Department of Internal Medicine,
1 Service of Urology,
2 Rheumatology and Rehabilitation Division, University Hospital, CHUV-1011 Lausanne, Switzerland
*Author for correspondence (e-mail: jhaeflig{at}chuv.hospvd.ch)
Accepted October 12, 2001
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SUMMARY |
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Key words: bladder, urothelium, islet-Brain1, JIP-1, JNK, c-Jun, adenovirus
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Introduction |
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The control of JNK activity is therefore a critical component of the cells response to stress (Davis, 2000). Recently, the JNK activity was shown to be controlled in part by the mammalian scaffold protein Islet-Brain1/c-Jun N-terminal kinase interacting protein-1 (IB1/JIP-1). This protein binds to three kinases, MLK3 (a MAPKKK), MKK7 (a MAPKK) and JNK (a MAPK) in a single transduction complex (Whitmarsh et al., 1998
; Yasuda et al., 1999
). These kinases together constitute an ordered unit of sequential signalling molecules, transducing a variety of stress signals (Davis, 2000
).
IB1/JIP1 was first studied in the brain and in the endocrine pancreas, where the protein was found to be highly expressed. In pancreatic ß cells the high content of IB1/JIP-1 limits the output of JNK signalling toward apoptosis. Furthermore, a single missense mutation (S/N) mutation in the IB1/JIP-1 gene was recently shown to be responsible for accelerated cell death and secondary diabetes in humans (Bonny et al., 1998; Bonny et al., 2000
; Waeber et al., 2000
). In the brain, IB1/JIP-1 is localised in neurons (Pellet et al., 2000
) and interacts functionally with p190 RhoGEF (Meyer et al., 1999
), the ApoE receptor 2 (ApoER2) (Stockinger et al., 2000
) and the low-density-lipoprotein (LDL)-related-receptor protein, megalin (Gotthardt et al., 2000
). Recently IB1/JIP-1 and its different isoforms have been demonstrated to directly interact with kinesins, a family of motor proteins required for axonal transport in neuronal cells. These data revealed that the scaffold IB1/JIP1 protein family are cargos for kinesins and could participate in the formation of multiprotein complexes linking motor proteins to their membranous cargo (Verhey et al., 2001
).
Bladder carcinoma originating from transitional cells is a highly prevalent disease, which has steadily increased in incidence over the past years (Jung and Messing, 2000). The functional role of JNK signal transduction in the bladder and in the urothelial cells is mostly unexplored. In neoplasic non-urothelial cells, cell transformation induced by Ras required the activation of the JNK pathway (Pulverer et al., 1991
; Smeal et al., 1991
). Furthermore, Ras-induced tumorigenicity is suppressed by mutation of the JNK phosphorylation sites on c-Jun (Behrens et al., 2000
). Lastly, an increase in c-Jun expression was shown to be correlated with rapid tumor proliferation and aggressive cell growth in transitional cell carcinoma (TCC) (Skopelitou et al., 1997
).
Here, we have evaluated the regulation of the JNK in urothelial cells in vivo. We have demonstrated that this signalling pathway is activated by parietal stress and is dependent upon the scaffold protein IB1/JIP-1. We prevented JNK activation by overexpressing IB1/JIP-1 and, conversely, a decreased IB1/JIP-1 content is associated with an increase in JNK activity. Moreover, JNK was overactivated in mice carrying a selective disruption of the IB1/JIP-1 gene as heterozygous carriers. Taken together, these data unravel a critical role for IB1/JIP-1 in the control of the JNK activity in urothelial cells.
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Materials and Methods |
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In situ hybridisation
For in situ hybridisation, after sacrifice, rats were infused with 30 ml diethyl pyrocarbonate (DEPC, Sigma)/PBS (phosphate-buffered saline). The bladder was rapidly excised, frozen in 2-methylbutane precooled in liquid nitrogen, then frozen in OCT medium (Miles Inc., IN, USA) and cryostat sectioned at 12 mm thickness. Sections were fixed in 4% paraformaldehyde in PBS for 10 minutes, rinsed in DEPC/PBS and analysed by in situ hybridisation as previously described (Braissant et al., 1996; Haefliger et al., 1999b
). Briefly, hybridisation with two non-overlapping rat IB1/JIP-1 digoxygenin-labeled antisense riboprobes (550 bp EcoR1-Apa1 and 377 bp Apa1-Xho of the rat IB1/JIP-1 cDNA) was carried out for 40 hours at 58°C in 5x standard saline citrate (SSC) and 50% formamide. Sections were washed for 30 minutes at room temperature in 2xSSC, then for one hour at 65°C in 2xSSC, one hour at 65°C in 0.1xSSC and then stained for alkaline phosphatase. Stained sections were dehydrated and mounted with Eukitt (O. Kindler Co., Germany). The specificity of hybridisation was ascertained using two sense IB1/JIP-1 RNA of the same length, GC content and specific activity as the corresponding antisense probe.
