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BMPR2 regulates the self-renewal of adult HSCs
the ability of the SMAD pathway to engage transcription- ally in response to BMP stimulation and that BMP prefer- entially signal through non-SMAD circuitries in hematopoietic cells. The BRE-reporter study is in agree- ment with the lack of hematopoietic phenotype seen upon deletion of SMAD1/SMAD5.
The p38 signaling pathway is an alternative signaling circuitry implicated downstream BMP receptors. Khurana et al. showed that p38 is phosphorylated in both human and mouse HSPC cultured in the presence of BMP4 in vitro.20 In agreement with this, we observed a reduction in phosphorylation of p38 in hematopoietic progenitor cells lacking BMPR-II. However, we could not detect a robust induction of phosphorylation in response to BMP4 in WT progenitor cells. This may be due to the length of BMP stimulation, as Khurana et al. measured p38 signaling following 5 days of continuous BMP4 exposure. We assayed p38 after 30 minutes of BMP4 stimulation, a time point to measure direct activation. Reduced phosphorylation of p38 is therefore in agree- ment with a more long-term loss of BMPR-II, and may thus be due to secondary effects.
Interestingly, we observed a significant increase in expression of TJP1 in purified BMPR-II-/- LT-HSC. TJP1 has previously been linked to regulation of self-renewal in embryonic stem cells where loss of TJP1 results in increased self-renewal.37 Expression of TJP1 is shared between HSC, ES cells, and neural stem cells, indicative of a universal role for TJP1 in self-renewal of stem cells.38 Additionally, TJP1 is downregulated in a multipotent hematopoietic cell line upon differentiation.39 Taken together, these data substantiate the link between TJP1 and HSC self-renewal. Contrary to what is seen in hematopoietic cells in vitro,39 our data suggests that loss of BMPR-II leads to disruption of HSC self-renewal via excessive expression of TJP1, which is in line with previ- ous findings in ES cells.37 It is possible that fine-tuned HSC regulation in vivo requires very specific levels of TJP1. Our findings further show that knockdown of TJP1 partly rescues the BMPR-II null phenotype. Following transplantation of BMPR-II-/- cells with TJP1 knockdown, we observed an increase in cell contribution to the donor LSK compartment. A similar trend was seen in the HSC compartment, but not in more differentiated populations. Our data suggests that the up-regulation of TJP1 is, at
least in part, one of the key mechanisms behind the observed BMPR-II-/- hematopoietic phenotype. Complete reversal of the phenotype may not have been achieved due to incomplete knockdown or that in addition to TJP1 there could be other mechanisms playing a part in gener- ating the phenotype.
In order to increase the therapeutic applicability of HSC, more detailed information is required regarding mecha- nisms controlling fate options such as self-renewal. In human hematopoiesis BMP have been shown to have an important role in adhesion to stroma, differentiation potential and ex vivo maintenance.19,20,36 Here, we identify BMPR-II and TJP1 as important players regulating murine LT-HSC self-renewal in vivo. In light of our findings, further work should focus on investigating the role for BMPR-II and in particular TJP1 in human HSC self-renewal.
Disclosure
No conflicts of interest to disclose.
Contributions
SW, UB, and SK designed experiments; SW, UB, MD, THMG, LS, and SA performed experiments; SW and UB analysed data. SW, UB, and SK wrote the paper; SK super- vised the study.
Acknowledgements
The authors would like to thank Leif Oxburgh from the Maine Medical Center for support in initial BRE-LacZ analy- ses, Elaine Dzierzak from the University of Edinburgh for pro- viding BRE-GFP reporter mice, Shamit Soneji and Stefan Lang at the Stem Cell Center Bioinformatics Core Facility at Lund University for input on the microarray and advice on statistical analyses, and Göran Karlsson at Lund University for continuous feedback on the project and manuscript.
Funding
This work was supported by funds from the European Commission (Stemexpand); Hemato-Linné and Stemtherapy program project grants from the Swedish Research Council; a project grant to SK from the Swedish Research Council; the Swedish Cancer Society; the Swedish Childhood Cancer Fund; a Clinical Research Award from Lund University Hospital; and a grant to SK from The Tobias Foundation awarded by the Royal Academy of Sciences.
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