Stat3 Activation by Src Induces Specific Gene Regulation and Is Required for Cell Transformation

Construction of plasmids.

An annealed oligonucleotide corresponding to the −35 to +11 region (relative to the transcriptional start site at +1) of the herpes simplex virus thymidine kinase (TK) promoter was cloned between the KpnI and BglII sites of the basic luciferase reporter pLuc (pGL2; Promega) to construct pLucTK (see Fig. 1 for structures of constructs). The Stat3 reporter pLucTKS3 was constructed from pLucTK by inserting seven copies of an annealed oligonucleotide corresponding to a Stat3-specific binding site from the human C-reactive protein (CRP) gene (48), called the CRP acute-phase response element (cAPRE) (nucleotides −123 to −85), into the SmaI site upstream of the TK minimal promoter. Another reporter, pLucTKSIE, contains two copies of an annealed oligonucleotide corresponding to a high-affinity mutant of the c-fos sis-inducible element (hSIE [mutant m67]) (41) inserted into the SmaI site of pLucTK. To construct pLucCRP, the authentic CRP promoter (nucleotides −123 to +3) was excised from plasmid −123/+3CRP-CAT (48) (a generous gift from D. Samols) by BamHI-XhoI restriction digestion, blunt-ended with Klenow, and inserted into the SmaI site of pLuc. The human Stat3β expression vector pSG5hStat3β is driven by the simian virus 40 promoter contained in pSG5 (Stratagene) and has been previously described (5). The Stat3 expression vector pVRStat3 was constructed by excising the mouse Stat3 cDNA from pBSStat3 by EcoRI and DraIII digestion, blunt-ending with T4 DNA polymerase, and cloning into a vector driven by the cytomegalovirus immediate early gene promoter (2a, 50). The reporter pLucSRE, which contains a serum response element (SRE) in the context of the c-fos promoter driving luciferase expression, as well as the N17-Ras and NT-Raf vectors have been described previously (30, 46). Expression vectors for v-Src, pMvSrc (17), and oncogenically activated c-H-Ras have been described previously (28).

Cell culture and transfections.

NIH 3T3 fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% iron-supplemented bovine calf serum (BCS). Transfections were carried out by the standard calcium phosphate method (9). NIH 3T3 fibroblasts were seeded at 5 × 10⁵ cells/100-mm-diameter plate in DMEM plus 5% BCS at 18 to 24 h prior to transfection. Total DNA for transfections was typically 20 μg per plate, including 4 μg of luciferase reporter construct (pLucTK, pLucTKS3, pLucCRP, pLucTKSIE, or pLucSRE), 0.2 μg of β-galactosidase (β-Gal) internal control vector, and the amounts of expression vector indicated in Results. Transfection was terminated 15 h later by aspirating the medium, washing the cells with 1× phosphate-buffered saline (PBS), and adding fresh DMEM. Where gamma interferon (IFN-γ) was present, it was added 5 h prior to harvest of transfected cells.

Preparation of cytosolic and nuclear extracts.

For transient expression assays, cytosolic extracts were prepared from cells at 48 h posttransfection. Briefly, after two washes with 1× PBS and equilibration for 5 min with 0.5 ml of PBS–0.5 mM EDTA, cells were scraped off of the dishes and the cell pellet was obtained by centrifugation (4,500 × g, 2 min, 4°C). Cells were resuspended in 0.4 ml of low-salt HEPES buffer (10 mM HEPES [pH 7.8], 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol) for 15 min, lysed by the addition of 20 μl of 10% Nonidet P-40, and centrifuged (10,000 × g, 30 s, 4°C) to obtain the cytosolic supernatant, which was used for luciferase assays (Promega) with a luminometer and for β-Gal activity detection by colorimetric assay at A₅₇₀. As an internal control for transfection efficiency, results were normalized to β-Gal activity. For electrophoretic mobility shift assays (EMSA), nuclear extracts were prepared from transiently transfected NIH 3T3 cells and volumes containing equal amounts of total protein were incubated with ³²P-labeled hSIE oligonucleotide probe, as previously reported (47). Supershift assays were performed by using rabbit polyclonal antibodies against the C-terminal amino acid residues (750 to 769) of full-length Stat3 that are absent in Stat3β (Santa Cruz Biotechnology).

Western blot analysis.

Whole-cell lysates were prepared in boiling sodium dodecyl sulfate (SDS) sample buffer in order to extract total Stat3 proteins from the cytoplasm and nucleus. Equivalent amounts of total cellular protein were electrophoresed on an SDS–7.5% polyacrylamide gel and transferred to a nitrocellulose membrane. Probing of nitrocellulose membranes with primary antibodies and detection of horseradish peroxidase-conjugated secondary antibodies by enhanced chemiluminesence (Amersham) were performed as previously described (14, 47). Probes used were rabbit polyclonal antibodies against the Stat3 C-terminal amino acids that are specific for full-length Stat3 (Santa Cruz Biotechnology) or mouse monoclonal antibody against the Stat3 N-terminal amino acid residues (1 to 178) that recognizes both full-length Stat3 and Stat3β (Transduction Laboratories).

