ID2 and HIF-1α collaborate to protect quiescent hematopoietic stem cells from activation, differentiation, and exhaustion
Brad L. Jakubison, … , Kimberly D. Klarmann, Jonathan R. Keller
Brad L. Jakubison, … , Kimberly D. Klarmann, Jonathan R. Keller
Published July 1, 2022
Citation Information: J Clin Invest. 2022;132(13):e152599. https://doi.org/10.1172/JCI152599.
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Research Article Hematology

ID2 and HIF-1α collaborate to protect quiescent hematopoietic stem cells from activation, differentiation, and exhaustion

Abstract

Defining mechanism(s) that maintain tissue stem quiescence is important for improving tissue regeneration, cell therapies, aging, and cancer. We report here that genetic ablation of Id2 in adult hematopoietic stem cells (HSCs) promotes increased HSC activation and differentiation, which results in HSC exhaustion and bone marrow failure over time. Id2Δ/Δ HSCs showed increased cycling, ROS production, mitochondrial activation, ATP production, and DNA damage compared with Id2+/+ HSCs, supporting the conclusion that Id2Δ/Δ HSCs are less quiescent. Mechanistically, HIF-1α expression was decreased in Id2Δ/Δ HSCs, and stabilization of HIF-1α in Id2Δ/Δ HSCs restored HSC quiescence and rescued HSC exhaustion. Inhibitor of DNA binding 2 (ID2) promoted HIF-1α expression by binding to the von Hippel-Lindau (VHL) protein and interfering with proteasomal degradation of HIF-1α. HIF-1α promoted Id2 expression and enforced a positive feedback loop between ID2 and HIF-1α to maintain HSC quiescence. Thus, sustained ID2 expression could protect HSCs during stress and improve HSC expansion for gene editing and cell therapies.

Authors

Brad L. Jakubison, Tanmoy Sarkar, Kristbjorn O. Gudmundsson, Shweta Singh, Lei Sun, Holly M. Morris, Kimberly D. Klarmann, Jonathan R. Keller

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Figure 10

HIF-1α regulates Id2 promoter activity, and ID2 stabilizes HIF-1α and promotes HSC survival via the VHL complex in vitro.

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HIF-1α regulates Id2 promoter activity, and ID2 stabilizes HIF-1α and pr...
(A) Luciferase (Luc) activity of pGL4.1 Id2promoter in HEK293 cells transfected with pcDNA3.1 or pcDNA3.1 HIF-1α3M (HIF-1α). (B) Schematic of the cloned Id2promoter with identified HRE-CACA boxes for luciferase reporter assays (left) and luciferase activity of the pGL4.1 Id2promoter with mutated HRE boxes transfected with pcDNA3.1 HIF-1α3M (right). (C) Western blot analysis of HIF-1α, ID2, VHL, and HSP90 in lysates from HEK293 cells transfected with either pcDNA3.1 or pcDNA3.1 mutated HIF-1α3M or PCMV or PCMV-ID2MycFlag. (D) Western blot analysis of VHL, ID2, and β-actin and IP analysis of ID2MycFlag and VHLHA in HEK293 lysates transfected with VHL or ID2 constructs. (E) Western blot analysis of CUL2, HIF-1α, VHL, ID2, and β-actin and IP analysis of VHL in HEK293 lysates transfected with increasing amounts of ID2MycFlag. (F) Western blot analysis of hydroxylated HIF-1α (OH–HIF-1α), ID2, and β-actin in HEK293 cell lysates transfected with increasing amounts of ID2. (G) Procedure to measure HSC expansion after knockdown of Vhl expression in Id2Δ/Δ Lin– cells using lentiviral control shRNA (shCtrl) and shRNA-VHL (shVHL-1, -2, -3) vectors. Graphs show the expression of Vhl transcripts in knockdown Lin– cells and total HSCs in culture. In B and G, data are presented as the mean ± SEM. Comparisons between mean values of 2 groups were evaluated using an unpaired, 1-tailed Student’s t test, and 1-way ANOVA with Dunnett’s correction was used for multiple means testing. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.