Cytoskeletal Responses and Aif-1 Expression in Caco-2 Monolayers Exposed to Phorbol-12-Myristate-13-Acetate and Carnosine

Abstract

The dis(re)organization of the cytoskeletal actin in enterocytes mediates epithelial barrier dys(re)function, playing a key role in modulating epithelial monolayer’s integrity and remodeling under transition from physiological to pathological states. Here, by fluorescence-based morphological and morphometric analyses, we detected differential responses of cytoskeletal actin in intestinal epithelial Caco-2 cell monolayers at two different stages of their spontaneous differentiation, i.e., undifferentiated cells at 7 days post-seeding (dps) and differentiated enterocyte-like cells at 21 dps, upon challenge in vitro with the inflammation-mimicking stimulus of phorbol-12-myristate-13-acetate (PMA). In addition, specific responses were found in the presence of the natural dipeptide carnosine detecting its potential counteraction against PMA-induced cytoskeletal alterations and remodeling in differentiated Caco-2 monolayers. In such an experimental context, by both immunocytochemistry and Western blot assays in Caco-2 monolayers, we identified the expression of the allograft inflammatory factor 1 (AIF-1) as protein functionally related to both inflammatory and cytoskeletal pathways. In 21 dps monolayers, particularly, we detected variations of its intracellular localization associated with the inflammatory stimulus and its mRNA/protein increase associated with the differentiated 21 dps enterocyte-like monolayer compared to the undifferentiated cells.

Keywords

• intestinal epithelial monolayers
• Caco-2 cells
• actin cytoskeleton
• allograft inflammatory factor 1
• carnosine