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Activation of Caspase-2 Mediates the Apoptosis Induced by GTP-Depletion in Insulin-Secreting (HIT-T15) Cells*

Cardiovascular Research Institute (J.H., R.-H.L., G.L.), National University Medical Institutes, National University of Singapore, Singapore 117597; and Pacific Northwest Research Institute (S.A.M.), Seattle, Washington 98122

Address all correspondence and requests for reprints to: Guodong Li, National University Medical Institutes, MD11 02-01, 10 Medical Drive, Singapore 117597, Singapore. E-mail: . nmiligd{at}nus.edu.sg

This study investigated the possible involvement of a specific caspase(s) (a family of aspartate-specific cysteine proteases) in programmed cell death of islet ß-cells due to sustained GTP depletion. Treatment (up to 48 h) with 3 µg/ml mycophenolic acid (MPA), which specifically depletes intracellular guanine nucleotides, reduced cell-cycle progression from G1 phase into S and G2/M phases (as assessed by flow cytometry) and, subsequently, induced apoptosis of HIT-15 cells (transformed pancreatic ß-cells). The latter was accompanied by a marked increase of caspase-2 activity (+343%) and moderate activation of caspase-9 (+150%) and caspase-3 (+145%). Importantly, only caspase-2 activation preceded induction of apoptosis. There was no change in activity of caspase-1, -4, -5, -6, and -8. Release of the mitochondrial protein cytochrome c into cytosol was also observed at a late stage. Cotreatment of cells with a permeable pan-caspase inhibitor (Z-VAD-FMK) blocked GTP depletion-induced cell death in a dose-dependent manner. A specific caspase-2 inhibitor (Z-VDVAD-FMK), but not a caspase-3 inhibitor (DEVD-CHO), was also capable of restoring cell viability. Interestingly, activation of caspase-2 leads to caspase-3 activation because the caspase-2 inhibitor abrogated caspase-3 activity. Our results indicate that, while activation of multiple caspases are involved in the execution phase of GTP depletion-induced apoptosis, caspase-2 appears to play the major role in the initiation of this program. This study revealed a novel, caspase-2 mediated form of apoptosis that may be consequent to impaired mitogenesis.

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