NF-κB and CREB Bridged by CBP/p300 Regulate CD59 Transcription 1 NF-κB and Enhancer-Binding CREB Scaffolded by CBP/p300 Regulate CD59 Expression to Protect Cells from Complement Attack*

The complement system can be activated spontaneously for immune surveillance or induced to clear invading pathogens, in which the membrane attack complex (MAC, C5b-9) plays a critical role. CD59 is the sole membrane complement regulatory protein (mCRP) that restricts MAC assembly. CD59 therefore protects innocent host cells from attacks by the complement system, and host cells require the constitutive and inducible expression of CD59 to protect themselves from deleterious destruction by complement. However, the mechanisms that underlie CD59 regulation remain largely unknown. In this study, we demonstrate that the widely expressed transcription factor Sp1 may regulate the constitutive expression of CD59, while CBP/p300 bridge NF-κB and CREB that surprisingly functions as an enhancer binding protein to induce the up-regulation of CD59 during in lipopolysaccharide (LPS)-triggered complement activation, thus conferring host http://www.jbc.org/cgi/doi/10.1074/jbc.M113.525501 The latest version is at JBC Papers in Press. Published on December 12, 2013 as Manuscript M113.525501 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from NF-κB and CREB Bridged by CBP/p300 Regulate CD59 Transcription 2 defense against further MAC-mediated destruction. Moreover, individual treatment with LPS, TNF-α and the complement activation products (sublytic MAC [SC5b-9] and C5a) could increase the expression of CD59 mainly by activating NF-κB and CREB signaling pathways. Together, our findings identify a novel gene regulation mechanism involving CBP/p300, NF-κB and CREB; this mechanism suggests potential drug targets for controlling various complement-related human diseases. INTRODUCTION The complement system is known as a major constituent of innate immunity and an important modulator of adaptive immunity; complement not only eliminates invading microbial pathogens, xenografts and host debris but also orchestrates immunological and inflammatory processes(1,2). The activation of the complement cascade leads to the direct lysis of invading pathogens by the membrane attack complex (MAC), phagocytosis opsonized by C3b/iC3b tagging and the production of anaphylatoxins C3a/C5a; all these effects synergistically promote the clearance of foreign intruders. To prevent deleterious bystander effects on innocent host cells during this process, more than 10 circulating and membrane-bound complement regulatory proteins (mCRPs, including CD59) have evolved to restrict the activation of complement activation at diverse stages. The versatile functions of the complement system are able to be finely tuned to establish a delicate balance between activation and regulation, but the tipping of this delicate balance has been attributed, at least in part, to various human disorders including immune, inflammatory, neurodegenerative, atherosclerosis, ischemic and age-related diseases; the initiation, progression, drug resistance and non-responsiveness of cancer; and persistent pathogen infection(1). Therefore, it is crucial to understand how mCRPs respond to the extracellular inflammatory environment and complement activation under various conditions. CD59 is a small, highly glycosylated and glycosylphosphatidylinositol (GPI)-anchored membrane protein. It has been well-defined as the sole mCRP in restricting MAC assembly and is widely expressed on all circulating cells and in almost all tissues; intriguingly, CD59 is weakly expressed in the central nervous system(3). Therefore, CD59 plays a crucial role in protecting autologous cells from destruction by complement. Deficient or reduced CD59 expression in pathogens or host cells may lead to the direct lysis of invading pathogens or autologous cells in various diseases, such as autoimmune hemocytopenia and systemic lupus erythematosus (SLE)(4,5). In contrast, high CD59 expression in abnormal host cells leads to the incapability of the complement system to destroy target cells and triggers comprehensive downstream pro-cell survival signaling (6). Therefore, these findings highlight the need to decipher the regulation of CD59 in human disorders. Some isolated studies have speculated that CD59 might be regulated by the transcription factors (TFs) Sp1(7), TP53(8) and ERK1/2/NF-κB(9), along with an enhancer in intron 1(10); however, the underlying mechanisms remain