Erythropoietin stimulates cancer cell migration and activates RhoA protein through a mitogen-activated protein kinase/extracellular signal-regulated kinase-dependent mechanism.

Erythropoietin (Epo) receptor (EpoR) is expressed in several cancer cell lines, and the functional consequence of this expression is under extensive study. In this study, we used a cervical cancer cell line in which EpoR was first found to be expressed and to correlate with the severity of the disease. We demonstrate that Epo is a chemoattractant for these cancer cells, enhancing their migration under serum-starved conditions. Using a Transwell migration system, we show that Epo enhances cancer cell migration in a dose- and time-dependent manner. The effect of Epo is dependent on the activity of two signaling pathways: the mitogen-activated protein kinase (MAPK) pathway and the RhoA GTPase pathway. We show that Epo activates both pathways in a Janus kinase-dependent manner and that this activation is required for Epo effects on cell migration. Furthermore, we use both pharmacological and genetic inhibitors to demonstrate that the activation of RhoA GTPase is dependent on the activity of the MAPK pathway, providing the first evidence for interaction between these two signaling cascades.

Lovastatin suppresses erythropoietin receptor surface expression through dual inhibition of glycosylation and geranylgeranylation.

Erythropoietin (Epo) is a cytokine that is required for the survival of erythroid progenitors through interaction with its receptor on the surface of these cells. Recent studies showed that erythropoietin receptor (EpoR) is expressed on many cancer cells. The factors that govern EpoR expression on the cell surface are poorly understood. Using both biotinlyation and radiolabeled Epo binding experiments, we show here that Epo starvation of the Epo-dependent erythroleukemia cell line, ASE2, leads to a time-dependent increase in both forms of EpoR, the maturing 64 kDa and the mature 66 kDa proteins. Mevalonate depletion inhibits the formation of the highly glycosylated mature form of EpoR without affecting the other form. Treatment of cells with lovastatin, a selective inhibitor of the rate-limiting enzyme in the mevalonate pathway leads to inhibition of cell surface EpoR that is induced by Epo starvation. The effect of lovastatin appears to be the consequence of inhibition of two processes, glycosylation and geranylgeranylation. Adding back geranylgeranyl pyrophosphate to lovastatin-treated cells completely prevents the lovastatin effect on EpoR expression. Dolichol, the sugar carrier in N-linked glycosylation that is derived from the mevalonate pathway, partially reverses lovastatin's effect. The glycosylation inhibitor tunicamycin also partially suppresses EpoR surface expression. Inhibiting protein geranylgeranylation mimics the effect of lovastatin and inhibits EpoR surface expression in a concentration-dependent manner. Finally, lovastatin inhibits Epo's stimulatory effects on cell proliferation. These results indicate that mevalonate derivatives are required for normal EpoR expression on the cell surface through two pathways, glycosylation and geranylgeranylation.

Erythropoietin receptor signal transduction requires protein geranylgeranylation.

Erythropoietin (Epo) acts through the erythropoietin receptor, a member of the type-1 cytokine receptor family, to influence survival, proliferation, and differentiation of erythroid progenitors. Epo stimulation of factor-dependent 32D cells results in phosphorylation of many proteins, including Janus kinase (Jak) 2, signal transducer and activator of transcription (Stat) 5, and extracellular signal-regulated kinase (Erk). Some of Epo-activated signaling proteins require isoprenylation, either farnesylation or geranylgeranylation, for post-translational modification. In this study, we sought to characterize the interplay between protein isoprenylation and Epo signal transduction. Using two different Epo-responsive cell lines, we found that depletion of mevalonate and its isoprenoid derivatives using the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin impairs Epo signaling as assessed by phosphorylation of cellular substrates and inhibition of apoptosis. Interestingly, the effect of mevalonate depletion was prevented by adding back geranylgeranyl pyrophosphate but not farnesyl pyrophosphate. Furthermore, selective inhibition of protein geranylgeranylation mimicked the effect of lovastatin, whereas selective inhibition of farnesylation had no effect. These results indicate that protein geranylgeranylation and not farnesylation is important for proper Epo signal transduction.