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Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma
Biochemical and Biophysical Research Communications 380 (2009) 387–391
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma Paul Gutwein a,*, Anja Schramme a, Beren Voss a, Mohamed Sadek Abdel-Bakky a, Kai Doberstein a, Andreas Ludwig b, Peter Altevogt c, Martin-Leo Hansmann d, Holger Moch e, Glen Kristiansen e,1, Josef Pfeilschifter a,1 a
Pharmazentrum frankfurt/ZAFES, Klinikum der, Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany Institute of Pharmacology and Toxicology, University Hospital RWTH, Aachen, Germany Tumor Immunology Programme, German Cancer Research Center, Heidelberg, Germany d Institute of Pathology, Goethe-Universität, Frankfurt am Main, Germany e Institute of Surgical Pathology, University Hospital Zurich, University of Zurich, Zurich, Switzerland b c
a r t i c l e
i n f o
Article history: Received 15 January 2009 Available online 23 January 2009
Keywords: JAM-A Kidney Renal cancer Metalloproteinases Tight junctions
a b s t r a c t Junctional adhesion molecule-A (JAM-A) is one component of tight junctions which are involved in important processes like paracellular permeability, cell polarity, adhesion, migration, and angiogenesis. Here we describe JAM-A expression in distal convoluted tubule, connecting tubule, and in cells of the collecting duct of the healthy human kidney. In addition, JAM-A was weakly expressed in cells of the proximal tubule. Using immunofluorescence, FACS and Western blot analysis we investigated JAM-A expression in tubular cells in vitro. Interestingly, treatment of HK-2 cells with IFN-c and TNF-a resulted in a metalloproteinase mediated downregulation of JAM-A. Importantly, in a tissue micro-array JAM-A protein expression was significantly downregulated in patients with clear cell renal cell carcinoma. Furthermore, knockdown of JAM-A with JAM-A specific siRNA induced the migration of RCC4 cells. In summary, downregulation of JAM-A is an early event in the development of renal cancer and increases the migration of renal cancer cells. Ó 2009 Elsevier Inc. All rights reserved.
Epithelial tight junctions (TJ) are dynamic structures and the molecular composition of TJ is complex consisting of multiple transmembrane proteins including claudin family members, occludin, and members of the junctional adhesion molecule (JAM) protein family [1]. JAMs are a family of transmembrane IgG glycoproteins expressed on a number of cells and the best characterized members are JAM-A, JAM-B, and JAM-C [2]. The expression of JAM-A has been shown on the surface of endothelial and epithelial cells of various tissues, including liver, pancreas, kidney, heart, brain, lymph nodes, intestine, lungs, placenta, and vascular tissue [3]. JAM-A is apically positioned at tight junctions and associates with the tight junction components cingulin and occludin, with the PDZ-domain-containing proteins ZO1, AF6, PARD3 or with CASK, all of which can act as scaffold for larger complexes. It has been suggested that the JAMs may participate in regulating the tight junctions and maintaining paracellular permeability [4]. In the same line, it has been shown that JAM-A regulates epithelial cell morphology by modulating the activity of the small GTPase Rap1 [5]. * Corresponding author. Fax: +49 69 6301 79 42. E-mail address: [email protected] (P. Gutwein). 1 Both the authors have contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.100
Global analysis of gene expression demonstrated that cancer cells have many differences in gene expression from their normal precursors. Particularly, many genes involved in cell–cell adhesion, including those contributing to tight junctions, are downregulated or overexpressed in different carcinomas [6]. In this context it has been recently shown that JAM-A functions as a key negative regulator of breast cancer cell invasion and possibly metastasis [7]. In this study we analyzed the expression of JAM-A in the human kidney and on a tissue micro-array with biopsies of 282 renal cancer patients by immunohistochemistry. We demonstrate for the first time, that JAM-A is strongly expressed in tubular cells in the normal human kidney and JAM-A expression was significantly downregulated in biopsies of patients with clear cell renal cell carcinomas (ccRCC). In addition, inhibition of JAM-A in a RCC cell line induced the migration of the cancer cells. Furthermore, we demonstrate that the pro-inflammatory cytokines IFNc and TNF-a induced a metalloproteinase mediated downregulation of JAM-A from the cell membrane of tubular cells. In summary, our study demonstrates that downregulation of JAM-A in the kidney contributes to the progression of renal cell carcinomas.
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Materials and methods Kidney sections. Specimens were taken from healthy parts of renal tissue from six different tumor nephrectomies (obtained from 2 female and 4 male patients with ages ranging from 34 to 66 years). Kidney tissues were originally submitted for diagnostic purposes and studied in accordance with national and local ethical principles. The use of human tissue samples has been approved by the local ethics committee (Ref-No. #11/10/04). Tissue micro-array construction. We constructed a tissue microarray (TMA) from renal cell carcinomas diagnosed at the Institute of Surgical Pathology, Universitätsspital Zurich, between 1993 and 2003. The TMA is described in detail in a recent publication [8]. Cell culture. The renal cancer cell line RCC4 was provided by Prof. Brüne (Institute of Biochemistry I/ZAFES, Faculty of Medicine, Goethe University Frankfurt, Frankfurt, Germany), the renal cancer cell lines ACHN3 and CAKI-6 were a kind gift from Prof. Jung (Department of Urology, Charité, Berlin, Germany) and the A498 renal cancer cell line was received from Dr. K. Joehrer (Department of Urology, Innsbruck Medical University, Innsbruck, Austria). The renal cell line CAKI-2 was provided by Dr. Grünberg (Center for Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland). The primary renal tubular cell line HRCEpiC was purchased from ScienCell Research Laboratories, Berlin, Germany. The proximal tubular cell line HK-2 has been described elsewhere [9]. The growth medium for ACHN3, RCC4, CAKI-2, and CAKI-6 cells was DMEM supplemented with fetal bovine serum (10%), sodium pyruvate (1 mM), penicillin (100 U/ml), and streptomycin (100 lg/ml). The A498 cells were cultivated in RPMI supplemented with fetal bovine (10%), penicillin (100 U/ml), and streptomycin (100 lg/ml). The growth medium for the HRCEpiC cells was EpiC Medium supplemented with fetal bovine serum (2%), penicillin (100 U/ml), streptomycin (100 lg/ml), and epithelial cell growth