largely obscure. The ubiquitously expressed transcription factor Sp1 binds to GC-rich elements that are widely distributed in the promoters of housekeeping genes and regulates the expression of thousands of genes involved in diverse cellular processes, such as cell growth, differentiation, apoptosis and immune responses(11); therefore, Sp1 has traditionally been regarded as a constitutive TF(12). However, NF-κB, which can be induced by both canonical and non-canonical signaling pathways, has critical regulatory functions in various processes including apoptosis, differentiation and especially immunity(13). Additionally, CREB regulates the expression of a wide range of genes that are responsible for glucose homeostasis, survival, proliferation, memory and learning(14) and is generally associated with the co-activator CREBbinding protein (CBP) and its close relative, p300(15). Through their different binding sites, CBP and p300 are functionally essential for CREB and many other TFs, such as NF-κB, TP53, signal transducers and activators of transcription (STATs) and activator protein 1 (AP-1); CBP and p300 provide a protein scaffold to connect diverse TFs to the transcription apparatus, which places them at the center of various signaling pathways in cell growth, transformation and development(16,17). Nevertheless, few studies have revealed the functions of these trans-acting factors in protecting host cells from complement-mediated damage. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from NF-κB and CREB Bridged by CBP/p300 Regulate CD59 Transcription 3 This study shows that the CD59 gene produces eight distinct transcripts, seven of which are newly identified, that share three different transcriptional initiation sites but the same open reading frame. Furthermore, we found that Sp1 mainly regulates the constitutive expression of CD59, whereas NFκB and CREB connected by CBP/p300 are responsible for the inducible expression of CD59. Furthermore and surprisingly, CREB is identified as an enhancer-binding protein for the first time. However, the potential TF roles of TP53 and CREB are negligible due to the very low abundance levels of their regulated transcripts. Finally, we investigated the signaling pathways that are responsible for up-regulating the transcription of CD59 in lipopolysaccharide (LPS)-triggered complement activation that subsequently confers cell protection from complement-induced and MAC-mediated destruction. Our findings clearly delineate a novel interaction between CBP/p300, NF-κB and CREB in gene transcriptional regulation; this interaction is important for the defense of host cells against complement attack via the up-regulation of CD59 transcription. EXPERIMENTAL PROCEDURES Cells Culture HeLa, A549, H1299 and U937 cells (Type Culture Collection Cell Bank, Chinese Academy of Sciences) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics; the IMR32 cell line (American Type Culture Collection) was grown in MEM supplemented with 10% FBS and 1% antibiotics. Reagents Recombinant human TNF-α was purchased from PROSPEC (East Brunswick, NJ); LPS and 8-BrcAMP were obtained from Sigma (St. Louis, MO); C5a, C5b-6, C7, C8, C9 and the rabbit anti-human C5b-9 antibody were obtained from CompTech (Tyler, Texas); the FITC-conjugated anti-mouse IgG, anti-β-actin (C4), anti-TFIIB (D-3), antiCD59 (H-7), anti-CD46 (M177), anti-CD55 (H-7), anti-phospho-ERK1/2 (E-4), anti-p65 (F-6), antip50 (E-10), anti-cRel (B-6), anti-RelB (D-4), antiSp1 (E-3), anti-CREB-1 (24H4B), antiphosphorylated CREB-1(S133) (sc-101663), antiTP53 (DO-2), anti-CBP (451) and anti-p300 (N-15) antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas); the anti-acetylTP53 (Lys382) (2525), anti-phospho-TP53 (Ser15) (16G8), anti-phospho-TP53 (Ser20) (9287), antiAkt (40D4) and anti-phospho-Akt (S473) (193H12) antibodies were obtained from Cell Signaling Technology (Danvers, MA); the Anti-NF-κB p100/p52 (ab7972) and anti-phosphorylated Sp1 (T453) (ab59257) antibodies were obtained from Abcam (Cambridge, MA); the anti-p65 (17-10060), anti-c-Rel (09-040), anti-RelB (EP613Y), antiCREB (AB3006) and anti-Phospho-CREB (Ser133) (17-1013) antibodies, all used only for chromatin immunoprecipitation (ChIP) assays, and the anti-acetyl-TP53 (Lys373) (06-916) antibody were obtained from Millipore (Billerica, MA); the FITC-conjugated mouse anti-human CD59 mAb (p282/H19) was obtained from BD Pharmingen (San Jose, California); the FITC-conjugated AffiniPure goat anti-rabbit IgG (H+L)(305-095003) was obtained from Jackson ImmunoResearch (West Grove, PA); and propidium iodide (PI) was obtained from Invitrogen (Carlsbad, CA). Normal human serum (NHS) was pooled from 12 healthy persons and aliquoted, then stored at -80°C until use. GeneRacer PCR GeneRacer PCRs were performed according to the manufacturer’s protocol (Invitrogen). The CD59specific reverse primer (RP; shown in Figure 1 A) was synthesized as 5’TCGTTAAAGTTACACAGGTCCTTC-3’. The first PCR was run with the GeneRacer first primer and RP. One microliter of the first-round PCR product was used to perform semi-nested PCR with the GeneRacer semi-nested primer and RP. The second PCR product was analyzed on a 12% PAGE gel, and the DNA was stained with ethidium bromide. Finally, the bands were excised from the PAGE gel, cloned into an appropriate Tvector and sequenced. Quantitative RT-PCR Total RNA was extracted using Trizol reagent (Invitrogen) and was reverse-transcribed to cDNA using a Reverse Transcription System (Promega). The input cDNA was standardized and then amplified for 45 cycles with SYBR Green Master Mix and gene-specific primers on an ABI Prism 7900HT machine (Applied Biosystems); endogenous β-actin was regarded as an internal control, and samples were analyzed in triplicate. The primers for amplifying β-actin were 5’TGGGACGACATGGAGAAAAT-3’ (F) and 5’by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from NF-κB and CREB Bridged by CBP/p300 Regulate CD59 Transcription 4 GCCAGAGGCGTACAGGGATA-3’ (R); the primers for amplifying CD59 transcripts T1-T8 were 5’-GATGCGTGTCTCATTACCAA-3’ (F) and 5’-AAGGATGTCCCACCATTTTC-3’(R); the primers for amplifying T1-T4 were 5’AGGCTGGAAGAGGATCTTGG-3’(F) and 5’AGGCTATGACCTGAATGGCA-3’(R); the primers for amplifying T5 were 5’CTTACAGTGAGGAGCCAGAG-3’(F) and 5’AATGAGACACGCATCAAAAT-3’ (R); and the primers for amplifying T6-T8 were 5’TGACTCACTGACCCTGATGGG-3’(F) and 5’CTATGACCTGAATGGCAGAAGA-3’ (R). Dual-Luciferase Reporter Assay The fragments upstream of exons 1, 1’ and 1’’ were cloned and inserted upstream of the luciferase gene in the pGL3 Basic Vector (Promega). The fragment from -888 to -1,155 bp upstream of exon 2 was inserted into the 3’ end of the luciferase gene in the relative pGL3 plasmid for enhancer activity identification, and the promoter-containing region was inserted at the 5’ end of the luciferase gene. Further, the critical nucleotides were mutated using a QuikChange site-directed mutagenesis kit (Stratagene) directly in the pGL3 plasmids containing the predicted trans-acting factor-binding sites as described in the text. The primer sequences for constructing the pGL3 plasmids with the above fragments inserted are not listed due to space considerations; the primer sequences used to generate the trans-acting factor-binding site mutations are shown in Supplementary Table I. The activity of the CD59 promoter was measured using a dual-luciferase reporter assay in HeLa, IMR32, A549 and H1299 cells. The indicated cells were transfected in 96-well plates with basic or modified pGL3 and pRL (Promega) using Lipofectamine 2000 (Invitrogen), without or with 1 mM 8-Br cAMP pre-treatment. After 24 h, the dual-luciferase activities were measured with the luciferase reporter assay system (Promega) using a Bio-Tek synergy HT microplate reader, and the firefly luciferase activities were normalized to the Renilla luciferase activities. These assays were repeated three or more times. To study the effects of Sp1, p60, p50, c-Rel, CREB, CBP and p300 deficiency on CD59 expression, we transfected siRNAs specific for these factors (GenePharma, Shanghai, China) into HeLa cells. The TP53 shRNA (Santa Cruz) was transfected into A549 cells. The siRNA sequences were as follows: 5’UUCUCCGAACGUGUCACGUTT-3’ (scrambled), 5’GCCCTATCCCTTTACGTCA3’ (p65), 5’-TATTAGAGCAACCTAAACATT-3’ (p50), 5’-GTGTGAAGGGCGATCAGCAGG-3’ (c-Rel), 5’-GGUGGAAAUGGACUGGCUTT-3’ (CREB), 5’-GGGAACATCACCTTGCTACCT-3’ (Sp1), 5’AACAGTGGGAACCTTGTTCCA-3’ (CBP) and 5’-AATTGGGACTAACCAATGGTG3’ (p300). Forty-eight hours after transfection, the cells were collected for dual-luciferase reporter assays and flow cytometry as described below. To further demonstrate the regulation of TP53 on T5, A459 cells were co-transfected with gradually increasing amounts of a wildtype TP53 expression vector (a kind gift from Dr. J. C. Bourdon, University of Dundee, UK), a pGL3 plasmid containing a -1 to -200 bp fragment upstream of CD59 exon 1’ and pRL; the dualluciferase activity was measured at 48 hours after