Calpastatin Prevents NF-κB–Mediated Hyperactivation of Macrophages and Attenuates Colitis

Abstract

Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated and reconstituted with either CAST knockout (KO) or wild-type (WT) bone marrow and subjected to dextran sulfate sodium (DSS)-induced colitis. CAST KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared to WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNFα, and IFNγ mRNA levels compared to WT controls. Mechanistic investigations using siRNA and KO bone marrow to generate CAST deficient macrophages showed that CAST deficiency during activation with bacterial PAMPs including heat-killed E. faecalis or CpG DNA led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNFα secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.

Calpain-2 Inhibitor Therapy Reduces Murine Colitis and Colitis-associated Cancer

“…The protocol was carried out for 4 days, which was consistent with our previously observation that DSS induced calpain activity peaks at 3–5 days of treatment. 10 Results indicated that at 0.75 mg/kg of calpain-2 inhibitor the maximum amount of calpain activity inhibition was achieved (Figure 1A). Vehicle control had no effect on calpain activity (data not shown).…”

“…Cell pellets were lysed and analyzed for IκB and NFκB by Western blot as previously described. 10 In other experiments, mouse CT-26.WT and human HT-29 colon cancer cells were plated in 96-well plates at 10 3 cells/well with 20 μg/mL inhibitor or DMSO, followed by analyses for IκB and NFκB by Western blot as described above for BMDM. For proliferation assays, fresh media with 20 μg/mL inhibitor or DMSO were added every 2 d. Each day 4 wells were quantified using Celltiter MTS reagent (Promega) and the resulting signal was used to generate a proliferation curve.…”

“…Enzyme-linked immunosorbant assay (ELISA) analyses of cecum hemoglobin has been previously described. 10 Invasion assays were performed using 12 well 8 μm filter inserts with 40 μl matrigel. The matrigel was added to cold wells and incubated at 37°C for 30 minutes.…”

“…10 Calpastatin was found to increase as a physiological response to calpain driven inflammation and mechanistic investigations revealed a particularly important role for calpastatin in limiting macrophage activation and inflammatory pathology during colitis. Calpains are Ca 2+ -activated cysteine proteases that cleave specific targets to modulate cellular functions relevant to inflammation such as proliferation, migration, and apoptosis.…”

“…12 Following activation by Ca 2+ , calpain cleaves a specific subset of cellular proteins, including cytoskeletal proteins and proteins involved in signal transduction. 13 In macrophages, calpain-2 is the predominant isoform and calpastatin was shown to prevent the hyperactivation of macrophages and NFκB driven inflammatory mediator production during colitis 10,14 …”