The cyclin-dependent kinase inhibitor p21 is required for TGF-beta1-induced podocyte apoptosis

Kidney Int. 2005 Oct;68(4):1618-29. doi: 10.1111/j.1523-1755.2005.00574.x.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antimetabolites, Antineoplastic / pharmacology
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Cell Line, Transformed
  • Gene Expression
  • Mice
  • Mice, Knockout
  • Mice, Transgenic
  • Podocytes / cytology*
  • Podocytes / drug effects
  • Podocytes / physiology*
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Proto-Oncogene Proteins p21(ras) / genetics*
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Puromycin Aminonucleoside / pharmacology
  • Temperature
  • Transforming Growth Factor beta / pharmacology*
  • Transforming Growth Factor beta1
  • bcl-X Protein / metabolism

Substances

  • Antimetabolites, Antineoplastic
  • Bcl2l1 protein, mouse
  • Proto-Oncogene Proteins c-bcl-2
  • Tgfb1 protein, mouse
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • bcl-X Protein
  • Puromycin Aminonucleoside
  • Proto-Oncogene Proteins p21(ras)