ARTICLE |
Correspondence to: Tuula Salo, Inst. of Dentistry, University of Oulu, Box 5281, 90014 Oulu, Finland. E-mail: Tuula.Salo@oulu.fi
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Summary |
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Collagen XVII (BP180) is a hemidesmosomal transmembrane component that has been hypothesized to participate in keratinocyte adhesion and motility. Using immunohistochemical (IHC) and in situ hybridization (ISH) methods, we showed downregulation of collagen XVII in basal cells in mild dysplasias and upregulation in suprabasal keratinocytes in moderate and severe dysplasias as well as in the central cells of grade II and III squamous cell carcinomas (SCCs). Overexpression of collagen XVII was found at the invasive front of the tumors. Collagen XVII and its cleaved ectodomain were characterized from culture extracts and precipitates of oral keratinocytes, tongue carcinoma cells, and tumor tissue extract. Malignant cell lines exhibited increased collagen XVII expression in immunoblotting analysis. In oral keratinocytes, collagen XVII gene expression was significantly induced by PMA but not by the inflammatory cytokines TGF-ß1, TNF-, EGF, IL-1ß, and IL-6. These results indicate altered expression of collagen XVII at different stages of carcinogenesis and suggest a correlation between overexpression of collagen XVII and tumor progression. The reduced collagen XVII expression at the early step of carcinogenesis may reflect disturbed keratinocyte adhesion to the basement membrane.
(J Histochem Cytochem 51:921929, 2003)
Key Words: BPAG2, adhesion molecules, cancer, mouth neoplasms
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Introduction |
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Hemidesmosomes are multi-protein junctional complexes that participate in adhesion of epithelial cells to the basement membrane and in maintenance of tissue integrity. Recent studies have revealed novel functions of the hemidesmosome components in signal transduction, including effects on cell behavior in processes such as wound healing, cell migration, and tumor invasion (6ß4 integrin, laminin-5, and collagen XVII has been reported, indicating their role in tumor development and invasion (
Collagen XVII, a hemidesmosome component, is a type II transmembrane protein composed of a globular cytoplasmic domain and a large extracellular domain containing multiple collagenous repeats (6 and ß1 integrins (
A 120-kD peptide is proteolytically released from the extracellular region of collagen XVII into the extracellular matrix (
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Materials and Methods |
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Tissue Samples
Thirty-five cases of tongue SCC, 25 cases of epithelial dysplasias, and four cases of fibrotic hyperplasia were obtained from the archives of the Department of Pathology, Oulu University Hospital. The epithelial dysplasias were graded histologically by an oral pathologist (TS) as mild (n=9), moderate (n=10), or severe (n=6) and the carcinoma samples as well (n=15), moderately (n=15), or poorly (n=5) differentiated. Five samples of normal buccal mucosa were collected from healthy volunteers as normal controls.
Antibodies
For immunostainings and immunoblotting, the following polyclonal collagen XVII antibodies were used: NC16a, raised against the juxtamembranous NC16 domain, i.e., amino acids 490567 (GenBank accession number M911669), Ecto-3, raised against the amino acids 13651413 (
Immunostaining
Immunoperoxidase staining of the paraffin sections was performed as described earlier (
To calculate a score for immunostaining, the intensity of the immunostaining signal and the number of positive cells were quantified by light microscopy (
Western Blotting Analysis
Protein samples, including immunoprecipitated media and cell extracts from oral keratinocytes (IHGKs) and SCC cells (SCC-25, HSC-3), as well as carcinoma tissue extract were separated on 12% SDS-PAGE gel and transferred onto nitrocellulose. Nonspecific antibody binding was blocked with 2% milk in Tris-buffered saline (TBS) (30 min). The polyclonal antibodies NC16a (1:1000), Ecto-5 (1:500), and Ecto-3 (1:500) were diluted in 2% milkTBS and incubated overnight with the membrane. After washes and incubation with the secondary antibody, the collagen XVII signal was detected by NBTBCIP substrate.
