Knockdown of Claudin-19 in the Retinal Pigment Epithelium Is Accompanied by Slowed Phagocytosis and Increased Expression of SQSTM1

PURPOSE: Besides regulating paracellular diffusion, claudin-19 modulates the expression of proteins essential for the retinal pigment epithelium (RPE). This study asks how RPE responds when the expression of claudin-19 is reduced. METHODS: In stem cell-derived RPE, claudin-19 and sequestosome-1/p62 (SQSTM1) were knocked down with siRNAs. Expression was monitored by quantitative RT-PCR and western blotting. Morphology and function were monitored by immunocytochemistry and transepithelial electrical resistance (TER). Phagocytosis of photoreceptor outer segments (POSs) was followed by fluorescence-activated cell sorting and western blotting. Pharmacology was used to assess the effects of AMP-activated protein kinase (AMPK) and SQSTM1 on phagocytosis. Enzymatic activity was measured using commercial assay kits. RESULTS: Knockdown of claudin-19 reduced the TER without affecting the integrity of the apical junctional complex, as assessed by the distribution of zonula occludens-1 and filamentous actin. AMPK was activated without apparent effect on autophagy. Activation of AMPK alone had little effect on phagocytosis. Without affecting ingestion, knockdown reduced the rate of POS degradation and increased the steady-state levels of LC3B and SQSTM1. Proteasome inhibitors also retarded degradation, as did knockdown of SQSTM1. The expression of metallothioneins and the activity of superoxide dismutase increased. CONCLUSIONS: Knockdown of claudin-19 slowed the degradation of internalized POSs. The study questions the role of activated AMPK in phagocytosis and suggests a role for SQSTM1. Further, knockdown was associated with a partial oxidative stress response. The study opens new avenues of experimentation to explore these essential RPE functions.