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Keratinocyte Growth Factor Receptor is Up-regulated in Cyclosporin A-induced Gingival Hyperplasia

¹ Department of Periodontology, Regional Dental College, Guwahati-32, India;
² Eastman Dental Institute for Oral Health Care Sciences, University College London, 256 Gray’s Inn Road, London WCIX 8LD, UK; and
³ Oral Health Research Centre, Parkside National Health Service Trust, London, UK;

* corresponding author, i.olsen{at}eastman.ucl.ac.uk

	ABSTRACT

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Keratinocyte growth factor stimulates the growth and activity of epithelial cells via the keratinocyte growth factor receptor. We have recently shown that the growth factor is markedly elevated in cyclosporin A-induced gingival hyperplasia tissue in vivo, but the effects of cyclosporin A on the receptor are not yet known. The present study was therefore carried out to determine whether expression of the keratinocyte growth factor receptor is up-regulated in gingival hyperplasia compared with normal gingiva. Using immunohistochemistry and the reverse-transcribed polymerase chain-reaction, we obtained results which showed that receptor antigen and gene transcript levels were both elevated in gingival hyperplasia tissue. In addition, flow cytometry and the reverse-transcribed polymerase chain-reaction showed that the receptor and mRNA were also higher in gingival epithelial cells following incubation with cyclosporin A in vitro. These findings suggest that the keratinocyte growth factor-receptor pathway of mesenchymal-epithelial interaction could play an important part in the molecular pathogenesis of gingival hyperplasia.

KEY WORDS: gingiva • hyperplasia • keratinocyte growth factor receptor • epithelial cells

	INTRODUCTION

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Cyclosporin A (CsA) is widely used as an immunosuppressant to prevent rejection following organ transplantation and also to treat many immune diseases (Rang et al., 1995^*). Gingival hyperplasia (GH) was first reported as a side-effect of this drug (Rateitschak-Pluss et al., 1983; Wysocki et al., 1983), the average incidence being approximately 25% (range, 8-70%) (Seymour and Heasman, 1988), depending on genetics, duration, drug dose, serum and salivary concentrations, oral hygiene, age, and gender (Marshall and Bartold, 1998). Although the precise mechanism underlying GH is not known, hyperplastic lesions in other tissues suggest that growth factors play an important part in the molecular pathology of these disorders (De Bellis et al., 1996, 1998). These include keratinocyte growth factor (KGF), which is produced by mesenchymal cells such as fibroblasts and functions primarily by stimulating epithelial tissues (Rubin et al., 1995a). We have shown that this factor is elevated in GH tissues in vivo (Das et al., 2001) and also in normal gingival fibroblasts in vitro by other hyperplasia-inducing drugs, including nifedipine and phenytoin (Das and Olsen, 2000, 2001). The effects of KGF are mediated by binding to the KGF receptor (KGFR), which is expressed by epithelial cells only and regulates epithelial cell growth and function. The KGF/KGFR pathway is thus likely to play a major part in paracrine interactions between mesenchymal and epithelial cells. In this study, we have therefore measured the relative levels of KGFR in CsA-induced GH compared with normal gingival tissue and the effects of CsA on KGFR in gingival epithelial cells in vitro.

	MATERIALS & METHODS

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Tissue Collection
Normal gingival tissue samples were obtained from nine healthy individuals (four males and five females; aged 30-45 yrs, mean 35 + 7.1) undergoing routine surgical crown lengthening, with little if any evidence of inflammation and no systemic medication. Redundant GH tissues were obtained from nine individuals (four males and five females; aged 12-65 yrs, mean 38.5 + 18.7) on a maintenance dose of 100 mg CsA twice daily for 2 to 3 yrs following organ transplant. Examination of cumulative attachment loss and radiographic bone support showed little if any evidence of destructive periodontal disease. The patients were instructed in good oral hygiene during this period and had undergone professionally delivered plaque removal (to a plaque score of less than 20%). Subgingival scaling was performed every 2 wks for approximately 2 mos prior to surgical removal of diseased gingiva as part of their clinical management. Informed consent was obtained for all samples from all individuals, following a protocol reviewed and approved by the Joint Research and Ethics Committee of the Eastman Dental Institute/Hospital. A portion of each tissue was frozen immediately in liquid nitrogen and used for RNA isolation. We processed a second portion to obtain epithelial cells; the remainder was fixed in formalin and paraffin-embedded for immunohistochemistry.

