Studying Simple Epithelial Keratins in Cells and Tissues

CHAPTER 17

Studying Simple Epithelial Keratins in Cells and Tissues Nam-On Ku,* Diana M. Toivola,* Qin Zhou,* Guo-Zhong Tao,* Bihui Zhong,{ and M. Bishr Omary* *Department of Medicine Palo Alto VA Medical Center and Stanford University Palo Alto, California 94304 {

Division of Gastroenterology The First Affiliated Hospital of Sun Yet-sen University Guangzhou 510080, China

I. Introduction II. Materials and Methods A. Isolation of Keratins B. Posttranslational Modifications of Keratins C. Keratin Staining in Tissues and Cells D. Simple Epithelial Keratins in Organ-Specific Injury Models III. Pearls and Pitfalls A. Isolation of Keratins B. Posttranslational Modifications of Keratins C. Keratin Staining in Tissues and Cells D. Simple Epithelial Keratins in Organ-Specific Injury Models IV. Concluding Remarks References

I. Introduction Intermediate filament (IF) proteins consist of a large family of nuclear (i.e., lamins) and cytoplasmic (e.g., keratins, neurofilaments, vimentin, desmin) cytoskeletal proteins (Fuchs and Weber, 1994; Herrmann et al., 2003). They are exMETHODS IN CELL BIOLOGY, VOL. 78 Copyright 2004, Elsevier Inc. All rights reserved. 0091-679X/04 $35.00

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pressed in a tissue-preferential manner such as keratins (K) in epithelial cells, neurofilaments in neuronal cells, vimentin in mesenchymal cells, and desmin in muscle. The major epithelial keratins (excluding the ‘‘hard’’ keratins found in epidermal appendages; see Chapter 15) include type I (K9–K20) and type II (K1–K8) IF proteins. This list of 20 keratins does not account for additional members (e.g., K6a-f ) that are expressed in keratinocytes, particularly during wound healing (see Chapter 16). All epithelial cells express at least one type I and one type II keratin and, regardless of their number in an epithelial cell, are typically found as noncovalent heteropolymers in a 1:1 type I to II molar ratio (Coulombe and Omary, 2002; Fuchs and Weber, 1994). The major keratins of simple-type epithelia (i.e., epithelia consisting of a single layer of cells) are K7 and K8 (type II) and K18, K19, and K20 (type I). However, some of these keratins may be found in other epithelia (e.g., K7 and K19 in stratified epithelia) (Moll, 1998; Smith et al., 2002). The relative solubility of simple epithelial keratins, compared with the minimal solubility of epidermal and other nonsimple-epithelial keratins, has helped facilitate the characterization of their regulation (Omary et al., 1998). This includes the identification of several phosphorylation and glycosylation sites of K8/K18/K19, defining their degradation by caspases during apoptosis and identifying several keratin-associated proteins such as heat shock protein 70 (hsp70) and 14-3-3 proteins (Fig. 1), glucose-regulated protein (Grp78), tumor necrosis factor (TNF) receptor type 2, and protein kinases including 40-kDa PKC"-related kinase, p38, and c-Jun N-terminal kinases (Coulombe and Omary, 2002). Although progress has been achieved in the biochemical studies of K8/K18/ K19/K20 by use of in vitro cell culture systems, studies in transgenic mice have added significantly to our understanding of K8/K18 function of protecting cells from necrotic and apoptotic forms of injury induced by mechanical and nonmechanical stresses. For example, studies of mice that lack K8 or that overexpress K18 Arg89!Cys demonstrated the importance of an intact K8/K18 network in protecting hepatocytes from a variety of stresses, including liver perfusion, partial hepatectomy, griseofulvin, or microcystin-LR toxicity, and apoptosis (Omary et al., 2002). Simple epithelial keratins are also essential for providing structural integrity during embryonic development. For example, although K18-null or K19-null mice have a normal life span, K8/K19 or K18/K19 double-null mice manifest embryonic lethality caused by placental defects with trophoblast giant cell cytolysis and abnormalities in the trophoblast layer (Hesse et al., 2000; Tamai et al., 2000). Studies in transgenic mice that overexpress phosphorylation-mutant K18 demonstrated the in vivo functional significance for keratin phosphorylation. In these studies, the function of the two major K18 phosphorylation sites, Ser52 and Ser33, was examined by overexpressing their corresponding K18 Ser!Ala mutants. In human cultured cells and tissues, Ser52 accounts for most of K18 phosphorylation during interphase, and this phosphorylation increases during mitosis and a variety of stresses. Although there was no significant impact on

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Fig. 1 Posttranslational modifications of K8/K18/K19. Phosphorylation and glycosylation occur at the head and/or tail domains of keratins (as is the case for all other intermediate filament [IF] proteins). Caspase-mediated cleavage occurs in the rod and tail domains of K18 and the rod domain of K19 and other IF proteins. VEVD or similar consensus sequences are found within the rod domain of many IF proteins, whereas DALD is a unique caspase site that is found only in the tail domain of K18. The sizes of the head, rod, and tail are not drawn to scale but consist, respectively, of 87, 315, and 80 amino acids (aa) for K8; 82, 308, and 39 aa for K18; and 71, 315, and 13 aa for K19. The schematic also highlights the association of K18 with 14-3-3 proteins and K8 with heat shock protein 70 (hsp70); 14-3-3 and hsp70 also interact with other proteins.

filament organization when K18 Ser52!Ala (S52A) was overexpressed in transgenic mice, these mice were significantly more predisposed to hepatotoxic injury compared with mice that overexpress wild-type (WT) K18 (Ku et al., 1998b). This provided direct evidence for the importance of keratin phosphorylation in cytoprotection. With regard to K18 Ser33, it becomes hyperphosphorylated during mitosis, with consequent K18 association with the 14–3–3 protein family (Ku et al., 1998a; Liao and Omary, 1996). Findings in transgenic mice that overexpress K18 Ser33!Ala (S33A) demonstrated that K18 Ser33 phosphorylation plays an important role in: (1) filament organization in the liver after mitosis and in the pancreas under basal conditions, (2) regulation of hepatocyte nuclear 14–3–3 redistribution during mitosis, and (3) modulation of hepatocyte mitotic progression, in a limited fashion, after partial hepatectomy (Ku et al., 2002b). This chapter focuses on describing techniques used to study simple epithelial keratins in cell culture systems and in mice. The methods that we will focus on include: (1) isolation of keratins, (2) keratin posttranslational modifications,

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(3) keratin staining in tissues and cells, and (4) simple epithelial keratins in organ-specific injury models.

II. Materials and Methods A. Isolation of Keratins

1. High Salt Extraction of Keratins Although simple epithelial keratins are significantly more soluble than epidermal keratins, most (95%) cellular K8/K18 is insoluble. In general, IF proteins can be solubilized in solutions containing a high concentration of urea or guanidinium hydrochloride, which produce soluble tetrameric or oligomeric subunits, or in solutions containing denaturing detergents such as sodium dodecylsulfate (SDS), which generate solubilized denatured monomers. A highly enriched keratin fraction can also be obtained with high salt extraction (HSE) (Fig. 2A) (Achtstaetter et al., 1986; Chou et al., 1993), which is a very useful tool for the isolation of most of the keratin content in tissues, primary cultured cells and cell

Fig. 2 (A) Flowchart of the high salt extraction method. (B) Example of keratins isolated by high salt extraction (HSE) then analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Keratins were isolated from the human colonic cell line HT29 or from the indicated mouse tissues. (C) Two-dimensional gel of HSE of the small intestine of nontransgenic FVB mice (Zhou et al., 2003). The boxed enclosure summarizes the abbreviations used in B and C.

