Cutting edge: proteolytic inactivation of poly(ADP-ribose) polymerase 1 by the Nlrp3 and Nlrc4 inflammasomes

Abstract

Caspase-mediated cleavage of the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP1) is a hallmark of apoptosis. However, it remains unclear whether PARP1 is processed during pyroptosis, a specialized cell-death program that occurs upon activation of caspase-1 in inflammasome complexes. In this article, we show that activation of the Nlrp3 and Nlrc4 inflammasomes induces processing of full-length PARP1 into a fragment of 89 kDa in a stimulus-dependent manner. Macrophages deficient for caspase-1 and those lacking the inflammasome adaptors Nlrp3, Nlrc4, and ASC were highly resistant to cleavage, whereas macrophages lacking the downstream inflammasome effector caspase-7 were partially protected. A modest, but statistically significant, reduction in Nlrp3 inflammasome-induced pyroptosis was observed in PARP1 knockout macrophages. Thus, protease-mediated inactivation of PARP1 is a shared feature of apoptotic, necrotic, and pyroptotic cells.

Figure 1. PARP1 is cleaved during ATP- and nigericin-induced pyroptotic cell death of activated macrophages

A–D, BMDMs were primed with LPS, Pam3 or mannan for 3 h and then stimulated with ATP (A, B) or nigericin (C, D) for 30 min. (A, C) Cell extracts were immunoblotted with antibodies against PARP1 and caspase-1. The bands corresponding with full-length PARP1 (113 kDa), the 89 kDa PARP1 fragment, procaspase-1 (45 kDa) and the large catalytic subunit (20 kDa) are indicated. (B, D) Membrane permeabilization was measured as a cell death parameter using the Live/Dead assay (Invitrogen). Cell death data represent the mean ± standard deviation of triplicates. Results are representative of at least three independent experiments. LPS, Lipopolysaccharide; Pam3, Pam3-GSK4

Figure 2. The Nlrp3 and Nlrc4 inflammasomes mediate PARP1 processing during pyroptosis

(A, B) Macrophages from wild-type, Nlrp3^−/−, Nlrc4^−/−, Pycard^−/− and Casp1^−/− mice were primed with LPS, Pam3 or mannan for 3 h and then stimulated with ATP for 30 min or (C) infected for 4 h with Salmonella. Cell extracts were immunoblotted with antibodies against PARP1. The bands corresponding with full-length PARP1 (113 kDa) and the 89 kDa PARP1 fragment are indicated. Results are representative of three independent experiments. LPS, Lipopolysaccharide; Pam3, Pam3-GSK4

Figure 3. PARP1 is processed by caspases-1 and -7 and PARP1 inactivation protects against pyroptosis

(A, B) Macrophages from wild type, Casp1^−/− and Casp7^−/− mice were primed with LPS, Pam3 or Mannan for 3 h and then stimulated with ATP for 30 min. Cell extracts were immunoblotted with antibodies against caspase-7 (A) and PARP1 (B). The bands corresponding with respectively procaspase-7 (33 kDa) and its large catalytic subunit (19 kDa), and full-length PARP1 (113 kDa) and the 89 kDa PARP1 fragment are indicated. (C) Recombinant PARP1 was incubated with 30 nM recombinant caspase-1 or casapse-7 in protease assay buffer (20 mm HEPES-KOH pH 7.5, 10 mm KCl, 1.5 mm MgCl₂, 1 mm EDTA, 1 mm DTT and Complete protease inhibitor (Roche)). PARP1 cleavage products were detected by Western blotting with PARP1 antibodies. (D) Macrophages from WT and PARP1^−/− mice were primed with LPS for 3 h and then stimulated with ATP for indicated times. Membrane permeabilization was measured as a cell death parameter using the Live/Dead assay (Invitrogen). Cell death data represent the mean ± standard deviation of triplicates. Results are representative of three independent experiments.