supplement (1%). The HK-2 cells were cultivated in RPMI supplemented with fetal bovine serum (10%), non-essential amino acids (1%), sodium pyruvate (1%), 10 mM Hepes, pH 7.4, Hydrocortison (0.1%), Insulin-Transferrin-Sodium-supplement (0.1%). Cytokines, chemicals, and antibodies. Recombinant human IFN-c and recombinant human TNF-a were obtained from R&D Systems (Wiesbaden, Germany). The F11R mouse monoclonal antibody (Western blot, immunohistochemistry, immunofluorescence) was from Abnova Corporation (Heidelberg, Germany) and the monoclonal antibody to JAM-A antibody (BV16; FACS analysis) was from Hycult Biotechnology (Uden, The Netherlands). The rabbit anti-Calbindin D-28K (EG-20) and the anti-Aquaporin-2 antibody were from Sigma (Taufkirchen, Germany). The rabbit anti-thiazide-sensitive NaCl cotransporter (NCC) polyclonal antibody was from Chemicon (Hampshire, UK). The Tamm–Horsfall protein (THP) antibody for immunohistochemistry was obtained from Biotrend GmbH (Cologne, Germany). The metalloproteinase inhibitors GI254023X was kindly provided by Dr. Andreas Ludwig (Institute of Pharmacology and Toxikology, University Hospital Aachen, Germany). The broad spectrum metalloproteinase inhibitors GM6001 and TAPI-2 were obtained from Calbiochem (Darmstadt, Germany). Immunohistochemistry. Paraffin tissue sections were deparaffinized in xylene, rehydrated through a graded ethanol series and washed in 10 mM phosphate-buffered 150 mM saline, pH 7.4. Antigen retrieval was performed by incubating the tissue sections for 20 min in 1 Target Retrieval Solution (DakoCytomation, Hamburg, Germany) in a microwave oven (500 W). Tissue sections were than processed as described in [10]. The sections were inspected with a Zeiss microscope coupled to a 12-bit digital image camera. TMA immunohistochemistry. The TMA blocks were freshly cut (3 lm) and mounted on superfrost slides (Menzel Gläser). Immunohistochemistry was conducted with the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson,
AZ) using Ventana reagents for the entire procedure. The primary JAM-A antibody was used at a concentration of 1:100. For detection we used the UltraVIEWTM DAB detection kit using the benchmarks CC1m-heat induced epitope retrieval. Slides were counterstained with hematoxylin, dehydrated and mounted. Evaluation of TMA immunohistochemistry. The immunohistochemistry was evaluated by a single GU pathologist (GK) in one go to minimize intraobserver variability. Intensity of immunoreactivity was semiquantitatively scored as negative, weakly, moderately or strongly positive, as illustrated by Fig. S2. This panel of figures was compiled before the systematic evaluation of the tumor cohort was commenced and was used as reference, which lay next to the microscope for continuous comparison. Fluorescence microscopy. For immunofluorescence analysis, tissue sections were deparaffinized as described above and antigen retrieval was performed incubating the tissue sections for 20 min in 1 Target Retrieval Solution (DakoCytomation, Hamburg, Germany) in a microwave oven (500 W). Tissue sections were than processed as described in detail elsewhere [10]. Cells were grown on coverslips and fixed with 4% paraformaldehyde/PBS. Fixed cells were than processed as described in [8]. FACS analysis. The renal cell lines HK-2, HRCEpiC, ACHN3, and CAKI-6 were incubated for 30 min with monoclonal antibody against JAM-A (diluted 1:50). All steps were performed as recently described [8]. Protein extraction and Western blot analysis. Cell extracts were prepared as described before [10] at time points indicated. Membrane fractions were isolated by incubating the cells for 10 min in hypolysis buffer (10 mM Tris–HCl, pH 8.0, 0.1 mM DTT, Protease Inhibitor complete (Roche Diagnostic, Penzberg, Germany) at 4 °C. Cell suspension was homogenized in a glass–glass homogenizer and membrane proteins were centrifuged at 1000g and 4 °C followed by a high-speed centrifugation at 100,000g. Proteins were separated under reducing conditions by electrophoresis using 12% SDS–PAGE (polyacrylamide gel electrophoresis) for detection of JAM-A. All following steps were performed as already described [8]. Blots were probed overnight with a monoclonal JAM-A antibody diluted 1:1500 in TTBS. siRNA transfection. For downregulation of JAM-A expression the following siRNA duplex (MWG Biotech AG, Ebersberg, Germany) was used: 50 -UUC GAG UAA GAA GGU GAU UUA TT-30 . As a negative control unspecific scrambled siRNA duplexes (50 -AGG UAG UGU AAU CGC CUU GTT-30 ) were used. Twenty-four hours before transfection 1 105 cells were seeded in six-well plates. Transfection of siRNA was carried out using Oligofectamine (InVitrogen, Karlsruhe, Germany) and 10 nM siRNA duplex per well. Transfection was performed as previously described [10]. Specific silencing of targeted genes was confirmed by at least two independent experiments. Cell migration assay. The effect of JAM-A knockdown on renal cell carcinoma cell migration was measured as the ability of cells to migrate through Transwell filters (6.5 mm diameter, 5 lm pore size). Transwell filters were coated with fibronectin (10 lg/ml in PBS). The migration assay has been described in detail in [8]. Each assay was performed in duplicates and the representative result from three independent experiments is shown. Data are presented by means ± SD. Statistical and significant differences were determined using one-way ANOVA with the Bonferroni multicomparison test. Results Constitutive JAM-A expression is found in segment-specific cells of DCT, CNT, and CD of the normal human kidney The expression of JAM-A in the human kidney was analyzed in serial sections of normal renal tissue by immunohistochemistry. As shown in Fig. 1A in the renal cortex and outer stripe, JAM-A expres-
P. Gutwein et al. / Biochemical and Biophysical Research Communications 380 (2009) 387–391
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Fig. 1. JAM-A localization in normal renal tissue. (A) JAM-A protein is constitutively expressed in tubular cells of the healthy kidney. Paraffin sections of normal human renal tissue were stained with a monoclonal antibody against JAM-A. (B) Renal tissue stained with a mouse isotype IgG control antibody was negative. JAM-A expression in the kidney cortex in the collecting duct (C), in connecting tubules and initial parts of the cortical collecting ducts (D) shown by the colocalization of JAM-A with Aquaporin-2 (C0 ) and Calbindin D-28K (D0 ). Using an antibody against Aquaporin-1 (E0 ) JAM-A expression was observed in proximal tubules (E). JAM-A is not expressed in the thick ascending limb of Henle (F) and in distal convoluted tubules (G) as shown by staining of serial sections with JAM-A and with a Tamm–Horsefall glycoprotein antibody (F0 ) or an antibody against anti-thiazide-sensitive NaCl cotransporter (NCC) antibody (G0 ). (H) Double immunofluorescent staining of JAM-A (green) with Aquaporin-2 (red), Calbindin D-28K (red), and NCC (red) confirm expression of JAM-A in collecting duct, in collecting tubules and initial parts of the cortical collecting ducts. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
