We established an in vitro cell culture system to determine novel activities of the retinoblastoma (Rb) protein during tumor progression. Rb depletion in p53-null mouse-derived soft tissue sarcoma cells induced a spherogenic phenotype. Cells retrieved from Rb-depleted spheres exhibited slower proliferation and less efficient BrdU incorporation, however, much higher spherogenic activity and aggressive behavior. We discovered six miRNAs, including mmu-miR-18a, -25, -29b, -140, -337, and -1839, whose expression levels correlated tightly with the Rb status and spherogenic activity. Among these, mmu-miR-140 appeared to be positively controlled by Rb and to antagonize the effect of Rb depletion on spherogenesis and tumorigenesis. Furthermore, among genes potentially targeted by mmu-miR-140, Il-6 was upregulated by Rb depletion and downregulated by mmu-mir-140 overexpression. Altogether, we demonstrate the possibility that mmu-mir-140 mediates the Rb function to downregulate Il-6 by targeting its 3'-untranslated region. Finally, we detected the same relationship among RB, hsa-miR-140 and IL-6 in a human breast cancer cell line MCF-7. Because IL-6 is a critical modulator of malignant features of cancer cells and the RB pathway is impaired in the majority of cancers, hsa-miR-140 might be a promising therapeutic tool that disrupts linkage between tumor suppressor inactivation and pro-inflammatory cytokine response.

Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.

Fibroblasts are critical for wound contraction; a pivotal step in wound healing. They produce and modify the extracellular matrix (ECM) required for the proper tissue remodeling. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a key regulator of ECM homeostasis and turnover. However, its role in wound contraction is presently unknown. Here we describe that Transforming growth factor type β1 (TGF-β1), one of the main pro-fibrotic wound-healing promoting factors, decreases RECK expression in fibroblasts through the Smad and JNK dependent pathways. This TGF-β1 dependent downregulation of RECK occurs with the concomitant increase of β1-integrin, which is required for fibroblasts adhesion and wound contraction through the activation of focal adhesion kinase (FAK). Loss and gain RECK expression experiments performed in different types of fibroblasts indicate that RECK downregulation mediates TGF-β1 dependent β1-integrin expression. Also, reduced levels of RECK potentiate TGF-β1 effects over fibroblasts FAK-dependent contraction, without affecting its cognate signaling. The above results were confirmed on fibroblasts derived from the Reck+/- mice compared to wild type-derived fibroblasts. We observed that Reck+/- mice heal dermal wounds more efficiently than wild type mice. Our results reveal a critical role for RECK in skin wound contraction as a key mediator in the axis: TGF-β1-RECK-β1-integrin.

Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow-derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin-administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline-administered wild-type mice. Interestingly, in bleomycin-administered S1pr2-/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2+/+ mice alleviated bleomycin-induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis.

The muscle regeneration process requires a properly assembled extracellular matrix (ECM). Its homeostasis depends on the activity of different matrix-metalloproteinases (MMPs). The reversion-inducing-cysteine-rich protein with kazal motifs (RECK) is a membrane-anchored protein that negatively regulates the activity of different MMPs. However, the role of RECK in the process of skeletal muscle differentiation, regeneration, and fibrosis has not been elucidated. Here, we show that during skeletal muscle differentiation of C2C12 myoblasts and in satellite cells on isolated muscle fibers, RECK is transiently up regulated. C2C12 myoblasts with reduced RECK levels are more prone to enter the differentiation program, showing an accelerated differentiation process. Notch-1 signaling was reduced, while p38 and AKT signaling were augmented in myoblasts with decreased RECK levels. Overexpression of RECK restores the normal differentiation process but diminished the ability to form myotubes. Transient up-regulation of RECK occurs during skeletal muscle regeneration, which was accelerated in RECK-deficient mice (Reck±). RECK, MMPs and ECM proteins augmented in chronically damaged WT muscle, a model of muscle fibrosis. In this model, RECK ± mice showed diminished fibrosis compared to WT. These results strongly suggest that RECK is acting as a potential myogenic repressor during muscle formation and regeneration, emerging as a new player in these processes, and as a potential target to treat individuals with the muscle-wasting disease.

