TGF-β induces p65 acetylation to enhance bacteria-induced NF-κB activation
2007; Springer Nature; Volume: 26; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7601546
ISSN1460-2075
AutoresHajime Ishinaga, Hirofumi Jono, Jae Hyang Lim, Soo-Mi Kweon, Haodong Xu, Un‐Hwan Ha, Haidong Xu, Tomoaki Koga, Chen Yan, Xin‐Hua Feng, Lin‐Feng Chen, Jian‐Dong Li,
Tópico(s)Histone Deacetylase Inhibitors Research
ResumoArticle1 February 2007free access TGF-β induces p65 acetylation to enhance bacteria-induced NF-κB activation Hajime Ishinaga Hajime Ishinaga Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Hirofumi Jono Hirofumi Jono Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Jae Hyang Lim Jae Hyang Lim Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Soo-Mi Kweon Soo-Mi Kweon Gonda Department of Cell and Molecular Biology, House Ear Institute, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Haodong Xu Haodong Xu Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Un-Hwan Ha Un-Hwan Ha Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Haidong Xu Haidong Xu Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Tomoaki Koga Tomoaki Koga Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Chen Yan Chen Yan Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Xin-Hua Feng Xin-Hua Feng Michael E DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Lin-Feng Chen Corresponding Author Lin-Feng Chen Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Jian-Dong Li Corresponding Author Jian-Dong Li Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Hajime Ishinaga Hajime Ishinaga Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Hirofumi Jono Hirofumi Jono Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Jae Hyang Lim Jae Hyang Lim Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Soo-Mi Kweon Soo-Mi Kweon Gonda Department of Cell and Molecular Biology, House Ear Institute, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Haodong Xu Haodong Xu Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Un-Hwan Ha Un-Hwan Ha Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Haidong Xu Haidong Xu Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Tomoaki Koga Tomoaki Koga Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Chen Yan Chen Yan Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Xin-Hua Feng Xin-Hua Feng Michael E DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Lin-Feng Chen Corresponding Author Lin-Feng Chen Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Jian-Dong Li Corresponding Author Jian-Dong Li Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA Search for more papers by this author Author Information Hajime Ishinaga1,‡, Hirofumi Jono1,‡, Jae Hyang Lim1, Soo-Mi Kweon2, Haodong Xu3, Un-Hwan Ha1, Haidong Xu1, Tomoaki Koga1, Chen Yan4, Xin-Hua Feng5, Lin-Feng Chen 6 and Jian-Dong Li 1 1Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA 2Gonda Department of Cell and Molecular Biology, House Ear Institute, University of Southern California, Los Angeles, CA, USA 3Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA 4Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA 5Michael E DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA 6Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA ‡These authors contributed equally to this work *Corresponding authors: Department of Microbiology and Immunology, Box 672, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA. Tel.: +1 585 275 7195; Fax: +1 585 276 2231; E-mail: [email protected] Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Tel.: +1 217 333 7764; Fax: +1 217 244 5858; E-mail: [email protected] The EMBO Journal (2007)26:1150-1162https://doi.org/10.1038/sj.emboj.7601546 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transforming growth factor-β (TGF-β) family members are multifunctional growth factors involved in regulating diverse biological processes. Despite the critical role for TGF-β in regulating cell