Arch Pharm Res Vol 29, No 3, 218-223, 2006 RrthilJe~ of ~armatal ~e~eartlj http://apr.psk.or.kr Stimulation of Cell Growth by Erythropoietin in RAW264.7 Cells: Association with AP-1 Activation Seu Run Seong, Jae Woong Lee, Yong Kyoung Lee, Tae II Kim, Dong Ju Son, Dong Cheol Moon, Young Won Yun 1, Do Young Yoon z, and Jin Tae Hong College of Pharmacy and 1Veteminaty Medicine, Chungbuk National University, Cheongju 361-763, Korea, and 2Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Korea (Received October 17, 2005) Erythropoietin (EPO), a hematopoietic factor, is required for normal erythrocyte developments, but it has been demonstrated to have many other functions, and its receptor is localized in other tissues. In the present study, we investigated whether EPO can promote other cell prolif- eration and possible molecular mechanisms. EPO restored the inhibition of the RAW264.7 and PC12 cell growth by fetal bovine serum (FBS) withdrawal in a dose dependent manner, but not that of other cell types tested. The restoring effect of EPO was completed when the RAW264.7 cells were cultured in the medium containing as low as 3% of FBS, and 10 U/mL EPO could replace FBS. The restoring effect of EPO in the RAW264.7 cells was associated with the increased of c-Fos and c-Jun expression as well as AP-1 activation. These data demonstrate that EPO can stimulate RAW264.7 cell as well as PC12 cell growth even when the cells were cultured without FBS or in the presence of small amounts of FBS in the medium, and this stim- ulating effect is associated with the activation of AP-1 transcription factor. Key words: EPO, Cell growth, AP-1, Erythropoietin INTRODUCTION In the human, erythropoietin (EPO) is a glycoprotein hormone that is produced by peritubular cells in the kidneys of the adult and in hepatocytes in the fetus (Fisher, 2003; Jelkmann, 2004). The primary function of the EPO is to promote red cell production from erythrocyte progenitors in hemopoietic tissues (Liboi et al., 1993; Sasaki et al., 2000). EPO binds to an erythroid progenitor cell surface receptor called EPO receptor. Acti- vation of EPO receptor leads to tyrosine phosphorylation of the EPO receptor and several proteins such as c-Jun and c-Fos, which is involved with cell proliferation (Adunyah et al., 1996; Damen, et al., 1995; Farrel et al., 2004). However, recent advances in analytical techniques have led to the demonstration of EPO-R mRNA, EPO-R protein, EPO binding to EPO-R, and intracellular signaling in a variety of nonhemopoietic cells and organs, including the brain, cardiovascular tissues (endothelium, vascular smooth muscle and cardiomyocytes), the liver, gastroin- Correspondence to: Jin Tae Hong, College of Pharmacy, Chung- buk National University,Cheongju 361-763, Korea Tel: 82-43-261-1813 FAX: 82-43-268-2732 E-mail: jinthong@chungbuk.ac.kr testinal tissues, pancreatic islands, the kidney, the testis, and the female reproductive organs. Thus, EPO is a more pleiotropic survival and growth factor than initially thought (Adunyah et al., 1996; Bergelson et al., 1998; Patel and Sytkowski, 1995). Similar to the finding by Sugawa et al. (2002), we pre- viously also found that recombinant human EPO markedly enhanced the proliferation and differentiation of astrocytes (Lee et al., 2004). Sigounas et al., also reported that EPO increase endothelial cell proliferation, and promoted IL-3 induced cell proliferation in a synergistic manner (1997). Proliferation of microglial cell by EPO was also observed by Vairano et al. (2002). Therefore, it is possible that EPO can facilitate cell proliferation of RAW264.7 cells which belongs to the macrophage lineage. A plethora of physiological and pathological stimuli such as growth factors induce and activate a group of DNA binding proteins that form AP-1 dimers (Hess et al., 2004; Shaulian and Karin, 2001). These proteins include the Jun and Fos of transcription factors. Recent studies using cells and mice deficient in individual AP-1 proteins have begun to shed light on their physiological functions in the control of cell proliferation (Shaulian and Karin, 2001). Bergelson found that EPO activates AP-1 transcription factor 218