Autophagy is a cellular degradation system contributing to homeostasis of tissue stem cells including haematopoietic stem cells (HSCs). It plays pleiotropic roles in HSC characteristics throughout life, but its stage-specific roles in HSC self-renewal are unclear. To investigate the effects of Atg5 deletion on stage-specific HSC functions, we compared the repopulating capacity of HSCs in Atg5f/f;Vavi-cre mice from postnatal day (P) 0-7 weeks of age. Interestingly, Atg5 deficiency led to no remarkable abnormality in the HSC self-renewal capacity at P0, but significant defects at P7, followed by severe defects. Induction of Atg5 deletion at P5 by tamoxifen administration to Atg5f/f;Rosa26-Cre-ERT2 mice resulted in normal haematopoiesis, including the HSC population, until around 1 year, suggesting that Atg5 in the early neonatal period was critical for haematopoiesis in adults. Mitochondrial oxidative stress was increased by Atg5 loss in neonatal HSC/progenitor cells. Although p62 had accumulated in immature bone marrow cells of Atg5f/f;Vavi-cre mice, p62 deletion did not restore defective HSC functions, indicating that Atg5-dependent haematopoietic regulation in the developmental period was independent of p62. This study proposes a critical role of autophagy in HSC protection against harsh environments in the early neonatal stage, which is essential for healthy long-term haematopoiesis.

We previously isolated derrisfolin A, a novel rotenoid derivative, from the stems of Derris trifoliata Lour. (Leguminosae). Here, we report that derrisfolin A induces the expression of endogenous regucalcin (RGN) protein in both pancreatic MIN6 β-cells and RAW264.7 macrophages. Induction of RGN expression by derrisfolin A or retrovirus-mediated gene transfer in MIN6 cells and RAW264.7 macrophages significantly decreased lipopolysaccharide (LPS)-induced mRNA expression of Nos2, Il1b, and Tnf via nuclear factor-κB activation; reduced LPS-induced apoptosis in MIN6 cells, accompanied by decreased production of nitric oxide, interleukin-1β, and tumor necrosis factor-α; and attenuated generation of LPS-induced reactive oxygen species, malondialdehyde, and 3-nitrotyrosine in MIN6 cells. Additionally, in co-cultures of MIN6 cells with RAW264.7 macrophages in the presence of LPS, induction of RGN expression by derrisfolin A or retrovirus-mediated gene transfer in RAW264.7 macrophages attenuated apoptosis and oxidative/nitrosative stress in MIN6 cells. These results suggest that the induction of RGN expression in MIN6 cells was effective in suppressing LPS-induced inflammatory cytotoxicity and that in co-culture conditions, the induction of RGN expression in RAW264.7 macrophages blocked LPS-induced paracrine effects of RAW264.7 macrophages on inflammatory cytotoxicity in MIN6 cells. Our findings suggest that derrisfolin A, a chemical inducer of RGN, might be useful for developing a new drug against macrophage-associated β-cell inflammation in type 2 diabetes.

Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)-the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the β-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinib-sensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.

Acute myeloid leukaemia (AML) is a heterogeneous neoplastic disorder in which a subset of cells function as leukaemia-initiating cells (LICs). In this study, we prospectively evaluated the leukaemia-initiating capacity of AML cells fractionated according to the expression of a nucleolar GTP binding protein, nucleostemin (NS). To monitor NS expression in living AML cells, we generated a mouse AML model in which green fluorescent protein (GFP) is expressed under the control of a region of the NS promoter (NS-GFP). In AML cells, NS-GFP levels were correlated with endogenous NS mRNA. AML cells with the highest expression of NS-GFP were very immature blast-like cells, efficiently formed leukaemia colonies in vitro, and exhibited the highest leukaemia-initiating capacity in vivo. Gene expression profiling analysis revealed that cell cycle regulators and nucleotide metabolism-related genes were highly enriched in a gene set associated with leukaemia-initiating capacity that we termed the 'leukaemia stem cell gene signature'. This gene signature stratified human AML patients into distinct clusters that reflected prognosis, demonstrating that the mouse leukaemia stem cell gene signature is significantly associated with the malignant properties of human AML. Further analyses of gene regulation in leukaemia stem cells could provide novel insights into diagnostic and therapeutic approaches to AML.

Sustained induction and activation of matrixins (matrix metalloproteinases or MMPs), and the destruction and deposition of extracellular matrix (ECM), are the hallmarks of cardiac fibrosis. The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a unique membrane-anchored endogenous MMP regulator. We hypothesized that elevated angiotensin II (Ang II), which is associated with fibrosis in the heart, differentially regulates MMPs and RECK both in vivo and in vitro. Continuous infusion of Ang II into male C57Bl/6 mice for 2weeks resulted in cardiac fibrosis, with increased expressions of MMPs 2, 7, 9 and 14, and of collagens Ia1 and IIIa1. The expression of RECK, however, was markedly suppressed. These effects were inhibited by co-treatment with the Ang II type 1 receptor (AT1) antagonist losartan. In vitro, Ang II suppressed RECK expression in adult mouse cardiac fibroblasts (CF) via AT1/Nox4-dependent ERK/Sp1 activation, but induced MMPs 2, 14 and 9 via NF-κB, AP-1 and/or Sp1 activation. Further, while forced expression of RECK inhibits, its knockdown potentiates Ang II-induced CF migration. Notably, RECK overexpression reduced Ang II-induced MMPs 2, 9 and 14 activation, but enhanced collagens Ia1 and IIIa1 expression and soluble collagen release. These results demonstrate for the first time that Ang II suppresses RECK, but induces MMPs both in vivo and in vitro, and RECK overexpression blunts Ang II-induced MMP activation and CF migration in vitro. Strategies that upregulate RECK expression in vivo have the potential to attenuate sustained MMP expression, and blunt fibrosis and adverse remodeling in hypertensive heart diseases.

