Abstract
Reverse transcriptase-polymerase chain reaction was used to study the biosynthesis of two different cholesteryl ester hydrolases by human and mouse macrophages. Oligonucleotide primers for bile salt-stimulated cholesterol esterase yielded positive reactions with RNA isolated from human peripheral blood monocytes, monocyte-derived macrophages, the human monocytic THP-1 cells, and phorbol ester-induced THP-1 macrophages. In contrast, oligonucleotide primers for hormone-sensitive lipase yielded positive reactions only with RNA isolated from non-differentiated human THP-1 monocytic cells and peripheral blood monocytes, but not those obtained from differentiated THP-1 macrophages or monocyte-derived macrophages. Thus, while human monocytes were capable of synthesizing both enzymes, human macrophages synthesized only bile salt-stimulated cholesterol esterase and not the hormone-sensitive lipase. The synthesis of bile salt-stimulated cholesterol esterase by human macrophages was confirmed by detection of bile salt-stimulated cholesteryl ester hydrolytic activity in conditioned media of differentiated THP-1 cells and human peripheral blood monocyte-derived macrophages. Moreover, incubating human macrophages with oxidized low density lipoprotein (LDL) or acetylated LDL increased bile salt-stimulated cholesterol esterase activity in the conditioned media of these cells. These results with human macrophages were contrasted with results of studies with mouse macrophages, which showed the presence of hormone-sensitive lipase mRNA but not the bile salt-stimulated cholesterol esterase mRNA. Taken together, these results demonstrated species-specific differences in expression of cholesteryl ester hydrolytic enzymes in macrophages. The expression of bile salt-stimulated cholesterol esterase by human macrophages, in a process inducible by modified LDL, suggests a role of this protein in atherogenesis.
Highlights
Reverse transcriptase-polymerase chain reaction was used to study the biosynthesis of two different cholesteryl ester hydrolases by human and mouse macrophages
Since previous studies by other investigators have reported the presence of a cholesterol esterase-like pseudogene in the human genome that can be transcribed into mRNA [29, 30], and that the major difference between the authentic cholesterol esterase gene and the pseudogene is the deletion of exon 2 through exon 7 in the latter sequence, PCR amplification was performed initially with oligonucleotide primers corresponding to sequences in exon 2 and exon 7 of the cholesterol esterase gene
The observation of similar RT-PCR products with RNA from human monocyte-derived macrophages and HepG2, a human hepatoma cell line that has been shown previously to synthesize the bile salt-stimulated cholesterol esterase [31], suggested that human monocyte-macrophages are capable of synthesizing a cholesterol esterase that is similar to the enzyme in pancreas
Summary
24 26 25 12 mone-sensitive lipase (HSL) found in steroidogenic tissues [11] In support of this hypothesis, HSL mRNA was found to be present in mouse macrophages [12]. HSL mRNA could not be demonstrated in human macrophages, even with the sensitive reverse transcription-polymerase chain reaction amplification technique [13]. These results suggest that another cholesterol ester hydrolase may be responsible for cholesteryl ester metabolism in human macrophages and arterial wall. In view of the recent observations that the pancreatic type of cholesterol esterase (the bile saltstimulated lipase/cholesterol esterase) is present in human serum [16], and that its serum activity may participate in lipoprotein remodeling [16, 17], this study was undertaken to determine if the bile salt-stimulated cholesterol esterase is expressed in macrophages
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