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First published online 15 January 2008
doi: 10.1242/jcs.017160

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STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes

¹ Institute of Experimental Biology, Masaryk University, 61137 Brno, Czech Republic
² Department of Cytokinetics, Institute of Biophysics ASCR, 61265 Brno, Czech Republic
³ Department of Psychiatry and Human Behavior, University of California, Irvine, CA 92697, USA
⁴ Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
⁵ Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
⁶ Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
⁷ Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA

View larger version (58K): [in this window] [in a new window]   Fig. 1. FGFR3 interacts with STAT1 and STAT3. FLAG-tagged wt, FGFR3-K650E or FGFR3-K508M were expressed in HeLa cells (A) or RCS chondrocytes (B) and the whole cell lysates were subjected to WB for indicated molecules (left panel). Actin serves as a loading control. The same lysates were subjected to STAT1 (middle panel) and STAT3 (right panel) immunoprecipitation (IP) followed by WB. Please note that the transfected FGFR3-FLAG was detected by FLAG antibody in A and by FGFR3 antibody in B. (C) FLAG-tagged wt or FGFR3-K650E, immunoprecipitated from CHO cells, or recombinant tyrosine kinase (TK) domain of FGFR3 were subjected to an in vitro kinase assay with recombinant STAT1 as a substrate. The level of STAT1 phosphorylation was determined by WB with antibody recognizing STAT1 only when phosphorylated at Y701. Samples including the FGFR inhibitor SU5402 (20 µM) or those with ATP omitted serve as negative controls for the kinase reaction. The levels of total STAT1 and FGFR3 serve as controls for the substrate and kinase quantity, respectively. Note that the FGFR3 antibody was raised against the extracellular domain of FGFR3 and thus cannot recognize FGFR3-TK. (D) The kinase assay was carried out as described above, using FGFR3-TK as a kinase, and recombinant STAT1 and/or recombinant FRS2 as substrates. The level of STAT1 phosphorylation was determined as described above, whereas FRS2 tyrosine phosphorylation was determined by WB with the 4G10 phosphotyrosine antibody.

View larger version (59K): [in this window] [in a new window]   Fig. 2. FGF2 treatment leads to serine but not tyrosine phosphorylation of STAT in RCS chondrocytes. (A,B) RCS chondrocytes were serum-starved for 12 hours, treated with FGF2, IL6 or IFN for the indicated times, and analyzed for ERK1/2, STAT1, STAT3 and STAT5 by WB with antibody recognizing the given molecule only when phosphorylated at a specific site. The arrow indicates Y694-phosphorylated STAT5. The same membrane was re-probed with antibody recognizing the given molecule regardless of its phosphorylation. (C) RCS chondrocytes were serum-starved for 12 hours, treated with FGF2 (100 ng/ml) in the presence of heparin (1 µg/ml) for the indicated times, and analyzed for STAT3 by WB with antibody recognizing STAT3 only when phosphorylated at S727. The appearance of S727-phosphorylated STAT3, triggered by FGF2, is indicated by an arrow. The lower band appears to be a crossreactivity of the antibody as indicated by the position of the total STAT3 detected on the re-probed membrane.

View larger version (56K): [in this window] [in a new window]   Fig. 3. FGF2 treatment does not trigger nuclear accumulation of STAT1-GFP or STAT3-YFP in RCS chondrocytes. (A,B) RCS chondrocytes were transfected with STAT1-GFP or STAT3-YFP vectors. Forty-eight hours after transfection, the cells were harvested and analyzed for STAT1 and STAT3 by WB. Note the amount of STAT fusion proteins in transfected cells (lane 2) in comparison to untransfected controls (lane 1). (C,D) RCS chondrocytes were transfected with STAT1-GFP (C) or STAT3-YFP (D), grown for 48 hours and treated with FGF2, IL6 and IFN for the indicated times, fixed and analyzed for STAT subcellular distribution by confocal microscopy. The DAPI staining indicates the cell nucleus. Note that FGF2 did not influence the distribution of either protein in contrast to positive controls (IFN- or IL6-treated cells) that show nuclear accumulation of all the STAT1-GFP or STAT3-YFP. Also note the incomplete IL6-mediated nuclear translocation of STAT3-YFP in cells pre-treated with FGF2 for 48 hours, suggesting an interference with canonical STAT3 signaling by the FGF2 stimulus. Scale bar: 10 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 4. FGF2 does not trigger STAT transcriptional activity. (A) Growing RCS chondrocytes were treated with FGF2 for the indicated times and cell nuclei were isolated and analyzed for active STAT1 (upper graph) or STAT3 (lower graph) by STAT ELISA-based EMSA assay as described in the Materials and Methods. STAT activation was quantified by spectrophotometry. Note that FGF2 did not significantly elevate active nuclear STAT1 or STAT3. Rather, a diminution of basal active nuclear STATs appeared with prolonged FGF2 treatment. The data represent an average from two samples with the indicated range. The dashed line indicates basal STAT nuclear activity. Cells treated for 30 minutes with IFN or IL6 serve as positive controls for STAT activation. (B) RCS chondrocytes were transfected with three different STAT firefly luciferase (F-Luc) reporter vectors and a control Renilla luciferase (R-Luc) vector, stimulated with FGF2, IFN or IL6 for 72 hours and analyzed for luciferase activity as described in the Material and Methods. Data represent an average from four wells with indicated standard deviation. Statistically significant differences relative to control are indicated (Student's t-test; **P<0.01).

