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1 Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
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First Department of Pathology, Hamamatsu University School of Medicine, Handayama, Hamamatsu 431-3192, Japan
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Abstract |
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Introduction |
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The physiological roles of JAM4 in kidney remain to be defined. When expressed in CHO cells, JAM4 shows a sealing effect co-operatively with MAGI-1 (Hirabayashi et al. 2003). JAM4 may play roles in the regulation of permeability in glomeruli. JAM4 is also detected on apical membranes of proximal tubular epithelial cells and at tight junctions of collecting ducts, suggesting that JAM4 may have other functions in kidney. Moreover, during the study, we found that JAM4 starts to be expressed in kidney before the formation of glomeruli and we speculated that it may be involved in the developmental process. Kidney development starts with the outgrowth of the ureteric bud from the posterior metanephric duct, or Wolffian duct (Pohl et al. 2000; Dressler 2002; Piscione & Rosenblum 2002). Once the ureteric bud epithelium invades the mesenchyme, it undergoes branching morphogenesis. The ureteric bud also promotes aggregation of the metanephric mesenchyme and subsequent tubulogenesis. A number of molecules, such as transcription factors, growth factors, and receptors, are involved in these steps. Extracellular matrix and cell adhesion molecules also influence branching morphogenesis. Among these molecules, L1 attracted our attention, because it is also a member of the immunoglobulin superfamily and, like JAM4, expressed in the mesonephric duct and the metanephros (Debiec et al. 1998).
L1 is composed of six-Ig-loops, followed by five fibronectin type III repeats, a transmembrane domain, and a cytoplasmic domain. Its expression in kidney is developmentally regulated. The antibody against L1 disturbs branching morphogenesis of the collecting duct. The signalling pathway of L1 is extensively studied in neurones and neurone-like cells, because L1 plays an important role in axonal growth (Crossin & Krushel 2000; Schmid et al. 2000). L1 cross-talks with FGF receptors, is phosphorylated by multiple kinases, and induces sequential activation of phosphatidylinositol 3-kinase (PI3K), Rac, and mitogen-activated kinase (MAPK) pathway. Because Rac effectors are implicated in actin reorganization, the activation of Rac by L1 results in the formation of lamellipodia and facilitates axonal growth (Bishop & Hall 2000). Although L1 signalling in kidney is unknown, the findings from neurones imply that L1 is involved in the rearrangement of actin, which is necessary for branching morphogenesis. In this study, by analogy with L1, we first tested whether JAM4 affects branching morphogenesis. Next, we attempted to find out which intercellular signal is activated by JAM4.
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Results |
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In the previous study, we reported that JAM4 is expressed in glomeruli and proximal renal tubules in adult rat kidney (Hirabayashi et al. 2003; Tajima et al. 2003). The biochemical fractionation shows that JAM4 is slightly enriched in kidney glomerular fraction from adult rat kidney (Fig. 1A). We prepared lysates of kidney at various developmental stages and examined the expression of JAM4. JAM4 was already detected on embryonal day 15 and increased gradually (Fig. 1B). Because glomeruli are not yet developed at this stage, we determined the localization of JAM4 in embryonal day 15 kidney and detected it in the ureteric epithelium (Fig. 2, arrows).
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The localization of JAM4 in the ureteric epithelium prompted us to examine whether JAM4 may play a similar role to L1 in developing kidney. Thereby, we tested the effect of JAM4 on tubulogenesis and branching. We here made use of Madin-Darby canine kidney (MDCK) cells, because branching tubule formation of MDCK cells is a well-established cell culture model. We first compared the growth patterns of parental MDCK and MDCK stably expressing JAM4 (MDCK-JAM4) cells in three-dimensional collagen gels and could not find any significant difference (Fig. 3A, the top panels). We subsequently added 20 ng/mL of hepatocyte growth factor (HGF) and cultured cells. 24 h later, both MDCK cells were organized into tubular structures (Fig. 3A, the middle panels). MDCK-JAM4 cells formed more branches than parental MDCK cells (Fig. 3Ba). After another 24 h-culture, the size of tubules increased (Fig. 3A, the bottom panels). The number of branches per the area decreased in both of parental MDCK and MDCK-JAM4 cells, but the difference between two types of MDCK cells remained (Fig. 3Bb).
