In Vivo, Villin Is Required for Ca2+-Dependent F-Actin Disruption in Intestinal Brush Borders

Abstract

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca2+-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca2+ differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca2+, whereas Ca2+ had no effect in villin-null isolates. Moreover, increase in intracellular Ca2+ by serosal carbachol or mucosal Ca2+ ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 ± 9.6%, compared with wild-type mice, 70 ± 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

A Novel Role for Villin in Intestinal Epithelial Cell Survival and Homeostasis

“…These data agree with the observations in UC patients that increasing rates of colonic epithelial apoptosis correlate with increasing severity of the disease (11). Our study also potentially provides an explanation for the higher death probability noted previously in DSS-treated villin knock-out mice compared with their wild-type littermates (24). No differences in the expression of brush border or tight junction proteins have been noted in the villin-null mice (24,25).…”

“…Our study also potentially provides an explanation for the higher death probability noted previously in DSS-treated villin knock-out mice compared with their wild-type littermates (24). No differences in the expression of brush border or tight junction proteins have been noted in the villin-null mice (24,25). No significant changes in the other actin-binding proteins were noted either (24,25).…”

“…No differences in the expression of brush border or tight junction proteins have been noted in the villin-null mice (24,25). No significant changes in the other actin-binding proteins were noted either (24,25). Hence, changes in the permeability or junctional complexes are unlikely to contribute to the severity of colitis in villin-null mice.…”

“…Likewise, in other inflammatory diseases such as chronic pancreatitis decreased villin expression has been reported (23). In one previous study it has been reported that the death probability was twice as high in dextran sodium sulfate (DSS)-treated villin-null mice compared with their wild-type littermates (24), although no explanation was provided for this observation nor was the specific molecular mechanism identified. In this study we demonstrate for the first time that the villin-null mice have higher levels of apoptosis compared with their wild-type littermates that correlate with the severity of colitis induced in DSS-treated villin-null mice.…”

Regulation of Cell Motility by Tyrosine Phosphorylated Villin

“…It has been assumed for several years that villin's actin-bundling and not actin-severing functions are important because nonphysiologically high Ca 2ϩ concentrations (200 M) are required to activate villin's actin-severing activity. However, studies done with the villin knockout mice suggest that in the absence of villin, the actin-bundling properties associated with villin can be substituted by other proteins in the microvilli (Pinson et al, 1998); on the other hand, the actin-severing activity of the microvilli is lost (Ferrary et al, 1999). In recent years, we have demonstrated that villin's actin-modifying functions can be regulated in vitro by tyrosine phosphorylation and phosphatidylinositol bisphosphate (PIP 2 ), suggesting that villin has the potential to function as a link between receptor activation and actin cytoskeleton reorganization even in the absence of high calcium (Arora and McCulloch, 1996;Zhai et al, 2001;.…”