MODULATION OF PROTEIN TYROSINE PHOSPHATASE 1B BY ERYTHROPOIETIN IN UT-7 CELL LINE

CALLERO MARIANA; PÉREZ GLADYS; VITTORI DANIELA; NESSE ALCIRA

Amsterdam, Holanda

Congreso; 11th Congress of the European Hematology Association; 2006

European Hematology Association

Background: The central role played by tyrosine phosphorylation of erythropoietin receptor (EpoR) in cell activation by erythropoietin (Epo) has focused attention on protein tyrosine phosphatases (PTPs) as candidates implicated in the pathogenesis of the resistance to therapy with human recombinant Epo. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of a number of signaling pathways, in particular, those involving tyrosine phosphorylation induced by growth factors, cytokines and hormones such as the downregulation of EpoR and insulin receptor. Binding of ligand to cell-surface EpoR results in the activation of JAK2 and phosphorylation of tyrosine residues in the cytosolic domain of the receptor. Termination of the EpoR signaling is attributed to the cytosolic SH-PTP1. However, it has been demonstrated that PTP1B also participates in down-regulation of the ligand-activated cell surface EpoR. Aim: To investigate the effect of Epo on PTP1B expression. Methods: The UT-7 human cell line was used as an Epo-dependent model. Epo was added to serum- and Epo-deprived cells for previous 18 h. After different periods of Epo incubation, cells were lysed and total proteins and RNA were obtained. cDNA was prepared from different RNA samples and PTP1B mRNA level was analysed by Real Time PCR. Total proteins or immunoprecipitates with anti-PTP1B were subjected to Western Blot using anti-PTP1B or anti-PTyr. Immunoprecipitates were also subjected to a PTP1B activity assay with pNPP. Then, the experiment was repeated including pretreatment with LY294002 (PI3K inhibitor) before Epo stimulation. Results: An increased and maximun level of PTP1B mRNA was already observed at 3 h of Epo stimulation (figure a). This increment correlates with the induction of PTP1B expression observed by Western Blot (figure b). However, after 9h with Epo, mRNA level returned to baseline while protein expression remained constant. PTP1B Tyr phosphorylation was detectable after 5 min of Epo stimulation and declined within 6 h PTP1B activity increased after 3 h of Epo incubation and diminished to the basal level within 6 h (figure c). Figure d shows that the pretreatment of UT-7 cells with LY294002 downregulated PTP1B expresion in a dose-dependent manner reaching the highest inhibition at 100 mM LY concentration. Conclusions: We have found an Epo-induced expression of PTP1B, associated with increased PTP1B Tyr phosphorylation, suggesting that besides modulating Epo/EpoR signaling, PTP1B suffers a feedback regulation by Epo.