Members of the Bcl-2 and Caspase Families Regulate Nuclear Degeneration during Chick Lens Fibre Differentiation

Abstract

The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis.

Caspase-dependent secondary lens fiber cell disintegration inαA-/αB-crystallin double-knockout mice

“…However, according to our preliminary caspase screening experiments with a wide spectrum of caspase inhibitors, caspase 6 plays a similar, or even more important, role than caspase 3 in secondary lens fiber cells maturation. A role for caspase 6 in lens secondary fiber cell maturation was suggested earlier (Wride et al, 1999;Foley et al, 2004), however, some data suggest that VEIDase activity in the lens is not attributed to caspase 6 (Zandy et al, 2005). Nevertheless, possible inhibition of caspase 6 by ␣-crystallin had not been previously described.…”

Blind cavefish and heat shock protein chaperones: a novel role for hsp90alpha in lens apoptosis

“…In this light, it must be considered that lens fiber cells undergo a unique process resembling apoptosis during normal differentiation, which is required for the elimination of organelles and optical clarification (Lang, 1997;Wride, 2000). Accordingly, bcl-2 and caspase family proteins are normally expressed in the chick lens, and bcl-2 overexpression and caspase inhibitors can block fiber cell differentiation (Wride et al, 1999). Therefore, hsp90α activation could function somewhere in the cell death pathway downstream of caspase-3 to drive the lens apoptotic process to completion.…”

Caspase Remodeling of the Spectrin Membrane Skeleton during Lens Development and Aging

“…In addition, members of the caspase family (1-4, 6) and the caspase substrates, DNA fragmentation factor and poly(ADPribose) polymerase, have been identified in the lens (46). Like spectrin, poly(ADP-ribose polymerase has also been reported to be cleaved late in lens development, after organelle loss (46).…”