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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00782

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Keratin-8 null mice have different gallbladder and liver susceptibility to lithogenic diet-induced injury

Guo-Zhong Tao^1,*, Diana M. Toivola¹, Bihui Zhong¹, Sara A. Michie¹, Evelyn Z. Resurreccion¹, Yoshitaka Tamai², Makoto M. Taketo³ and M. Bishr Omary^1,

¹ Palo Alto VA Medical Center, Palo Alto, Mail code 154J, 3801 Miranda Avenue, Palo Alto, CA 94304 and Stanford University School of Medicine Digestive Disease Center, Stanford, CA 94305, USA
² Banyu Tsukuba Research Institute (Merck), Tsukuba 300-2611, Japan
³ Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

View larger version (29K): [in a new window]   Fig. 1. Characterization of normal mouse gallbladder keratins and comparison with keratins of the liver and small intestine. (A) Keratins were isolated using high salt extraction from BALB/c mouse gallbladder (GB), liver and small intestine (SI), then analyzed by SDS-PAGE and Coomassie Blue staining (upper panel) or by immune blotting using antibodies specific to K8, K18, and K19 (lower panel). (B) Keratin high salt extract preparations were obtained as in A then analyzed using two-dimensional gels followed by Coomassie Blue staining. Separation in the first (horizontal) dimension was carried out using isoelectric focusing (IEF) followed by separation in the second (vertical) dimension using SDS-PAGE. Arrowheads highlight desmin isoforms that are derived from smooth muscle cells present in the gallbladder and intestine but not the liver.

View larger version (39K): [in a new window]   Fig. 2. Comparison of keratin expression profiles in gallbladders of different mouse keratin genotypes. (A) Gallbladders from six mice of each indicated genotype were isolated and pooled followed by high salt extraction of the relatively insoluble proteins (primarily keratins) as described in Materials and methods. The extracts were analyzed by SDS-PAGE followed by visualization of the protein bands by Coomassie Blue staining (upper panel). Arrowhead indicates position of desmin. Duplicate gels were transferred to membranes followed by blotting with the indicated anti-keratin antibodies (lower panel). (B) Gallbladders were isolated from K8-null (–/–), K8 heterozygous (+/–), or K8 WT (+/+) mice (2 mice/genotype). The gallbladders from each genotype were pooled then used to prepare total tissue homogenates that were analyzed by immunoblotting using antibodies specific to K8, K18, K19 and actin. (C) Keratin expression at the protein and mRNA levels was analyzed. (a) Gallbladders were isolated from three K19 WT and three K19-null mice, followed by analyses of equal amount of total lysate protein samples using SDS-PAGE and immunoblotting using the indicated keratin-specific antibodies. (b) Total RNA was isolated from K19-null (striped bars) or K19 WT (open bars) gallbladders (pooled, n=3 for each genotype) followed by RT-PCR and real time PCR analysis. Note that both K18 and K20 are up-regulated at the protein and mRNA levels in K19-null gallbladders.

View larger version (96K): [in a new window]   Fig. 7. Effect of a lithogenic diet on keratin phosphorylation in the gallbladder and liver. (A) Liver sections from K8+/+ (a,b) or K8–/– (c,d) mice that were fed control diet (a,c) or LD (b,d) were stained by indirect immunofluorescence using anti-K8/18 antibodies. Note the absence of keratins in K8–/– liver, except for staining of biliary ductal cells owing to the presence of K7 and K19 in these cells. (B,C) Liver (B) and gallbladder (C) sections from K8+/+ mice that were fed a control or lithogenic diet were triple stained using toto-3 (blue; nuclear DNA staining), anti-K8/K18 or anti-K18-pS33 (red), and anti-K8 pS436 or pS79 (green). L, lumen. Asterisks in b and d of B indicate areas of injury. Note the induction of liver and gallbladder keratin phosphorylation at the three different K8/K18 phosphorylation sites tested, after exposure to the lithogenic diet. Scale bars in A,B,C: 20 µm.

View larger version (69K): [in a new window]   Fig. 3. Indirect immunofluorescence keratin staining of gallbladders of K8+/+ and K8–/– mice. Gallbladders were removed from age and sex matched K8+/+ or K8–/– mice, followed by fixing then staining using the indicated keratin-specific antibodies. L, lumen; scale bar (in a): 10 µm. Note the absence of K8 and the limited compensatory induction of K7 in K8–/– mouse gallbladder.

View larger version (83K): [in a new window]   Fig. 4. Histologic analysis of LD-induced injury in WT and K8-null mouse gallbladder and liver. K8 mice (–/–, +/+) were fed a normal or lithogenic diet for 5 weeks. Gallbladders and livers were resected, fixed, paraffin embedded, sectioned, and then stained with Hematoxylin and Eosin. (a-d) Gallbladder sections from mice fed a normal (a,b) or a lithogenic (c,d) diet. E, single layer of cuboidal epithelial cells; M, muscularis; I, small foci of mononuclear inflammatory cells. (e-h) Liver sections from mice fed a normal (e,f) or a lithogenic (g,h) diet. (f) A focal area of inflammation (arrow) and a small area of hemorrhage (arrowhead). (g) A small area of inflammation and necrosis (arrow). Panel h shows a markedly vacuolated cytoplasm with displacement of the nuclei to the cell periphery, Kupffer cell nodule (arrow) and hepatocyte erythrophagocytosis (arrowhead). The histologic findings of the liver and gallbladder of K8+/– mice that were fed a normal diet or LD (not shown) were very similar to those shown for the corresponding K8+/+ mice. Scale bar (in a): 100 µm for a-d and 200 µm for e-h.

View larger version (22K): [in a new window]   Fig. 5. Serologic assessment of liver injury among K8 mouse genotypes after a lithogenic diet. Blood was collected from K8 mice that were fed a lithogenic diet (the same mice as used for the experiments of Table 3), or from five age-matched mice that were fed a normal diet. Sera were isolated and used to measure alkaline phosphatase (AP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The y axis shows the mean fold increase in serum enzymes compared with mice fed control diet. *P<0.05 when comparing K8–/– with K8+/– or K8+/+.

View larger version (35K): [in a new window]   Fig. 6. Effect of a lithogenic diet on keratin expression levels in the gallbladder and liver. (A) Total tissue homogenates of gallbladders (GB) from K8–/–, K8+/– and K8+/+ mice that were fed a normal or lithogenic diet were analyzed by immunoblotting using antibodies to the indicated keratins and to actin. (B) Total tissue homogenates of livers from the same mice used in A were analyzed by immunoblotting using antibodies to actin, tubulin and the indicated keratins. Note that the LD induces overexpression of keratins in WT mouse gallbladder but not liver.

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