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

SIMPLE SUMMARY: The functionality of the enterocyte monolayer is directly impaired by inflammatory insults targeting the major cellular processes, including the cytoskeletal dynamics involving actin elements. Enterocyte’s actin cytoskeleton plays key roles in maintaining the epithelial monolayer’s integrity, and its remodeling is critically intertwined with the transition from physiological to pathological states of the gastrointestinal epithelial barrier challenged by inflammation onsets. Hence, understanding the behavior of the actin cytoskeleton in enterocytes forming the epithelial monolayer is a primary aim for clarifying fundamental aspects of inflammatory mechanisms in the gastrointestinal tract. Here, we analyzed the changing aspects of cytoskeletal actin in the human Caco-2 epithelial cell model at two different stages of differentiation: undifferentiated cells and spontaneously differentiated enterocyte-like cells. An in vitro inflammation-mimicking stimulus (phorbol-12-myristate-13-acetate) was used for challenging intestinal epithelial cells in association with the naturally occurring carnosine dipeptide, which showed its potential counteraction against alterations of the actin cytoskeleton in the enterocyte-like monolayers. Through such experiments, for the first time, we described in enterocyte-like monolayers the expression, localization, and variations of the allograft inflammatory factor 1, a protein functionally related to both inflammatory and cytoskeletal pathways, that we suggest considering as interesting features potentially marking the intestinal epithelial monolayers. 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.