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Lysyl oxidase cDNA of myofibroblast from mouse fibrotic liver
C Jourdan-Le Saux1, C Gleyzal, J M Garnier
1Unité de Pathologie cellulaire, Institut Pasteur de Lyon, CNRS URA 1459, France.
Abstract:
In order to study the regulation of lysyl oxydase (LO) in fibrosis, mRNAs were extracted from an enriched population of myofibroblasts (MF) isolated from liver of schistosomiasis infected mouse. Four mRNAs (5.5kb, 4.5kb, 2.4kb and 2.0kb) hybridizing with a LO cDNA probe were transcribed in fibrotic liver, but only the two largest mRNAs were found in MF. A cDNA library was constructed, allowing the cloning of twenty four cDNAs. The largest clone of 4689bp should correspond to the 5.5kb mRNA. Its sequence was essentially similar to the NIH-3T3 fibroblasts LO-ras recision gene (rrg4) cDNA, with the same exon/intron structure, but with some differences at the sites of initiation of transcription which were shown to occur mainly at -392 and -358 nucleotides before the putative start of translation. These two main sites of initiation did not explain the origin of the 4.5kb and 5.5kb mRNAs, and as no spliced variants were found among the 24 clones, some regulation should also involve the 3'end region.
Insights
Researchers investigated lysyl oxidase (LO) regulation in liver fibrosis. They found distinct mRNA patterns in myofibroblasts, suggesting complex regulation involving transcription initiation and 3' end regions.
Area of Science:
- Molecular Biology
- Fibrosis Research
- Gene Expression Analysis
Background:
- Fibrosis, a pathological scarring process, involves the dysregulation of extracellular matrix production.
- Lysyl oxidase (LO) is a key enzyme in collagen cross-linking, crucial for matrix stiffening during fibrosis.
- Understanding LO gene regulation is vital for developing antifibrotic therapies.
Purpose of the Study:
- To investigate the transcriptional regulation of lysyl oxidase (LO) in the context of liver fibrosis.
- To identify specific mRNA transcripts and regulatory mechanisms of LO in myofibroblasts (MF).
Main Methods:
- Isolation of myofibroblasts (MF) from schistosome-infected mouse livers.
- Extraction and analysis of mRNA using a LO cDNA probe.
- Construction and screening of a cDNA library to clone LO transcripts.
- DNA sequencing and comparative analysis of cloned cDNAs with known LO genes.
Main Results:
- Four LO mRNA transcripts (5.5kb, 4.5kb, 2.4kb, 2.0kb) were detected in fibrotic liver, with only the larger two (5.5kb, 4.5kb) present in MF.
- A 4689bp cDNA clone, corresponding to the 5.5kb mRNA, showed high similarity to the NIH-3T3 fibroblasts LO-ras recision gene (rrg4) cDNA.
- Identified alternative transcription initiation sites at -392 and -358 nucleotides, but these did not fully explain the observed mRNA sizes.
- Absence of spliced variants suggested regulation might involve the 3' end of the mRNA.
Conclusions:
- Lysyl oxidase expression in fibrotic liver myofibroblasts is regulated by distinct mRNA species.
- Alternative transcription initiation sites contribute to LO gene regulation, but do not account for all observed mRNA heterogeneity.
- Post-transcriptional regulation, potentially involving the 3' untranslated region, plays a significant role in controlling LO mRNA levels in fibrosis.