Karin Golan

S1P promotes murine progenitor cell egress and mobilization via S1P1-mediated ROS signaling and SDF-1 release

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1(+)/c-Kit(+)/Lin(-) (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P(1), or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit(+)/Lin(-) cells expressing surface S1P(1) in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin(+) mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P(1) axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility.

Enhanced c-Met activity promotes G-CSF–induced mobilization of hematopoietic progenitor cells via ROS signaling

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

Monocytes-macrophages that express α-smooth muscle actin preserve primitive hematopoietic cells in the bone marrow

Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E2 (PGE2) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA+ activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.

CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions

The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact–dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34+ progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP–protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells.

MT1-MMP and RECK are involved in human CD34+ progenitor cell retention, egress, and mobilization

The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1-MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF-mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti-MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP-deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF-induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.

Reactive Oxygen Species Regulate Hematopoietic Stem Cell Self-Renewal, Migration and Development, As Well As Their Bone Marrow Microenvironment

SIGNIFICANCE Blood forming, hematopoietic stem cells (HSCs) mostly reside in the bone marrow in a quiescent, nonmotile state via adhesion interactions with stromal cells and macrophages. Quiescent, proliferating, and differentiating stem cells have different metabolism, and accordingly different amounts of intracellular reactive oxygen species (ROS). Importantly, ROS is not just a byproduct of metabolism, but also plays a role in stem cell state and function. RECENT ADVANCES ROS levels are dynamic and reversibly dictate enhanced cycling and myeloid bias in ROS(high) short-term repopulating stem cells, and ROS(low) quiescent long-term repopulating stem cells. Low levels of ROS, regulated by intrinsic factors such as cell respiration or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity, or extrinsic factors such as stem cell factor or prostaglandin E2 are required for maintaining stem cell self-renewal. High ROS levels, due to stress and inflammation, induce stem cell differentiation and enhanced motility. CRITICAL ISSUES Stem cells need to be protected from high ROS levels to avoid stem cell exhaustion, insufficient host immunity, and leukemic transformation that may occur during chronic inflammation. However, continuous low ROS production will lead to lack of stem cell function and opportunistic infections. Ultimately, balanced ROS levels are crucial for maintaining the small stem cell pool and host immunity, both in homeostasis and during stress situations. FUTURE DIRECTIONS Deciphering the signaling pathway of ROS in HSC will provide a better understanding of ROS roles in switching HSC from quiescence to activation and vice versa, and will also shed light on the possible roles of ROS in leukemia initiation and development.

An MTCH2 pathway repressing mitochondria metabolism regulates haematopoietic stem cell fate

The metabolic state of stem cells is emerging as an important determinant of their fate. In the bone marrow, haematopoietic stem cell (HSC) entry into cycle, triggered by an increase in intracellular reactive oxygen species (ROS), corresponds to a critical metabolic switch from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Here we show that loss of mitochondrial carrier homologue 2 (MTCH2) increases mitochondrial OXPHOS, triggering HSC and progenitor entry into cycle. Elevated OXPHOS is accompanied by an increase in mitochondrial size, increase in ATP and ROS levels, and protection from irradiation-induced apoptosis. In contrast, a phosphorylation-deficient mutant of BID, MTCH2s ligand, induces a similar increase in OXPHOS, but with higher ROS and reduced ATP levels, and is associated with hypersensitivity to irradiation. Thus, our results demonstrate that MTCH2 is a negative regulator of mitochondrial OXPHOS downstream of BID, indispensible in maintaining HSC homeostasis.

PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation related also control retention of endothelial protein C receptor–positive (EPCR+) LT-HSCs in the bone marrow and their recruitment to the blood via two pathways mediated by protease activated receptor 1 (PAR1). Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to EPCR shedding mediated by tumor necrosis factor-α–converting enzyme (TACE), enhanced CXCL12-CXCR4–induced motility and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with activated protein C (aPC) that retains EPCR+ LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing integrin VLA4 affinity and adhesion. Inhibition of NO production by aPC-EPCR-PAR1 signaling reduces progenitor cell egress from the bone marrow, increases retention of bone marrow NOlow EPCR+ LT-HSCs and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR in controlling NO production to balance maintenance and recruitment of bone marrow EPCR+ LT-HSCs, with potential clinical relevance for stem cell transplantation.

B-cell depletion reactivates B lymphopoiesis in the BM and rejuvenates the B lineage in aging

Aging is associated with a decline in B-lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. These changes decrease the bodys ability to mount protective antibody responses. We show here that age-related changes in the B lineage are mediated by the accumulating long-lived B cells. Thus, depletion of B cells in old mice was followed by expansion of multipotent primitive progenitors and common lymphoid progenitors, a revival of B-lymphopoiesis in the bone marrow, and generation of a rejuvenated peripheral compartment that enhanced the animals immune responsiveness to antigenic stimulation. Collectively, our results suggest that immunosenescence in the B-lineage is not irreversible and that depletion of the long-lived B cells in old mice rejuvenates the B-lineage and enhances immune competence.

Physiologic corticosterone oscillations regulate murine hematopoietic stem/progenitor cell proliferation and CXCL12 expression by bone marrow stromal progenitors.

The role of corticosterone (Cort), the immune system’s major stress hormone, in the regulation of hematopoietic stem and progenitor cells (HSPCs) and their dynamic bone marrow (BM) microenvironment is currently unknown. We report that corticotropin-releasing factor receptor 1 (CRFR1) mutant mice with chronically low Cort levels showed aberrant HSPC regulation, having higher HSPC numbers and upregulation of the chemokine CXCL12, phenotypes that were restored by Cort supplementation. Expanded stromal progenitors known to support HSPCs were also observed in these low-Cort-containing mice. A similar phenotype was induced in wild-type (WT) mice by Metyrapone, a Cort synthesis inhibitor. Conversely, high Cort exposure induced HSPC apoptosis, reduced long-term BM repopulation and decreased stromal progenitor cell numbers. We documented circadian oscillations of Cort in WT BM but not in CRFR1 mutant mice, leading to diminished circadian BM CXCL12 fluctuations and increased number of circulating HSPCs in these mice. Finally, low Cort induced expansion of stromal progenitors, CXCL12 expression, HSPC proliferation and BM repopulation capacity, involving Notch1 signaling. This was associated with upregulation of the Notch ligand, Jagged1, in BM myeloid cells. Our results suggest that daily physiologic Cort oscillations are critical for balanced HSPC proliferation and function involving Notch1 signaling and their supportive BM microenvironment.