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lines (e.g., Fig. 2B). Of note, all vendors listed in this chapter are based in the United States. a. Reagents . Phosphate-buVered saline (PBS, pH 7.4): 36 g NaCl plus 80 ml of 5 M Na2HPO4 (titrated to pH 7.4) brought up to 4 L with distilled (d) H2O. . High salt buVer (HSB): 10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1.5 M KCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X100, plus a protease inhibitor mix consisting of 1 mM phenylmethylsulfonyl fluoride (PMSF) added fresh (from a 1 M stock solution prepared in methanol) and a premixed protease inhibitor cocktail consisting of 10 M leupeptin, 10 M pepstatin, and 25 g/ml aprotinin (added fresh). The premixed protease inhibitor cocktail is prepared as a 1000 stock (e.g., 10 mM leupeptin) and stored in aliquots (at 20  C). . Triton X100 buVer: 1% Triton X100 plus 5 mM EDTA in PBS (pH 7.4), plus PMSF and the protease inhibitor cocktail as above. . 4 Nonreducing sample buVer (4 NRSB): 10 ml 2 stacking gel buVer, 16 ml glycerol, 6.4 ml 0.1% bromophenol blue, and 3.2 g SDS brought to a total volume of 40 ml with dH2O. . 2 Stacking gel buVer: 1.75 ml concentrated phosphoric acid (or 25.6 ml of 1 M), and 5.7 g Tris base brought to 50 ml with dH2O.

b. Steps 1. Cells grown in suspension are harvested by centrifugation (5 minutes, 1000 rpm), whereas adherent cells are scraped into an Eppendorf tube. Cells are washed 2 with PBS (22  C). The cell pellet is resuspended in 1 ml of Triton X100 buVer, mixed, and incubated over ice (2 minutes), then repelleted (10 minutes; 14,000 rpm; 4  C) and processed as in step ‘‘3’’ below. 2. For tissue fragments, add 1 ml of ice cold Triton X100 buVer per 0.5 cm3 of isolated tissue, homogenize by douncing 50 strokes—or by using a PowerGen 125 tissue homogenizer (Fisher Scientific) (for tissues diYcult to dounce such as the intestine) for 1 minute (keep over ice during mechanical homogenization), then spin (10 min; 14,000 rpm; 4  C) and discard the supernatant. 3. The pellet, which consists mainly of nuclei and nonsolubilized membrane and cytoskeletal proteins, is resuspended in 1 ml of HSB, dounced (100 strokes over ice), mixed (30 minutes) with a rotator placed in a cold room, then repelleted (20 minutes; 14,000 rpm; 4  C). 4. Discard the supernatant, then add 1 ml PBS with 5 mM EDTA, dounce 100 strokes (as a pellet washing step), then spin 10 minutes as previously. Add 200–400 l of 2 NRSB, vortex to dissolve the pellet as much as possible, heat to 95  C (2–4 minutes), and spin (2 minutes; 14,000 rpm; 22  C).

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5. Load 5–10 l onto SDS-polyacrylamide gel electrophoresis (PAGE) gel and store the remaining sample (which may be aliquoted depending on the extent of anticipated freeze-thawing) at 20  C. An example of HSE keratin profiles, on isolation from cells and tissues, is shown in Fig. 2B. If two-dimensional (2D) gel analysis (isoelectric focusing then SDS-PAGE) is performed after HSE, 2D sample buVer (which contains 9.5 M urea and ampholyte) is added to the pellet at the last step of the procedure followed by vortexing and heating to 37  C (ureacontaining samples should not be boiled). An example of 2D gel analysis of keratins is shown in Fig. 2C.

2. Keratin Immunoprecipitation Immunoprecipitation has been successfully used as an aid to study K8/K18/ K19/K20 biochemical properties, such as their posttranslational modifications and associated proteins. These keratins are more amenable to such studies, compared with epidermal keratins, because of their relative solubility in detergents that maintain antigen (keratin)–antibody binding. These studies include the characterization of keratin posttranslational modifications (Ku and Omary, 1994, 1995, 1997; Liao et al., 1997) and associated proteins (Ku et al., 1998a; Liao and Omary, 1996; Liao et al., 1995a). For example, heat shock protein 70 (hsp70) and 14-3-3 protein (Fig. 3) were identified as keratin-associated proteins after coimmunoprecipitation then microsequencing of individual coprecipitated protein bands (after in-gel protease digestion and isolation of released peptides). The following protocol oVers a general guideline for keratin immunoprecipitation, although optimization may be required for other specific IF proteins and antibodies. A list of monoclonal antibodies (mAb) and other immunologic reagents useful for studying simple epithelial keratins are listed in Table I. a. Materials . Cell line: HT29 cells, an adherent human colonic carcinoma cell line (American Type Culture Collection), were cultured at 37  C in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 100 g/ ml streptomycin, and 2 mM l-glutamine. . Beads: protein A Sepharose 4B (Cat. No. 10-1041) or protein G Sepharose 4B (Cat. No. 10-1242) (Zymed). . Antibody: monoclonal mouse anti-human K18 antibody (L2A1) (Table I) (Chou and Omary, 1993). . Lysis buVer: 1% Nonidet P-40 (NP-40) or 1% Empigen BB (Calbiochem) in PBS (pH 7.4) containing 5 mM EDTA, 0.1 mM PMSF, and the protease inhibitor cocktail used in the HSE Section. . Washing buVer: 0.1% NP-40 or 0.1% Empigen in PBS (pH 7.4) containing 5 mM EDTA.

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Fig. 3 Coimmunoprecipitation of K8/K18 and their associated proteins. (A) HT29 cells were cultured at 37  C (16 hours) as normal control, at 42  C (16 hours), or in the presence of rotavirus (12 hours), respectively (Liao et al., 1995c). Rotavirus infection was performed by addition of trypsinactivated rotavirus into serum-free medium (2.8  107 plague forming units/dish) and incubated for 1 hour. The virus-containing medium was then removed followed by incubation in normal medium for 12 hours. Cells were solubilized with NP-40 lysis buVer, followed by precipitation with monoclonal antibody (mAb) L2A1. The immunoprecipitates (ip) were analyzed by 10% SDS-PAGE followed by Coomassie staining. Note that heat shock protein (hsp)70 coimmunoprecipitated with K8/K18, and a protein band of hyperphosphorylated K8 (HK8) was induced after heat treatment or rotavirus infection. (B) Freshly isolated human colon biopsies were cultured at 37  C with or without okadaic acid (OA; 1 g/ml; 2 hours), homogenized with NP-40 lysis buVer, followed by immunoprecipitation with mAb L2A1. Note K8/K18 and 14-3-3 protein association (confirmed by microsequencing of the isolated bands and by immunoblotting using antibodies to 14-3-3 proteins, not shown) after OA treatment, which induces K18 S33 phosphorylation and 14-3-3 binding with K18 (Liao and Omary, 1996; Ku et al., 1998a). OA also induces generalized K8/K18 hyperphosphorylation, with retardation of their gel migration. **Highlights the immunoglobulins.

b. Preparation of NP-40 Cell Lysate 1. Rinse cells in a tissue culture dish (10 cm, at 80%–90% confluence) with 5 ml of prewarmed (37  C) PBS-EDTA, then scrape the cells into 1 ml of prewarmed PBS-EDTA buVer. 2. Pellet the cells into an Eppendorf tube by a brief centrifugation (15 seconds; 10,000 rpm). 3. Add 1.3 ml of prechilled lysis buVer and vortex, then gently shake on a rotator (2 hours, 4  C).

