Negative regulation of the NFAT1 factor by CD45: Implication in HIV-1 longterminal repeat activation

HIV-1 gene regulation is greatly dependent on the presence of the -104/-81 enhancer region which is regulated by both NF-kappaB and NFAT transcription factors. We have found that a greater induction in HIV-1 long terminal rep eat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulati on of a CD45-deficient cell line (J45.01) in comparison to the parental Jur kat cells. Unlike NF-kappaB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transc riptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-indu ced NFAT activation, NFAT translocation and calcium influx peaked at simila r time points for both Jurkat and J45.01 cell lines. The NFAT-dependent pro moters from the IL-2 and TNF-alpha genes were also more potently activated by PMA/Iono in J45.01 cells. Interestingly, higher levels of intracellular calcium were consistently demonstrated in PMA/Iono-induced CD45-deficient c ell lines (J45.01 and HPB45.0). Furthermore, PMA/Iono induction of calcium mobilization in both Jurkat and J45.01 cell lines was observed to be EGTA-s ensitive. Mechanistic studies revealed that CD3 xi and ZAP-70 were more hea vily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was p resent at higher levels in J45.01 nuclear extracts and that the NFAT1 membe r was predominant. In conclusion, our results indicate that NFAT activation by stimuli acting in a more distal fashion from the TCR-mediated signaling pathway can be down-regulated by CD45 and that this CD45-dependent regulat ion in turn affects HIV-1 long terminal repeat activation.