6066–6079 Nucleic Acids Research, 2008, Vol. 36, No. 19 doi:10.1093/nar/gkn607 Published online 27 September 2008 Sumoylation of LAP1 is involved in the HDAC4-mediated repression of COX-2 transcription Wen-Ling Wang 1 , Yi-Chao Lee2,3, Wen-Ming Yang 4 , Wen-Chang Chang1,2,3,5 and Ju-Ming Wang2,4,5,* 1Institute of Basic Medical Sciences, 2Department of Pharmacology, College of Medicine, 3Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, 4Institute of Molecular Biology, National Chung Hsing University, Taichung and 5Institute of Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan Received March 1, 2008; Revised September 4, 2008; Accepted September 8, 2008 ABSTRACT CEBPB, one of the CEBP family members, is a crucial regulator of gene expression during innate immunity, inflammatory responses and adipogenesis. In this study, the EGF-induced increase of CEBPB mRNA is shown to be coincident with the decrease of COX-2 mRNA. We identified that all of the individual CEBPB isoforms, LAP1, LAP2 and LIP, attenuate EGF-induced COX-2 promoter activity. Although increased sumoylation of both LAP1 and LAP2 is observed during the lagging stage of EGF treatment, only the sumoylated LAP1, but not the sumoylated LAP2, is responsible for COX-2 gene repression. In addition, EGF treatment can regulate the nucleocy- toplasmic redistribution of HDAC4 and SUMO1. We further demonstrated by loss-of- and gain-of-func- tion approaches that HDAC4 can be a negative reg- ulator while inactivating COX-2 transcription. The sumoylation mutant LAP1, LAP1K174A, exhibits an attenuated ability to interact with HDAC4, and increased COX-2 promoter activity. Furthermore, the in vivo DNA binding assay demonstrated that LAP1K174A and CEBPDK120A, sumoylation-defec- tive CEBPD mutants, attenuate the binding of HDAC4 on the COX-2 promoter. In light of the above, our data suggest that the suCEBPD and suLAP1 are involved in the repression of COX-2 transcription through the recruitment of HDAC4. INTRODUCTION There are two known isoforms of cyclooxygenase (COX), which is also known as prostaglandin H synthase and prostaglandin endoperoxide synthase, COX-1 and COX-2 (1,2). COX-1 functions as a housekeeping gene and is constitutively expressed in most tissues. Conversely, COX-2 is an inducible enzyme that is induced by cytokines (3), growth factors (4), phorbol esters (5), endotoxins (6) and oncogenes (7,8) in diﬀerent cell types. Previous studies have shown that COX-2 is expressed in a large number of human cancers and is involved in cancer development and progression (9,10). Overexpression of COX-2 plays impor- tant roles in hyperproliferation, transformation, cell growth, invasion and metastasis of tumor cells. For the transcriptional activation of the COX-2 gene, several tran- scriptional activators, including nuclear factor-kappa B (NF-kB) (11), CCAAT/enhancer-binding protein b (CEBPB) (12), CEBP delta (CEBPD) (13,14), cyclic AMP-responsive element binding protein (CREB) (15) and activating protein 1 (AP1) (16), have been reported. However, the participating components in the repression of the COX-2 gene and mechanism have been less studied. The CEBPs belong to a subfamily of the basic region of leucine zipper (bZIP) transcription factors. Six members have been identiﬁed in mammalian cells, including CEBP alpha (CEBPA), CEBPB, CEBPD, CEBP epsilon (CEBPE), CEBP gamma (CEBPG) and CEBP zeta (CEBPZ). All CEBPs, except for CEBPZ, consist of three structural domains: an N-terminal domain contain- ing both positive and negative regulatory regions, a cano- nical basic domain and a C-terminal leucine zipper domain. The basic region binds to speciﬁc CCAAT motifs located in CEBPs targeted gene promoter, whereas the leucine zipper domain is responsible for heterodimer/ homodimer formation between various CEBP members (17). CEBPB and CEBPD are involved in the regulation of COX-2 transcription (12–14). The binding of CEBPB or CEBPD on the CEBP or cyclic AMP-responsive element (CRE) motifs of the human COX-2 promoter is increased by inﬂammatory stimulation (18,19). Three variants of CEBPBs have been detected in many cell types: a 46- kDa full-length liver-enriched transcription-activating protein (LAP1), a 42-kDa LAP2 and a 20-kDa *To whom correspondence should be addressed. Tel: +886 6 2757575 31067; Fax: +886 6 2083663; Email: yumingw@mail.ncku.edu.tw Correspondence may also be addressed to Wen-Chang Chang. Tel: +886 6 2353535 5496; Fax: +886 6 2749296; Email: wcchang@mail.ncku.edu.tw 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.