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HGF-mediated formation of branched tubules requires many steps including cell proliferation, migration, and rearrangement of cell junctions (Rosario & Birchmeier 2003; Birchmeier et al. 2003). To test which step JAM4 modulates, we first examined HGF-induced cell proliferation, but could not detect any significant difference between MDCK-JAM4 cells and parental MDCK cells (data not shown). We next evaluated HGF-induced scattering. HGF stimulated scattering of both of MDCK-JAM4 and wild MDCK cells in a dose-dependent manner (Fig. 4). However, MDCK-JAM4 cells started to scatter at 1 ng/mL of HGF, whereas parental MDCK cells showed scattering at more than 3 ng/mL of HGF. This finding suggests that MDCK-JAM4 cells are more sensitive to the scattering-inducing activity of HGF.
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The signalling of HGF is mediated by its receptor, the tyrosine kinase Met (Rosario & Birchmeier 2003; Birchmeier et al. 2003). HGF triggers the phosphorylation of Met, resulting in the recruitment of scaffolding proteins, and subsequently activates multiple pathways including PI3K, Akt, Ras, MAPK, Rac, CDC42, and Rap1. Among these signals, the activation of Rac is the most closely related to the scattering, because it is directly involved in the formation of lamellipodia and enhances the cell motility. We confirmed that MDCK-JAM4 cells formed lamellipodia in response to 1 ng/mL of HGF, whereas parental MDCK cells did not respond to the same dose (Fig. 5A, white arrows, and data not shown). Several adhesion molecules are known to activate Rac. Thereby, we speculated that the expression of JAM4 modulates the HGF-mediated activation of Rac in MDCK cells (Crossin & Krushel 2000; Braga 2002; Yap & Kovacs 2003). We utilized the PAK-CDC42 and Rac interactive binding domain fused to glutathione S-transferase (GST-PAK-CRIB) to detect active Rac. However, GST-PAK-CRIB captured active Rac from the non-stimulated MDCK and MDCK-JAM4 cells (data not shown). Due to this high basal level activation, we could not obtain conclusive results to show the difference in the HGF-mediated activation of Rac. We next tested COS-7 cells, because in the course of previous studies, we noticed that COS-7 cells expressing JAM4 cells show characteristic morphology. COS-7 cells expressing FLAG-JAM4 formed robust protrusions (Fig. 5B). FLAG-JAM-A did not induce this morphological change, indicating that FLAG-tag did not cause it. This finding means that JAM4 itself incites signalling in COS-7 cells without any additional factor. Thereafter, we focused on COS-7 cells for the simplicity.
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COS-7 cells expressing JAM4 did not form typical lamellipodia. To determine whether Rac is indeed involved in the formation of protrusions, we co-transfected dominant negative Rac1 or CDC42. The dominant negative form of Rac1, but not of CDC42 suppressed protrusions (Fig. 6A). Because the protrusions were reminiscent of eyelashes that were induced by R-Ras and Rap subfamily proteins, we also tested the dominant negative form of Rap1, but it had no effect (Heo & Meyer 2003). Even though we plated cells on dishes coated by the recombinant extracellular domain JAM4, we could not observe typical lamellipodia (data not shown). We suspected that Rac activation may not make COS-7 cells form lamellipodia. To test this possibility, we expressed dominant active Rac1 in COS-7 cells. COS-7 cells formed not lamellipodia but protrusions (Fig. 6B).
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For further confirmation, we performed the pull-down assay using GST-PAK-CRIB. Only a small amount of Rac was trapped from control COS-7 cells (Fig. 7A). The active form of Rac increased in COS-7 cells expressing FLAG-JAM4. The amount of active CDC42 did not change (Fig. 7B). We attempted to identify how JAM4 activates Rac. Because phosphatidyl inositol 3-kinase is one of the upstream regulators of Rac, we treated COS-7 cells expressing JAM4 with 50 nM of wortmannin, but protrusions were still observed (data not shown) (Bishop & Hall 2000; Burridge & Wennenberg 2004). Ras is also involved in the activation of Rac. We co-transfected the dominant negative form of Ki-Ras but did not find any suppression on protrusions (data not shown).
