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S. Warsi et al.
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Figure 6. TJP1 knockdown in BMPR-II deficient cells increases primitive cell engraftment. Engraftment of green fluorescent protein-positive (GFP+) cells percent in bone marrow following transplantation of transduced cells in (A) CD45.2+ (donor) GFP+ LSK population, (B) CD45.2+ GFP+ HSC (LSKCD48-CD150+) population, (C) CD45.2+ GFP+ Lin- population, and (D) CD45.2+ GFP+ population, analyzed at 16 weeks post transplant (n=8-9). In data set (A) and (B) outliers (one and two respec- tively) were detected using Grubb’s test (α=0.01) and removed; this did not alter outcomes of statistical analyses. *P<0.05.
Here we aimed to elucidate the endogenous role of BMP signaling in adult murine HSC, by conditional dele- tion of BMPR-II specifically in hematopoietic cells. Unlike deletion of SMAD1 and SMAD5, we report here that BMPR-II is essential for self-renewal of adult HSC. It is likely that this non-SMAD signal in HSC is mediated by BMPR-II associated with the BMP type-I receptor ALK2 or possibly ALK3, based on our transcriptional profiling of receptor expression in WT LT-HSC and that we find no significant change in expression levels of other BMP receptors in primitive hematopoietic cells from BMPR-II-/- mice. Additionally, there is limited knowledge about BMPR-II being able to activate downstream signaling pathways independently of type-I receptors. Despite the absence of significant differences in our measurements of above mentioned transcript levels, a trend of increased Alk3 seemed to be observed following BMPR-II deletion. This will require further studies to fully decipher the rela- tion between BMP receptors and their cross-regulation, and to understand their relative function in the context of HSC regulation. Though it is possible that cross-talk and feedback regulation occurs within the BMP signaling pathway, BMPR-II deletion does not seem to have a reg- ulatory effect at transcript level on other BMP family receptors in HSC.
In this study we show that BMPR-II deficient HSC fail
to efficiently generate additional HSC upon transplanta- tion, thus causing a significant reduction in hematopoietic regeneration following serial BM transplantation. Loss of BMPR-II did not affect homing capacity of HSC to the BM, suggesting that the reduced regenerative capacity observed upon transplantation derives from compro- mised self-renewal ability of LT-HSC. During steady state hematopoiesis, BMPR-II-/- mice display essentially normal hematopoietic parameters, lending further evidence to a specific role for BMPR-II in self-renewal of LT-HSC. Furthermore, as cell cycle distribution among LT-HSC is more or less unaffected by loss of BMPR-II and the hematopoietic system recovers almost normally follow- ing stress, our data suggest that a possible effect on cell cycle progression plays only a small part in HSC regula- tion by BMP. Instead, LT-HSC deficient of BMPR-II fail to maintain stemness during conditions when self-renewal divisions are required. This is in agreement with previous data, which shows that BMP stimulation does not affect proliferation of HSC in vitro.23
By investigating the transcriptional activity of the SMAD pathway, our data reveals that a majority of hematopoietic cells fail to respond transcriptionally to BMP and thus do not employ SMAD-dependent tran- scriptional response, despite phosphorylation of SMAD. We hypothesize that other regulatory mechanisms limit
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haematologica | 2021; 106(8)