C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

PURPOSE: In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. METHODS: For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. RESULTS: Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. CONCLUSIONS: DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

Characterization of DeltaNp63 isoforms in normal cornea and telomerase-immortalized human corneal epithelial cells.

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 expression in the human corneal epithelium.

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 was 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=0.014) 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 24h (P<0.001) and TUNEL positive cells at 24 and 48 h (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 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.

Corin gene minor allele defined by 2 missense mutations is common in blacks and associated with high blood pressure and hypertension.

BACKGROUND: The natriuretic peptide system contributes to blood pressure regulation. Atrial and brain natriuretic peptides are cleaved into smaller biologically active molecules by corin, a transmembrane serine protease expressed in cardiomyocytes. METHOD AND RESULTS: This genotype-phenotype genetic association study included replication samples and genomic control to correct for population stratification. Sequencing of the human corin gene identified 2 nonsynonymous, nonconservative single nucleotide polymorphisms (Q568P and T555I) in near-complete linkage disequilibrium, thus describing a single minor I555 (P568) corin gene allele. This allele was present in the heterozygote state in &12% of blacks but was extremely rare in whites (<0.5% were homozygous for the minor allele). In our primary population sample, the Dallas Heart Study, after adjustment for potential confounders, including population stratification, the corin I555 (P568) allele remained independently associated with increased risk for prevalent hypertension (odds ratio, 1.63; 95% CI, 1.11 to 2.38; P=0.013). The corin I555 (P568) allele also was associated with higher systolic blood pressure in subjects not using antihypertensive medication in unadjusted (133.7+/-20.7 versus 129.4+/-17.4 mm Hg; P=0.029) and adjusted (132.5+/-1.6 versus 128.9+/-0.6 mm Hg; P=0.029) analyses. The independent association of the minor corin allele with increased risk for prevalent hypertension was confirmed in the Multi-Ethnic Study of Atherosclerosis (odds ratio, 1.50; 95% CI, 1.09 to 2.06; P=0.014). In addition, the association of the minor corin I555 (P568) allele with higher systolic blood pressure was confirmed in adjusted analysis in the Chicago Genetics of Hypertension Study (125.8+/-1.9 versus 121.4+/-0.7 mm Hg; P=0.03). CONCLUSIONS: The corin I555 (P568) allele is common in blacks and is associated with higher blood pressure and an increased risk for prevalent hypertension.

The damage-responsive Drosophila gene sickle encodes a novel IAP binding protein similar to but distinct from reaper, grim, and hid.

In flies and mammals, critical regulators of cell death function by antagonizing Inhibitor of Apoptosis Proteins (IAPs), which themselves directly block caspase action. The three currently known IAP antagonists in Drosophila map to the H99 genomic interval required for all programmed cell death. Here we describe a fourth member of this genetic group, sickle (skl), which maps just outside of the H99 deletion. At its N terminus, Skl shares residues in common with other IAP antagonists in flies (Rpr, Grim, and Hid) and in mammals (Smac/DIABLO and Omi/Htra2). Like other activators of apoptosis mapping in the Reaper region, full-length skl induced apoptosis when overexpressed, and the N terminus of this protein specifically bound to the BIR2 domain of DIAP1. However, unlike the N termini of Grim, Hid, and Rpr, the N terminus of Skl did not induce apoptosis. skl transcripts accumulate in cells that are fated to die in some but not all regions of the embryo. Genotoxic stimuli induced skl expression, but skl was not responsive to all signals that trigger premature apoptosis. skl is potentially a fourth IAP antagonist in the "Reaper region" and a new candidate transducer of apoptotic damage signaling in Drosophila.