Down-Regulation of CXXC5 De-Represses MYCL1 to Promote Hepatic Stellate Cell Activation

Liver fibrosis is mediated by myofibroblasts, a specialized cell type involved in wound healing and extracellular matrix production. Hepatic stellate cells (HSC) are the major source of myofibroblasts in the fibrotic livers. In the present study we investigated the involvement of CXXC-type zinc-finger protein 5 (CXXC5) in HSC activation and the underlying mechanism. Down-regulation of CXXC5 was observed in activated HSCs compared to quiescent HSCs both in vivo and in vitro. In accordance, over-expression of CXXC5 suppressed HSC activation. RNA-seq analysis revealed that CXXC5 influenced multiple signaling pathways to regulate HSC activation. The proto-oncogene MYCL1 was identified as a novel target for CXXC5. CXXC5 bound to the proximal MYCL1 promoter to repress MYCL1 transcription in quiescent HSCs. Loss of CXXC5 expression during HSC activation led to the removal of CpG methylation and acquisition of acetylated histone H3K9/H3K27 on the MYCL1 promoter resulting in MYCL1 trans-activation. Finally, MYCL1 knockdown attenuated HSC activation whereas MYCL1 over-expression partially relieved the blockade of HSC activation by CXXC5. In conclusion, our data unveil a novel transcriptional mechanism contributing to HSC activation and liver fibrosis.

Differential H4K16ac levels ensure a balance between quiescence and activation in hematopoietic stem cells

Hematopoietic stem cells (HSCs) are able to reconstitute the bone marrow while retaining their self-renewal property. Individual HSCs demonstrate heterogeneity in their repopulating capacities. Here, we found that the levels of the histone acetyltransferase MOF (males absent on the first) and its target modification histone H4 lysine 16 acetylation are heterogeneous among HSCs and influence their proliferation capacities. The increased proliferative capacities of MOF-depleted cells are linked to their expression of CD93. The CD93+ HSC subpopulation simultaneously shows transcriptional features of quiescent HSCs and functional features of active HSCs. CD93+ HSCs were expanded and exhibited an enhanced proliferative advantage in Mof+/− animals reminiscent of a premalignant state. Accordingly, low MOF and high CD93 levels correlate with poor survival and increased proliferation capacity in leukemia. Collectively, our study indicates H4K16ac as an important determinant for HSC heterogeneity, which is linked to the onset of monocytic disorders.

The Space of Disse: The Liver Hub in Health and Disease

Since it was first described by the German anatomist and histologist, Joseph Hugo Vincenz Disse, the structure and functions of the space of Disse, a thin perisinusoidal area between the endothelial cells and hepatocytes filled with blood plasma, have acquired great importance in liver disease. The space of Disse is home for the hepatic stellate cells (HSCs), the major fibrogenic players in the liver. Quiescent HSCs (qHSCs) store vitamin A, and upon activation they lose their retinol reservoir and become activated. Activated HSCs (aHSCs) are responsible for secretion of extracellular matrix (ECM) into the space of Disse. This early event in hepatic injury is accompanied by loss of the pores—known as fenestrations—of the endothelial cells, triggering loss of balance between the blood flow and the hepatocyte, and underlies the link between fibrosis and organ dysfunction. If the imbalance persists, the expansion of the fibrotic scar followed by the vascularized septae leads to cirrhosis and/or end-stage hepatocellular carcinoma (HCC). Thus, researchers have been focused on finding therapeutic targets that reduce fibrosis. The space of Disse provides the perfect microenvironment for the stem cells niche in the liver and the interchange of nutrients between cells. In the present review article, we focused on the space of Disse, its components and its leading role in liver disease development.

