Focus: Detecting Caspase Activities in Tissue

Detecting Caspase Activities in Mouse Liver

Here we describe the use of the Caspase-Glo^® Assays (G8090, G8200, and G8210) to evaluate apoptosis in mouse liver tissue. The assays were performed using a hypotonic extraction buffer during liver tissue homogenization.

From the article: Liu, D. et al. (2004) J. Biol. Chem. 279, 48434–42.
Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

Introduction

This study investigated the role of cSN50 peptide on mouse liver apoptosis. cSN50 is an inhibitor of the nuclear import of stress-responsive transcription factors (SRTFs). Mouse animal model of acute liver injury treated with the cSN50 peptide before and after challenge with lipopolysaccharide (LPS) and 2-amino-2-deoxy-d-galactosamine (d-Gal) displayed a decrease in liver apoptosis compared to animals that were treated with vehicle only before and after challenge with LPS and d-Gal. This work included experiments demonstrating initiator caspase-8 and -9 and effector caspase-3/7 activities in liver tissue homogenates of mice undergoing LPS/d-Gal treatment.

Materials and Methods

Female C57BL/6 mice were injected intraperitoneally with LPS and d-Gal. The mice were then divided into two groups. The experimental group received the cSN50 peptide through seven intraperitoneal injections before and after being challenged with LPS and d-Gal. The control mice received injections of 5% DMSO (vehicle only).

To detect caspase-3/7, -8 and -9 activities in mouse livers, the authors used the Caspase-Glo^® 3/7, 8 and 9 Assays (Cat.# G8090, G8200 and G8210, respectively) with a slightly modified procedure. This modified procedure employed a hypotonic extraction buffer (25mM HEPES [pH 7.5], 5mM MgCl₂, 1mM EGTA, 1mM Pefabloc^® Reagent, and 1µg/ml each pepstatin, leupeptin and aprotinin) during Dounce homogenization of livers. After homogenization the extracts were cleared by centrifugation at 13,000rpm for 15 minutes at 4°C. Protein concentrations of the cleared extracts were adjusted to 1mg/ml prior to storage at –80°C. To perform the caspase assays, the authors mixed an equal volume of diluted (10µg/ml) extract with the appropriate Caspase-Glo^® Reagent in 96-well, white-walled plates. The assays were incubated for one hour at room temperature before reading on a plate-reading luminometer.

Results and Discussion

Data from the caspase-3/7 assays indicate that C57BL/6 mice treated with vehicle alone (5% DMSO; no peptide inhibitor) and then challenged with LPS and d-Gal showed increased levels of activated caspase-3 and -7 in liver tissue as assessed by the Caspase-Glo^® 3/7 Assay. The largest increase in caspase-3/7 activity occurred 6 hours after the challenge with LPS and d-Gal (Figure 1). Most interestingly, mice that had been treated with the cSN50 peptide did not display this same increase in liver cell apoptosis.

Figure 1. Caspase activity in liver tissue from cSN50 peptide treated and untreated mice challenged with d-Gal. Wildtype C57BL/6 mice were treated with cSN50 peptide (0.7mg in 200ul of 5% DMSO) or diluent before and after intraperitoneal administration of LPS with d-Gal according to the protocol described in Liu, D. et al. (2004). Caspase activities in liver were measured after LPS and d-Gal challenge in diluent controls (open bar) and cDN50 peptide-treated animals (solid bar). Error bars indicate the +/– standard error of the mean value in four mice that are represented by each data point. p values represent the significance of the difference between the control and the cSN50 peptide-treated groups (two-way ANOVA) RLU = relative light units. Reprinted with the kind permission of Dr. Jacek Hawiger, Vanderbilt University, and The Journal of Biological Chemistry.

The authors also describe their analysis of intrinsic and extrinsic apoptosis pathway induction by studying the activities of the initiator caspases, caspase-9 (intrinsic pathway) and caspase-8 (extrinsic pathway). The activities of both caspases were monitored in liver extracts at various time points (0–6 hours) after the LPS and d-Gal challenge. Both caspase-8 and -9 activities dramatically increased at 6 hours after treatment with LPS and d-Gal in the control mice that did not receive cSN50 peptide. Like caspase-3/7, caspase-8 and -9 activities decreased when mice were treated with the cSN50 peptide. The authors noted that, although inflammatory cytokine TNF-α levels increased within an hour of LPS/d-Gal treatment, caspase-8 activity in the liver did not increase until 6 hours into the treatment.

Based on liver caspase activity data and other supporting data the authors conclude that nuclear import of SRTFs is important for the initiation of apoptosis and liver injury caused by the release of cytokines by macrophages. This finding is ultimately important for understanding the biology and pathogenesis of massive liver apoptosis mediated through macrophage expression of inflammatory cytokines.