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. 2001 Jul 31;98(16):9014-9.
doi: 10.1073/pnas.161281298. Epub 2001 Jul 24.

Rac1 mediates STAT3 activation by autocrine IL-6

T R Faruqi  1 , D Gomez, X R Bustelo, D Bar-Sagi, N C Reich
Affiliations

Rac1 mediates STAT3 activation by autocrine IL-6

T R Faruqi et al. Proc Natl Acad Sci U S A. .

Abstract

The activity of the small GTPase, Rac1, plays a role in various cellular processes including cytoskeletal rearrangement, gene transcription, and malignant transformation. In this report constitutively active Rac1 (Rac V12) is shown to stimulate the activation of STAT3, a member of the family of signal transducers and activators of transcription (STATs). The activity of Rac1 leads to STAT3 translocation to the nucleus coincident with STAT3-dependent gene expression. The expression of Vav (Delta1-187), a constitutively active guanine nucleotide exchange factor for the Rho GTPases, or activated forms of Ras or Rho family members, leads to STAT3-specific activation. The activation of STAT3 requires tyrosine phosphorylation at residue 705, but is not dependent on phosphorylation of Ser-727. Our studies indicate that Rac1 induces STAT3 activation through an indirect mechanism that involves the autocrine production and action of IL-6, a known mediator of STAT3 response. Rac V12 expression results in the induction of the IL-6 and IL-6 receptor genes and neutralizing antibodies directed against the IL-6 receptor block Rac1-induced STAT3 activation. Furthermore, inhibition of the nuclear factor-kappaB activation or disruption of IL-6-mediated signaling through the expression of IkappaBalpha S32AS36A and suppressor of cytokine signaling 3, respectively, blocks Rac1-induced STAT3 activation. These findings elucidate a mechanism dependent on the induction of an autocrine IL-6 activation loop through which Rac1 mediates STAT3 activation establishing a link between oncogenic GTPase activity and Janus kinase/STAT signaling.

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Figures

Figure 1
Figure 1
Effect of H-Ras, K-Ras, Rac1, and Raf expression on the transcriptional activation of STAT3. Rat1 cells were cotransfected with (GAS)3-Luc, SV40-LacZ, and pCDNA3 (control) or STAT3 either alone (open bars) or with (A) H-Ras V12 and K-Ras V12 or (B) Rac V12 and Raf CAAX (solid bars). Cell lysates were prepared 48 h after transfection and used to measure luciferase and β-gal activity. The data presented are means ± SEM (n = 3) and represent one of three similar experiments.
Figure 2
Figure 2
Effect of Rho family members and oncogenic Vav (Δ1–187) expression on STAT activation. (A) Rat1 cells were cotransfected with (GAS)3-Luc, SV40-LacZ, pCDNA3 (control), and STATs 1, 2, 3, 5a, and 6 either alone (open bars) or with Rac V12 or oncogenic Vav (Δ1–187) (solid bars). (B) Rat1 cells were similarly cotransfected with either constitutively active forms of Rac1, RhoA, Cdc42, and RhoG alone (open bars) or with STAT3 (solid bars). Cell lysates were prepared 48 h after transfection and used to measure both luciferase activity and β-gal activity. The data presented are means ± SEM (n = 3) and represent one of four similar experiments.
Figure 3
Figure 3
Functional analysis of mutants of STAT3 and Rac1 on transcriptional activation. Rat1 cells were cotransfected with (GAS)3-Luc, SV40-LacZ, and pCDNA3 (control) alone (open bars); (A) with wild-type STAT3, STAT3 Y705F, or STAT3 S727A in the presence of Rac V12 (solid bars); or (B) with Rac V12, Rac V12L37, or Rac V12H40 in the presence of STAT3 (solid bars). Cell lysates were prepared 48 h after transfection and used to measure luciferase and β-gal activity. The data shown are means ± SEM and are representative of similar experiments.
Figure 4
Figure 4
Effect of Rac V12 expression on STAT3 activation and specific gene induction. (A) HeLa cells were either untransfected or transfected with either pCDNA3 (as a transfection control) or Rac V12. Total RNA was collected and examined by reverse transcription–PCR for IL-6, IL-6 receptor, and GAPDH mRNA expression. The ethidium bromide-stained 2% agarose gel shows the IL-6 and GAPDH cDNA fragments (Left) and IL-6 receptor and GAPDH cDNA fragments (Right) amplified from the same amounts of cDNA. (B) HeLa cells were transfected with STAT3-GFP alone or with Rac V12 and were incubated for 18 h in serum-free DMEM in the presence of neutralizing IL-6 receptor antibody (1 μg/ml). Cells either were left untreated (a, d, and e) or were treated with soluble IL-6 receptor protein (100 ng/ml) (to enhance IL-6 responsiveness) and IL-6 (20 ng/ml) for 20 min (b and c). Cells were fixed and examined by fluorescent microscopy. (C) HeLa cells and HT1080 cells were cotransfected with (Gas)3-Luc, SV40-LacZ, and either pCDNA3 (control) or STAT3 alone or with Rac V12 and placed in serum-free DMEM in the absence (open bars) or presence (solid bars) of neutralizing IL-6 receptor antibodies (1 μg/ml). Cell lysates were prepared 48 h after transfection and were used to measure both luciferase and β-gal activity. The data shown are means ± SEM and are representative of similar experiments.
Figure 5
Figure 5
Effects of blocking IL-6 or NF-κB signaling. (A) Rat1 cells were cotransfected as described for Fig. 4C with pCDNA3 (control), STAT3, or Rac V12 in the absence or presence of SOCS-3. Cells were either untreated (open bars) or treated with IL-6 (20 ng/ml) (solid bars) for 6 h before harvest. (B) Rat1 cells were cotransfected with (NF-κB)5-Luc, SV40-LacZ, along with either control pCDNA3 plasmid (open bar) or increasing amounts of plasmid DNA encoding Rac V12 (solid bars) and Rac V12L37 (shaded bars). Cell lysates were prepared 48 h after transfection and were used to measure both luciferase and β-gal activity. The data shown are means ± SEM and are representative of two to four similar experiments. (C) Rat1 cells were cotransfected with STAT3-GFP alone (a), or with Rac V12 in the absence (b) or presence of IκBα S32AS36A (c). Cells were placed in serum-free DMEM for 24 h, fixed, and examined by fluorescent microscopy.
Figure 6
Figure 6
Proposed model for Rac1-mediated STAT3 activation. We show that constitutively active small GTPases such as Rac1 stimulate the JAK/STAT3 pathway from an “inside-out” signaling pathway involving the induction of an autocrine IL-6 activation loop. The left side of the diagram is a simple illustration of an oncogenic mutation rendering constitutively active Ras or Rac1 (Ras*/Rac1*) signaling to one of its known downstream targets, the NF-κB transcription factor. NF-κB activation leads to its translocation to the nucleus, DNA binding, and subsequent induction of NF-κB-dependent genes such as IL-6. After IL-6 binds to its receptor, the JAKs are activated and in turn tyrosine phosphorylate STAT3 (-pY). Signal transduction by the JAK/STAT pathway for STAT3 is depicted on the right side of the diagram. Phosphorylated STAT3 molecules form dimers and translocate to the nucleus to bind to promoter elements of responsive genes. Hence, gain-of-function mutations that render small GTPases constitutively active mediate STAT3 activation through the induction of an autocrine IL-6 activation loop.
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