The CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages

Abstract

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21(-/-) mice. Analysis of isolated macrophages, which are one of the central producers of IL-1beta, reveals that deficiency for p21 led to more IL-1beta mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1beta pro-protein is associated with elevated secretion of active IL-1beta by p21(-/-) macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1beta secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1beta by p21(-/-) macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21((WAF1/CIP1)) in inhibiting inflammation.

Figure 1. p21^−/− mice develop severe LPS-induced endotoxic shock

(A) Survival was monitored over seven days in Wt (n=14) or p21^−/− (n=20) mice intraperitoneally injected with LPS (10mg/kg of body weight). Data shown are representative of two independent combined experiments. Values represent the percent survival compared by chi square analysis. (B) Circulating levels of IL-1β were measured in serum from Wt or p21^−/− mice following LPS injection as in (A). Serum was isolated at 0 (WT, n=12; p21^−/−, n=12) and 24 (WT, n=6; p21^−/−, n=13) hours post injection and IL-1β levels were analyzed using a Luminex based assay. Data shown are representative of two combined independent experiments. Values show mean ± SE (Student's t-test). (C) Survival following treatment with LPS as in (A) was monitored over five days in Wt (n=10) or p21^−/− (n=10) mice or a cohort of p21^−/− mice (n=10) treated with anakinra as described in the Materials and methods. Data shown are representative of an individual experiment. Values represent the percent survival compared by chi square analysis.

Figure 2. Increased production of IL-1β in p21^−/− macrophages

(A) IL-1β mRNA levels were analyzed by RT-PCR (performed in duplicate and normalized to GAPDH) following LPS (10 ng/mL) stimulation and RNA harvest from triplicate BMDM cultures per genotype. Shown is the fold increase for IL-1β expression normalized to unstimulated expression levels. (B) Pro-IL-1β protein and GAPDH levels were assayed in protein extracts isolated 12 hours following LPS stimulation (10 ng/mL) by immunoblot. (C) IL-1β secretion by BMDM from WT or p21^−/− mice stimulated with LPS (10 ng/mL) and ATP (5 mM) as described in the Materials and methods was measured by ELISA. Supernatants were collected from at least three individual wells per genotype per timepoint and data were normalized to the number of cells per well. (D) Secretion of biologically active IL-1β by Wt or p21^−/− macrophages stimulated as in (C) was assayed as described in the Materials and methods. (E) IL-1β secretion by Wt or p21^−/− peritoneal macrophages treated as described in (C) was analyzed by ELISA. (F) IL-1β secretion by human macrophages transfected with non-specific or p21 siRNA as described in the Materials and methods and stimulated with LPS and ATP as in (C) was measured by ELISA. Knockdown of p21 was confirmed by western blot analysis (insert). (A-F) Data shown are representative of at least two independent experiments. Values represent the mean ± SE per time point, compared by Student's t-test (*p<0.04).

Figure 3. The CDK binding domain of p21 is required to suppress IL-1β synthesis

(A) Schematic of the cyclin, CDK, and PCNA binding domains on p21 protein. (B) Secretion of IL-1β by p21^−/− BMDM pretreated with saline or p21-mimetic peptide (10 μM) for two hours, stimulated with LPS (10 ng/mL) in media containing saline or p21-mimietic peptide for 12 hours, and activated with ATP as described in the Materials and methods was measured by ELISA. Data shown are representative of two combined independent experiments. Values represent the mean ± SE per time point normalized by cell count (Student's t-test).