Page 176 - 2021_06-Haematologica-web

S. Warsi et al.
ostasis and adult regeneration of a variety of organ sys- tems. Several lines of evidence suggest that BMP play a role in adult HSC regulation, but conclusive evidence for direct BMP-requirement by HSC is still lacking. For instance, ALK3-mediated signaling is required by the HSC osteoblastic niche, with loss of ALK3 leading to increased HSC numbers.17 By contrast, decreased levels of BMP4 in the BM results in reduced HSC numbers, as shown in a hypomorphic BMP4 mutant mouse model.18 Additionally, BMP4 maintains cord blood-derived human hematopoietic stem and progenitor cells (HSPC) during ex vivo culture, by acting as a survival factor.19 Recently, Khurana et al. showed that BMP4 exposure in vitro maintains the expression of ITGA4 in murine HSC, thereby preventing culture-induced loss of homing capacity.20 However, SMAD1 and SMAD5 are dispensable for adult HSC, leading to the conclusion that BMP signaling is not endogenously required by adult HSC.21,22 Interestingly, BMPR-II is reportedly highly expressed in adult HSC, suggesting that BMP may signal via alternative circuitries in HSC.23 Indeed, several path- ways can be activated by BMP, including components of the MAPK pathway, such as p38 and JNK.24,25 A role for p38 has been suggested in maintenance of ITGA4 expression in HSC in vitro, but a conclusive role for BMP in the regulation of HSC in vivo has never been shown.20 Therefore, in order to investigate the complete role of BMP signaling in HSC in vivo, we conditionally deleted BMPR-II in hematopoietic cells using the Cre/loxP system. We report here that BMPR-II is essential for self-renewal of HSC with mutants displaying significantly reduced regenerative capacity upon BM transplantation. Steady state hematopoiesis is normal in mice deficient of BMPR-II and the differentiation capac- ity upon transplantation is likewise unaltered, indicating a specific role for BMPR-II in HSC self-renewal. In addition, we map the transcriptional activity of SMAD-mediated signaling in hematopoietic cells by using a BRE-GFP reporter mouse,26 which suggests a failure to engage SMAD-dependent transcriptional response upon BMP exposure. Furthermore, our findings indicate that loss of BMPR-II results in up-regulation of tight junction protein 1 (TJP1) and that knockdown of TJP1 partly rescues the BMPR-II knockout phenotype. TJP1 is a protein previously implicated in self-renewal regulation of several types of stem cells, including both embryonic and adult stem cells. Together, our findings show that BMP signaling, via BMPR-II, is endogenously required by adult HSC to main- tain self-renewal capacity in vivo.
Methods
Mice
Mice on C57Bl/6 background with loxP flanking one allele of exon 4-5 of the BMPR-II gene (MMRRC, University of North Carolina, Chapel Hill, NC, USA)27 were bred to homozygosity and mated with Vav-Cre28 transgenic mice to generate conditional Vav-Cre;BMPR-IIfl/fl mice. Detection of Cre, floxed (fl), wild-type (WT), and excised alleles was done by polymerase chain reaction (PCR) as previously described.22,27 Mice were housed and bred in ventilated cages in the BMC animal facility. All experiments involving animals were approved by the regional Animal Ethical Committee in Lund.
Transplantation assays
For competitive transplantation assays, 0.2x106 unfractionated
BM cells from BMPR-IIfl/fl;Vav-Cre, BMPR-IIfl/+;Vav-Cre, and WT lit- termates (BMPR-IIfl/fl or BMPR-IIfl/+) (CD45.2) were transplanted with 0.2x106 congenic CD45.1 BM cells by tail vein injection to lethally irradiated (900 cGy) congenic CD45.1/2 recipients (three recipients per donor). Donor, competitor, and recipient cells were monitored by peripheral blood (PB) samplings at several time points at 4-16 weeks. Sixteen weeks post-transplantation mice were killed, BM was analyzed and 2x106 cells were transplanted to secondary recipients, monitored as above. After another 16 weeks secondary mice were killed and tertiary transplantations were performed using 20x106 BM cells. Tertiary recipients were monitored as above for 16 weeks, after which final analyses of BM and PB were performed. For transplantations using purified HSC, ten LSK/CD48-/CD150+ cells from BMPR-IIfl/fl;Vav-Cre or WT littermates were transplanted together with 0.2x106 whole BM support cells (CD45.1/2) to CD45.1 recipients. Reconstitution was monitored as above and BM was analyzed at 16 weeks. Homing assays were performed by transplantation of 15x106 unfractionated BM cells to congenic CD45.1 recipients; BM analy- sis was done 20 hours post-transplantation. For competitive hom- ing 10x106 BM cells from donors were transplanted with an equal number of WT competitor cells.
Knockdown of TJP1
FFor knockdown of TJP1, lentiviral plasmid pGFP-C-shLenti
containing short hairpin RNA (shRNA) targeting TJP1 or scram- bled shRNA (OriGene) was used to produce lentiviral particles at the Stem Cell Center Vector Core Facility (Lund University). C-kit-enriched BM cells (CD45.2; BMPR-IIfl/fl;Vav-Cre or WT) were placed into virus-loaded plates at a multiplicity of infection (MOI) of 30-50 and incubated over night (37°C, 5% CO2). Transduced cells were collected and transplanted into lethally irradiated CD45.1 recipient mice (two recipients/donor). An aliquot of cells was cultured for flow cytometry analysis of transduction efficiency after 48 hours. BM of transplanted ani- mals was analyzed at 16 weeks. Additional information can be found in the Online Supplementary Appendix.
Results
BMPR-II is highly expressed in long-term hematopoietic stem cells
In order to map the extent of BMPR-II expression in dis- tinct populations of primitive adult hematopoietic cells, we performed quantitative PCR (qPCR) analyses on sorted long-term HSC (LT-HSC) (LSK-CD34-FLT3-), short-term HSC (ST-HSC) (LSK-CD34+FLT3-), lymphoid-primed mul- tipotent progenitors (LMPP) (LSK-CD34+FLT3+), as well as various progenitor populations.29 Robust expression of BMPR-II was detected in all subsets, although LT-HSC exhibited the highest expression on average between test- ed populations (Figure 1A). Similarly, we examined expression of type-I receptors ALK2, ALK3, and ALK6 in HSC populations (Figure 1B). In LT- and ST-HSC, both ALK2 and ALK3 were expressed, but expression of both receptors was more abundant in LT-HSC. In LMPP, ALK2 was the dominating receptor. ALK6 was undetectable in all hematopoietic subsets tested.
Normal steady state hematopoiesis despite reduced progenitor activity upon deletion of BMPR-II
Given the robust expression of BMPR-II in LT-HSC, we hypothesized that its deletion would blunt most signaling events initiated by BMP in HSC, allowing us to probe the
2204
haematologica | 2021; 106(8)