Altered microRNA expression links IL6 and TNF-induced inflammaging with myeloid malignancy in humans and mice

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Activated but Not Quiescent Hematopoietic Stem Cells Rely Readily on Glycolysis As Their Main Source of Energy

Elucidating mechanisms that regulate hematopoietic stem cells (HSCs) quiescence is critical for improving bone marrow transplantation. It is postulated that quiescent HSCs rely mostly on glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) as their energy source. We have identified a population of HSCs with low mitochondrial activity (LSKCD150+CD48- within the 25% lowest mitochondrial membrane potential; MMP-low) that we show are highly enriched in quiescent HSCs (~95% ±2.65 in G0) as measured by pyronin Y/Hoechst staining in contrast to LSKCD150+CD48- within 25% highest MMP (MMP-high) that are in majority in G1/S/G2/M phase of cell cycle (55.4%±16; p<0.05; n=3). MMP-low HSCs exhibit greater in vivo competitive repopulation ability (3.7 fold; p=0.021; n=10 mice) at 16 weeks as compared to MMP-high fractions, show higher self renewal ability and are enriched in label-retaining H2B-GFP+ cells (~3 fold; p<0.01; n=3). Conversely, label-retaining GFP+HSCs maintain lower MMP than non-label-retaining cells. Altogether these results reinforce the notion that MMP-low HSCs are quiescent whereas MMP-high HSCs are primed/activated. Using single cell RNA-sequencing (scRNA-seq) analysis to interrogate the transcriptome by a Fluidigm C1 platform within MMP-high versus MMP-low HSCs we found major metabolic pathways including OXPHOS, exhibit significantly greater expression within the MMP-high than in the MMP-low HSC fraction. Seahorse analysis confirmed that MMP-high but not -low hematopoietic stem and progenitor cells (HSPCs) use OXPHOS as their source of energy (3.9 fold; n=16 mice). Unexpectedly, glycolytic gene expression was also enriched in the primed MMP-high HSCs and relatively low in quiescent MMP-low HSCs. qRT-PCR analysis further confirmed that the expression of glycolytic enzymes and other genes including glucose transporter 1 (Glut1, slc2a1) that is the main glucose transporter on HSCs is greater in MMP-high relative to -low HSCs. These unforeseen findings raised the potential that despite the current consensus in HSC biology, glycolysis may more readily support activated rather than quiescent HSCs. We thus measured the glucose uptake in MMP-low vs -high HSCs under metabolic (pyruvate, glucose and glutamine)-restricted conditions. Using 2NBDG, a fluorescently tagged glucose analog, we found that MMP-high HSCs uptake 3.3-fold more glucose and contained three times more 2NBDG+ cells as compared to MMP-low HSCs (p<0.001 for each; n=3). Pharmacological inhibition of Glut1 reduced glucose uptake in MMP-high but not -low HSCs suggesting the specific sensitivity of MMP-high HSCs to the glucose inhibition. In addition, activation of tricarboxylic acid cycle TCA cycle with combined methyl-pyruvate and dimethyl-alpha-ketoglutarate led to a greater glucose uptake (~3.5 fold) in MMP-high as compared to MMP low HSCs (~2.2 fold; p<0.0001). To address the degree to which glycolysis is necessary, FACS-purified MMP-low and -high HSCs were incubated with 2-Deoxy D-Glucose (2DG) - a glucose analog - that inhibits glycolysis via its action on hexokinase. While glucose deprivation with 50 mM dose of 2DG within 12 hours did not have much effect on MMP-low HSCs, over 60% of MMP-high HSCs died (p<0.001; n=3), suggesting that MMP-high but not -low HSCs rely readily on glycolysis for their survival. Also, the inhibition of mitochondrial transport of pyruvate that is the end product of glycolysis (α-cyano-4-hydroxycinnamate (CHC)), decreased survival in MMP-high HSCs by 80% with negligible effect on MMP-low HSCs (p<0.01; n=3). Finally 50 FACS-purified HSCs (LSKCD150+CD48-MMP-low or -high) were transplanted in lethally irradiated mice along with 200,000 unfractionated bone marrow cells in a competitive repopulation assay and mice were subsequently treated with 2DG or control vehicle every other day for two weeks. Two months later, 2DG treatment led to significantly enhanced reconstitution levels in MMP-high HSCs with no or little effect on MMP low reconstitution levels (ongoing). These combined results suggest that primed MMP-high rather than quiescent MMP-low HSCs rely on glycolysis as their main source of energy. These findings are consistent with the concept that glycolysis is key in sustaining rapidly dividing cells such as embryonic stem cells and cancer cells. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