Immunofluorescence
For light microscopy studies, rats were sacrificed and then perfused through the left ventricle with 30 ml of PBS. The bladder was rapidly excised and quickly frozen in 2-methylbutane precooled in liquid nitrogen. The bladders were frozen in OCT medium (Miles Inc., IN) and cryo-sectioned at about 5-8 µm thickness. Sections were rinsed in PBS, incubated for 45 minutes in a buffer containing 3% BSA (Bovin Serum Albumin) and 0.1% triton and then exposed for 20 hours to antibodies against IB1/JIP-1 diluted in PBS. Primary antibodies were detected using fluorescein-isothiocyanate-labeled anti-rabbit immunoglobulins (Biosystem, France). Sections were then rinsed in PBS, stained with Evans blue, viewed with a Leica microscope (Leica, West Germany) and photographed on Kodak Tmax 400 films.
RNA isolation and northern blot analysis
The bladder was homogenised in a 4 M guanidine hydrothiocyanate buffer containing 25 mM sodium citrate and 100 mM ß-mercaptoethanol using a Kinametic polytron blender (Kinametica, Switzerland). Total RNA was extracted by the acid guanidium isothiocyanate method, and yields were evaluated by absorbance at 260 nm. 10-15 mg total RNA were fractionated by size on 1% agarose gels containing 8% formaldehyde (Fluka, Switzerland) and 1xMOPS buffer (3-[N-Morpholino]propanesulfonic acid, Fluka, Switzerland). Capillary transfer in the presence of 10xSSC transferred RNAs overnight to gene screen membrane (Du Pont de Nemours GmbH, NEN Division, Germany). Membranes were UV cross-linked and vacuum-baked for two hours at 80°C. After prehybridisation, total mRNA levels were determined by hybridisation with randomly primed (Boehringer-Mannheim, Germany) cDNA that were specific for IB1 (Bonny et al., 1998), and glyceradheyde-3-phosphate dehydrogenase (GAPDH) (Haefliger et al., 1999a
) was labeled with [
-32P]dCTP (Amersham, Switzerland).
Antibodies
Polyclonal IB1/JIP-1 antiserum was described previously (Bonny et al., 1998). These antibodies were affinity purified from crude serum using a Hitrap NHS-activated affinity sepharose column (Amersham Pharmacia Biotech AB Switzerand), which was coupled to the immunogenic protein. Polyclonal antibodies (JNK, p-JNK, p38, p-p38, ERK, p-ERK, c-Jun) were purchased from New England Biolabs (Bioconcept, Switzerland).
Western blotting
Immediately after sacrifice, rats were infused through the heart with 30 ml PBS, the bladders were excised and rapidly frozen in liquid nitrogen, or urothelial cells were scraped from freshly removed bladder using a scalpel, and frozen. The bladders were homogenised with a polytron in the passive lysis buffer (Promega, Switzerland). The urothelial cells were lysed in Mono Q buffer (1% Triton X-100, 0.5% Nonidet P-40,20 mM Tris-HCl, pH 7.5, 0.2 mM sodium vanadate, 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 5 mM PMSF). Western blots of nuclear extract were performed using the same samples prepared for the EMSA. The DC protein assay reagent kit (Bio-Rad Laboratories, Switzerland) determined protein content. Aliquots of bladder or urothelial cells (150 mg for IB1/JIP-1, 50 mg for MAPK, 30 mg for c-Jun) were fractionated by electrophoresis in a 10% polyacrylamide gel and immunoblotted onto Immobilon PVDF membranes (Millipore Co, MA) overnight at a constant voltage of 30 V. Membranes were incubated for one hour at room temperature in PBS containing 5% milk and 0.1% Tween 20 (blocking buffer) and then incubated for two hours at room temperature with antibodies directed against IB1/JIP-1. Antibodies against JNK, p-JNK, p38, p-p38, ERK, p-ERK and c-Jun were incubated overnight at 4°C in blocking buffer. Specific antigen-antibody complexes were detected with the HRP western blot detection system (Amersham, Switzerland). Each lane shows a sample from a different rat.
