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First published online 15 January 2008
doi: 10.1242/jcs.017160
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Research Article |
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
* Author for correspondence (e-mail: krejcip{at}sci.muni.cz)
Accepted 30 October 2007
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Summary |
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Key words: FGFR3, STAT, Cartilage, Chondrocyte, Fibroblast growth factor, Growth arrest
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Introduction |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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). Probably the best documented effect of FGFR3 signaling in cartilage is inhibition of chondrocyte proliferation, although the molecular mechanism of this phenotype is only beginning to emerge.
Several lines of evidence suggest that members of the signal transducers and activators of transcription (STAT) family of transcription factors may participate in the growth-inhibitory feature of FGFR3 signaling in chondrocytes. First, activation of FGFR3 by the TD K650E mutation or FGF treatment, has been reported to result in phosphorylation and nuclear localization of STAT1 in several non-chondrocyte cell models as well as in RCS chondrocytes or human primary chondrocytes, which is accompanied by induction of the p21Waf1 (also known as CDKN1A) inhibitor of cell cycle and growth inhibition in 293T and RCS cells (Su et al., 1997; Lievens and Liboi, 2003; Nowroozi et al., 2005; Sahni et al., 1999; Legeai-Mallet et al., 1998). Second, in the cartilage of ACH- and TD-affected human fetuses as well as in mice carrying the ACH (G369C) or TD (K644E) mutations in FGFR3, STAT1 and STAT5 accumulate and show nuclear localization, suggesting their activation (Legeai-Mallet et al., 1998; Legeai-Mallet et al., 2004; Chen et al., 1999; Li et al., 1999). Third, in two experimental studies, the loss of STAT1 partially rescued the growth-inhibitory action of FGF signaling in both in vitro and in vivo chondrocyte environments (Sahni et al., 1999; Sahni et al., 2001).
Since STAT1-mediated induction of p21Waf1 represents the mechanism of cell growth inhibition by 
-interferon (IFN) (Chin et al., 1996; Bromberg et al., 1996), it is believed that this mechanism also occurs with FGFR3-mediated growth inhibition of cartilage (Sahni et al., 1999; Legeai-Mallet et al., 2004). There are, however, several concerns regarding this model. First, since the loss of FGFR3 leads to significant skeletal overgrowth by increased chondrocyte proliferation (Deng et al., 1996), the loss of Stat1 should resemble the phenotype of Fgfr3-null mice, yet this is not the case (Durbin et al., 1996). Second, in primary chondrocytes isolated from Stat1-null mice, RCS chondrocytes and PC12 cells, ERK and p38 MAP kinases appear to be candidates for FGF-mediated induction of p21Waf1 and growth inhibition (Murakami et al., 2004; Raucci et al., 2004; Krejci et al., 2004; Nowroozi et al., 2005). Third, crossing of Stat1-null mice with those carrying the ACH mutation in FGFR3 did not rescue the ACH phenotype although increased chondrocyte proliferation was observed (Murakami et al., 2004).
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; Sahni et al., 1999; Nowroozi et al., 2005), but the nature of the STAT-kinase involved is unknown. FGFR3 itself is a tyrosine kinase and can recruit SRC kinase (a known STAT activator) (Klint et al., 1999; Cao et al., 1996), but there is no evidence demonstrating direct FGFR3- or SRC-mediated tyrosine phosphorylation of STAT in chondrocytes. This study was undertaken to extend our knowledge regarding yet unclear aspects of STAT signaling in FGFR3 signaling in a chondrocyte environment. |
Results |
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Second, we used a cell-free kinase assay to test whether FGFR3 can directly phosphorylate STAT1 and STAT3 on their activatory tyrosines 701 and 705, respectively. Wild-type or FGFR3-K650E were expressed in CHO cells, immunoprecipitated and subjected to a kinase assay (Krejci et al., 2007), using recombinant STAT1 or STAT3 as a substrate. Fig. 1C shows that FGFR3-wt phosphorylates STAT1 on tyrosine 701 weakly, in comparison to FGFR3-K650E, which causes strong STAT1(Y701) phosphorylation; no phosphorylation of STAT3(Y705) was found (data not shown). When the recombinant, kinase-active intracellular domain of FGFR3 was used as a kinase, STAT1 was phosphorylated to a similar extent as with FGFR3-K650E, suggesting that the cell-borne FGFR3 does not phosphorylate STAT1 via a co-immunoprecipitated intermediate. Next, we tested whether FGFR3 can phosphorylate STAT1 in the presence of its physiological substrate fibroblast growth factor receptor substrate 2 (FRS2). Fig. 1D shows that STAT1(Y701) phosphorylation does occur in the presence of recombinant FRS2, albeit to a lesser extent.
