A non-tight junction function of claudin-7—Interaction with integrin signaling in suppressing lung cancer cell proliferation and detachment
- Zhe Lu†1, 2,
- Do Hyung Kim†2,
- Junming Fan2,
- Qun Lu2, 3,
- Kathryn Verbanac3, 4,
- Lei Ding2,
- Randall Renegar2 and
- Yan-Hua Chen2, 3Email author
© Lu et al. 2015
Received: 5 October 2014
Accepted: 18 May 2015
Published: 17 June 2015
Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells.
Claudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin β1. Tumor growth in vivo were analyzed using athymic nude mice.
Claudin-7 co-localizes and forms a stable complex with integrin β1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin β1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin β1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin β1 expression and increases cell-matrix adhesion. Integrin β1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells.
In this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin β1.
Lung cancer is the leading cause of cancer death for both men and women in the United States and nearly 85 % of patients who develop lung cancer die from it. The overall 5-year survival rate of lung cancer is as low as 15 % . Lung cancer usually forms in the cells lining air passages. The vast majority of lung cancers are carcinomas-malignancies that arise from epithelial cells. Tight junctions (TJs) are the most apical component of the junctional complex and provide the critical support of cell-cell adhesion in epithelial cells . The disruption of cell adhesion promotes cancer progression, invasion and metastasis [3, 4]. TJs serve as a barrier regulating the ions and small molecules through the paracellular pathway . Both downregulation and upregulation of TJ proteins can alter the organized arrangement of TJ strands, allowing more nutrients and growth factors to cancerous cells, thus acquiring the cells with invasive behavior .
Claudins are tetraspan proteins with molecular weights of 20–27 kDa . They are the major structural and functional components of TJs. Abnormal expressions and mislocalization of claudins are frequently observed in epithelial-derived cancers [8–11]. Moldvay et al. have analyzed the expression profile of different claudins in lung cancers and found that claudin-7 is downregulated in several types of lung cancers including the squamous cell carcinoma at the mRNA level . Our previous study demonstrates that claudin-7 is strongly expressed in benign bronchial epithelial cells with a predominant cell-cell junction staining pattern while it is either altered with discontinued weak expression or completely absent in lung cancers . However, the exact roles of claudin-7 in lung tumorigenesis are largely unknown.
Although claudins are well-known apical TJ proteins, recent antibody-based studies indicated that several claudins, including claudin-7, are not only localized at the apical TJs but also have a strong basolateral membrane distribution in the epithelia of various tissues [14–16]. These observations suggest that claudins could be involved in cell-matrix interactions. The principal proteins at the basolateral membrane responsible for anchoring cells to extracellular matrix proteins are integrins . Integrins are heterodimers with α and β subunits and play essential roles in cell attachment, survival, migration and invasion [18, 19].
In this study, we identified that claudin-7 co-localized and formed a protein complex with integrin β1 in human lung cancer cells. Suppression of claudin-7 not only promoted cell proliferation, but also disrupted the localization and downregulated the expression of integrin β1 at both mRNA and protein levels, resulting in the severe defective cell attachment. Introducing integrin β1 into claudin-7-deprived cells partially rescued the defect in cell attachment. Thus, claudin-7 exhibits a non-TJ function in regulating cell attachment through integrin β1.
Increased cell proliferation and cell cycle progression in claudin-7 KD cells
We proceeded to examine several proteins involved in cell proliferation, cell survival and apoptosis by western blot. Our results revealed increased levels of phospho-ERK1/2, phospho-Bcl-2 and survivin and decreased level of cleaved PARP in claudin-7 KD cells (Fig. 2c). The claudin-7 KD2 cells also displayed an increased level of phospho-ERK1/2 and a decreased level of cleaved PARP as shown in Additional file 3: Figure S3B. Similar results were obtained in H358 claudin-7 KD cells in that these cells were higher in cell proliferation and had increased phospho-ERK1/2 level and decreased cleaved PARP level (Additional file 4: Figure S4).