Semiquantitative analysis of collagen XVII in extracts of IHGK, SCC-25, and HSC-3 cells was performed by ECL Western blotting. The samples were run on a 12% SDS-PAGE gel and transferred onto Immobilon-P nitrocellulose membrane (Millipore; Bedford, MA). Nonspecific binding of the antibody was blocked by incubation with 10% milk in TBS for 60 min. The membrane was washed with TBS and incubated overnight with polyclonal XVIIcol antibody NC16a diluted 1:500. After washes in TBS, the filter was incubated with peroxidase-conjugated swine anti-rabbit immunoglobulins (1:500; DAKO, Glostrup, Denmark) for 60 min, washed again, and incubated with avidinbiotinylated horseradish peroxidase (ABComplex/HRP; DAKO) for 60 min. The filter was treated with ECL detection solution for 1 min, and collagen XVII was visualized by X-ray film exposure (Hyperfilm-ECL; Amersham Life Science, Poole, UK). The relative amount of collagen XVII in the samples was calculated by scanning the bands on the X-ray film and dividing the scanning unit values by the respective values of the total loaded protein visualized by Coomassie staining (ScionImage PC; Scion Corporation, Annapolis, MD).
In Situ Hybridization
To prepare non-radioactive ISH probes with the DIG RNA Labeling kit (Boehringer Mannheim; Mannheim, Germany), a cDNA fragment, Ecto-4, covering the amino acids 13651497 of the collagen XVII ectodomain was amplified from keratinocyte mRNA with reverse transcriptase polymerase chain reaction (PCR) (Titan reverse transcriptase PCR; Roche Molecular Biochemicals, Mannheim, Germany) with the sense primer 5'-CGCGGATCCGCTGACTTTGCTGGAGATCT-3' and the antisense primer 5'-CGCGGAATTCCGGCTTGACAGCAATACT-3' and, after digestions with BamHI and EcoRI (restriction sites underlined), cloned into the pGEM.4Z vector (Promega; Madison,WI). The probe in the sense orientation was used as a control for nonspecific hybridization. ISH was carried out as described earlier (
Cell Culture Experiments
IHGK cells (HPV-16-transformed oral keratinocyte cells; a kind gift from Prof. Dolphin Oda, Seattle, WA) (
To assess the regulation of collagen XVII gene expression, subconfluent cells were incubated with TGF-ß1 (1/5/10 ng/ml), PMA (30/60/100 ng/ml), TNF- (5/20/50 ng/ml), EGF (0.1/1/10 ng/ml), IL-1ß (10/20/50 ng/ml), or IL-6 (10/20/50 ng/ml). After 24-hr incubation, total RNA was isolated from the cells using a Trizol kit (Gibco BRL; Life Technologies, Roskilde, Denmark). Each experiment was repeated six times.
Protein Extractions
The cultured cells and the conditioned media were processed separately for protein extractions. For immunoprecipitation of the shed ectodomain of collagen XVII, the medium was collected, cooled, centrifuged to remove cell debris, and supplemented with 1 mM Pefablock (Merck; Darmstadt, Germany) and 1 mM N-ethylamide (SigmaAldrich; Deisenhofen, Germany). Before immunoprecipitation, 0.2 g of protein ASepharose (Amersham Pharmacia Biotech; Uppsala, Sweden) was prepared as indicated by the supplier, washed once with PBS, once with PBS0.5% Tween, and once again with PBS. Then 50 µl of the NC16a antibody was added to protein ASepharose in 0.5 ml of PBS and rotated for 3 hr at 4C. The protein ASepharose/antibody complex was washed three times as above, and 30 ml of medium was added to the antibody complexes and rotated at 4C overnight. After three washes as above, the pellet was packed into a small column using 50 mM Tris-HCl, pH 7.0, at RT as column buffer. After extensive washing with the same buffer, the ectodomain of collagen XVII was eluted using 6 ml of 0.1 M glycine buffer, pH 3.0, at RT, and the pH of the fractions was neutralized with 1 M Tris-HCl, pH 9.0, at RT, immediately after elution.