FGF signaling does not activate STATs in RCS chondrocytes
For the following experiments we used RCS chondrocytes, a FGFR3-expressing chondrocytic cell line that represents the best characterized cell model for FGFR3-related skeletal dysplasias to date. RCS chondrocytes respond to FGFR3 activation with growth arrest similar to in vivo chondrocytes (Aikawa et al., 2001; Rozenblatt-Rosen et al., 2002; Dailey et al., 2003; Raucci et al., 2004; Krejci at al., 2005). First, we used WB with phosphorylation-specific antibodies to test whether FGFR3 activation, via FGF2 treatment, leads to STAT phosphorylation. Fig. 2A,B shows that RCS treatment with FGF2 did not lead to activatory tyrosine phosphorylation of STAT1(Y701), STAT3(Y705) or STAT5(Y694) in contrast to IFN or interleukin 6 (IL6) treatment that induced significant tyrosine phosphorylation of all three STATs. Similar results were found in cells treated with FGF2 together with heparin or with FGF1 (data not shown). Since FGF activates ERK1/2 in RCS cells (Fig. 2A), and MAP kinases are capable of phosphorylating STATs at a conserved P(M)SP motif located between residues 720 and 730 (Decker and Kovarik, 2000), we probed RCS cells for STAT1(S727) and STAT3(S727) phosphorylation. Fig. 2C shows that treatment with FGF2 leads to phosphorylation of STAT3(S727); no phosphorylation of STAT1(S727) was found (data not shown).
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Detection of activated STATs by WB can yield false-negative results due to a limited sensitivity of the method. We therefore used more sensitive ways to look for the STAT activation in FGF2-treated RCS chondrocytes. First, since the nuclear accumulation of the STATs represent a marker of their activation, we expressed STAT1 or STAT3 tagged with green or yellow fluorescent protein (GFP or YFP, respectively) in RCS cells and examined the influence of FGF2 on subcellular localization of both fusion proteins by confocal microscopy. The bio-activity of both STAT fusion proteins has been extensively examined and found not to be altered by the tag (Köstner et al., 1999; Herrmann et al., 2003). In untreated cells, STAT1-GFP and STAT3-YFP were found equally distributed between the cell nucleus and cytoplasm (Fig. 3C,D). Since RCS cells used for STAT imaging were grown in the presence of 10% fetal bovine serum, the presence of nuclear STATs may reflect basal activation by endogenous or serum-borne cytokines. Alternatively, the STATs may have a nuclear presence because of constant circulation of inactive STATs between the nucleus and cytoplasm, according to models proposed recently by Reich and Liu (Reich and Liu, 2006). In RCS cells treated with positive controls for STAT activation, i.e. with IFN or IL6 for 30 minutes, we observed rapid nuclear accumulation of the entire cytoplasmic complement of STAT1 or STAT3, respectively. By contrast, both short-term (10, 30 minutes, 1, 2 and 4 hours) and long-term (8, 24 and 48 hours) FGF2 treatment did not significantly affect the subcellular distribution of either STAT (Fig. 3C,D and data not shown). We also tested whether long-term FGF2 treatment affects the ability of IFN and IL6 to cause a nuclear translocation of STAT1 or STAT3, respectively. Fig. 3D shows that IL6-mediated nuclear translocation of STAT3 appears inhibited by a 48-hour pre-treatment of cells with FGF2. Nuclear translocation of STAT1 induced by 30-minute treatment with IFN was not affected by FGF2 (data not shown).
