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First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02409

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Urokinase-induced activation of the gp130/Tyk2/Stat3 pathway mediates a pro-inflammatory effect in human mesangial cells via expression of the anaphylatoxin C5a receptor

¹ Hannover Medical School, Carl-Neuberg Straße 1, 30625 Hannover, Germany
² Department of Immunology, Georg-August-University, Kreuzbergring 57, 37073 Göttingen, Germany
³ Ehime University School of Medicine, Shitsukawa, Shigenobucho, Onsengun, Ehime 791-0295, Japan
⁴ Medical Faculty of the Charité–Franz Volhard Clinic, HELIOS Klinikum-Berlin and Max Delbrück Center for Molecular Medicine, Wiltbergstrasse 50, 13125 Berlin, Germany

View larger version (28K): [in this window] [in a new window]   Fig. 1. uPA upregulates expression of C5aR in MCs. (A) RT-PCR analysis for C5aR mRNA was performed using the TaqMan method. RNA was isolated from quiescent MCs incubated for the indicated times with 20 nM uPA or in medium without uPA (control). GAPDH served as a house keeping gene. The results are presented as mean ± s.e.m. of four separate and independent experiments. (B) The quiescent MCs were stimulated for 20 hours at 37°C with the indicated concentrations of uPA, and C5aR protein was visualized in the membrane fractions by immunoblotting with anti-C5aR antibodies. MCs incubated in medium without uPA served as a control. The data on the upper panel are representative of four separate and independent experiments. Quantification of the results of these experiments by densitometry presented as mean ± s.e.m. is shown in the lower panel. Significance between control unstimulated and stimulated cells was determined by Student's t-test (*P<0.05; **P<0.01). (C) FACS analysis of MCs stimulated (solid line) or not (dashed line) for 20 hours at 37°C with 20 nM uPA using a FITS-labeled murine monoclonal anti-C5aR antibody. FITS-labeled isotype-matched antibody was used as a negative control (grey area). The results are representative of two separate and independent experiments. (D) The quiescent MCs were stimulated for 20 hours with 20 nM uPA or in medium without uPA (control). The cells were then fixed and stained with primary anti-C5aR antibodies and Alexa Fluor 488-conjugated secondary antibodies. Results are representative of three separate experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 2. uPAR mediates uPA-dependent upregulation of C5aR expression. The quiescent MCs were stimulated for 20 hours with 20 nM uPA in the presence or absence of 5 µg/ml anti-uPAR monoclonal antibody, and C5aR expression was investigated at the mRNA (A) and protein (B) levels using RT-PCR TaqMan analysis and western blotting, respectively. MCs incubated in medium without uPA and anti-uPAR antibody served as a control. The results in A are presented as mean ± s.e.m. for three separate and independent experiments. The data on the upper panel in B are representative of three separate and independent experiments. Quantification of the results of these experiments by densitometry presented as mean ± s.e.m. is shown in the lower panel. Significance between control unstimulated and stimulated cells was determined by Student's t-test (*P<0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 3. uPA potentiates C5aR-related responses in MCs. (A) Quiescent MCs were incubated in 96-well microtiter plates with 20 nM uPA or 20 nM C5a alone or in combination with both stimuli in the presence or absence of 5 µg/ml anti-uPAR monoclonal antibody or irrelevant mouse IgG for 24 hours. The cells incubated in medium without stimuli served as a control. After 16 hours labeling with BrdU DNA synthesis was used as measure of the proliferation rate. Results are given as mean ± s.e.m. of four independent experiments performed in six parallel wells for each condition. (B) MCP-1 release was evaluated in supernatants from MC monolayers that were incubated as described in A for 48 hours. MCs incubated in medium without stimuli and producing 868±95 pg/ml MCP-1 served as a control. The data are given as mean ± s.e.m. of four separate and independent experiment performed in triplicate for each condition. Significance between control unstimulated and stimulated cells, as well as between the cells stimulated in the presence or not of 5 µg/ml anti-uPAR monoclonal antibody was determined by Student's t-test (*P<0.05; **P<0.01).

View larger version (43K): [in this window] [in a new window]   Fig. 4. uPA induces activation and nuclear translocation of Stat3 in MCs. (A) Quiescent MCs were treated with 10 nM uPA for the indicated times and phosphorylated Stat3 protein was visualized in cell lysates by immunoblotting with specific anti-(P)-Tyr-Stat3 antibodies. MCs incubated in medium without uPA served as a control. The middle panel demonstrates the amount of total Stat3 loaded on the gel for each sample. Results are representative of three independent experiments. Quantification (mean ± s.e.m.) of the results of these experiments by densitometry is shown below. Significance between control unstimulated and stimulated cells was determined by Student's t-test (*P<0.05). (B) A subconfluent MC monolayer was treated with 10 nM uPA at 37°C or left untreated, fixed and stained using primary anti-(P)-Tyr-Stat3 antibody and the corresponding Alexa Fluor-488 secondary antibody. The data are representative of three separate and independent experiment performed in duplicate for each condition.