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Table I Antibodies Directed to Simple-Type Epithelial Keratins Antibody name

Host

Species reactivity*

K7

RCK-105

Mouse

h, m

ICC,WB

K8

M20 Troma I E3264 GT3{ DC10 L2A1 GT6 4.62 KA4 E2634 Ks20.8 ITKs20.10 Q6 8.13x LJ4 2D6 5B3 IB4 M30

Mouse Rat Rabbit Rabbit Mouse Mouse Rabbit Mouse Mouse Rabbit Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse

h m h h h h h h h h h, h h h h, h, h, h, h,

ICC, WB ICC, WB ICC, WB ICC, WB, P ICC, WB ICC, WB, P ICC, WB, P ICC, WB ICC, WB ICC, WB ICC, WB ICC, WB ICC{ WB, P ICC, WB, P ICC, WB, P ICC, WB, P ICC, WB ICC, WB, P ICC, WB

Keratin

K18

K19

K20

Pan keratin K8 phospho-S73 (h) K8 phospho-S431 (h) K18 phospho-S33 (h) K18 43-kDa fragment (cleaved at D396)

m

m m m m m

Applications

Vendor ICN Biochemicals, Progen Labvision DSHB Spring Bioscience Epitomics Labvision Labvision Epitomics Sigma Sigma Spring Bioscience Labvision Progen Labvision Sigma Labvision Labvision Labvision Labvision Roche Applied Science

Antibodies for keratins with applications for immunocytochemistry (ICC), Western blotting (WB) and immunoprecipitation (P) are shown. ICC indicates that the antibody works on acetone-fixed frozen sections unless otherwise stated. Websites for the companies from which the antibodies may be purchased are DSHB, www.uiowa.edu/dshbwww, Epitomics, www.epitomics.com, ICN, www.mpbio.com, Labvision (Neomarkers), www.labvision.com, Progen, www.progen.de, Roche Applied Science, www.roche-applied-science.com, Sigma, www.sigma.com, Spring Bioscience, www.springbio.com { PFA fixation only for mAb Q6. *h, human; m, mouse. { Recognizes human non-phospho-K8 S73–containing epitope selectively (Tao et al., manuscript in preparation). x Reacts with type II keratins K1/K5/K6/K7/K8 and type I keratins K10/K11/K18.

4. Pellet the cell lysate (14,000 rpm; 20 minutes; 4  C), then carefully transfer the supernatant of solubilized material (NP-40 cell lysate), without disturbing the pellet, to a clean tube. c. Cell Lysate Preclearing 1. Transfer 50 l of the protein A (or protein G) beads slurry to an Eppendorf tube and add 500 l cold lysis buVer. 2. Spin at 10,000 rpm for 30 seconds and wash one additional time with 500 l of cold lysis buVer.

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3. Add 600 l of NP-40 cell lysate to the Eppendorf tube and shake at 4  C for 30–60 minutes. 4. Spin (10,000 rpm; 10 minutes; 4  C) then transfer the cleared lysate to a new tube. d. Immunoprecipitation 1. Add 5–10 g of antibody to the Eppendorf tube containing the cold precleared NP-40 lysate. Incubate at 4  C for 1 hour, then add 50 l of prewashed protein A slurry and incubate for 2 hours at 4  C on a rotator. 2. Spin (5000 rpm; 30 seconds; 4  C) then carefully remove the supernatant and wash the beads three times with 1.0 ml of washing buVer. 3. Add 50 l of 2 nonreducing Laemmli sample buVer. Vortex then heat (95  C, 2 minutes). 4. Spin (14,000 rpm; 1 minute) and collect the supernatant, analyze a fraction (typically 10–30 l) by SDS-PAGE, then use Coomassie staining or transfer the gels of interest to membranes for immunoblotting.

B. Posttranslational Modifications of Keratins Keratins are highly dynamic and reorganize in response to many stimuli such as mitosis and apoptosis. Potential mechanisms that may regulate keratin filament organization include keratin-associated proteins and posttranslational modifications, such as phosphorylation, glycosylation, and proteolysis. The current state of knowledge of the posttranslational modifications of K8/K18/K19 (summarized in Fig. 1) was made possible by the approaches and some of the techniques described in this section. General information regarding overall keratin phosphorylation can be obtained by gel analysis of the in vivo or in vitro labeled keratins. The 2D gel analysis also provides information regarding the relationship of the charged phosphoisoforms to the neutral O-GlcNAc–containing species that may or may not be phosphorylated (Liao et al., 1996). K8/K18/K19/K20 isoforms are separated in the first dimension by use of gels containing 1.6% Bio-Lyte 5/7 ampholyte and 0.4% BioLyte 3/10 ampholyte (Bio-Rad), as detailed further in manufacturer’s instructions. K8/K18 phosphorylation is also detected by immunostaining (see Section II.C) or immunoblotting with site-specific phospho-antibodies (Table I). In addition to the K8/K18/K19 serine phosphorylation sites known to date (Fig. 1), K8/K19 pervanadate-mediated tyrosine phosphorylation was demonstrated by immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid (PAA) analysis of K8 and K19 isolated from metabolically labeled cells, although the modified K8/ K19 phosphotyrosine residues remain to be identified (Feng et al., 1999). Several methods have been used to identify in vivo phosphorylation sites, including the microsequencing of chemically or protease-generated peptides

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(Steinert, 1988). Alternatively, in vivo or in vitro labeled tryptic or chymotryptic keratin peptides, generated from purified keratins and isolated after peptide mapping (electrophoresis then chromatography; van der Geer and Hunter, 1994), can be subjected to manual Edman degradation to identify the position of the radiolabeled residue(s) within the isolated radiolabeled peptide (Ku and Omary, 1994, 1995). The peptide mapping technique can be applied to both phosphorylation and glycosylation sites, which are predicted on the basis of the location of the radiolabeled residue and the likely modified keratin peptide (predicted from known keratin sequences). The predicted modification sites can then be confirmed by mutation of the potentially involved residue(s), transfection of the wild type (WT) and predicted modification-mutant cDNAs, in vivo labeling, then peptide mapping to confirm the absence of a radiolabeled peptide in the maps generated from the mutant vs the WT constructs (Ku and Omary, 1994, 1995). Mass spectrometry techniques (see Chapter 14) may also be used to identify O-GlcNAc or phosphorylation sites, as done for the characterization of neurofilament phosphorylation (Betts et al., 1997) and the glycosylation of a variety of proteins (Dell and Morris, 2001). Improvements in the sensitivity of these techniques could make the characterization of IF posttranslational modifications less tedious. In addition, a reverse immunologic approach can be used to identify phosphorylation residues (Omary et al., 1998). This approach entails generating monoclonal antibodies against hyperphosphorylated keratins purified from cells treated with phosphatase inhibitors, such as okadaic acid. Antibodies are screened for specific binding to phosphorylated keratins. Potential target phosphorylation sites are predicted and then confirmed by combining the information generated from: (1) binding of the antiphosphokeratin antibodies to keratins phosphorylated in vitro with a panel of kinases and (2) the consensus sequence of the kinase(s) and the known keratin sequences. Alternatively, the phosphorylation sites recognized by the generated antibodies can be identified by epitope mapping of the antibodies of interest with synthetic peptides and confirmed by generating sitespecific keratin mutants and then immunoblotting (using a dot blot technique) with the test antibodies. This approach was used to identify phosphorylation at K8 Ser-73, which plays a significant role in keratin filament organization in response to stress, apoptosis, and mitosis (Liao et al., 1997). Type I keratins, including K18 and K19, undergo caspase-mediated proteolysis during apoptosis, whereas their type II partner, K8, manifests remarkable resistance to apoptosis-associated degradation. The methods used to identify the in vivo cleavage sites have relied on microsequencing of keratin fragments obtained from apoptotic cells (Ku et al., 1997), by testing keratin fragmentation with purified recombinant caspases (Caulin et al., 1997), by epitope mapping of an antibody (called M30, Table I) that recognizes K18 only after caspase cleavage (Leers et al., 1999). The potential caspase cleavage sites are confirmed by mutation (e.g., K18 D237E) followed by transfection of WT and mutant cDNAs then testing for caspase-mediated cleavage after inducing apoptosis in transfected cells (Caulin et al., 1997; Ku and Omary, 2001).