proliferation, differentiation, migration and development, its role in regulating NF-κB-dependent inflammatory response still remains unclear. Here, we show that TGF-β1 induces acetylation of NF-κB p65 subunit to synergistically enhance bacterium nontypeable Haemophilus influenzae-induced NF-κB activation and inflammatory response in vitro and in vivo. The TGF-β1-induced acetylation of p65 is mediated via a Smad3/4-PKA-p300-dependent signaling pathway. Acetylation of p65 at lysine 221 by TGF-β1 is critical for synergistic enhancement of bacteria-induced DNA-binding activity, NF-κB activation, NF-κB-dependent transcription of TNF-α and IL-1β and interstitial polymorphonuclear neutrophil infiltration in vitro and in vivo. These studies provide new insights into the novel regulation of NF-κB by TGF-β signaling. Introduction Transforming growth factor-β (TGF-β) family members are multifunctional growth factors involved in regulating diverse biological processes. Despite the critical role for TGF-β in regulating cell proliferation, differentiation, migration and development, its role in regulating NF-κB-dependent inflammation still remains elusive (Wahl, 1994; ten Dijke and Hill, 2004; Feng and Derynck, 2005; Massague et al, 2005). Targeted disruption of the mouse TGF-β1 gene results in excessive inflammatory responses (Shull et al, 1992). Systemic TGF-β administration suppresses inflammation (Wahl, 1994). Consistent with these in vivo findings, TGF-β1 inhibits LPS-induced NF-κB activation in both intestinal epithelial cells and microglial cells (Haller et al, 2003; Le et al, 2004). These data thus suggest that TGF-β acts as a suppressor for NF-κB-dependent inflammatory response. However, there is now growing evidence that TGF-β induces and promotes inflammatory response via activation of NF-κB. For instance, TGF-β1 overexpression in keratinocytes in transgenic mice results in inflammatory skin lesions (Li et al, 2004); local TGF-β administration promotes inflammation (Wahl, 1994). In addition, in vitro studies also indicate that TGF-β–Smad signaling mediates activation of NF-κB in human airway epithelial cells (Jono et al, 2002; Mikami et al, 2006). The molecular mechanisms underlying TGF-β-mediated NF-κB-dependent inflammatory responses remain totally unknown. TGF-β initiates signaling through the ligand-dependent activation of a heteromeric complex of type II and type I receptors (Feng and Derynck, 2005; Massague et al, 2005). The type II receptor kinase then phosphorylates the type I receptor in a conserved glycine-serine domain, resulting in activation of the type I receptor. The activated type I receptor subsequently recognizes and phosphorylates the Smad subgroup known as receptor-activated Smads (R-Smad), including Smad 2 and 3. This causes dissociation of R-Smad from the receptor, stimulates the assembly of a heteromeric complex between the phosphorylated R-Smad and the Co-Smad, Smad4, and then induces the translocation of the Smad complex to the nucleus, where the Smad complex regulates the expression of target genes. In addition to its direct interaction with Smad DNA-binding element, growing evidence suggests that Smads also regulate gene transcription by direct interaction and functional cooperation with other transcription factors, such as NF-κB. NF-κB is known to be activated via phosphorylation and degradation of IκB by IκB kinases (IKKs), which in turn leads to the nuclear translocation of NF-κB and the subsequent transcription of NF-κB-dependent genes such as TNF-α (Bonizzi and Karin, 2004; Hayden and Ghosh, 2004). Recently, interesting studies have suggested that degradation of IκBα and nuclear translocation of NF-κB are not sufficient to promote a maximal NF-κB transcriptional activity. Rather, the NF-κB complex must undergo additional post-translational modification involving site-specific phosphorylation and acetylation (Chen et al, 2001, 2002, 2005). The p65 subunit of NF-κB is a