View larger version (22K): [in this window] [in a new window]   Fig. 5. uPA induces activation of Tyk2 in MCs. (A) Quiescent MCs were treated with 20 nM uPA for the indicated times and phosphorylated Tyk2 protein was visualized in cell lysates by immunoblotting with specific anti-phospho-Tyk2 antibodies (upper panel). MCs incubated in medium without uPA served as a control. The middle panel demonstrates the amount of total Tyk2 loaded on the gel for each sample. Results are representative of three independent experiments. Quantification (mean ± s.e.m.) of the results for these experiments by densitometry is shown below. Significance between control unstimulated and stimulated cells was determined by Student's t-test (*P<0.05). (B) MCs were left uninfected or were infected with wild-type Ad5Tyk2 or with the mutant form, Ad5Tyk2C, and then stimulated for 20 minutes with 20 nM uPA. Phosphorylated Stat3 protein was visualized in cell lysates by immunoblotting with specific anti-(P)-Tyr-Stat3 antibodies. Noninfected MCs incubated in medium without uPA served as a control. The middle panel demonstrates the equal amount of total Stat3 loaded on the gel for each sample. Results are representative of three independent experiments. Quantification (mean ± s.e.m.) of the results by densitometry is shown for three experiments is shown below. Significance between control unstimulated and stimulated cells was determined by Student's t-test (*P<0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 6. uPAR mediates uPA-dependent Tyk2/Stat3 pathway activation in MCs. A. Quiescent MCs were infected with LV-uPARsi and time-dependent downregulation of uPAR expression was monitored by immunoblotting, using clone R3 monoclonal anti-uPAR antibody. MCs infected with LV-uPARsi or mock viruses were treated with 10 nM uPA for the indicated times at day 3 after infection and phosphorylated Tyk2 (B) or Stat3 (C) proteins were visualized in cell lysates by immunoblotting with specific anti-phospho-Tyk2 and anti-(P)-Tyr-Stat3 antibody, respectively. MCs incubated in medium without uPA served as a control. The upper panels is representative of three independent experiments. Quantification (mean ± s.e.m.) of the results by densitometry is shown below. Significance between control unstimulated and stimulated cells was determined by Student's t-test (*P<0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 7. TheTyk2/Stat3 pathway mediates uPA-induced upregulation of C5aR in MCs. Quiescent MCs that were uninfected or infected with wild-type Ad5Tyk2, the Tyk2 mutants, Ad5Tyk2C and Ad5Tyk2KE, or with the Ad5STAT3F mutant form of Stat3 were stimulated with 20 nM uPA for 20 hours, and RT-PCR analysis for C5aR mRNA was performed using the TaqMan method. MCs incubated in medium without uPA served as a control. Results are presented as mean ± s.e.m. of three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 8. uPAR utilizes gp130 adaptor protein to mediate C5aR expression in MCs. (A) Quiescent MCs were stimulated with 10 nM uPA for the indicated times or left unstimulated (control), and anti-gp130 (upper panel) or anti-uPAR (lower panel) antibody was used to coimmunoprecipitate gp130 and uPAR from the cell lysates. The immunoprecipitates were then analyzed with anti-uPAR (upper panel) or anti-gp130 antibody (lower panel). (B) RT-PCR analysis of C5aR mRNA was performed using the TaqMan method. RNA was isolated from quiescent MCs incubated for 6 hours with 20 nM uPA or in medium without uPA (control) in the presence or absence of 5 µg/ml of anti-gp130 antibody. Results are presented as mean ± s.e.m. of two independent experiments performed in duplicates for each condition.

View larger version (20K): [in this window] [in a new window]   Fig. 9. uPAR is required for C5aR expression in mesangium upon LPS-induced renal inflammation in mice. LPS-induced nephritis was induced in wild-type and uPAR–/– mice by an intraperitoneal injection of 50 µg LPS in 200 µl PBS. Animals injected with PBS only, served as a control. 8 hours after challenge the mice were killed, RNA was isolated from kidney glomeruli and used for expression analysis by the TaqMan method. The mRNA levels were analyzed for uPA (A), uPAR (B), C5aR (C) and MCP-1 (D). -tubulin served as a house-keeping gene. Results are expressed as mean ± s.e.m. (n=6 mice for each group). Significance between PBS controls and LPS-treated animals was determined by Student's t-test (*P<0.05; **P<0.01).

View larger version (41K): [in this window] [in a new window]   Fig. 10. uPAR is required for C5aR expression in mesangium upon LPS-induced renal inflammation in mice. (A) LPS-induced nephritis was induced in wild-type and uPAR–/– mice as described in Fig. 9. 8 hours after challenge the mice were killed and 6 µm cryosections of kidney cortex were stained for C5aR protein expression using anti-C5aR primary antibody and Cy3-conjugated secondary antibodies. Representative microphotographs are shown (6 mice for each group). Bar, 50 µm. (B) Statistical analysis of C5aR expression in mesangium of wild-type and uPAR–/– mice with LPS-induced nephritis was performed by evaluating the total number of positively stained glomeruli and the staining intensity (in arbitrary units) as described in the Materials and Methods. Results are expressed as mean ± s.e.m. (n=6 mice for each group). Significance between PBS controls and LPS-treated animals was determined by Student's t-test (*P<0.05; **P<0.01). +, arbitrary units (based on staining intensity using criteria described in Materials and Methods).

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