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; Besser et al., 1999), together with an internal control reporter encoding Renilla reniformis luciferase driven by the thymidine-kinase promoter. Fig. 4B shows that FGF2 did not activate any of the three STAT reporters used. Interestingly, we observed a slight decrease in the basal luciferase activity of two out of three reporters in cells treated with FGF2 for 72 hours, again suggesting an inhibition of endogenous STAT activity by chronic FGF2 treatment.
Addition of active STATs to FGF signal does not enhance FGF2-mediated growth arrest
Although the FGF-FGFR3 signaling does not appear to activate STAT1 or STAT3 in RCS chondrocytes (Figs 2, 3, 4), both STATs are present and thus may contribute to FGF2-mediated growth arrest when activated. We tested this hypothesis by determining whether addition of active STAT1 or STAT3 sensitizes RCS cells to FGF-mediated growth arrest. First, we added active STATs to the FGF signaling via co-stimulation of RCS cells with IFN or IL6. Cells were grown in presence of FGF2, IFN and/or IL6 for 72 hours and counted to test if IFN and/or IL6 contribute to the FGF2-mediated growth arrest. Despite clear STAT activation in cells treated with IFN and/or IL6, there was no contribution from either cytokine to FGF2-mediated growth arrest (Fig. 5A,B).
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Next, we transfected RCS cells with a constitutively active SRC kinase mutant (Y529F; ca-SRC), that is known to activate STAT3 via phosphorylation at Y705 (Shi and Kehrl, 2004). As evidenced by a STAT luciferase reporter assay, ca-SRC-expressing cells show significant amounts of STAT activation, although they respond to FGF2 treatment identically to untransfected cells (Fig. 5C,D,E). Finally, we expressed a constitutively active STAT3 mutant (A662C/N664C; ca-STAT3) in RCS chondrocytes and determined its contribution to FGF2-mediated growth arrest. Fig. 5G shows significant activation of the STAT luciferase reporter in cells expressing ca-STAT3. However, this activation does not sensitize RCS chondrocytes to FGF2-mediated growth arrest (Fig. 5G,H). Similar experiments could not be carried out with wt and ca-STAT1 since we were unable to express STAT1 in sufficient quantities in RCS chondrocytes (data not shown).
siRNA-mediated downregulation of STAT1 or STAT3 does not rescue FGF-mediated growth arrest
Taken together, activation of STAT1 or STAT3 does not contribute to FGF2-mediated growth arrest in RCS chondrocytes (Fig. 5). Since it has been suggested that STATs function in FGF-mediated chondrocyte growth arrest independent of their activation (Raucci et al., 2004), we asked whether downregulation of STAT1 or STAT3, via RNA interference, rescues the FGF2-mediated growth arrest of RCS chondrocytes. Cells were transfected with siRNAs directed against STAT1, STAT3 or ERK1/2 and analyzed for target downregulation as well as for siRNA specificity 24 hours later. At an 80 nM siRNA scale, a significant silencing of all three targets was observed (Fig. 6A), although the transfection with siRNA targeting ERK1/2 also led to a slight STAT1 downregulation. Next, cells were transfected with siRNAs targeting STAT1, STAT3 or ERK1/2, grown for 24 hours and probed for their response to FGF2 in a 72-hour-long growth experiment. Fig. 6B shows that untransfected RCS cells responded to various levels of FGF2 with the usual amount of growth arrest. This phenotype did not appear to be significantly rescued by STAT1 or STAT3 downregulation (Fig. 6B). By contrast, downregulation of ERK1/2 led to at least partial reversal of the growth arrest with all FGF2 concentrations used.
