G-CSF Signaling Counteracts TGF-β—PAI-1-Dependent Retention of Hematopoietic Stem Cells in the Niche

Hematopoietic stem cells (HSCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but are usually stayed in the niche. Recently, we have reported that niche cell-producing TGF-β induces intracellular PAI-1 expression in HSCs, which inhibits the proteolytic activity of proprotein convertase Furin-dependent maturation of MT1-MMP, a protease involved in the mobilization of HSPCs, thereby keeping HSCs retaining in the BM niche. Pharmacological inhibition or genetic disruption of the TGF-β−PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSCs from the TGF-β-expressing niche cells, demonstrated a crucial role for TGF-β−PAI-1 signal in the retention of HSCs in the BM. On the other hand, HSCs frequently egress from the BM to circulation and replenish blood supply in order to maintain the homeostasis of blood system. At this moment, however, how environmental stimuli, such as a blood G-CSF concentration rise, counteract the TGF-β-PAI-1 signaling and releases the motility restriction of HSCs remains unknown.

To identify the molecular mechanism that govern the dynamic motion of HSCs in the niche, we chose to investigate the cellular activities of HSCs in response to G-CSF. Here, we provide evidence that G-CSF inhibits Smad-dependent signaling by regulating cAMP-PKA-dependent signaling pathway in HSCs. G-CSF-induced activation of this pathway inhibits Smad-dependent expression of PAI-1 through an increase in phosphorylation levels of CREB protein. Mechanistically, increased pCREB competitively inhibited the Smad from binding to the p300/CBP, which ultimately resulting in a transcriptional suppression of Smad-p300/CBP transcription regulatory complex-dependent PAI-1 gene expression. In line with this, administration of G-CSF to mice strongly downregulated intracellular PAI-1 expression in HSCs, which resulted in enhanced Furin-dependent MT1-MMP maturation. The enhanced expression of MT1-MMP results in MT1-MMP-mediated CD44 cleavage as well as CXCR4 and VLA-4 activation, which in turn stimulates detachment of HSPCs from the niche and active trafficking into the blood stream. Expectedly, pharmacological inhibition of intracellular PAI-1 activity synergized with G-CSF-induced HSC mobilization.

Our study reveals a novel role for G-CSF signaling in controlling TGF-β−PAI-1-dependent retention of HSCs to recruit them into the circulation, which potentially improve clinical HSC mobilization and transplantation protocols.