Calpain regulates enterocyte brush border actin assembly and pathogenic Escherichia coli-mediated effacement.
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.
Potter
David A
DA
Division of Hematology/Oncology, Walther Oncology Center and Veterans Affairs Medical Center and the Department of Biochemistry and Molecular Biology, Indiana University, Indianapolis, Indiana 46202, USA. dapotter@iupui.edu
Srirangam
Anjaiah
A
Fiacco
Kerry A
KA
Brocks
Daniel
D
Hawes
John
J
Herndon
Carter
C
Maki
Masatoshi
M
Acheson
David
D
Herman
Ira M
IM
D000199:Actins / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D015536:Down-Regulation; D020895:Enterocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008871:Microvilli / Q000378:metabolism* / Q000648:ultrastructure; D010750:Phosphoproteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099149:Ezrin
D000199:Actins; D002135:Calcium-Binding Proteins; D002154:Calpain; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D000099149:Ezrin; C033668:calpastatin