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Iqbal Ahmad, Carolina Del Debbio, Sowmya Parameswaran; Notch mediated activation of mammalian Müller stem cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2213. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In adult mammalian retina, a subset of Müller glia (MG) possesses stem cell properties, which is unmasked upon injury and/or exposure to exogenous factors (Das et al., 2006, Developmental Biology, 299(1):283-302.). We have observed, that one of the non-cell autonomous activation of Müller stem cells involves the canonical Notch signaling pathway (Del Debbio et al., 2011, Plos One, 5(8):e12425). Here, we have tested the hypothesis that Notch signaling activates Müller stem cells by regulating cyclin-dependent kinase (CDK) inhibitor p27 kip1 (Notch-Hes1-p27kip1) and its proteasomal degrader SKP2 (Notch-CSL-Skp2-p27kip1).
The Notch regulatory axes were examined in neurosphere assay of Müller stem cells. Briefly, enriched MG was cultured in the presence of Jagged1 peptide to activate Notch signaling, followed by the examination of the axes at transcriptional and protein levels. The role of these axes in the activation of Müller stem cells was determined by the loss of function approach.
We observed an increase in the number of Müller neurosphere in the presence of Jagged1 with corresponding increase and decrease in Hes1 and p27 kip1 transcript levels, respectively, compared to controls, suggesting the existence and involvement of Notch-Hes1-p27kip1 axis. The levels of SKP2 transcripts increased and that of P27KIP1 protein decreased in response to Notch signaling suggesting that Notch-CSL-Skp2-p27 kip1 axis may lead to proteasomal degradation of P27KIP1. The shRNA-mediated silencing of p27 kip1 and Skp2 expression facilitated and compromised Notch-mediated generation of Müller neurospheres, respectively, suggesting the functional involvement of the axes in the activation of Müller stem cells.
Taken together, our results suggest that Notch signaling regulates the activation of Müller stem cells directly by influencing the transcription and indirectly by facilitating the proteasomal degradation of P27KIP1.
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