The full-length collagen XVII was extracted from the cell layer as described earlier (
RT-PCR
Four ng of human total mRNA extracted from immortalized keratinocytes of oral cavity (IHGK) and skin (HaCat), SCC cells (SCC-25, HSC-3), endothelial cells (EA.hy926), and myeloma cells (RPMI 8226) was reverse-transcribed using Superscript II RnaseH- Reverse Transcriptase (Gibco BRL) and random hexamer primers. A human cDNA fragment of 230 bp corresponding to the NC16a domain of collagen XVII (amino acids 490567) was amplified using the sense primer 5'-GAGGAGGTGAGGAAGCTGAA-3' and the antisense primer 5'-TCGGAGATTTCCATTTTCC-3'. The PCR reaction was performed in a thermal reactor with thermostable Dynazyme DNA polymerase (Finnzymes Oy; Espoo, Finland), using 35 cycles. The PCR products were analyzed by electrophoresis on 1% agarose gel containing 1.0 µg/ml ethidium bromide.
Ribonuclease Protection Assay (RPA)
RPA was carried out using an RPA IIITM Ribonuclease Protection Assay Kit (Ambion; Austin, TX) according to the manufacturer's instructions. [-32P]-UTP-labeled 228-bp NC16a cRNA probe (nucleotides 41984596) was hybridized with 10 µg of total RNA. The protected double-stranded RNA fragment was run on a 5% denaturing polyacrylamide gel and visualized on an X-ray film (Kodak; Rochester, NY). A 115-bp 18S cRNA probe (Ambion) was included in the hybridization reaction as an internal control for semiquantitative analysis of the protected collagen XVII fragment. Statistical analysis was performed by Student's t-test and statistical significance was set at p<0.05.
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Results |
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Enhanced Expression of Collagen XVII in Epithelial Dysplasias and SCCs
Distribution of collagen XVII in oral precancers and SCCs was studied by immunoperoxidase staining with NC16a, a polyclonal antibody against the extracellular juxtamembranous NC16a domain. In healthy oral mucosa, immunostaining of collagen XVII was seen at the cell membranes surrounding the basal keratinocytes (Fig 1A). In samples of non-dysplastic fibrotic hyperplasia of oral mucosa the staining pattern was similar to that of the healthy controls (not shown). Surprisingly, in all mild dysplasias, collagen XVII staining was mostly missing from the basal keratinocytes, and faint positive signals were seen only in some small areas (Fig 1B). In all moderate and severe dysplasias, in some areas an intense staining signal was detected throughout all layers of the epithelium, but there were also areas without any staining, even in the basal cells (Fig 1C and Fig 1D). In grade I oral SCCs, the immunoreaction of collagen XVII was intense and mainly located in the peripheral carcinoma cells of tumor nests (Fig 1E). Different staining patterns were observed in grade II and III tumors, as the signal was also detected in the central carcinoma cells (Fig 1G). In all tumors, the malignant cells at the invasive front, the most peripheral area of the tumor invading into the surrounding tissue, displayed intense staining (Fig 1H).
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Immunostaining scores were calculated for each tissue section by grading the positive cells and the staining intensity to quantify the immunostainings. Statistical analyses revealed a significantly higher score for collagen XVII level in suprabasal keratinocytes of moderate and severe displasia compared to mild dysplasias or normal mucosa (Fig 3A). The scores were significantly lower for the basal cells of mild and severe dysplasias than for normal mucosa (Fig 3A). The score for basal cells in moderate dysplasias was clearly but not significantly lower than that of the normal control group (p=0.063) (Fig 3A). The immunostaining scores for the basal cells of moderate and severe dysplasias were significantly higher compared to mild dysplasias (Fig 3A). In grade II and III SCCs, the score for the central cells of tumor islands was significantly higher compared to grade I tumors (Fig 3B). There was no significant difference between the scores for the peripheral carcinoma cells in grade I and grade II/III tumors (Fig 3B).
