Interplay among p21Waf1/Cip1, MUSASHI-1 and Kr\xfcppel-like factor 4 in activation of Bmi1-CreER reserve intestinal stem cells after gamma radiation-induced injury

Emilia J. Orzechowska,Agnieszka B. Bialkowska,Takahito Katano,Vincent W. Yang

doi:10.1038/s41598-020-75171-w

Emilia J. Orzechowska, Agnieszka B. Bialkowska + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-75171-w

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Abstract

Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

Highlights

Radiation has an important role in abdominal cancer treatment, especially as an adjuvant therapy
Since preexisting experimental data focused on Hopx-CreER-marked rISCs31, we investigated the role of MSI1 in Bmi1CreER-marked regenerating crypts and elucidated its function upon γ radiation-induced injury
According to a hierarchical model, active intestinal stem cells (ISCs) serve as a source of constant replenishment of cells in homeostasis, and reserve ISCs (rISCs) become activated following depletion of aISCs upon injury[12,13,14]

Summary

Introduction

Radiation has an important role in abdominal cancer treatment, especially as an adjuvant therapy. MSI1 and MSI2 were shown to drive mTORC1 activation, most likely through the PTEN-PIK3-AKT axis, which is required for the regenerative p rocess[28,31] These findings indicate the importance of the MSI1 protein as well as the Hopx-CreER and Bmi1-CreER subpopulations of rISCs in the response to γ radiation. Our results demonstrated that γ irradiation-induced expression of p 21Waf1/Cip[1] in Bmi1-CreER-marked cells during the early phase of the postirradiation period is retarded by MSI1, an established negative regulator of p21Waf1/Cip[1] mRNA translation[21,25,26]. We and others showed that Bmi1-CreER-marked cells represent one of the populations of rISCs that exit a quiescent state and start proliferating following γ irradiation to regenerate the IE2,16,18. To determine whether an increase in p 21Waf1/Cip[1] expression occurs in the Bmi1-CreERmarked cells, we performed immunofluorescence (IF) staining and analyzed the time-course expression pattern of p21Waf1/Cip[1] in the Y FP+ cells

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Conclusion