STAT3 activation in response to IL-6 is prolonged by the binding of IL-6 receptor to EGF receptor Yuxin Wanga,b, Anette H. H. van Boxel-Dezaireb, HyeonJoo Cheonb, Jinbo Yanga,b,1, and George R. Starkb,1 aSchool of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, People’s Republic of China; and bDepartment of Molecular Genetics, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195 Contributed by George R. Stark, August 28, 2013 (sent for review July 19, 2013) The activation of STAT3 by tyrosine phosphorylation, essential for (10), indicating the involvement of IL-6 in the growth and dif- normal development and for a normal inflammatory response to ferentiation of the preimplantation mouse embryo (11). One of invading pathogens, is kept in check by negative regulators. Abnor- the most notable activities of the IL-6 family of cytokines, from mal constitutive activation of STAT3, which contributes to the the viewpoint of embryonic development, is the expression of the pathology of cancer and to chronic inflammatory diseases such as IL-6 family member leukemia inhibitory factor (LIF) in the mu- rheumatoid arthritis, occurs when negative regulation is not fully rine uterine endometrial glands of pregnant mice, coincident with effective. SOCS3, the major negative regulator of STAT3, is induced the onset of blastocyst implantation (12, 13). Mice in which the by tyrosine-phosphorylated STAT3 and terminates STAT3 phosphor- IL-6 or LIF genes have been knocked out show much less severe ylation about 2 h after initial exposure of cells to members of the IL-6 defects than might have been anticipated from the widespread family of cytokines by binding cooperatively to the common receptor effects of these cytokines (14, 15), perhaps reflecting the shared subunit gp130 and JAKs 1 and 2. We show here that when the use of common gp130 signaling pathways by other gp130- epidermal growth factor receptor (EGFR) is present and active, STAT3 dependent cytokines. In addition, intriguingly, mice lacking SOCS3 is rephosphorylated about 4 h after exposure of cells to IL-6 or die between days 11 and 13 of gestation (16), showing the impor- oncostatin M and remains active for many hours. Newly synthesized tance of down-regulation of STAT3 activation in development. IL-6 drives association of the IL-6 receptor and gp130 with EGFR, The STAT transcription factors, and especially STAT3, play leading to EGFR-dependent rephosphorylation of STAT3, which is not vital roles in tumor initiation and progression (17, 18). Aberrant inhibited by the continued presence of SOCS3. This second wave of activation of STAT3 leads to the overexpression of oncogenes STAT3 activation supports sustained expression of a subset of IL-6- that drive proliferation and metastasis and inhibit apoptosis (19). induced proteins, several of which play important roles in in- As an activator of the JAK-STAT signaling pathway, IL-6 can act flammation and cancer, in which both IL-6 secretion and EGFR in both a proinflammatory and anti-inflammatory manner. De- levels are often elevated. regulation of IL-6 production can cause severe disease, such as rheumatoid arthritis, psoriasis, arteriosclerosis, and cancer (20, fter ligand-induced dimerization of the IL-6 receptor (IL-6R), 21). Through the JAK/STAT signaling pathway, IL-6 mediates Athe associated kinases JAK1 and JAK2 cross-phosphorylate the production of IL-17 and IL-10 while regulating TH17, which tyrosine residues of adjacent glycoprotein 130 (gp130) subunits of is important in autoimmunity (22). IL-6 elevation and the fl the complex. The SH2 domain of STAT3 binds to newly phos- chronic in ammation that follows are protumorigenic. IL-6 phorylated tyrosines, followed by the phosphorylation of Y705 of exerts powerful antiapoptotic functions through the induction of STAT3 (1). Two phosphorylated STAT3 monomers then dimerize B-cell lymphoma 2 (BCL-2), B-cell lymphoma-extra large (BCL- – and translocate to the nucleus, where they activate the transcription xL), and related inhibitors (23). Therefore, anti IL-6/IL-6R mono- of many downstream genes whose products mediate the diverse clonal antibodies are used to inhibit aberrant and prolonged IL-6 effects of STAT3 in development and disease (2). Suppressor of fi cytokine signaling 3 (SOCS3), the major negative regulator of IL-6- Signi cance IMMUNOLOGY dependent signaling, is highly induced by activated STAT3, strongly inhibiting further STAT3 phosphorylation and thus collaborating An important part of the response to invading pathogens is with additional negative regulatory mechanisms to prevent excessive activation of the transcription factor STAT3 in response to IL-6. activation of potentially deleterious gene expression (3). SOCS3 This normal response is terminated rapidly by the major neg- down-regulates the