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First published online 17 January 2006
doi: 10.1242/jcs.02773

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The major vault protein is responsive to and interferes with interferon--mediated STAT1 signals

Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, A-1090 Vienna, Austria

View larger version (12K): [in a new window]   Fig. 1. Role of a STAT-binding site in constitutive activation of the human MVP gene promoter. Hep3B cells were transiently transfected with a full-length and a 5'-end deletion MVP promoter construct containing wild-type (pMVP1670 and pMVP124, respectively) and mutated (pMVP1670-STATmut, pMVP124-STATmut) STAT-binding sites (box with cross). Luciferase values were normalized to the protein content and are given relative to the full-length promoter construct pMVP1670 arbitrarily set as 1. Means and s.d. of four independent experiments are shown. Data were compared by Mann-Whitney U-test: **P<0.01; ***P<0.005. The inset gives a sequence comparison of the putative STAT-binding site (–33 to –25 relative to the TSP) in the human MVP promoter (STATwt) with the consensus GAS sequence and the STAT consensus sequence (STATcons) used by the MatInspector program. Bold face indicates accordance with the STAT consensus, accordance with the GAS sequence is indicated by asterisks. Mutated bases (GGAC) in the STATmut reporter constructs are underlined.

View larger version (40K): [in a new window]   Fig. 2. Effect of IFN- on MVP gene promoter activity and binding of STAT1. (A) Hep3B cells were transiently transfected with the indicated wild-type (pMVP1670, pMVP124) and mutated (pMVP1670-STATmut, pMVP124-STATmut) MVP promoter constructs or the vector control pGL3-Basic. Transfected cells were treated with 250 IU/ml IFN- for 6 hours prior to performing luciferase assays. Values were normalized to the protein content and are given relative to the promoter activity of untreated cells transfected with the respective promoter construct. Means and s.d. of 4 independent experiments are shown. Data were compared by Mann-Whitney U-test: *P<0.05; **P<0.01. Significant differences between untreated (dotted line) and treated cells are indicated next to the bars and between wild-type and mutated constructs next to the brackets. (B) EMSA was performed as described in the Materials and Methods using as probes a labeled oligonucleotide containing a high-affinity STAT1 site (SIE) or the wild-type (STAT-wt) and mutated (STAT-mut) GAS site of the human MVP promoter. Extracts of untreated and IFN--treated Hep3B cells were incubated with the indicated probes, unlabeled oligonucleotides (competitors) or a STAT1 antibody. ns, non-specific antibody; UK, unknown complexes. (C) ChIP assays of untreated and IFN--treated Hep3B cells were performed as described in the Materials and Methods. DNA/protein complexes were immunoprecipitated with an anti-STAT1 antibody (STAT1-Ab) or no antibody (no Ab) for control. PCR products from immunoprecipitated material were compared with those obtained from the input material (Input).

View larger version (53K): [in a new window]   Fig. 3. IFN- induces upregulation of MVP mRNA and protein expression. (A) mRNA contents of Hep3B cells treated with IFN- (250 IU/ml) for 6 hours, 24 hours and 48 hours were analyzed by northern blots in comparison to ß-actin and GAPDH mRNAs as controls. Signals for MVP were normalized to the 28s RNA band and are given as fold induction compared with untreated control cells. (B) MVP expression of human cancer cell lines treated for 24 hours and 48 hours with IFN- (250 IU/ml) were analyzed by western blot. ICAM-1 was used as a positive control for the IFN- response, and ß-actin as a control for equal loading. (C) A representative western blot experiment (out of four experiments performed) as shown in B was quantified using ImageQuant software. Changes in MVP expression by IFN- are given as fold induction compared with control cells at 24 hours. (D) Western blot analysis of protein lysates obtained from cells pretreated for 30 minutes with DMSO (Co) or the indicated concentrations of curcumin or AG490 as indicated followed by incubation without or with IFN- (250 IU/ml) for 24 hours. MVP expression is compared with ICAM-1 as a positive control for the IFN- response and ß-actin to demonstrate equal loading. (E) Western blot analysis was performed using whole cell extracts derived from Hep3B cells transiently transfected with a DN-STAT1 expression or control plasmid. Forty-eight hours after transfection, cells were treated with 250 IU/ml IFN- for 24 hours. All experiments shown in this figure were performed at least twice with comparable results.

