Methylprednisolone-Treatment Induces Dephosphorylation of Syk in B-CLL Cells In Vitro.

Glucocorticoids (GCs) are often implemented in the treatment regimens of B-cell Chronic Lymphocytic Leukemia (B-CLL). Apart from caspase-involvement and downregulation of Lyn, the exact mechanisms of action in apoptosis induction in B-CLL remain uncertain. We studied methylprednisolone (MP)-induced apoptosis in B-CLL and found a greater sensitivity of ZAP−70+ B-CLL cells for MP-induced cell death, which was accompanied by a glucocorticoid receptor-dependent decrease of ZAP-70 expression. Micro-array data correlated this downregulation of ZAP-70 with an upregulation of the phosphatase PTP1B (Protein Tyrosine Phosphatase 1B). This observation was confirmed on genomic and protein level by RT-qPCR and Western blotting. Next, functional assays with the phosphatase-inhibitor ortho sodium vanadate and with the PTP1B-inhibitor 3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(−4-(thiazol-2-ylsulfamyl)-phenyl)-amide were performed. Both drugs were able to inhibit the GC-induced ZAP-70 downregulation, which is suggestive for the role of PTP1B. In order to explore this further, we studied the effect of MP on the phosphorylation of Syk by Western blotting and flow cytometry. We did not take phosphorylated ZAP-70 into account as phosphorylation of ZAP-70 is very unclear in CLL cells. We observed GC-induced dephosphorylation of Syk (tyrosine residues 352 and 526), associated with a decrease of total Syk. The kinetics were completely in accordance with those of the ZAP-70 downregulation, occurring only after 12 hours after the start of treatment. Next, we performed immunoprecipitation experiments to investigate the reciprocal influence of Syk and ZAP-70 and found co-immunoprecipitation of Syk, ZAP-70 and hsp90, suggesting a close relation of ZAP-70 and Syk in performing their tasks. In spite of the unclear status of ZAP-70 phosphorylation, ZAP-70 positive B-CLL cells allow for more effective IgM-signaling than their ZAP-70 negative counterparts, probably by blocking inhibitors of signalling through Syk. Moreover, ZAP-70 positive B-CLL cells undergo stronger and prolonged BCR-induced phosphorylation of tyrosine residues 352 and 526 of Syk, which are already prominent in unstimulated conditions. The kinetics and concentration overlapping downregulation of ZAP-70 and dephosphorylation of Syk after MP treatment suggests a relation between these two protein tyrosine kinases in performing their tasks. This was further strengthened by the observation of a co-immunoprecipitation of ZAP-70, Syk and hsp90. In conclusion, in vitro GC treatment of ZAP-70+ B-CLL cells is associated with a downregulation of ZAP-70 and a dephosphorylation of Syk tyrosines 352 and 526 via a GR-dependent way. The upregulation of PTP1B at both genomic and protein level, and the results of the functional assays, are suggestive for a role of this phosphatase during treatment with GCs.