RAPAMYCIN SELECTIVELY BLOCKS INTERLEUKIN-2-INDUCED PROLIFERATING CELL NUCLEAR ANTIGEN GENE-EXPRESSION IN T-LYMPHOCYTE

The macrolide rapamycin arrests T lymphocytes stimulated by interleukin-2 (IL-2) at G(1)/S. We have recently found that IL-2 induced an increase in the binding of discrete transcription factors of the ATF/cAMP-responsive element binding factor (CREB) family at G(1)/S, and that this effect was inhibited by rapamycin (Feuerstein, N., Huang, D., Hinrichs, S. H., Orten, D. J., Aiyar, N., and Prystowsky, M. B. (1995) J. Immunol, 154, 68-79), We now show, by using high resolution two-dimensional gel electrophoresis, that rapamycin inhibited selectively the synthesis of three discrete IL-2-induced soluble proteins (35 kDa/pI similar to 5, 68 kDa/pI similar to 4, 110 kDa/pI similar to 4.3), Analysis of nuclear proteins demonstrated that rapamycin selectively blocked the expression of proliferating cell nuclear antigen (PCNA), an obligate cofactor of DNA polymerase-delta, an important component for DNA replication, Rapamycin inhibited the IL-2-induced PCNA mRNA, and the murine PCNA promoter activity in IL-2-stimulated cells, Inducible CRE-binding proteins were shown previously to be required for PCNA promoter activity in IL-2-stimulated T lymphocytes, Using DNA binding gel mobility shift assay we demonstrated that rapamycin potently inhibited the binding of CREB/ATF transcription factors to CRE elements in the murine proximal PCNA promoter, These results suggest that PCNA is a preferred target in a rapamycin-sensitive transduction pathway, and that the mechanism by which rapamycin inhibits PCNA gene expression may involve the inhibition of the interaction of CREB/ATF transcription factors with CRE elements in the proximal PCNA promoter.