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sion was mainly detectable in tubular cells. To confirm the specificity of JAM-A immunostaining, we used a mouse isotype control antibody, which did not show any immunoreactivity (Fig. 1B). Using antibodies against Aquaporin-1 (a marker for proximal tubules), Aquaporin-2 (a marker for the collecting duct), Calbindin D-28K (a marker for distal convoluted tubules, connecting tubules and early part of cortical collecting ducts), Tamm–Horsfall glycoprotein (THP) (expressed in the thick ascending limb of Henle (TAL)) and the anti-thiazide-sensitive NaCl cotransporter antibody (NCC) (expressed in distal convoluted tubules) we determined the tubular cells expressing JAM-A. We observed a weak basolateral expression of JAM-A in proximal tubules (Fig. 1E and E0 ). JAM-A was predominantly expressed in the collecting duct (Fig. 1C and C0 ), but expression could also be shown in connecting tubules and in early part of the collecting duct (Fig. 1D and D0 ). In contrast JAM-A expression was not detectable in tubular cells of the thick ascending limb of henle (Fig. 1F and F0 ) and in distal convoluted tubules (Fig. 1G and G0 ). With double immunofluorescence staining using Aquaporin-2, the Calbindin D-28K and the NCC antibody (Fig. 1H) we could confirm the results obtained by the immunohistochemical analyses. Pro-inflammatory cytokines downregulate the expression of JAM-A in primary tubular cells and HK-2 cells To investigate the regulation and expression of JAM-A in tubular cells we performed FACS analysis, Western blot, and immunofluorescent staining (Fig. S1) in proximal tubular HK-2 cells and in priTable 1 Expression of JAM-A in different tumor subtypes of renal cell carcinoma. JAM-A immunoreactivity
Clear cell RCC Papillary RCC Chromophobe RCC
0
1+
2+
3+
73 2 1
82 9 3
60 22 5
15 9 1
mary renal tubular cells (HRCEpiC). JAM-A was constitutively expressed in HK-2 and HRCEpiC cells (Fig. S1, C). In addition, JAM-A was mainly located in the membrane and particularly at cell–cell contacts of both cell lines (Fig. S1, B). The Western blot analysis and FACS staining demonstrated that HK-2 cells possess a stronger JAM-A expression than the primary cell line HRCEpiC (Fig. S1, A+C). Interestingly, combined treatment of HK-2 cells with the pro-inflammatory cytokines TNF-a and IFN-c resulted in a reduction of JAM-A expression (Fig. S1, A+B). The immunofluorescence staining and the Western blot analysis of membrane fractions demonstrated that JAM-A is strongly reduced from the cell membrane after cytokine mix treatment (Fig. S1, D+E). Importantly, pre-incubation with the broad spectrum metalloproteinase inhibitor GM6001 and TAPI-2 restored the JAM-A expression to almost basal levels in the HK-2 cells (Fig. S1, D). In contrast pre-incubation with the ADAM10 specific inhibitor GI254023X did not inhibit the downregulation of JAM-A in HK-2 cells (Fig. S1, D+E). Similar results were obtained in HRCEpiC cells (data not shown). Clinicopathological correlations of JAM-A protein expression To investigate the expression of JAM-A in renal cell carcinoma (RCC) we performed an immunohistochemical analysis on a TMA with 282 biopsies of patients with RCC. Of 282 renal carcinomas, 76 (27.0%) were JAM-A negative, 94 cases (33.3%) were weakly positive, 87 cases (30.9%) showed a moderate JAM-A expression and 25 cases (8.9%) were strongly positive. In comparison to distal tubuli (mostly 3+), renal tumors show a downregulation of JAM-A in 91.1% of cases. JAM-A showed significantly different expression rates according to tumor histology (Table 1, Fig. S2), papillary and chromophobe carcinomas showed higher JAM-A levels than ccRCC. In Spearman rank correlations of all tumors, JAM-A expression did not correlate to pT category (correlation coefficient (CC = 0.029, p = 0.63) but showed a trend towards a positive correlation to higher tumor grades (CC = 0.116, p = 0.053). However, in the subgroup of ccRCC, JAM-A was significantly correlated to pT category (CC = 0.157, p = 0.017) and tumor grade (CC = 0.256,
Fig. 2. JAM-A inhibition induced the migration of RCC4 cells. (A) Western blot analysis of JAM-A expression in different renal cell lines. Blot was reprobed with an antibody specific for b-actin as a loading control. (B) Inhibition of JAM-A protein expression 24, 48, and 72 h after the transfection of siRNA specific against JAM-A shown by Western blot analysis. b-Actin was used as a loading control. (C) Inhibition of JAM-A expression correlated with an increase in the number of migrated RCC4 cells. Data represents means ± SD from representative experiments (n = 3). ***p > 0.001 considered statistically significant compared to control RCC4.
P. Gutwein et al. / Biochemical and Biophysical Research Communications 380 (2009) 387–391
p = 0.001). In univariate COX survival analyses, no prognostic value of JAM-A could be demonstrated, neither in all tumors (relative risk = 1.060, p = 0.574) nor in the subgroup of ccRCC (relative risk = 1.112, p = 0.348). Knockdown of JAM-A in RCC induced the migration of renal cell carcinoma cells To investigate the expression and role of JAM-A in renal cancer cells, we characterized the JAM-A protein levels in different renal tumor cells (Fig. 2A). Western blot analysis revealed high expression of JAM-A in the RCC4, whereas JAM-A was not detectable in CAKI-2 cells (Fig. 2A). In contrast A498, ACHN3, and CAKI-6 cells showed a moderate JAM-A expression. To investigate the role of JAM-A in renal cancer cell migration we performed migration assays with RCC4 cells. The efficient knockdown of JAM-A (Fig. 2B) resulted in an significant increase in the number of migrated RCC4 cells in comparison to the controls (Fig. 2C). Discussion Tight junctions (TJ) have attracted considerable attention in relation to kidney diseases [11]. It is known that tight junctions interact directly with neighboring cells and thereby form a selective barrier that regulate the passage of molecules [12]. Beside this TJ proteins configure a trafficking and signaling platform that regulate cell growth, proliferation, differentiation and dedifferentiation [13]. TJ consist of occludin, members of the claudin family and the junctional adhesion molecules (JAMs). The classical JAMs are JAM-A, JAM-B, and JAM-C which are the best characterized members of the JAM family [14]. It is known that JAM-A expression can be regulated