The asialoglycoprotein receptor (AGPR) is responsible for the catabolism of acute phase proteins. The effects of inflammation-related cytokines on the expression of AGPR were investigated in HepG2 cells derived from a human hepatoblastoma cell line. Binding studies, using a [125I]-labeled asialo-orosomucoid ligand, revealed that AGPR numbers on cell surfaces were increased by interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). In cells treated with IL-1, IL-6, or TNF, immunohistochemical staining with an anti-AGPR monoclonal antibody demonstrated augmented expression. Pulse labeling analysis, using [35S]-labeled methionine, showed newly synthesized AGPR in both the precursor and the mature forms. When IL-1, IL-6, and TNF were added to the culture medium, total synthesis of AGPR (sum of the mature and precursor forms) was increased. In addition, the relative proportion of the synthesized precursor form of AGPR was higher in cytokine-treated than in untreated cells, suggesting that these cytokines augment the synthesis of AGPR, particularly in the stage prior to glycosylation.
Monoclonal antibodies (MoAbs) were produced by immunizing BALB/c mice with asialoglycoprotein receptor (AGPR) purified from human liver. The purity of AGPR was confirmed by SDS-polyacrylamide gel electrophoresis and by amino acid sequence. An enzyme-linked immunoassay revealed 24 monoclonal antibodies which reacted with human AGPR. By Western blot analysis, all antibodies recognized the 46 kDa human AGPR under the non-reduced conditions and four MoAbs recognized reduced protein. Two MoAbs reacted with AGPR derived from rat, rabbit and mouse liver under both non-reduced and reduced conditions, suggesting that we could obtain antibodies which reacted with AGPR epitopes shared by different species. In immunohistochemical studies, 30201-MoAb reacted with human liver tissue but not with other tissues. This antibody immunoprecipitated two major bands of 46 kDa and 39 kDa from [35S]-methionine metabolically labeled human hepatoma HepG2 cells. The determinant recognized by 30201-MoAb is a common epitope of AGPR which is present in different species and in both the precursor and maturation forms of the receptor.
The effect of ethanol on the expression of asialoglycoprotein receptor protein and its mRNA was studied in a human hepatoma cell line, HepG2. The number of asialoglycoprotein receptors on the cell surface was decreased to 60% of the control level, without a loss in affinity, by incubating the cells with 100 mM ethanol. The decrease in cell surface asialoglycoprotein receptors was paralleled by a decrease in total receptor numbers, including intracellular and surface receptors. The internalization of asialoglycoprotein was also diminished, to 44% of that in control cells. The decreases in cell surface receptors and total receptor numbers were partially restored by 2 mM 4-methylpyrazole, suggesting that ethanol metabolites participated in the diminution of asialoglycoprotein receptor expression. However, the steady-state expression of asialoglycoprotein receptor mRNA was increased in ethanol-treated cells and further augmented by a longer ethanol exposure. These paradoxical results, i.e., the decrease of asialoglycoprotein receptor protein and the increase of its mRNA expression, suggest that the reduction in the asialoglycoprotein receptor protein is a post-transcriptional event and that a possible feedback regulatory mechanism may control asialoglycoprotein receptor gene transcription and/or impair the degradation of its mRNA.
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