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Regulation of the mRNA for monocyte-derived neutrophil-activating peptide in differentiating HL60 promyelocytes
1Cancer Research Laboratory, University of Western Ontario, London, Canada.
Abstract:
A cDNA library was constructed from HL60 human promyelocyte poly(A)+ RNA harvested 3 h after induction of macrophage differentiation with 12-O-tetradecanoyl phorbol-13-acetate in the presence of cycloheximide. We isolated from this library a 1.6-kilobase full-length clone designated b4 whose corresponding mRNA was greatly increased in abundance in cytoplasmic RNA under these conditions. Dideoxy sequencing revealed that this mRNA encoded MONAP (monocyte-derived neutrophil-activating peptide), a 10-kilodalton monokine with neutrophil-specific chemotactic and enzyme-releasing activities. The 3' untranslated region of this mRNA was found to be 1.2 kilobases long and possessed nine copies of the AUUUA sequence known to be associated with regulation of mRNA stability. Actinomycin D chase experiments yielded evidence that cytoplasmic stabilization was one of the means of regulation of MONAP expression. Analysis of cytoplasmic poly(A)- RNA revealed the presence of several discrete truncated species that shared a common 5' end and appeared to be intermediates of degradation. S1 mapping showed that the 3' ends of these molecules were distributed throughout the 3' untranslated region, preferentially in A + U-rich regions, broadly correlating with the distribution of AUUUA sites. Nuclear run-on experiments indicated that transcriptional induction accounted for less than 15% of the accumulation of MONAP mRNA. This mRNA was induced in HL60 cells by treatment with several differentiation-inducing agents: 12-O-tetradecanoyl phorbol-13-myristate alone, sodium butyrate, vitamin D3, and dimethyl sulfoxide. It was also induced in quiescent diploid lung fibroblasts stimulated to divide by serum, and it was constitutively overexpressed by some human tumor lines.
Insights
This study identifies monocyte-derived neutrophil-activating peptide (MONAP) mRNA, crucial for macrophage differentiation. Its expression is primarily regulated by mRNA stabilization, not transcription, in HL60 cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- HL60 cells are human promyelocytes used to study differentiation.
- Macrophage differentiation involves complex gene expression regulation.
- Monocyte-derived neutrophil-activating peptide (MONAP) is a key mediator in inflammatory responses.
Purpose of the Study:
- To isolate and characterize the mRNA encoding MONAP.
- To investigate the regulatory mechanisms controlling MONAP expression during HL60 cell differentiation.
- To identify factors influencing MONAP mRNA stability and abundance.
Main Methods:
- cDNA library construction and screening from differentiated HL60 cells.
- Dideoxy sequencing to identify the encoded protein.
- Actinomycin D chase experiments to assess mRNA stability.
- Nuclear run-on assays to evaluate transcriptional regulation.
- S1 mapping to analyze mRNA degradation intermediates.
Main Results:
- A 1.6-kilobase clone encoding MONAP was isolated.
- MONAP mRNA abundance significantly increased during induced macrophage differentiation.
- mRNA stability, particularly cytoplasmic stabilization, was identified as a primary regulatory mechanism.
- Transcriptional induction contributed less than 15% to MONAP mRNA accumulation.
- MONAP mRNA was induced by various differentiation agents and overexpressed in some tumor lines.
Conclusions:
- MONAP expression is predominantly regulated post-transcriptionally via mRNA stabilization.
- The 3' untranslated region, containing AUUUA motifs, plays a critical role in MONAP mRNA stability.
- MONAP induction is a conserved response during differentiation and can be dysregulated in cancer.