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In this section, we will focus on the in vivo and in vitro detection of keratin phosphorylation, glycosylation, and proteolysis. For each posttranslational modification, this section is subdivided into: ‘‘Reagents,’’ ‘‘In Vivo modification,’’ and ‘‘In Vitro modification.’’

1. Phosphorylation In vivo metabolic labeling of cells is performed with 32PO4 in phosphate-free medium, supplemented with 0.1% (v/v) normal medium to prevent phosphate depletion during labeling and to facilitate uptake of the labeled phosphate. After in vivo labeling, the immunoprecipitated keratins can be used for PAA analysis to confirm the presence of one or more of the radiolabeled PAA residues phosphoserine, phosphotyrosine, and phosphothreonine. The PAA analysis is carried out by acid hydrolysis of the 32P-labeled keratins, followed the separation of PAA by one-dimensional or two-dimensional thin-layer electrophoresis (van der Geer and Hunter, 1994). Alternatively, global changes in keratin phosphorylation can be detected by immunoblotting with commercially available antibodies against phosphoserine, phosphotyrosine, or phosphothreonine (Feng et al., 1999). For in vitro phosphorylation studies, the amount of purified kinase and components of the kinase buVer are variable, depending on the kinase and its cofactor requirements. We describe, as an example, in vitro human K8 phosphorylation by p42 MAP kinase (Erk2) (Fig. 4A), which is a likely physiological kinase for human K8 Ser431 (Ku et al., 2002a). a. Reagents [32P]-orthophosphoric acid and [-32P]-ATP (3,000 Ci/mmol) (DuPont New England Nuclear); phosphate-free Dulbecco’s modified Eagles medium (DMEM) and dialyzed FCS (Invitrogen); yeast adenosine triphosphate (ATP) (A2383, Sigma); p42 MAP kinase (New England Biolabs); 1 in vitro kinase buVer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, and 0.01% Brij 35). The kinase buVer may vary. b. In Vivo Phosphorylation 1. Incubate cells (in 100-mm dishes) with 5 ml of phosphate-free DMEM containing 10% dialyzed FCS 2 mM, l-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin (30 minutes, 37  C). 2. Add 50 l of normal medium and 250–500 Ci/ml [32P]-orthophosphoric acid and incubate for 5 hours at 37  C. The labeling time can vary, depending on the nature of the experiment, with a 3- to 5-hour labeling period typically providing steady-state incorporation. 3. Wash cells with PBS three times. 4. Isolate K8/K18 as described in Section II.A, and then analyze by SDS-PAGE and autoradiography.

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Fig. 4 In vivo and in vitro posttranslational modifications of K8/K18. (A) In vivo and in vitro phosphorylation of K8/K18. For in vivo phosphorylation, cultured cells that express WT K8 and WT K18, or WT K8 with Ser52!Ala K18, were labeled with 32PO4 followed by solubilization, immunoprecipitation of K8/K18, SDS-PAGE, then Coomassie staining (lanes 1, 2). Note that mutation of K18 Ser52, the major K18 phosphorylation site, results in near total abolishment of K18 phosphorylation. For in vitro phosphorylation, BHK-21 cells were cotransfected with WT K18 and one of the K8 constructs, WT or S431A. After 3 days, K8/K18 immunnoprecipitates were prepared then used for in vitro phosphorylation by p42 MAP kinase (Erk2), boiled, and then analyzed by SDSPAGE and autoradiography (lanes 3, 4). Note that mutation of K8 Ser431 results in abolishment of K8 phosphorylation, but minor phosphorylation is detected in a distinct hyperphosphorylated K8 (HK8) caused by K8 Ser73 phosphorylation (Ku et al., 2002a). (B) In vitro galactosylation of K8/K18. K8/K18 were immunoprecipitated from cells that express WT K8/K18 or WT K8 and K18 S29/30/ 48A. The immunoprecipitates were in vitro labeled with UDP-[3H]galactose and galactosyltransferase,

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c. In Vitro Phosphorylation 1. Prepare K8/K18 immunoprecipitates as described in Section II.A.2. 2. For 10 l of immune complex–associated beads (total bead volume), add 15 l of 1 kinase buVer and heat for 2 minutes (90  C) to inactivate endogenous kinase activity. 3. After cooling to room temperature, add 5 Ci of [-32P]-ATP, 20 M ATP, and 1 unit of p42 MAP kinase (total reaction volume ¼ 25 l) and incubate 15 minutes at 22  C. 4. Add 25 l of 2 Laemmli sample buVer containing 4% SDS and 20% glycerol and boil for 2 minutes. 5. Pellet then analyze the labeled K8/K18 immunoprecipitates by SDS-PAGE and autoradiography.

2. Glycosylation Detection and confirmation of keratin glycosylation is performed by use of a combination of techniques, including in vivo labeling and in vitro galactosylation (Chou et al., 1992; Greis et al., 1996; Hart, 2003; King and Hounsell, 1989; Ku and Omary, 1995). Metabolic labeling of cells is performed with 3H-glucosamine, and the nature of the attached saccharides is assessed after acid hydrolysis of the purified protein. In vitro galactsylation is carried out with immunopurified keratins, UDP3H-galactose, and galactosyltransferase. This adds a radiolabeled galactose to accessible terminal GlcNAc on the protein backbone (Fig. 4B) and provides an indirect assessment of a protein’s O-GlcNAc modification. a. Reagents D-[6-3H]-glucosamine (40 Ci/mmol) and uridine diphosphate (UDP)-[4,5-3H]galactose (36.7 Ci/mmol) (DuPont New England Nuclear); glucose-free RPMI 1640 medium (Invitrogen); Amplify fluorographic reagent (Amersham

followed by SDS-PAGE, Coomassie staining, and fluorography. Mutation of K18 Ser29/30/48 (to Ala), the major glycosylation sites of K18, abolishes most of K18 glycosylation. (C) In vivo K18 fragmentation during anisomycin-induced apoptosis. HT29 cells were treated with anisomycin (10 g/ml) for 24 hours. The cells were solubilized in 1 Laemmli sample buVer (total lysate) or in 1% NP-40 followed by K8/K18 immunoprecipitation (ip). Total lysates and K8/18 ip were analyzed by SDS-PAGE and Commassie staining or were immunoblotted with anti-K18 (N-terminal epitope) antibody. p29 represents an N-terminal K18 fragment that is generated after K18 cleavage at Asp237. (D) In vitro K18 fragmentation by caspase-3. K8/K18 ip were incubated with buVer alone or with buVer containing recombinant caspase-3 for 3 hours. The K8/K18 ip were separated by SDS-PAGE, then analyzed by Commassie staining, or by blotting with Ab DC10 that recognizes K18 (aa 1-237) or Ab M30 that specifically recognizes the exposed Asp396 of K18 (i.e., on release of the K18 tail fragment, aa 397–429). Both K18 Asp396 and Asp237 are caspase-3 in vitro digestion sites that generate p43 and p29 (arrowheads), respectively, after sequential cleavage. Arrow ¼ nonspecific bond.