Figure 2d shows the cell cycle analysis before and after 24 h of aphidicolin treatment. Aphidicolin blocks cells in S phase and prevents G1 cells from entering mitosis. Cyclin dependent kinase 2 (Cdk2) is required for G1 to S phase transition . After 2.5 and 18 h of releasing the cells from aphidicolin, the cdk2 expression levels were higher in claudin-7 KD cells (Fig. 2e), indicating that more KD cells were transitioning from G1 to S phase. There were no significant differences in expression levels of cyclin B1 and cyclin E between control and claudin-7 KD cells.
We used TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining method to detect the apoptosis. Although the total population of apoptotic cells in both control and claudin-7 KD cells was low, the claudin-7 KD cells showed a lower percentage of apoptotic cells than the control cells as shown in Additional file 5: Figure S5.
Impaired cell attachment and decreased integrin β1 expression in claudin-7 KD cells
To determine whether integrin β1 is the key regulator mediating cell-matrix adhesions, we used mouse anti-human integrin β1 adhesion-blocking antibody to treat both control and claudin-7 KD cells. Mouse IgG treatment was used as a negative control (Fig. 4e). Anti-integrin β1 antibody treatment significantly interfered with the cell attachment in both control and KD cells when compared to the mouse IgG treatment (Fig. 4e, right). The cell attachment assay revealed that the attached cells were significantly reduced in both control and claudin-7 KD cells after integrin β1 antibody treatment. This adhesion-blocking experiment indicates that integrin β1 is one of the key regulators in mediating cell attachment in HCC827 cells.
Importantly, H358 claudin-7 KD cells also formed spheroids when they were plated on the uncoated coverslips while control cells adhered well (Additional file 8: Figure S8A). Results from cell attachment assay indicated that only about 15 % H358 claudin-7 KD cells were attached to the culture plate when compared to the control cells (Additional file 8: Figure S8B). The expression levels of integrin β1 and phospho-FAK were also reduced in H358 claudin-7 KD cells (Additional file 9: Figure S9A). Immunofluorescent staining showed the partial co-localization of claudin-7 with integrin β1 (Additional file 9: Figure S9B). In addition, claudin-7 interacted with integrin β1 and formed a protein complex in H358 cells as well (Additional file 9: Figure S9C).
Rescue claudin-7 KD cell defects by exogenous claudin-7 or integrin β1 expression
To investigate whether slowing down the cell proliferation rate is able to rescue the phenotype of KD cells, we treated control and claudin-7 KD cells with genistein or aphidicolin for 24 h. Our results showed that claudin-7 KD cells responded to both drug treatments in that the cell size became enlarged when compared to untreated cells (Fig. 5d&e). However, culturing KD cells on type IV collagen-coated plates or treating KD cells with genistein or aphidicolin did not increase integrin β1 protein expression level in claudin-7 KD cells (Fig. 5f).
Claudin-7 suppressed tumor growth in vivo
In this study, we demonstrated that knockdown of claudin-7 significantly reduced integrin β1 expression and altered its localization in HCC827 and NCI-H358 lung cancer cells. These claudin-7 KD cells displayed the severe cell-matrix adhesion defect that was also observed in breast cancer cell line T-47D. Confocal immunofluorescence microscopy revealed the partial co-localization of claudin-7 with integrin β1 at the basolateral membrane. Co-immunoprecipitation experiments indicated that claudin-7 interacted with integrin β1 and formed a protein complex in the control cells. The cell-matrix adhesion defect can be rescued by transfecting claudin-7 or integrin β1 into claudin-7 KD cells. Our data also demonstrated that knockdown of claudin-7 promoted cell proliferation and propelled cell cycle progression. The in vivo experiments indicated that claudin-7 KD cells produced larger and heavier tumors in nude mice when compared to the control cells. It is known that the fast-growing tumor has a higher cell proliferation rate as well as a higher cell apoptosis rate due to lack of nutrition and hypoxia within the tumor tissue [21–23]. In the cell culture system, cancer cells have sufficient nutrition and oxygen. This could explain why a higher expression level of cleaved PARP was observed in Claudin-7 KD cells-induced tumors (Fig. 7b) while claudin-7 KD cells in culture had a decreased level of cleaved PARP (Fig. 2c). Our results suggest that claudin-7 could potentially be a tumor suppressor in lung cancer inhibiting tumor cell growth both in vitro and in vivo.
