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  • br CDKs as Direct Coactivators of Proinflammatory Transcript

    2020-07-30


    CDKs as Direct Coactivators of Proinflammatory Transcription Factors CDKs fuel inflammation by triggering the function of proinflammatory transcription factors such as NF-κB, STAT3, and AP-1. This is achieved at two levels because CDKs can affect gene expression by targeting global transcription by phosphorylation of the RNA Pol II CTD and also in a highly specific manner upon direct association and regulation of proinflammatory transcription factors, as summarized in Figure 2A. A high-throughput kinome siRNA screen revealed that TNF-induced NF-κB activation was strongly impaired in the absence of either CDK2, CDK5, or CDK7 [28]. The knockdown of these CDKs does not affect other pathways, such as IL-6-induced SOCS3 (suppressor of cytokine signaling 3) expression, showing the specificity of CDK2, CDK5, and CDK7 for the NF-κB pathway. Support for a functional role of CDKs in NF-κB activation comes from a study in which the CDK inhibitors roscovitine and olomoucine strongly reduce lipopolysaccharide (LPS)-induced NF-κB activity in murine RAW264.7 macrophages [29]. In addition, CDK9 is required for inflammatory gene expression, as revealed in a seminal paper showing that RNAi against CDK9 blocks TNF-induced and NF-κB-dependent induction of IL-8 expression [30]. Moreover, CDK9 contributes to IL-6-regulated gene transcription 31, 32, 33. IL-6-induced binding of STAT3 to the promoter controlling the p21CIP1-encoding gene is followed by recruitment of CDK9 to ensure RNA Pol II CTD Ser2 phosphorylation and transcription elongation [33]. An independent study revealed IL-6-inducible association between tyrosine phosphorylated STAT3 and CDK9 at the γ-fibrinogen promoter in liver apexbio dilution [32]. CDK9 is also important for the TNF-inducible expression of a subset of NF-κB target genes. This process depends on inducible ATM (ataxia telangiectasia mutated)-mediated phosphorylation of the NF-κB subunit p65 at Ser276, which allows binding of CDK9 and further components of positive transcription elongation factor b (P-TEFb) 34, 35. This allows CDK9 to phosphorylate the CTD at Ser2 to promote transcription elongation. Recent results show that IL-1 triggers a signaling cascade which leads to the inducible recruitment of CDK6 to distinct chromatin regions of inflammatory genes [36]. The functional relevance of CDK6 or its functional homolog CDK4 for proinflammatory gene expression was revealed in knockdown experiments. Downregulation of CDK6 or CDK4 with specific short hairpin RNAs (shRNAs) suppresses the expression of 34–45% of all IL-1-induced genes, showing their broad relevance for the expression of inflammatory genes. Intriguingly, a comparison of genes expressed selectively in the cell apexbio dilution cycle G1 phase and in response to IL-1 showed strong overlap [36], suggesting that cytokine release is preferentially mediated by cells in the G1 phase. Chromatin immunoprecipitation sequencing (ChIP-Seq) experiments revealed the constitutive and IL-1-induced association of CDK6 with many genomic regions, most of which overlap with binding sites for p65. The importance of CDK6 for NF-κB-triggered gene expression stems from its ability to ensure proper IL-1-induced binding of p65 to its cognate sites in the chromatin. The molecular mechanisms allowing CDK6 to mediate p65 recruitment are not well understood and may involve the assistance from further factors such as TRIP6 (thyroid hormone receptor interacting protein 6) [36]. CDK6 is also important for TNF-induced NF-κB-dependent transcription, which is restricted by the transcription factor hypoxia-inducible factor 1α (HIF-1α). Depletion of HIF-1α augments NF-κB p65 transcriptional activity in a CDK6-dependent manner [37]. Downregulation of HIF-1α results in increased association of CDK6 and the coactivator CBP (CREB-binding protein) to the IL8 promoter, thus augmenting TNF-induced gene expression. Although CDK6 phosphorylates p65 at Ser536 [38], its kinase activity seems not to be required for the recruitment of p65, as revealed in reconstitution experiments with a kinase-inactive CDK6 mutant.