Regulation of ΔNp63α by NFκΒ

Abstract

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NF-κΒ in presence of cisplatin. We find that NF-κΒ promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NF-κΒ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NF-κΒ with siRNA-mediated silencing NF-κΒ expression attenuates chemotherapy induced degradation of ΔNp63α . These data demonstrate that NF-κΒ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NF-κΒ may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.

Figure 1

NFκB/p65 interacts with and regulates ΔNp63α in response to cisplatin. (A) Loss of ΔNp63α and nuclear accumulation of NFκB/p65 in JHU-022 cells in response to treatment with cisplatin. At the indicated time periods after treatment with cisplatin, JHU-022 cells were fractionated into nuclear and cytoplasmic fractions and analyzed by immunobloting with the indicated antibodies. (B) Complex formation between ΔNp63α with NFκB/p65. (i) JHU-022 cells were transfected with NFκB/p65 or empty Flag-vector and treated with cisplatin or vehicle as indicated. Whole cell lysates were immunoprecipitated with either anti-flag or anti-NFκB/p65 matrix, as indicated, and subjected to western blot analysis using anti-p63 antibody, as indicated. (ii) JHU-022 cells were transfected with Flag-ΔNp63α or empty Flag-vector and treated with cisplatin or vehicle as indicated. Immunoprecipitation was performed using anti-flag matrix and the membrane was blotted with NFκB/p65 antibody, as indicated. (C) Immunoprecipitates of JHU-022 cell lysates using either anti-p63 (i) or anti-NFκB/p65 (ii) antibody were subjected to immunoblot analysis using either anti-NFκB/p65 or anti-p63 antibody, as indicated.

Figure 2

NFκB/p65 mediates the reduction of ΔNp63α levels by cisplatin. (A and B) JHU-022 and p53/p63 deficient H1299 cells were transfected with increasing concentrations (0, 0.5, 1 and 1.5 µg) of NFκB/p65 expression plasmids as indicated; western blot was performed using the indicated antibodies. p53/p63 deficient H1299 cells were also transfected with 1 µg of ΔNp63α expression plasmid. (C) JHU-022 cells were transfected with increasing concentrations of either NFκB/p65 RNAi plasmid and subjected to western blot analysis using anti-p63 antibodies to assess the endogenous levels of p63. (D) JHU-022 cells were transfected with or without NFκB/p65 plasmid 24 h after transfection, the cells were treated with or without 75 µM cisplatin for 8 h, and cell lysates were subjected to western blot using anti-p63 antibodies.

Figure 3

NFκB/p65 promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. (A) JHU-022 cells were transfected with or without an expression plasmid encoding NFκB/p65; 24 h after transfection, cells were treated with 10 µM MG132 for the indicated time periods. Whole cell lysates were subjected to western Blot analysis using anti-NFκB/p65 or anti-p63 antibodies. (B) JHU-022 cells were transfected with constant amount of ΔNp63α expression plasmid with or without expression plasmids encoding NFκB/p65, or empty vector, as indicated. 24 h after transfection, the cells were treated with 100 µg/ml cycloheximide. At the indicated time points, whole cell lysates were analyzed for ΔNp63α by immunoblotting. Actin was used for loading control. (C) JHU-022 cells were transfected with constant amount of NFκB/p65 RNAi expression plasmid. 24 h after transfection, the cells were treated with 100 µg/ml cycloheximide. At the indicated time points, whole cell lysates were analyzed for endogenous ΔNp63α by immunoblotting. Actin was used for loading control. (D) JHU-022 cells were co-transfected with ΔNp63α and Ub-HA expression plasmids, with or without increasing concentrations of an expression vector encoding NFκB/p65. At 36 h following transfection, cells were treated with MG132 for 10 h. Cell lysates were immunoprecipitated with anti-HA-matrix and subjected to western blot analysis with an antibody that recognizes ΔNp63α to assess the ubiquitination levels of ΔNp63α.

Figure 4

NFκB/p65 counteracts the repressive transcriptional activity of ΔNp63α. (A) JHU-022 cells were transfected with either p21 or Bax luciferase promoter construct along with Renilla luciferase plasmid, with or without NFκB/p65 or NFκB/p65Δ450 and/or ΔNp63α, as indicated. The amount of DNA per transfection was kept constant by using empty pCDNA3.1 vector. At 24 h post-transfection, the luciferase activity was determined. The transfection efficiency was standardized against Renilla luciferase. Results shown are representative of three independent experiments. *indicated p ≤ 0.001. (B) JHU-022 cells were transfected with increasing concentrations of NFκB/p65 expression plasmid and western blot analysis was performed with the indicated antibodies to assess the endogenous levels of the indicated proteins. (C) (i) JHU-022 cells were transfected with empty vector or expression vector encoding NFκB/p65Δ450, and western blot analysis was performed with the indicated antibodies to assess the endogenous levels of the indicated proteins. (ii) A diagramatic representation of the NFκB/p65 domains.