行政院國家科學委員會專題研究計畫成果報告

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行政院國家科學委員會專題研究計畫成果報告

第一型類胰島素生長因子及其接受器在人類正常頰黏膜與口腔黏膜下纖維化症的表現

計畫類別：個別型計畫

計畫編號： NSC93-2314-B-040-021-

執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日執行單位：中山醫學大學牙醫學系

計畫主持人：蔡崇弘共同主持人：張育超

報告類型：精簡報告

處理方式：本計畫可公開查詢

中華民國 94 年 10 月 12 日

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The upregulation of insulin-like growth factor-1 in oral submucous fibrosis

Chung-Hung Tsai

^a

, Shun-Fa Yang

^b

, Yi-Juai Chen

^c

, Ming-Yung Chou

^c

, Yu-Chao Chang

^d,^*

aDepartment of Oral Pathology, Chung Shan Medical University, Taichung, Taiwan, ROC

b Institute of Biochemistry, Chung Shan Medical University, Taichung, Taiwan, ROC

cDepartment of Oral and Maxillofacial Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC

d School of Dentistry, College of Oral Medicine, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Road, Taichung, Taiwan, ROC

Received 3 May 2005; accepted 16 May 2005

Summary Insulin-like growth factor-1 (IGF-1) is a member of a family of two interacting polypeptide hormone ligands with close homology to proinsulin. IGF-1 can influence mesenchymal cell migration, proliferation, and extracellular matrix deposition, thus implicating it in the progression of fibrotic disorders. Currently, there is limited information about the regulation of IGF-1 expression in areca quid-associated oral submucous fibrosis (OSF). The aim of this study was to compare IGF-1 expression in normal human buccal mucosa and OSF specimens and further explore the potential mechanism that may lead to induce IGF-1 expression. Twenty OSF specimens and 10 normal buccal mucosa were examined by immunohistochemistry. The activity of IGF-1 from cells cultured from OSF and normal buccal mucosa were by using reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Furthermore, the effect of arecoline, the major areca nut alkaloid, was added to explore the potential mechanism that may lead to induce IGF-1 expression. IGF-1 expression was significantly higher in OSF specimens (p < 0.05) and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. OSF demonstrated significantly higher IGF-1 protein expression than normal buccal mucosa fibroblast (BMF) both in mRNA and protein levels (p < 0.05). In addition, arecoline was also found to elevate IGF-1 mRNA and protein expression in a dose-dependent manner (p < 0.05). Taken together, the data presented here demonstrated that IGF-1 expression is significantly upregulated in KEYWORDS

Arecoline;

Buccal mucosal fibroblasts;

Oral submucous fibrosis;

Insulin-like growth factor-I

1368-8375/$ - see front matter

doi:10.1016/j.oraloncology.2005.05.006

* Corresponding author. Tel.: +886 4 24718668x55011; fax: +886 4 24759065.

E-mail address:cyc@csmu.edu.tw(Y.-C. Chang).

http://intl.elsevierhealth.com/journals/oron/

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OSF from areca quid chewers and arecoline may be responsible for the enhanced IGF-1 expression in vivo.

Introduction

Oral submucous fibrosis (OSF) has been identified as a precancerous condition.¹ One of the clinical symptoms of OSF is trismus, a limitation of mouth opening. These may eventually impair the ability to eat and speak. Areca quid chewing has been rec- ognized as one of the most important risk factors for OSF.^2,3

The main histopathological characteristic of OSF is the deposition of collagen in the oral mucosa.^4,5 It has been found that arecoline, a major areca nut alkaloid, could stimulate human buccal mucosal fibroblasts (BMFs) proliferation^6,7and collagen synthesis.⁶A reduced degradation of the a1(I) collagen trimer synthesized by OSF fibroblasts may induce the alteration of the ratio of a1(I):a2(I) chains.⁸ The attendant increase of lysyl oxidase activity may also contribute to abnormal deposition of collagen in OSF.⁹ Recently, our studies have shown that the upregulation of tissue inhibitor of metalloproteinase-1,¹⁰ vimentin,¹¹ cyclooxygenase-2,¹² plasminogen activator inhibitor-1,¹³ interleukin- 6,¹⁴ and keratinocyte growth factor-1¹⁵ may contribute to the extracellular components accu- mulation in OSF. Despite above evidences, the pathogenesis of OSF related areca quid chewing still remains to be elucidated.

