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.


CONCLUSIONS
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.
Wada
Takehiko
T
Division of Nephrology, University of Washington, Seattle, Washington 98195, USA.
Pippin
Jeffrey W
JW
Terada
Yoshio
Y
Shankland
Stuart J
SJ
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D015870:Gene Expression; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D050199:Podocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism*; D011692:Puromycin Aminonucleoside / Q000494:pharmacology; D013696:Temperature; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D051020:bcl-X Protein / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; C494812:Bcl2l1 protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D051020:bcl-X Protein; D011692:Puromycin Aminonucleoside; D016283:Proto-Oncogene Proteins p21(ras)