PERK Maintains Hematopoietic Stem Cells Pool Integrity By Promoting Proliferation
Authors: Zheng Manxi , Peng QinLu , Kropp Erin M , Liu Suxuan , Yin Zhengyou , Shen Zhejuan , Matono Sho , Iwawaki Takao , Inoki Ken , Mei Yang , Li Qing , Liu Lu 
Published: December 2024

The integrity of the hematopoietic stem cell (HSC) pool relies on efficient long-term self-renewal and the timely removal of damaged or differentiation-prone HSCs. Previous studies have demonstrated the critical role of PERK branch of the unfolded protein response (UPR) to maintain HSC pool integrity in response to severe ER stress in vitro via promoting programmed cell death. However, whether PERK contributes to maintaining the HSC integrity under physiological conditions in vivo remains unclear.
To determine the role of UPR PERK signaling in HSCs and hematopoiesis, we crossed the conditional PERK knockout (KO) with either Mx1-Cre or Vav1-Cre to delete PERK in hematopoietic tissues. We found that PERK KO had minimal effect on steady state hematopoiesis and the PERK KO mice displayed comparable frequency and number of SLAM HSCs and all other hematopoietic populations. Competitive repopulation assay was then performed to assess the impact of PERK loss on function of HSCs by transplanting CD45.2 PERK KO or control HSCs, together with CD45.1 wild type competitor cells, into lethally irradiated CD45.1 recipients. Control and PERK KO transplants showed similar levels of CD45.2 percentage in all hematopoietic populations indicating comparable reconstitution potentials. These findings suggested that PERK signaling is dispensable for steady-state hematopoiesis and HSC functions.
Since PERK is activated in response to ER stress and ER associated degradation (ERAD) loss frequently leads to ER stress and HSC dysfunction, we next investigated the role of PERK in ERAD deficiency-induced HSC impairment. First, we confirmed that KO of the key component of Sel1L-Hrd1 ERAD complex, Sel1L, leads to increased ER stress as manifested by increased levels of protein aggregates and ER mass. Importantly, all three UPR branches including PERK were activiated and levels of transcriptional targets of UPR pathways were increased. However, this UPR activation did not lead to increased cell death as these stressed HSCs formed comparable number of colonies ex vivo as the control HSCs. Additionally, ERAD loss did not confer a higher sensitivity of HSCs to further Tunicamycin or Thapasigargin induced cell death, suggesting activation of PERK is not associated with increased cell death in HSCs. Next, we investigated whether PERK activation mediates the HSC impairments induced by Sel1L knockout by generating double knockout of Sel1L/PERK (DKO). We found that PERK KO rescued the Sel1L KO-induced depletion of HSC pool at steady state and reduction of HSC reconstitution and self-renewal in primary and secondary transplants.
To confirm the results in Sel1L KO, we generated a Hrd1 conditional knockout mouse strain by deleting the exon 1-14 of Hrd1. When we crossed this line with either Mx1-cre or Vav1-cre, we observed similar phenotype of HSC impairments and hematopoietic defects as seen in Sel1L KO. Consistently, we detected activation of PERK in Hrd1 knockout mice. Similarly, the Hrd1/PERK double knockout mice revealed a significant rescue of HSC pool size depletion and HSC reconstitution ability in transplants with Hrd1/PERK DKO cells. Taken together, these findings suggest that PERK activation mediates the effects of increased ER stress induced by ERAD deficiency via knockout of Sel1L or Hrd1 and deletion of PERK rescues the HSC defects of Sel1L KO or Hrd1 KO.
To dissect the underlying mechanism of the effects of PERK, we first investigated whether its function depends on the kinase activity. We generated a knock-in (KI) strain with an in situ inducible point mutation of PERK, K618A (kinase dead). The depletion of HSCs induced by Hrd1 KO was completely rescued by PERK K618A KI at steady state. More importantly, PERK K618A KI improved the reconstitution potential of Hrd1 KO HSCs in competitive repopulation assay. Finally, our previous studies showed that ERAD loss induces activation of mTOR and increased proliferation rate in HSCs, effects completely abolished when PERK is deleted. Therefore, our results indicate that PERK contributes to mTOR activation to increase HSC proliferation upon ERAD deficiency and requires its kinase activity to execute these functions.
In summary, we identified PERK as a crucial regulator of HSCs that preserves their integrity by promoting proliferation in response to ER stress.