CONICET | Buscador de Institutos y Recursos Humanos

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. Prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and endoplasmic reticulum membrane, which has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines and hormones. In previous reports we have shown that PTP1B expression, activity and phosphorylation are reciprocally modulated by Epo in undifferentiated UT-7 cell line. However, no information is available with respect to the modulation of this phosphatase in non-Epo depending cells or at late stages of erythroid differentiation. Aim: To characterize the relationship between Epo and PTP1B expression and to investigate a probable link between changes in PTP1B and intracellular calcium levels modulated by Epo. Methods: The TF-1 human cell line was used as a model of cells that can be grown with GM-CSF or Epo while the Epo-dependent UT-7 cells were induced to erythroid differentiation with hemin. Controls: TF-1 cells cultured in the presence of GM ad undifferentiated UT-7 cells cultured with Epo. Epo was added to Epo/serum-deprived cells for 18 h and after different periods, cells were lysed and total proteins and RNA were obtained to be analyzed by Western blotting and Real Time PCR, respectively. Immunoprecipitates with anti-PTP1B were subjected to a PTP1B activity assay using p-nitrophenylphosphate as substrate. Experiments with ionophore A23187 and calpeptin were performed to determine calpain involvement in PTP1B modulation by Epo. Flow cytometry was used to quantify calcium response to Epo. Results: A significantly increased level of PTP1B mRNA was observed in TF-1 and differentiated UT-7 cells either permanently maintained with Epo or Epo-stimulated for 24 h (P<0.05, n=4). This increment correlated with the highest level of PTP1B activity under the same conditions. Besides, an additional 46 kDa PTP1B isoform was observed in TF-1 cells and in hemoglobinized UT-7 cells cultured in the presence of Epo. After subcellular fractionation, this protein was found to be attached to the membrane fraction as the 50 kDa PTP1B isoform. Cell pre-treatment with calcium ionophore showed that a calcium-dependent protease was involved in the PTP1B cleavage. The addition of calpeptin to the experiments prevented PTP1B degradation, demonstrating that calpain was responsible for this Epo modulation. Flow cytometry analyses showed an Epo-induced intracellular calcium increase which was maintained after 60 min-Epo stimulation in TF-1 and differentiated UT-7 cells but not in UT-7 control undifferentiated cells. Conclusions: Results demonstrated an Epo-induced cleavage of PTP1B, resulting in a 46 kDa isoform attached to cell membrane which is associated with increased PTP1B enzymatic activity. Calpain was found to be responsible for this differential cleavage being the protein activation presumably a consequence of the intracellular calcium concentration change induced by Epo. These findings suggest a novel pathway of Epo - induced PTP1B regulation involving calcium, an already well known intracellular mediator.

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