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; Sahni et al., 1999; Legeai-Mallet et al., 1998; Legeai-Mallet et al., 2004; Chen et al., 1999; Li et al., 1999), the molecular mechanism of this role remains obscure. In this study, we attempted to characterize some of the unclear aspects of STAT participation in FGF signaling, namely the mechanism of FGF-mediated STAT activation and the contribution of STATs to FGF-mediated chondrocyte growth inhibition.
STAT1 activation by FGFR3
Activation of FGFR3 has been reported to lead to tyrosine phosphorylation of STAT1, STAT3 or STAT5, depending on the cell system (Su et al., 1997; Sahni et al., 1999; Nowroozi et al., 2005), but the nature of a STAT-kinase involved in this process is unknown. Since FGFR3 itself is a tyrosine kinase, we asked whether it can directly phosphorylate STAT1 or STAT3. We found that FGFR3 is capable of phosphorylating STAT1(Y701) in a cell-free kinase assay (Fig. 1). Although one cannot rule out that this phosphorylation is an artifact of the method where large quantities of both kinase and substrate are present, several findings suggest that direct FGFR3-mediated phosphorylation of STAT1(Y701) is physiologically relevant. First, FGFR3 did not phosphorylate STAT3(Y705) despite the similarity in the motif sequences (GYIKT for STAT1 versus PYLKT for STAT3) that are both recognized by known STAT kinases (Kisseleva et al., 2002). Second, the difference in magnitude of STAT1 phosphorylation observed here between wt and FGFR3-K650E corresponds to the difference in 293T cells, where FGFR3-K650E caused 20 times stronger STAT1 phosphorylation than wt FGFR3 (Su et al., 1997). Third, FGFR3 phosphorylated STAT1 in the presence of its physiological substrate FRS2 (Fig. 1).
We conclude that FGFR3 can potentially function as a STAT1 kinase in chondrocytes, directly activating STAT1 through its phosphorylation at Y701. In a kinase assay, FGFR3 equally phosphorylated both STAT1 and FRS2 (Fig. 1D). In contrast to normal cells, TD chondrocytes may have different substrate selectivities since FGFR3-K650E is active in the endoplasmic reticulum unlike wt FGFR3, which is active at the cell membrane (Lievens and Liboi, 2003; Raffioni et al., 1998). It is thus likely that cytoplasmic STAT1 is phosphorylated predominantly by FGFR3-K650E whereas membrane-anchored FRS2 is a preferred substrate for wt FGFR3. In RCS chondrocytes, the immature FGFR3 forms immunoprecipitated preferentially with STAT1, supporting this hypothesis (Fig. 1B). In contrast to STAT1, we found that FGFR3 does not phosphorylate STAT3 in a kinase assay. This implies that STAT3 activation by FGFR3 signaling, when it occurs, involves intermediates, such as JAK2 and SRC kinases, recently shown to activate STAT3 in response to FGF in endothelial cells (Deo et al., 2002).
In further support of physiological interaction between STATs and FGFR3, we found that FGFR3 co-immunoprecipitates with both STAT1 and STAT3 from HeLa cells and RCS chondrocytes (Fig. 1A,B). The strength of the STAT-FGFR3 interaction correlated positively with the amounts of FGFR3 activation. Surprisingly, however, we found that a significant amount of both STATs associate with the FGFR3-K508M mutant suggesting that FGFR3 activation (i.e. autophosphorylation) is not a prerequisite for STAT binding (Fig. 1), but may facilitate binding. Although we cannot rule out the possibility that FGFR3-K508M is heterophosphorylated by endogenous FGFRs, our data suggest that FGFR3 pre-assembles with STAT in FGF-naive cells.