by pro-inflammatory cytokines. For instance, combined treatment of human umbilical vein endothelial cells with interferon-c (IFN-c), tumor necrosis factor (TNF-a) decreased the junctional localization of JAM-A [15]. In our study we demonstrate that the combined treatment of proximal and primary tubular cells with IFN-c and TNF-a reduced JAM-A expression from the membrane through a metalloproteinase dependent way. It has been already shown that adhesion molecules can be processed by metalloproteinases. For instance L1-CAM can be cleaved by the metalloproteinase ADAM10 in a subpopulation of colorectal cancer cells at the invasive front of tumors [16]. In addition ectodomain shedding by ADAM10 seems to be a key mechanism regulating the expression and function of CD44 [17]. In contrast our study showed that other metalloproteinases beside ADAM10 are involved in the shedding of JAM-A in renal proximal tubular cells. However, further analysis have to be performed to characterize the metalloproteinase(s) responsible for the processing of JAM-A. In biopsies of patients with ccRCC we observed a downregulation of JAM-A expression compared to normal tissue. It has been already described that TJ are drastically reduced in patients diagnosed with renal cell carcinomas [18]. The loss of TJ function in carcinomas is regarded as an important step in the process that leads to loss of cell–cell adhesion and tumor metastasis [19]. Therefore, we hypothesized that the downregulation of JAM-A in RCC leads to a disassembly of TJ and to the loss of cell–cell contacts encouraging the migration of tumor cells. Downregulation of TJ molecules, such as ZO-1 has been reported to correlate with tumor progression [20]. In addition low expression of claudin-1 has been correlated in lung adenocarcinoma patients with shorter overall survival [21]. In addition, overexpression of claudin-1 inhibited cell migration and invasion of a human lung adenocarcinoma cell line [21]. In our study we could also show that the downregulation of JAM-A significantly induced the migration of renal cell carcinoma cells. In the same line Naik et al. have recently shown that JAM-A is a key negative regulator of cell migration and invasion by affect-
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ing cytoskeletal rearrangements [7]. Currently, we are investigating if JAM-A can influence the expression of genes involved in cancer migration and invasion. In summary we could show that JAM-A knockdown induced the migration of renal cancer cells and JAM-A was downregulated in biopsies of patients diagnosed with ccRCC. In addition we show for the first time that downregulation of JAM-A in HK-2 cells is mediated by metalloproteinases, assuming that downregulation of JAM-A in renal cell carcinoma may be due to enhanced shedding by metalloproteinases. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.01.100. References [1] A. Hartsock, W.J. Nelson, Adherens and tight junctions: structure, function and connections to the actin cytoskeleton, Biochim. Biophys. Acta 1778 (2008) 660–669. [2] P.F. Bradfield, S. Nourshargh, M. Aurrand-Lions, B.A. Imhof, JAM family and related proteins in leukocyte migration (Vestweber series), Arterioscler. Thromb. Vasc. Biol. 27 (2007) 2104–2112. [3] M. Aridor, L.A. Hannan, Traffic jam: a compendium of human diseases that affect intracellular transport processes, Traffic 1 (2000) 836–851. [4] L.A. Williams, I. Martin-Padura, E. Dejana, N. Hogg, D.L. Simmons, Identification and characterisation of human junctional adhesion molecule (JAM), Mol. Immunol. 36 (1999) 1175–1188. [5] K.J. Mandell, B.A. Babbin, A. Nusrat, C.A. Parkos, Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity, J. Biol. Chem. 280 (2005) 11665–11674. [6] L. Gonzalez-Mariscal, S. Lechuga, E. Garay, Role of tight junctions in cell proliferation and cancer, Prog. Histochem. Cytochem. 42 (2007) 1–57. [7] U.P. Naik, M.U. Naik, K. Eckfeld, P. Martin-DeLeon, J. Spychala, Characterization and chromosomal localization of JAM-1, a platelet receptor for a stimulatory monoclonal antibody, J. Cell Sci. 114 (2001) 539–547. [8] P. Gutwein, A. Schramme, N. Sinke, M.S. Abdel-Bakky, B. Voss, N. Obermuller, K. Doberstein, M. Koziolek, F. Fritzsche, M. Johannsen, K. Jung, H. Schaider, P. Altevogt, A. Ludwig, J. Pfeilschifter, G. Kristiansen, Tumoural CXCL16 expression is a novel prognostic marker of longer survival times in renal cell cancer patients, Eur. J. Cancer (2008) [Epub ahead of print]. [9] A. Bhandari, S. Koul, A. Sekhon, S.K. Pramanik, L.S. Chaturvedi, M. Huang, M. Menon, H.K. Koul, Effects of oxalate on HK-2 cells, a line of proximal tubular epithelial cells from normal human kidney, J. Urol. 168 (2002) 253–259. [10] A. Schramme, M.S. Abdel-Bakky, P. Gutwein, N. Obermuller, P.C. Baer, I.A. Hauser, A. Ludwig, S. Gauer, L. Schafer, E. Sobkowiak, P. Altevogt, M. Koziolek, E. Kiss, H.J. Grone, R. Tikkanen, I. Goren, H. Radeke, J. Pfeilschifter, Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney, Kidney Int. 74 (2008) 328–338. [11] D.B. Lee, E. Huang, H.J. Ward, Tight junction biology and kidney dysfunction, Am. J. Physiol. Renal Physiol. 290 (2006) F20–F34. [12] O.W. Blaschuk, T.M. Rowlands, Plasma membrane components of adherens junctions (Review), Mol. Membr. Biol. 19 (2002) 75–80. [13] D. Bilder, Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors, Genes Dev. 18 (2004) 1909–1925. [14] G. Bazzoni, The JAM family of junctional adhesion molecules, Curr. Opin. Cell Biol. 15 (2003) 525–530. [15] H. Ozaki, K. Ishii, H. Horiuchi, H. Arai, T. Kawamoto, K. Okawa, A. Iwamatsu, T. Kita, Cutting edge: combined treatment of TNF-alpha and IFN-gamma causes redistribution of junctional adhesion molecule in human endothelial cells, J. Immunol. 163 (1999) 553–557. [16] N. Gavert, M. Conacci-Sorrell, D. Gast, A. Schneider, P. Altevogt, T. Brabletz, A. Ben Ze’ev, L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers, J. Cell Biol. 168 (2005) 633–642. [17] O. Nagano, H. Saya, Mechanism and biological significance of CD44 cleavage, Cancer Sci. 95 (2004) 930–935. [18] G. Kim, S.A. Rajasekaran, G. Thomas, E.A. Rosen, E.M. Landaw, P. Shintaku, C. Lassman, J. Said, A.K. Rajasekaran, Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes, Histol. Histopathol. 20 (2005) 35–44. [19] N. Sawada, M. Murata, K. Kikuchi, M. Osanai, H. Tobioka, T. Kojima, H. Chiba, Tight junctions and human diseases, Med. Electron Microsc. 36 (2003) 147–156. [20] K.B. Hoover, S.Y. Liao, P.J. Bryant, Loss of the tight junction MAGUK ZO-1 in breast cancer: relationship to glandular differentiation and loss of heterozygosity, Am. J. Pathol. 153 (1998) 1767–1773. [21] Y.C. Chao, S.H. Pan, S.C. Yang, S.L. Yu, T.F. Che, C.W. Lin, M.S. Tsai, G.C. Chang, C.H. Wu, Y.Y. Wu, Y.C. Lee, T.M. Hong, P.C. Yang, Claudin-1 is a metastasis suppressor and correlates with clinical outcome in lung adenocarcinoma, Am. J. Respir. Crit. Care Med. 179 (2) (2009) 123–133.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma Paul Gutwein a,*, Anja Schramme a, Beren Voss a, Mohamed Sadek Abdel-Bakky a, Kai Doberstein a, Andreas Ludwig b, Peter Altevogt c, Martin-Leo Hansmann d, Holger Moch e, Glen Kristiansen e,1, Josef Pfeilschifter a,1 a