View larger version (43K): [in this window] [in a new window]   Fig. 5. Addition of active STATs does not sensitize RCS chondrocytes to FGF2-mediated growth arrest. (A) RCS chondrocytes were treated with FGF2, IL6 and IFN for 30 minutes and analyzed for activatory phosphorylation of STAT1, STAT3 and ERK1/2 by WB. The levels of total STAT1, STAT3 and ERK1/2 serve as loading controls. (B) RCS chondrocytes were treated with FGF2, IL6 and IFN for 72 hours and counted. The data represent an average from at least three wells with the indicated standard deviation. The arrow indicates maximal growth arrest in cells treated with 20 ng/ml of FGF2. Note that all FGF2 treatments led to statistically significant growth inhibition (Student's t-test; P<0.01; results not shown on the graph) relative to untreated control, and that IL6 and/or IFN co-treatment with FGF2 led to statistically significant differences relative to FGF2 alone (Student's t-test; *P<0.05, **P<0.01). (C) RCS chondrocytes were transfected with vector carrying constitutively activated SRC kinase (ca-SRC), grown for 24 hours and analyzed for the indicated molecules by WB. (D) RCS chondrocytes were transfected with empty vector or vector carrying ca-SRC together with STAT firefly luciferase (F-Luc) reporter vector (pTATA-TK-Luc-4xM67) and a control Renilla luciferase (R-Luc) vector (pRL-TK), grown for 96 hours and analyzed for luciferase activity as described in the Material and Methods. The data represent an average of four wells with indicated standard deviation. Statistically significant differences relative to control are indicated (Student's t-test; *P<0.05, **P<0.01). (E) RCS chondrocytes were transfected with vector carrying ca-SRC, treated with FGF2 for 72 hours and counted. Note that all FGF2 treatments led to statistically significant growth inhibition (Student's t-test; P<0.01; not shown on the graph) relative to untreated controls, but overexpression of ca-SRC did not alter the FGF2-mediated growth arrest. The data represent an average from three wells with the indicated standard deviation. (F,G,H) RCS chondrocytes were transfected, analyzed for transgene expression, luciferase activity and FGF2-mediated growth arrest as described above except that vectors expressing wt or constitutively active STAT3 (ca-STAT3) were used instead of ca-SRC. Note that all FGF2 treatments led to statistically significant growth inhibition (Student's t-test; P<0.01; not shown on the graph) relative to untreated controls, but overexpression of wt or ca-STAT3 did not alter the FGF2-mediated growth arrest.

View larger version (25K): [in this window] [in a new window]   Fig. 6. RNAi-mediated downregulation of STAT1 and STAT3 does not rescue the FGF-mediated RCS growth arrest. (A,B) RCS chondrocytes were subjected to ERK1/2, STAT1 and STAT3 RNAi as described in the Materials and Methods. 24 hours later, the cells treated with specific siRNA (arrow) were either analyzed for ERK1/2, STAT1, STAT3 and actin levels by WB (A), or treated with FGF2 for 72 hours and counted (B). The data represent an average from three wells with the indicated standard deviation. Note that only ERK1/2 downregulation lead to statistically significant rescue (Kruskal-Wallis test, *P<0.05) of the FGF2-mediated growth arrest.

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