principal target of phosphorylation by various kinases. These kinases function both in the cytoplasm and in the nucleus and are differentially induced by various stimuli including LPS and TNF-α. Both the Rel-homology domain and the carboxy-terminal transactivation domain of p65 contain key phosphoacceptor sites that are specifically targeted by various kinases. The most physiologically inducible phosphorylation sites reported for p65 occurs at serine 536, serine 276 and serine 529. Serine 536 is phosphorylated by IKK complex (Sakurai et al, 1999), whereas Ser 529 was phosphorylated by casein kinase II (Wang et al, 2000). In addition, phosphorylation of Ser 276 is mediated by catalytic protein kinase A (PKA) subunit or mitogen- and stress-activated protein kinase 1 (Zhong et al, 1998; Vermeulen et al, 2003). Additional p65 phosphorylation events have been described. Their functional significance, however, remains unclear. Acetylation, like phosphorylation, is also important for regulation of the nuclear function of NF-κB (Chen et al, 2002, 2005; Kiernan et al, 2003; Hoberg et al, 2006). Endogenous p65 is acetylated in a stimulus-coupled manner after activation of cells with TNF-α or other stimuli at multiple sites. The p300 and CBP appear to play a major role in acetylation of p65. The p300/CBP possesses a histone acetyltransferase (HAT) enzymatic activity that regulates gene expression in part through acetylation of the N-terminal tails of histones (Ogryzko et al, 1996; Chen et al, 2002). In addition to modifying histones, p300 and CBP also directly acetylates p65. Acetylation of NF-κB leads to changes in its biological activity, such as alterations in DNA-binding activity and transcriptional activity (Ogryzko et al, 1996; Kouzarides, 2000; Chen et al, 2002; Gu et al, 2004). Three main acetylation sites have been identified within p65—lysines 218, 221 and 310. Site-specific acetylation of p65 regulates discrete biological action of the NF-κB complex. For example, acetylation of lysine 221 increases the DNA-binding affinity of p65 for κB site, whereas acetylation of lysine 221 alone or in combination with lysine 218 impairs the assembly of p65 with IκBα. Acetylation of lysine 310 of p65 is required for full transactivation by NF-κB complex. The relationship between p65 phosphorylation and acetylation has been also explored recently. There is evidence indicating that the acetylation of p65 is importantly regulated by prior phosphorylation of serine 276 and 536 (Chen et al, 2005; Hoberg et al, 2006). Such phosphorylated and acetylated forms of p65 display enhanced transcriptional activity. Given that TGF-β plays a critical role in regulating inflammatory response via NF-κB in infectious diseases, it is still unclear whether TGF-β regulates NF-κB-dependent inflammatory response via either phosphorylation or acetylation of NF-κB. The Gram-negative bacterium nontypeable Haemophilus influenzae (NTHi) is an important human pathogen in both adults and children (Foxwell et al, 1998; Murphy, 2000). In adults, it exacerbates chronic obstructive pulmonary diseases (COPD), the fourth leading cause of death in the United States (Murphy, 2006), whereas in children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss (Murphy, 2000). Like most other bacterial infections, NTHi infections are characterized by inflammation. We have previously shown that NTHi activates NF-κB via a Toll-like receptor 2-dependent NIK–IKKα/β–IκBα signaling pathway (Shuto et al, 2001). Based on the essential involvement of NF-κB in NTHi-induced inflammatory responses and TGF-β, an important regulator of inflammation, is highly upregulated in airways of COPD patients (de Boer et al, 1998), we hypothesized that TGF-β signaling may play a critical role in regulating NTHi-induced inflammation via modulation of NF-κB activity. Here, we show that TGF-β1 induces acetylation of NF-κB p65 subunit at lysine 221 to synergistically enhance NTHi-induced NF-κB