ERAD Activity Distinguishes the Functional Heterogeneity of Hematopoietic Stem Cells

Proteostasis must be precisely maintained in hematopoietic stem cells (HSCs) through a network of quantity and quality control mechanisms to ensure their self-renewal and sustained hematopoiesis. However, whether variability of proteostatic features could prospectively distinguish the heterogeneity of phenotypical HSCs remains unclear. Among the various proteostasis maintaining mechanisms, the disruption of ER associated degradation (ERAD) results in one of the most pronounced impairments of HSC function. We speculate that variations in ERAD activity may account for the proteostatic diversity that defines the functional heterogeneity of HSCs. However, the absence of in vivo tools to monitor ERAD activity represents a significant barrier to exploring this hypothesis.

To monitor ERAD activity in vivo, we first developed an indicator of ERAD activity based on the stability of the Sel1L-Hrd1 ERAD substrate, NHK. We generated a dual-fluorescent reporter, ZsGreen-P2A-NHK-DsRed Express 2, to visualize ERAD activity. This reporter allows us to measure ERAD activity by the ratio of DsRed (representing the level of NHK) and ZsGreen (representing the level of non ERAD substrate). After confirming that this reporter could reliably facilitates real-time tracking of ERAD activity in various cell types, we generated an inducible (floxed) transgenic mouse strain expressing the ERAD activity indicator at the ROSA 26 locus (ERADi). Hematopoietic specific expression of ERADi revealed that expression of ERADi did not change the frequency or total number of any hematopoietic populations. We further validated the sensitivity and reliability of this indicator by crossing ERADi with Sel1L knockout (KO) or Hrd1 KO strains. The fluorescence intensity ratio of DsRed to ZsGreen in either Sel1L or Hrd1 KO was much higher in all hematopoietic populations, suggesting a robust reduction of ERAD activity. This reporter line therefore can reliably indicate the activity of ERAD in vivo.

To assess if ERAD activity can distinguish the functionality of hematopoietic cell, we first purified ERAD^high and ERAD^low whole bone marrow (WBM) cells, and transplanted these cells along with the competitors into lethally irradiated recipients. Interestingly, only ERAD^high WBM cell were able to reconstitute multilineage hematopoiesis, suggesting that functional HSCs reside exclusively in the population with higher ERAD activity. We then purified ERAD^high or ERAD^low HSCs and found that the HSCs with high ERAD activity have much higher reconstitution potential than ERAD low HSCs, suggesting that ERAD activity can prospectively distinguish the functional heterogeneity of HSCs.

To study the underling mechanism that regulate the ERAD diversity in HSCs. RNA-Seq and RT-qPCR were performed to detect the transcriptional differences in ERAD high/low HSPCs(Hematopoietic Stem and Progenitor Cells). Strikingly, neither gene expression of unfolded protein response (UPR) pathway, which has been believed to govern the ERAD activity by transcriptional regulation, nor the ERAD pathway genes were differently expressed. In addition, HSC dormancy related genes were highly expressed in ERAD^high HSPCs. More importantly, we found that the negative regulator of JAK2 signaling, SOCS2, was highly expressed in ERAD^high HSPCs. The involvement of the SOCS2-JAK2 pathway in ERAD regulation was further validated in a mouse model that express a constitutively active JAK2 mutant, JAK2 V617F. ERAD activity as measured by ERADi was significantly inhibited in HSPCs of the JAK2 V617F mice. Further biochemical assays indicated that JAK2 modulates ERAD activity through its potential interaction with the ERAD complex.

In summary, we developed the first ERAD reporter that reliably measures ERAD in vivo. Using this reporter, we found that functional heterogeneity in HSCs can be prospectively distinguished by ERAD activity. In addition, we discovered a novel role of the SOCS2-JAK2 signaling as an important regulator of ERAD in HSCs.