Page 138 - Haematologica Vol. 109 - July 2024

ARTICLE - Ppm1b regulates HSC homeostasis
Z. Lu et al.
the commitment gene markers were identified, including myeloid-biased (pMy), lymphoid-biased (pLy), megakaryo- cytic-biased (pMk), and erythroid-biased (pEry) clusters (Figure 5B). The remaining cell populations, characterized by the maintenance of stemness and lacking a specific pro- pensity for lineage differentiation according to enrichment analysis, were annotated as “non-primed”. This cluster was further confirmed by the elevated expression of Hlf, a mas- ter transcription factor predominantly expressed in an HSC subpopulation with extensive self-renewal capacity (Figure 5C).38 Consistent with the findings in flow cytometric anal- ysis, the relative frequency of the “non-primed” cluster was also reduced in the Ppm1bCKO group (Online Supplementary Figure S6C).
Several genes associated with HSC homeostasis were identi- fied in the non-primed cluster between CKO and WT groups (Figure 5D, Online Supplementary Table S2). Nf4a1 has been reported to restrict HSC proliferation in part through activa- tion of a C/EBPα-driven antiproliferative network by directly binding to a hematopoietic-specific Cebpa enhancer, a mem- ber of the Nr4a subfamily of nuclear receptors.39 Fos serves as a gatekeeper in the cell cycle progression of dormant HSC, whose sustained expression inhibits HSC entry into the cell cycle.40 These two genes showed obvious upregulation in CKO HSC compared to that of the corresponding controls (Figure 5D). On the other hand, it has been demonstrated that down-regulated Txnip is required for the hematopoi- etic reconstitution and HSC self-renewal capacity.41 In line with the previous report that low FLT3 expression led to a blockage at G0 phase in HSC,42 CKO HSC showed reduced FLT3 expression and enhanced quiescence. Furthermore, GO enrichments with these differentially expressed genes revealed that the physiological activity was down-regulated in CKO HSC, such as cytokine-mediated signaling, mRNA metabolism, and translation, which phenocopies the qui- escent status (Figure 5E). These transcriptomic changes further confirmed the impact of Ppm1b on HSC expansion.
Loss of Ppm1b impaired the functional expansion of hematopoietic stem cells through Wnt/b-catenin pathway
Given that the phosphorylation of b-catenin functions as a downstream key node of Wnt signaling, and it has been pre- viously identified as a potential subtract of PPM1B, we spec- ulated that Ppm1b regulates HSC via Wnt/b-catenin signaling. Indeed, the phosphorylation of b-catenin was dramatically increased in Ppm1bCKO HSC compared to that of WT controls (Figure 6A). The target genes of Wnt/b-catenin signaling such as Cylin D1 and Myc, were also down-regulated upon Ppm1b deletion (Figure 6B). Proximity ligation assay in sorted LSK cells indicated that Ppm1b interacted with b-catenin (Figure 6C). Moreover, Ppm1b inhibitor HN252 treatment led to a considerable decrease in the active b-catenin (non-phos- phorylated) in the LSK cells (Online Supplementary Figure S7A, B), which is further confirmed by the downregulation
of its target genes (Online Supplementary Figure S7C). These data suggest that Ppm1b mediates the b-catenin activity via the dephosphorylation in HSC.
To confirm Wnt/b-catenin signaling is responsible for the impaired expansion of HSC with Ppm1b deficiency, we per- formed rescue experiments with a potent Wnt signaling ac- tivator BML-284 (BML)43 and found that BML could activate the expression of Wnt-targeted genes in Ppm1b deficient cells (Online Supplementary Figure S7D). Lineage negative BM cells were sorted from CKO and WT mice and labeled with CFSE, followed by in vitro culture with BML treatment. Compared to the high MFI of CFSE in Ppm1bCKO LSK cells after 48 hr ex vitro culture, BML treatment led to a signif- icant reduction in CFSE staining (Figure 6D). This was fur- ther confirmed by the increase in LSK cells derived from Ppm1bCKO mice upon BML treatment (Online Supplementary Figure S7E), indicating that BML reversed the suppressed HSC expansion with Ppm1b deficiency.
To verify the functional roles of Ppm1b/Wnt/b-catenin axis in HSC expansion in vivo, we pretreated Ppm1bCKO and WT mice with BML for four days and then challenged them with a sublethal dose of 5-FU. Similar to the findings in ex vitro culture, the frequencies and cell number of HSC in Ppm- 1bCKO mice were remarkably increased by BML treatment, whereas the HSC in Ppm1bfl/fl mice remained unchanged (Figure 6E, Online Supplementary Figure S7F). Furthermore, BML treatment led to a significant decrease in LSK cells at G0 phases but a notable increase at the G2/S/M-phase in Ppm1bCKO mice, compared to that of the control group (Fig- ure 6F). However, BML had no detectable impact on the cell cycle of LSK cells in WT control mice (Figure 6F). These data confirm that loss of Ppm1b impaired the HSC homeostasis via the blockage of Wnt/b-catenin signaling.
Discussion
In stable state conditions, most HSC are in a state of quies- cence with only a small proportion undergoing self-renewal or differentiation.44,45 The balance between the quiescence and activation of HSC is tightly regulated to protect the HSC pool from exhaustion, which is also required for the capabilities of long-term hematopoiesis.46 In this study, we identified the critical role of Ppm1b in the regulation of HSC homeostasis and B-cell development in vitro and in vivo. Ppm1b inhibition suppressed the expansion of HSC due to the blockage of the cell cycle at G0 phase. Moreover, Ppm1b deficiency led to a reduction in the early B-cell progenitors committed from CLP, which consequently impaired B-cell differentiation (Figure 7). Altogether, our work demonstrated that Ppm1b emerges as a key regulator in normal hematopoiesis.
The canonical Wnt/b-catenin signaling has been document- ed to be crucial for the self-renewal and differentiation of HSC.47 However, its regulatory role in HSC remains a sub- ject of debate.48 Weissman et al. found that constitutive
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