Journal s~f Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Stat3 Is Present in the Developing and Adult Rat Cerebellum and Participates in the Formation of Transcription Complexes Binding DNA at the sis-Inducible Element Anna M. Planas, Meritxeil Berruezo, Caries Justicia, Sonia Barrón, and *Isjdre Ferrer Departament de Farmacologia i Toxicologia, 1/RB, CS/C; and *Unitat de Neuropatologia, Servei d’Anatomia Patolôgica, Hospital Princeps d’Espanya, Barcelona, Spain Abstract: Stat3 is a member of a recently identified family of proteins named STATs for their ability to act as signal transducers and activators of transcription. Stimulation of epidermal growth factor or cytokine receptors can cause activation of Stat3 and transduction of specific responses to the nucleus. Here the presence of Stat3 has been ex- amined in the rat cerebellum at different times during postnatal development and adulthood by means of im- munohistochemistry and western blotting. In addition, DNA binding activity at the sis-inducible element that is present in the promoter of the c-fos gene has been stud- ied by electrophoretic mobility shift assay. The results have shown that Stat3 p92 is abundant in the rat cerebel- lum. Stat3 was found in the external granule cell layer and also within the molecular layer from postnatal day (P) 0 to P7. From P15 to the adult, the internal granule cell layer and Purkinje cells were also stained for Stat3. Nuclear extracts were found to contain DNA binding ac- tivity to the sis-inducible element during development and adulthood. Supershift assays demonstrated that Stat3 mediates the formation of one protein—DNA complex. The present results suggest that Stat3 participates in in- tracellular signaling and is involved in maintaining basal c-fos expression in the cerebellum of the developing and adult rat under physiological conditions. Key Words: Stat3 —sis-Inducible element—Transcription factor— Brain— Rat. J. Neurochem. 68, 1345—1351 (1997). The family of signal transducers and activators of transcription (STATs) can be stimulated following li- gand binding to various growth factor and cytokine receptors (Darnell et a!., 1994; lhle et al., 1994, 1995; Schindler and Darnell, 1995; IhIe, 1996). For instance, binding of the epidermal growth factor (EGF) to its receptor (EGFR) can activate an intracellular signaling pathway mediated by members of the STAT protein family (Ruff-Jamison et al., 1994; Zhong et al., 1 994a). Binding of the ligand to the extracellular do- main of one of these cytokine receptors activates mem- bers of the membrane-anchored family of Janus ki- nases (Jaks) (Shuai et al., 1993; Silvennoinen et al., 1993; Stahl and Yancopoulos, 1993; Beadling et al., 1994; Briscoe et al., 1994; Brunn et al., 1995; Guschin et al., 1995; Ihle et al., 1995). These are tyrosine ki- nases that become themselves phosphorylated on tyro- sine (Shuai et al., 1993) and can then phosphorylate STAT proteins in the cytoplasm, and, as a result, ho- modimers and/or heterodimers (Shuai et al., 1994; Zhong et al., 1994a,b; Brunn et al., 1995) of STAT proteins are formed. Complexes of this kind can subse- quently translocate to the nucleus and interact with specific DNA sequences (Darnell et al., 1994; Ihle et al., 1994, 1995). Activated Stat3 complexes can bind to the sis-inducible element (SIE) (Darnell et al., 1994; Horvath et al., 1995; Seidel et al., 1995), which is present in the c-lbs gene promoter (Wagner et al., 1990), and can then regulate c-fos expression in the mouse liver in response to EGF (Ruff-Jamison et al., 1994). Also, Stat3 complexes can bind at other DNA sites; for instance, in the rat liver Stat3 binds the in- terleukin-6 response element of the a 2-macroglobulin gene in a model of acute-phase response following administration of complete Freund’s adjuvant (Rip- perger et al., 1995) or interleukin-6 (Fujitani et al., 1994). En sympathetic neurons the cytokine response element has a consensus STAT site, and binding activ- ity to this site is stimulated by the neuropoietic cyto- kine leukemia inhibitory factor (Symes et al., 1994). Stat3 mRNA has been detected in the rodent brain (Zhong et al., 1994a), and we have recently found Stat3 protein in cortical neurons and Stat3 induction in reac- tive microglia following transient focal ischemia in the Received August 19, 1996; revised manuscript received Novem- ber 25, 1996; accepted November 25. 1996. Address correspondence and reprint requests to Dr. A. M. Planas at Departament de Farmacologia i Toxicologia, IIBB, CSIC, Jordi Girona, 18-26, 08034-Barcelona, Spain. Abbreviations used: EGF, epidermal growth factor; EGFR, epi- dermal growth factor receptor; P, postnatal day; PBS, phosphate- buffered saline; SDS, sodium dodecyl sulfate; SIE, sis-inducible element; STAT, signal transducer and activator of transcription. 1345