Pharmazentrum frankfurt/ZAFES, Klinikum der, Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany Institute of Pharmacology and Toxicology, University Hospital RWTH, Aachen, Germany Tumor Immunology Programme, German Cancer Research Center, Heidelberg, Germany d Institute of Pathology, Goethe-Universität, Frankfurt am Main, Germany e Institute of Surgical Pathology, University Hospital Zurich, University of Zurich, Zurich, Switzerland b c
a r t i c l e
i n f o
Article history: Received 15 January 2009 Available online 23 January 2009
Keywords: JAM-A Kidney Renal cancer Metalloproteinases Tight junctions
a b s t r a c t Junctional adhesion molecule-A (JAM-A) is one component of tight junctions which are involved in important processes like paracellular permeability, cell polarity, adhesion, migration, and angiogenesis. Here we describe JAM-A expression in distal convoluted tubule, connecting tubule, and in cells of the collecting duct of the healthy human kidney. In addition, JAM-A was weakly expressed in cells of the proximal tubule. Using immunofluorescence, FACS and Western blot analysis we investigated JAM-A expression in tubular cells in vitro. Interestingly, treatment of HK-2 cells with IFN-c and TNF-a resulted in a metalloproteinase mediated downregulation of JAM-A. Importantly, in a tissue micro-array JAM-A protein expression was significantly downregulated in patients with clear cell renal cell carcinoma. Furthermore, knockdown of JAM-A with JAM-A specific siRNA induced the migration of RCC4 cells. In summary, downregulation of JAM-A is an early event in the development of renal cancer and increases the migration of renal cancer cells. Ó 2009 Elsevier Inc. All rights reserved.
Epithelial tight junctions (TJ) are dynamic structures and the molecular composition of TJ is complex consisting of multiple transmembrane proteins including claudin family members, occludin, and members of the junctional adhesion molecule (JAM) protein family [1]. JAMs are a family of transmembrane IgG glycoproteins expressed on a number of cells and the best characterized members are JAM-A, JAM-B, and JAM-C [2]. The expression of JAM-A has been shown on the surface of endothelial and epithelial cells of various tissues, including liver, pancreas, kidney, heart, brain, lymph nodes, intestine, lungs, placenta, and vascular tissue [3]. JAM-A is apically positioned at tight junctions and associates with the tight junction components cingulin and occludin, with the PDZ-domain-containing proteins ZO1, AF6, PARD3 or with CASK, all of which can act as scaffold for larger complexes. It has been suggested that the JAMs may participate in regulating the tight junctions and maintaining paracellular permeability [4]. In the same line, it has been shown that JAM-A regulates epithelial cell morphology by modulating the activity of the small GTPase Rap1 [5]. * Corresponding author. Fax: +49 69 6301 79 42. E-mail address: [email protected] (P. Gutwein). 1 Both the authors have contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.100
Global analysis of gene expression demonstrated that cancer cells have many differences in gene expression from their normal precursors. Particularly, many genes involved in cell–cell adhesion, including those contributing to tight junctions, are downregulated or overexpressed in different carcinomas [6]. In this context it has been recently shown that JAM-A functions as a key negative regulator of breast cancer cell invasion and possibly metastasis [7]. In this study we analyzed the expression of JAM-A in the human kidney and on a tissue micro-array with biopsies of 282 renal cancer patients by immunohistochemistry. We demonstrate for the first time, that JAM-A is strongly expressed in tubular cells in the normal human kidney and JAM-A expression was significantly downregulated in biopsies of patients with clear cell renal cell carcinomas (ccRCC). In addition, inhibition of JAM-A in a RCC cell line induced the migration of the cancer cells. Furthermore, we demonstrate that the pro-inflammatory cytokines IFNc and TNF-a induced a metalloproteinase mediated downregulation of JAM-A from the cell membrane of tubular cells. In summary, our study demonstrates that downregulation of JAM-A in the kidney contributes to the progression of renal cell carcinomas.
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Materials and methods Kidney sections. Specimens were taken from healthy parts of renal tissue from six different tumor nephrectomies (obtained from 2 female and 4 male patients with ages ranging from 34 to 66 years). Kidney tissues were originally submitted for diagnostic purposes and studied in accordance with national and local ethical principles. The use of human tissue samples has been approved by the local ethics committee (Ref-No. #11/10/04). Tissue micro-array construction. We constructed a tissue microarray (TMA) from renal cell carcinomas diagnosed at the Institute of Surgical Pathology, Universitätsspital Zurich, between 1993 and 2003. The TMA is described in detail in a recent publication [8]. Cell culture. The renal cancer cell line RCC4 was provided by Prof. Brüne (Institute of Biochemistry I/ZAFES, Faculty of Medicine, Goethe University Frankfurt, Frankfurt, Germany), the renal cancer cell lines ACHN3 and CAKI-6 were a kind gift from Prof. Jung (Department of Urology, Charité, Berlin, Germany) and the A498 renal cancer cell line was received from Dr. K. Joehrer (Department of Urology, Innsbruck Medical University, Innsbruck, Austria). The renal cell line CAKI-2 was provided by Dr. Grünberg (Center for Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland). The primary renal tubular cell line HRCEpiC was purchased from ScienCell Research Laboratories, Berlin, Germany. The proximal tubular cell line HK-2 has been described elsewhere [9]. The growth medium for ACHN3, RCC4, CAKI-2, and CAKI-6 cells was DMEM supplemented with fetal bovine serum (10%), sodium pyruvate (1 mM), penicillin (100 U/ml), and streptomycin (100 lg/ml). The A498 cells were cultivated in RPMI supplemented with fetal bovine (10%), penicillin (100 U/ml), and streptomycin (100 lg/ml). The growth medium for the HRCEpiC cells was EpiC Medium supplemented with fetal bovine serum (2%), penicillin (100 U/ml), streptomycin (100 lg/ml), and epithelial cell growth supplement (1%). The HK-2 cells were cultivated in RPMI supplemented with fetal bovine serum (10%), non-essential amino acids (1%), sodium pyruvate (1%), 10 mM Hepes, pH 7.4, Hydrocortison (0.1%), Insulin-Transferrin-Sodium-supplement (0.1%). Cytokines, chemicals, and antibodies. Recombinant human IFN-c and recombinant human TNF-a were obtained from R&D Systems (Wiesbaden, Germany). The F11R mouse monoclonal antibody (Western blot, immunohistochemistry, immunofluorescence) was from Abnova Corporation (Heidelberg, Germany) and the monoclonal antibody to JAM-A antibody (BV16; FACS analysis) was from Hycult Biotechnology (Uden, The Netherlands). The rabbit anti-Calbindin D-28K (EG-20) and the anti-Aquaporin-2 antibody were from Sigma (Taufkirchen, Germany). The rabbit anti-thiazide-sensitive NaCl cotransporter (NCC) polyclonal antibody was from Chemicon (Hampshire, UK). The Tamm–Horsfall protein (THP) antibody for immunohistochemistry was obtained from Biotrend GmbH (Cologne, Germany). The metalloproteinase inhibitors GI254023X was kindly provided by Dr. Andreas Ludwig (Institute of Pharmacology and Toxikology, University Hospital Aachen, Germany). The broad spectrum metalloproteinase inhibitors GM6001 and TAPI-2 were obtained from Calbiochem (Darmstadt, Germany). Immunohistochemistry. Paraffin tissue sections were deparaffinized in xylene, rehydrated through a graded ethanol series and washed in 10 mM phosphate-buffered 150 mM saline, pH 7.4. Antigen retrieval was performed by incubating the tissue sections for 20 min in 1 Target Retrieval Solution (DakoCytomation, Hamburg, Germany) in a microwave oven (500 W). Tissue sections were than processed as described in [10]. The sections were inspected with a Zeiss microscope coupled to a 12-bit digital image camera. TMA immunohistochemistry. The TMA blocks were freshly cut (3 lm) and mounted on superfrost slides (Menzel Gläser). Immunohistochemistry was conducted with the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson,
AZ) using Ventana reagents for the entire procedure. The primary JAM-A antibody was used at a concentration of 1:100. For detection we used the UltraVIEWTM DAB detection kit using the benchmarks CC1m-heat induced epitope retrieval. Slides were counterstained with hematoxylin, dehydrated and mounted. Evaluation of TMA immunohistochemistry. The immunohistochemistry was evaluated by a single GU pathologist (GK) in one go to minimize intraobserver variability. Intensity of immunoreactivity was semiquantitatively scored as negative, weakly, moderately or strongly positive, as illustrated by Fig. S2. This panel of figures was compiled before the systematic evaluation of the tumor cohort was commenced and was used as reference, which lay next to the microscope for continuous comparison. Fluorescence microscopy. For immunofluorescence analysis, tissue sections were deparaffinized as described above and antigen retrieval was performed incubating the tissue sections for 20 min in 1 Target Retrieval Solution (DakoCytomation, Hamburg, Germany) in a microwave oven (500 W). Tissue sections were than processed as described in detail elsewhere [10]. Cells were grown on coverslips and fixed with 4% paraformaldehyde/PBS. Fixed cells were than processed as described in [8]. FACS analysis. The renal cell lines HK-2, HRCEpiC, ACHN3, and CAKI-6 were incubated for 30 min with monoclonal antibody against JAM-A (diluted 1:50). All steps were performed as recently described [8]. Protein extraction and Western blot analysis. Cell extracts were prepared as described before [10] at time points indicated. Membrane fractions were isolated by incubating the cells for 10 min in hypolysis buffer (10 mM Tris–HCl, pH 8.0, 0.1 mM DTT, Protease Inhibitor complete (Roche Diagnostic, Penzberg, Germany) at 4 °C. Cell suspension was homogenized in a glass–glass homogenizer and membrane proteins were centrifuged at 1000g and 4 °C followed by a high-speed centrifugation at 100,000g. Proteins were separated under reducing conditions by electrophoresis using 12% SDS–PAGE (polyacrylamide gel electrophoresis) for detection of JAM-A. All following steps were performed as already described [8]. Blots were probed overnight with a monoclonal JAM-A antibody diluted 1:1500 in TTBS. siRNA transfection. For downregulation of JAM-A expression the following siRNA duplex (MWG Biotech AG, Ebersberg, Germany) was used: 50 -UUC GAG UAA GAA GGU GAU UUA TT-30 . As a negative control unspecific scrambled siRNA duplexes (50 -AGG UAG UGU AAU CGC CUU GTT-30 ) were used. Twenty-four hours before transfection 1 105 cells were seeded in six-well plates. Transfection of siRNA was carried out using Oligofectamine (InVitrogen, Karlsruhe, Germany) and 10 nM siRNA duplex per well. Transfection was performed as previously described [10]. Specific silencing of targeted genes was confirmed by at least two independent experiments. Cell migration assay. The effect of JAM-A knockdown on renal cell carcinoma cell migration was measured as the ability of cells to migrate through Transwell filters (6.5 mm diameter, 5 lm pore size). Transwell filters were coated with fibronectin (10 lg/ml in PBS). The migration assay has been described in detail in [8]. Each assay was performed in duplicates and the representative result from three independent experiments is shown. Data are presented by means ± SD. Statistical and significant differences were determined using one-way ANOVA with the Bonferroni multicomparison test. Results Constitutive JAM-A expression is found in segment-specific cells of DCT, CNT, and CD of the normal human kidney The expression of JAM-A in the human kidney was analyzed in serial sections of normal renal tissue by immunohistochemistry. As shown in Fig. 1A in the renal cortex and outer stripe, JAM-A expres-
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Fig. 1. JAM-A localization in normal renal tissue. (A) JAM-A protein is constitutively expressed in tubular cells of the healthy kidney. Paraffin sections of normal human renal tissue were stained with a monoclonal antibody against JAM-A. (B) Renal tissue stained with a mouse isotype IgG control antibody was negative. JAM-A expression in the kidney cortex in the collecting duct (C), in connecting tubules and initial parts of the cortical collecting ducts (D) shown by the colocalization of JAM-A with Aquaporin-2 (C0 ) and Calbindin D-28K (D0 ). Using an antibody against Aquaporin-1 (E0 ) JAM-A expression was observed in proximal tubules (E). JAM-A is not expressed in the thick ascending limb of Henle (F) and in distal convoluted tubules (G) as shown by staining of serial sections with JAM-A and with a Tamm–Horsefall glycoprotein antibody (F0 ) or an antibody against anti-thiazide-sensitive NaCl cotransporter (NCC) antibody (G0 ). (H) Double immunofluorescent staining of JAM-A (green) with Aquaporin-2 (red), Calbindin D-28K (red), and NCC (red) confirm expression of JAM-A in collecting duct, in collecting tubules and initial parts of the cortical collecting ducts. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
