NAD+ consumption by PARP-1 increases p65 acetylation and NF-κB activation.
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NAD+ addition and PARP-1 inhibition prevent p65 acetylation and NF-κB activation.
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Sirtuin inhibition increases p65 acetylation and NF-κB activation.
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PARP-1 depletion does not affect p65 nuclear translocation.
Abstract
NF-κB is a transcription factor that integrates pro-inflammatory and pro-survival responses in diverse cell types. The activity of NF-κB is regulated in part by acetylation of its p65 subunit at lysine 310, which is required for transcription complex formation. De-acetylation at this site is performed by sirtuin 1(SIRT1) and possibly other sirtuins in an NAD+ dependent manner, such that SIRT1 inhibition promotes NF-κB transcriptional activity. It is unknown, however, whether changes in NAD+ levels can influence p65 acetylation and cellular inflammatory responses. Poly(ADP-ribose)-1 (PARP-1) is an abundant nuclear enzyme that consumes NAD+ in the process of forming (ADP-ribose)polymers on target proteins, and extensive PARP-1 activation can reduce intracellular NAD+ concentrations. Here we tested the idea that PARP-1 activation can regulate NF-κB transcriptional activity by reducing NAD+ concentrations and thereby inhibiting de-acetylation of p65. Primary astrocyte cultures were treated with the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) to induce PARP-1 activation. This resulted in sustained acetylation of p65 and increased NF-κB transcriptional activity as monitored by a κB-driven eGFP reporter gene. These effects of MNNG were negated by a PARP-1 inhibitor, in PARP-1−/− cells, and in PARP-1−/− cells transfected with a catalytically inactive PARP-1 construct, thus confirming that these effects are mediated by PARP-1 catalytic activity. The effects of PARP-1 activation were replicated by a SIRT1 inhibitor, EX-527, and were reversed by exogenous NAD+. These findings demonstrate that PARP-1-induced changes in NAD+ levels can modulate NF-κB transcriptional activity through effects on p65 acetylation.