Amyloid-β (Aβ), a primary component of amyloid plaques, has been widely associated with the pathogenesis of Alzheimer's disease. The Ca2+-binding protein regucalcin (RGN) plays multiple roles in maintaining cell functions by regulating intracellular calcium homeostasis, various signaling pathways, and gene expression systems. Here, we investigated the functional role of RGN against Aβ-induced cytotoxicity in neuronally differentiated PC12 cells. Overexpression of RGN reduced Aβ-induced apoptosis by reducing mitochondrial dysfunction and caspase activation. It also attenuated Aβ-induced reactive oxygen species production and oxidative damage and decreased Aβ-induced nitric oxide (NO) overproduction, upregulation of inducible NO synthase by nuclear factor-κB, and nitrosative damage. Interestingly, the genetic disruption of RGN increased the susceptibility of neuronally differentiated PC12 cells to Aβ toxicity. Thus, RGN possesses antioxidant activity against Aβ-induced oxidative and nitrosative stress and may play protective roles against Aβ-induced neurotoxicity in Alzheimer's disease.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

Despite extensive efforts, oncogenic KRAS remains resistant to targeted therapy. Combined downstream RAL-TBK1 and MEK inhibition induces only transient lung tumor shrinkage in KRAS-driven genetically engineered mouse models (GEMMs). Using the sensitive KRAS;LKB1 (KL) mutant background, we identify YAP1 upregulation and a therapy-induced secretome as mediators of acquired resistance. This program is reversible, associated with H3K27 promoter acetylation, and suppressed by BET inhibition, resensitizing resistant KL cells to TBK1/MEK inhibition. Constitutive YAP1 signaling promotes intrinsic resistance in KRAS;TP53 (KP) mutant lung cancer. Intermittent treatment with the BET inhibitor JQ1 thus overcomes resistance to combined pathway inhibition in KL and KP GEMMs. Using potent and selective TBK1 and BET inhibitors we further develop an effective therapeutic strategy with potential translatability to the clinic.

Developmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear.

Reactive oxygen species (ROS) activate uncoupler protein 1 (UCP1) in brown adipose tissue (BAT) under physiological cold exposure and noradrenaline (NA) stimulation to increase thermogenesis. However, the endogenous regulator of ROS in activated BAT and its role in pathological conditions remain unclear. We show that serum levels of selenoprotein P (SeP; encoded by SELENOP) negatively correlate with BAT activity in humans. Physiological cold exposure downregulates Selenop in BAT. Selenop knockout mice show higher rectal temperatures and UCP1 sulfenylation during cold exposure. SeP treatment to brown adipocytes eliminated the NA-induced mitochondrial ROS by upregulating glutathione peroxidase 4 and impaired cellular thermogenesis. A high-fat/high-sucrose diet elevates serum SeP levels and diminishes the elevated NA-induced thermogenesis in BAT-Selenop KO mice. Therefore, SeP is the intrinsic factor inducing reductive stress that impairs thermogenesis in BAT and may be a potential therapeutic target for obesity and diabetes.

Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.

Necroptosis is a regulated form of cell death involved in various pathological conditions, including ischemic reperfusion injuries, virus infections, and drug-induced tissue injuries. However, it is not fully understood when and where necroptosis occurs in vivo. We previously generated a Forster resonance energy transfer (FRET) biosensor, termed SMART (the sensor for MLKL activation by RIPK3 based on FRET), which monitors conformational changes of MLKL along with progression of necroptosis in human and murine cell lines in vitro. Here, we generate transgenic (Tg) mice that express the SMART biosensor in various tissues. The FRET ratio is increased in necroptosis, but not apoptosis or pyroptosis, in primary cells. Moreover, the FRET signals are elevated in renal tubular cells of cisplatin-treated SMART Tg mice compared to untreated SMART Tg mice. Together, SMART Tg mice may provide a valuable tool for monitoring necroptosis in different types of cells in vitro and in vivo.

The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed "X-SPINK1". Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3+/- mice rescued perinatal lethality, but the resulting Spink3-/-;XXSPINK1 mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3-/-;XXSPINK1 mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis.

Synthetic cyclin-dependent kinase (CDK) 4/6 inhibitors exert antitumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis, and increased immunogenicity. These agents currently have an indication in advanced breast cancers and are in clinical trials for many other solid tumors. HCC is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC; however, this is revivable, as RB family members are not frequently mutated or deleted in this malignancy.