activation and inflammatory response via a Smad3/4-PKA-dependent mechanism in vitro and in vivo. These studies may provide novel insights into the regulation of NF-κB by TGF-β signaling. Results TGF-β1 synergizes with bacterium NTHi to induce NF-κB activation and inflammatory response in vitro and in vivo To determine whether TGF-β1 regulates NTHi-induced NF-κB activation and inflammatory response, we first measured NF-κB-dependent promoter activity by using luciferase reporter plasmid in a variety of human epithelial cells. As shown in Figure 1A, TGF-β1 enhanced NTHi-induced NF-κB activation in a synergistic manner in human epithelial HeLa cells, airway epithelial A549 cells and middle ear epithelial HMEEC-1 cells as well as human primary bronchial epithelial NHBE cells. Because of the important role for NF-κB in regulating a variety of key inflammatory mediators, we next determined whether TGF-β1 synergistically enhances NTHi-induced transcription of TNF-α and IL-1β by performing real-time quantitative PCR (Q-PCR) analysis. As shown in Figure 1B, TGF-β1 synergistically enhanced NTHi-induced expression of TNF-α and IL-1β in HeLa cells. Similar result was also observed in A549 and primary NHBE cells. We further confirmed whether TGF-β1 also enhances NTHi-induced expression of TNF-α and IL-1β in vivo. As shown in Figure 1C, TGF-β1 synergistically enhanced induction of TNF-α and IL-1β by NTHi in the lungs of mice. Consistent with this result, TGF-β1 also synergistically enhanced polymorphonuclear neutrophil (PMN) accumulation in broncho-alveolar lavage (BAL) fluids from the lungs of the NTHi-inoculated mice (Figure 1D and Supplementary Figure S1). Collectively, these data demonstrate that TGF-β1 synergistically enhances bacterium NTHi-induced NF-κB-dependent inflammatory response in vitro and in vivo. Figure 1.TGF-β1 synergizes with bacterium NTHi to induce NF-κB activation and inflammatory response in vitro and in vivo. (A) TGF-β1 synergistically enhanced NTHi-induced NF-κB-dependent promoter activity in human HeLa, airway A549, middle ear HMEEC-1 and primary bronchial epithelial NHBE cells, as assessed by NF-κB-dependent promoter assays. (B) TGF-β1 synergistically enhanced NTHi-induced expression of TNF-α and IL-1β in HeLa cells, as assessed by real-time Q-PCR analysis. Similar results were also observed in A549 and primary NHBE cells. (C) TGF-β1 synergistically enhanced NTHi-induced expression of TNF−α and IL-1β in the lung of BALB/c mice in vivo. Values are means±s.d. (n=5). (D) TGF-β1 synergistically enhanced NTHi-induced PMN infiltration in the lung of BALB/c mice in vivo. Data are representative of three independent experiments. Download figure Download PowerPoint TGF-β1 synergistically enhances NTHi-induced NF-κB activation via a mechanism dependent on increased DNA-binding activity, but independent of p65 nuclear translocation Because phosphorylation and degradation of IκBα and nuclear translocation of NF-κB are critical for NF-κB activation, we next sought to determine whether TGF-β1 synergistically enhances NTHi-induced NF-κB activation by increasing phosphorylation and degradation of IκBα. As shown in Figure 2A, TGF-β1 did not enhance NTHi-induced IκBα phosphorylation and degradation. We then determined whether TGF-β1 enhances NTHi-induced NF-κB activation by causing delay in the cytoplasmic reappearance of IκBα. No further delay in the cytoplasmic reappearance of IκBα was observed with TGF-β1 treatment (Figure 2B). Therefore, it is clear that TGF-β1-induced enhancement of NF-κB activation by NTHi does not occur at the level of IκBα. We next determined whether TGF-β1 enhances NTHi-induced NF-κB activation by inducing its nuclear translocation of p65 by performing Western blot analysis using nuclear extract. As shown in Figure 2C, p65 was markedly translocated to the nucleus when the cells were stimulated with NTHi alone. Simultaneous treatment with TGF-β1 and NTHi resulted in no synergistic