, 1997) and Salmonella typhi, (Pier et al., 1998) use CFTR as a receptor for their binding and uptake into epithelial cells. CFTR has been reported as a pattern recognition molecule for bacteria in lungs (Schroeder et al., 2002). Interestingly, certain agents such as oestrogen known to up-regulate CFTR protein expression increase the rate of bacterial infection in humans (Maslow et al., 1988; Guseva et al., 2003). Thus, CFTR present on gingival epithelia may be involved in the binding and uptake of bacteria responsible for the formation of pathogenic plaque biofilm and development of periodontal disease. Furthermore, CFTR has been reported to mediate fluid formation during infection (Ajonuma et al., 2005a, 2008a, 2008b), thereby probably contributing to the resultant inflammation. The present findings of CFTR expression in human gingiva and up-regulated expression in periodontal disease may have important implications for further understanding of periodontal disease pathogenesis and future development of novel treatment strategies.
Louis Ajonuma designed and performed experiments, analysed results and participated in preparing the manuscript. Qian Lu helped in preparing patient samples for RT-PCR and immunostaining. Becky Pik Ki Cheung helped in preparing samples and performing RT-PCR. W. Keung Leung participated in results interpretation and manuscript preparation. Lakshman P. Samaranayake participated in manuscript preparation. Lijian Jin helped in the design of experiments, data analysis and manuscript preparation.
This study was supported by the
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Received 12 May 2009/16 July 2009; accepted 4 September 2009
Published as Cell Biology International Immediate Publication 4 September 2009, doi:10.1042/CBI20090019
© 2010 The Author(s) Journal compilation. © 2010 Portland Press Ltd
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 3 Mean CFTR immunoreactive intensity in biopsies from periodontally healthy subjects and chronic periodontitis patients