FGF treatment of RCS chondrocytes leads to potent activation of ERK1/2 (Krejci et al., 2004; Raucci et al., 2004) (Fig. 2A), which can phosphorylate STAT at a conserved P(M)SP motif located between residues 720 and 730 (Decker and Kovarik, 2000). We found that such phosphorylation of STAT3(S727) occurred in FGF2-treated RCS cells (Fig. 2C). Although we did not detect similar phosphorylation of STAT1, probably because of the low level of STAT1 in RCS cells, it is probable that STAT1(S727) is also phosphorylated by ERK1/2 in RCS cells (Decker and Kovarik, 2000). Serine 727 phosphorylation of STAT3 is necessary for its full transcription factor activity, depending on the promoter and/or cell type (Wen et al., 1995; Kim and Baumann, 1997). The functional significance of STAT3(S727) phosphorylation in FGF signaling in RCS chondrocytes is, however, unclear since it occurs without concomitant activatory phosphorylation at Y705.
Contribution of STATs to FGF-mediated growth arrest in the RCS chondrocytes
Although murine models have played an important role in delineating the features of FGFR3-related skeletal dysplasias, unraveling the precise molecular mechanisms of aberrant FGF signaling in the chondrocyte environment requires an in vitro experimental model. Although human primary chondrocytes can easily be obtained from the long bone epiphyses of TD fetuses, such cells rapidly lose their differentiated, cartilage-like phenotype upon in vitro cultivation, including their growth inhibitory response to a FGF stimulus. As early as 72 hours in culture, human primary chondrocytes begin responding to FGF treatment by proliferating, which is typical for undifferentiated mesenchymal cells but opposite to the outcome of FGF signaling in cartilage (P.K. and W.R.W., unpublished) (Legeai-Mallet et al., 1998). This rapid loss of cartilage-like properties of FGF signaling renders cultured human chondrocytes impractical for experimental studies addressing the mechanisms of chondrocyte-specific FGF signaling. Although several other cell models have been used to explore the mechanism of FGF signaling in a chondrocyte environment (Henderson et al., 2000; Yamanaka et al., 2003), RCS chondrocytes represent the best studied chondrocyte cell model to date. Using RCS chondrocytes, several essential features of FGF signaling in chondrocytes have been elucidated, including the mechanisms of FGF-mediated chondrocyte growth arrest, cytoskeletal alterations, loss of chondrocyte extracellular matrix, mechanisms of FGF and C-natriuretic peptide signaling crosstalk and others (Aikawa et al., 2001; Ben-Zvi et al., 2006; Rosenblatt-Rosen et al., 2002; Dailey et al., 2003; Raucci et al., 2004; Krejci et al., 2004; Krejci et al., 2005; Krejci et al., 2007; Priore et al., 2006). Since they respond to FGF treatment with potent growth arrest that is accompanied by the activation of STAT1 (Sahni et al., 1999), we used RCS chondrocytes in this study.
In contrast to previously published data (Sahni et al., 1999; Ben-Zvi et al., 2006), we were unable to detect STAT1 activation following FGF treatment of RCS chondrocytes using four different experimental approaches, i.e. WB with the STAT phosphorylation-dependent antibodies, imaging of nuclear translocation of active STAT, STAT ELISA-based EMSA assay and STAT luciferase reporter assay (Figs 2, 3, 4). We presently do not know the reason for this difference.