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Biosciences); galactosyltransferase (G5507, Sigma); 1 galactosyltransferase buVer (20 mM MnCl2, 100 mM sodium cacodylate [pH 6.5]; 1 washing buVer (0.5% NP-40 in PBS [pH 7.4]). b. In Vivo Glycosylation 1. For confluent cells in one 100-mm dish, dry 500 Ci of D-[6-3H]-glucosamine with a SpeedVac concentrator and resuspend with 50 l of normal medium. 2. Incubate the cells (one 100-mm dish) in 5 ml of glucose-free RPMI 1640 medium containing 10% dialyzed FCS 2 mM l-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin for 30 minutes at 37  C. 3. Add 50 l of the radioactive-containing medium (final concentration; 100 Ci/ml [3H]-glucosamine) and incubate for 12 hours at 37  C. 4. Wash cells with PBS three times. 5. Isolate K8/K18 as described in Section II.A and then analyze by SDS-PAGE and fluorography (for this, we use Amplify fluorographic reagent as recommended by the supplier). c. In Vitro Galactosylation 1. Prepare K8/K18 immunoprecipitiates as described in Section II.A.2. 2. For 10 l of immune complex–associated beads, dry 0.6 Ci of UDP-[3H]galactose in a SpeedVac concentrator and resuspend with 20 l of 1 galactosyltransferase buVer. 3. Add 25 m of galactosyltransferase and incubate for 2 hours at 37  C. 4. Wash the immunoprecipitates with 0.4 ml of 1 washing buVer two times. 5. Boil beads in 50 l of 2 Laemmli sample buVer for 1 minute. 6. Analyze the labeled samples by SDS-PAGE and fluorography.

3. Cleavages by Caspases We describe in vivo K18 fragmentation during anisomycin-mediated apoptosis and in vitro K18 cleavage by purified recombinant caspase 3 (Fig. 4C,D) (Ku and Omary, 2001; Ku et al., 1997). Similar findings were reported in other K8/K18-expressing systems with etoposide (125–250 g/ml, >24 hours) or daunomycin (2–6 g/ml, >24 hours) treatment of cultured cells to induce apoptosis or by use of caspases 3, 6, or 7 for in vitro K18 cleavage (Caulin et al., 1997). Notably, K18 and K19 degradation can be easily detected in BHK hamster kidney cells that are transfected with K8/K18 or K8/K19 cDNA constructs with LipofectAMINE (Invitrogen), without adding any proapoptotic reagents (Ku et al., 1997). K18 fragmentation is also observed in transgenic mice after intraperitoneal injection of Fas antibody (0.15 g antibody/g body weight) or injection of TNF- (15 ng/g) with actinomycin D (1 g/g) (Ku et al., 2003b) (see also Section II.D.1).

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a. Reagents Anisomycin (CalBiochem); recombinant caspase-3 (Upstate Biotechnology Inc.); 1 caspase buVer (25 mM Hepes, 1 mM dithiothreitol [pH 7.5]). b. In Vivo Keratin Fragmentation 1. Incubate 80%–90% confluent cells in 10 ml of normal culture medium containing 10 g/ml anisomycin for 16–24 hours at 37  C. 2. Wash cells with PBS three times (need to include apoptotic floater cells). 3. Isolate K8/K18 as described in Section II.A and then analyze by SDS-PAGE and Coomassie staining or by immunoblotting. c. In Vitro Fragmentation 1. Prepare K8/K18 immunoprecipitates as described in Section II.A.2. 2. For 10 l of immune complex–associated beads, add 20 l of 1 caspase buVer with 1 unit of recombinant caspase-3 and incubate for 3 hours at 37  C. 3. Add 25 l of 2 Laemmli sample buVer containing 4% SDS and 20% glycerol and boil for 3 minutes. 4. Analyze the fragmented K8/K18 by SDS-PAGE and immunoblotting.

C. Keratin Staining in Tissues and Cells Immunofluorescence staining combined with confocal microscopy are indispensable tools in studying keratin filament organization, disassembly, degradation, phosphorylation, and interaction with other proteins. Because keratins are very abundant proteins composed of filament networks that undergo dynamic alterations during physiological and stress conditions, they are easy to visualize. Antibodies to posttranslationally modified keratins, such as the phospho-specific keratin antibodies and the M30 antibody to the caspase degraded K18 (Table I and Fig. 5) (Ku and Omary, 1997; Ku et al., 1998a; Leers et al., 1999; Liao et al., 1995b, 1997) are helpful specific tools to aid our understanding of simple epithelial keratin dynamics and regulation.

1. Reagents O.C.T. compound (optimum cutting compound) and Vinyl Tissue-Tek Cryomolds (Sakura); sterile Lab-Tek Chamber slides with cover (Nalgen Nunc Int.); gelatin (Kodak); chrom alum (chromium potassium sulfate, 12-hydrate; EMD Chemicals); charged superfrost/plus microscope slides and premium cover glasses thickness 1 (17 mm; Fisher scientific); 100% acetone or methanol stored at 20  C; freshly made 0.5% para-formaldehyde (PFA) in PBS (heat PFA powder in H2O to 60  C and add NaOH until dissolved, cool to 22  C, add 5 strength solution of

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Fig. 5 Analysis of keratins in mouse and human tissues by fluorescence microscopy. Sections of normal mouse ileum (a,b), human cirrhotic liver (c,d), and mouse pancreas (e,f ) treated with saline (e) or 48 hours after 7 hourly injections of 50 g/kg caerulein (f ) were double- or triple-stained for keratins ( nuclear staining) then viewed with a BioRad MRC1024 confocal microscope. K8/K18 (red) in (a–d) were detected with a rabbit anti-keratin antibody and a CY5-conjugated goat anti-rabbit antibody and further pseudo-colored red with the Adobe photoshop program. K20 (green) was detected with Ab Ks20.8 Ab, phospho-K8 (green) in (b) and (c) was probed with mAb LJ4, and the K18

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PBS and water to a final 1 PBS solution, pH 7.4); buVer A (2.5% [w/v] BSA [bovine serum albumin, Sigma] in PBS [pH 7.4]; buVer B (2% normal serum [that matches the host species of the secondary antibody] in buVer A); buVer C (0.2 mg/ ml ribonuclease A [Sigma] in buVer B); fluorescent-labeled secondary antibodies diluted in buVer B; nuclear fluorescent probes (Toto-3 iodide, Yo-Pro-1 iodide) prepared in buVer C, and ProLong antifade mounting media (Molecular Probes).

2. Steps a. Staining of Frozen Tissue Sections 1. Sacrifice test animals by CO2 inhalation and immediately excise desired tissues, immerse (with orientation if needed, such as for intestinal tissues) in an O.C.T.–filled cryomold (placed over a block of dry ice), then allow to quickly freeze on dry ice. Store blocks at 80  C. 2. Precoat microscope slides with gelatin: add 5 g gelatin to 1 L of water (55  C) and dissolve, then add 0.5 g chrom alum. Filter the warm solution with Whatman filter paper and store at 4  C. Wipe slides free of dust, dip in the coating solution, drain oV excess solution, stand vertical to dry, and store at 22  C. 3. Cut thin sections (6 m) with a cryostat and place on gelatin-coated cover slides. .

