The IjB kinase (IKK)-NF-jB 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-jB 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 IjBa, and is terminated through IjBa 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-jB family member p65/ RelA, in part mediated by GSK3b, results in transcriptional silencing of NFKBIA and IKK-independent, constitutive activation of NF-jB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-jB activation with important implications for genotoxic cancer treatment.
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.
, and -L-5-methyl-2-deoxycytidine (EC 50 , 0.9 M). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the -L-cytidine derivatives was also assessed. In accordance with the cell culture data, -L-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 M. The cytotoxicities of some of the 4-NHOH-modified -L-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH 2 group. The 50% cytotoxic concentrations for -L-Hyd4C in HepG2 and HL-60 cells were 2,500 M and 3,500 M, respectively. In summary, our results demonstrate that at least -L-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.Complete and sustained suppression of viral replication remains the most important goal in the treatment of patients with chronic hepatitis B virus (HBV) infection. Nucleoside and nucleotide analogues have greatly improved the disease outcome for these patients and have also prevented hepatic decompensation or the development of hepatocellular carcinoma. While -L-2Ј,3Ј-dideoxy-3Ј-thiacytidine (3TC; lamivudine) has been approved for the treatment of HBV infections, a series of further -Lnucleosides are under clinical investigation as inhibitors of hepadnavirus infections. These include -L-2Ј,3Ј-dideoxy-3Ј-The most important advantage associated with some, but not all, of these -L-nucleosides seems to be a much higher selectivity for the viral target than for the cellular targets, resulting in reduced cytotoxicity. This quality of -L-nucleosides stimulated the search for new -L-nucleosides but also the synthesis of enantiomeric isomers of effective -D-nucleosides (7).Some of the unmodified pyrimidine and purine -L-nucleosides were synthesized for the first time more than 35 years ago (12, 28, 34) but were considered to be metabolized very poorly in mice (14). A systematic biochemical investigation of the -L-enantiomers was initiated later and demonstrated the antihuman immunodeficiency virus (HIV) activity of -L-2Ј,3Ј-dideoxycytidine (-L-ddC) in a cellular system (18). In addition, it was shown that -L-dT might be phosphorylated by herpes simplex virus type 1 (HSV-1) thymidine kinase and further, by cellular enzymes, to -L-dTTP (35).As a consequence, the effects of -L-dTTP on various cellular DNA polymerases, TdT, the Klenow fragment of Escherichia coli DNA polymerase I, and viral DNA polymerases such as HSV-1 DNA polymerase and HIV reverse transcriptase were investigated (6,32,38). We showed that -L-TTP does not influence the activity of any of the cellular DNA polymerases (␣, , ␥, ␦, or ε) or of HIV reverse transcriptase, but we demonstrated for the first time a strong inhibition of human and duck HBV DNA polymerases by -L-dTTP (concentrations resulting in 50% inhibition of HBV DNA polymerase activity [IC 50 ], 0.46 M and 1.0 M, respectively) (37). Investigations on the cellular level and in vivo experim...
Although the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in ‘+4/+5’ secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF–κB functions in SI epithelial self-renewal, mice or SI crypt organoids (‘mini-guts’) with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.
The IκB kinase (IKK) -NF-κB pathway is activated as part of the DNA damage response and controls both resistance to apoptosis and inflammation. How these different 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 first, rapidly activated phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of IκB and is terminated through IκBα (NFKBIA) reexpression. The second phase is activated days later in senescent cells but is independent of IKK and the proteasome. An altered phosphorylation status of p65, in part driven by GSK3β, results in transcriptional silencing of NFKBIA and IKKindependent, 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.
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