Integrins are cell surface receptors that lack intrinsic tyrosine kinase activity. FAK is constitutively associated with integrin β1 of the integrin receptors. The binding of integrin β1 to extracellular matrix proteins leads to the activation of FAK through its auto-phosphorylation at tyrosine 397 site. It has been reported that claudin-2 facilitates the cell-matrix adhesions by increasing the cell surface expression of α2β1 and α5β1-integrin complexes in breast cancer cells, promoting the formation of breast cancer liver metastases . In this study, we found that knockdown of claudin-7 reduced integrin β1 at both mRNA and protein levels. How claudins affect integrin expression is largely unknown. We were unable to detect claudin-7 in the nucleus by immunofluorescence staining, however, we cannot rule out the possibility that claudin-7 translocated into the nucleus at a level that was beyond the immunofluorescence detection sensitivity. Western blot on nuclear extracts is needed to further determine whether claudin-7 is present in the nucleus and can potentially affect integrin β1 transcriptional expression. On the other hand, claudin-7 could indirectly regulate integrin β1 transcription by regulating the transcription factors of integrin β1, such as Pax6 , Hox D3 , hypoxia-inducible factor (HIF)  and c-Myc . One approach to investigate the potential transcriptional factors involved is to apply cDNA microarrays in the future.
Since suppression of claudin-7 not only disrupted cell-matrix adhesions but also caused a higher cell proliferation, we investigated which of these two consequences was the primary effect of claudin-7 knockdown. Frequent cell division can promote cell detachment. During mitosis, cells undergo large morphologic changes; they become detached, form retraction fibers while the cell margin moves inward, and then cells are finally rounded up and prepared for division . This indicates that cell-matrix detachment could be a consequence of rapid cell division. Therefore, we inhibited cell proliferation using aphidicolin or genistein and found that claudin-7 KD cells partially improved the cell attachment measured by the prolonged trypsinization time although KD cells still took less time to be lifted from the plates than the control cells (data not shown). However, we did not detect the increased level of integrin β1 after drug treatment. Therefore, the improved cell attachment could be due to the increased cell size with the larger attachment surface.
Type IV collagen is the structural backbone of the basement membrane of several solid organs including lung and has the ability to interact with the cell surface adhesion molecules such as integrins. Our results show that several collagens were significantly decreased at mRNA levels in claudin-7 KD cells. Culturing claudin-7 KD cells on collagen IV coated-plates partially reversed the morphology of claudin-7 KD cells in that they became larger in size, more spread out, and had improved cell attachment. However, cell number count revealed that claudin-7 KD cells still displayed a higher proliferation rate than the control cells when culturing on type IV collagen-coated plate. Type IV collagen has been noted to have disparate effects on adhesion and proliferation in other cell lines. Adhesion of melanoma cells to type IV collagen is enhanced by 50 %, but proliferation is inhibited by 40 % when compared to controls . The proliferation of rat cortical progenitor cells is also inhibited on type IV collagen . In the case of hepatoma cells, both adhesion and proliferation increase on type IV collagen . Our results suggest that cell attachment depends on both cell proliferation and integrin β1 level, whereas cell proliferation does not depend on cell attachment. Both enhanced cell proliferation rate and decreased integrin β1 level are the primary results of claudin-7 suppression in HCC827 and H358 lung cancer cells.
