Association between the calcium-binding protein calretinin and cytoskeletal components in the human colon adenocarcinoma cell line WiDr.

Calretinin (CR) is a Ca(2+)-binding protein (CaBP) of the EF-hand family expressed in a cell-type-specific manner and thought to act as a Ca(2+) buffer. Based upon previous studies, CR can undergo Ca(2+)-induced conformational changes, suggesting that it may also belong to the subfamily of Ca(2+)-sensor proteins that are characterized by their ability to interact with target ligands. To elucidate the role of CR, we used the undifferentiated colon adenocarcinoma cell line WiDr, which expresses significant amounts of CR. It has been shown previously that combined treatment with an inducer of differentiation sodium butyrate (NaBt) and a cell growth inhibitor hexamethylene bisacetamide (HMBA) or treatment with CR antisense oligonucleotides is down-regulating CR in parallel with a decrease of cell growth, suggesting a possible involvement of CR in maintaining the undifferentiated phenotype of WiDr cells. Furthermore, CR is absent from normal colon cells and from well-differentiated colon adenocarcinoma cell lines (e.g., Caco-2). Since members of the EF-hand family of proteins are interacting with cytoskeletal components, we investigated the possible association of CR with the cytoskeleton in WiDr cells. With double immunofluorescence stainings and immunoprecipitation experiments, we show close association of CR with intermediate filaments or microtubules in WiDr cells. Treatment with NaBt either disrupted or strongly diminished this interaction, respectively. The same effect was observed after elevation of Ca(2+) by applying the ionophore A-23187. These data suggest that CR may contribute to the transformation of enterocytes by interfering with the differentiation process, i.e., acting at both levels: cell shape dynamics and mitosis.