P190-B RhoGAP and intracellular cytokine signals balance hematopoietic stem and progenitor cell self-renewal and differentiation

Ashwini Hinge,Punam Malik,Bruce J Aronow,Eucabeth Mose,Sachin Kumar,Juying Xu,Jose Javier,Reuben Kapur,Marie-Dominique Filippi,Edward F Srour

doi:10.1038/ncomms14382

Abstract

The mechanisms regulating hematopoietic stem and progenitor cell (HSPC) fate choices remain ill-defined. Here, we show that a signalling network of p190-B RhoGAP-ROS-TGF-β-p38MAPK balances HSPC self-renewal and differentiation. Upon transplantation, HSPCs express high amounts of bioactive TGF-β1 protein, which is associated with high levels of p38MAPK activity and loss of HSC self-renewal in vivo. Elevated levels of bioactive TGF-β1 are associated with asymmetric fate choice in vitro in single HSPCs via p38MAPK activity and this is correlated with the asymmetric distribution of activated p38MAPK. In contrast, loss of p190-B, a RhoGTPase inhibitor, normalizes TGF-β levels and p38MAPK activity in HSPCs and is correlated with increased HSC self-renewal in vivo. Loss of p190-B also promotes symmetric retention of multi-lineage capacity in single HSPC myeloid cell cultures, further suggesting a link between p190-B-RhoGAP and non-canonical TGF-β signalling in HSPC differentiation. Thus, intracellular cytokine signalling may serve as ‘fate determinants’ used by HSPCs to modulate their activity.

Highlights

The mechanisms regulating hematopoietic stem and progenitor cell (HSPC) fate choices remain ill-defined
Our findings suggest the existence of a signalling network, p190-B-TGF-b-p38MAPK activity that controls HSPC activity independent of cell proliferation—and may control a fate decision leading to Hematopoietic stem cells (HSCs) accelerated differentiation during division
Numb can be asymmetrically distributed to daughter cells in HSPCs51, HSC functions are preserved in absence of aPKC expression[54], suggesting other pathways contribute to asymmetric divisions

Summary

D18 D21

Known as ‘in vitro paired daughter cell assay’. Under these conditions, single LSK-SLAM can divide symmetrically and produce two daughter cells that have multiple myeloid lineage potential (hereafter nemM daughter cell)[9]. Since exogenous aTGF-b is released in the BM fluid of Tg-Cre þ mice (Supplementary Fig. 8B), making assessment on HSPC functions in vivo complicated by multiple confounding factors including effects from the BM microenvironment and all hematopoietic lineages, LSK-SLAM were isolated 3 weeks after poly-IC injection and used for in vitro experiments. SB203580, a p38MAPK activity inhibitor, completely rescued symmetric division of 2T-WT LSK-SLAM in the in vitro paired daughter cell assay (Fig. 8c) and it prevented effect of rTGF-b1 on 0T LSK-SLAM divisions since single LSK-SLAM treated with rTGF-b1 plus SB203580 generated two nemM daughter cells at a frequency similar to LSK-SLAM treated with vehicle, compared with rTGF-b1 treatment alone (Fig. 8d) This suggests p38MAPK activity mediates TGF-b effects on HSPC differentiation in vitro. P values were calculated by Fisher exact 2 Â 2 contingency table by comparing per cent of symmetric and asymmetric divisions of p38MAPK inhibitor versus DMSO

Discussion

Findings

Methods