Immunostaining Analysis of KGFR Expression
Five-mm-thick sections were cut from the normal gingiva and GH paraffin-embedded specimens, deparaffinized, and immunostained with rabbit anti-human KGFR (anti-bek) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with biotinylated swine anti-rabbit IgG (Dako, Glostrup, Denmark), imunoreactivity was detected by means of the avidin-biotin complex (Sigma, Poole, UK). Sections were counterstained with Mayer’s hematoxylin. In control sections, the primary antibody was replaced by non-immune rabbit serum.

Epithelial Cell Culture
The outer layer of the normal gingival tissue (n = 6) was mechanically dissected from the connective tissue, cut into approximately 1-mm³ pieces, and transferred to 25-cm² tissue culture flasks (Nunc, Taastrup, Denmark) containing 5 mL of keratinocyte basal medium-2 (KBM-2) (BioWhittaker, Wokingham, UK) supplemented with 7.5 mg/mL bovine pituitary extract (BPE), 0.1 µg/mL recombinant human epidermal growth factor (EGF), 5 mg/mL insulin, 0.5 mg/mL hydrocortisone, 10 mg/mL transferrin, 0.5 mg/mL epinephrine, and 50 µg/mL gentamycin. The explants were incubated at 37°C in a humidified atmosphere of 5% CO₂ in air. When individual colonies of adherent epithelial cells reached an average size of between 50 and 200 cells, estimated visually by means of a phase-contrast microscope, the cells were detached from the monolayer by trypsin-ethylenediaminetetracetic acid (EDTA) (Gibco Life Technologies Ltd., Paisley, UK) and re-cultured. The purity of the cells was assessed by immunostaining with an antibody against cytokeratins 5, 6, 8, and 17 (clone MNF 116) (Dako).

Flow Cytometry (FCM) Analysis of KGFR Expression
The gingival epithelial cells were grown to 80% confluence in KBM-2 and then for 48 hrs in the absence of EGF and BPE. The medium was replaced by fresh medium without EGF and BPE, the cells re-cultured for 3 days in the absence and presence of 500 ng/mL CsA, then detached with the use of 20 mM EDTA in PBS, centrifuged, and fixed with 3% paraformaldehyde. Aliquots of 10⁵ cells were reacted with the rabbit anti-human KGFR, washed with PBS, and incubated with fluorescein isothiocyanate (FITC)-labeled swine anti-rabbit antibody (Dako). The average fluorescence intensity (AFI) of the cells, indicating the relative level of KGFR expression, was analyzed by means of a FACScan flow cytometer (Becton Dickinson Labware, Oxford, UK) and the CELLQuest Software program, as previously described (Das and Olsen, 2001).

Extraction of RNA and RT-PCR Analysis of KGFR
Total RNA was isolated from normal gingiva (n = 9) and GH (n = 7) tissue samples according to the one-step method of Chomczynski and Sacchi (1987). RNA samples obtained from normal human skin and periodontal ligament tissue were used as positive and negative controls, respectively. To isolate RNA from the cell samples, we centrifuged the trypsin-detached suspensions and then treated the pellet as for the tissue samples. RNA was measured by absorbance at 260 nm, and protein contamination was assessed by the measurement of absorbance at 280 nm.

The first strand of cDNA was synthesized from the total RNA with the use of an oligonucleotide (oligo dT) primer (Promega, Madison, WI, USA) and cloned Moloney murine leukemia virus reverse-transcriptase (Stratagene, Cambridge, UK). The resulting cDNA was then amplified with the use of primer pairs specific for the KGFR and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences, at the same time and under the same PCR conditions (Das and Olsen, 2001). The expression of the GAPDH gene is considered to be constitutive and was used in this study as an internal standard for estimating the relative levels of KGFR mRNA. The primer pairs used in this study generated PCR products of 141 and 600 bp for the KGFR and GAPDH gene sequences, respectively.

The intensities of the bands corresponding to KGFR and GAPDH mRNA were measured by image analysis with the Scion Image Software Program (Scion Corporation, Frederick, MD, USA). The relative amounts of KGFR mRNA transcripts present in each sample were calculated from the ratio of the KGFR band intensity to the GAPDH band intensity, and are shown as KGFR/GAPDH x 100.