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sion was mainly detectable in tubular cells. To confirm the specificity of JAM-A immunostaining, we used a mouse isotype control antibody, which did not show any immunoreactivity (Fig. 1B). Using antibodies against Aquaporin-1 (a marker for proximal tubules), Aquaporin-2 (a marker for the collecting duct), Calbindin D-28K (a marker for distal convoluted tubules, connecting tubules and early part of cortical collecting ducts), Tamm–Horsfall glycoprotein (THP) (expressed in the thick ascending limb of Henle (TAL)) and the anti-thiazide-sensitive NaCl cotransporter antibody (NCC) (expressed in distal convoluted tubules) we determined the tubular cells expressing JAM-A. We observed a weak basolateral expression of JAM-A in proximal tubules (Fig. 1E and E0 ). JAM-A was predominantly expressed in the collecting duct (Fig. 1C and C0 ), but expression could also be shown in connecting tubules and in early part of the collecting duct (Fig. 1D and D0 ). In contrast JAM-A expression was not detectable in tubular cells of the thick ascending limb of henle (Fig. 1F and F0 ) and in distal convoluted tubules (Fig. 1G and G0 ). With double immunofluorescence staining using Aquaporin-2, the Calbindin D-28K and the NCC antibody (Fig. 1H) we could confirm the results obtained by the immunohistochemical analyses. Pro-inflammatory cytokines downregulate the expression of JAM-A in primary tubular cells and HK-2 cells To investigate the regulation and expression of JAM-A in tubular cells we performed FACS analysis, Western blot, and immunofluorescent staining (Fig. S1) in proximal tubular HK-2 cells and in priTable 1 Expression of JAM-A in different tumor subtypes of renal cell carcinoma. JAM-A immunoreactivity
Clear cell RCC Papillary RCC Chromophobe RCC
0
1+
2+
3+
73 2 1
82 9 3
60 22 5
15 9 1
mary renal tubular cells (HRCEpiC). JAM-A was constitutively expressed in HK-2 and HRCEpiC cells (Fig. S1, C). In addition, JAM-A was mainly located in the membrane and particularly at cell–cell contacts of both cell lines (Fig. S1, B). The Western blot analysis and FACS staining demonstrated that HK-2 cells possess a stronger JAM-A expression than the primary cell line HRCEpiC (Fig. S1, A+C). Interestingly, combined treatment of HK-2 cells with the pro-inflammatory cytokines TNF-a and IFN-c resulted in a reduction of JAM-A expression (Fig. S1, A+B). The immunofluorescence staining and the Western blot analysis of membrane fractions demonstrated that JAM-A is strongly reduced from the cell membrane after cytokine mix treatment (Fig. S1, D+E). Importantly, pre-incubation with the broad spectrum metalloproteinase inhibitor GM6001 and TAPI-2 restored the JAM-A expression to almost basal levels in the HK-2 cells (Fig. S1, D). In contrast pre-incubation with the ADAM10 specific inhibitor GI254023X did not inhibit the downregulation of JAM-A in HK-2 cells (Fig. S1, D+E). Similar results were obtained in HRCEpiC cells (data not shown). Clinicopathological correlations of JAM-A protein expression To investigate the expression of JAM-A in renal cell carcinoma (RCC) we performed an immunohistochemical analysis on a TMA with 282 biopsies of patients with RCC. Of 282 renal carcinomas, 76 (27.0%) were JAM-A negative, 94 cases (33.3%) were weakly positive, 87 cases (30.9%) showed a moderate JAM-A expression and 25 cases (8.9%) were strongly positive. In comparison to distal tubuli (mostly 3+), renal tumors show a downregulation of JAM-A in 91.1% of cases. JAM-A showed significantly different expression rates according to tumor histology (Table 1, Fig. S2), papillary and chromophobe carcinomas showed higher JAM-A levels than ccRCC. In Spearman rank correlations of all tumors, JAM-A expression did not correlate to pT category (correlation coefficient (CC = 0.029, p = 0.63) but showed a trend towards a positive correlation to higher tumor grades (CC = 0.116, p = 0.053). However, in the subgroup of ccRCC, JAM-A was significantly correlated to pT category (CC = 0.157, p = 0.017) and tumor grade (CC = 0.256,
Fig. 2. JAM-A inhibition induced the migration of RCC4 cells. (A) Western blot analysis of JAM-A expression in different renal cell lines. Blot was reprobed with an antibody specific for b-actin as a loading control. (B) Inhibition of JAM-A protein expression 24, 48, and 72 h after the transfection of siRNA specific against JAM-A shown by Western blot analysis. b-Actin was used as a loading control. (C) Inhibition of JAM-A expression correlated with an increase in the number of migrated RCC4 cells. Data represents means ± SD from representative experiments (n = 3). ***p > 0.001 considered statistically significant compared to control RCC4.
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p = 0.001). In univariate COX survival analyses, no prognostic value of JAM-A could be demonstrated, neither in all tumors (relative risk = 1.060, p = 0.574) nor in the subgroup of ccRCC (relative risk = 1.112, p = 0.348). Knockdown of JAM-A in RCC induced the migration of renal cell carcinoma cells To investigate the expression and role of JAM-A in renal cancer cells, we characterized the JAM-A protein levels in different renal tumor cells (Fig. 2A). Western blot analysis revealed high expression of JAM-A in the RCC4, whereas JAM-A was not detectable in CAKI-2 cells (Fig. 2A). In contrast A498, ACHN3, and CAKI-6 cells showed a moderate JAM-A expression. To investigate the role of JAM-A in renal cancer cell migration we performed migration assays with RCC4 cells. The efficient knockdown of JAM-A (Fig. 2B) resulted in an significant increase in the number of migrated RCC4 cells in comparison to the controls (Fig. 2C). Discussion Tight junctions (TJ) have attracted considerable attention in relation to kidney diseases [11]. It is known that tight junctions interact directly with neighboring cells and thereby form a selective barrier that regulate the passage of molecules [12]. Beside this TJ proteins configure a trafficking and signaling platform that regulate cell growth, proliferation, differentiation and dedifferentiation [13]. TJ consist of occludin, members of the claudin family and the junctional adhesion molecules (JAMs). The classical JAMs are JAM-A, JAM-B, and JAM-C which are the best characterized members of the JAM family [14]. It is known that JAM-A expression can be regulated by pro-inflammatory cytokines. For instance, combined treatment of human umbilical vein endothelial cells with interferon-c (IFN-c), tumor necrosis factor (TNF-a) decreased the junctional localization of JAM-A [15]. In our study we demonstrate that the combined treatment of proximal and primary tubular cells with IFN-c and TNF-a reduced JAM-A expression from the membrane through a metalloproteinase dependent way. It has been already shown that adhesion molecules can be processed by metalloproteinases. For instance L1-CAM can be cleaved by the metalloproteinase ADAM10 in a subpopulation of colorectal cancer cells at the invasive front of tumors [16]. In addition ectodomain shedding by ADAM10 seems to be a key mechanism regulating the expression and function of CD44 [17]. In contrast our study showed that other metalloproteinases beside ADAM10 are involved in the shedding of JAM-A in renal proximal tubular cells. However, further analysis have to be performed to characterize the metalloproteinase(s) responsible for the processing of JAM-A. In biopsies of patients with ccRCC we observed a downregulation of JAM-A expression compared to normal tissue. It has been already described that TJ are drastically reduced in patients diagnosed with renal cell carcinomas [18]. The loss of TJ function in carcinomas is regarded as an important step in the process that leads to loss of cell–cell adhesion and tumor metastasis [19]. Therefore, we hypothesized that the downregulation of JAM-A in RCC leads to a disassembly of TJ and to the loss of cell–cell contacts encouraging the migration of tumor cells. Downregulation of TJ molecules, such as ZO-1 has been reported to correlate with tumor progression [20]. In addition low expression of claudin-1 has been correlated in lung adenocarcinoma patients with shorter overall survival [21]. In addition, overexpression of claudin-1 inhibited cell migration and invasion of a human lung adenocarcinoma cell line [21]. In our study we could also show that the downregulation of JAM-A significantly induced the migration of renal cell carcinoma cells. In the same line Naik et al. have recently shown that JAM-A is a key negative regulator of cell migration and invasion by affect-
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ing cytoskeletal rearrangements [7]. Currently, we are investigating if JAM-A can influence the expression of genes involved in cancer migration and invasion. In summary we could show that JAM-A knockdown induced the migration of renal cancer cells and JAM-A was downregulated in biopsies of patients diagnosed with ccRCC. In addition we show for the first time that downregulation of JAM-A in HK-2 cells is mediated by metalloproteinases, assuming that downregulation of JAM-A in renal cell carcinoma may be due to enhanced shedding by metalloproteinases. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.01.100. References [1] A. Hartsock, W.J. Nelson, Adherens and tight junctions: structure, function and connections to the actin cytoskeleton, Biochim. Biophys. Acta 1778 (2008) 660–669. [2] P.F. Bradfield, S. Nourshargh, M. Aurrand-Lions, B.A. Imhof, JAM family and related proteins in leukocyte migration (Vestweber series), Arterioscler. Thromb. Vasc. Biol. 27 (2007) 2104–2112. [3] M. Aridor, L.A. Hannan, Traffic jam: a compendium of human diseases that affect intracellular transport processes, Traffic 1 (2000) 836–851. [4] L.A. Williams, I. Martin-Padura, E. Dejana, N. Hogg, D.L. Simmons, Identification and characterisation of human junctional adhesion molecule (JAM), Mol. Immunol. 36 (1999) 1175–1188. [5] K.J. Mandell, B.A. Babbin, A. Nusrat, C.A. Parkos, Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity, J. Biol. Chem. 280 (2005) 11665–11674. [6] L. Gonzalez-Mariscal, S. Lechuga, E. Garay, Role of tight junctions in cell proliferation and cancer, Prog. Histochem. Cytochem. 42 (2007) 1–57. [7] U.P. Naik, M.U. Naik, K. Eckfeld, P. Martin-DeLeon, J. Spychala, Characterization and chromosomal localization of JAM-1, a platelet receptor for a stimulatory monoclonal antibody, J. Cell Sci. 114 (2001) 539–547. [8] P. Gutwein, A. Schramme, N. Sinke, M.S. Abdel-Bakky, B. Voss, N. Obermuller, K. Doberstein, M. Koziolek, F. Fritzsche, M. Johannsen, K. Jung, H. Schaider, P. Altevogt, A. Ludwig, J. Pfeilschifter, G. Kristiansen, Tumoural CXCL16 expression is a novel prognostic marker of longer survival times in renal cell cancer patients, Eur. J. Cancer (2008) [Epub ahead of print]. [9] A. Bhandari, S. Koul, A. Sekhon, S.K. Pramanik, L.S. Chaturvedi, M. Huang, M. Menon, H.K. Koul, Effects of oxalate on HK-2 cells, a line of proximal tubular epithelial cells from normal human kidney, J. Urol. 168 (2002) 253–259. [10] A. Schramme, M.S. Abdel-Bakky, P. Gutwein, N. Obermuller, P.C. Baer, I.A. Hauser, A. Ludwig, S. Gauer, L. Schafer, E. Sobkowiak, P. Altevogt, M. Koziolek, E. Kiss, H.J. Grone, R. Tikkanen, I. Goren, H. Radeke, J. Pfeilschifter, Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney, Kidney Int. 74 (2008) 328–338. [11] D.B. Lee, E. Huang, H.J. Ward, Tight junction biology and kidney dysfunction, Am. J. Physiol. Renal Physiol. 290 (2006) F20–F34. [12] O.W. Blaschuk, T.M. Rowlands, Plasma membrane components of adherens junctions (Review), Mol. Membr. Biol. 19 (2002) 75–80. [13] D. Bilder, Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors, Genes Dev. 18 (2004) 1909–1925. [14] G. Bazzoni, The JAM family of junctional adhesion molecules, Curr. Opin. Cell Biol. 15 (2003) 525–530. [15] H. Ozaki, K. Ishii, H. Horiuchi, H. Arai, T. Kawamoto, K. Okawa, A. Iwamatsu, T. Kita, Cutting edge: combined treatment of TNF-alpha and IFN-gamma causes redistribution of junctional adhesion molecule in human endothelial cells, J. Immunol. 163 (1999) 553–557. [16] N. Gavert, M. Conacci-Sorrell, D. Gast, A. Schneider, P. Altevogt, T. Brabletz, A. Ben Ze’ev, L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers, J. Cell Biol. 168 (2005) 633–642. [17] O. Nagano, H. Saya, Mechanism and biological significance of CD44 cleavage, Cancer Sci. 95 (2004) 930–935. [18] G. Kim, S.A. Rajasekaran, G. Thomas, E.A. Rosen, E.M. Landaw, P. Shintaku, C. Lassman, J. Said, A.K. Rajasekaran, Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes, Histol. Histopathol. 20 (2005) 35–44. [19] N. Sawada, M. Murata, K. Kikuchi, M. Osanai, H. Tobioka, T. Kojima, H. Chiba, Tight junctions and human diseases, Med. Electron Microsc. 36 (2003) 147–156. [20] K.B. Hoover, S.Y. Liao, P.J. Bryant, Loss of the tight junction MAGUK ZO-1 in breast cancer: relationship to glandular differentiation and loss of heterozygosity, Am. J. Pathol. 153 (1998) 1767–1773. [21] Y.C. Chao, S.H. Pan, S.C. Yang, S.L. Yu, T.F. Che, C.W. Lin, M.S. Tsai, G.C. Chang, C.H. Wu, Y.Y. Wu, Y.C. Lee, T.M. Hong, P.C. Yang, Claudin-1 is a metastasis suppressor and correlates with clinical outcome in lung adenocarcinoma, Am. J. Respir. Crit. Care Med. 179 (2) (2009) 123–133.