Purine Metabolism and Hematopoietic Stem and Progenitor Stem Cells Under Stress

Hematopoietic stem cells (HSCs) play a key role in the lifelong maintenance of hematopoiesis through self-renewal and multi-lineage differentiation. Adult HSCs reside within a specialized microenvironment of the bone marrow (BM), called "niche", in which they are maintained in a quiescent state in cell cycle. Most of HSCs within BM show quiescence under the hypoxic niche. Since the loss of HSC quiescence leads to the exhaustion or aging of stem cells through excess cell division, the regulation of quiescence in HSCs is essential for hematopoietic homeostasis. On the other hand, cellular metabolism has been suggested to play a critical role in many biological processes including the regulation of stem cell properties and functions. However, the metabolic condition and adaptation of stem cells remain largely unaddressed. First, we have analyzed HSC metabolism using metabolomics approaches. With step-wise differentiation of stem cells, the cell metabolism associated with each differentiation stage may be different. A feature of quiescent HSCs is their low baseline energy production; quiescent HSCs rely on glycolysis and exhibit low mitochondrial membrane potential (ΔΨm). Likewise, HSCs with a low ΔΨm show higher reconstitution activity in BM hematopoiesis, compared to cells with high ΔΨm. By contrast, upon stress hematopoiesis, HSCs actively divide and proliferate. However, the underlying mechanism for the initiation of HSC division still remains unclear. In order to elucidate the mechanism underlying the transition of cell cycle state in HSCs, we analyzed the change of mitochondria activity in HSCs after BM suppression induced by 5-fluoruracil (5-FU). Upon 5-FU treatment, cycling progenitors are depleted and then quiescent HSCs start to divide. We found that HSCs initiate cell division after exhibiting enhanced ΔΨm, as a result of increased intracellular Ca2+ level. We hypothesize that extracellular adenosine, derived from hematopoietic progenitors, inhibits the calcium influx and mitochondrial metabolism. While further activation of Ca2+-mitochondria pathway led to loss of the stem cell function after cell division, the appropriate suppression of intracellular Ca2+ level by nifedipine, a blocker of L-type voltage-gated Ca2+ channels, prolonged cell division interval in HSCs, and simultaneously achieved both cell division and HSC maintenance (self-renewal division). Thus, our results indicate that the adenosine-Ca2+-mitochondria pathway induces HSC division critically to determine HSC cell fate. Next, to examine the mitochondria oxidative metabolism and purinergic pathways, we introduced the study on a tumor suppressor, Folliculin (FLCN). Conditional deletion of FLCN in HSC compartment using the Mx1-Cre or Vav-iCre system disrupted HSC quiescence and BM homeostasis dependently on the lysosomal stress response induced by TFE3. Together all, we propose that the change in cellular metabolism involving mitochondria is crucial for HSC homeostasis in the stress settings. Disclosures No relevant conflicts of interest to declare.

CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells

Key Points Chemokine ligands CXCL1-4, 6, 10, 11, and 13 are upregulated in human quiescent HSCs with CXCR2 and CXCL4 regulating their survival. Genetic ablation of Cxcr2 or Cxcl4 in murine models induces initial expansion but eventual exhaustion of HSC in transplantation assays.

