Limits...
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.

Show MeSH

Related in: MedlinePlus

ALT levels and MB formation after DDC feeding. WT, K8, K18, and K8/18 mice (4–5 mice per genotype; 3–4-mo-old sex-matched mice) were fed a DDC-containing diet for 6 wk followed by harvesting of the livers and blood. (A) ALT levels (means ± SD), with a significant increase noted in K8 as compared with WT mice. (B) Livers from mice described in A were fixed and stained with hematoxylin and eosin. Histology of the livers from WT (a) and K8 (b) mice is shown, but K18 and K8/18 liver histology was very similar to WT (not depicted). Note the significant MB formation in K8 livers (arrows). Bar, 200 μm. (C) MBs were quantified (see Materials and methods) using the hematoxylin and eosin–stained liver sections described in B. The P values for comparing the genotypes WT with K8, K8 with K18, and K8 with K8/18 are shown. Error bars represent SD. (D) Conventional (a and b) and immune (c–f) electron microscopy of livers from K8 mice that were fed a DDC-containing diet show typical-appearing MB deposits. In a, the MB is encircled with a black line, and in b, arrows highlight IF bundles that are in close proximity to MBs. For immune electron microscopy, keratins were labeled with 10 nm gold particles (c–e). The particles are difficult to see in c but are clearly evident in the higher magnification images d and e. As a specificity control, the grids were also treated with nonimmune serum (f; note the absence of gold particles as contrasted with e). Arrows in e and f point to keratin IF bundles. Bars (a), 2 μm; (b) 500 nm; (c) 300 nm; (d) 100 nm; (e) 300 nm. N, nucleus.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171301&req=5

fig3: ALT levels and MB formation after DDC feeding. WT, K8, K18, and K8/18 mice (4–5 mice per genotype; 3–4-mo-old sex-matched mice) were fed a DDC-containing diet for 6 wk followed by harvesting of the livers and blood. (A) ALT levels (means ± SD), with a significant increase noted in K8 as compared with WT mice. (B) Livers from mice described in A were fixed and stained with hematoxylin and eosin. Histology of the livers from WT (a) and K8 (b) mice is shown, but K18 and K8/18 liver histology was very similar to WT (not depicted). Note the significant MB formation in K8 livers (arrows). Bar, 200 μm. (C) MBs were quantified (see Materials and methods) using the hematoxylin and eosin–stained liver sections described in B. The P values for comparing the genotypes WT with K8, K8 with K18, and K8 with K8/18 are shown. Error bars represent SD. (D) Conventional (a and b) and immune (c–f) electron microscopy of livers from K8 mice that were fed a DDC-containing diet show typical-appearing MB deposits. In a, the MB is encircled with a black line, and in b, arrows highlight IF bundles that are in close proximity to MBs. For immune electron microscopy, keratins were labeled with 10 nm gold particles (c–e). The particles are difficult to see in c but are clearly evident in the higher magnification images d and e. As a specificity control, the grids were also treated with nonimmune serum (f; note the absence of gold particles as contrasted with e). Arrows in e and f point to keratin IF bundles. Bars (a), 2 μm; (b) 500 nm; (c) 300 nm; (d) 100 nm; (e) 300 nm. N, nucleus.

Mentions: We tested whether the occurrence of pre-MB aggregates in old K8 mice could render younger K8 mice more susceptible to MB formation by feeding the four mouse genotypes a DDC-containing diet. Mice fed with DDC or griseofulvin for 3–4 mo are established models of MB formation (Yuan et al., 1996; Cadrin et al., 2000; Denk et al., 2000; Stumptner et al., 2000). However, in our experimental design, we analyzed the livers at an early time point of 6 wk after DDC feeding, when MBs are typically not seen. In DDC-fed mice, there was a significant increase in serum alanine aminotransferase (ALT; an indicator of hepatocyte necrosis) in K8 as compared with WT, K18, or K8/18 mice (Fig. 3 A) but no significant difference in liver damage when assessed by hematoxylin and eosin staining (not depicted). Other serologic tests (see Animal feeding and tissue experiments) were comparable in all four genotypes (not depicted). However, MBs were found almost exclusively in the K8 mice, as confirmed by hematoxylin and eosin staining (Fig. 3, B and C), standard and immune electron microscopy (Fig. 3 D), and immunofluorescence staining using antikeratin and antiubiquitin antibodies (Fig. 4 A). K8 livers had 35 ± 19 hepatocytes with MBs per 10 high power fields compared with 2.6 ± 3.7, 1.5 ± 1.7, and 0.2 ± 0.4 for WT, K18, and K8/18 livers, respectively (Fig. 3 C). Ubiquitin localized in most, if not all, of the keratin aggregates (Fig. 4 A), whereas p62 (which is a component of MBs; Zatloukal et al., 2000, 2004) was seen in some of the aggregates (not depicted). After DDC feeding, keratin protein levels increased dramatically, as expected in all genotypes (Fig. 4 B), in concert with an increase in K8 and K18 mRNA levels (not depicted). In addition, phosphorylation of K8 S79 and K18 S33 increased similarly in all four genotypes (not depicted) as described previously for WT mice (Stumptner et al., 2000). Therefore, K8-overexpressing mice are highly primed to develop MBs after DDC challenge.


