Abstract
This study identifies calpain as being instrumental for brush border (BB) microvillus assembly during differentiation and effacement during bacterial pathogenesis. Calpain activity is decreased by 25-80% in Caco 2 lines stably overexpressing calpastatin, the physiological inhibitor of calpain, and the effect is proportional to the calpastatin/calpain ratio. These lines exhibit a 2.5-fold reduction in the rate of microvillus extension. Apical microvillus assembly is reduced by up to 50%, as measured by quantitative fluorometric microscopy (QFM) of ezrin, indicating that calpain recruits ezrin to BB microvilli. Calpain inhibitors ZLLYCHN2, MDL 28170, and PD 150606 block BB assembly and ezrin recruitment to the BB. The HIV protease inhibitor ritonavir, which inhibits calpain at clinically relevant concentrations, also blocks BB assembly, whereas cathepsin and proteasome inhibitors do not. Microvillus effacement is inhibited after exposure of calpastatin-overexpressing cells to enteropathogenic Escherichia coli. These results suggest that calpain regulates BB assembly as well as pathological effacement, and indicate that it is an important regulator involved in HIV protease inhibitor toxicity and host-microbial pathogen interactions.
Highlights
Mation during spreading [4]
M-Calpain is Down-regulated in Calpastatin-overexpressing Caco 2 Cells—To determine whether calpain plays a role in epithelial cell brush border (BB) assembly, the ⌬3 isoform of calpastatin was over-expressed in Caco-2a enterocytes. 10 of 30 selected clonal lines were analyzed for calpastatin overexpression
The calpastatin over-expressing lines (Fig. 6, B and C) exhibit nearly intact microvilli, suggesting a role for calpain in enteropathogenic E. coli (EPEC)-mediated effacement. These studies demonstrate the Ca2ϩ and calpain dependence of intestinal epithelial cell BB assembly and BB disassembly initiated by a common enteric pathogen, EPEC
Summary
Vol 278, No 32, Issue of August 8, pp. 30403–30412, 2003 Printed in U.S.A. Calpain Regulates Enterocyte Brush Border Actin Assembly and Pathogenic Escherichia coli-mediated Effacement*. Microvillus effacement is inhibited after exposure of calpastatin-overexpressing cells to enteropathogenic Escherichia coli These results suggest that calpain regulates BB assembly as well as pathological effacement, and indicate that it is an important regulator involved in HIV protease inhibitor toxicity and in intestinal microvilli, are Ca2ϩ-sensitive, the role that Ca2ϩ plays in the assembly and stability of microvilli is undetermined. A recently developed approach to study the role of calpain in cytoskeletal remodeling has been to create stable transfectants of established cell lines that overexpress calpastatin, calpain’s physiological inhibitor [4]. To determine the role of calpain in BB assembly, calpain activity levels, and corresponding BB microvillus elongation rates as well as ezrin and F-actin recruitment were measured for Caco 2 enterocyte cell lines stably overexpressing calpastatin. These findings indicate dynamic roles for calpain in epithelial morphogenesis and modulation of host-bacterial interactions during bacterial pathogenesis
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