enhancement of p65 translocation. Likewise, TGF-β1 also did not prolong nuclear presence of p65 (Figure 2D). These results thus suggest that TGF-β1-induced synergistic enhancement of NF-κB activation did not occur at the level of p65 nuclear translocation. Because the DNA-binding activity of the NF-κB complex is critical for NF-κB to exert its transcriptional activity, we investigated the effect of TGF-β1 on NTHi-induced DNA-binding activity of NF-κB by performing electrophoretic mobility shift assay (EMSA). As shown in Figure 2E, TGF-β1 treatment resulted in synergistic enhancement of DNA binding of NF-κB in cells treated with NTHi. Further analysis using supershift assay reveals that p65 and p50 appear to be the major subunits of the NF-κB band that was synergistically enhanced by TGF-β1 treatment (Figure 2F). To further confirm whether TGF-β enhances p65 DNA binding to an NF-κB-dependent gene promoter such as TNF-α promoter in the context of chromatin, chromatin immunoprecipitation (ChIP) assays were performed. As shown in Figure 2G, TGF-β indeed synergistically enhanced NTHi-induced DNA binding of p65 to the TNF-α promoter. Together, these data suggest that TGF-β1 synergistically enhances NTHi-induced NF-κB activation via a mechanism dependent on increased DNA-binding activity, but independent of p65 nuclear translocation. Figure 2.TGF-β1 synergistically enhances NTHi-induced NF-κB activation via a mechanism dependent on increased DNA-binding activity, but independent of p65 nuclear translocation. (A) TGF-β1 did not enhance NTHi-induced phosphorylation and degradation of IκBα in HeLa cells, as assessed by Western blot analysis. (B) TGF-β did not affect cytoplasmic reappearance of IκBα after NTHi treatment in HeLa cells. (C) TGF-β did not enhance NTHi-induced p65 nuclear translocation in HeLa cells. (D) TGF-β did not alter prolonged intranuclear retention of p65 after translocation to the nucleus in HeLa cells. (E) TGF-β1 enhanced NTHi-induced DNA-binding activity of NF-κB, as assessed by performing EMSA in HeLa cells. (F) p65 and p50 appear to be the major subunits of the NF-κB band that was synergistically enhanced by TGF-β1 60 min after treatment, as assessed by supershift assays. For competition experiments, nonlabeled probes (200 times) were used. IgG was used as a control. (G) TGF-β synergistically enhanced NTHi-induced DNA binding of p65 to the TNF-α promoter as assessed by performing ChIP assays. Isotype-matched control IgG was used as a control. Data are representative of three or more independent experiments. Download figure Download PowerPoint TGF-β1 synergistically enhances NTHi-induced NF-κB activation via induction of p65 acetylation at lysine 221 Having identified the synergistic enhancement of TGF-β1 on NTHi-induced DNA-binding activity of NF-κB, still unknown is how TGF-β1 enhances NTHi-induced NF-κB activation by increasing DNA-binding activity. Post-translational modifications, particularly phosphorylation and acetylation, have been shown to play a critical role in NF-κB activation by enhancing the DNA-binding activity of p65 to the κB site (Chen et al, 2005). Thus, we hypothesized that these modification of p65 may be involved in mediating the enhancement of NTHi-induced DNA-binding activity of NF-κB by TGF-β1. We tested our hypothesis by assessing the effect of TGF-β1 on NTHi-induced p65 phosphorylation and p65 acetylation. As shown in Figure 3A, TGF-β1 enhanced NTHi-induced p65 acetylation but not phosphorylation. It should be noted that TGF-β, like NTHi, also induced phosphorylation of p65 at Ser276 (middle panel). Because trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), broadly inhibits the action of the HDACs function and results in hyperacetylation of the core histones and nonhistone proteins (Adam et al, 2003; Kiernan et al, 2003), we next evaluated the effect of TSA on p65 acetylation induced by NTHi and TGF-β1. As expected, p65 acetylation was enhanced by TSA