Dynamic Changes in Intracellular ROS Levels Regulate Airway Basal Stem Cell Homeostasis through Nrf2-Dependent Notch Signaling
Nrf2 Directly Regulates Notch, which Activates ABSC Proliferation
(A) qPCR from FACS sorted mABSCs of the repairing airway epithelium of WT and Nrf2−/− mice at 48 hpi for Notch pathway components compared to uninjured airway epithelium (dashed line) (n = 13).
(B) Dual luciferase assay to study the interaction between Nrf2 and Notch by CBL-Luc Notch reporter luciferase activity in ABSCs in the presence of DBZ, the Nrf2 activator SFN, and a constitutively active Notch plasmid (Nact) (n = 6).
(C) Adenoviral-Cre-GFP was transduced into FACS-sorted Keap1fl/fl ABSCs. Keap1-deficient ABSCs were treated with DBZ and the number and size of the spheres was quantified. Dotted line represents WT untreated control.
(D) FACS-sorted mABSCs isolated from the WT, Nrf2−/−, K5-NICD, and KNN mice were cultured in the sphere assay. The diameter and number of spheres were quantified from the brightfield images ( Figure S7 J). The dotted line represents WT control. Data are presented as mean ± SEM (n = 3).
(E) IF for K5, K8, and BrdU of the repairing airway epithelium at 48 hpi in WT, Nrf2−/−, K5-NICD, DBZ-treated WT, and KNN mice.
(F) Quantification of BrdU in the repairing airway epithelium in uninjured WT, K5-NICD mice; Nrf2−/−, K5-NICD, DBZ-treated WT, and KNN mice at 48 hpi. Data are presented as mean ± SEM (n = 8).
Significance was calculated by two-tailed, paired Student’s t test. Values are as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
(G) mABSCs were transfected with two different known activating mutations of Nrf2 (Nrf2 V36 del [Nrf2a] and Nrf2 E821 [Nrf2b]). Notch expression was measured using qPCR. Notch expression was measured relative to WT untransfected ABSC control expression. Data are presented as mean ± SEM (n = 3).
(H) Cartoon showing the mouse Notch1 promoter and the ARE consensus sequences. The Notch ARE sequence used in the EMSA corresponds to ARE1, which is located between −204 and −196 bps upstream of the transcriptional start site. The Notch1 sequence used to generate probes for the EMSA is shown. The blue arrow shows the specific protein complex with the Notch1 probe, and the red arrowhead shows the super-shifted band after adding the Nrf2 antibody.
(I) Schematic model of ROS flux tightly regulating ABSC self-renewal and repair after injury. ABSCs demonstrate heterogeneity with regard to ROS status. ROShi ABSCs are proliferation-deficient and not shown. Quiescent ABSCs have a low level of ROS and after injury the ROSlo ABSCs undergo a flux change in their ROS levels to ROSmod. This flux activates Nrf2, which directly increases Notch1 expression and promotes self-renewal and proliferation for repair. Nrf2 also induces antioxidants that reduce ROS levels and this brings the ABSC back to the quiescent state with inhibition of proliferation in a tightly regulated fashion.
See also Figure S7 .