Reduction of Pnn by RNAi induces loss of cell-cell adhesion between human corneal epithelial cells.

PURPOSE Pinin (Pnn/DRS/memA) plays an important role in regulating cell-cell adhesion of corneal epithelial cells. In the nucleus, Pnn interacts with both transcriptional repressor and pre-mRNA processing machinery. Here we investigated the consequences of "knocking down" Pnn expression with short hairpin RNAi (shRNAi) on the corneal epithelial cell phenotype. METHODS Cultured human corneal epithelial (HCE-T) cells were cotransfected with a shRNAi-expressing construct containing an inverted repeat of a Pnn specific 21 nucleotide sequence (Pnn shRNAi) and a GFP vector as a marker of transfected cells. After 24-48 h, cells were fixed and immunostained with antibodies against Pnn, keratin, desmoplakin, desmoglein, E-cadherin, ZO-1, SR-proteins, and SRm300. To demonstrate specificity of the Pnn knock down, a rescue vector was designed by incorporating three conservative nucleotide substitutions within the Pnn-shRNAi targeting sequences of the full length Pnn-GFP construct, thus generating a Pnn construct to produce mRNA that Pnn shRNAi could not target (Pnn-CS3-GFP). RESULTS HCE-T cells were cotransfected with Pnn shRNAi and GFP vectors and after 24 and 48 h exhibited significantly reduced immunostaining for Pnn. Western blot analyses of Pnn and E-cadherin protein expression in cells transfected with Pnn-shRNAi and GFP vectors revealed marked reduction in levels of both proteins compared to those observed in cells transfected with GFP alone. The cells receiving Pnn-shRNAi appeared to be less adherent to neighboring nontransfected cells, often exhibited altered cell shape, downregulated cell adhesion and cell junction molecules, and escaped from the epithelium. The Pnn shRNAi transfected cells exhibited fewer keratin filaments anchored to desmosomes and a concurrent increase in the perinuclear bundling of filaments. SR proteins and SRm300 showed an altered distribution in the Pnn knock down cells. Cotransfection of Pnn-CS3-GFP with Pnn shRNAi demonstrated that the conservatively mutated Pnn could maintain cell-cell adhesion. CONCLUSIONS Our results indicate that knocking down Pnn expression leads to a loss of epithelial cell-cell adhesion, changes in cell shape, and movement of Pnn shRNAi transfected cells out of the epithelium. We suggest that Pnn plays an integral role in the establishment and maintenance of epithelial cell-cell adhesion via its activity within nuclear multi-protein complexes.