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C. Leveau et al.
els of protein aggregation compared to control HSC (Figure 7A). In keeping with this, in vitro-expanded fsn HSC had an elevated level of BIP protein (Figure 7B). These results suggest that Ttc7a loss could modify HSC suscep- tibility to ER stress. Therefore, to determine the impact of Ttc7a loss in the response of HSC to ER stress, we ana- lyzed the proliferative capacity of Ttc7a-deficient HSC and progenitor cells (i.e., HSC, MPP, HPC-2 and HPC-1) upon chemical induction of ER stress in vitro. To do so, lin- eage-negative cells from 4-week old fsn and ctrl mice were cultured for 48 h in the presence or absence of tuni- camycin, which blocks the synthesis of N-linked glyco- proteins, leading to an accumulation of unfolded proteins and the induction of ER stress.32 As expected, on day 2, tunicamycin treatment reduced the proliferation ability of control cells in a dose-dependent manner (Figure 7C). In contrast, at a low dose of tunicamycin, the proliferative capacity of Ttc7a-deficient HSC was significantly greater than that of control HSC (Figure 7C). These differences were particular to HSC, as Ttc7a-deficient MPP, HPC-2 and HPC-1 subsets had a similar response to tunicamycin as their control counterparts (Online Supplementary Figure S7A). The alterations in the ER stress response in Ttc7a- deficient HSC were not due to protein aggregation (Figure 7A and Online Supplementary Figure S7B), nor to low expression of the ER stress sensors and effectors (Ire1α, Perk, Atf6, etc.), as no differences were observed in our transcriptomic analysis (Online Supplementary Figure S7C and Online Supplementary Table S1). Surprisingly, the reduction in cell proliferation of ctrl HSC in response to tunicamycin was independent of apoptosis, in contrast to that of other progenitor cells. Apoptosis of Ttc7a-deficient HSC was reduced compared to that of unstimulated ctrl cells, and remained unchanged upon tunicamycin treat- ment (Online Supplementary Figure S7D). No differences were observed in other progenitor populations (Online Supplementary Figure S7D). Altogether, these data suggest that ex vivo purified Ttc7a-deficient HSC had a higher level of ER stress compared to their control counterparts.
Interestingly, we observed that the expression of Hsp70, a chaperone protein associated with broad cellular stress- es, was also higher in fsn HSC than in controls (Online Supplementary Figure S7E). In order to determine whether Ttc7a regulates the cellular response to stress in vivo, we monitored the proliferative response of Ttc7a-deficient cells following the induction of stress by 5-FU. The deple- tion of cycling cells by 5-FU stimulates HSC to replenish peripheral leukocytes,33,34 inducing a broad stress response in HSC, not limited to ER stress (e.g., oxidative stress, pro- liferative stress). We injected 5-FU into Ctrlfsn and Ctrlctrl mice 3 months after BM transplantation and monitored the replenishment of peripheral leukocytes for 19 days. The Ttc7a-deficient and ctrl leukocyte counts fell until day 9 post-injection, and then increased. On day 15, Ttc7a- deficient leukocytes were growing significantly more strongly than ctrl cells, with a peak on day 16 (Figure 7D). Interestingly, the spleen of Ctrlfsn mice enlarged further after 5-FU treatment (Figure 7E, compared with Figure 3D), with higher lymphoid and myeloid counts (Figure 7F- G). To assess the proliferative response of Ttc7a-deficient HSC following stress injury, we assayed BrdU uptake by LSK subsets between day 6 and day 7 after 5-FU injection. The greater BrdU uptake in Ttc7a-deficient HSC, MPP, HPC-2 and HPC-1 (Figure 7H), suggested that Ttc7a con- trols the cell cycle progression of HSC under stress condi-
tions. Strikingly, BrdU uptake did not differ in committed progenitors, CLP, CMP, GMP and MEP (Figure 7I). These results suggest that Ttc7a is involved in the regulation of the proliferative response of HSC under stress conditions but not in that of committed progenitor cells.
Discussion
The present study revealed a previously unrecognized role for Ttc7a in the negative regulation of HSC function. Using murine transplantation models and Ttc7a-deficient HSC, we found that Ttc7a intrinsically regulates the main- tenance and proliferation of HSC in vivo, and the subse- quent homeostasis of downstream cell populations. We also found that Ttc7a expression in HSC is closely associ- ated with the transcriptional response to ER stress.
HSC are the only cells capable of self-renewing and dif- ferentiating into all mature blood lineages. The quiescence of HSC must be tightly regulated in order to control pro- liferation, maintain normal homeostasis, and prevent stem cell exhaustion.35,36 Various intrinsic and cell-extrinsic regu- latory factors of the HSC cell cycle have been described, such as phosphatase and tensin homologue (Pten) signal- ing, Wnt signaling and cytokine signaling.37 Indeed, in mice that lack growth factor independent 1 (Gfi1),38 Pten, forkhead box proteins 1, 3, 4, or M139 or other proteins,20,36 excessive HSC proliferation is associated with stem cell exhaustion and the loss of self-renewal. In contrast, we found that mice reconstituted with Ttc7a-deficient pro- genitors exhibit a characteristic phenotype with enhanced HSC function, higher HSC-derived peripheral blood cell counts and no evidence of stem cell exhaustion when compared with Ttc7a-proficient HSC. Indeed, Ttc7a-defi- cient HSC were able to repopulate the hematopoietic sys- tem better in serial transplantation experiments, indicating that the self-renewal of Ttc7a-deficient HSC is not com- promised by repeated rounds of proliferation. Although this situation clearly differs from the above-mentioned knock-out mice, a few similar observations have been reported after the deletion of the cyclin-dependent kinase 4 inhibitor C (CDKN2C),40 the ubiquitin-mediated protein degradation Cbl41 and Itch,41 and the transcription factors Hif1a42 and Egr1.43 The loss of these proteins enabled the maintenance of HSC, despite an increase in the cells’ pro- liferative capacity. However, the specific mechanisms by which these proteins regulate HSC function remain large- ly unknown.
Our findings support a role for the ER stress response in the enhanced function of Ttc7a-deficient HSC. Since long- lived HSC are particularly sensitive to stress stimuli, their response must be tightly controlled in order to prevent either a loss of function or the clonal persistence of onco- genic mutations. It has been shown that HSC are enriched in components of the UPR pathway. Upon exposure to acute stress in vitro, HSC are more prone to apoptosis, via upregulation of the canonical UPR genes, than related pro- genitors that have lost their self-renewal capacity.21,44 Along these lines, the ectopic expression of developmental pluripotency-associated 5 (Dppa5) was associated with enhanced HSC function, via suppression of the ER stress response (by downregulating the expression of ER stress chaperones) and the subsequent apoptotic signals.29 However, UPR activation can also have an anti-apoptotic outcome in HSC. It has been shown that stimulation of
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haematologica | 2020; 105(1)