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Rapid induction of mouse virus-like (VL30) element transcripts by erythropoietin in murine erythroid progenitor cells
1Department of Pharmacology, University of Missouri-Columbia, School of Medicine, Columbia 65212.
Abstract:
We examined the activation of genes induced by erythropoietin (Epo) in erythroid progenitor cells that were isolated from the spleens of mice infected with anemia-inducing strain of the Friend virus. These erythroid progenitor cells, termed FVA cells, undergo in vitro differentiation to erythrocytes under the influence of Epo within 2 to 3 days. We used a differential hybridization procedure to screen a cDNA library constructed from FVA cells that were treated with Epo 2U/mL in the absence of serum for 2 hours. Of 20,000 recombinant phages, 47 plaques hybridized preferentially to cDNA probe prepared from Epo-stimulated cells. We found at least three different Epo-responsive genes (ERGs) and one of them corresponds to the mouse virus-like (VL30) element, similar to already reported BVL-1. The induction of VL30, which was evident within 30 minutes after Epo exposure, reached a maximum by 1 hour and remained stable for up to 4 hours. The treatment of FVA cells with cycloheximide (CHX) 10 micrograms/mL, which in itself activates the expression of VL30 caused a superinduction of the Epo signal. Changes in intracellular Ca2+ concentrations, either raised by ionomycin or depleted by EGTA, had no effect on the Epo-induced VL30 expression. In addition, protein kinase C (PKC) inhibitors such as staurosporine (3 mumol/L) or H7 (20 mumol/L) and a tyrosine kinase inhibitor, genistein (200 mumol/L), did not inhibit the Epo-induced expression of VL30. TPA (100 ng/mL), a PKC agonist, did not induce VL30 expression. Although the physiologic role of VL30 in the differentiation of erythroid progenitor cells is not known, our findings demonstrate that VL30 is an early ERG, and may be a useful indicator of the initial molecular actions of Epo.
Insights
Erythropoietin (Epo) induces virus-like 30 (VL30) element expression in erythroid progenitor cells. This early gene response, identified via differential hybridization, may indicate Epo's initial molecular actions during cell differentiation.
Area of Science:
- Molecular Biology
- Hematology
- Virology
Background:
- Erythropoietin (Epo) is a key hormone regulating red blood cell production.
- Friend virus-infected erythroid progenitor cells (FVA cells) offer a model for studying Epo-induced differentiation.
- Understanding early gene activation by Epo is crucial for deciphering erythropoiesis.
Purpose of the Study:
- To identify and characterize genes rapidly activated by Epo in FVA cells.
- To investigate the role of VL30 as an early Epo-responsive gene (ERG).
- To explore signaling pathways involved in Epo-induced VL30 expression.
Main Methods:
- Differential hybridization screening of a cDNA library from Epo-treated FVA cells.
- Analysis of VL30 gene induction kinetics following Epo exposure.
- Investigation of intracellular calcium and protein kinase C signaling pathways.
Main Results:
- Identified at least three Epo-responsive genes (ERGs), including the mouse virus-like 30 (VL30) element.
- VL30 expression was rapidly induced by Epo within 30 minutes, peaking at 1 hour.
- VL30 induction was not affected by intracellular calcium levels or inhibited by PKC/tyrosine kinase inhibitors.
Conclusions:
- VL30 is an early Epo-responsive gene (ERG) in erythroid progenitor cells.
- Epo-induced VL30 expression is independent of Ca2+ and PKC signaling pathways.
- VL30 may serve as a valuable marker for the initial molecular effects of Epo.