Background
DNA damage promotes carcinogenesis. This is clearly seen when DNA repair mechanisms are compromised. The eukaryotic cell takes many measures to prevent DNA damage, including formation of physical barriers to prevent the entry of carcinogens and other substances into the organism. In the gastrointestinal tract, a layer of polarised epithelial cells, held together by tight junctions and covered by a layer of mucus, forms a surprisingly efficient barrier. However, this barrier is often compromised already in dysplastic tissue [
1]. This is likely to be a factor driving carcinogenesis by allowing carcinogens to enter the underlying tissue.
In the intestine, molecules may pass the monolayer of epithelial cells, either by the transcellular route involving transcytosis or by the paracellular route crossing the tight junctions. Tight junctions are primarily located at the apical end of the lateral plasma membrane [
2]. In addition to controlling the paracellular diffusion, tight junctions prevent the diffusion of membrane proteins and lipids between the apical and the basolateral plasma membrane domains [
3]. Tight junctions are formed mainly by three types of integral proteins: occludins, junctional adhesion molecules and claudins [
4]. In total, 24 different mammalian claudins have been described [
5]. The composition of claudins in the tight junction complex is thought to dictate the permeability of the epithelium by regulating the permeability of the tight junctions [
4,
6]. The expression levels of various claudins in a tissue can change according to physiological and pathological conditions, thereby altering the permeability of the epithelial barrier [
7].
Connections of tight junctions between cells are made by interactions between the extracellular loops of the claudins. However, the molecular mechanisms and the exact stoichiometry behind the assembly of tight junctions are poorly understood [
8]. The majority of claudins are known to increase the epithelial tightness by sealing the tight junctions. Claudins like claudin-1, -4, -5, -7, -8, -11, -14, and -19 are considered sealing claudins, as increased expression of these leads to increased epithelial tightness [
6,
9‐
12]. Other claudins are able to form paracellular anion- and cation pores as well as water channels [
5,
13]. Claudin-2, -10, and -16 are examples of pore-forming claudins known to decrease epithelial tightness when expression is increased.
The serine protease matriptase plays an important but, as yet, poorly defined role in the generation and maintenance of epithelial barriers. Matriptase, encoded by the
ST14 gene, is expressed in most epithelial cells [
14] and its proteolytic activity is tightly regulated by at least two membrane-bound inhibitors, HAI-1 (
SPINT1) and HAI-2 (
SPINT2) [
15‐
17]. Ablation of the
ST14 gene in mice generates a phenotype with compromised epithelial barrier function and fatal outcome [
18,
19]. In addition, dysregulated matriptase expression as investigated in transgenic mice has been shown to have a very strong oncogenic potential [
15]. A study using the colonic adenocarcinoma cell line Caco-2, which spontaneously form tight monolayers of polarized cells, when grown on filters, showed that siRNA-induced down-regulation of matriptase resulted in compromised epithelial barrier function [
20].
We have previously shown that the mRNA level of
matriptase and its inhibitors are significantly down-regulated during colorectal carcinogenesis [
21,
22]. Still, the molecular mechanism whereby the proteolytic activity of matriptase affects the epithelial barrier is unknown. In the search for downstream effectors of matriptase, we made an
in-silico array based study to identify genes co-regulated with the matriptase gene,
ST14. The analysis showed that besides the already known inhibitor of matriptase, HAI-1 encoded by
SPINT1, CLDN7, encoding
claudin-7, is the gene most tightly co-regulated with the
ST14 gene. Claudin-7 belongs to the class of claudins promoting epithelial tightness [
10‐
12] and is found in most epithelia, for instance in the airways [
23], the intestines [
24,
25], the Loop of Henle and the collecting duct of the kidney [
8]. Claudin-7 is involved in regulation of the permeability of Cl
- and Na
+ ions. Recently, claudin-7 knockout mice were generated and shown to have a normal phenotype at birth. However, within days they developed chronic dehydration, leading to a fatal outcome 12 days after birth [
12].
In the present study, we investigated the levels of claudin-7 mRNA during colorectal carcinogenesis and found that claudin-7 mRNA is significantly decreased in mild/moderate dysplasia, severe dysplasia and carcinomatous tissue. The decrease in claudin-7 level could also be confirmed at the protein level.