.

For acetone fixation: allow sections to air dry (1 hour), then fix in 20  C acetone (10 minutes), and air dry (1 hour). Slides can be stored at 20  C and when used refixed immediately (while frozen) in 20  C acetone for 10 minutes, air dried (1 hour), or dried with drie-rite desiccant (22  C; W A Hammond Drierite Co Ltd). For PFA fixation: air dry sections (5 minutes) and fix in 0.5% PFA (for labeling that requires PFA, such as FITC-phalloidin) for 20 minutes (22  C), then wash slides with PBS.

4. For subsequent incubations, place slides in a moisture chamber (a box with lid with the bottom covered with wet paper towels). 5. Wash 3 with PBS (for PFA fixation: permeabilize cell membranes with 0.2% NP-40 in PBS, 5 minutes [22  C], then wash 3 with PBS). 6. Block with buVer A for 10 minutes, then with buVer B for 10 minutes. apoptotic caspase-generated 43-kDa fragment (green) in (d) was detected with mAb M30 (Table I). Nuclei in (a–f ) were stained with propidium iodide and pseudo-colored blue. Note that although K8/ K18 (red, a) are expressed throughout the ileal epithelium, K20 (green) is present in the villus cells (yellow when colocalized with K8/K18) but is absent in the basal glands. Arrows in (b) and (c) show phosphorylated K8 (S79 in mouse and S73 in human) in a mitotic cell in a mouse ileal gland and in hepatocytes of a human cirrhotic liver, respectively. Arrowheads in (d) point to keratin and M30positive aggregates indicative of apoptosis. Note dramatic induction of filaments (panel f ). F, fibrosis; G, intestinal glands; L, lumen. Scale bars: a, c, d ¼ 50 m, b ¼ 10 m, e, f ¼ 20 m.

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7. Incubate with primary antibodies 45–60 minutes (e.g., mAb L2A1 and rabbit antibody E2634, Table I) diluted in buVer B. In general, use 1:50–1:100 dilutions of mouse mAb ascites and 1:500–1:2000 of rabbit polyclonal antibodies. Depending on nature and extent of experiments, antibody titration may be necessary. 8. Wash by incubating with PBS 3 (5 minutes/incubation), then block with buVer C (10 minutes). 9. Incubate with the fluorescently labeled secondary antibodies (1:50–1:200) and the nuclear marker (e.g., Toto-3; for double or triple staining) diluted in buVer C for 30 minutes, then wash 3 with PBS. For example, we use Texas red-conjugated goat anti-rabbit (Molecular Probes, T-2767) and FITC-conjugated goat anti-mouse antibody (BioSource, AMI4408). Preabsorbed secondary antibodies should be purchased when double/triple staining is carried out. 10. Mount sections under coverslips with fresh Prolong anti-fade mounting media, then allow to dry. 11. View in microscope with appropriate filters and lasers for the fluorophores used (see Fig. 5 for tissue staining examples). Stained slides can be stored at 4  C for several weeks. b. Staining of Cells 1. Sterilize coverslips by soaking them in 99% ethanol for 5 minutes, dry one at a time in an open flame, and place in a sterile 2-ml tissue culture dish or use sterile plastic chamber slides. 2. Plate cells onto the coverslips or plastic chamber slides at 104 cells/ml and culture until cells are attached and dividing. 3. If applicable, treat cells with stimulatory agent of choice (e.g., phosphatase inhibitor). Remove media and wash cells 3 with PBS. 4. If treatment generates floater (detached) cells, collect and centrifuge the medium at 1000 rpm, 5 minutes, further wash cells 3 with PBS by centrifugation as earlier. Incubate floater cells in all further steps in Eppendorf tubes. Alternatively, transfer cells to slides with a cytospin centrifuge (e.g., Shandon Cytospin, Thermo Electron Corp). 5. Fix cells with methanol (20  C, 10 minutes), then air dry. For PFA fixation, use 0.5% PFA for 20 minutes (22  C). 6. Stain as described for the frozen tissues.

D. Simple Epithelial Keratins in Organ-Specific Injury Models Simple-type glandular epithelia express K8 (primarily) and K7 (at low levels, typically in ductal cells) as the type II keratins and variable levels of type I keratins (K18, K19, or K20), depending on the cell type, as found in several digestive organs, including hepatocytes (K8 and K18), gallbladder (K8 and K19>K18),

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pancreas (K8 and K18>K19/K20), small intestine and colon (K8 and K19>K18/ K20) (Fig. 2B). This section addresses a variety of techniques that can be used to examine organ-specific injury models (Table II). Their usefulness is in comparing the phenotype of transgenic mouse lines and in testing the eVect of a keratin transgene or a keratin-null in a tissue-specific fashion. General principles of animal experiments to keep in mind, independent of organs, include the following. .

. .

.

Pilot experiments need to be carried out to optimize dosing to avoid potential rapid lethality caused by variables related to mouse genotypes and strain diVerences. Sex- and age-matched mice (2- to 3-month-old) need to be used. Organs and blood are collected after euthanizing the mice by CO2 inhalation. Blood is collected by intracardiac puncture (0.5–1.0 ml with a 22-G needle) followed immediately by organ harvesting. Dissected organs are generally divided into several pieces, depending on the experiment and used for: (1) fixation with 10% formalin followed by paraYn embedding, sectioning, then hematoxylin/eosin staining; (2) snap freezing in optimum cutting-temperature compound, sectioning, then fixing in cold acetone or PFA for subsequent immunofluorescence staining (see Section II.C); and (3) snap freezing in liquid nitrogen for subsequent biochemical analysis (see Section II.A).

1. Liver Injury and Regeneration Models The drug doses described herein are based on studies with K8 or K18 transgenic mouse models in an FVB strain background. a. Toxin Administration 1. Griseofulvin: Mice are fed a powdered Lab Diet (PMI Feeds, Inc) with 1.25% griseofulvin (Sigma) for 7–17 days. The amount of griseofulvin was chosen on the basis of preliminary experiments that showed that this dosing and duration lead to fatality within 10 days in the K18 Arg 89!Cys overexpressing transgenic mice that are highly susceptible to drug-induced liver injury (Ku et al., 1996). This injury model is diVerent than the longer duration feeding regimen, which induces Mallory body formation (see Chapter 8). 2. Acetaminophen (McNeil): Mice are deprived of solid food for 3 hours followed by administration of acetaminophen by gavage (400 g/g body wt, diluted with H2O to a final volume of 0.4–0.5 ml), then switching the water source to 5% dextrose in water. After 2 hours, the mice are allowed to feed ad lib and observed intermittently for 72 hours, followed by tissue harvesting and analysis (Ku et al., 1996). 3. Microcystin-LR (MLR) (Alexis Corp): MLR is a hepatotoxin that inhibits type 1 and type 2A serine/threonine phosphatases and causes extensive intrahepatic hemorrhage (see also Chapter 14). MLR is prepared in dimethylsulfoxide as a

Table II Simple Epithelial Keratin-Related Disease Animal Models Organ

Disease

Specifics

Griseofulvin

Acute/chronic hepatitis

Acetaminophen Microcystin-LR

Acute hepatitis Acute hepatitis

Feed mice for 7–17 days (1.25% griseofulvin in food) Gavage, 400 g/g* Single IP{30 ng/g