In this study, we systematically evaluated the requirement of both STAT1 and STAT3 for the FGF-FGFR3-mediated growth inhibition of RCS chondrocytes. As discussed above, we were unable to detect STAT1 and STAT3 activation by FGF despite combining several different approaches (Figs 2, 3, 4). Next, we determined whether addition of active STAT1 or STAT3 to the FGF signal, by means of cytokine treatment, SRC-mediated STAT activation, or expression of a constitutively active STAT mutant could sensitize RCS chondrocytes to the FGF-mediated growth arrest and found that none of the experimental interventions had an effect (Fig. 5). Finally, we tested whether STAT1 and STAT3 contribute to the FGF-mediated growth arrest independently of their activation, as proposed earlier (Raucci et al., 2004), using siRNA-mediated acute knockdown. Although the information value of the RNAi experiment is diminished because of the slight downregulation of STAT1 by siRNA targeting ERK1/2, we found no contribution of STAT1 or STAT3 downregulation to the FGF-mediated growth arrest in RCS chondrocytes (Fig. 6). Altogether, we did not find a direct contribution of either STAT1 or STAT3 to the growth inhibitory signaling of FGF-FGFR3 in RCS chondrocytes. Our data thus confirm that the ERK1/2 arm, but not the STAT arm of FGF signaling is directly responsible for the growth arrest phenotype in chondrocytes, as demonstrated earlier (Raucci et al., 2004; Krejci et al., 2004). Our findings cannot entirely reflect what is occurring in the cartilage growth plate, where several signaling systems influence chondrocyte proliferation in a complex spatiotemporal relationship. Also, since RCS chondrocytes express wt FGFR3, our findings might not reflect the signaling of mutated FGFR3, particularly that of FGFR3-K650E, which is a strong STAT1 activator (Su et al., 1997) (Fig. 1). Lastly, STATs may contribute to the FGF signaling in cartilage by acting in FGF-regulated processes not directly related to proliferation.
While attempting to detect FGF-mediated activation of STATs, we surprisingly found that prolonged FGF treatment inhibits STAT1 and STAT3 activation by IFN or IL6, respectively. This inhibition was evident using several approaches to detect STAT activation (Figs 3, 4), suggesting that chronic FGF stimulus interferes with cytokine-induced STAT activation in RCS chondrocytes. Some cytokine-STAT signaling systems, such as the IL6 family of cytokines that signals via the gp130 receptor, represent important positive regulators of endochondral growth (Sims et al., 2004). Our data opens the possibility that FGF signaling exerts some of its effects on cartilage through interfering with canonical STAT pathways. This hypothesis is currently under investigation.
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Signal transduction studies, FGFR3 kinase assays and STAT ELISA-based EMSA assay
Cells were serum starved for 12 hours before treatment with 10 ng/ml FGF2, 20 ng/ml IL6 (R&D Systems, Minneapolis, MN) and 40 ng/ml IFN (Calbiochem, San Diego, CA). When heparin (Gibco-BRL) was used, the concentration was 1 µg/ml. The FGFR3 kinase assays were carried out as described before (Krejci et al., 2007). Briefly, the kinase reactions were performed in 50 µl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na3VO4, 1.2 mM DTT) supplemented with 2.5 µg PEG, 10 µM ATP and recombinant human STAT1 (500 ng), STAT3 (250 ng; Active Motif, Carlsbad, CA), or FRS2 (500 ng; Abnova, Taipei City, Taiwan) as a substrate. The recombinant FGFR3 intracellular domain (E322-T806; Cell Signaling) was used at 300 ng per reaction. To obtain C-terminally FLAG-tagged human wt FGFR3 or FGFR3-K650E, vectors expressing both kinases were transfected into CHO cells and the kinases were purified by immunoprecipitation with FLAG antibody as described previously (Krejci et al., 2007). Immunocomplexes were washed with kinase buffer and subjected to the kinase assay. SU5402 was obtained from Calbiochem. The solutions used for STAT and FGFR3 co-immunoprecipitation experiments have been described elsewhere (Bonofiglio et al., 2005). STATs were immunoprecipitated from 500 µg of total HeLa lysate using 2 µg of STAT1 or STAT3 antibody (Santa Cruz). For STAT ELISA-based EMSA assay, growing RCS cultures were treated as desired and cell nuclei were isolated using a Nuclear Extract Kit (Active Motif) as directed by the manufacturer. The ELISA-based EMSA assay (TransAM Stat Assay kit; Active Motif) was used to quantify the amount of active STAT1 and STAT3 in nuclear extracts. Briefly, the active STATs were purified from a nuclear lysate upon binding an immobilized oligonucleotide containing a 5'-TTCCCGGAA-3' motif, and detected by ELISA.
