TGF-β-induced intracellular PAI-1 is responsible for retaining hematopoietic stem cells in the niche

Abstract

Hematopoietic stem and progenitor cells (HSPCs) 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 usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-β-induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-β signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-β-PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSPCs from the TGF-β-expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1-deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.

Graphical abstract

Figure 1.

TGF-β signaling induces PAI-1 expression and controls cell trafficking–related molecules expression in HSPCs. Representative flow cytometric profiles and MFI (n = 6) for TGF-β RI (A), TGF-β RII (B), p-Smad3 (C), and PAI-1 (D) expressions in freshly isolated immature hematopoietic cells. (E) Schema for in vitro experiments. Representative flow cytometric profiles and MFI (n = 6) for PAI-1 (F), MT1-MMP (G and K), CD44 (H), CXCR4 (I), and VLA-4 (J) expressions in WT or PAI-1 KO LSK cells treated with either TGF-β or LY364947 in vitro. Means ± SD are shown in each bar graph. FACS, fluorescence-activated cell sorter; IgG, immunoglobulin G; LSK, Lin⁻c-kit⁺Sca-1⁺; LS⁻K, Lin⁻c-kit⁺Sca-1⁻; LS⁻K⁻, Lin⁻c-kit⁻Sca-1⁻; MFI, mean fluorescence intensity; NS, not significant; RI, receptor I; RII, receptor II.

Figure 2.

PAI-1 regulates mobilization of functional multipotent HSPCs. (A) Schema for mobilization experiments. (B) Representative flow cytometric profiles and MFI (n = 6) for MT1-MMP and CD44 expressions in LSK CD34⁻ cells. (C) Representative flow cytometric profiles of circulating LSK cells. (D-E) Percentages of mobilized LSK cells (D) and the number of colony-forming cells (E) in PB (n = 6). (F) Schema for long-term competitive reconstitution experiments. (G) Percentages of donor cells in PB of primary and secondary recipients at 12 weeks after transplantation. Pooled data (n = 5) of 3 independent experiments are shown. (H) Representative flow cytometric profiles of donor-derived multilineage reconstitution in primary recipients. Means ± SD are shown in each bar graph. CFU, colony-forming unit; MNC, mononuclear cell; NS, not significant; Tx, transplantation.

Figure 3.

PAI-1 regulates HSCs localization in the BM niche. (A) Schema for immunofluorescence analysis. (B-C) Representative pictures of the BM cavity of vehicle- or TM5509-treated mice. BM sections were stained with anti-CD150 (blue), anti-CD41 (green), anti-TGF-β (red), and anti-CD48 and -lineage markers (purple) antibodies. Arrowheads indicate TGF-β–expressing megakaryocytes. Arrows indicate Lin⁻CD48⁻CD150⁺ HSCs. Bars represent 100 μm. (D) Percentages of TGF-β–expressing megakaryocytes in close contact to HSCs. More than 100 cells in random fields on a slide were counted for 3 independent experiments. Means ± SD are shown in each bar graph. Meg, megakaryocyte.

Figure 4.

iPAI-1 determines the activity of MT1-MMP–dependent HSPC migration. Percentages of migrated Lin⁻c-kit⁺ cells in in vitro (A) or in vivo (B) experiments (n = 6 each). rPAI-1, recombinant plasminogen activator inhibitor-1. Means ± SD are shown in each bar graph.

Figure 5.

Intracellular, but not extracellular, PAI-1 regulates the motility of HSPCs. (A) Schema for long-term reciprocal transplantation experiments. (B) Representative flow cytometric profiles and MFI (n = 12) for MT1-MMP expression in LSK CD34⁻ cells of primary recipients. (C-D) Percentages of circulating LSK cells (C) and the number of circulating CFCs (D) in PB samples obtained from the primary recipients of reciprocal transplantation experiments (n = 12). (E-F) Percentages of PKH26⁺ cells (E) and the number of CFCs (F) homed to the BM of secondary recipients (n = 12). (B-F) Means ± SD of 3 independent experiments are shown.

Figure 6.

iPAI-1 modulates MT1-MMP expression through the regulation of Furin. (A) Representative immunofluorescence microscopic images of PAI-1, Furin, and trans-Golgi complex in LSK CD34⁻ cells. (B) D-PLA imaging shows the specific interaction between iPAI-1 and Furin in LSK CD34⁻ cells. As a negative control for D-PLA, slides were treated with either the combination of anti-PAI-1 Ab and mouse IgG or anti-Furin Ab and rabbit IgG followed by the D-PLA secondary Abs. No fluorescent foci were detected by this treatment. (C) The docking simulations between Furin (white) and the active form of PAI-1 (yellow) show the tightly bound PAI-1 covering the active site of Furin, which prevents other substrates from approaching the active site (top). Compare with a structure of Furin (white) with a Furin inhibitor (green) bound at the active site of Furin (bottom). (D) Representative flow cytometric profiles and MFI (n = 6) for Furin expression in LSKCD34⁻ cells isolated from WT or PAI-1 KO mice. (E) Representative flow cytometric profiles and MFI (n = 6) for Furin expression in LSK cells treated in vitro with either TGF-β or LY364947. (F-H) Representative flow cytometric profiles and MFI (n = 6) for MT1-MMP (F and H) and Furin (G) expression in PAI-1-overexpressed (OE), PAI-1-deleted (ΔPAI-1), or PAI-1/Furin double-deleted (ΔPAI-1/ΔFurin) hematopoietic cell lines. (D-H) Means ± SD are shown. (I-J) Relative messenger RNA (I) and representative flow cytometric profile (J) for MT1-MMP expression in LSK cells treated with LY364947 in the presence of actinomycin D in vitro. Bar graphs represent means ± SD (n = 5) of MT1-MMP expression. DAPI, 4',6-diamidino-2-phenylindole.

Figure 7.

TGF-β–induced iPAI-1 activation is responsible for the retention of HSPCs in the BM niche. (A) Schema for in vivo experiments. Representative flow cytometric profiles and MFI (n = 6) for p-Smad3 (B), iPAI-1 (C), MT1-MMP (D), and CD44 (E) expressions in BM LSK CD34⁻ cells isolated from vehicle- or LY364947-treated WT mice. (F-G) Representative flow cytometric profiles and percentages (n = 6) of mobilized LSK cells in PB of vehicle- or LY364947-treated WT (F), stable PAI-1 transgenic mouse (PAI-1 stab. Tg) (G), or PAI-1 KO (H) mice. Means ± SD are shown. (I) A proposed working model schematically representing the main message of this work. In a steady state, TGF-β signaling stays active in niche-residing HSPCs. TGF-β–induced iPAI-1 inhibits Furin-dependent maturation of MT1-MMP, thereby causing HSPCs to remain within the BM niche. Conversely, when the TGF-β–iPAI-1 signal is suppressed, for example, in response to environmental stimuli, Furin-dependent MT1-MMP maturation becomes enhanced. 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 bloodstream. VCAM-1, vascular cell adhesion molecule-1.