Insulin-like growth factor-1 (IGF-1) is a 70 amino acid, 7.6 kd, single-chain nonglycosylated polypeptide with structural similarity to insulin. It may act as an autocrine or paracrine growth hormone¹⁶and mediates most of the peripheral IGF-1 directly stimulates fibroblast proliferation and perhaps collagen synthesis.^17,18 IGF-1 is consistently and dra- matically upregulated in a variety of fibrotic diseases, such as idiopathic pulmonary fibrosis,¹⁹ systemic sclerosis²¹bleomycin-induced pulmonary fibrosis,²⁰and carbon tetrachloride (CCl4)-induced hepatic fibrosis.²²

The biologic roles of IGF-1 induce cell proliferation and collagen synthesis, which may be important in fibroproliferative process in areca quid-associated OSF. The purpose of this study was to test whether IGF-1 expression regulated within OSF specimens and to further explore possi-

ble pathogenic mechanisms that might lead to enhanced expression of IGF-1 in vivo. More specifically, we also set out to explore where expression of IGF-1 can be triggered in human buccal mucosa fibroblasts (BMFs) stimulated by arecoline in vitro.

Materials and methods

Immunohistochemistry

Formalin-fixed, paraffin-embedded specimens of 10 normal buccal mucosa from non-areca quid chewers, and twenty OSF specimens from areca quid chewers, were drawn from the files of the Department of Pathology, Chung Shan Medical University Hospital. Diagnosis was based on histo- logical examination of hematoxylin- and eosin- stained sections. Five micron sections were stained with the monoclonal anti-IGF-1 antibody (Santa Cruz Biotechnology, CA, USA) (1:100 dilution) using a standard avidin–biotin–peroxidase complex method.²³AEC (DAKO, Carpinteria, USA) was then used as the substrate for localizing the antibody binding. Negative controls included serial sections from which either the primary or secondary anti- bodies were excluded. The preparations were counterstained with hematoxylin, mounted with Permount (Merck, Darmstadt, Germany) and examined by light microscopy.

Cell culture

Nine healthy individuals without areca quid chewing habits were selected from the Department of Oral Surgery (Chung Shan Medical University Hospi- tal, Taichung, Taiwan) with the informed consent for this study. Biopsy specimens were derived from histologically normal areas of surgical third molar extraction from patients. The OSF specimens were obtained from 22 male patients with areca quid chewing habits during surgical biopsy. Clinical diagnosis was confirmed by histopathological The upregulation of insulin-like growth factor-1 in oral submucous fibrosis 941

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examination of the biopsy specimens. Fibroblasts were cultured using an explant technique as described previously.²⁴The tissues were minced using sterile techniques and wash twice in phosphate buffer saline (PBS) supplemented with antibiotics (100 U/ml penicillin, 100 lg/ml streptomycin and 0.25 lg/ml of fungizone). Explants were placed into 60 mm Petri dishes and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gib- co Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS) (Gibco Laboratories, Grand Island, NY, USA) and antibiotics as described above. Cell cultures between the third and eighth passages were used in this study.

Expression of IGF-1 mRNA in OSF and BMF

Total RNA was prepared using TRIzol reagent (Gibco Laboratories, Grand Island, NY, USA) following the manufacturer’s instructions. Single- stranded DNA was synthesized from RNA in a 15 ll reaction mixture containing 100 mg random hexamer and 200 units of Moloney murine leuke- mia virus reverse transcriptase (Gibco Laborato- ries, Grand Island, NY, USA). The reaction mixture was diluted with 20 ll of water and 3 ll of the diluted reaction mixture was used for the polymerase chain reaction (PCR). PCR reaction mixture contains 10 pmol of forward and reverse primers and two units of Tag DNA polymerase.

Amplification was performed at 25 cycles for GAPDH and 30 cycles for IGF-1 in a thermal cycle.

Each cycle consisted of 1 min of denaturation at 94C, 1 min of annealing at 57 C, and 1 min of extension at 72C. The sequences of primers used were as follows:²⁵

[(A) GAPDH]

Forward: 5⁰-TCCTCTGACTTCAACAGCGACACC-3⁰ Reverse: 5⁰-TCTCTCTTCCTCTTGTGCTCTTGG-3⁰

[(B) IGF-1]

Forward: 5⁰-AAATCAGCAGTCTTCCAACC-3⁰ Reverse: 5⁰-CTTCTGGGTCTTGGGCATGT-3⁰

The PCR products were analyzed by agarose gel electrophoresis and a 395 bp band for IGF-1 was noted. When the band densities were measured and compared with the density of the band obtained for the housekeeping gene GAPDH, relative proportions of mRNA synthesis could be determined within each experiment. The intensity of each band after normalization with GAPDH mRNA was quantified by the photographed gels with a

densitometer (AlphaImager 2000; Alpha Innotech, San Leandro, CA, USA).

