M1689 The Endocrine Disruptor Bisphenol a (BPA) Decreases Paracellular Permeability in the Female Rat Colon At Environmentally Relevant Doses and Increases Tight Junction Protein Expression in CACO-2 Cells

Abstract

Early epithelial restitution is an important repair modality in the gut mucosa and occurs as a consequence of epithelial cell migration. Our previous studies have shown the canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and regulates early mucosal restitution. Increased TRPC1 channel activity increases store-operated Ca2+ entry (SOCE) and enhances IEC migration during restitution. However, the exact upstream signals initiating TRPC1 activation after mucosal injury remain elusive. Recently, stromal interaction molecule 1 (STIM1) has been identified as a store Ca2+ sensor and can rapidly translocate to the plasma membrane (PM) where it interacts with and modulates Ca2+ channels. We hypothesized that STIM1 is crucial for stimulation of IEC migration after wounding by activating TRPC1 channel activity.Methods: Studies were conducted in IEC-Cdx2L1 cells, which represent differentiated IECs. STIM1 subcellular distribution was analyzed by surface biotinylation assays and immunohistochemical staining, while its functions were investigated by specific siRNA (siSTIM1) and ectopic overexpression of constitutively active STIM1 EF-hand mutants. [Ca]cyt was measured by fluorescence digital imaging analysis. Cell migration was measured in a model that mimics cell division-independent epithelial restitution. Results: IECs highly expressed STIM1 in cultured cells and in the intestinal mucosa of the rat. STIM1 translocation to the PM increased significantly within 10 min after wounding, peaked between 30 to 60 min, and then gradually returned to normal. Maximum increases in levels of STIM1 at PM were ~six times the prewounding control, which was associated with a dramatic increase in SOCE. There were no changes in total STIM1 protein levels after wounding. STIM1 directly interacted with TRPC1 in the PM as measured by immunoprecipitation assays. Ectopic expression of constitutively active STIM1 EF-hand mutants increased SOCE (by ~60%) and also stimulated IECmigration (by ~35%) after wounding. In contrast, STIM1 silencing by siSTIM1 decreased SOCE, inhibited IEC migration, and delayed epithelial restitution in cells overexpressing TRPC1. Levels of Ca2+ influx due to SOCE were decreased by ~70% in STIM1-silenced populations, whereas cell migration was inhibited by ~45%. Conclusions: These findings indicate that 1) wounding increases STIM1 translocation to the PM in IECs and 2) STIM1 at the PM interacts with and activates TRPC1, thus stimulating epithelial restitution as a result of increase in SOCE.