treatment (Figure 3B, left panel). Consistent with this result, TSA treatment further enhanced the synergistic activation of NF-κB induced by NTHi and TGF-β (Figure 3B, right panel). Figure 3.TGF-β1 synergistically enhances NTHi-induced NF-κB activation via induction of p65 acetylation at lysine 221. (A) Synergistic enhancement of p65 acetylation was observed in HeLa cells treated with both NTHi and TGF-β1 (1 ng/ml; right panel), whereas TGF-β1 induced p65 phosphorylation at Ser276 but not Ser536 (left and middle panels). Acetylation of p65 was detected by immunoblotting (IB) of the anti-p65 (α-p65) immunoprecipitates (IP) with anti-acetylated lysine antibodies. Levels of p65 present in each of the lysates are shown in the lower panel. (B) TSA enhanced NTHi-induced p65 acetylation and NF-κB activation. (C) TGF-β1 enhanced p65 acetylation induced by coexpressing WT p300 with WT p65, but not with p65-KR mutant (lysine 218/lysine 221/lysine 310 acetylation site mutant) in HeLa cells. (D) TGF-β1 enhanced DNA-binding activity of NF-κB in HeLa cells transfected with WT p65 but not with p65-KR mutant. (E) TGF-β1 enhanced NF-κB activation in HeLa cells transfected with WT p65 but not with p65-KR mutant. (F) TGF-β1 enhanced NTHi-induced NF-κB activation in p65−/− MEFs reconstituted with WT p65 but not in p65-KR-reconstituted p65−/− MEFs. (G) TGF-β1 enhanced TNF-α and IL-1β expression in HeLa cells transfected with WT p65 but not with p65-KR. (H) Mutation of lysine 221, but not 218 or 310, markedly decreased p65 acetylation in response to TGF-β compared to WT p65. (I) TGF-β1 enhanced NF-κB-dependent transcriptional activity in cells transfected with p65-K218R and p65-K310R, but not with p65-K221R mutant compared to cells transfected with WT p65. (J) Synergistic NF-κB activation was observed in p65-K310R-reconstituted MEFs but not in p65-K221R-reconstituted MEFs in response to NTHi and TGF-β1 compared to p65−/− MEFs reconstituted with WT p65. Values are means±s.d. (n=3). Data are representative of three or more independent experiments. Download figure Download PowerPoint Owing to the important role of lysines 218, 221 and 310 as major acetylation sites in p65 (Chen et al, 2002), we next examined whether mutation of all three of these lysine residues (p65KR (p65K218/221/310R)) alters p65 acetylation, DNA binding and NF-κB activation by TGF-β1 and NTHi. As shown in Figure 3C, coexpressing wild-type (WT) p65, but not p65KR mutant, with WT p300 together with TGF-β1 treatment markedly induced p65 acetylation. Consistent with this result, TGF-β1 also enhanced NF-κB DNA-binding activity and NF-κB-dependent transcriptional activity in cells transfected with WT p65 but not with p65-KR mutant (Figure 3D and E). To further confirm the functional involvement of these three acetylation sites in synergistic activation of NF-κB by NTHi and TGF-β1, we assessed the synergistic induction of NF-κB in p65−/− MEFs that were reconstituted with either WT p65 expression plasmid or p65-KR mutant. As shown in Figure 3F, synergistic NF-κB activation was observed in WT p65-reconstituted MEFs but not in p65-KR-reconstituted MEFs in response to NTHi and TGF-β1. To confirm whether these three acetylation sites are also involved in synergistic induction of TNF-α and IL-1β, we evaluated induction of TNF-α and IL-1β in cells transfected with either WT p65 or p65-KR mutant upon TGF-β1 treatment. As shown in Figure 3G, TGF-β1 synergistically enhanced induction of TNF-α and IL-1β in cells transfected with WT p65 but not with p65-KR mutant. Taken together, our results suggest that these three acetylation sites are critical for mediating synergistic enhancement of p65 acetylation by TGF-β1, which, in turn, leads to enhancement of DNA-binding activity and NF-κB-dependent transcription of proinflammatory cytokines TNF-α and IL-1β. We next determined which individual lysine residue is acetylated in response to TGF-β. As shown in Figure 3H, mutation of lysine 