Poly(ADP-ribose) polymerase-1-induced NAD(+) depletion promotes nuclear factor-κB transcriptional activity by preventing p65 de-acetylation

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.

Fig. 1

MNNG-induced PARP-1 activation is blocked by DPQ, but not NAD⁺. Immunoblots show formation of poly(ADP-ribose)-conjugated proteins (PAR). A, Ten-minute incubations with 100 µM MNNG induce robust PAR formation that is prevented by the PARP-1 inhibitor, DPQ (25 µM). Time points denote intervals following washout of MNNG. B, NAD⁺ (2.5 mM) does not prevent PAR formation. Representative of n = 3.

Fig. 2

NF-κB subunit p65 acetylation is affected by PARP-1 activation and NAD⁺ levels. A, MNNG (100 µM for 10 min) induces acetylation of NF-κB p65 in wild-type (wt) astrocytes. B, MNNG does not induce p65 acetylation in PARP-1 deficient astrocytes. C, MNNG induced p65 acetylation in wt astrocytes is blocked by both the PARP inhibitor DPQ (25 µM) and by medium supplementation with NAD⁺ (2.5 mM). Time points denote intervals following washout of MNNG. D, Graph shows quantified data of p65 acetylation in wt astrocytes (A and C) (^#p < 0.05 compared to control, *p < 0.05 compared to MNNG at each time point, n = 3–4.).

Fig. 3

NF-κB subunit p65 acetylation is increased by SIRT1 inhibition. A, Diagram showing the proposed relationships between PARP-1, SIRT1, NAD⁺, and NF-κB. NF-κB is normally sequestered in the cytosol. In canonical NF-κB activation [2], phosphorylation of the IκB subunit permits the p65/p50 dimer to translocate to the nucleus, where it is acetylated and binds with other proteins to form an activated transcription complex on gene promoter regions. The activated transcription complex is normally deactivated by NAD⁺-dependent deacetylation of the p65 subunit, catalyzed by SIRT-1. NAD⁺ levels are reduced by PARP-1 activation, thereby preventing this de-acetylation step and promoting gene transcription. This effect can be negated by either PARP-1 inhibition (DPQ) or by NAD⁺ repletion. The SIRT-1 inhibitor EX-527 also blocks de-acetylation of NF-κB, but this effect cannot be negated by NAD⁺. B, Astrocytes treated with the SIRT1 inhibitor EX-572 (10 µM) show accumulation of acetylated p65, and this effect is not blocked by NAD⁺. Representative of n = 3. C, Photomicrographs show NF-κB transcriptional activity in astrocytes transfected with a κB reporter gene driving eGFP expression. eGFP expression is increased by EX-527, and this effect is not blocked by NAD⁺. D, Graph shows quantification of eGFP expression. (*p < 0.05, n = 3).

Fig. 4

PARP-1 enzymatic activity is required for NF-κB transcriptional activation. A, NF-κB transcription activity detected in astrocytes transfected with an eGFP-expressing κB reporter gene. Photomicrographs were prepared at 1 and 24 h after MNNG exposures (100 µM for 10 min). MNNG triggers NF-κB activation in wild-type cells, but not in PARP-1^−/− cells or in wild-type cells treated with 25 µMDPQ or 2.5 mM NAD⁺. B, PARP-1^−/− cells exhibit NF-κB transcriptional activation when transfected normal human PARP-1 (hPARP-1) or with hPARP-1 containing the S372E phosphomimetic mutation, but not when transfected with hPARP-1 containing the S372A and T373A mutations that prevent enzymatic activation. C and D, eGFP expression quantification. For C, ^#p < 0.05 compared to control, *p < 0.05 compared to MNNG, n = 3. For D, *p < 0.05 compared to PARP-1^−/− cultures transfected with hPARP-1, n = 3. Where no bar is visible, there was no detectable eGFP expression.

Fig. 5

PARP-1 depletion does not affect NF-κB p65 expression or nuclear translocation. A, Immunoblots showing p65 expression in wild-type and PARP^−/− cells. B, Confocal images demonstrate NF-κB p65 subunit nuclear translocation 30 min after MNNG stimulation (100 µM for 10 min) in both wild-type and PARP-1^−/− cells. Representative of n = 3.