p21: Protector of Progenitor Pools

Sci. Signal.  12 Nov 2013:
Vol. 6, Issue 301, pp. ec273
DOI: 10.1126/scisignal.2004897

Progression of the cell cycle is regulated by cyclin-dependent kinases (CDKs) and their inhibitors (CKIs). By limiting cell proliferation, the CKI p21Waf1/Cip1 (hereafter called p21, encoded by Cdkn1a) prevents senescence of neural stem cell (NSC) pools that generate neuronal and oligodendroglial cells in the brain. Porlan et al. found that p21 maintained NSCs by suppressing the expression of Bmp2, encoding bone morphogenetic protein 2 (BMP2), which promotes gliogenesis. Analysis of single NSCs dissociated from the subependymal zone (SEZ) of 2-month-old mice and cultured on Matrigel showed that nuclear p21 abundance decreased during differentiation and that p21-negative differentiated cells were more likely to adopt the astroglial fate. Culture NSCs derived from Cdkn1a–/– mice, but not those of wild-type mice, exhibited a flattened morphology, abundant S100β (a marker of immature astrocytes), and decreased expression of stem cell–associated Notch targets Hes1 and Hes5. Cdkn1a–/–-derived NSCs also showed an increase in Bmp2 expression as well as increased BMP2 abundance in both cell lysates and conditioned media. Treatment of wild-type cells with recombinant BMP2 reduced the formation of multipotent neurospheres. Phosphorylation of SMAD1, 5, and 8 (downstream markers of activated BMP receptors) was increased in Cdkn1a–/– cells compared with wild-type cells. However, the number of cells exhibiting phosphorylated SMADs and S100β was reduced to similar numbers observed in wild-type cultures by either the addition of the BMP antagonist Noggin or immunodepletion of BMP2 from the conditioned medium. The phenotypes observed in the absence of p21 in culture (increased differentiation, Bmp2 expression, and interference by Noggin) were also observed in the intact SEZ of Cdkn1a–/– mice. Cdkn1a–/– cells cocultured with wild-type cells in plates separated by a semipermeable membrane inhibited the formation of neurospheres in the wild-type culture, and this could be prevented by treatment with Noggin. Together, the data suggest that increased expression and secretion of BMP2 in the absence of p21 is responsible for the loss of “stemness” in NSCs. Expression of full-length p21 or a CDK binding-deficient mutant p21, which did not trigger cell cycle arrest, in Cdkn1a–/– cells repressed the expression of a Bmp2 reporter construct. Repression of the reporter was not prevented by a pharmacological CDK inhibitor but was abrogated by an inhibitor of the transcription factor E2F1. Chromatin immunoprecipitation indicated that p21 bound the Bmp2 promoter in a region containing the putative E2F1 binding site. Thus, by repressing Bmp2 expression through an interaction with E2F1, p21 prevents premature loss of the NSC population in a manner that is independent of its regulation of the cell cycle.

E. Porlan, J. M. Morante-Redolat, M. A. Marqués-Torrejón, C. Andreu-Agulló, C. Carneiro, E. Gómez-Ibarlucea, A. Soto, A. Vidal, S. R. Ferrón, I. Fariñas, Transcriptional repression of Bmp2 by p21Wif1/Cip1 links quiescence to neural stem cell maintenance. Nat. Neurosci. 16, 1567–1575 (2013). [PubMed]