IKK-dependent, NF-κB-independent control of autophagic gene expression

Abstract

The induction of mammalian autophagy, a cellular catabolic bulk-degradation process conserved from humans to yeast, was recently shown to require IκB kinase (IKK), the upstream regulator of the nuclear factor (NF)-κB pathway. Interestingly, it was shown that this response did not involve NF-κB. Thus, the mechanism by which IKK promotes stimulus-induced autophagy is largely unknown. Here, we investigate the role of IKK/NF-κB in response to nutrient deprivation, the well-understood autophagy-inducing stimulus. IKK and both the classic and non-canonical pathways of NF-κB are robustly induced in response to cellular starvation. Notably, cells lacking either catalytic subunit of IKK (IKK-α or IKK-β) fail to induce autophagy in response to cellular starvation. Importantly, we show that IKK activity but not NF-κB controls basal expression of the proautophagic gene LC3. We further demonstrate that starvation induces the expression of LC3 and two other essential autophagic genes ATG5 and Beclin-1 in an IKK-dependent manner. These results indicate that the IKK complex is a central mediator of starvation-induced autophagy in mammalian cells, and suggest that this requirement occurs at least in part through the regulation of autophagic gene expression. Interestingly, NF-κB subunits are dispensable for both basal and starvation-induced expression of proautophagic genes. However, starvation-induced activation of NF-κB is not inconsequential, as increases in expression of antiapoptotic NF-κB target genes such as Birc3 are observed in response to cellular starvation. Thus, IKK likely has multiple roles in response to starvation by regulating NF-κB-dependent antiapoptotic gene expression as well as controlling expression of autophagic genes through a yet undetermined mechanism.

Figure 1. IKK/NF-κB signaling is activated by nutrient stress

(A) Wildtype mouse embryonic fibroblasts (WT mEFs) were starved in Hanks' Balanced Salt Solution (HBSS, Sigma: 55021C) for a two-hour timecourse and cells were harvested at the indicated time points for whole cell lysates (WCL). WCL were subjected to westernblot analysis for phosphorylated IκBα (phospho, Cell Signaling Technology (CST) 9246; total, CST: 4812), phosphorylated p65 (phospho, CST: 3033; total CST: 4764), and LC3 processing (CST: 3868). (B) Nuclear extracts were prepared for WT mEFs treated with a starvation timecourse for the indicated time points. NF-κB DNA binding was assessed by EMSA using a consensus κB oligonucleotide (Promega: E3292) (upper panel). Nuclear extracts from WT mEFs starved for 240 minutes were used for supershift anlysis of NF-κB complexes using antibodies against p65 and p50 (CST: 3034; Santa Cruz: 7178, respectively) (C) WT mEFs were starved for a 16-hour time course and total RNA was collected from cells at the indicated timepoints. Real Time PCR was used to assess the levels of the classic NF-κB target gene NFKBIA (Applied Biosystems Taqman gene expression assays, ABI: Mm00477798_m1). (D) WCLs were prepared from WT mEFs treated for starvation time course and activation of non-canonical NF-κB was interrogated by processing of p100 to p52 by westernblot analysis (CST: 4882).

Figure 2. IKK is required for basal and starvation-induced expression of pro-autophagic genes

(A) WT or IKK deficient mEFs (IKKβ -/-, IKKα-/-) were grown in basal or starvation media (HBSS) for 90 minutes and WCLs were prepared. Induction of autophagy was measured by LC3 processing. (B) WT, IKKβ -/-, and IKKα-/- cells were grown in basal or starvation media for 12 hours total RNA was collected for cDNA synthesis. Expression of LC3, ATG5, BECN1, and NFKBIA were measured using Taqman gene expression assays (ABI: LC3, Mm00458724_m1; BECN1, Mm01265461_m1; ATG5, Mm00504340_m1). Samples were normalized to GUSB expression (ABI: GUSB, Mm03003537_m1). Statistically significant differences were measured by Student's t-test (*<.05) (C) WT mEFs were treated with vehicle control or IKK inhibitor NEMO Binding Domain (NBD) peptide (100μM) 1 hour prior to starvation. Cells were then starved in the presence NBD peptide or control for 12 hours. Gene expression was measured by Real Time PCR analysis. Data is represented as fold-change over expression of cells grown in basal media (*<.05).

Figure 3. Basal and starvation-induced autophagic gene expression is NF-κB-independent

(A) p65 null (p65 -/-), p65/c-Rel double null (p65/c-Rel DKO) cells were grown in basal or starvation media for 18 hours and pro-autophagic gene expression was measured. (B) WT, RelB, and NFKB2 null (RelB-/-, NFKB2-/-) mEFs were grown in basal or starvation media for 12 hours and pro-autophagic gene expression was measured. Data is represented as fold-change over basal expression, *<.05.