The Cyclin-dependent Kinase Inhibitor P21 is Required for TGF-beta1-induced Podocyte Apoptosis

Background: Reduced podocyte number is a critical determinant in the development of glomerulosclerosis. Transforming growth factor-beta1 (TGF-beta1) induces podocyte apoptosis, but the cell cycle events are not known. The cyclin-dependent kinase (CDK) inhibitor p21 increases in podocytes in diseases where TGF-beta increases. Accordingly, we studied the role of p21 in podocyte apoptosis.

Methods: Immortalized and primary p21+/+ and p21-/- mouse podocytes were used. Apoptosis was measured by Hoechst 33342 staining and caspase-3 activity following the exposure to TGF-beta1 or puromycin aminonucleoside. p21 and specific Bcl-2-related family proteins levels were measured by Western blot analysis. To prove a role for p21, we reconstituted p21 expression in p21-/- podocytes utilizing an adenovirus vector.

Results: TGF-beta1 increased the protein levels of p21 in p21+/+ podocytes, and this coincided with apoptosis. In contrast, TGF-beta1 did not induce apoptosis in p21-/- podocytes. Restoring p21 expression increased apoptosis in p21-/- podocytes following exposure to TGF-beta1. TGF-beta1 increased the protein levels of an anti-apoptotic Bcl-2 in p21-/- podocytes, but not in p21+/+ podocytes. Moreover, TGF-beta1 did not increase Bcl-2 expression in p21-/- podocytes in which p21 expression was restored. Finally, puromycin aminonucleoside also induced apoptosis in p21+/+ podocytes, but not in p21-/- podocytes.

Conclusion: Podocyte apoptosis induced by TGF-beta1 and puromycin aminonucleoside requires p21, and Bcl-2 plays a crucial role downstream of p21 in mediating this effect. These results suggest that p21 may play a critical role in the decrease in podocyte number in disease status accompanied by increased TGF-beta1.