Negative regulation of the NFAT1 factor by CD45: implication in HIV-1 long terminal 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 repeat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulation of a CD45-deficient cell line (J45.01) in comparison to the parental Jurkat cells. Unlike NF-kappaB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transcriptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-induced NFAT activation, NFAT translocation and calcium influx peaked at similar time points for both Jurkat and J45.01 cell lines. The NFAT-dependent promoters 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 cell 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-sensitive. Mechanistic studies revealed that CD3zeta and ZAP-70 were more heavily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was present at higher levels in J45.01 nuclear extracts and that the NFAT1 member 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 regulation in turn affects HIV-1 long terminal repeat activation.