Interferon Alpha Mediated Activation of the Bone Marrow Stem Cell Vascular Niche

Endothelial cells (ECs) significantly influence the response of an organism to inflammation and infection. In the bone marrow, these cells form a major part of the bone marrow vascular niche, which regulates stem cell function and influences stem cell fate. The primary response to infection involves synthesis of immune-modulatory cytokines, such as interferon alpha (IFNα). We, and others, have shown that in contrast to the anti-proliferative effect of IFNα on hematopoietic stem cells (HSCs) in vitro, in vivo, IFNα induces cell cycle entry of quiescent HSCs (Essers et al. 2009). Given the contrasting outcome of in vitro and in vivo exposure of HSCs to IFNα, it is probable that niche cells and molecular maintenance signals from the niche are required for IFNα-induced activation of quiescent HSCs. Here, we now show that although interferon signaling itself in niche cells is not required for HSC activation, niche components do respond to IFNα stimulation. Of these, bone marrow ECs are rapidly and indirectly stimulated following IFNα treatment in vivo. The vascular system, lined by ECs, is a central primary responder following inflammatory insult with a multifaceted role, including transport of immune cells and promotion of a rapid return to homeostasis. We have found that IFNα stimulation in vivo results in an increased bone marrow vascularity, visualized by fluorescence imaging of cryo-sectioned murine femurs immunostained with the vascular basement membrane protein, laminin. IFNα-mediated activation of ECs involves the expression of key inflammatory and endothelial-stimulatory markers, including VE-cadherin and ESAM, and also an increased vascular leakiness in the bone marrow, demonstrated by the Evans blue assay. In accordance with this finding of vascular activation, we confirmed that VEGF, which is an established regulator of vascular dynamics, is rapidly up regulated in the bone marrow supernatant of treated mice. Furthermore, we can also demonstrate a key role for VEGF in our observed IFNα-mediated stimulation of ECs by abrogation of this activation using co-treatment with Avastin (bevacizumab) in vivo. We are now investigating the feedback from this activation of ECs on the hematopoietic system itself. In summary, these data indicate a rapid and indirect stimulation of the bone marrow vascular niche in an inflammatory setting. In addition, they support a previously undescribed cellular instruction from an activated hematopoietic system to a niche component. Disclosures No relevant conflicts of interest to declare.

A Stress-Responsive Transcriptional Factor NRF2 Activates Hematopoietic Stem Cells