Assessment of IGF-1 activity in OSF and BMF

Confluent cells were trypsinized, counted, and pla- ted at a concentration of 1· 10⁵cells in 60 mm culture dish and allowed to achieve confluence. The conditioned medium samples were collected after 2 day cultured period. Levels of IGF-1 antigen were determined by enzyme-linked immunosorbent assay (ELISA) (human IGF-1, Quantikine, DG100, R&D Systems, Inc. MN, USA). Briefly, 20 ll of conditioned media were directly transferred to the microtest strip wells of the ELISA plate. All further procedures were performed following the manufacturer’s instructions. The absorbance at 495 nm was measured in a microtest plate spectrophotom- eter and IGF-1 levels were determined with a cali- bration curve using human IGF-1 as a standard.

Effect of arecoline on IGF-1 mRNA and protein in BMF

BMF were seeded 1· 10⁵cells per well into 10 cm culture dish and incubated for 24 h. Then the medium was changed to a medium containing 10%

heated activated FCS and various concentrations of arecoline (Sigma, St. Louis, MO, USA) (0–80 lg/

ml). Total RNA was isolated after 6 h incubation period for RT-PCR as described above. Condition medium were collected after 24 h incubation period for ELISA as described earlier.

Statistical analysis

Triplicate or more separate experiments were performed throughout this study. For testing of differences in the IGF-1 between the BMF and OSF, the Wilcoxon rank sum test was applied. The signifi- cance of the results obtained from control and treated groups was statistically analyzed by the Student t-test. A p-value of <0.05 was considered to be statistically significant.

Results

The connective tissue from normal buccal mucosa consists of loosely woven collagen bundles in the lamina propria revealing a fine reticular pattern next to the epithelium and a coarser pattern dee- per in the lamina propria. The connective tissue of normal human buccal mucosa demonstrated

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with very faint IGF-1 expression (Fig. 1). All OSF samples exhibited the features of either moder- ately advanced or advanced submucous fibrosis.

IGF-1 expression was observed mainly in the cyto- plasm of fibroblasts, endothelial cells, and inflammatory cells throughout the connective tissue (Fig. 2). However, IGF-1 staining was also expressed in the epithelium both OSF and normal buccal mucosa specimens. The intensity of IGF-1 within epithelium between normal buccal mucosa and OSF was not statistical significant (p > 0.05).

RT-PCR assay was used to compare IGF-1 mRNA gene expression of the fibroblasts cultured from BMF and OSF. As shown in Fig. 3, OSF specimens exhibited significantly higher IGF-1 mRNA expression than BMFs. From the AlphaImager 2000, the intensity of IGF-1 mRNA from OSF was elevated about 3.4 fold as compared with BMFs (p < 0.05).

The individual values of IGF-1 from BMF and OSF cultures from ELISA were shown in Table 1. The amount of IGF-1 protein in BMF was about 10.96 ± 1.63 ng/10⁶cells. The amount of IGF-1 protein in OSF was about 35.44 ± 6.51 ng/10⁶cells. In addition, IGF-1 was found increased about 3.2 fold in OSF as compared with BMF (p < 0.05).

To examine the effect of arecoline on the IGF-1 expression, human BMFs were treated with areco-

line and the levels of mRNA and protein were measured. The effects of arecoline on the IGF-1 gene expression in three different cell strains were similar, and their intracellular variations were limited.

RT-PCR were used to verify whether arecoline could affect IGF-1 mRNA gene expression by human BMFs.Fig. 4reveals a dose-dependent change following treatment of BMF with arecoline for 6 h.

Arecoline was found to elevate IGF-1 mRNA gene expression in a dose-dependent manner (p < 0.05).

From the AlphaImager 2000, the amount of IGF-1 was elevated about 2.1, 2.3, 3.2, and 3.4 fold at Figure 2 In OSF specimen, KGF-1 was evident as intense, diffuse brown coloring throughout the connective tissue and detected at relatively high levels in the epithelium.

Figure 3 Comparison of the IGF-1 mRNA level from BMFs and OSFs using RT-PCR assay. GAPDH gene was performed in order to monitor equal RNA loading.

Fibroblasts derived from OSF are significantly upregulated IGF-1 mRNA gene expression than BMFs.

Table 1 Summary of IGF-1 protein levels from BMF and OSF by using ELISA

Subjects BMF (n = 9) OSF (n = 22)

Media Range Media Range

IGF-1 11.69

(ng/10⁶cells)

7.84–14.6 (ng/10⁶cells)

29.52^* (ng/10⁶cells)

13.5–83.78 (ng/10⁶cells) Mean ± SD 10.96 ± 1.63

(ng/10⁶cells)

35.44 ± 6.51^* (ng/10⁶cells)

* Statistically significant between BMF and OSF, p < 0.05.

