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The scaffold protein IB1/JIP-1 controls the activation of JNK in rat stressed urothelium

Department of Internal Medicine,
¹ Service of Urology,
² Rheumatology and Rehabilitation Division, University Hospital, CHUV-1011 Lausanne, Switzerland

View larger version (130K): [in a new window]   Fig. 1. The scaffold protein IB1/JIP-1 is present in urothelial cells of rat bladder. In situ hybridisation using two non-overlapping cDNA probes specific for IB1/JIP-1 (A,B) demonstrated the presence of IB1/JIP-1 transcripts in the urothelium of rat bladder. These transcripts are translated into immunodetectable protein in non-dilated and dilated bladder respectively (C,D). Smooth muscle cells (smc); Urothelium (u); Lumen (l). Bar represents 15 µm in A and C and 25 µm in B and D.

View larger version (60K): [in a new window]   Fig. 2. Regulated expression of the scaffold protein IB1/JIP-1. (A) Northern blot analysis of bladders revealed that the level of IB1/JIP-1 transcripts is drastically decreased in dilated bladder after seven hours of dilation. In the same samples, levels of GAPDH mRNA were slightly increased (1.5 fold) in the dilated bladder. Each lane is from a different animal and was loaded with 10 µg mRNA. (B) Quantitative assessment of sixteen experiments (one per rat) confirmed that the levels of IB1/JIP-1 mRNA were decreased by three fold compared with control values. Values represent ratios of densitometric measurements of IB1/JIP-1 and GAPDH mRNAs and are expressed as mean±s.e.m. (n=16± s.e.m.; *P<0.01). (C) IB1/JIP-1 content was reduced in dilated bladder after seven hours of dilation as assessed by western blot analysis. Equal protein loading was evaluated by measuring the level of tubulin. (D) Quantitative evaluation of four immunoblots revealed that the levels of IB1/JIP-1 were reduced by 50% in samples of dilated bladder (seven hours after ligation) compared with their controls. Values represent ratios of densitometric measurements of IB1/JIP-1 and tubulin. (mean±s.e.m.; n=5; P<0.05).

View larger version (62K): [in a new window]   Fig. 3. Parietal stressed is associated with a selective increase phosphorylated MAPK ERK and JNK but not p38 in urothelial cells. Western blot analysis of freshly scraped urothelial cells using specific antibodies against the phosphorylated and the non phosphorylated form of the MAPKs p38 (A), ERK (B), JNK (C) demonstrates an increase in the phosphorylated form of the MAPKs ERK and JNK but not in p38 in urothelium of dilated bladder after seven hours of obstruction, compared with empty bladder. Each lane is from a different animal and quantitative assessment of five experiments (one per rat) confirmed that the levels of P-JNK and P-ERK were increased six-fold and four-fold respectively over control values. Values represent ratios of densitometric measurements of either P-JNK or JNK, P-ERK and ERK or P-p38 and p38 and are expressed as mean±s.e.m.

View larger version (66K): [in a new window]   Fig. 4. Phosphorylated JNK activity is functionally active and associated with an increase in AP-1 binding activity. (A) JNK activity was evaluated in urothelial cells. Endogenous JNK was first purified using GST-Jun, and then a solid phase kinase assay was performed. [-33P]-phosphorylated substrates (P-GST-Jun) were separated on a polyacrylamide gel. Western blotting monitored JNK expression in the same samples. The gel was stained with Coomassie blue to evaluate the loading of substrate (GST-Jun). P-GST-Jun showed a progressive increase during the development of the dilated bladder. Maximum expression occurred seven hours after ligature. All lanes were loaded with 50 µg of protein. (B) JNK activity in urothelium was monitored seven hours after ligature. Quantitative assessment demonstrated that urothelial cells of dilated bladder increased c-Jun phosphorylation about five fold compared to the empty bladder. On the same samples, IB1/JIP-1 content was decreased in freshly scraped urothelial cells from the dilated bladder. Values represent densitometric measurements of six independent experiments and are expressed as mean±s.e.m. ***=P<0.001 level. (C) Electrophoretic mobility shift assay (EMSA) showed that AP-1 binding activity is increased in stressed urothelium seven hours after ligature. Each lane shows a sample from a different rat. All lanes were loaded with 7 µg. (D) Specificity of the AP-1 shift. Nuclear extracts of freshly scraped urothelial cells were pre-incubated with specific c-Jun antibody (c-Jun) or two different irrelevant antibodies (IgG). AP-1 shift was competed with the incubation of a cold-specific competitor (specific) but not with non-specific oligonucleotides (NS). (E) Western blot analysis of c-Jun expression on the same nuclear extract of freshly scraped urothelial cells used for EMSA experiment seven hours after ligature demonstrated a 400% increase of c-Jun expression in dilated bladder compared with the control.

View larger version (39K): [in a new window]   Fig. 5. The IB1/JIP-1 content is critical for mediating JNK activity. Western blot analysis and a solid phase kinase assay using GST-Jun as the substrate revealed a decrease in IB1/JIP-1 expression associated and an increase of JNK activity (P-GST-Jun) in empty bladders of mice heterozygous (KO+/–) compared with wild-type mice (WT). The JNK content was evaluated by western blotting. Equal loading of substrate was confirmed by Coomassie blue staining of the gel (GST-jun). Quantitative assessments of IB1/JIP-1 and c-Jun phosphorylation in urothelium of mice, which had had the IB1/JIP-1 gene selectively disrupted, demonstrated an inverse relationship between the levels of IB1/JIP-1 content and the JNK activity compared to basal level in wild-type mice.

View larger version (46K): [in a new window]   Fig. 6. Gene transfer of IB1/JIP-1 can prevent the JNK activity. (A) Western blot analysis of scraped urothelial cells infected with different adenovirus constructs showed that IB1/JIP-1 content is largely increased in cells overexpressing IB1/JIP-1 (adCMV-sIB1), whereas a decreased expression was observed when cells were infected with the adCMV-asIB1 compared to control adCMV-GFP. Protein loading was evaluated by tubulin. (B) JNK activity analysis in urothelial cells of dilated bladder revealed that cells infected with adCMV-asIB1 increased their JNK activity; in contrast, cells infected with adCMV-sIB1 decreased their JNK activity. Quantitative assessment demonstrated that the JNK activity is increased about three-fold in urothelial cells of dilated bladder infected with adCMV-asIB1 compared with non infected bladder (NI) or infected with control adenovirus (adCMV-GFP). In contrast, a 50% reduction in JNK activity is observed in cells infected with adCMV-sIB1. In the same samples, an increase in P-JNK was detected by immunoblot in urothelial cells infected with adCMV-asIB1; in contrast, a decrease in P-JNK content was observed in cells infected with adCMV-sIB1 compared to controls (NI, adCMV-GFP). (C) Electrophoretic mobility shift assay (EMSA) revealed a decrease in AP-1 binding activity in stress urothelium transduced with adCMV-sIB1/JIP-1 compared to non-infected dilated urothelium (NI) or cells transduced with the control adenovirus. (D) Western blot analysis of c-Jun expression on the same nuclear extract of dilated urothelial cells demonstrated a decrease in c-Jun expression in bladder transduced with adCMV-sIB1 compared to non infected bladder (NI) or infected with the control adenovirus.