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Hormone sensitive lipase mRNA in both monocyte and macrophage forms of the human THP-1 cell line
W J Johnson1, S Y Jang, D W Bernard
1Abramson Research Center, Joseph Stokes, Jr, Research Institute, Children's Hospital of Philadelphia, PA 19104, USA. johnsonw@email.chop.edu
Abstract:
The identity of the neutral cholesteryl ester hydrolase (CEH) in human monocyte/macrophages is uncertain. Prior studies indicate that hormone sensitive lipase (HSL) is a major CEH in mouse macrophages, and that HSL mRNA is present in human THP-1 monocytes. In the present study, HSL mRNA expression was examined in THP-1 cells as a function of differentiation status and cholesterol enrichment. By RT-PCR with primer pairs that span exon boundaries, HSL mRNA was demonstrated in THP-1 monocytes and phorbol-ester differentiated THP-1 macrophages. cDNA identities were confirmed by sequencing. By Northern blotting, with HSL cDNA as probe, THP-1 monocytes were found to contain HSL mRNA of approximately 3 and 3.9 kb. In THP-1 macrophages, the 3 kb mRNA was greatly diminished, while the level of the 3.9 kb mRNA was maintained. mRNA of approximately 3 and 3.9 kb are those expected of the 86-kDa (adipocyte) and 117-kDa (testicular) HSL isoforms, respectively. The presence of the testicular isoform mRNA was confirmed in THP-1 cells by amplification and sequencing of an isoform-specific cDNA. Additionally, Northern-blot comparisons showed that the 3 and 3.9 kb mRNA in THP-1 comigrated with the HSL mRNA in 3T3-L1 adipocytes and rat testis, respectively. The level of the 3.9 kb mRNA did not vary greatly with cholesterol enrichment. Thus, the HSL gene is transcribed in THP-1 cells both before and after differentiation into macrophages; after differentiation, the predominant mRNA is that for the 117-kDa isoform. This isoform is a CEH, and may mediate some CE turnover in THP-1 cells.
Insights
The identity of neutral cholesteryl ester hydrolase (CEH) in human macrophages remains unclear. This study shows hormone-sensitive lipase (HSL) gene expression in THP-1 cells, with the 117-kDa isoform predominant after differentiation, suggesting its role in CE turnover.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The neutral cholesteryl ester hydrolase (CEH) in human monocyte/macrophages is not definitively identified.
- Previous research suggests hormone-sensitive lipase (HSL) acts as a major CEH in mouse macrophages.
- HSL mRNA has been detected in human THP-1 monocytes.
Purpose of the Study:
- To investigate HSL mRNA expression in THP-1 cells.
- To determine how differentiation and cholesterol levels affect HSL mRNA expression.
- To identify the specific HSL isoforms present in THP-1 cells.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) to detect HSL mRNA.
- Sequencing of cDNA to confirm identity.
- Northern blotting to analyze mRNA size and levels.
- Isoform-specific cDNA amplification and sequencing.
Main Results:
- HSL mRNA was detected in both THP-1 monocytes and differentiated macrophages.
- THP-1 monocytes expressed approximately 3 kb and 3.9 kb HSL mRNA.
- Differentiated macrophages showed diminished 3 kb mRNA and maintained 3.9 kb mRNA.
- The 3.9 kb mRNA corresponds to the 117-kDa HSL isoform, predominant after differentiation.
- Cholesterol enrichment did not significantly alter the 3.9 kb mRNA levels.
Conclusions:
- The HSL gene is transcribed in THP-1 cells before and after differentiation into macrophages.
- Following differentiation, the 117-kDa HSL isoform mRNA becomes predominant.
- This 117-kDa isoform is a CEH and may play a role in cholesteryl ester turnover in THP-1 cells.