Vectors, cell transfection and RNA interference
Vectors carrying C-terminally FLAG-tagged human wt or FGFR3-K650E have been described elsewhere (Krejci et al., 2007). pRK7 vector containing C-terminally FLAG-tagged kinase-dead FGFR3 was created by subcloning an FGFR3 segment containing a kinase-inactivating K508M substitution from PFR3K508M (Raffioni et al., 1998) into the C-terminally FLAG-tagged human wt FGFR3. The vectors containing STAT1 or STAT3 C-terminally fused to GFP or YFP (Köstner and Hauser, 1999; Herrmann et al., 2003) were obtained from H. Hauser and G. Müller-Newen, respectively. Vectors expressing wt and constitutively active (ca-)STAT1 (A656C/N658C) were obtained from D. Frank and T. Ouchi, respectively (Liddle et al., 2006; Sironi and Ouchi, 2004). Vectors containing C-terminally FLAG-tagged wt STAT3 or ca-STAT3 mutant (A662C/N664C) were obtained from Addgene (Cambridge, MA). Vector containing ca-SRC kinase mutant (pUSEamp-SRC-Y529F) was obtained from Upstate Biotechnology. The following vectors were used for STAT luciferase reporter assays: pZLuc-TK-3xLy6e, pTATA-TK-Luc-4xM67 (Addgene), pTAL-Luc-4xGas and pRL-TK (Promega, Madison, WI). Before transfection, the extracellular matrix was degraded by treatment with 0.3% bacterial collagenase (type II; Invitrogen, Carlsbad, CA). Cells were transfected with FuGENE6 (Roche Diagnostics, Penzberg, Germany) or Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's protocol.
RNA interference
Cells were transfected with siRNA using X-tremeGENE according to manufacturer's protocol (Roche). The siRNA transfection was carried out in 2 ml of transfection medium (Opti-MEM; Invitrogen) in six-well plates (Costar, Cambridge, MA). The siRNA concentration was 80 nM, the RNA/X-tremeGENE ratio (µg/µl) was 1:5 and the amount of transfected cells was 1x105. The medium was changed 4 hours after transfection and the cells were cultured for 24 hours and either harvested for WB or treated with FGF2 for 72 hours and counted. The siRNAs against ERK1/2, STAT1 and STAT3 were purchased from Dharmacon (Lafayette, CO).
STAT luciferase reporter assay
RCS chondrocytes were transfected with a plasmid containing ca-SRC or wt-STAT3 or ca-STAT3, and a STAT firefly luciferase reporter plasmid and a control Renilla reniformis luciferase plasmid in a 3:1.5:0.5 ratio using FuGENE6, according to the protocol described above. Total amount of plasmid DNA was 5 µg used to transfect 1x105 cells. When only the STAT reporter plasmid and the Renilla luciferase plasmid were used, the total amount of transfected DNA was 4 µg and the plasmid ratio was 3:1. Twenty-four hours after transfection, cells were treated with FGF2, IFN or IL6 for an additional 72 hours. The luciferase activity was determined using a Dual-Luciferase Reporter Assay kit (Promega).
Confocal microscopy
Cells were transfected with STAT1-GFP or STAT3-YFP as described above and grown for 24 hours, treated as desired, fixed with 4% paraformaldehyde and mounted in medium (Vectashield; Vector Laboratories, Burlingame, CA) containing DAPI for nuclear staining. Confocal fluorescence and two photon laser scanned images were taken on a Leica TCS-SP MP confocal and multiphoton inverted microscope (Heidelberg, Germany) equipped with an argon laser (488 nm blue excitation), a diode-pumped solid-state laser (561 nm yellow-green excitation), a helium-neon laser (633 nm) and a two photon laser setup consisting of a Spectra-Physics Millenia X 532 nm green diode pump laser and a Tsunami Ti-Sapphire picosecond pulsed infrared laser tuned at 768 nm for UV excitation. Nomarski differential interference contrast (DIC) images were scanned using the argon laser for excitation.
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