View larger version (46K): [in a new window]   Fig. 4. Time course of MVP de novo synthesis induced by IFN-. (A) Hep3B cells were treated for the indicated time intervals with IFN- (250 IU/ml) prior to a 30 minute pulse with [35S]methionine. The upper panel illustrates the overall incorporation of [35S]methionine in cell lysates, the middle panel shows Coomassie Blue staining of the same gel to demonstrate equal loading of the lanes. The lower panel depicts the labeled, immunoprecipitated MVP using the antibody LRP56 (MVP) as compared with an unrelated mouse IgG1 antibody (IgG). Quantification of three experiments by ImageQuant software is shown as a bar chart on the right. The amount of newly synthesized MVP was normalized to the total protein content and subsequently to the overall 35S incorporation. Values are given as mean fold induction compared with untreated control cells. (B) MVP, ICAM-1 and ß-actin expression in Hep3B cells treated with IFN- (250 IU/ml) for the indicated time periods were analyzed in parallel by western blot. All experiments shown in this figure were performed at least twice with comparable results.

View larger version (16K): [in a new window]   Fig. 5. Impact of IFN- treatment on MVP stability. An autoradiograph of a pulse-chase experiment of Hep3B cells treated with IFN- (250 IU/ml) for 15 hours prior to the pulse and further during the chase of 12 and 24 hours is shown. Labeled MVP was immunoprecipitated and resolved by 8% SDS-PAGE. A mouse IgG1 antibody was used as a background control for the immunoprecipitation. Means of two independent experiments are shown in the bar chart.

View larger version (49K): [in a new window]   Fig. 6. Assembly of IFN--induced MVP into intact vault particles. (A) Cell lysates obtained from the indicated cell lines incubated for 24 hours in the absence or presence of IFN- (250 IU/ml) were fractionated as described in Materials and Methods. Total cell lysates (L) are opposed to a 100,000 g particle (P) and a high-speed supernatant fraction (S). Similar amounts of protein were separated by SDS-PAGE, immunoblotted and analyzed for presence of MVP. One of three experiments leading to comparable results is shown. (B) KB-3-1 cells were grown on chamber slides, treated with IFN- (250 IU/ml) for the indicated times, fixed and immunostained for MVP (green). Nuclei were counterstained with DAPI (blue). Bar, 5 µm.

View larger version (41K): [in a new window]   Fig. 7. Influence of MVP on cell surface marker protein expression. (A) Expression of CD13 (detected by two different antibodies Ab1, Ab2) and CD36 in a representative MVP-expressing (thick line) as compared with a vector control H65 cell clone (thin line) was measured by FACS analysis. (B) Presence of ICAM-1 on the surface of two MVP-expressing (pMVP) and control (pSport6) H65 clones each was determined by FACS analysis and compared with the integrins vß5 and vß3. Thick and thin lines represent probes with specific primary and unspecific control antibodies, respectively. (C) Basal and IFN--induced ICAM-1 expression of stably MVP-transfected (pMVP) and vector control (pSport6) H65 cell clones was analysed by western blotting. (D) The presence of ICAM-1 mRNA was determined by RT-PCR in H65 cell clones with and without ectopic MVP expression. (E) Human U373 glioblastoma cells were infected with adenoviruses expressing shRNA targeting human MVP or Renilla luciferase as a non-expressed control for 5 days and treated with 250 IU/ml IFN- for 24 hours. Whole cell lysates were analysed by western blot and probed with the indicated antibodies. All experiments were performed at least twice with comparable results.

View larger version (41K): [in a new window]   Fig. 8. MVP expression inhibits STAT1 signaling. Localization of STAT1 in cytosol and nuclei of MVP-transfected (pMVP) and control (pSport6) H65 clones in response to 1000 IU/ml IFN- (1 hour treatment) as indicated was determined by western blot (A) and immunocytochemical (B) analyses. Bar, 10 µm. S6 ribosomal protein (rpS6) was used as cytosolic marker. (C,D) Phosphorylation of STAT1 at Y701 in response to 1000 IU/ml IFN- in the investigated MVP-transfected H65 (C) and the EGFP-MVP transfected VM-48 (D) cell clones was determined by western blot analysis and compared with total STAT1 and MVP expression as indicated. H65 cells were treated with IFN- for 60 minutes and VM-48 cells for 15 and 30 minutes for optimal read-out. All experiments were performed at least twice with comparable results.

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