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)

ALT levels and MB formation after DDC feeding. WT, K8, K18, and K8/18 mice (4–5 mice per genotype; 3–4-mo-old sex-matched mice) were fed a DDC-containing diet for 6 wk followed by harvesting of the livers and blood. (A) ALT levels (means ± SD), with a significant increase noted in K8 as compared with WT mice. (B) Livers from mice described in A were fixed and stained with hematoxylin and eosin. Histology of the livers from WT (a) and K8 (b) mice is shown, but K18 and K8/18 liver histology was very similar to WT (not depicted). Note the significant MB formation in K8 livers (arrows). Bar, 200 μm. (C) MBs were quantified (see Materials and methods) using the hematoxylin and eosin–stained liver sections described in B. The P values for comparing the genotypes WT with K8, K8 with K18, and K8 with K8/18 are shown. Error bars represent SD. (D) Conventional (a and b) and immune (c–f) electron microscopy of livers from K8 mice that were fed a DDC-containing diet show typical-appearing MB deposits. In a, the MB is encircled with a black line, and in b, arrows highlight IF bundles that are in close proximity to MBs. For immune electron microscopy, keratins were labeled with 10 nm gold particles (c–e). The particles are difficult to see in c but are clearly evident in the higher magnification images d and e. As a specificity control, the grids were also treated with nonimmune serum (f; note the absence of gold particles as contrasted with e). Arrows in e and f point to keratin IF bundles. Bars (a), 2 μm; (b) 500 nm; (c) 300 nm; (d) 100 nm; (e) 300 nm. N, nucleus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171301&req=5

fig3: ALT levels and MB formation after DDC feeding. WT, K8, K18, and K8/18 mice (4–5 mice per genotype; 3–4-mo-old sex-matched mice) were fed a DDC-containing diet for 6 wk followed by harvesting of the livers and blood. (A) ALT levels (means ± SD), with a significant increase noted in K8 as compared with WT mice. (B) Livers from mice described in A were fixed and stained with hematoxylin and eosin. Histology of the livers from WT (a) and K8 (b) mice is shown, but K18 and K8/18 liver histology was very similar to WT (not depicted). Note the significant MB formation in K8 livers (arrows). Bar, 200 μm. (C) MBs were quantified (see Materials and methods) using the hematoxylin and eosin–stained liver sections described in B. The P values for comparing the genotypes WT with K8, K8 with K18, and K8 with K8/18 are shown. Error bars represent SD. (D) Conventional (a and b) and immune (c–f) electron microscopy of livers from K8 mice that were fed a DDC-containing diet show typical-appearing MB deposits. In a, the MB is encircled with a black line, and in b, arrows highlight IF bundles that are in close proximity to MBs. For immune electron microscopy, keratins were labeled with 10 nm gold particles (c–e). The particles are difficult to see in c but are clearly evident in the higher magnification images d and e. As a specificity control, the grids were also treated with nonimmune serum (f; note the absence of gold particles as contrasted with e). Arrows in e and f point to keratin IF bundles. Bars (a), 2 μm; (b) 500 nm; (c) 300 nm; (d) 100 nm; (e) 300 nm. N, nucleus.
Mentions: We tested whether the occurrence of pre-MB aggregates in old K8 mice could render younger K8 mice more susceptible to MB formation by feeding the four mouse genotypes a DDC-containing diet. Mice fed with DDC or griseofulvin for 3–4 mo are established models of MB formation (Yuan et al., 1996; Cadrin et al., 2000; Denk et al., 2000; Stumptner et al., 2000). However, in our experimental design, we analyzed the livers at an early time point of 6 wk after DDC feeding, when MBs are typically not seen. In DDC-fed mice, there was a significant increase in serum alanine aminotransferase (ALT; an indicator of hepatocyte necrosis) in K8 as compared with WT, K18, or K8/18 mice (Fig. 3 A) but no significant difference in liver damage when assessed by hematoxylin and eosin staining (not depicted). Other serologic tests (see Animal feeding and tissue experiments) were comparable in all four genotypes (not depicted). However, MBs were found almost exclusively in the K8 mice, as confirmed by hematoxylin and eosin staining (Fig. 3, B and C), standard and immune electron microscopy (Fig. 3 D), and immunofluorescence staining using antikeratin and antiubiquitin antibodies (Fig. 4 A). K8 livers had 35 ± 19 hepatocytes with MBs per 10 high power fields compared with 2.6 ± 3.7, 1.5 ± 1.7, and 0.2 ± 0.4 for WT, K18, and K8/18 livers, respectively (Fig. 3 C). Ubiquitin localized in most, if not all, of the keratin aggregates (Fig. 4 A), whereas p62 (which is a component of MBs; Zatloukal et al., 2000, 2004) was seen in some of the aggregates (not depicted). After DDC feeding, keratin protein levels increased dramatically, as expected in all genotypes (Fig. 4 B), in concert with an increase in K8 and K18 mRNA levels (not depicted). In addition, phosphorylation of K8 S79 and K18 S33 increased similarly in all four genotypes (not depicted) as described previously for WT mice (Stumptner et al., 2000). Therefore, K8-overexpressing mice are highly primed to develop MBs after DDC challenge.

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