Protein kinase assays
JNK activity was measured using glutathione S-transferase-c-Jun(1-79) bound to glutathione-Sepharose 4B (Bonny et al., 2000). Urothelial cells scraped from freshly removed bladder were lysed in 0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.6, 0.25 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1mM dithiothreitol, 1mM PMSF (phenylmethylsulfonyl fluoride), 2 mM sodium vanadate, 20 µg/ml aprotinin and 5 µg/m leupeptin. Lysates were centrifuged at 14000 g for 10 minutes to remove any nuclei, and supernatants (50 µg of protein) were mixed with 19 µl of glutathione S-transferase-c-Jun. The mixture was rotated at 4°C for one hour, washed twice in lysis buffer and twice in kinase buffer (20 mM Hepes, pH 7.5, 10 mM MgCl2, 20 mM ß-glycerophosphate, 10 mM p-nitrophenylphosphate, 1 mM dithiothreitol and 50 µm sodium vanadate). Beads were suspended in 40 µl of kinase buffer with 10 µCi of [
-32P]ATP and incubated at 30°C for 25 minutes. Samples were boiled in loading buffer, and phosphorylated proteins were resolved on 10% SDS polyacrylamide gels. To verify the selectivity of the JNK assay, cell lysates were fractionated by Mono Q ion exchange chromatography, and each fraction assayed as described above. Fractions were immunoblotted with rabbit antiserum recognising JNK. Only fractions containing immunoreactive JNK phosphorylated the glutathione S-transferase-c-Jun(1-79) protein.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts of freshly scraped urothelial cells from control or dilated bladders were prepared as previously described (Andres et al., 1999). The protein concentration was measured using a Bio-Rad protein measurement kit. For EMSA, the double-stranded DNA probe corresponding to the AP-1 consensus sequence was labeled with [
-32P]dCTP by the Klenow enzymes. The labeled probe was incubated with 5 mg of the nuclear protein and 1 mg of poly d(I-C) in a total volume of 20 ml containing 10% glycerol, 15 mM Hepes-NaOH, pH 7.8, 5 mM MgCl2, 5 mM dithiothreitol and 50 mM KCl. After incubation at 4°C for 30 minutes, the reaction mixtures were resolved on a 4% non-denaturing polyacrylamide gel electrophoresis in 0.5xTBE buffer (1xTBE; 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0), and the products were detected by autoradiography. Antiserum against c-Jun (H-79) was purchased from Santa Cruz biotechnology, Inc. (CA). For the competition experiments, each unlabeled oligonucleotide was added to the reaction mixture prior the addition of the labeled probe. For the antibody experiments, antiserum was added to the extracts and the mixture was incubated for two hours on ice prior to the addition of the labeled oligonucleotides.
Generation of recombinant adenoviruses
To overexpress IB1 or inhibit its expression, we have constructed recombinant adenoviruses comprising the complete cDNA of rat IB1/JIP-1 (Bonny et al., 1998) in the sense orientation or in the antisense orientation. Adenoviruses expressing the green fluorescent protein (GFP) alone were used as a control. To generate the adenoviruses, the cDNAs were inserted into the plasmid pXC15 (Schaack et al., 1995
), and adenoviruses were then generated by homologous recombination in 293 cells following cotransfection by the calcium phosphate procedure of the plasmid pJM17 (Hitt et al., 1998
) and pXC15 constructs. Viruses were further plaque-purified three times on HR911 cells (IntroGene, Leiden, The Netherlands). Two rounds of CsCl centrifugation purified large stock viruses. After the second centrifugation, the virus band (1.5 ml) was collected and dialysed at 4°C against three changes (at least 200 volumes each) of 10 mM Hepes pH 8.0, 150 mM NaCl in a Slide-A-Lyzer (0.5-3.0 ml capacity), gamma irradiated 10K dialysis cassette (Pierce, Switzerland). The recombinant adenoviruses allowed expression of IB1/JIP-1, GFP and/or the IB1/JIP-1 antisense RNA under the control of the strong immediate early cytomegalovirus (CMV) promoter.
Statistical analysis
Densitometric analysis of signals (autoradiograms) were performed using a Molecular Dynamics scanner (Sunnyvale, USA), which integrates areas and corrects for background. Data were expressed as mean ±s.e.m. and were compared using the Students t test. Statistical significance was defined at a value P<0.05 (*), P<0.01 (**) and, P<0.001 (***).