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Increased Collagen XVII mRNA Expression in Epithelial Dysplasias and SCCs
RNA expression was studied by non-radioactive ISH with an Ecto-4 RNA probe corresponding to the 133 most C-terminal amino acids of collagen XVII. The epithelium of healthy oral mucosa contained no detectable mRNA signal (Fig 2A). In mild dysplasias, too, the signal for collagen XVII was mainly negative, except for some areas showing a weak signal in the basal epithelial cells (Fig 2B). In contrast, an extremely strong mRNA signal was observed locally in all epithelial layers of moderate and severe dysplasias (Fig 2C and Fig 2D). In SCCs the collagen XVII mRNA expression pattern was in line with the results of the immunostainings. The signal was detected in the central and peripheral cells of tumor islands (Fig 2E and Fig 2G). The most intense mRNA signal for collagen XVII was seen in the carcinoma cells at the invasive tumor front (Fig 2G).
Collagen XVII mRNA Expression in Cultured Oral Keratinocytes and SCC Cells
Oral keratinocytes (IHGK) and two tongue SCC cell lines (SCC-25, HSC-3) were examined for collagen XVII mRNA synthesis by RT-PCR, the amplified sequence covering the extracellular NC16a domain. Collagen XVII mRNA could be demonstrated in the oral keratinocyte cell line (IHGK) and in both of the oral SCC cell lines (SCC-25, HSC-3) (Fig 4A). Skin keratinocytes (HaCat) served as a positive control. No signal was detected in the mRNA of two negative control cell lines, immortalized endothelial cells (EA.hy926), and plasmacytoma cells (RPMI 8226) (Fig 4A).
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Characterization of Two Forms of Collagen XVII from Cultured Oral Keratinocytes and from SCC Cells and Carcinoma Tissue
Collagen XVII could be extracted from oral keratinocytes (IHGK), two tongue SCC cell lines (SCC-25 and HSC-3), and homogenized SCC tissue. The shed ectodomain was immunoprecipitated from the conditioned media with the NC16a antibody. For immunoblotting, three specific polyclonal antibodies were used: NC16a, Ecto-3, and Ecto-5. All three antibodies detected the full-length protein of 180 kD in the cell extracts, while the 120-kD soluble ectodomain was found in the cell media precipitates (Fig 4B). A signal for the 120-kD peptide was obtained in IHGK, SCC-25, and HSC-3 cell extracts and also in the carcinoma tissue by the NC16a antibody (Fig 4B), which has strong affinity for the shed peptide (
Upregulation of Collagen XVII in SCC Cells
The relative level of collagen XVII synthesis in cultured oral keratinocytes and in two tongue SCC cell lines, SCC-25 and HSC-3, was determined by Western blotting analyses (Fig 4C). In HSC-3 cells, Western blotting with NC16a antibody showed an approximately 1.3-fold collagen XVII expression level compared to IHGK cells (Fig 4C). In SCC-25 cells, the level of collagen XVII was approximately 1.6-fold compared to IHGK cells (Fig 4C).
Collagen XVII Expression Is Induced by PMA
The regulation of collagen XVII gene expression was studied by treating IHGK cells with different concentrations of cytokines TNF-, EGF, TGF-ß, IL-1ß, IL-6, and PMA. The level of collagen XVII mRNA synthesis was measured semiquantitatively by the ribonuclease protection assay (RPA) (Fig 5A). Incubation of keratinocytes with TNF-
(20 ng/ml) or TGF-ß1 (1 ng/ml) led to a 1.35-fold collagen XVII mRNA level compared to the control level (Fig 5B). Treatment with PMA (100 ng/ml) caused a 1.5-fold induction in collagen XVII mRNA expression, which is significantly higher than the steady-state level (p<0.05) (Fig 5B). There were no significant changes in collagen XVII mRNA synthesis in the cells treated with EGF, IL-1ß, or IL-6 (Fig 5B).