Methods
In-silicoco-expression array database study
The COXPRESSdb version 4.0 located at
http://coxpresdb.jp/[
26] was used to identify genes co-regulated with matriptase. All expression data for this database are based on affymetrix GeneChip, information which has been released by NCBI GEO. The analysis was performed using default settings entering
ST14 as gene symbol in the gene search tool. In the results, the
ST14 icon under the human gene search was selected. Expression similarity for
ST14 in relation to all other genes in the database was calculated using Pearson's correlation coefficient and ranked. The opposite correlation coefficients where also calculated and ranked. To evaluate the strength of co-expression a mutual ranking (MR) value was calculated using the formula MR
(AB) = (rank
(A→B)* rank
(B→A))
0.5. The lower the MR value the higher the co-expression.
Human tissue samples
The tissues used in this study have previously been described [
22]. In short, the KAM cohort is based on a screening performed in the Norwegian Colorectal Cancer Prevention study (NORCCAP) in the county of Telemark, Norway [
27] with the ID number NCT00119912 at Clinicaltrials.gov. In addition, a series of colorectal cancer (CRC) cases was recruited to the cohort from Telemark Hospital in Skien and Ulleval University Hospital in Oslo. The KAM study is approved by the Regional Committee for Medical Research Ethics and the Norwegian Data Inspectorate. In the present study, we analysed 121 cases with carcinoma, 100 cases with adenomas and 18 healthy individuals. From individuals with adenomas, control tissue was sampled 30 cm above the anus. From patients with carcinomas, two control samples were taken from the surgically removed specimen. One sample was taken adjacent to the cancer (normal adjacent) and the other sample was taken as distant from the cancer as possible (normal distant). The histology of the adenomas was examined independently by two histopathologists, who categorised the degree of dysplasia as either mild/moderate (n = 87) or severe (n = 13). Consensus was reached in all cases. Cases of dysplasia were also classified as either low- or high-risk according to the size and/or differentiation state of the adenoma. A high-risk adenoma is defined as an adenoma measuring ≥ 10 mm in diameter and/or with villous components or showing severe dysplasia [
27]. Carcinomas were classified according to Dukes staging. The distribution of gender and age among individuals included in the study is shown in Table
1.
Table 1
Characteristics of cases and healthy individuals participating in this study
Number of men | 6 | 61 | 7 | 66 |
Number of women | 12 | 26 | 6 | 55 |
Mean age ± S.D. | 56.8 ± 4.5 | 56.9 ± 3.6 | 54.9 ± 3.1 | 69.5 ± 12.0 |
Real-time reverse transcriptase polymerase chain reaction
The tissue samples were frozen as soon as possible after surgery and stored in liquid nitrogen until RNA purification. Total RNA was purified from tissue as recommended by the manufacturer using E.Z.N.A. Total RNA Kit II (cat. no. R6834-02, Omega Bio-Tek) and the RNase Free DNase kit I (cat. no. E1091-01). cDNA synthesis was performed on approximately 200 ng RNA per 20 μl using the High-Capacity cDNA Archive Kit (cat. no. 4375222, Applied Biosystems). Quantitative real time RT-PCR for
claudin-7 was performed on the ABI7300 sequence detection system (Applied Biosystems) in Universal PCR Master Mix (cat. no. 4326614, Applied Biosystems) using 250 nM probe and 300 nM primers. Primers and probe were:
CLDN7 forward 5'-ATGATGAGCTGCAAAATGTACGA-3';
CLDN7 reverse 5'-GCACCAGGGAGACCACCAT-3';
CLDN7 probe 5'- FAM-CGCCCTGTCCGCGGCCTT-BHQ-3'. Primers were designed within different exons and with the probe covering the border of exon 1 and exon 2 to prevent amplification of genomic DNA. Primers and probes were obtained from TAGCopenhagen (Denmark). The endogenous
β-actin control was obtained pre-developed (cat. no.4310881E) from Applied Biosystems. In a validation experiment using a control sample, a dilution series was assayed by the comparative C
t method [
28]. When C
t values were plotted against the logarithmic value of the amount of cDNA added, it was shown that the assays were quantitative over a range of 4096-fold dilution and that the PCR reactions had similar efficiencies provided that a threshold of 0.1 is used for
claudin-7 and 0.175 is used for
β-actin. The threshold is a fixed fluorescence signal level above the baseline and the C
t value of a sample is determined as the fractional cycle number where the sample's fluorescence signal exceeds the threshold. All samples were quantified in triplicates. The standard deviation on the triplicates was 6% or less. The standard deviation on repeated measurements of the same sample (the control) in separate experiments was 26%, indicating the day-to-day variation of the assay. Negative controls (where the RNA was not converted into cDNA) and positive controls were included in all runs. Samples for which either the
β-actin or
claudin-7 values fell outside the upper or lower limits of the standard curve were excluded from the study.