KEAP1-NRF2 system is a major regulator of cellular redox balance and xenobiotic metabolism. NRF2 is an inducible transcription factor, and KEAP1 is its negative regulator. Under normal conditions, NRF2 is poly-ubiquitinated by KEAP1-CUL3 complex and is degraded through proteasome. KEAP1 is a stress sensor and is inactivated by oxidative stress and metabolites of xenobiotics, which results in relief of NRF2 from the KEAP1-mediated suppression and thereby induces cytoprotective genes. In addition to this cytoprotective function of KEAP1-NRF2 system, emerging evidence suggests that their roles extend to cell proliferation and differentiation. Hematopoietic stem cells (HSCs) are maintained quiescent under normal conditions, and are stimulated to proliferate and differentiate in response to environmental alterations. However, the underlying mechanisms of driving HSCs from quiescence into proliferation are still elusive. Here, we investigated whether the KEAP1-NRF2 system contributes to the stress-responsive proliferation of HSCs. In this study, we analyzed long-term HSCs (LT-HSCs), defined as Lin- Sca-1+ c-Kit+ (LSK) CD48- CD150+ cells, which possess high bone marrow (BM) reconstitution capacity. Short-term HSCs (ST-HSCs) and multipotent progenitor (MPP) cells were also defined as LSK CD48- CD150- and LSK CD48+ CD150- cells, respectively. First, we examined Keap1 conditional knockout mice, Keap1F/F::Mx1-Cre (Keap1 CKO1), and compared to control mice, Keap1F/+ or Keap1F/+::Mx1-Cre. In the Keap1 CKO1 mice, ST-HSCs and MPPs were increased, whereas LT-HSCs were not changed in comparison to control mice. However, Keap1-deficient LT-HSCs exhibited less engraftment and reconstitution in the competitive bone marrow transplantation (BMT) assay, in which donor-derived LT-HSCs were transplanted with BM competitor cells (Fig. 1). In particular, the Keap1-deficient LT-HSCs were almost completely lost after secondary BMT. Importantly, the attenuated reconstitution capacity of LT-HSCs in the absence of Keap1 was clearly recovered by the additional Nrf2 deletion (Fig. 1), which indicates that constitutive activation of NRF2 causes dysfunction of LT-HSCs. To elucidate the cause of the functional impairment in Keap1-deficient LT-HSCs, we examined homing potentials and apoptosis. However, there were no significant differences in either of the parameters. Next, we evaluated cell cycle status of LT-HSCs and found that Keap1-deficient LT-HSCs contained less quiescent cells, compared to control cells (G0 phase; Control = 63.6 ± 3.5%, Keap1 CKO1 = 55.2 ± 4.0%). This implies that Nrf2 activation enhances cell cycle entry of quiescent HSCs, which attenuates the stem cell activity. All these results observed in Keap1 CKO mice were reproduced in another Keap1 conditional knockout mice, Keap1F/F::Vav1-Cre (Keap1 CKO2). Of note, the reduction of quiescent LT-HSCs was nicely recovered by the additional Nrf2 deletion under the Keap1 CKO2 background (Fig. 2, G0 phase; Control2 = 55.1 ± 5.9%, Keap1 CKO2 = 43.0 ± 3.8%, Keap1 CKO2::Nrf2-/- = 65.3 ± 3.7%). These results show that NRF2 activation drives quiescent HSCs into cell cycling. Since NRF2 exerts its activity in response to exogenous stimuli, we assessed whether the NRF2-mediated proliferation of LT-HSCs are induced by transient treatment with an NRF2 inducer, CDDO-Im. CDDO-Im treatment promoted cell cycling of LT-HSCs, which was ablated by Nrf2 deficiency, suggesting that NRF2 activation, even transiently, leads to cell cycle entry of quiescent HSCs. Finally, in order to elucidate contribution of NRF2 to LT-HSCs under steady-state conditions, we assessed Nrf2-deficient (Nrf2-/-) mice. No differences were observed in the BM of Nrf2-/- mice, but after the competitive BMT of LT-HSCs, Nrf2-deficient LT-HSCs exhibited less contribution to the BM reconstitution in the recipients. However, it is noteworthy that the rate of engrafted donor cells tends to be higher in the recipients transplanted with Nrf2-deficient LT-HSCs than in the recipients transplanted with wild-type cells after the secondary BMT, implying that NRF2, at least in part, contributes to cell cycle entry of LT-HSCs under steady-state conditions and thereby Nrf2 deficiency prevents LT-HSCs from proliferation-induced exhaustion. These results show that KEAP1-NRF2 system plays an important role in the stress-responsive proliferation and differentiation of quiescent HSCs. Disclosures No relevant conflicts of interest to declare.

Kindlin-3–mediated integrin adhesion is dispensable for quiescent but essential for activated hematopoietic stem cells

Hematopoietic stem cells (HSCs) generate highly dividing hematopoietic progenitor cells (HPCs), which produce all blood cell lineages. HSCs are usually quiescent, retained by integrins in specific niches, and become activated when the pools of HPCs decrease. We report that Kindlin-3–mediated integrin activation controls homing of HSCs to the bone marrow (BM) and the retention of activated HSCs and HPCs but not of quiescent HSCs in their BM niches. Consequently, Kindlin-3–deficient HSCs enter quiescence and remain in the BM when cotransplanted with wild-type hematopoietic stem and progenitor cells (HSPCs), whereas they are hyperactivated and lost in the circulation when wild-type HSPCs are absent, leading to their exhaustion and reduced survival of recipients. The accumulation of HSPCs in the circulation of leukocyte adhesion deficiency type III patients, who lack Kindlin-3, underlines the conserved functions of Kindlin-3 in man and the importance of our findings for human disease.