Abstract
Abstract: :
Purpose: To investigate the mechanisms by which Pinin (Pnn) regulates cell adhesion and pre–mRNA processing, we employed the RNA interference technique. Pnn is a 140kD phosphoprotein, previously shown to play an important role in regulating cell–cell adhesion of corneal epithelial cells. It binds to keratin and its association with preformed desmosomes enhances desmosomal stability and keratin filament organization. Over expression of Pnn is associated with increased E–cadherin expression. We have shown that nuclear Pnn localizes to nuclear speckles and binds to several SR proteins including SRp75, SRm300, and SRrp130, proteins thought to play a role in pre–mRNA processing. Pnn can also bind to the transcriptional co–repressor CtBP and thereby potentially regulate the transcription of genes including E–cadherin. We investigated whether "knocking down" Pnn expression in corneal epithelial cells would lead to altered epithelial integrity. Methods: Cultured human corneal epithelial cells (HCE–T, RCB1384, K Sasaki) were cotransfected with a short hairpin RNA–expressing vector (shRNA) containing an inverted repeat of Pnn–specific 21 nucleotide sequences and a GFP vector as a transfection marker. After 24–72 hours, cells were fixed and immunostained with antibodies against Pnn, keratins, desmoplakin, desmoglein, E–cadherin, SR–proteins, and SRm300. Western blot analyses of proteins with which Pnn interacts or influences, were performed. Results: Transfection of Pnn shRNA–expressing vector resulted in reduced levels of both Pnn and E–cadherin. Epithelial monolayer integrity was disrupted with lower levels of desmoplakin and desmoglein observed at cell–cell junctions. Aberrant keratin intermediate filament organization was observed at the cell boundary and keratin filaments appeared to condense in the perinuclear region. Decreased Pnn expression was also associated with fewer, larger and denser nuclear speckle staining for SRm300, reminiscent of the speckle morphology observed after a transcriptional block. Conclusions: "Knocking down" Pnn expression by shRNA technology does indeed result in decreased cell–cell adhesion and aberant cytoskeletal organization and suggests that Pnn may be involved in the organization of nuclear multi–protein complexes influencing transcription and pre–mRNA processing. (Supported by NIH grant EY07883).
Keywords: cornea: basic science • cell adhesions/cell junctions • gene/expression