Immunolocalization of a novel cholesteryl ester hydrolase in the endoplasmic reticulum of murine and human hepatocytes.

We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.

A conserved mechanism for controlling the translation of beta-F1-ATPase mRNA between the fetal liver and cancer cells.

To characterize the mechanisms governing the biogenesis of mitochondria in cancer, we studied the mitochondrial phenotype and the mechanisms controlling the expression of the beta subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) gene in the rat FAO and AS30D hepatomas. When compared with normal adult rat liver, the relative cellular content of the mitochondrial beta-F1-ATPase and glutamate dehydrogenase, as well as of mitochondrial DNA, was severely reduced in both cell lines. A paradoxical increase in the cellular abundance of beta-F1-ATPase mRNA was observed in cancer cells. Run-on transcription assays and the estimation of mRNA half-lives revealed that the increased abundance of beta-F1-ATPase mRNA results from the stabilization of the transcript in cancer. In vitro translation assays revealed a specific inhibition of the synthesis of the beta-precursor when translation reactions were carried out in the presence of extracts derived from cancer cells. The inhibitory effect was recapitulated using an RNA chimera that contained the 3'-untranslated region of beta-F1-ATPase mRNA. Hepatoma extracts also contained an increased activity of the developmentally regulated translation-inhibitory proteins that bind the 3'-untranslated region of beta-F1-ATPase mRNA. The results indicate that the expression of this gene in hepatoma cells is controlled by the same mechanisms that regulate its expression in the liver during fetal development.