To conclude, in this study, we have discovered a novel function of claudin-7 as a basolateral protein in regulating cell proliferation and cell-matrix adhesion in lung cancer cells. This study extends our current understanding of the roles of claudins in carcinogenesis. Both claudins and integrins play essential regulatory roles in tumor proliferation, invasion and metastasis. Understanding the molecular mechanisms of the lung cancer cell-matrix adhesions could lead to the identification of novel therapeutic strategies to target tumor cells. This novel basolateral function of claudin-7 engaging integrin β1 in human lung cancer cells could provide a previously unidentified therapeutic target in the future.
Our study suggests a tumor suppression role of claudin-7 in lung cancer growth and identifies a new function of claudin-7 in maintaining epithelial cell attachment through interaction with integrin β1.
Materials and methods
Antibodies and reagents
The rabbit polyclonal anti-claudin-1 (Cat. 51–9000), −2 (Cat. 516100), −3 (Cat. 341700), −4 (Cat. 364800), ZO-1 (Cat. 61–7300), and mouse monoclonal anti-myc (Cat. 46–0603) antibodies were from Invitrogen (Carlsbad, CA). The rabbit polyclonal anti-claudin-7 antibody (Cat. 18875) was obtained from Immuno-Biological Laboratories (Japan). The anti-E-cadherin antibody (Cat. 610182, clone: 36) was purchased from BD Biosciences (San Jose, CA). The rabbit polyclonal anti-phospho-Bcl-2 (Cat. A7025, Ser56) was from Assay Biotechnology (San Francisco, CA). The rabbit polyclonal anti-phospho-FAK (Cat. 3283S) and anti-phospho-ERK1/2 (Cat. 9101S) were from Cell Signaling Technology (Beverly, MA). The mouse monoclonal and the goat polyclonal anti-integrin β1 antibodies were purchased from BD Biosciences (Cat. 610467) and Santa Cruz Biotechnology (Cat. sc-6622, Santa Cruz, CA), respectively. The mouse anti-integrin β1 adhesion-blocking antibody was from Chemicon (Cat. MAB22253Z, Millipore, MA).
Cell culture, lentivirus shRNA knockdown of claudin-7, transfection of claudin-7 tagged with myc
Lung adenocarcinoma cell line HCC827, lung carcinoma cell line NCI-H358 (H358) and mammary ductal carcinoma cell line T-47D were purchased from ATCC (Manassas, VA) and grown in RPMI 1640 culture medium containing 10 % FBS, 100 units/ml of penicillin, and 100 μg/ml streptomycin in a humidified air (5 % CO2 atmosphere) at 37 °C. Each of three lentivirus claudin-7 shRNA vectors (sequences #1: 5′-TTCCAAGGAGTATGTGTGA-3′; #2: 5′-GGCTATGGGAGTGTCTAGA-3′; #3: 5′-TCCCTACCAACATTAAGTA-3′) or one off-target shRNA control vector were transfected into HCC827, H358 or T-47D cells. The cells infected with #2 lentivirus claudin-7 shRNA vector are designated as KD cells, and the cells infected with #3 shRNA vector are designated as KD2 cells. All lentivirus vectors contain a GFP expression sequence. After 48 h incubation, transfected cells were selected by 1 μg/ml puromycin.
The pcDNA3.1 myc or pcDNA3.1-claudin-7-myc (mouse claudin-7 cDNA) was transfected into HCC827 claudin-7 KD cells using Amaxa Nucleofector device (Amaxa Biosystems, Cologne, Germany). Geneticin (G418) was added to select the transfected cells.
Cell number count and cell cycle analysis by flow cytometry
Five × 103 HCC827 or H358 control or claudin-7 KD cells were seeded into each well of 24-well culture plates and then trypsinized on days 2, 4, 6 after plating, respectively. One × 105 6 micron AlignFlow Plus beads (Molecular Probes, Eugene, OR) were added to each sample and relative ratio of beads versus cells was obtained by the flow cytometer. The total cell number for each sample was then calculated.
