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Rapamycin selectively blocks interleukin-2-induced proliferating cell nuclear antigen gene expression in T
N Feuerstein1, D Huang, M B Prystowsky
1Medical College of Pennsylvania, Philadelphia 19129, USA.
Abstract:
The macrolide rapamycin arrests T lymphocytes stimulated by interleukin-2 (IL-2) at G1/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 G1/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 approximately 5, 68 kDa/pI approximately 4, 110 kDa/pI approximately 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 rampamycin inhibits PCNA gene expression may involve the inhibition of the interaction of CREB/ATF transcription factors with CRE elements in the proximal PCNA promoter.
Insights
The macrolide rapamycin inhibits T lymphocyte proliferation by blocking the expression of proliferating cell nuclear antigen (PCNA). This occurs by preventing the binding of CREB/ATF transcription factors to the PCNA promoter, impacting DNA replication.
Area of Science:
- Immunology
- Molecular Biology
- Cell Cycle Regulation
Background:
- The macrolide rapamycin inhibits T lymphocyte proliferation at the G1/S phase.
- Interleukin-2 (IL-2) stimulation increases ATF/cAMP-responsive element binding factor (CREB) family transcription factor binding, an effect blocked by rapamycin.
Purpose of the Study:
- To elucidate the molecular mechanisms by which rapamycin inhibits IL-2-stimulated T lymphocyte proliferation.
- To identify specific protein synthesis and gene expression targets affected by rapamycin in T lymphocytes.
Main Methods:
- High-resolution two-dimensional gel electrophoresis to analyze protein synthesis.
- Analysis of nuclear proteins, including proliferating cell nuclear antigen (PCNA).
- Quantitative analysis of PCNA mRNA levels and promoter activity.
- DNA binding gel mobility shift assays to assess transcription factor binding.
Main Results:
- Rapamycin selectively inhibited the synthesis of three IL-2-induced soluble proteins.
- Rapamycin blocked the expression of PCNA, a key factor in DNA replication.
- Rapamycin reduced PCNA mRNA levels and promoter activity in IL-2-stimulated cells.
- Rapamycin inhibited the binding of CREB/ATF transcription factors to the PCNA promoter.
Conclusions:
- Proliferating cell nuclear antigen (PCNA) is a key target in rapamycin-sensitive signaling pathways.
- Rapamycin inhibits PCNA gene expression by interfering with CREB/ATF transcription factor binding to the PCNA promoter.
- These findings provide insight into the mechanism of rapamycin's immunosuppressive effects.