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Modifications in cytokeratin and actin in cultured liver cells derived from griseofulvin-fed mice
M Cadrin1, N M Anderson, L H Aasheim
1Département de Chimie-Biologie, Université du Québec a Trois-Rivières, Canada.
Background:
Hepatocytes from mice fed griseofulvin (GF) for 8 months form Mallory bodies (MBs), which represent a pathologic state of intermediate filaments (IFs). The cellular mechanisms that lead to MB formation are not known.
Experimental Design:
This study was aimed to investigate if MB formation could be related to modification in cytokeratin (CK) metabolism. Primary cultures of hepatocytes from control and GF livers were studied. Immunofluorescence microscopy was used to study the organization of the cytoskeleton in these cells. The hepatocytes were labeled with [35S]methionine or [32P]orthophosphate to study, respectively, the level of amino acid incorporation into IF proteins (CK 8 and CK 18) and their phosphorylation levels. The response to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate stimulation of the phosphorylation of CK 8 and CK 18 was also elicited in contrast to control hepatocytes.
Results:
We found that there was a change in the organization of actin and the IF network in the hepatocytes from GF-treated animals. This was associated with an increase in labeled amino acid incorporation into CK 8 and CK 18 as well as in actin. Although there was no significant difference in the absolute level of CK phosphorylation, we found modifications in the phosphorylated isomers of CK 8, the more phosphorylated isomers becoming more prominent. The treatment of the hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate did not induce changes in the level of CK phosphorylation in GF-pretreated hepatocytes.
Conclusions:
These results suggest that the modification of the IF network and MB formation are the consequences of increased CK synthesis and the modification of phosphorylation. They could alter the normal interaction of the IFs with different cellular components, which results in conformational changes of CKs and the reorganization of the IF network to the form of MBs.
Insights
Griseofulvin exposure in mice causes changes in intermediate filament organization and increased cytokeratin synthesis, leading to Mallory body formation in hepatocytes. These findings reveal cellular mechanisms behind this pathological state.
Area of Science:
- Cell Biology
- Hepatology
- Biochemistry
Background:
- Griseofulvin (GF) induces Mallory bodies (MBs) in mouse hepatocytes, a pathological state of intermediate filaments (IFs).
- The precise cellular mechanisms driving MB formation remain unknown.
- This study investigates the role of cytokeratin (CK) metabolism in GF-induced MB formation.
Purpose of the Study:
- To investigate the relationship between Mallory body formation and alterations in cytokeratin (CK) metabolism.
- To analyze changes in the organization of the cytoskeleton and IF network in hepatocytes.
- To assess CK synthesis and phosphorylation levels in response to griseofulvin exposure.
Main Methods:
- Primary hepatocyte cultures from griseofulvin-treated and control mice.
- Immunofluorescence microscopy to visualize cytoskeletal organization.
- Radiolabeling ([35S]methionine, [32P]orthophosphate) to quantify CK 8 and CK 18 synthesis and phosphorylation.
- Assessment of CK phosphorylation in response to 12-O-tetradecanoyl-phorbol-13-acetate stimulation.
Main Results:
- Altered organization of actin and the IF network observed in hepatocytes from GF-treated mice.
- Increased incorporation of labeled amino acids into CK 8, CK 18, and actin.
- No significant change in absolute CK phosphorylation levels, but modifications in phosphorylated CK 8 isomers were noted.
- 12-O-tetradecanoyl-phorbol-13-acetate did not alter CK phosphorylation in GF-pretreated hepatocytes.
Conclusions:
- Mallory body formation is linked to increased CK synthesis and altered CK phosphorylation.
- These metabolic changes likely disrupt normal IF interactions with cellular components.
- Conformational changes in CKs and IF network reorganization lead to MB formation.