HCC827 control or claudin-7 KD cells untreated or treated with 5 μg/ml aphidicolin for 24 h were harvested, washed with PBS, and fixed in 70 % ethanol. Prior to analysis, cells were washed with PBS and then resuspended in the propidium iodide staining solution. Samples were analyzed on the flow cytometer.
RNA isolation and real-time RT-PCR
Total RNA was isolated from HCC827 control and claudin-7 KD cells using Qiagen RNeasy Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions and reverse transcripted into cDNA using RT2 First Strand Kit (Qiagen). Real-time RT-PCR was performed using the RT2 SYBR Green qPCR Mix (Qiagen). The relative changes in gene expression were analyzed using 2-ΔΔCt method .
HCC827 or H358 control or claudin-7 KD cells grown on poly-D-lysine coated glass coverslips (BD Biosciences, Bedford MA) were fixed in 100 % methanol for 8 min at −20 °C and washed with PBS for 5 min before blocked in 5 % bovine serum albumin for 60 min at room temperature. After blocking, cells were incubated with primary antibodies. All antibodies were diluted in PBS containing 2.5 % BSA. After washing, cells were incubated with corresponding secondary antibody for 45 min at room temperature. Coverslips were mounted with ProLong Antifade Kit (Molecular Probes Inc., OR). Samples were photographed using a Zeiss Axiovert S100 or Zeiss LSM 510 laser confocal scanning microscopy (Carl Zeiss Inc., Thornwood, NY) and analyzed by MetaMorph software (Molecular Devices, Sunnyvale, CA).
HCC827 or H358 control cells were washed three times with ice-cold PBS and then lysed in RIPA buffer. After centrifugation, the supernatants were incubated with either anti-claudin-7 or anti-integrin β1 antibody at 4 °C overnight. Protein A or protein G beads were then added to the mixture, followed by incubation at 4 °C for 3 h. The beads were washed twice with RIPA buffer, once with high salt buffer (0.5 M NaCl), and once with Tris buffer (10 mM Tris, pH 7.4). Bound proteins were eluted from the beads in SDS sample buffer and analyzed by western blot.
Measurement of transepithelial electrical resistance (TER)
For TER measurement, HCC827 control or claudin-7 KD cells were plated on collagen-coated Transwell filters with a pore size of 0.4 μm (Corning Costar, Cambridge, MA). A millicell-ERS volt-ohm meter (Millipore, Bedford, MA) was used to determine the TER value. All the TER values were normalized for the area of the filter and obtained after background subtraction.
In vivo tumor xenograft model
Five-week old male athymic nude mice were obtained from Charles River Laboratory (Wilmington, MA) and used for human tumor xenografts. Two × 106 HCC827 control or claudin-7 KD cells were suspended in the culture medium and injected subcutaneously into the left and right flanks of each nude mouse, respectively. All the mice were sacrificed eight weeks after injection. The tumors were removed and weighed. The animal experiments were performed according to the animal use protocol (AUP) approved by East Carolina University.
Statistical analysis was performed using Origin50 (OriginLab, MA) or SigmaPlot (SPSS Science, Chicago, IL) software. For each in vitro experiment, at least three independent experiments were performed. All data was recorded and expressed as means ± SE. The differences between two groups were analyzed using the unpaired Student’s t-test. The data from the nude mice tumor xnenograft experiment was analyzed using Wilcoxon Rank-Sum Test. All statistical tests were two-sided and a value of P < 0.05 was considered significant (*).
We thank Joani Zary-Oswald, Rodney Tatum, and Beverly Jeansonne for their technical assistance. This work was supported by a research award from ECU Division of Research and Graduate Studies and National Institute of Health grants ES016888 and HL085752 (Y.-H Chen), and The National Science Foundation of China 31200581 and the foundation from Hangzhou Science Technology Bureau 20120633B26 (Z. Lu).
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