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Discussion |
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The molecular link between JAM4 and Rac1 remains to be identified. L1 and E-cadherin activate Rac1 signalling through PI3K, although E-cadherin also uses an unidentified pathway independent of PI3K (Schmid et al. 2000; Kovacs et al. 2002). However, the PI3K inhibitor did not block JAM4-mediated Rac1 activation. MAGI-1 is reported to interact with guanine nucleotide exchange factors for Rap1 and Rho proteins, but so far the interaction between MAGI-1 and a regulator of Rac1 has not been detected (Mino et al. 2000; Dobrosotskaya 2001). Moreover, COS-7 cells are unlikely to express endogenous MAGI-1. Thereby, JAM4 may directly interact with a regulator of Rac, and the study to search for JAM4-interacting molecules is important to elucidate how JAM4 induces the activation of Rac. The reason why Rac does not induce lamellipodia in COS-7 cells is also unclear. Several molecules including PAK, LIM-kinase, and WAVE/Scar are known to play roles in the formation of lamellipodia in the downstream of Rac (Burridge & Wennenberg 2004). Such molecules may be missing in COS-7 cells, resulting in the formation of protrusions distinct from lamellipodia.
The physiological significance of the JAM4-mediated enhancement of HGF signals also remains to be clarified. JAM4 is expressed in both of ureteric epithelial cells and epithelial cells derived from the metanephric mesenchyme in developing kidney. Met, the receptor for HGF, is expressed in both of the ureteric tips and the metanephric mesenchyme, and HGF is expressed only in the mesenchyme (Woolf et al. 1995). HGF induces via Met the branching in the ureteric buds. Thereby, we can speculate that JAM4 facilitates branching in ureteric epithelial cells. In contrast, roles of JAM4 in epithelial cells derived from the metanephric mesenchyme are elusive, because our knowledge about HGF/Met signals in the mesenchyme is limited. Experiments using gene targeting mice will be necessary to obtain the whole picture about the physiological significance of JAM4 in kidney development.
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Experimental procedures |
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pFLAG-JAM4, pFLAG-JAM-A, and pLN FLAG-JAM4 were previously described (Hirabayashi et al. 2003). pEF-BOS-Myc-RacDN (Thr to Asn at 17), pEF-BOS-Myc-RacDA (Gly to Val at 12), pEF-BOS-Myc-CDC42DN (Thr to Asn at 17), pEF-BOS-HA-Rap1DN (Ser to Asn at 17), pEF-BOS-Myc-Ki-RasDN (Thr to Asn at 17), pGex-PAK-CRIB, and pFastBac-IgG were prepared by site-directed mutagenesis and generous gifts of Dr Yoshimi Takai (Osaka University) (Takaishi et al. 1994, 1997; Kawakatsu et al. 2002). pFastBac-IgG-JAM4 was constructed using pFastBac-IgG and contains the amino acid residues 1–236 of JAM4.
Antibodies and other reagents
Mouse anti-Myc-tag monoclonal antibody 9E10 was obtained from American Type Culture Collection. Mouse anti-HA-tag monoclonal antibody was a gift of Dr Yoshimi Takai (Osaka University). Anti-Rac (Upstate Biotechnology), anti-CDC42 (Santa Cruz Biotech.), mouse anti-FLAG (Sigma-Aldrich Fine Chemicals), mouse anti-phosphotyrosine (BD Pharmingen), and rhodamine-conjugated, FITC-conjugated, and Cy5-conjugated second antibodies for dual labelling (Chemicon International Inc.), hepatocyte growth factor (Toyobo, Osaka, Japan), rhodamine-phalloidin (Molecular Probes, Inc), and wortmannin (Sigma-Aldrich Fine Chemicals) were purchased from commercial sources.
