Molecular Biology of the Cell Vol. 14, 2959 –2971, July 2003 Keratin 20 Helps Maintain Intermediate Filament Organization in Intestinal Epithelia Qin Zhou,* Diana M. Toivola,* Ningguo Feng,* Harry B. Greenberg,* Werner W. Franke, ‡ and M. Bishr Omary* *Department of Medicine, VA Palo Alto Health Care System, Palo Alto, California 94304, and the Digestive Disease Center, Stanford University School of Medicine, Palo Alto, CA 94305; and ‡ German Cancer Research Center, Heidelberg, Germany Submitted August 8, 2002; Accepted February 28, 2003 Monitoring Editor: Keith Mostov Of the 20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg803 His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regu- lated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia. INTRODUCTION Most mammalian cells contain a complex cytoskeleton com- posed of three major protein families: actin-containing mi- crofilaments, tubulin-containing microtubules, and interme- diate filaments (IF), and their associated proteins (Fuchs and Cleveland, 1998; Ku et al., 1999). The so-called “soft” keratins (i.e., those excluding the “hard’ keratins found in epidermal appendages such as hair) make up the IF proteins of epithe- lial cells. Soft keratins (K) consist of 20 members (K1–20) that include types I (K9-K20) and II (K1-K8) IF proteins. All epithelial cells express one or more type I and type II kera- tins as noncovalent heteropolymers. Regardless of the num- ber of keratins found in a given epithelial cell, the ratio of type I to type II keratins is 1:1 (reviewed by Moll et al., 1982; Fuchs and Weber, 1994; Herrmann and Aebi, 2000; Cou- lombe and Omary, 2002). For example, hepatocytes express K8/18 exclusively, whereas intestinal epithelial cells express K7/8 (type II) and K18/19/20 (type I) at variable levels. Because cell culture models have not been revealing in understanding keratin function, transgenic animal models were generated for several keratins as a handle toward understanding their function. These models provided clear evidence for keratins as serving an essential role in protect- ing cells from a variety of mechanical and nonmechanical forms of stress, particularly in the skin and liver (Fuchs and Weber, 1994; Magin et al., 2000; Coulombe and Omary, 2002; Omary et al., 2002; Oshima, 2002). The disease phenotypes of these mouse models also led to the identification of several human diseases as keratin-associated diseases (Fuchs and Cleveland, 1998; Omary et al., 2002). For example, transgenic mice (R89C mice) that overexpress human (h) K18 that is mutated at a highly conserved Arg (Arg893 Cys; R89C) develop mild chronic hepatitis and a marked predisposition to drug-induced liver injury, in association with hepatocyte and pancreatic acinar cell keratin filament disruption (Ku et al., 1995, 1996; Toivola et al., 1998, 2000a; Omary et al., 2002). Arg89 of K18 was chosen because it is mutated in K14 and K5 (the equivalent residue is Lys in some type II keratins) in some patients with the severe form of the blistering skin Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E03– 02– 0059. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03– 02– 0059. ‡ Corresponding author. E-mail address: mbishr@stanford.edu. Abbreviations used: g, genomic; h, human; HSE, high salt ex- tract(ion); IF, intermediate filament; K, keratin; m, mouse; M, mutant; mAb, monoclonal antibody; WT, wild-type. © 2003 by The American Society for Cell Biology 2959