PKCβΙΙ Augments NF-κB-Dependent Transcription at the CCL11 Promoter via p300/CBP-Associated Factor Recruitment and Histone H4 Acetylation

Abstract

The transcription factor NF-κB plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators, including histone acetyltransferases (HATs) such as CREB-binding protein/p300 and p300/CBP-associated factor (p/CAF). The molecular mechanisms regulating cofactor recruitment are poorly understood. In this study, we describe a novel role for protein kinase C (PKC) βΙΙ in augmenting NF-κB-mediated TNF-α-induced transcription of the target gene CCL11 in human airway smooth muscle cells by phosphorylating the HAT p/CAF. Studies using reporters, overexpression strategies, kinase-dead and HAT-defective mutants, and chromatin immunoprecipitation showed that PKCβII activation was not involved in NF-κB translocation, but facilitated NF-κB-mediated CCL11 transcription by colocalizing with and phosphorylating p/CAF, and thereby acetylating histone H4 and promoting p65 association with the CCL11 promoter. The effect was dependent on p/CAF’s HAT activity. Furthermore, mouse embryonic fibroblasts from PKCβ knockout mice showed markedly reduced TNF-α-induced CCL11 expression and NF-κB reporter activity that was restored on PKCβII overexpression, suggesting a critical role for this pathway. These data suggest a novel important biological role for PKCβΙΙ in NF-κB-mediated CCL11 transcription by p/CAF activation and histone H4 acetylation.

Mass Spectrometric Studies on Epigenetic Interaction Networks in Cell Differentiation

“…This correlation was also underscored by a previous observation in which PKC␤ was found to be down-regulated in differentiated F9 cells induced by ATRA (54). More recently, PKC␤ has been shown to play a critical role in NF-B-mediated CCL11 transcription by phosphorylation and activation of the p300/CBP and its associated factor (p/CAF), resulting in histone hyperacetylation (55). In contrast, a down-regulated PKC␤, as demonstrated in the PMA-induced differentiated U937 cells, would be correlated with a down-regulation and/or inactivation of p300/CBP.…”

TNF and IFN Synergistically Enhance Transcriptional Activation of CXCL10 in Human Airway Smooth Muscle Cells via STAT-1, NF- B, and the Transcriptional Coactivator CREB-binding Protein

“…␤ 2 -Microglobulin was used validated for use in this assay and used as the housekeeping gene. 1 ng of reverse-transcribed cDNA was subjected to real-time PCR using Excite Real-time Mastermix with SYBR Green (Biogene, Cambridge, UK) and the ABI Prism 7700 detection system (Applied Biosystems, Warrington, Cheshire, UK) as described previously (25). CXCL10 expression was normalized to the housekeeping gene by dividing the mean of the CXCL10 triplicate value by the mean of the ␤ 2 -microglobulin triplicate value.…”

“…Transient Transfection of CXCL10 Promoter-driven Luciferase Constructs-All transient transfections were conducted by using FuGENE 6 according to the recommended protocol of the manufacturer and as described previously (25). A 1:3 ratio was used; 0.4 g/well plasmid per 1.2 l FuGENE 6, together with 0.8 ng/well of Renilla luciferase reporter/well as an internal control.…”

“…Western Blot Analysis-HASM cells were treated for times indicated in the text, cells were washed, and proteins were extracted as described previously (25). Anti-human pSTAT-1, total STAT-1, or pJAK1 and pJAK2 polyclonal antibody were used at 1:1,000 dilution.…”

“…The ChIP assay was performed using the ChIP-IT kit (Active Motif, Rixensart, Belgium) following the manufacturer's protocol and as described previously (25). 4 g of antibody or IgG control was used for each immunoprecipitation and stimulation parameter.…”