PROTECTIVE AND APOPTOTIC CASPASES CONTROL THE SURVIVAL OF THE RAT INITIAL SEGMENT THROUGH CLEAVAGE OF THE REACTIVE OXYGEN SPECIES GENERATOR PHOSPHOLIPASE A2

Abstract

The role of caspases is no longer restricted to cell death pathways. Caspases have been implicated in development, proper functioning of the immune system, and even in the protection of cells. The initial segment of the epididymis is a tissue that undergoes cell death within 12–18 hours after withdrawal of luminal testicular factors by efferent duct ligation (EDL). The signaling proteins controlling this region-specific cell death remain unknown. Attempts to identify caspases involved through Western Blotting have demonstrated that the initiator caspase 9 and the executioner caspase 3 are cleaved and activated in both the normal and EDL tissue and that the presence of these active caspases is confined to the most proximal region of the initial segment. Western Blotting of nuclear and cytoplasmic extracts has confirmed that cleaved caspase 3 is located in both the cytoplasm and nucleus of normal and EDL treated initial segment tissue. Immunohistochemistry has confirmed that upon 18 hour EDL, in addition to cytoplasmic cleaved caspase 3, there is an an increase in nuclear-localized cleaved caspase 3 staining suggesting that caspase 3 plays a role in initial segment cell death. To determine if cleaved caspase 3 plays a role in the protection of the initial segment in normal tissue, the caspase inhibitor Z-VAD FMK was injected into the initial segment of EDL and normal initial segment tissue. A trend was visible that seemed to suggest an increase in cell death in Z-VAD FMK treated initial segments following a 12 hour EDL. In addition, many cells within normal initial segment tissue injected with Z-VAD FMK appeared to undergo cell death and sloughing into the lumen, suggesting that inhibition of caspases could promote cell death. Lastly, Western Blotting confirmed that phospholipase A2, an enzyme that metabolizes phospholipids and generates reactive oxygen species through the generation of oxidative biproducts, is inactivated by caspases in the normal initial segment tissue through cleavage, and that this cleavage is inhibited in initial segments that have undergone EDL. These data suggest that prodeath caspases contribute to cell death in the initial segment and that a potentially separate population of caspases performs a protective function in the initial segment that involves cleavage and inhibition of the reactive oxygen species generator, phospholipase A2. Supported by NIH-NICHD HD045890 (poster)