Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells Takeshi Senga 1 , Takashi Iwamoto 2 , Sean E Humphrey 4 , Takashi Yokota 3 , Elizabeth J Taparowsky 4 and Michinari Hamaguchi* ,1 1 Department of Molecular Pathogenesis, Nagoya University School of Medicine, 65 Tsurumai Showa Nagoya 466-8550, Japan; 2 Radioisotope Research Center Medical Division, Nagoya University School of Medicine, 65 Tsurumai Showa Nagoya 466-8550, Japan; 3 Division of Stem Cell Biology, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8640, Japan; 4 Department of Biological Sciences, Purdue University, West Lafayette, Indiana, IN 47907-1392, USA Stat3 mediates cellular responses associated with proliferation, survival and differentiation, but the mechanisms underlying the diverse effects of this signaling molecule remain unknown. M1 mouse myeloid leukemia cells arrest growth and differentiate into macrophages following treatment with interleukin 6 (IL-6) or leukemia inhibitory factor (LIF), and recent studies have shown that Stat3 plays a central role in this process. Utilizing representational difference analysis, we demonstrate that expression of the mouse BATF gene is upregulated as an early response to IL-6/LIF stimulation and Stat3 activation in this cell system. Immunoblots using antibodies to BATF detected an increase in BATF protein in response to LIF/IL-6 stimulation. BATF is a member of the AP-1 family of basic leucine zipper transcription factors and functions to inhibit the transcriptional and biological functions of AP-1 activity in mammalian cells. BATF forms complexes with c-Jun in M1 cells and forced expression of BATF in the absence of Stat3 signaling results in a reduced rate of cellular growth. These results indicate that Stat3 mediates cellular growth by modulating AP-1 activity through the induction of BATF. Oncogene (2002) 21, 8186 – 8191. doi:10.1038/sj.onc. 1205918 Keywords: IL-6/LIF; gp130; Stat3; BATF; AP-1 Members of the interleukin-6 (IL-6) cytokine family, which include leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), interleukin-11 (IL-11) and cardiotrophin-1 (CT-1) regulate cell growth, survival and differentiation and are involved in variety of biological responses, including the immune response, inflammation, hema- topoiesis and oncogenesis. These cytokines use gp130 as a common receptor subunit. The binding of ligands to gp130 activates the JAK/Stat signal transduction pathway where Stat3 plays a central role in transmit- ting signals from the membrane to the nucleus (Hirano et al., 2000). An increasing number of candidate target genes of Stat3 have been characterized and many of these genes encode proteins that regulate cell cycle progression and/or modulate the apoptotic response. The expres- sion of positive cell cycle regulatory genes such as cyclin D1/D2/D3/A, cdc25A, c-myc, pim-1, bcl-2 and bcl-x are up-regulated by Stat3 activation (Fukada et al., 1996, 1998; Bromberg et al., 1999; Shirogane et al., 1999). In contrast, Stat3 has been shown to contribute to cell cycle arrest by inducing the expression of cyclin- dependent kinase inhibitors such as p19 INK4D and p21 CIP1 (Bellido et al., 1998; O’Farrell et al., 2000). The cellular circumstances, as well as the precise molecular mechanisms, underlying the diverse signaling effects described for Stat3 have yet to be determined. To identify the genes involved in signal transduction via gp130, we have performed representational differ- ence analysis (RDA) using M1 cells which differentiate into macrophages upon Stat3 activation by LIF/IL-6 (Lisitsyn et al., 1993; Hubank et al., 1994). Recently, we reported that the Ral guanine nucleotide dissocia- tion stimulator, RalGDS, was induced in this system in a manner dependent on Stat3 activation (Senga et al., 2001). We have analysed an additional 85 differentially represented DNA fragments and found that eight corresponded to the mouse BATF cDNA. BATF is a 125 amino acid nuclear basic leucine zipper protein and a member of the AP-1 family of transcription factors (Dorsey et al., 1995). BATF forms DNA binding heterodimers with Jun proteins and negatively regulates AP-1 mediated transcription in vitro and in vivo (Dorsey et al., 1995; Echlin et al., 2000; Williams et al., 2001). While the physiological function of BATF is still unknown, the fact that BATF is restricted to hematopoietic tissues suggests a role for the protein in myeloid and/or lymphoid lineage development. To confirm that BATF mRNA is induced upon LIF- stimulation, we examined expression by RNA blot analysis. BATF mRNA was induced 1 h after LIF- stimulation, peaked at 3h and declined thereafter (Figure 1a). Stimulation of M1 cells with IL-6 had the same effect (Figure 1b). Transcription of JDP-1 and Received 21 January 2002; revised 24 July 2002; accepted 26 July 2002 *Correspondence: M Hamaguchi; E-mail: mhamagu@tsuru.med.nagoya-u.ac.jp Oncogene (2002) 21, 8186 – 8191 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc