RESUMO
The IκB kinase (IKK)-NF-κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double-strand breaks elicit two subsequent phases of NF-κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first-phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re-expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF-κB family member p65/RelA, in part mediated by GSK3ß, results in transcriptional silencing of NFKBIA and IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.
Assuntos
Senescência Celular/fisiologia , Quinase I-kappa B/metabolismo , Inibidor de NF-kappaB alfa/biossíntese , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/genética , Animais , Apoptose/genética , Linhagem Celular , Proliferação de Células/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Feminino , Inativação Gênica/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
The IκB kinase (IKK)-NF-κB signaling pathway plays a multifaceted role in inflammatory bowel disease (IBD): on the one hand, it protects from apoptosis; on the other, it activates transcription of numerous inflammatory cytokines and chemokines. Although several murine models of IBD rely on disruption of IKK-NF-κB signaling, these involve either knockouts of a single family member of NF-κB or of upstream kinases that are known to have additional, NF-κB-independent, functions. This has made the distinct contribution of NF-κB to homeostasis in intestinal epithelium cells difficult to assess. To examine the role of constitutive NF-κB activation in intestinal epithelial cells, we generated a mouse model with a tissue-specific knockout of the direct inhibitor of NF-κB, Nfkbia/IκBα. We demonstrate that constitutive activation of NF-κB in intestinal epithelial cells induces several hallmarks of IBD including increased apoptosis, mucosal inflammation in both the small intestine and the colon, crypt hyperplasia, and depletion of Paneth cells, concomitant with aberrant Wnt signaling. To determine which NF-κB-driven phenotypes are cell-intrinsic, and which are extrinsic and thus require the immune compartment, we established a long-term organoid culture. Constitutive NF-κB promoted stem-cell proliferation, mis-localization of Paneth cells, and sensitization of intestinal epithelial cells to apoptosis in a cell-intrinsic manner. Increased number of stem cells was accompanied by a net increase in Wnt activity in organoids. Because aberrant Wnt signaling is associated with increased risk of cancer in IBD patients and because NFKBIA has recently emerged as a risk locus for IBD, our findings have critical implications for the clinic. In a context of constitutive NF-κB, our findings imply that general anti-inflammatory or immunosuppressive therapies should be supplemented with direct targeting of NF-κB within the epithelial compartment in order to attenuate apoptosis, inflammation, and hyperproliferation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Apoptose , Doenças Inflamatórias Intestinais/metabolismo , Intestino Delgado/metabolismo , Inibidor de NF-kappaB alfa/deficiência , Celulas de Paneth/metabolismo , Células-Tronco/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Intestino Delgado/patologia , Camundongos Knockout , Inibidor de NF-kappaB alfa/genética , Organoides/metabolismo , Organoides/patologia , Celulas de Paneth/patologia , Células-Tronco/patologia , Fator de Transcrição RelA/metabolismo , Via de Sinalização WntRESUMO
The IκB kinase (IKK) is considered to control gene expression primarily through activation of the transcription factor NF-κB. However, we show here that IKK additionally regulates gene expression on post-transcriptional level. IKK interacted with several mRNA-binding proteins, including a Processing (P) body scaffold protein, termed enhancer of decapping 4 (EDC4). IKK bound to and phosphorylated EDC4 in a stimulus-sensitive manner, leading to co-recruitment of P body components, mRNA decapping proteins 1a and 2 (DCP1a and DCP2) and to an increase in P body numbers. Using RNA sequencing, we identified scores of transcripts whose stability was regulated via the IKK-EDC4 axis. Strikingly, in the absence of stimulus, IKK-EDC4 promoted destabilization of pro-inflammatory cytokines and regulators of apoptosis. Our findings expand the reach of IKK beyond its canonical role as a regulator of transcription.
Assuntos
Quinase I-kappa B/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Quinase I-kappa B/genética , Complexos Multiproteicos/genética , Proteínas/genética , RNA Mensageiro/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
The transcription factor NF-κB controls key features of hair follicle (HF) development, but the role of NF-κB in adult HF cycle regulation remains obscure. Using NF-κB reporter mouse models, strong NF-κB activity was detected in the secondary hair germ of late telogen and early anagen HFs, suggesting a potential role for NF-κB in HF stem/progenitor cell activation during anagen induction. At mid-anagen, NF-κB activity was observed in the inner root sheath and unilaterally clustered in the HF matrix, which indicates that NF-κB activity is also involved in hair fiber morphogenesis during HF cycling. A mouse model with inducible NF-κB suppression in the epithelium revealed pelage hair-type-dependent functions of NF-κB in cycling HFs. NF-κB participates in telogen-anagen transition in awl and zigzag HFs, and is required for zigzag hair bending and guard HF cycling. Interestingly, zigzag hair shaft bending depends on noncanonical NF-κB signaling, which previously has only been associated with lymphoid cell biology. Furthermore, loss of guard HF cycling suggests that in this particular hair type, NF-κB is indispensable for stem cell activation, maintenance, and/or growth.