PO-131 Over expression of extracellular matrix protein 1 (ECM1) in muscle-invasive bladder cancer

Abstract

IntroductionThe connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells to directly exchange ions and small molecules. The connexin channels assemble into distinct plasma membrane domains known as gap junctions. Intercellular communication via gap junctions has an important role in regulating cell growth, differentiation and in maintaining tissue homeostasis. The most ubiquitously expressed connexin isoform in human tissues, connexin 43 (Cx43), acts as a tumour suppressor in multiple tissue types and is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the regulation of Cx43 degradation remains poorly understood.Material and methodsThe cervical cancer cell lines HeLa and C33a were used as model systems. Silencing and ectopic overexpression of proteins were performed by siRNA and plasmid transfection, respectively. The Cx43 ubiquitination status was determined using immunoprecipitation and western blotting. Gap junction intercellular communication (GJIC) was measured using the scrape loading dye transfer assay. Cells were imaged using confocal microscopy.Results and discussionsHere, we identify a member of the NEDD4 (neural precursor cell-expressed developmentally downregulated gene 4) family of E3 ubiquitin ligases, termed ITCH, as a novel regulator of Cx43 degradation and GJIC. Depletion of ITCH resulted in increased Cx43 protein levels, gap junction size and GJIC. Ectopic overexpression of ITCH, but not a catalytically inactive ITCH mutant, led to decreased Cx43 protein levels and loss of gap junctions. The data further indicate that ITCH acts in concert with two other members of the NEDD4 family that previously have been shown to regulate Cx43 degradation, termed NEDD4 and SMURF2. Simultaneous depletion of NEDD4, SMURF2, and ITCH by siRNA was found to result in a significantly lower Cx43 ubiquitination level and reduced Cx43 degradation under basal conditions. The triple knock-down of these three E3 ubiquitin ligases was also found to strongly counteract the TPA (12-O-tetradecanoylphorbol 13-acetate)-induced degradation of Cx43.ConclusionThese data identify ITCH as a novel regulator of Cx43 degradation and GJIC. The data also indicate that ITCH acts together with NEDD4 and SMURF2 to mediate the basal and TPA-induced turnover of Cx43.