Modulation of immediate early gene expression by tristetraprolin in the differentiation of 3T3-L1 cells Nien-Yi Lin a , Chung-Tien Lin a, * , Ching-Jin Chang b,c, * a Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan b Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1 Sec 4 Roosevelt Road, Taipei 106, Taiwan c Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Received 18 October 2007 Available online 29 October 2007 Abstract Tristetraprolin (TTP) is a zinc-finger-containing AU-rich elements (ARE)-binding protein. AREs presented in the 3 0 untranslated region (UTR) of mRNAs from many proto-oncogenes, cytokines, and growth factors may be targets for regulation of messenger RNA stability. In this study, we observed that many immediate early genes (IEGs) were induced during the early differentiation of 3T3-L1 preadipocytes and their ARE-containing transcripts were degraded rapidly. Immunoprecipitation followed by RT-PCR analysis showed that two of IEG mRNAs, COX-2 (cyclooxygenase-2) and MKP-1 (mitogen-activated protein kinase phosphatase), were the tar- get of TTP. Biotinylated MKP-1 AREs also could bring down TTP and the other ARE-binding protein HuR. RNA EMSA and com- petition assays showed that each of three AREs located in 3 0 UTR of MKP-1 mRNA has differential binding affinity to TTP. Sequence analysis of 3 0 UTR of IEG mRNAs suggested that TTP may prefer binding to UUAUUUAUU sequence. Taken together, our results implied that TTP may target specific ARE-containing IEGs’ mRNAs such as COX-2 and MKP-1 mRNAs to modulate their expression post-transcriptionally. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Immediate early gene; Tristetraprolin; 3T3-L1; AU-rich element; MKP-1 The established preadipocyte cell line 3T3-L1 has been used in examining the process of adipogenesis in vitro. When treated with an empirically-derived prodifferentiative regimen that includes cAMP, insulin, and glucocorticoids in the presence of fetal bovine serum, they undergo differ- entiation to mature fat cells over a period of 4–6 days. The first step in the process of adipogenesis is the re-entry of growth-arrested preadipocytes into the cell cycle and the completion of several rounds of clonal expansion [1–3]. Several transcriptional factors are expressed coordinately to exert the terminal differentiation. Many immediate early genes (IEGs) such as c-jun, c-fos, egr-1, egr-2, nur77, cox-2, cyr61, pip92, btg2, ttp, and mkp-1, which expressed briefly in the trigger of differentiation hormones, have been observed [4]. Most IEGs contain adenylate/uridylate-rich elements (AREs) in the 3 0 UTR of their mRNAs to control their RNA turnover [5]. AREs can range in size and generally contain one or more copies of the pentameric sequence AUUUA, and have been divided into three classes [6]. Sev- eral ARE-binding proteins have been identified to regulate mRNA turnover [7]. HuR can respond to certain extracel- lular stimuli to mediate specific mRNAs stabilization [8]. Knockdown of HuR could attenuate the differentiation process in 3T3-L1 cells [9]. In contrast, TTP is important for the destabilization of tumor necrosis factor and GM- CSF mRNAs, as shown in knockout mice [10,11] and in 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.10.119 * Corresponding authors. Address: Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1 Sec 4 Roosevelt Road, Taipei 106, Taiwan (C.-J. Chang). Fax: +886 2 7359931 (C.-T. Lin), +886 2 23635038 (C.-J. Chang). E-mail addresses: ctlin@ntu.edu.tw (C.-T. Lin), chingjin@gate. sinica.edu.tw (C.-J. Chang). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 365 (2008) 69–74