phosphorylation of STAT3 about 90 min after ative regulator suppressor of cytokine signaling 3 (SOCS3). an initial exposure to IL-6 (4), using two opposing surfaces to bind Abnormal prolonged activation of STAT3 contributes to the to JAK2 and gp130 simultaneously (5). However, even in the pathology of cancer and to chronic inflammatory diseases. We continued presence of SOCS3, STAT3 is rephosphorylated in re- show that prolonged activation of STAT3 results from associ- sponse to IL-6 after about 4 h through a mechanism that has been ation of the IL-6 receptor with the epidermal growth factor obscure until now. Rephosphorylation of STAT3 might occur be- receptor, which in turn activates STAT3 in a way that is not cause the interaction between SOCS3 and the IL-6 receptor is inhibited by SOCS3. Prolonged STAT3 activation supports somehow prevented or because continued STAT3 phosphor- sustained expression of a subset of proteins that play impor- ylation occurs through a mechanism that is immune to SOCS3. tant roles in inflammation and in cancer, where both IL-6 The epidermal growth factor receptor (EGFR) is well known to secretion and epidermal growth factor receptor levels are catalyze the tyrosine phosphorylation of STAT3 in response to EGF often elevated. (6), and the intrinsic kinase activity of the receptor, but not any of Author contributions: Y.W., A.H.H.v.B.-D., H.C., J.Y., and G.R.S. designed research; Y.W. the JAKs, is required for this reaction (7). On activation by EGF, performed research; Y.W., A.H.H.v.B.-D., H.C., J.Y., and G.R.S. analyzed data; and Y.W. the EGFR dimerizes to facilitate cross-phosphorylation of several and G.R.S. wrote the paper. tyrosine residues, including Y1068, the binding site for STAT3 (8). The authors declare no conflict of interest. Multiple tyrosine phosphorylations of EGFR initiate downstream Freely available online through the PNAS open access option. signaling cascades that facilitate proliferation, regeneration, and 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. tumorigenesis (9). cn. During the early phase of mouse development, IL-6 mRNA is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. differentially expressed and translated into the mature protein 1073/pnas.1315862110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1315862110 PNAS | October 15, 2013 | vol. 110 | no. 42 | 16975–16980 Downloaded by guest on October 5, 2021 signaling in the treatment of autoimmunity and cancer (21). Be- that factors that are newly synthesized and secreted mediate the cause EGFR activation can mediate both IL-6 production (24, 25) second wave. We used a cytokine array to identify such factors by and STAT3 activation, we investigated the role of EGFR in reg- analyzing media conditioned for 4 h after washout from cells ulating STAT3 activation in response to IL-6. treated with IL-6 for 30 min, finding that IL-6 is the dominant We confirm the observation of others (3, 26) that, after the induced factor (Fig. 2C). The supernatant media were also an- initial phosphorylation of STAT3 in response to IL-6 and sub- alyzed by ELISA to quantify IL-6 production. The lack of IL-6 in sequent inhibition by SOCS3, a second wave of activation leads to media collected 10 min after washout shows that the removal of the rephosphorylation of STAT3. We now find that rephosphor- the IL-6 added initially is efficient (Fig. 2D, Left). IL-6 was se- ylation requires new IL-6 synthesis and secretion, leading to ligand- creted by 1 h after washout and remained in the media at dependent association of IL-6R and EGFR. STAT3 phosphory- a similar level for at least 4 h (Fig. 2D, Left). An analysis by real- lation continues to be driven for many hours by this two-receptor time PCR showed that IL-6 mRNA was induced within the first complex, which is immune to inhibition by SOCS3, thus increasing 30 min of initial treatment with IL-6 and then declined (Fig. 2D, and prolonging the expression of many STAT3-dependent proteins Right). An antibody against human IL-6 totally ablated the sec- that are involved in tumorigenesis and immune regulation. ond wave of STAT3 activation in DLD1 cells, showing that no other secreted protein is responsible (Fig. 2E). The same phe- Results nomenon was observed in HepG2 cells (Fig. 2F). In summary, we IL-6 Signaling Is Prolonged in a Biphasic Pattern. To examine the show that treatment with IL-6 induces the synthesis and secre- second wave of STAT3 activation in detail, we treated DLD1 tion of more IL-6 and that this induction is necessary for the (colon cancer cells) with IL-6 for 30 min, followed by washout, second wave of STAT3 activation. and measured STAT3 phosphorylation as a function of time (Fig. 1 A and B). Phosphorylation lasts for many hours, with EGFR Is Required for the Second Wave of STAT3 Phosphorylation.