Western blot analysis
25-50 mg of frozen tissue was lysed in 500 μl PBS containing 1% Triton X-100, 0.5% deoxycholate, and protease inhibitors (10 mg/l benzamidine, 2 mg/l pepstatin A, 2 mg/l leupeptin, 2 mg/l antipain, and 2 mg/l chymostatin). Protein concentrations were measured using BCA™ Protein Assay Kit (cat. no. 23225, Pierce). Samples were mixed with 2 × SDS PAGE sample buffer containing DTT and boiled for 5 min. The proteins were resolved on 7% acrylamide gels and transferred to Immobilon-P PVDF membranes (Millipore cat. no. IPVH00010). The blots were blocked with 10% non-fat dry milk in PBS containing 0.1% Tween-20 (PBST). PVDF membranes were probed with 0.5 μg/ml rabbit anti-claudin-7 antibody (cat. no. 34-9100, Invitrogen) in 1% non-fat dry milk in PBST at 4°C overnight. Membranes were washed 3 × 5 min wash with PBST and incubated 1 hour with 2 ng/ml goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (cat no. 1858415, Pierce) diluted in 1% non-fat dry milk in PBST. After 3 × 5 min wash with PBST the signal was developed using the ECL reagent Supersignal West Femto Maximum Sensitivity Substrate (cat. no. 34095, Pierce) according to the protocol supplied by the manufacturer, and developed with a Fuji LAS1000-camera (FujiFilm, Sweden). Blots were stripped in 5 ml Restore™ Western Blot Stripping Buffer (cat. no. 21059, Pierce) for 15 min at 37°C. The above mentioned procedure was repeated, using 0.3 μg/ml primary antibody against ß-actin (cat. no. 8226, Abcam) for one hour at room temperature and 2 ng/ml goat anti-mouse secondary antibody conjugated with HRP (cat. no. 1858413, Pierce) for one hour.
Immunohistochemistry
Paraformaldehyde-fixed, paraffin-embedded tissue sections were used for the immunohistochemical analysis. The sections were deparaffinated and antigen retrieval was performed in 10 mM Tris, 0.5 mM EGTA, and pH 9 by microwave treatment for 18 min at 600 Watt. Subsequently, after cooling for 20 min they were rinsed with distilled water. The sections were then treated with 3% H2O2 in distilled water for 10 min and rinsed thoroughly in distilled water, washed in TBS buffer pH 7.6 for 5 min before a 10 min incubation with 1% BSA diluted in TBS buffer. Subsequently, the sections were incubated at room temperature in rabbit anti-claudin-7 antibody (cat. no. 34-9100, Invitrogen) 2 μg/ml in 1% BSA in TBS buffer for 60 min. The sections were then washed 3 × 5 min in TBS buffer, before visualization in EnVision™+ System HRP/rabbit (cat. no. K-4011, DAKO) for 30 min. After an additional 3 × 5 min wash in TBS buffer, DAB (cat. no. K-4011, DAKO) was applied for 10 min. The slides were then rinsed in distilled water and counterstained with Mayer's Haematoxylin for 90 sec and rinsed in water for 5 min before dehydration and mounting in Pertex™ Mounting Medium (cat. no. 00801, Histolab).
Statistical analysis
GraphPad Prism 4 was used for the statistic calculations. The data were not adjusted for gender since the incidence ratio of CRC between the genders is 1:1 in Norway. For all statistical analysis the data was log transformed. Kruskal Wallis and Dunn's Multiple Comparison test were used for statistical comparison of samples from healthy individuals, samples of mild/moderate dysplasias, severe dysplasias and CRC samples. Paired Student's t-test was used for comparison of affected tissue with the matching control sample.
