Rapid induction of mouse virus-like (VL30) element transcripts by erythropoietin in murine erythroid progenitor cells

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.