Cells
MDCK, phoenix ampho, and COS-7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% Fetalclone III (Hyclone), 100 U/mL penicillin, and 100 µg/mL streptomycin under 5% CO2 at 37 °C. COS-7 cells were transfected with DEAE-dextran method. MDCK-JAM4 cells were prepared using retrovirus system (Hirabayashi et al. 2003). Briefly, phoenix ampho cells were transfected using calcium phosphate co-precipitation method with pLN FLAG- JAM4. The medium was collected 48 h later as a virus stock. MDCK cells were cultured in the medium containing retrovirus for 48 h and then in selective medium with 1 mg/mL of G418 (Calbiochem) or 2 µg/mL puromycin (Invitrogen). The resulting resistant colonies were tested for the expression of FLAG-JAM4 and cloned.
Collagen culture
MDCK cells were isolated with trypsin and triturated into a single cell suspension. Cells were diluted to 1.5 x 104 cells/mL in a type I collagen neutral solution (Cellegen, Koken, Tokyo, Japan). 300 µL of the cell solution was plated in a 1.9 cm2-well and incubated at 37 °C to allow collagen to gel, and then added with 1 mL of the medium. Medium was changed every 24 h, and after 5 days, the medium containing 20 ng/mL of HGF was added. Cells were observed by a confocal microscopy (Olympus EV 300-BX).
Scattering assay
2.4 x 104 MDCK cells were plated in a 3.8 cm2-well in the medium containing 10% Fetalclone III. 24 h later, the medium was changed to that containing 0.1% Fetalclone III. After another 24 h-culture, cells were rinsed with phosphate buffered saline (PBS) and the medium containing various doses of HGF was added. 24 h later, cells were fixed and stained with rhodamine-phalloidin. MDCK-JAM4 cells were also immunostained with anti-FLAG antibody to confirm the expression of FLAG-JAM4.
Pull-down assay
COS-7 cells from one 10 cm-plate were harvested and homogenized in 200 µL of the buffer A (20 mM Tris/HCl pH 7.4 containing 50 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 10 mg/L leupeptin, 10 mg/L aprotinin, 1 mM dithiothreitol, and 10 µM (p-amidinophenyl) methanesulphonyl fluoride). After centrifugation at 100 000 g for 15 min at 4 °C, the supernatant was collected and incubated with 20 µg of GST-PAK-CRIB immobilized on glutathione Sepharose 4B beads for 1 h. After washing with buffer A three times, the beads were collected and proteins attached on the beads were detected by Western blotting.
Preparation of coverslips coated with the recombinant protein containing the extracellular domain of JAM4
Baculoviruses bearing cDNA encoded by pFastBac-IgG-JAM4 were prepared using Bac-to-Bac Baculovirus expression system (Invitrogen) according to the manufacturer's protocol. Sf9 cells were cultured in Sf-900 II SFM (Invitrogen) and infected with the viruses. 72 h later, the medium was collected and incubated with protein A Sepharose CL-4B beads (Amersham Biosciences). The bound proteins were eluted with 0.1 M glycine/HCl pH 2.5 and neutralized with 1/10 volume of 1.5 M Tris followed by dialysis against PBS. The coverslips were coated with 50 µg/mL of protein overnight and washed with PBS. Human IgG was used as control.
Immunofluorescence microscopy and immunohistochemistry
COS-7 and MDCK cells were immunostained as previously described (Hirabayashi et al. 2003). When COS-7 cells were plated on coverslips coated with the extracellular domain of JAM4, cells were replated 24 h after transfection. For the immunohistochemistry, kidney was obtained from embryonal day 15 rat and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Postfixed kidney was processed as previously described (Hirabayashi et al. 2003).
Other procedures
Glomeruli were obtained from adult rat using sieving techniques. Briefly, renal cortex were minced and passed through 250 µm-, 150 µm-, and 75 µm-stainless meshes, sequentially. Glomeruli trapped by the last mesh were confirmed by microscopy and used as samples. Kidneys from rat embryos were homogenized in PBS. Lysates were analysed with SDS-PAGE and immunoblotted by anti-JAM4 antibody.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yuhammch{at}med.tmd.ac.jp
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References |
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Received: 29 April 2004
Accepted: 9 June 2004
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