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Keratin 8 overexpression promotes mouse Mallory body formation.

Nakamichi I, Toivola DM, Strnad P, Michie SA, Oshima RG, Baribault H, Omary MB - J. Cell Biol. (2005)

Bottom Line: Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression.MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC).Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stanford University, and Veterans Affairs Palo Alto Health Care System, CA 94305, USA.

ABSTRACT
Keratins 8 and 18 (K8/18) are major constituents of Mallory bodies (MBs), which are hepatocyte cytoplasmic inclusions seen in several liver diseases. K18- but not K8- or heterozygous mice form MBs, which indicates that K8 is important for MB formation. Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression. We used transgenic mice that overexpress K8, K18, or K8/18 to test the importance of K8 and/or K18 in MB formation. MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Livers of young K8 or K8/K18 overexpressors had no histological abnormalities despite increased keratin protein and phosphorylation. In aging mice, only K8-overexpressing livers spontaneously developed small "pre-MB" aggregates. Only K8-overexpressing young mice are highly susceptible to MB formation after short-term DDC feeding. Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation. K8 overexpression is sufficient to form pre-MB and primes animals to accumulate MBs upon DDC challenge, which may help explain MB formation in human liver diseases.

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Liver histology and keratin mRNA, protein levels, and immune staining in young WT, K8, K18, and K8/18 mice. (A) Real-time PCR of the indicated keratin mRNA (normalized to ribosomal L7 mRNA) in livers of the four genotypes. For mK8 and mK18 mRNA, the mRNA levels are normalized to those in WT mice (mean ± SD; n = 3/genotype). For mice that overexpress hK18, the levels are normalized to hK18 overexpressors (N/A, not applicable; because WT and K8 mice do not express hK18). (B) Total liver homogenates were prepared, and equal amounts of protein were separated by SDS-PAGE followed by blotting using Abs to the indicated keratin or phosphokeratin epitopes. Actin is included as a loading control. H, human; m, mouse. (C) Livers from 10-wk-old sex-matched mice were fixed, sectioned, and stained with hematoxylin (H) and eosin (E; a, d, g, and j). Alternatively, liver keratins were visualized by immunofluorescence staining using Abs to K8/18 (pankeratin; b, e, h, and k) or to K18 pS33 (c, f, i, and l). Arrows (f and l) highlight basal K18 pS33 staining to contrast with the surrounding enhanced staining (asterisks). Insets show separately performed double staining using anti-mK18 (e) and anti-K18 pS33 (f) to highlight some cells (asterisks) with increased K18 and K18 pS33. Bars, 50 μm.
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fig1: Liver histology and keratin mRNA, protein levels, and immune staining in young WT, K8, K18, and K8/18 mice. (A) Real-time PCR of the indicated keratin mRNA (normalized to ribosomal L7 mRNA) in livers of the four genotypes. For mK8 and mK18 mRNA, the mRNA levels are normalized to those in WT mice (mean ± SD; n = 3/genotype). For mice that overexpress hK18, the levels are normalized to hK18 overexpressors (N/A, not applicable; because WT and K8 mice do not express hK18). (B) Total liver homogenates were prepared, and equal amounts of protein were separated by SDS-PAGE followed by blotting using Abs to the indicated keratin or phosphokeratin epitopes. Actin is included as a loading control. H, human; m, mouse. (C) Livers from 10-wk-old sex-matched mice were fixed, sectioned, and stained with hematoxylin (H) and eosin (E; a, d, g, and j). Alternatively, liver keratins were visualized by immunofluorescence staining using Abs to K8/18 (pankeratin; b, e, h, and k) or to K18 pS33 (c, f, i, and l). Arrows (f and l) highlight basal K18 pS33 staining to contrast with the surrounding enhanced staining (asterisks). Insets show separately performed double staining using anti-mK18 (e) and anti-K18 pS33 (f) to highlight some cells (asterisks) with increased K18 and K18 pS33. Bars, 50 μm.

Mentions: Keratin mRNA and protein expression were analyzed in four transgenic mouse lines: single (K8 or K18) or double overexpressors (K8/18) and control wild-type (WT) nonoverexpressors. These lines were generated by mating mK8 overexpressors (EA5 +/−) together or with hK18 overexpressors (TG2 +/+; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507093/DC1). Analysis of keratin transcription by real-time PCR demonstrated a 3.2-fold increase of mK8 RNA in the K8 overexpressors and a similar increase (2.5-fold) in mice that overexpressed K8/18 as compared with WT mice (Fig. 1 A). K8 overexpression had a slightly more prominent effect on K18 mRNA induction (1.8 ± 0.2) as compared with the effect of K18 overexpression on K8 (1.3 ± 0.3). As expected, hK18 mRNA was detected in the K18 and K8/18 overexpressors (Fig. 1 A). Keratin protein overexpression paralleled the mRNA changes (Fig. 1 B). For example, mK8 protein increased ∼2.5-fold in the K8 and K8/18 overexpressors (see Protein analysis). K8 overexpression has a more profound impact on K18 protein induction as compared with K18 overexpression on K8 protein induction, and K8 + K18 protein levels are highest in the K8/18 double and K8 overexpressors (Fig. 1 B). Therefore, of the K8, 18, or 8/18 lines, the normal 1:1 K8 to K18 liver protein ratio is best approximated in the K8/18 overexpressors while yielding a nearly 2.5-fold increase in overall keratin protein.


