Page 131 - Haematologica Vol. 109 - July 2024

ARTICLE - Ppm1b regulates HSC homeostasis
active centers and lower substrate specificity. Emerging evidence of phosphatases in controlling HSC homeostasis and functions has been gradually elucidated in recent years. Src homology 2 domain-containing phosphatase 2 (Shp2) plays a critical role in controlling the survival and mainte- nance of HSC.14 Ptpn21 has been reported to control HSC homeostasis.15 STS1/STS2 functions in normal and stress hematopoiesis via the regulation of HSC fitness.16
The protein phosphatase 2C (PP2C) family is a member of the metal-dependent protein phosphatases (PPM) of serine/ threonine phosphatases. PP2C family members are known to be negative regulators of cell stress response pathways, and involved in cell cycle control via the dephosphorylation of cyclin-dependent kinases. PP2C family members such as PPM1K and PPM1D, have been demonstrated to play functional roles in maintaining the stemness of HSC.17,18 Protein phosphatase, Mg2+/Mn2+ dependent 1B (Ppm1b, also known as PP2Cb), is involved in the regulation of multiple signaling pathways by targeting substrate protein dephos- phorylation,19,20 including MAPK signaling, TGF-b signal- ing, TNF signaling, AMPK signaling, and cell cycle. Ppm1b showed high expression in embryonic stem cells,21 and its deficiency led to early pre-implantation lethality.22 Our previous study suggested that Ppm1b may participate in the regulation of hematopoietic processes such as B-/T-cell receptor signaling, FOXO signaling, and the pathogenesis of leukemia.23 It had consistently been reported that Ppm1b regulates terminal erythropoiesis by interacting with an erythroid Kruppel-like factor.24 However, the role of Ppm1b in hematopoiesis is still largely unknown.
In this study, we generated and characterized a conditional knockout (KO) mouse model in which Ppm1b was specifically depleted in hematopoietic cells, and demonstrated that loss of Ppm1b impaired the normal HSC pool and B-cell development. Furthermore, Ppm1b regulates the functional expansion of HSC via Wnt/b-catenin signaling.
Methods
Mice
Ppm1b floxed mice (Ppm1bfl/fl) were generated by inserting loxp sites flanking exon 2, which, when deleted, results in a frame-shift and forms a premature stop codon in the reading frame. Vav-cre mice were purchased from the Jackson Laboratory. The animals were kept in individually ventilated cages with filtered germ-free air, and maintained with sterilized water and irradiated food. The Institutional Animal Care and Use Committees at Shandong University (#19026) approved all animal experiments in our study.
Z. Lu et al. is provided in the Online Supplementary Appendix. Flow
cytometric analysis was performed with an ACEA NovoCyte flow analyzer, and the results were further analyzed with FlowJo and NovoExpress software.
In vitro hematopoietic stem cell expansion
The purified lineage-negative cells were cultured in Stem- Span expansion medium (StemCell) supplemented with 10 ng/mL mouse TPO (Peprotech) and 10 ng/mL mouse SCF (Peprotech) for different time periods as indicated in the figure legends (Figures 1-7).
Bone marrow transplantation
Non-competitive and competitive bone marrow trans- plantation (BMT) was performed as described previously.27 Briefly, for non-competitive BMT, 2x106 BM mononuclear cells (BMMC) (CD45.2) from Ppm1bCKO or Ppm1bfl/fl mice were injected retro-orbitally into lethally irradiated wild-type (WT) recipient mice (CD45.1). For competitive BMT, 1x106 BMMC cells from Ppm1bCKO or Ppm1bfl/fl mice (CD45.2) mixed with an equal number of WT CD45.1 BMMC were injected retro-orbitally into lethally-irradiated CD45.1 recipient mice. For serial competitive BMT, 2x106 donor BMMC from primary transplant recipient mice were transplanted into lethally irradiated CD45.1 congenic recipients. PB was analyzed every four weeks by flow cytometry to assess engraftment.
Limiting dilution assays
Different doses (1x104, 2x104, 4x104, 8x104) of BMMC from Ppm1bCKO or Ppm1bfl/fl mice (CD45.2) plus 2x105 competitor BMMC (CD45.1) were transplanted retro-orbitally into le- thally irradiated WT recipient mice (CD45.1). Sixteen weeks after transplantation, the frequency of donor-derived cells (CD45.2) was evaluated by flow cytometry. ELDA (bioinf. wehi.edu.au/software/elda/) and L-Calc software (StemCell) were used to analyze HSC frequency.
Proximity ligation assay
Proximity ligation assay (PLA) was performed using the DuoLink reagents as previously described.28 Briefly, LSK cells were sorted by an Aria III flow cytometer and plated in 8-mm glass coverslips. Cells were fixed with 4% parafor- maldehyde and permeabilized with 0.5% Triton X-100 sep- arately, followed by incubating with blocking solution. Cells were then incubated with Ppm1b and b-catenin overnight at 4°C. After incubation with PLA probes, probe ligation, and amplification separately, cells were visualized using an Axio Observer fluorescent A1 microscope.
Results
Ppm1b inhibition led to the compromised proliferation of phenotypic hematopoietic stem cells
By qRT-PCR, we confirmed that Ppm1b is highly expressed
Flow cytometric assays
Single-cell suspensions of tissues, cultured cells, or pe- ripheral blood (PB) were prepared and stained as previ- ously described.25,26 Detailed information for antibodies
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