Figure 9. The involvement of CEBPD and LAP1 in the EGF-regulated COX-2 transcription. In the resting stage or starving stage, suCEBPD and suLAP1 could bind to the CRE and CEBP motifs of COX-2 promoter and interact with nuclear HDAC4 to form a repressive com- plex. In the early 1–3 h of EGF treatment, the instantly induced non- sumoylation of CEBPD can be positive regulators that replace the suCEBPD and suLAP1. This replacement will increase the p300 bind- ing and form an intact initiation of the activation complex to stimulate COX-2 transcription. After 3 h of EGF treatment, combination of decreased binding of nonsuCEBPD and p300, with the increased bind- ing of suLAP1, suCEBPD and increased importation of HDAC4 will re-inactivate COX-2 transcription through the new formation of the HDAC4/suCEBPD/suLAP1 complex. (20,53,54). Therefore, the normal physical situation for CEBPD- and CEBPB-mediated COX-2 transcription needs to be further elucidated. In tumorigenesis, the over- expression of LAP1/LAP2 has been reported in several human tumors with the implication that it is correlated with COX-2 expression, moreover leading to the conclu- sion that CEBPB might be a crucial positive regulator in COX-2 transcription. However, LIP draws less attention because it comes from one transcript of the CEBPB gene and is a strong repressor of COX-2 transcription. On the other hand, our unpublished data demonstrates that induc- tion of CEBPD expression is insensitive to external stimu- lation in cancer cell lines because of hypermethylation of the CEBPD promoter. Consequentially, this may result in the inaccurate conclusion that CEBPD may potentially be the most negative regulator just because its expression does not match the increase of COX-2 expression in tumors. Combining previous reports and our studies, we speculated that the CEBPD serves as a bifunctional regu- lator, at least in part, whereas LAP1/LAP2 may function as repressors acting through spatial and temporal changes to execute the switching of COX-2 transcription in response to external stimuli (Figure 9). In cancer cells, the overexpressed LAP1/LAP2 or de-sumoylated LAP1/ LAP2 can replace the silenced CEBPD and become a potential activator of COX-2 transcription. The overexpression of HDAC1 represses COX-2 pro- moter activity in phorbol 12-myristate 13-acetate (PMA)-treated HFb cells and in lipopolysaccharide Nucleic Acids Research, 2008, Vol. 36, No. 19 6077 (LPS)-treated RAW 264.7 cells (44). CEBPB is suggested to function in the gene repression of PPARb/d through a HDAC1-mediated mechanism via a speciﬁc C/EBP- responsive element (24). However, this current study demonstrates that the exogenous overexpression of HDAC1 alone has no eﬀect on COX-2 promoter activity (Figure 6A) even with the cotransfected LAP1 (Figure 7A). A similar experiment was performed in our ongoing study in which the overexpression of HDAC1 can repress the PPARG2 promoter activity, participating in the suCEBPD-mediated repression in HepG2 cells (52). These results suggest that the HDAC1-mediated transcrip- tional repression of genes may occur in cell type-depen- dent or gene-speciﬁc manners (49). Class II HDACs show a nucleocytoplasmic shuttling phenomenon through inter- action with 14-3-3 protein (55,56). Under long-term EGF treatment, the accumulation of HDAC4 shuttling in the nucleus was observed. Interestingly, the SUMO1 or SUMO1-conjugated proteins can be transiently exported to the cytoplasm during the short-term EGF treatment. Most importantly, the increasing amount of suLAP1 is coincident with the SUMO1 re-importation and HDAC4 accumulation (Figure 5A). These observations are also consistent with the ChIP and re-ChIP results that repre- sent the in vivo transcription factor-binding pattern on the COX-2 promoter in a time-dependent manner (Figure 8A and B). Compared with LAP1, LAP2 is a shorter protein lacking the N-terminal 23 amino acids in human. The integrity of N-terminal LAP1 not only aﬀects the SUMO2/3 conjugation, but is also essential for the speciﬁc interaction of SWI/SNF complexes (23,57). Among the CEBPB isoforms, the suLAP1 is the only eﬀecter involved in the HDAC4-mediated repression of COX-2 transcrip- tion. This implies that HDAC4 could be a functional dis- tinguisher between the SUMO1-conjugated LAP1 and LAP2, at least in COX-2 gene transcription. Additionally, HDAC4 possesses the SUMO E3 ligase activity. This raises an interesting issue of whether LAP1 is a HDAC4 substrate for its activity of SUMO E3 ligase. The over- expressed HDAC4 transfectant shows a consistent sumoy- lation of LAP1 (Supplementary Figure S7). This implies that the sumoylation of LAP1 is in a HDAC4-indepen- dent manner. Our data also demonstrate that the wild- type LAP1 and CEBPD bear higher aﬃnities for HDAC4 interaction than their sumoylation mutants. Moreover, the PCR products of COX-2 promoter are detectable in the CEBPD- or LAP1-re-ChIP samples from the ChIP sample of HDAC4 (Figure 8C). These results suggest that the suCEBPD and suLAP1 are able to coordinate with HDAC4, and modulate COX-2 tran- scription in A431 cells (Figure 9). However, the integra- tion of the EGF-induced signaling pathway and the HDAC4-nucleocytoplasmic shuttling remains unclear. This precise study not only conﬁrms previous reports suggesting that the CEBPB could be a repressor but also dissects the function of CEBPB isoforms in COX-2 tran- scription upon the long-term EGF treatment-induced re-inactivation and the EGF-starved stage. HDAC4 plays a role as a kinetic regulator in a time-dependent manner to coordinate the regulation of the COX-2 gene. suCEBPD, on the lysine 120 residue, and suLAP1, on the