Figure 1 Very faint immunoreactivity of KGF-1 was observed in normal human buccal mucosal connective tissue and the strong signal was seen in the epithelium.

The upregulation of insulin-like growth factor-1 in oral submucous fibrosis 943

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concentrations of 10, 20, 40, and 80 lg/ml, respec- tively, as compared with control (Fig. 5).

As shown inFig. 6, arecoline was found to upreg- ulate IGF-1 activity in BMF cultures. The effect of arecoline on IGF-1 protein in BMF during 24 h incubation period is shown in Fig. 6. Arecoline was found to elevate IGF-1 expression in a dose-dependent manner (p < 0.05).

Discussion

The relationship between areca quid chewing and OSF is well established from epidemiological studies,^2,3although the underlying biochemical mechanisms are not completely understood. It has been described that areca quid as well as its component affect fibroblasts proliferation^6,7increase collagen synthesis.⁶IGF-1 is a profibrogenic growth factor, promoting fibroblast proliferation and extracellular matrix deposition, where increased expression may be critical to the pathogenic of OSF. From the results of immunohistochemistry, our findings demonstrated, for the first time, that positive staining for IGF-1 in connective tissue was found to be upregulated in OSF specimens compared to normal buccal mucosa. Strong immunostaining for IGF-1 was detected throughout the connective tissues, mainly fibroblasts, endothelial cells and inflammatory cells.

Fibroblasts are the principal cell type residing in connective tissue and are responsible for the formation and turnover of extracellular matrix. Fibro- blast function is, in turn, regulated by bioactive molecules acting in the local tissue environment.

To the best of our knowledge, we first found that fibroblasts derived from OSF demonstrated significantly higher IGF-1 expression than BMF. Similar results were found other fibrotic pathogenesis like lung fibrosis,^19,20systemic sclerosis,²¹and hepatic fibrosis.²²This result may be the reason why there is an excessive increase of collagen in OSF. This phenomena promoted us to elucidate that OSF may be due to increased synthesis and deposition of extracellular matrix.

Areca quid chewing-related oral mucosal lesions are potential hazards to a large population world- wide. Many of the undesirable effects of areca nut have been attributed to arecoline. Data from our in vitro experiment show that arecoline is capable of stimulating IGF-1 expression in human BMFs.

The data presented here may partly explain why arecoline could increase oral fibroblasts proliferation^6,7,26 and collagen synthesis.^6,26 During areca quid chewing, arecoline could stimulate synthesis

Arecoline ng protein/106 cells

6 8 10 12 14 16 18 20 22 24 26

IGF-1

0 10 20 40 80 (µg/ml)

*

Figure 6 Expression the protein level of IGF-1 in arecoline-treated human BMFs by using ELISA. \Signifi- cant differences from control values with p < 0.05.

Arecoline concentration

Fold change

0 1 2 3 4

0 10 20 40 80 (µg/ml)

Figure 5 Densitometric analysis of the IGF-1 bands was calculated from their mRNA activity. Optical density values represent the means of three different BMF ± standard deviations.

Figure 4 Expression of IGF-1 mRNA gene in arecoline- treated human BMFs by RT-PCR assays. Cells were exposed for 6 h containing arecoline concentrations as indicated. M = DNA molecular size marker. GAPDH gene was performed in order to monitor equal RNA loading.

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and deposition of extracellular matrix by elevating IGF-1 levels.

As far as we known, this is the first systematic attempt to evaluate the role of IGF-1 expression in areca quid associated-OSF in human at both in vivo and in vitro levels. We have demonstrated for the first time that IGF-1 expression is upregulated in OSF than normal buccal mucosa. Data from our in vitro experiments showed that arecoline was capable of stimulating IGF-1 mRNA and protein expression in human BMFs. This suggests that one of the pathogenic mechanisms of OSF in vivo may be the synthesis of IGF-1 by resident cells in re- sponse to areca nut challenge. However, the ge- netic and environmental determinants of IGF-1 expression are still incompletely understood. Fur- ther research is required, however, including detection of IGF-1 gene transcripts, specifically whether OSF evolves solely as a result of increased/altered de novo synthesis and deposition of IGF-1 by areca nut constitutes.

Acknowledgement

This study was supported by a research grant from National Science Council, Taiwan (NSC93-2314-B- 040-021).

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行政院國家科學委員會專題研究計畫 成果報告