Focus formation assays.

NIH 3T3 fibroblasts were transfected as described above and then harvested by trypsinization at 48 h posttransfection. Cytosolic extracts were prepared from 25% of the transfected cells to measure β-Gal activity, which was used for determination of transfection efficiency. The remaining 75% of the cells were seeded into new dishes and fed 1× DMEM every 3 days. Foci were counted at 16 or 21 days posttransfection by using phase-contrast microscopy.

Stat3-mediated gene regulation is induced by Src.

We previously demonstrated that Stat3 is constitutively activated in Src-transformed fibroblasts, as measured by enhanced tyrosine phosphorylation and DNA-binding activity of Stat3 (47). To determine whether Stat3 activation by Src could lead to regulation of gene expression, we transiently transfected NIH 3T3 cells with v-Src expression vector and a reporter construct, pLucTKS3 (Fig. 1B), containing multimerized Stat3-specific binding sites inserted upstream of a TK minimal promoter. This Stat3-specific binding site, which is derived from the human CRP gene and is called cAPRE here, contains the core sequence TTCCCGAA (36, 48). Assays for luciferase reporter gene expression (Fig. 2A) show dose-dependent gene induction, up to 15-fold over basal levels, mediated through activation of endogenous cellular Stat3 by increasing amounts of transfected v-Src expression vector. To confirm that this gene induction is dependent on Stat3, we used an expression vector encoding the Stat3 splice variant, Stat3β (Fig. 1A) (5), to disrupt Stat3 signaling. Figure 2B shows that induction of pLucTKS3 by v-Src requires the presence of cAPRE and is reduced to basal levels by cotransfection with Stat3β expression vector. To further characterize the dominant negative properties of Stat3β, Fig. 3A shows that increasing amounts of transfected Stat3β vector results in dose-dependent inhibition of Src-induced Stat3 reporter expression. In contrast to Stat3β, cotransfection of an expression vector encoding full-length Stat3 results in increased levels of gene induction over that observed with v-Src alone (Fig. 3B). Together, these results establish that activation of Stat3 by v-Src leads to Stat3-mediated gene regulation.

Specificity of Stat3-mediated gene regulation.

An authentic promoter construct, pLucCRP (Fig. 1B), harboring cAPRE embedded in the natural context of the CRP gene’s promoter (48), was used to confirm the results obtained with the chimeric pLucTKS3 reporter. Figure 4A shows that v-Src induces expression from pLucCRP and that Stat3β functions as a dominant negative modulator of transcription from this promoter, in agreement with the results presented above. As a control, Fig. 4B shows that Stat3β overexpression has no effect on the ability of v-Src to induce another reporter construct, pLucSRE (Fig. 1B), containing the c-fos SRE that is dependent on Ras and Raf-1 signaling for activation (46). In similar experiments with 20 μg of Stat3β vector and 200 ng of v-Src vector, the same conditions used in subsequent focus formation assays (see below), there was no effect of Stat3β on the ability of Src to induce pLucSRE expression (data not shown). These results demonstrate that Stat3β acts through Stat3-binding sites and that Stat3β overexpression does not nonspecifically inhibit v-Src function or Stat3-independent signaling pathways leading to SRE induction.

To further characterize the specificity of Src-induced STAT signaling and Stat3β function, we used a reporter construct based on the c-fos SIE inserted upstream of the TK promoter. This reporter, pLucTKSIE (Fig. 1B), contains a high-affinity mutant of the SIE (hSIE) that binds both Stat1 and Stat3 (41, 47). Figure 5A shows that expression from pLucTKSIE was induced by v-Src, which activates Stat3 but not Stat1 (47), and to a lesser extent by IFN-γ, which activates Stat1 but not Stat3 (4, 47). In both cases, gene induction was blocked by expression of Stat3β, indicating that Stat3β can disrupt Stat1 and Stat3 signaling. For comparison of the specificity of the various reporter constructs for Stat1 and Stat3, pLucTKS3, pLucCRP, and pLucTKSIE were activated by either v-Src or IFN-γ. Results shown in Fig. 5B demonstrate that the pLucTKS3 and pLucCRP constructs, both of which harbor cAPRE, are induced specifically by v-Src and not by IFN-γ. This finding confirms that cAPRE responds only to Stat3 signaling and not to Stat1 signaling. Together, our results demonstrate that activation of endogenous cellular Stat3 signaling by v-Src leads to Stat3-specific induction of gene expression, which is disrupted in a dominant-negative manner by Stat3β.

Induction of Stat3 and Stat3β DNA-binding activity by Src.

Because Stat3β retains an intact DNA-binding domain as well as the SH2 domain and tyrosine required for dimerization, activation of Stat3β by v-Src is predicted to induce dimerization and DNA binding. To measure DNA-binding activities, we prepared nuclear extracts from transiently transfected NIH 3T3 cells and performed EMSA with the ³²P-labeled SIE probe that binds both Stat1 and Stat3 with high affinity. Consistent with earlier studies of fibroblasts stably transformed by v-Src (47), Fig. 6A (lanes 1, 2, and 11 to 15) shows that specific DNA-binding activity of endogenous Stat3, but not Stat1, is induced in an Src-dependent manner in transiently transfected NIH 3T3 cells. Moreover, the Src-induced levels of specific DNA-binding activity detected in transiently transfected cells overexpressing Stat3 or Stat3β are greater than 10-fold higher than that of cells containing only endogenous Stat3 (Fig. 6A, lanes 3 to 10). As reported earlier (5, 34), Stat3β appears to have a higher basal level of DNA-binding activity than does Stat3 in the absence of any external stimulus.

Previous studies of cells stimulated with IL-5 demonstrated that Stat3β can form homodimers, which migrate more slowly in EMSA than Stat3 homodimers, as well as heterodimers with Stat3, which exhibit an intermediate degree of migration (5). By supershift analysis with an antibody that recognizes a C-terminal epitope present in full-length Stat3 but not in Stat3β, we confirmed that activation of Stat3 or Stat3β by v-Src induces mostly homodimers when either one is overexpressed alone (Fig. 6B, lanes 1 to 10) and predominantly Stat3-Stat3β heterodimers when both proteins are overexpressed together (lanes 11 to 16). Combined with our results presented above, these data suggest that Stat3β disrupts Stat3-specific gene regulation by binding to Stat3 response elements as either a homodimer or a heterodimer and preventing transcriptional activation.

Cotransfection of Stat3β vector blocks Src transformation.

To investigate the role of Stat3 signaling in cell transformation, we tested the effect of Stat3β on transformation of NIH 3T3 cells by v-Src. As a sensitive and quantitative measure of cell transformation by v-Src, we used a focus formation assay, which in the case of v-Src correlates very well with growth in soft agar and tumorigenesis (18). Focus formation assays were performed by using cells transfected with expression vectors for v-Src alone or v-Src together with Stat3β (Fig. 7A). Results show that Stat3β consistently inhibited Src-induced focus formation by 50% with small amounts (2 μg) of Stat3β expression vector, with greater than 80% inhibition observed in cotransfections with larger amounts (20 μg) of Stat3β expression vector. As a control, cotransfection of empty vector alone did not significantly affect focus formation by v-Src, whereas expression of Stat3β alone resulted in levels of focus formation comparable to the background of spontaneous transformation. For comparison with v-Src, the effect of Stat3β overexpression on focus formation induced by the activated c-H-Ras oncoprotein, which does not activate Stat3 signaling (14), was also examined. Stat3β overexpression had either no effect (at 2 μg of vector) or much-less-pronounced effects (at 20 μg of vector) on Ras-induced transformation compared to Src-induced transformation (compare Fig. 7A and B). These results indicate that Stat3β inhibits transformation by Src to a significantly greater extent than it does transformation by Ras.

Cell lines stably overexpressing Stat3β are resistant to Src transformation.

To confirm the results obtained in the cotransfection experiments described above, and to exclude a possible toxic effect of Stat3β overexpression on cell viability, a different approach was taken with cell lines stably overexpressing Stat3β. Following transfection with Stat3β expression vector, G418-resistant colonies were selected, expanded, and further characterized. Western blot analysis with antibodies directed against the N-terminal portion of Stat3 identified three independent clones that stably overexpressed Stat3β compared to control NIH 3T3 cells (Fig. 8A). Transient transfection of these cells with pLucTKS3 reporter together with v-Src vector confirmed that Stat3-mediated signaling was abrogated by Stat3β overexpression, while Stat3-independent signaling leading to pLucSRE induction was not affected (Fig. 8B). In agreement with the results presented in Fig. 2 to 4, these results indicate that Stat3β specifically disrupts Stat3 signaling without impairing v-Src function or other signaling pathways. These findings further demonstrate that cells which stably overexpress Stat3β and are deficient in Stat3-mediated signaling remain viable and proliferate. To assess whether these Stat3β overexpressing clones could be transformed by Src, focus formation assays were performed following transfection with v-Src expression vector. Results presented in Fig. 9 show that all three Stat3β-overexpressing clones were refractory to Src-induced cell transformation, with up to 90% inhibition of focus formation. Together, the data shown in Fig. 7 to 9 demonstrate that overexpression of Stat3β blocks cell transformation by v-Src, presumably by disrupting Stat3-dependent regulation of the cellular gene expression that is required for v-Src transformation.