221, but not 218 or 310, markedly decreased p65 acetylation in response to TGF-β compared to WT p65. Consistent with this result, TGF-β1 synergistically enhanced NF-κB activation in cells transfected with WT p65, p65-K218R and p65-K310R, but not with p65-K221R mutant (Figure 3I). To further confirm the functional involvement of lysine 221 in synergistic activation of NF-κB by NTHi and TGF-β1, we assessed the synergistic induction of NF-κB in p65−/− MEFs that were reconstituted with either WT p65 expression plasmid or p65-K221R mutant or p65-K310R mutant. As shown in Figure 3J, synergistic NF-κB activation was observed in p65-K310R-reconstituted MEFs but not in p65-K221R-reconstituted MEFs compared to p65−/− MEFs reconstituted with WT p65. Thus, it is evident that lysine 221 residue is critical for mediating synergistic enhancement of p65 acetylation by TGF-β1 and the subsequent NF-κB DNA-binding and transcriptional activity. p300 is involved in enhancement of p65 acetylation by TGF-β1 Based on recent studies that p300 acetyltransferase plays a major role in acetylation of p65 (Chen et al, 2002, 2005; Kiernan et al, 2003; Hoberg et al, 2006), we next determined whether p300 is involved in synergistic enhancement of p65 acetylation by TGF-β1. As shown in Figure 4A, TGF-β1 enhanced p65 acetylation in epithelial cells cotransfected with WT p65 and WT p300, but not in cells cotransfected with WT p65 and p300 HAT mutant. This result indicates that synergistic enhancement of p65 acetylation by TGF-β1 may involve the HAT activity of p300. To confirm whether p300 HAT activity is also involved in synergistic NF-κB activation by NTHi and TGF-β1, we evaluated the effect of overexpressing of WT p300 and p300 HAT mutant on NTHi- and TGF-β1-induced NF-κB activation. As shown in Figure 4B, overexpression of WT p300 enhanced NTHi- and TGF-β1-induced NF-κB activation, whereas overexpressing p300 HAT mutant inhibited synergistic activation of NF-κB by NTHi and TGF-β1. Thus, these data suggest that p300 is involved in synergistic NF-κB activation by NTHi and TGF-β1 via a mechanism dependent on p65 acetylation. Figure 4.p300 is involved in enhancement of p65 acetylation by TGF-β1. (A) TGF-β1 enhanced p65 acetylation in HeLa cells co-transfected with WT p65 and WT p300 but not p300 HAT mutant. Data are representative of three independent experiments. (B) WT p300 enhanced NTHi-induced NF-κB activation, whereas p300 HAT mutant inhibited synergistic NF-κB activation by NTHi and TGF-β1 in HeLa cells. Values are means±s.d. (n=3). Download figure Download PowerPoint Smad3 and Smad4 are required for enhanced NF-κB activation by TGF-β1 via a mechanism independent of direct interaction with NF-κB We next sought to determine the involvement of Smad3 and Smad4 in TGF-β1-induced synergistic enhancement of NF-κB activation. As shown in Figure 5A, overexpression of a dominant-negative mutant of either Smad3 or Smad4 and Smad3 or Smad4 knockdown using siRNA inhibited synergistic activation of NF-κB by NTHi and TGF-β1. It should be noted that Smad4 DN did not inhibit TNF-α-induced NF-κB activation, suggesting that the inhibitory effect of Smad4 DN is specific for NTHi-induced NF-κB activation (Supplementary Figure S2). Consistent with these results, no synergistic enhancement of NF-κB activation was observed in Smad3 null cells and Smad4-deficient MDA-MB-468 cells (Figure 5C and D), whereas cotransfecting Smad3 null cells with WT Smad3 and cotransfecting MDA-MB-468 cells with WT Smad4 expression plasmid rescued the responsiveness to TGF-β1. We further investigated whether Smad3 and Smad4 are also required for the synergistic induction of p65 acetylation by TGF-β and NTHi. As shown in Figure 5E, Smad3 or Smad4 knockdown inhibited p65 acetylation induced by TGF-β and NTHi. We next determined whether there is a direct interaction between NF-κB components and Smads by performing supershift
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