Drosophila activators of apoptosis mapping to the Reaper region function, in part, by antagonizing IAP proteins through a shared RHG motif. We isolated Reaper from the Blowfly L. cuprina, which triggered extensive apoptosis in Drosophila cells. Conserved regions of Reaper were tested in the context of GFP fusions and a second killing activity, distinct from the RHG, was identified. A 20 amino-acid peptide, designated R3, conferred targeting to a focal compartment and promoted membrane blebbing. Killing by the R3 fragment did not correlate with translational suppression or with reduced DIAP1 levels. Likewise, R3-induced cell deaths were only modestly suppressed by silencing of Dronc and involved no detectable association with DIAP1. Instead, a second IAPbinding domain, distinct from the R3, was identified at the C terminus of Reaper that bound to DIAP1 but failed to trigger apoptosis. Collectively, these findings are inconsistent with single effector models for cell killing by Reaper and suggest, instead, that Reaper encodes conserved bifunctional death activities that propagate through distinct effector pathways.

Previous reports have suggested that specific isoforms of the potential stem cell marker p63 may regulate corneal epithelial homeostatic renewal through control of cell proliferation. In this study, we characterized the presence of DeltaNp63 isoforms in telomerase-immortalized human corneal epithelial cells (hTCEpi) in comparison to normal human corneal epithelium to validate the hTCEpi cell line as a viable model for the study of p63 isoforms. We further examined roles for DeltaNp63 in proliferation and differentiation. For in vitro studies, hTCEpi cells were cultured in serum-free culture media and grown under 0.15 mM calcium or sequential 1.15 mM calcium/air-lifted culture. Fresh donor human corneal tissue was used to assess expression and localization in situ. mRNA and protein levels were assessed by real-time PCR, Immunofluorescence (IF) and Western blotting (WB). DeltaNp63 expression levels throughout the cell cycle were assessed by double-labeling with DeltaNp63 and Ki-67. In situ, DeltaNp63 localized to nuclei throughout the human corneal epithelium and was lost only in superficial cells. WB confirmed the presence of all three DeltaNp63 isoforms in the central corneal epithelium and in hTCEpi cells. DeltaNp63 mRNA levels decreased when grown on collagen substrate and under increased calcium/air-lifted culture. mRNA and protein levels increased as cells approached confluence, with a significant decrease in post-confluent culture. DeltaNp63 expression levels did not vary with the cell cycle, as assessed by Ki-67 labeling. Collectively, the presence of all three DeltaNp63 isoforms in hTCEpi cells and in intact cornea validates the use of this cell line for the study of individual isoforms in the corneal epithelium; and these data suggest that expression of DeltaNp63 isoforms are not altered as a function of the cell cycle or cell division in subconfluent hTCEpi cells cultured in serum-free media, but demonstrate reduced expression upon contact-inhibited growth down-regulation and differentiation. Significantly, the localization of DeltaNp63 in central corneal epithelial cells with a loss of expression in superficial cells suggests that DeltaNp63 may play a role in mediating desquamative events at the ocular surface.

Insulin-like Growth Factor Binding Protein-3 (IGFBP3) is a high affinity binding protein shown to regulate cell growth, differentiation, and apoptosis in a variety of cellular systems. The primary aim of this study was to characterize IGFBP3 expression in the human corneal epithelium and in a corneal epithelial cell line and to establish a potential role for IGFBP3 mediated apoptotic signaling in corneal epithelial cells.Using a telomerase-immortalized human corneal epithelial (hTCEpi) cell line cultured in serum-free media and fresh human eye bank donor tissue, expression and localization of IGFBP3 were established in situ and in vitro by indirect immunofluorescence and western blotting. Real time PCR was used to measure IGFBP3 mRNA levels following Trichostatin-A (TSA) treatment and as a function of confluence. IGFBP3 protein levels were assessed in resting human tears and in conditioned media by western blotting; as was the ability of recombinant human IGFBP3 protein to associate with the cell surface. Apoptotic signaling was assessed in vitro using TSA and recombinant human (rh)IGFBP3. Apoptosis was measured by Viability/Cytotoxicity, Annexin V, and TUNEL assays.IGFBP3 localized to the plasma membrane of human corneal epithelial cells in situ and was upregulated in surface cells in the central cornea. IGFBP3 was secreted in conditioned media of growing cells, with a robust upregulation following confluence (P<0.001) and differentiation. IGFBP3 was undetectable in human tears. Addition of TSA to the culture media resulted in an upregulation of IGFBP3 mRNA (P<0.001) and protein. In addition, TSA treatment led to a significant increase in Annexin V positive cells at 18 and 24 hours (P<0.001) and TUNEL positive cells at 24 and 48 hours (P<0.001). The addition of rhIGFBP3 to the cell culture media appeared to induce occasional membrane blebbing, but cells failed to become positive with Annexin V or TUNEL.Taken together, these results demonstrate that cell membrane-associated IGFBP3 is produced by corneal epithelial cells and associates with the plasma membrane of superficial cells in situ and in cultured cells, but is not present in human tears. The differential localization and effect(s) on apoptosis suggest that the effects of IGFBP3 are likely tissue compartment and receptor specific and may be regulated by glycosylation.