Page 160 - 2021_06-Haematologica-web

G. Wu and C.C. Zhang
(CAR cKO mice before treatment with tamoxifen) to specifically inhibit Notch signaling (Figure 6H). In the competitive repopulation assay, the recipient CD45.1 mice were treated with tamoxifen to induce CAR cKO in the donor cells, with vehicle treatment as controls. With overexpressed dnMAML, the donor WT and CAR cKO BM cells showed the same repopulation abilities, where- as WT BM cells had a stronger repopulation ability than CAR cKO BM cells in the control condition (Figure 6I). In order to test whether enhanced Notch1 signaling rescues the phenotype of CAR deficiency, CAR cKO mice inject- ed with 5-FU (300 mg/kg) were treated with valproic acid, a Notch1 activator.35 These mice survived signifi- cantly longer than CAR cKO mice without valproic acid treatment (Online Supplementary Figure 14). In addition, valproic acid restored the repopulation ability of CAR cKO HSC (Online Supplementary Figure 15).
LNX contains a PDZ domain that may bind to the PDZ binding motif in the intracellular domain of CAR.36, 37 There are two forms of LNX: LNX1 and LNX2.36, 37 In order to test the roles of LNX1 and LNX2 in the function of CAR, the dominant negative (DN) forms of the LNX proteins (DN-LNX1 and DN-LNX2) with the ring finger domains and the NPXY motifs deleted 21 were over expressed in 5-FU treated Lin- BM cells from UBC-Cre- ERT2/CARloxp/loxp mice (CAR cKO mice before treatment with tamoxifen), and a competitive repopulation assay was performed (Figure 6H). The cells expressing control DNA had significantly weaker repopulation ability in CAR cKO mice than in WT mice in both myeloid (Mac1+) and lymphoid (B220+) populations (Figure 6J). Repopulation of WT and CAR cKO mice was similar when donor cells expressed DN-LNX2, while donor cells expressing DN-LNX1 less effectively repopulated CAR cKO mice (Figure 6J). At 170 days after transplantation, repopulation percentages were similar in all groups (Figure 6J), indicating that lack of CAR had no effect on the total HSC pool. In order to further explore the rela- tionship among CAR, LNX2 and Numb, we overex- pressed CAR, LNX2 or DN-LNX2 in Lin- cells and evalu- ated the protein levels of Numb in LSK cells with flow cytometry. Overexpression of CAR or LNX2 decreased the Numb level in LSK cells, whereas DN-LNX2 elevated Numb expression (Online Supplementary Figure 16). These data suggest that LNX2, but not LNX1, is involved in the function of CAR (Online Supplementary Figure 17).
Discussion
Substantial efforts have been dedicated toward uncover- ing the mechanisms that regulate HSC niche maintenance.7, 8 Recent studies demonstrated the essential roles of BM endothelial cells and osteolineage cells in regulating HSC regeneration following myelotoxicity38-40 and identified two BM endothelial cell-derived paracrine factors, PTN and EGF, and osteolineage cell-derived Dkk1 as regulators of HSC regeneration in vivo.38,41,42 Here we identified CAR as a membrane protein on HSC that is rapidly upregulated after stress, whose transient expression induces quiescent HSC to enter the cell cycle to counteract the hematopoietic injury. Without CAR, the process was delayed, and the mice in which CAR was conditionally deleted were more sensitive to injury than WT mice. Lack of CAR neither changed the pool nor altered the self-renewal of HSC.
Mechanistically we showed that elevated CAR upon stress activates Notch1 signaling by down regulating expression of Notch antagonist Numb to facilitate entry of quiescent HSC into the cell cycle. To our knowledge, this is the first report demonstrating the role of CAR in HSC regeneration.
The Notch pathway plays an important part in many developmental processes and appears to regulate many adult stem cell fate decisions.43, 44 While it was reported that Notch signaling is dispensable for HSC self-renewal and maintenance during hematopoiesis,45, 46 Notch1 can pro- mote the expansion of LT-HSC while preserving self- renewal ability.47, 48 The expression of the canonical Notch ligands Jagged-1 and Jagged-2 by endothelial cells support hematopoietic regeneration.10, 29 Varnum-Finney et al. showed that Notch2 enhances the rate of formation of short-term repopulating multi-potential progenitor cells as well as LT-HSC after 5-FU treatment.6 Our results indicate that expression of CAR in quiescent LT-HSC increases Notch1 expression and induces HSC to enter the cell cycle immediately after injury and has no effect on HSC that have entered the cell cycle. Of note, this Notch1-mediated proliferation upon stress does not alter self-renewal of HSC.
Numb is a membrane-associated, evolutionarily con- served adaptor protein that regulates cell fate determina- tion via its ability to antagonize Notch signaling.49, 50 Normal HSC differentiation and self-renewal occur in the absence of Numb during homeostasis.51 We observed that Numb expression decreased after injury and then returned to nor- mal levels after the injury was repaired (Figure 6A), and CAR accelerated Numb protein degradation before Numb mRNA levels decreased (Figure 6B). A low level of Numb protein might be necessary for functional Notch signaling during regeneration. Low Numb mRNA levels during regen- eration may be due to inhibition by Notch signaling.52 LNX proteins are E3 ligases that act on Numb 53 and that may bind to the PDZ motif of CAR.36, 37 Here we demonstrated that CAR expressed on HSC activates Notch signaling via LNX2, suggesting the PDZ motif of CAR plays a critical role in hematopoietic regeneration. CAR and CAR can form trans-interaction with a low affinity,54 and this trans- interaction may stimulate CAR downstream signaling. However, in CAR cKO recipient mice, CAR expressed on donor hematopoietic cells still plays a critical role in regen- eration in repopulation assay (Online supplementary Figure 8). Analysis of BM cells after 5-FU injection, HSC were more prone to increase CAR expression compared to other cells (Figure 1C). These results indicate that CAR expressed in the BM microenvironment is not important in HSC regeneration, and an unknown ligand may act on CAR and on HSC to regulate transition from quiescence to prolifera- tion during hematopoietic stress. These possibilities war- rant future investigations.
How HSC detect signals sent upon injury remains unknown. Low oxygen tension (hypoxia) is thought to be a characteristic of the quiescent HSC niche,55 and oxygen levels may change markedly after radiation and chemother- apy.56 CAR expression is repressed by hypoxia inducible factor-1a (Hif-1a).57 We therefore propose that the oxygen increase resulting from hematopoietic injury may suppress Hif-1a expression, leading to induction of CAR expression. This transient elevation of CAR levels activates Notch1 sig- naling by down regulating expression of Notch antagonist Numb. Notch signaling could promote expansion of HSC by preventing hematopoietic cell differentiation upon
2188
haematologica | 2021; 106(8)