	RESULTS

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Analysis of KGFR in vivo
KGFR was observed in the epithelium in all normal gingiva and GH tissue sections, although relatively higher staining intensity was apparent in the GH tissue sections (Fig. 1). In the normal sections, the labeling was localized primarily in the spinous and granular cell layers, whereas only little if any KGFR was detected in the basal and cornified cell layers (the inner and outermost layers of the epithelium, respectively). In contrast, in the GH tissue samples, KGFR was detected in the basal as well as the spinous and granular cell layers. As with the normal gingival tissue, the GH sections had no KGFR in the cornified layer (Fig. 1). KGFR was also found to be cytoplasmic as well as surface-associated, especially in the basal layer. In addition, relatively little KGFR was observed in the connective tissue areas of both the normal and GH samples. Control sections of these tissues, which had received no primary antibody, were negative.

View larger version (127K): [in this window] [in a new window]   Figure 1. Immunohistochemical analysis of KGFR in gingival tissue. The brown enzyme reaction product indicates the presence of KGFR in gingival tissue. (A) Section of normal gingiva showing the presence of KGFR in the spinous and granular cell layers. Note the absence of KGFR in the basal and cornified cell layers. (B) GH tissue, in which KGFR is seen in the basal as well as the spinous and granular layers of the epithelium, although not in the cornified cell layer. Note the relatively higher staining intensity in the GH section (original magnification, 500x).

KGFR Gene Activity in vivo
In an initial experiment, the relationship between the number of amplification cycles and the intensities of the PCR products was determined for both the GAPDH and KGFR gene sequences by PCR amplification in 5-cycle steps from 10 to 40 cycles (data not shown). Accordingly, all subsequent PCR reactions were carried out within the linear range, using 30 and 25 cycles for the tissue- and cell-derived cDNAs, respectively.

All the RNA samples used in this study showed the single band corresponding to the GAPDH gene sequence (600 bp), indicating that the extracted RNA samples were intact and not degraded (Fig. 2A). An additional band corresponding to the KGFR gene sequence (141 bp) was also observed in the normal and GH tissues, while the control skin and periodontal ligament tissue samples were clearly positive and negative, respectively. Image analysis profiles showed that the KGFR band intensity was higher in the GH than in the normal tissue (relative KGFR mRNA levels of 58 and 19, respectively) (Fig. 2B). The average relative transcript level of the 7 GH tissues examined was found to be 76.9 + 13.5 (range, 40 to 179), approximately 270% greater than the average of the 9 normal samples (28.5 + 2.5; range, 10 to 42). The Mann-Whitney U test (SPSS Inc., Chicago, IL, USA) showed these differences to be very highly significant (p < 0.001).

View larger version (30K): [in this window] [in a new window]   Figure 2. Expression of KGFR mRNA in gingival tissues. (A) Agarose gel showing the PCR products of the GAPDH and KGFR genes. RNA obtained from the normal gingiva (NG), GH, skin, and periodontal ligament (PDL) tissues was reverse-transcribed and amplified with the use of GAPDH- and KGFR-specific oligonucleotide primers. The amplified products were subjected to electrophoresis, and the mobilities of the products were compared with that of the 100-bp DNA ladder used as a size marker (not shown). Note the presence of a band corresponding to the molecular size of GAPDH (600 bp) in all samples, including controls. The band corresponding to KGFR (141 bp) is seen in all samples except the PDL (negative control). (B) Image analysis profile of the agarose gel in (A), showing the presence of bands corresponding to KGFR mRNA in all of the samples except the PDL (negative control). The numbers in brackets are the relative levels of KGFR mRNA in the corresponding samples. Note the relatively greater amounts of transcripts in the GH compared with the normal gingival tissues.

View larger version (34K): [in this window] [in a new window]   Figure 3. Effects of CsA on KGFR expression by gingival epithelial cells. (A) Representative fluorescence profiles showing KGFR expression by epithelial cells cultured in the absence and presence of CsA. The dashed lines represent the fluorescence profiles of cells that received no primary antibody. The vertical black line shows the AFI of the non-treated gingival epithelial cells cultured in the absence of CsA. Note the higher AFI of the CsA-treated cells compared with the control cells, as shown in the upper righthand corner. (B) Representative experiment showing RT-PCR analysis of normal gingival epithelial cells cultured in the absence (A) and presence of CsA (B) for 3 days. Each panel shows the electrophoretic mobility of the RT-PCR products and the corresponding image analysis of these bands. Note the presence of GAPDH bands (600 bp) of similar intensities in both panels and the relatively greater amounts of the KGFR gene product (141 bp) in panel B compared with panel A. Numbers in brackets indicate the relative KGFR mRNA level in each panel.