Keratin 8 overexpression promotes mouse Mallory body formation.

Nakamichi I, Toivola DM, Strnad P, Michie SA, Oshima RG, Baribault H, Omary MB - J. Cell Biol. (2005)

Liver histology and keratin mRNA, protein levels, and immune staining in young WT, K8, K18, and K8/18 mice. (A) Real-time PCR of the indicated keratin mRNA (normalized to ribosomal L7 mRNA) in livers of the four genotypes. For mK8 and mK18 mRNA, the mRNA levels are normalized to those in WT mice (mean ± SD; n = 3/genotype). For mice that overexpress hK18, the levels are normalized to hK18 overexpressors (N/A, not applicable; because WT and K8 mice do not express hK18). (B) Total liver homogenates were prepared, and equal amounts of protein were separated by SDS-PAGE followed by blotting using Abs to the indicated keratin or phosphokeratin epitopes. Actin is included as a loading control. H, human; m, mouse. (C) Livers from 10-wk-old sex-matched mice were fixed, sectioned, and stained with hematoxylin (H) and eosin (E; a, d, g, and j). Alternatively, liver keratins were visualized by immunofluorescence staining using Abs to K8/18 (pankeratin; b, e, h, and k) or to K18 pS33 (c, f, i, and l). Arrows (f and l) highlight basal K18 pS33 staining to contrast with the surrounding enhanced staining (asterisks). Insets show separately performed double staining using anti-mK18 (e) and anti-K18 pS33 (f) to highlight some cells (asterisks) with increased K18 and K18 pS33. Bars, 50 μm.
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fig1: Liver histology and keratin mRNA, protein levels, and immune staining in young WT, K8, K18, and K8/18 mice. (A) Real-time PCR of the indicated keratin mRNA (normalized to ribosomal L7 mRNA) in livers of the four genotypes. For mK8 and mK18 mRNA, the mRNA levels are normalized to those in WT mice (mean ± SD; n = 3/genotype). For mice that overexpress hK18, the levels are normalized to hK18 overexpressors (N/A, not applicable; because WT and K8 mice do not express hK18). (B) Total liver homogenates were prepared, and equal amounts of protein were separated by SDS-PAGE followed by blotting using Abs to the indicated keratin or phosphokeratin epitopes. Actin is included as a loading control. H, human; m, mouse. (C) Livers from 10-wk-old sex-matched mice were fixed, sectioned, and stained with hematoxylin (H) and eosin (E; a, d, g, and j). Alternatively, liver keratins were visualized by immunofluorescence staining using Abs to K8/18 (pankeratin; b, e, h, and k) or to K18 pS33 (c, f, i, and l). Arrows (f and l) highlight basal K18 pS33 staining to contrast with the surrounding enhanced staining (asterisks). Insets show separately performed double staining using anti-mK18 (e) and anti-K18 pS33 (f) to highlight some cells (asterisks) with increased K18 and K18 pS33. Bars, 50 μm.
Mentions: Keratin mRNA and protein expression were analyzed in four transgenic mouse lines: single (K8 or K18) or double overexpressors (K8/18) and control wild-type (WT) nonoverexpressors. These lines were generated by mating mK8 overexpressors (EA5 +/−) together or with hK18 overexpressors (TG2 +/+; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507093/DC1). Analysis of keratin transcription by real-time PCR demonstrated a 3.2-fold increase of mK8 RNA in the K8 overexpressors and a similar increase (2.5-fold) in mice that overexpressed K8/18 as compared with WT mice (Fig. 1 A). K8 overexpression had a slightly more prominent effect on K18 mRNA induction (1.8 ± 0.2) as compared with the effect of K18 overexpression on K8 (1.3 ± 0.3). As expected, hK18 mRNA was detected in the K18 and K8/18 overexpressors (Fig. 1 A). Keratin protein overexpression paralleled the mRNA changes (Fig. 1 B). For example, mK8 protein increased ∼2.5-fold in the K8 and K8/18 overexpressors (see Protein analysis). K8 overexpression has a more profound impact on K18 protein induction as compared with K18 overexpression on K8 protein induction, and K8 + K18 protein levels are highest in the K8/18 double and K8 overexpressors (Fig. 1 B). Therefore, of the K8, 18, or 8/18 lines, the normal 1:1 K8 to K18 liver protein ratio is best approximated in the K8/18 overexpressors while yielding a nearly 2.5-fold increase in overall keratin protein.

Bottom Line: Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression.MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC).Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stanford University, and Veterans Affairs Palo Alto Health Care System, CA 94305, USA.

ABSTRACT
Keratins 8 and 18 (K8/18) are major constituents of Mallory bodies (MBs), which are hepatocyte cytoplasmic inclusions seen in several liver diseases. K18- but not K8- or heterozygous mice form MBs, which indicates that K8 is important for MB formation. Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression. We used transgenic mice that overexpress K8, K18, or K8/18 to test the importance of K8 and/or K18 in MB formation. MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Livers of young K8 or K8/K18 overexpressors had no histological abnormalities despite increased keratin protein and phosphorylation. In aging mice, only K8-overexpressing livers spontaneously developed small "pre-MB" aggregates. Only K8-overexpressing young mice are highly susceptible to MB formation after short-term DDC feeding. Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation. K8 overexpression is sufficient to form pre-MB and primes animals to accumulate MBs upon DDC challenge, which may help explain MB formation in human liver diseases.

Show MeSH
Related in: MedlinePlus