Fas antibody

Acute hepatitis

Single IP 0.15 g/g

TNF- (with Act. D{)

Acute hepatitis

Gallbladder and liver

Concanavalin A Lithogenic diet

Pancreas

Caerulein

Acute hepatitis Gallstones and steatohepatitis Acute pancreatitis (generally mild)

Single IP 15 ng/g TNF- (with 1 g/g Act. D{) Single IVx30 g/g Feed mice for 5 weeks

Liver

Model/Toxin

Choline-deficient, ethionine-supplemented diet (CD diet)

Acute pancreatitis (generally severe)

Small Intestine

Gamma irradiation Rotavirus infection

Colon

Dextran sulfate sodium

Stem cell apoptosis Diarrhea and crypt cell injury Colitis

*Reagents are administrated per gram of mouse body weight. IP, Intraperitoneal. { Act. D, actinomycin D. {

Hourly seven IP injections; no age or sex preference Feed mice for up to 3 days; more eVective in females; causes 50% lethality in 16–20 g weight mice Whole-body irradiation at 8 Gy Oral or intestinal loop infection Feed mice for up to 12 days (2% dextran sulfate sodium in water)

References Ku et al. (1996) Ku et al. (1996) Toivola et al. (1998) Ku et al. (1998) Gilbert et al. (2001) Ku et al. (2003b) Ku et al. (2003b) Caulin et al. (2000) Tao et al. (2003) Toivola et al. (2000a,b) Zhong et al. (2004) Zhong and Omary (2004) Toivola et al. (2000a,b) Zhong et al. (2004) Zhong and Omary (2004) Martin et al. (1998) Burns et al. (1995) Ludert et al. (1996) Siegmund et al. (2002) Ku et al. (unpublished results)

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1 mg/ml stock solution and then diluted in PBS (pH 7.4). Mice are fasted overnight then given 30 ng/g mouse wt intraperitoneally (IP), followed by processing of the livers after 3 hours (Ku et al., 1998b; Toivola et al., 1998). 4. Proapoptotic reagents: Apoptosis is induced by IP injection of Fas antibody (0.15 g/g body wt) (PharMingen) to overnight-fasted mice. Alternatively, purified TNF- (15 ng/g) (Sigma) with actinomycin D (1 /g) (Sigma) is injected. Mice are killed by CO2 inhalation 4 hours after injection (Ku et al., 2003b). Concanavalin A (Caulin et al., 2000) or Fas antibody (Gilbert et al., 2001) can also induce apoptosis-mediated liver injury as tested in K8-null mice. b. Partial Hepatectomy Partial hepatectomy is performed by sequentially removing the lateral, left, and right median lobes from mice (Wilson et al., 1953; Yokoyama et al., 1953). Mice are euthanized 1, 2, 3, 7, or 10 days afterwards, depending on the nature of the experiment. For control sham-hepatectomy, mice undergo anesthesia, abdominal wall and peritoneal incision, liver exposure, and then closure of the incision (Ku et al., 2002b). Peak mitotic activity is noted 2–3 days after liver resection.

2. Gallbladder and Liver: High-Fat Lithogenic Diet-induced Injury Male mice (4–6 weeks old) are fed a lithogenic diet (Cat. No. 960393, ICN Biomedicals) containing 1.23 g cholesterol, 0.48 g sodium cholate, 17.84 g butter fat, 0.98 g corn oil, 48.33 g sucrose, 19.33 g casein, and essential vitamins and minerals for 5 weeks (Tao et al., 2003). All mice are allowed free access to water. This diet induces gallbladder stone formation and causes liver injury from fat deposition. A dissection microscope needs to be used to optimally visualize stone or stone-debris formation.

3. Pancreatitis Models Pancreatic injury can be induced by various mouse models (Table II) (Banerjee et al., 1994; Lerch and Adler, 1994) with subsequent eVects on keratin expression, degradation, phosphorylation, and filament organization (Toivola et al., 2000a,b; Zhong and Omary, 2004; Zhong et al., 2004). Caerulein, an analog of the secretagogue cholecystokinin, causes pancreatic autolysis and mild edematous, nonlethal pancreatitis (Lerch and Adler, 1994). The choline-deficient, ethionine-supplemented diet (CD diet) causes severe acute hemorrhagic pancreatitis with fat necrosis and results in significant fatality in young female mice, which are most susceptible to this injury (Lerch and Adler, 1994). Pancreatitis can also be induced by other models such as the coxsackievirus B4, which causes acute or chronic pancreatitis depending on the virus strain (Caggana et al., 1993; Toivola et al., unpublished observations). The pancreatitis models that have been used to study keratin biology in the pancreas (Table II) will be described.

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a. Caerulein-Induced Acute Pancreatitis 1. Starve mice from solid food overnight, but allow water intake ad libitum. Susceptibility to injury in this model seems to be sex and age independent, but there are strain diVerences (Zhong and Omary, 2004). 2. Inject IP 0.9% saline (in control mice) or 50 g/kg of mouse wt caerulein diluted in 0.9% NaCl (Research Plus; or #C-9026, Sigma) once every hour for 6 hours (seven total injections). 3. Sacrifice mice 1–240 hours after the first injection and collect blood by cardiac puncture, then quickly excise and process the pancreas. In this model, caerulein induces reversible keratin degradation, hyperphosphorylation, and overexpression. An example of K20 overexpression with dramatic formation of K20-containing cytoplasmic filaments is shown in Fig. 5e,f. b. Choline-Deficient, Ethionine Supplemented Diet Induced Acute Pancreatitis 1. Starve young female mice (14–19 g, typically 4–5 weeks old) from solid food for 12–16 hours, but allow water intake ad libitum. 2. Mix CD powdered chow (#TD90262, Harlan Teklad), thoroughly with 0.5% dl-ethionine (#E-5139, Sigma). 3. Allow mice to feed with the CD diet for 3 days, then switch to a normal diet for 1–7 days. The diet is placed in cups at a corner of the cage and refurbished daily. Note that dl-ethionine is carcinogenic; thus, excess food and bedding need to be discarded as hazardous waste. 4. Depending on the age of the mice, CD diet induced lethality, when present, typically occurs in 1–2 days, but no later than 5 days, after discontinuation of the diet. Complete disruption of keratin filament networks together with vacuole formation and cell death is most prominent 60 hours after the onset of the diet (Toivola et al., 2000a,b; Zhong and Omary, 2004; Zhong et al., 2004).

4. Small Intestine Injury Models a. Irradiation Two-month-old male mice are examined 4, 24, 48, 72 hours after whole-body gamma irradiation, given as an acute 8-Gy dose. Cell injury and apoptosis occur primarily in the crypt region of the epithelium (Martin et al., 1998). b. Rotavirus-Induced Diarrhea Model in Suckling Mice Five-day-old mice are orally inoculated with 104 diarrhea dose 50 (DD50) of a virulent wild-type murine rotavirus strain ECw. Presence of diarrhea in the suckling mice is confirmed by gently pressing the lower abdomen of the mice and resolves 10 days after infection (Burns et al., 1995).

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c. In Vivo Small Bowl Loop Rotavirus Infection Mice are given ketamine and xylazine for anesthesia. A ventral abdominal incision is made in 10-day-old suckling mice or 4- to 6-week-old mice. Two loops of small bowel (jejunum or ileum) approximately 0.5 to 1 cm in length are isolated with fine suture material. The loops are injected with virus by use of a Hamilton syringe, and the abdomen is closed with suture or glue. After 4–10 hours, injected mice are euthanized followed by harvesting of the isolated loops for imaging and biochemical studies. Analgesia (0.005–0.05 mg/kg of bupronorphine) and hydration (0.5 ml saline) are administrated IP postoperatively (Ludert et al., 1996).