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Discussion |
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These results show downregulation of collagen XVII in basal cells in mild dysplasias and upregulation in suprabasal keratinocytes in moderate and severe dysplasias as well as in the central cells of grade II and III carcinomas. Collagen XVII upregulation in malignant cells was confirmed by in vitro studies. Regulation of collagen XVII gene expression by cytokines was studied in oral keratinocytes, revealing clear induction by a tumor promoter, PMA.
In normal oral mucosa, collagen XVII was expressed exclusively in the basal keratinocytes, representing its role as a hemidesmosomal component. Immunostaining was observed on the lateral and basal surfaces of the basal cells, which is typical of collagen XVII and 6ß4 integrin, the transmembrane components of the hemidesmosome (
In moderate and severe oral dysplasias, collagen XVII expression was notably upregulated, because areas of cytoplasmic immunoreaction and mRNA signal covering all the layers of the epithelium were detected. Earlier, a similar staining pattern has been reported for collagen XVII in Bowen's disease (6ß4 integrin, in the areas of invasive growth of head and neck SCCs (
We demonstrated by RT-PCR collagen XVII mRNA expression in two tongue SCC cell lines, HSC-3 and SCC-25, as well as in immortalized oral IHGK keratinocytes. Collagen XVII protein could also be extracted from these cell lines and was characterized by immunoblotting with three antibodies. For the first time, full-length collagen XVII and the shed ectodomain of 120-kD were extracted from SCC tissue. Both the transmembrane 180-kD collagen XVII and the soluble ectodomain have previously been extracted from skin using a similar method (
In this study, treatment with the inflammatory cytokines TNF-, EGF, TGF-ß1, IL-1ß and IL-6, did not result in a significant increase in collagen XVII mRNA expression in oral keratinocytes. However, stimulation with a phorbol ester, PMA, caused a clear induction of collagen XVII mRNA. PMA is a potent tumor promoter capable of inducing several genes that have a role in carcinogenesis and tumor invasion (
2 chain gene promoter is one of the targets for PMA activation, occurring via interaction with the activator protein 1 complexes (
Upregulation of collagen XVII and other hemidesmosome-associated proteins in central carcinoma cells not expressing hemidesmosomes may imply an alternative function of these molecules, in addition to their known role in keratinocytematrix adhesion. In addition, 6ß4 integrin has a significant role in signaling pathways stimulating cell migration and invasion (
5ß1 and
vß1 integrins, which are expressed by keratinocytes during wound healing and, interestingly, also by carcinoma cells of head and neck SCCs (
The results of this study indicate that collagen XVII expression is abnormal at various stages of multi-step carcinogenesis. The reduced expression in mild dysplasias is likely to reflect the disturbed adhesion of basal keratinocytes to the extracellular matrix. On the other hand, collagen XVII upregulation in the absence of hemidesmosome formation in moderate and severe dysplasias and in grade II and III tumors may reflect an alternative function in addition to the role in cell adhesion. The observation of upregulated collagen XVII gene expression in response to the tumor promoter PMA indicates potential involvement in carcinogenesis and tumor progression.
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Acknowledgments |
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Supported by a grant from Finnish Dental Society Apollonia to MP and TS, Finnish Cancer Foundation and OYS KEVO grants to TS, grants from the Alexander von Humboldt Foundation, the Academy of Finland and Oulu University Hospital to KT and by grant no. SFB293-B3 from the German Research Council (DFG) to LBT.
We thank Ms Sirpa Kangas, Ms Anja Mattila, Ms Sanna Juntunen, Ms Eeva-Liisa Kiljander, Ms Maritta Harjapää, Ms Riitta Vuento, and Ms Merja Tyynismaa for expert technical assistance.
Received for publication November 14, 2002; accepted February 5, 2003.
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