Discussion
In the present study we found a correlation between the
claudin-7 mRNA level, as determined by real-time RT-PCR, and the claudin-7 protein level, as determined by immunohistochemistry in normal mucosa, adenomas and carcinomas of the colon. This suggests that
claudin-7 mRNA level reflects the protein level. Our results suggest that a decrease in the level of
claudin-7 occurs already in mild/moderate dysplasias as an early event in carcinogenesis and that the decreased level is maintained in severe dysplasias and in the CRC tissue. To our knowledge this is the first analysis of
claudin-7 mRNA expression in colorectal mucosa from healthy individuals and from individuals with colorectal dysplasia. Previous studies have compared normal and cancerous colorectal tissue from the same individual. The group of Nakayama et al. [
24] found a lower expression of claudin-7 in 80% of invasive CRCs (n = 90) than in non-neoplastic tissue, which corresponds well with our findings. However, our observations are in contrast to the studies by Kuhn et al. [
25] and Darido et al. [
30], who both found higher expression of claudin-7 protein in CRC tissue as compared to normal tissue from the same individual, using immunohistochemistry alone.
For other types of cancer, a number of studies have compared claudin-7 expression in malignant tissue and normal tissue from patients. They find that claudin-7 is down-regulated in head and neck cancer [
31], nasopharyngeal cancer [
32], squamous cell carcinomas of the oesophagus [
33] and in breast cancer [
29,
34,
35]. However, claudin-7 seems to be up-regulated in gastric cancer [
36] and ovarian cancer [
37]. In squamous cell carcinomas of the oesophagus, reduced expression of claudin-7 correlates with invasion and metastasis [
33]. Likewise, reduced claudin-7 levels correlates with the histological grading of breast carcinomas [
29,
34].
Our
in-silico analysis suggests that dysregulated matriptase may affect the epithelial tightness during carcinogenesis by modulating the expression of claudin-7. We have previously shown that the mRNA expression levels of
matriptase (ST14) and
HAI-1 (
SPINT1) are dysregulated during colorectal carcinogenesis in a cohort very similar to the one used in this study [
21,
22]. Comparison between the mRNA levels of
claudin-7 (this study) and
matriptase mRNA levels in the similar cohort [
21] shows that they have a virtually identical pattern, confirming that mRNA expression of
matriptase and
claudin-7 are closely correlated.
This suggests that the genes encoding matriptase and claudin-7 may be regulated by the same transcription factors. Alternatively, the expression levels of matriptase may affect the expression levels of claudin-7. We attempted to analyse whether an over-expression of matriptase in the colonic adenocarcinoma cell line Caco-2 influenced the claudin-7 mRNA level. However, these experiments were inconclusive as manipulation of matriptase expression is cytotoxic (Vogel et al., unpublished results). Further investigations are needed to clarify this point.
It has recently been shown that siRNA silencing of matriptase in Caco-2 cells resulted in up-regulation of the claudin-2 protein level [
20]. Claudin-2 is a pore-forming claudin closely related to claudin-7 [
38]. Up-regulation of claudin-2 thus results in increased epithelial leakiness. Claudin-2 is heavily up-regulated in colorectal cancer tissue compared to normal tissue from the same individual [
39,
40]. There have been no reports about the expression level of claudin-2 in dysplastic tissue. The increased epithelial permeability seen in colorectal dysplastic tissue is thus probably the result of dysregulation of a number of claudins, some of which may depend on matriptase expression or activity.
In esophageal squamous cell carcinoma cells, over-expression of claudin-7 resulted in more adhesive and less invasive cells, whereas knockdown of
claudin-7 using a small interfering RNA approach led to enhanced invasion into a three-dimensional matrix [
41]. This suggests that
claudin-7 down-regulation does indeed contribute to drive carcinogenesis.
Acknowledgements
We thank Christel Halberg for kind technical help and assistance performing the mRNA analysis and Margit Bæksted for performing the immunohistochemical stainings. This work was supported by The Norwegian Cancer Society, Telemark University College, the Norwegian Colorectal Cancer Prevention (NORCCAP) study, Eastern Norway Regional Health Authority, The Cluster of Cell Biology at the University of Copenhagen, The Harboe Foundation, The Augustinus Foundation, The Brothers Hartmanns Foundation, The A.P. Møllers Foundation for the Advancement of Medical Science and the Lundbeck Foundation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SF and LV conceived the idea of the study. SF performed the in-silico analysis. EHK, IMBL, EJ, TI and KMT established the NORCCAP and Ulleval colorectal cancer cohort (KAM cohort). LV and EHK extracted the RNA. LV validated primers and probes. JB did the western blotting. ESR performed the immunohistochemical stainings. SSP took the pictures. IMBL performed and evaluated the sections of immunohistochemical analysis. JB analysed the data, prepared the figures and performed the statistical analysis. JB drafted the first manuscript. All authors helped with the draft, read and approved the final version.