5. Colitis Injury Model Dextran sulfate sodium (DSS) causes colitis by interfering with the intestinal epithelial cell barrier with subsequent induction of cytokines and other inflammatory mediators (Blumberg et al., 1999). For lethality experiments, mice are given 2% DSS (ICN Pharmaceuticals, Inc) that is dissolved in the drinking water. DSS is administered for up to 12 days followed by switching to normal drinking water. Mice are assessed daily for up to 4 weeks (Ku et al., unpublished observation; Siegmund et al., 2002). Lethality typically begins toward the end of the DSS administration period. For nonlethality studies, mice are given 2% DSS for 10 days or less followed by removal of the colons for histology, immunofluorescence, and biochemical studies.

III. Pearls and Pitfalls A. Isolation of Keratins

1. High-Salt Extraction 1. Although the HSE method retains most of the keratins (>90%) in the cell, a small portion of soluble keratins are discarded as part of the supernatant fractions. 2. Occasionally, nonkeratin proteins and nucleic acid genomic material may coprecipitate with the keratins because of insuYcient homogenization and douncing or inadequate solubilization buVer to cell/tissue pellet volume ratio. This will result in a high background after gel analysis. Also, keratin degradation may occur during the isolation procedure, so protease inhibitors are added in the buVer (EDTA is essential to minimize keratin degradation during HSE and immunoprecipitation), and the procedures should be performed as eYciently as possible. 3. In some occasions, keratin analysis of individually isolated fractions including cytosolic (detergent free) membrane or ‘‘loosely’’ associated cytoskeleton, cytoskeletal, or remaining insoluble fractions may need to be carried out. For this, we previously described a sequential extraction method (see Omary et al., 1998 for details).

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2. Immunoprecipitation 1. We routinely test the anti-keratin antibodies by immunoprecipitation with both protein A and G, and then select what works better. In addition, we routinely covalently conjugate our antibodies to protein A or G Sepharose beads (with a kit purchased from Pierce) to minimize the amount of antibody release from the beads after adding the sample buVer. 2. Nonspecific protein binding can be a problem, which is generally overcome by the preclearing step or washing with salt or detergent-containing buVers. Inclusion of a control immunoprecipitation where the test antibody is replaced by a nonrelevant immunoglobulin is important. 3. Immunoprecipitation of NP-40 (or other nonionic detergent) solubilized cells can isolate, if quantitative, 10%–15% of the total cellular keratins, whereas Empigen extracts 50% of the total keratin pool. However, these quantities can be significantly less, depending on antibody aYnity and experimental conditions (e.g., interphase vs. mitotic cells; incubation of cells with phosphatase inhibitors that increase keratin solubility). 4. When testing keratin-associated proteins by coimmunoprecipitation, it is essential to use two or more antikeratin antibodies to avoid ‘‘pseudo-association’’ by means of molecular mimicry of antibody binding (e.g., see Zhou et al., 2000). 5. We typically use nonreducing sample buVer for gel analysis, particularly for immunoprecipitate analysis of K8/K18/K19, which do not contain any cysteines.

B. Posttranslational Modification of Keratins 1. [32P]-orthophosphoric acid usually is sold in 1-ml volume, independent of the purchased millicurries, hence, it may be necessary to custom request the volume as needed to ensure high specific activity. 2. D-[6-3H]-glucosamine and uridine diphosphate (UDP)-[4,5-3H]-galactose are dried, just before application, by use of a SpeedVac concentrator to remove the stabilizing organic solvent, typically 90% ethanol.

C. Keratin Staining in Tissues and Cells 1. Most antibodies for keratins and other IF proteins work best when the cells or tissues are fixed with water-free 20  C methanol or acetone (10 minutes), whereas some antibodies require formalin fixation or the use of antigen retrieval protocols (Table I). Double labeling of a specimen with antibodies or reagents, which requires acetone (e.g., keratin antibodies), or PFA (e.g., fluorescent-phalloidin, which binds to F-actin), can be done by use of

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mild PFA fixation (0.5% for 20 minutes), which gives reasonable staining results. 2. Isotype-specific secondary antibodies or fluorophore-conjugated antibodies can be used to double-label with two mouse monoclonal antibodies simultaneously. Online resources for fluorophores and help with selection of secondary antibodies can be found at http://fluorescence.bio-rad.com/, http:// www.probes.com/handbook/, and http://www.jacksonimmuno.com/.

D. Simple Epithelial Keratins in Organ-Specific Injury Models 1. In assessing injury models in mice, there is significant variability in the extent of injury. This variability needs to be taken into consideration so that adequate numbers of mice are analyzed, because comparison among genotypes may be missed or overinterpreted if not enough mice are examined. 2. For the CD diet that induces pancreatitis, the extent of injury becomes limited if old (the 4- to 5-week age window is critical for lethality-type experiments) mice are used.

IV. Concluding Remarks We have described tools to study simple epithelial keratins in cell culture and in mice. The biochemical approaches we described in Section II.A and II.B can be used to expand on our current knowledge of the regulation of K7, K8, K18, K19, and K20 and are routinely used in our laboratory. In addition, such approaches can be extended to other keratins and IF proteins (see also Chapters 5, 13, and 14) for which analysis of their posttranslational modifications and regulation by associated proteins has been somewhat limited. Although the lower solubility of nonsimple epithelial keratins can be challenging and may hinder the usefulness of such approaches, we believe that they can still be applicable. In addition to the biochemical approaches, analysis of keratin organization at the light microscope level as highlighted in Section II.C is critical in understanding the relationship of keratin organization to its regulation. It is clear from cell culture systems, and more importantly from animal model studies, that mutation at a single residue (e.g., K18 Arg89!Cys) can have a tremendous eVect on keratin function, organization, and posttranslational modifications (Ku et al., 2003b). Hence, assessment of keratin organization in tissues and cells, as described in Section II.C, provides a powerful tool, particularly with the increasing ability to generate specialized antibody probes. For example, probes to specific phosphorylation sites of K8 and K18 (Table I), and to K19 and K20 (unpublished results), and to the K18 caspase-generated cleavage site (Table I) provide essential and highly sensitive adjuncts to study keratin regulation at the single cell level.

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As K8 and K18 mutations become recognized as a likely risk factor for a variety of human digestive diseases (Omary et al., 2004), the usefulness of the injury models described in Section II.D will become even more essential. The relationship of K8 and K18 to human disease includes liver disease (K8 and K18 mutations: Ku et al., 2001, 2003a), inflammatory bowel disease (K8 mutations: Owens et al., 2004), and chronic pancreatitis (K8 mutations: Cavestro et al., 2003). It remains to be determined whether mutations in K7, K19, and K20 will cause or predispose patients to digestive or nondigestive (e.g., in the case of K19) diseases that relate to their cell preferential expression. Regardless, understanding the pathogenesis of the currently known K8 and K18 mutations and the function of simple epithelial keratins should be facilitated by the use of approaches described in this chapter in combination with animal and tissue culture models of disease-related mutations. Acknowledgments We thank Kris Morrow for his assistance with figure preparation. Our work is supported by NIH grants DK52951 and DK47918 and Department of Veterans AVairs Merit Award (M.B.O.). We are also grateful to VA Research Enhancement Award Program (Q.Z.) and Crohn’s & Colitis Foundation of America Research Award support (G-Z.T.).

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