Expression of a mutant ER-retained polytope membrane protein in cultured rat hepatocytes results in Mallory body formation

Abstract

Mallory bodies represent cytokeratin-rich inclusion bodies which occur characteristically but not exclusively in human alcoholic liver disease and experimentally in mice during chronic intoxication with drugs. We report the first in vitro cell system of Mallory body induction. In clone 9 rat hepatocytes stably transfected to express an ER-retained T126M-aquaporin-2 (AQP2), on the mean 40% of the cells contained cytokeratin-rich inclusion bodies. By electron microscopy, their structure corresponded to that of genuine Mallory bodies. Such inclusion bodies were not detectable in clone 9 rat hepatocytes stably expressing a Golgi apparatus/lysosome-retained E258K-aquaporin-2. Proteasome inhibition increased the number of Mallory body-containing T126M-AQP2-expressing clone 9 hepatocytes to 60% on average. Proteasome inhibition in non-transfected, cytokeratin meshwork-forming clone 9 hepatocytes resulted in Mallory body formation on average in 6% of cells. Collectively, these data suggest that in the described in vitro cell system, Mallory body formation is induced by the presence of non-native protein conformers and point to the involvement of the proteasomal digestive system. The here reported in vitro system will be useful in studies about the biogenesis and progression of Mallory bodies, their relationship to aggresomes, and the role of inclusion bodies in the pathogenesis of cell damage.

Endomannosidase processes oligosaccharides of α1-antitrypsin and its naturally occurring genetic variants in the Golgi apparatus

“…All samples Confocal immunofluorescence microscopy. Cells grown as monolayer on glass cover slips were formaldehydefixed and saponin-permeabilized as described [22]. For double immunofluorescence, cells were incubated with rabbit anti-rat endomannosidase (0.4 μg/ml) for 2 h at ambient temperature followed by rhodamine red-X-conjugated Fab fragments of goat anti-rabbit IgG (1000-fold diluted) for 1 h. After rinses, free rabbit IgG was blocked with unlabeled goat anti-rabbit Fab (12 μg/ml) followed by rabbit anti-human α1-antitrypsin antibody (2 μg/ml) for 1 h and Alexa 488-conjugated (Fab′) 2 fragments of goat anti-rabbit IgG (1000-fold diluted) for 45 min.…”

The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

“…24 Expression of the ER-retained T126M AQP2 in clone 9 hepatocytes caused the formation of Mallory body-type inclusion bodies probably because of ER stress. 26 Despite the progress made in the genetic, molecular, and functional characterization of NDI-causing AQP2 mutants and in the elucidation of the pathophysiology of this disease, specific aspects of the subcellular and molecular pathology remain to be investigated. In particular, the degradation pathway of mutant AQP2 is unknown.…”

“…The mutant AQP2 forms were generated by a PCR-based site-directed mutagenesis strategy as described previously. 26 Transfection of clone 9 rat hepatocytes with the linearized AQP2-pcDNA3.1 constructs was performed using lipofectamine 2000 and clonal cell lines were established as described previously. 26 For the detection of rat AQP9, reverse transcriptase (RT)-PCR was performed using RNA isolated from rat liver or from clone 9 hepatocytes.…”

“…26 For double-immunofluorescence microscopy, cells were simultaneously incubated with anti-AQP2 antibodies in combination with either anti-Golgi mannosidase II or anti-LAMP 1 antibodies for 2 hours at ambient temperature, washed three times with PBS containing 1% bovine serum albumin followed by simultaneous incubation with the respective fluorescent secondary antibodies for 1 hour. Finally, coverslips were rinsed with PBS and embedded in Moviol (Harco, Harlow, UK).…”

Ubiquitin-Proteasome Pathway in the Pathogenesis of Liver Disease

“…Several studies, where tissue culture cells were transfected with plasmids containing cDNA coded for cytokeratin 8 or cytokeratin 18 to cause overexpression, led to the formation of cytokeratin aggresomes. But these aggresomes failed to form the characteristic filaments seen in Mallory bodies [14,39,73].…”