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).

Schematic representation of TGF-β1-induced synergistic NF-κB activation via Smad3/4–PKA–p300-dependent p65 acetylation in human epithelial cells. As indicated, TGF-β induces p65 acetylation at lysine 221 via a Smad3/4–PKA–p300-dependent mechanism, which in turn leads to enhancement of DNA-binding activity, NF-κB activation and NF-κB-dependent inflammatory response in response to bacterium NTHi.

PKA acts downstream of TGF-β–Smad3/4 signaling pathway in mediating TGF-β1-induced synergistic enhancement of NF-κB activation. Overexpression of dominant-negative mutant of either Smad3 or Smad4 (A) and Smad3 or Smad4 knockdown (B) inhibited TGF-β1-induced PKA activation in HeLa cells. (C) TGF-β1 induced PKA activation in Smad4-deficient MDA-MB-468 reconstituted with WT Smad4 as indicated. Data (A–C) are representative of three independent experiments. (D) PKAc knockdown using siRNA-PKAc reduced NF-κB activation induced by overexpression of WT Smad3 and Smad4 in HeLa cells. Values are means±s.d. (n=3).

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.

Synergistic enhancement of NTHi-induced p65 acetylation by TGF-β1 is mediated by PKA in vitro and in vivo. (A) TGF-β1 induced PKA activation in HeLa cells. TGF-β1-induced PKA activation was inhibited by either 1 μM PKI (B) or PKAc knockdown using siRNA-PKAc (C). (D) Synergistic p65 acetylation by NTHi and TGF-β1 was inhibited by PKI in HeLa cells. (E) Coexpression of WT PKA and p300 with WT 65 but not with p65-KR mutant markedly induced p65 acetylation in HeLa cells. (F) PKAc knockdown using siRNA-PKAc inhibited synergistic enhancement of NTHi-induced DNA-binding activity by TGF-β1 in HeLa cells. (G) WT PKA enhanced DNA-binding activity in HeLa cells transfected with WT 65 but not with p65-KR mutant. (H) Synergistic NF-κB activation by NTHi and TGF-β1 was inhibited by either PKI (left) or siRNA-PKAc in HeLa cells (right). (I) WT PKA enhanced NF-κB activation in HeLa cells transfected with WT p65, but not with p65-KR. (J) Synergistic enhancement of NTHi-induced TNF-α and IL-1β expression by TGF-β1 was inhibited by PKI in HeLa cells. (K) PKI inhibited synergistic enhancement of NTHi-induced expression of TNF-α in the lungs of mice. (L) PKI markedly inhibited the TGF-β-mediated enhancement of PMN accumulation in BAL fluids from the lungs of the NTHi-inoculated mice. Values are means±s.d. (n=3 for H–K). Data are representative of three independent experiments.

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.

Smad3 and Smad4 are required for enhanced NF-κB activation by TGF-β via a mechanism independent of direct interaction with NF-κB. Overexpression of dominant-negative mutant of Smad3 or Smad4 (A) and Smad3 or Smad4 knockdown using siRNA (B) inhibited synergistic enhancement of NTHi-induced NF-κB activation by TGF-β in HeLa cells. Similar result was also observed in A549 cells. (C, D) TGF-β1 did not enhance NTHi-induced NF-κB activation in Smad3 null cells (C) and Smad4-deficient MDA-MB 468 cells (D). Transfecting Smad3 null cells with WT Smad3 and MDA-MB 468 cells with WT Smad4 rescued the responsiveness to TGF-β-1. (E) Smad3 or Smad4 knockdown inhibited p65 acetylation induced by TGF-β and NTHi in HeLa cells. (F) Smad3 and Smad4 does not appear to directly interact with NF-κB DNA-binding complex as assessed by supershift assay in HeLa cells. (G) No supershifted band was observed by anti-Smad3 or anti-Smad4 antibodies when TNF-α promoter containing an NF-κB-binding site with surrounding sequence was used as a probe to perform Smad supershift assay. IgG was used as a control in (F) and (G). Values are means±s.d. (n=3 for A–D). Data are representative of three independent experiments.

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.