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Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis
K Cohen-Solal1, J L Villeval, M Titeux
1INSERM U 362, Institut Gustave Roussy, Villejuif, France.
Abstract:
Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.
Insights
Thrombopoietin (TPO) gene regulation is not affected by platelet levels. Studies show Mpl ligand mRNA levels and isoforms remain unchanged in thrombocytopenia and thrombocytosis, suggesting other factors regulate TPO bioactivity.
Area of Science:
- Hematology
- Molecular Biology
- Gene Regulation
Background:
- Mpl ligand (thrombopoietin, TPO) is the primary regulator of platelet production.
- Mpl ligand mRNA is primarily found in the liver and kidney in mice.
Purpose of the Study:
- To investigate the regulatory mechanisms of the Mpl ligand gene.
- To determine if Mpl ligand mRNA levels or isoform ratios change during thrombocytopenia or thrombocytosis.
Main Methods:
- Developed experimental models of severe thrombocytopenia and chronic thrombocytosis in mice.
- Utilized Northern blot analysis to assess Mpl-L mRNA levels in liver and kidney.
- Employed ribonuclease protection assays to analyze Mpl-L mRNA splice variant ratios.
Main Results:
- Mpl ligand mRNA levels in the liver and kidney did not change in response to thrombocytopenia, regardless of platelet count.
- No alterations in the ratios of wild-type to variant Mpl-L mRNA isoforms were observed.
- Mpl ligand bioactivity was highest in mice with severely reduced platelets and megakaryocytes, suggesting their involvement in regulation.
Conclusions:
- Regulation of Mpl ligand does not occur at the transcriptional level or through modulation of mRNA isoform ratios.
- Platelets and megakaryocytes likely play a role in regulating circulating Mpl ligand bioactivity, independent of mRNA levels.