	DISCUSSION

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GH is a well-documented side-effect of certain groups of drugs with anti-epileptic, calcium-channel-blocking, and immunosuppressive activity. The reason for the localization of this effect to the gingiva is unknown, although it is possible that the gingival tissue may be exposed to higher concentrations of these drugs than other tissues, both directly from the bloodstream as well as from the oral cavity through the crevicular epithelium (Marshall and Bartold, 1998). Although the underlying mechanism of GH is not known, there is substantial evidence that the drugs act directly or indirectly on the growth and function of both gingival fibroblasts and gingival epithelial cells via cytokines and growth factors (Nares et al., 1996; Atilla and Kutukculer, 1998). The expression of these mediators and their corresponding receptors is thus likely to be of fundamental importance in the pathogenesis of GH.

KGFR has been shown to play a prominent part in the proliferation and differentiation of epithelial cells both in vivo and in vitro (Pierce et al., 1994; Werner et al., 1994; Marchese et al., 1995, 1997; Rubin et al., 1995b) and has also been implicated in several hyperplastic pathologies (Finch et al., 1997; De Bellis et al., 1998). However, little is known about the role of KGFR in oral tissues (Partridge et al., 1996), and KGFR expression in GH has not been examined previously. The present study has shown that there are apparently increased levels of KGFR immunoreactivity in GH tissues which, like other hyperplasias (Finch et al., 1997; De Bellis et al., 1998), is associated with increased proliferation of epithelial cells and thickened epithelium. The presence of KGFR in the basal and suprabasal epithelial layers of the GH samples, like that of the Ki 67 antigen, a DNA synthesis marker (Saito et al., 1999^*), is indicative of the extensive proliferative activity in this tissue.

KGFR was also found to be absent in the cornified layer of both the GH and normal gingival tissue samples, as previously reported in skin and the soft and hard palates (LaRochelle et al., 1995^*; Finch et al., 1997). This may be due to lysosomal breakdown before the cells enter this layer and undergo terminal differentiation, as noted by Pierce et al. (1994). The presence of low levels of positive staining in the connective tissue of the gingiva is consistent with that observed in the stroma of inflamed intestinal mucosa (Brauchle et al., 1996^*) and most probably arises because of the use of the particular anti-bek antibody, which has been shown to react not only with KGFR but also with FGFR 2 (Finch et al., 1997) expressed by mesenchymal cells (Miki et al., 1992).

RT-PCR analysis showed that KGFR gene transcription is, as in benign prostate hyperplasia (De Bellis et al., 1998), relatively higher in GH tissue compared with normal gingiva, and that this difference was statistically significant. The elevated levels of KGFR mRNA in GH tissue in vivo suggest that the GH-inducing drugs control the expression of KGFR. Moreover, the present experiments have also demonstrated, for the first time, that CsA up-regulates both KGFR antigen and transcript levels in normal gingival epithelial cells in vitro. Although KGFR is primarily involved in epithelial cell proliferation, interaction with KGF also induces the expression of several mediators by keratinocytes which, in turn, regulate the biological activities of mesenchymal cells. For example, KGF stimulates epithelial production of TGF-ß and activin (Hubner and Werner, 1996), factors which are actively involved in the proliferation of fibroblasts and also in the production of extracellular matrix by epithelial as well as mesenchymal tissues (Pierce and Mustoe, 1995). TGF-ß not only up-regulates matrix synthesis but also blocks ECM breakdown by down-regulating the expression of protease inhibitors such as TIMP-1 (Edwards et al., 1987). Thus, CsA-induced up-regulation of KGFR would facilitate enhanced responsiveness to KGF, leading to both increased cellular proliferation and connective tissue overproduction, key features of the molecular pathology of GH.

	ACKNOWLEDGMENTS

 
The authors are grateful to the staff of the Periodontology Clinic and Victor Goldman Unit, Eastman Dental Hospital, London, for generously providing redundant surgical materials. S.J. Das acknowledges The Association of Commonwealth Universities for a Commonwealth Scholarship and for the financial support of this work.

	FOOTNOTES

 
^* References marked with an asterisk (*) were inadvertently omitted from the reference list and were unavailable at press time. Interested readers should contact the corresponding author directly. The references will appear as an erratum in the November issue. The authors regret this inconvenience.

Received December 27, 2001; Last revision July 11, 2002; Accepted July 15, 2002

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