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Results |
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Discussion |
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We investigated a rat model of complete bladder outlet obstruction (Tammela et al., 1993) to study in vivo the activity of MAPKs, as mechanical stress is known to activate the JNK pathway in many systems (Komuro et al., 1996
; Li et al., 1999
; Matsuda et al., 1998
; Ishida et al., 1999
). In this model, ligation of the urethra induces an increased voiding pressure, which enhances bladder wall stress. In these conditions, we observed a drastic increase in the activated state of JNK associated with an increased of AP-1 binding activity. Furthermore, this urothelial AP-1 complex is constructed by c-Jun, the physiological substrate of JNK. These data suggested that the active JNK could potentiate, through c-Jun phosphorylation, the ability of AP-1 to activate transcription, which, in turn, would increase the c-Jun expression, as observed here in mechanically stressed urothelium. This increased c-Jun synthesis could result in the increased AP-1 binding activity observed (Karin, 1995
). Mechanical stress reduced the IB1/JIP-1 content in the urothelium, and using an adenovirus gene transfer approach, we could demonstrate that IB1/JIP-1 is a critical regulator of JNK activity. Moreover, we showed that increased levels of IB1/JIP-1 induced by viral gene transfer could prevent the stress-induced activation of JNK and regulate c-Jun expression and AP-1 activity. Conversely, JNK activity was increased in urothelial cells where the IB1/JIP-1 content was experimentally reduced using an antisense RNA strategy. Moreover, reduced IB1/JIP-1 content in urothelial cells of heterozygous mice carrying a disruption of the IB1/JIP-1 gene leads to a basal increase in JNK activity. Therefore, IB1/JIP-1 plays a crucial role in the dilated urothelium by controlling the effects of JNK signalling under mechanical stress. Taken together, these data unravel for the first time a critical role for IB1/JIP-1 in the control of the JNK signalling pathway in vivo. The stoichiometry of the interaction between scaffold proteins and protein kinases may be critical for the regulation of protein kinase activity; high levels of IB1/JIP1 would be predicted to inhibit JNK activation by titrating the protein kinase components into separate IB1/JIP1 complexes, whereas lower levels are likely to form functional complexes leading to JNK activation (Yasuda et al., 1999
). It is important to emphasize that the in vitro protein kinase assay may not be a true reflection of JNK activity measured in the cells. JNK interacts tightly with IB1/JIP-1 and with less affinity with its substrate c-Jun. This means that in cells from dilated bladder where there is reduced IB1/JIP1 expression, more JNK is likely to be pulled down, resulting in increased c-Jun phosphorylation, which may not reflect the situation in vivo.
We previously reported that IB1/JIP-1 regulates cytokine-induced apoptosis in insulin-producing cells (Bonny et al., 2000). In sympathetic neurons, the NGF withdrawal induced apoptosis, which could be prevented by the overexpression of the JBD (JNK binding domain) of IB1/JIP-1 (Harding et al., 2000
). These data indicated that IB1/JIP-1 plays an anti-apoptotic function in ß cells and neurons by controlling the JNK signalling pathway. In the bladder, the most prominent response to acute bladder distention is a urothelial cells proliferation (Tammela et al., 1993
) associated with an increase in smooth muscle cells volume (hypertrophy) (Levin et al., 1990
) as well as lamina propria and connective tissue proliferation (Tong et al., 1992
). In the same model it was demonstrated that Cox-2 is upregulated and may participate in cell proliferation (Park et al., 1999
). By TUNEL and Hoechst/propidium iodide staining, we did not observe an increase apoptotic rate (data not shown), but it remains technically difficult to evaluate this apoptotic rate in vivo. Further studies will be required to evaluate, in vitro, if a decrease of IB1 content, in this specialised epithelium, increased cell death.
Although JNK is implicated in cancer, the potential contribution of the JNK signalling is still unclear. Recent investigations concurred that JNK is required for stress-induced apoptosis in primary cells (Garay et al., 2000; Ho et al., 2000
). In contrast, antisense JNK oligonucleotides inhibit the growth of tumor cells and can induce apoptosis (Bost et al., 1999
; Potapova et al., 2000
). It is possible that proteins that inhibit JNK activity could act as an oncogene by supressing the regulated apoptosis induced by the JNK pathway activation (Adler et al., 1999
; Kurokawa et al., 2000
), but this speculative role remains to be evaluated in further studies in vitro. All these data revealed a potential role for IB1/JIP-1 in bladder oncogenesis; IB1/JIP-1 may therefore represent an alternative target for gene therapy of cell proliferation or tumorogenesis in the bladder as a new regulator of the JNK signalling pathway.
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ACKNOWLEDGMENTS |
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