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Cyclosporine induces the expression of the cyclin inhibitor p21
1Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee 53226, USA. akkhanna@mcw.edu
Background:
Current immunosuppression strategies involve inhibition of T cell activation and/or lymphocyte proliferation. During T cell cycle progression/activation, the expression of cyclins and cyclin dependent kinases is increased. In this study, we examined whether cyclosporine A (CsA) suppresses the cell cycle progression through the induction of the cell cycle inhibitor p21. Because CsA induces the expression of transforming growth factor (TGF)-beta, and TGF-beta induces p21 expression, we also determined whether CsA's induction of p21 is dependent on or independent of TGF-beta.
Method:
Using reverse transcription assisted polymerase chain reaction and western blot analysis, we studied the induction of p21 mRNA and protein in human T cells and A-549 cells (human lung adenocarcinoma cells) by CsA. The stimulation of p21 promoter activity was studied by luciferase assay using p21-luc, chimeric plasmid DNA containing a p21 promoter segment, and luciferase reporter gene. The dependence of CsA's induction of p21 was studied using anti-TGF-beta antibody and TGF-beta altered A-549 cells.
Results:
CsA induced p21 mRNA protein expression and stimulated its promoter activity in lymphoid (T cells) and nonlymphoid (human lung adenocarcinoma, A-549 cells).CsA's induction of p21 was inhibited both by a neutralizing anti-TGF-beta antibody and in TGF-beta-altered A-549 cells, consistent with its effects on p21 requiring TGF-beta.
Conclusion:
These data support the hypothesis that at least one component of CsA's antiproliferative effects may occur through the induction of p21 and that this induction is dependent on TGF-beta. Should p21 induction be a viable immunosuppressive strategy, inducing this molecule independent from the fibrogenic cytokine TGF-beta might reduce the toxicity associated with current immunosuppression.
Insights
Cyclosporine A (CsA) suppresses T cell proliferation by increasing the cell cycle inhibitor p21, a process dependent on transforming growth factor-beta (TGF-beta). Inducing p21 independently of TGF-beta may reduce immunosuppression toxicity.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Current immunosuppression targets T cell activation and proliferation.
- Cyclosporine A (CsA) is an immunosuppressant.
- Cell cycle progression involves cyclins and cyclin-dependent kinases.
Purpose of the Study:
- To investigate if CsA suppresses cell cycle progression via p21 induction.
- To determine if CsA's p21 induction is dependent on transforming growth factor-beta (TGF-beta).
Main Methods:
- Reverse transcription PCR and Western blot to assess p21 mRNA and protein.
- Luciferase assay to study p21 promoter activity.
- Use of anti-TGF-beta antibody and TGF-beta altered cells to assess dependence.
Main Results:
- CsA induced p21 mRNA, protein expression, and promoter activity in T cells and A-549 cells.
- CsA's induction of p21 was inhibited by anti-TGF-beta antibody.
- Effects on p21 required TGF-beta, as shown in TGF-beta altered cells.
Conclusions:
- CsA's antiproliferative effects involve p21 induction, which is dependent on TGF-beta.
- Inducing p21 independently of TGF-beta could mitigate immunosuppression toxicity.