C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs

Abstract

The genesis of carcinoma cells often involves epithelial-to-mesenchymal transitions and the acquisition of apoptosis resistance, but it is unclear whether these alterations are controlled coordinately or independently. Our previously reported effects of adenovirus E1a in human tumor cells raised the possibility that the E1a-interacting corepressor protein C-terminal-binding protein (CtBP) might selectively repress epithelial cell adhesion and proapoptotic genes. Here, we report that CtBP-knockout cells were hypersensitive to apoptosis. Correspondingly, microarray analysis of CtBP-knockout vs. CtBP-rescued mouse embryo fibroblasts revealed that many epithelial-specific and proapoptotic genes were indeed regulated by CtBP. Neither the apoptosis nor the repression activities of CtBP required histidine-315, suggesting that the proposed dehydrogenase activity is not essential for CtBP function. The results presented herein establish two functional roles of CtBP: to corepress epithelial genes, thus permitting epithelial-to-mesenchymal transitions, and to modulate the cellular threshold for apoptotic responses.

Figure 1

Epithelial phenotype-related proteins are regulated by CtBP. Equal amounts of total cellular protein from CtBP1,2^−/− cells (−/−), cells rescued with vector alone (vec−/−), cells rescued with wild-type CtBP1 [res.(wt)] or the H315Q mutant [res.(H315Q)], or heterozygous control cells (−/+) were assayed for expression of desmoglein-2 (DSG-2), plakoglobin (PG), keratin-8 (K8), E-cadherin (E-cad), or occludin (Occl) by Western blotting. Confirmation of mouse CtBP (left) or human CtBP (right) expression in the mixed populations of CtBP-rescued cells and an actin-loading control is also shown; CtBP expression in >95% of the rescued cells was additionally verified by immunofluorescence (data not shown). (Lower) Epithelial genes indicated by the microarray analysis to be both down-regulated in the CtBP1,2^−/+ and the CtBP-rescued cells, relative to the CtBP1,2^−/− cells, are shown. The complete microarray results are published as supporting information on the PNAS web site.

Figure 2

CtBP-knockout cells are hypersensitive to apoptosis and deficient in tumorigenicity after transformation by H-rasV12. (a) Anoikis. Cell lines described in Fig. 1 were assayed for anoikis-related caspase activation (Upper) or for the percentage of apoptotic nuclei (judged by DAPI staining, Lower) after 3 h of suspension (SUSP) or under control, attached conditions (ATT); caspase activities (DEVD cleavage) in a–e are in arbitrary fluorescence units (FU). (b) Etoposide. Cells were tested after 4 h of treatment with 50 μM etoposide. (c) UV irradiation. Cells were UV-irradiated (40 J/m²) and incubated for another 4 h. (d) Staurosporine. Cells were treated with the indicated doses of staurosporine for 4 h. (e) FASL. Cells were treated with 100 ng/ml FASL in the presence of enhancer antibody for 4 h under attached or suspended (Susp) conditions. (f) CtBP-knockout cells are less tumorigenic than heterozygous cells, after transformation by ras. Two stable clones resulting from expression of HA-H-rasV12 in CtBP1,2^−/− (CtBP90.5 and CtBP90.7) or CtBP1,2^−/+ cells (CtBP86.5 and CtBP86.9) were generated and found to express similar levels of HA-H-rasV12 (data not shown). These were tested for tumorigenicity in nude mice (Upper, data represent the average of the cell clones, each injected at six sites) and for growth kinetics under normal culture conditions (Lower).

Figure 3

CtBP-knockout up-regulates proapoptotic genes. (a) RT-PCR analysis of the indicated genes was performed under conditions of linear response to cDNA input amount. RNAs were derived from cells treated with 50 μM etoposide for 4 h. Similar results were observed without etoposide induction (data not shown). (b) Western blot analysis of selected proapoptotic genes from etoposide-treated cells. (c) CtBP does not affect transcriptional activation by p53 or p53-related proteins. Synthetic promoters containing wild-type or mutant p53/p63/p73-binding sites linked to a luciferase reporter (0.8 μg) were cotransfected with CtBP or with empty control vector (0.4 μg); this ratio of reporter to CtBP plasmid repressed the E-cadherin promoter by >90% (data not shown). Cell lysates were assayed for luciferase activity. (d) Proapoptotic genes identified by the microarray analysis as down-regulated in the CtBP1,2 ^−/+, and the CtBP-rescued cells (relative to the CtBP^−/− cells) are shown; different members of the same gene family were included.

Figure 4

Dehydrogenase activity of CtBP is not required for transcriptional repression. (a) The PERP or β-actin control promoters linked to luciferase (1.5 μg) were cotransfected with the indicated CtBP mutants, and activity of the luciferase reporters was assayed. (b) GST-E1a fusion proteins coupled to glutathione-Sepharose beads were incubated with His-tagged human CtBP or H315Q mutant. The bound material was dissolved in sample buffer and analyzed by Western blotting by using an antibody against the His-tag. Equal amounts of GST and GST-E1a proteins were used in each pull-down assay, as shown (Lower). (c) An SV40 promoter adjacent to four gal4-binding sites (1.5 μg) was assayed for repression by the gal4-CtBP fusion proteins (0.5 μg), as indicated.