Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas
Introduction
Type 1 diabetes is an autoimmune disease in which mononuclear cell infiltration of the pancreatic islets of Langerhans (‘insulitis’) leads to destruction of the insulin-producing β cells [1]. Chromatin condensation, DNA fragmentation and Fas expression observed in β cells during insulitis implicate β-cell apoptosis as one mode of β-cell death [2], [3], [4]. The insulitis lesion is a rich source of pro-apoptotic cytokines such as IL-1β, IFN-γ and TNF-α, and of the radical NO produced by cytokine-induced iNOS [5]. The combination of IL-1β and IFN-γ provokes β-cell dysfunction, DNA-damage and apoptosis by activation of networks of transcription factors and genes [6].
Cytokine-induced expression of the Fas receptor sensitises β cells to FasL-induced apoptosis [7], [8]. Studies with well-established models of apoptosis demonstrate that triggering of Fas by FasL rapidly induces cell death by recruitment of the adaptor protein Fas-associated death domain (FADD), formation of the death-inducing signalling complex (DISC) and activation of the caspase cascade [9]. Caspases cleave proteins after an aspartate residue, specificity residing in a four-residue sequence upstream of the cleavage site which, for the main effector caspase, caspase-3, is DEVD [10], [11].
To investigate the role of caspase-3 in β-cell death, we asked whether cytokine- and/or FasL-induced apoptosis is associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Moreover, we compared the content of active caspase-3 in cytokine-treated islets and insulinoma cells with that of Dex-treated thymocytes.
Section snippets
Reagents
Super-FasL, general caspase inhibitor (ZVAD), caspase-3 substrate (DEVD-pNA) and caspase-3 inhibitor (DEVD-fmk) used for colorimetric detection of caspase-3-like activity were purchased from Alexis (San Diego, CA). Bioluminescent caspase-3 activity assay (CleavaLite™) was from Chemicon International (Temecula, CA). Active caspase-3 set containing fluorogenic substrate (AC-DEVD-AFC), recombinant caspase-3 and caspase-3 inhibitor (AC-DEVD-CHO) as well as polyclonal rabbit anti-caspase-3 antibody
Comparison of colorimetric, fluorogenic and bioluminescent cleavage assays for detection of caspase-3-like activity
To establish a sensitive method for detecting caspase-3-like activity in insulinoma cells and islets, we compared cleavage assays based on the peptide substrates DEVD-pNA, DEVD-AFC and CleavaLite™, a bioluminescent substrate containing the DEVD motif (Fig. 1). Active recombinant human caspase-3 was applied as source of enzyme activity and signals monitored at concentrations ranging in log2-steps from 200 to 0.78 ng/ml. The resulting curve was analysed to obtain assay parameters. All assays
Discussion
In autoimmune diabetes a variety of mechanisms contribute to β-cell death [1], [6]. Prominent are pro-inflammatory cytokines, that may be directly cytotoxic to β cells or may act indirectly to upregulate Fas receptor and sensitise for FasL–Fas-induced apoptosis [13]. To investigate the activation of caspase-3 by the cytokine combination IL-1β and IFN-γ alone or in presence of FasL, we first compared assays to detect caspase-3-like activity in NIT-1 insulinoma cells and NOD mouse islets. The
Acknowledgements
The advice of Dr. Birgit Finke for the fluorometic detection method is gratefully acknowledged. We thank Mrs. Christel Salzsieder for excellent technical assistance.
This work was supported by grants from the Deutsche Forschungsgemeinschaft AU 151/1-1, 1-2 and from the Ministerium für Bildung, Wissenschaft und Kultur Mecklenburg-Vorpommern IDK 97 007 80/SOM and IDK 97 007 80/HSP III.
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