Hypoxia activates SREBP2 through Golgi disassembly in bone marrow-derived monocytes for enhanced tumor growth.

Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression.

Leukopenia, macrocytosis, and thrombocytopenia occur in young adults with Down syndrome.

Down syndrome (DS) is a common congenital disorder caused by trisomy 21. Due to the increase in maternal age with population aging and advances in medical treatment for fatal complications in their early childhood, the prevalence and life expectancy of DS individuals have greatly increased. Despite this rise in the number of DS adults, their hematological status remains poorly examined. Here, we report that three hematological abnormalities, leukopenia, macrocytosis, and thrombocytopenia, develop as adult DS-associated features. Multi- and uni-variate analyses on hematological data collected from 51 DS and 60 control adults demonstrated that young adults with DS are at significantly higher risk of (i) myeloid-dominant leukopenia, (ii) macrocytosis characterized by high mean cell volume (MCV) of erythrocytes, and (iii) lower platelet counts than the control. Notably, these features were more pronounced with age. Further analyses on DS adults would provide a deeper understanding and novel research perspectives for multiple aging-related disorders in the general population.

The endothelial Dll4-muscular Notch2 axis regulates skeletal muscle mass.

Adult skeletal muscle is a highly plastic tissue that readily reduces or gains its mass in response to mechanical and metabolic stimulation; however, the upstream mechanisms that control muscle mass remain unclear. Notch signalling is highly conserved, and regulates many cellular events, including proliferation and differentiation of various types of tissue stem cell via cell-cell contact. Here we reveal that multinucleated myofibres express Notch2, which plays a crucial role in disuse- or diabetes-induced muscle atrophy. Mechanistically, in both atrophic conditions, the microvascular endothelium upregulates and releases the Notch ligand, Dll4, which then activates muscular Notch2 without direct cell-cell contact. Inhibition of the Dll4-Notch2 axis substantively prevents these muscle atrophy and promotes mechanical overloading-induced muscle hypertrophy in mice. Our results illuminate a tissue-specific function of the endothelium in controlling tissue plasticity and highlight the endothelial Dll4-muscular Notch2 axis as a central upstream mechanism that regulates catabolic signals from mechanical and metabolic stimulation, providing a therapeutic target for muscle-wasting diseases.

Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis.

Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated T cells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.

NFAT indicates nucleocytoplasmic damped oscillation via its feedback modulator.

Cell signaling and the following gene regulation are tightly regulated to keep homeostasis. NF-κB is a famous key transcription factor for inflammatory cell regulations that obtain a closed feedback loop with IκB. Similarly, we show here, NFAT is also tightly regulated via its downstream target, down syndrome critical region (DSCR)-1. In primary cultured endothelium, either shear stress or VEGF treatment revealed quick NFAT1 nuclear localization following the DSCR-1 transactivation, which in turn induced NFAT1 cytoplasm sequestration. Interestingly, both NFAT and DSCR-1 can be competitive substrates for calcineurin phosphatase and DSCR-1 is known to unstable protein, which caused NFAT1-nucleocytoplasmic damped oscillation via sustained shear stress or VEGF stimulation in endothelial cell (EC)s. To understand the molecular mechanism underlying the NFAT1 oscillation, we built a mathematical model of spatiotemporal regulation of NFAT1 combined with calcineurin and DSCR-1. Theoretically, manipulation of DSCR-1 expression in simulation predicted that DSCR-1 reduction would cause nuclear retention of dephosphorylated NFAT1 and disappearance of NFAT1 oscillation. To confirm this in ECs, DSCR-1 knockdown analysis was performed. DSCR-1 reduction indeed increased dephosphorylated NFAT1 in both the nucleus and cytoplasm, which eventually led to nuclear retention of NFAT1. Taken together, these studies suggest that DSCR-1 is a responsible critical factor for NFAT1 nucleocytoplasmic oscillation in shear stress or VEGF treated ECs. Our mathematical model successfully reproduced the experimental observations of NFAT1 dynamics. Combined mathematical and experimental approaches would provide a quantitative understanding way for the spatiotemporal NFAT1 feedback system.

Loss of Down syndrome critical region-1 leads to cholesterol metabolic dysfunction that exaggerates hypercholesterolemia in ApoE-null background.

Down syndrome critical region (DSCR)-1 functions as a feedback modulator for calcineurin-nuclear factor for activated T cell (NFAT) signals, which are crucial for cell proliferation and inflammation. Stable expression of DSCR-1 inhibits pathological angiogenesis and septic inflammation. DSCR-1 also plays a critical role in vascular wall remodeling associated with aneurysm development that occurs primarily in smooth muscle cells. Besides, Dscr-1 deficiency promotes the M1-to M2-like phenotypic switch in macrophages, which correlates to the reduction of denatured cholesterol uptakes. However, the distinct roles of DSCR-1 in cholesterol and lipid metabolism are not well understood. Here, we show that loss of apolipoprotein (Apo) E in mice with chronic hypercholesterolemia induced Dscr-1 expression in the liver and aortic atheroma. In Dscr-1-null mice fed a high-fat diet, oxidative- and endoplasmic reticulum (ER) stress was induced, and sterol regulatory element-binding protein (SREBP) 2 production in hepatocytes was stimulated. This exaggerated ApoE-/--mediated nonalcoholic fatty liver disease (NAFLD) and subsequent hypercholesterolemia. Genome-wide screening revealed that loss of both ApoE and Dscr-1 resulted in the induction of immune- and leukocyte activation-related genes in the liver compared with ApoE deficiency alone. However, expressions of inflammation-activated markers and levels of monocyte adhesion were suspended upon induction of the Dscr-1 null background in the aortic endothelium. Collectively, our study shows that the combined loss of Dscr-1 and ApoE causes metabolic dysfunction in the liver but reduces atherosclerotic plaques, thereby leading to a dramatic increase in serum cholesterol and the formation of sporadic vasculopathy.

Loss of Down Syndrome Critical Region-1 Mediated-Hypercholesterolemia Accelerates Corneal Opacity Via Pathological Neovessel Formation.

OBJECTIVE: The calcineurin-NFAT (nuclear factor for activated T cells)-DSCR (Down syndrome critical region)-1 pathway plays a crucial role as the downstream effector of VEGF (vascular endothelial growth factor)-mediated tumor angiogenesis in endothelial cells. A role for DSCR-1 in different organ microenvironment such as the cornea and its role in ocular diseases is not well understood. Corneal changes can be indicators of various disease states and are easily detected through ocular examinations. Approach and Results: The presentation of a corneal arcus or a corneal opacity due to lipid deposition in the cornea often indicates hyperlipidemia and in most cases, hypercholesterolemia. Although the loss of Apo (apolipoprotein) E has been well characterized and is known to lead to elevated serum cholesterol levels, there are few corneal changes observed in ApoE-/- mice. In this study, we show that the combined loss of ApoE and DSCR-1 leads to a dramatic increase in serum cholesterol levels and severe corneal opacity with complete penetrance. The cornea is normally maintained in an avascular state; however, loss of Dscr-1 is sufficient to induce hyper-inflammatory and -oxidative condition, increased corneal neovascularization, and lymphangiogenesis. Furthermore, immunohistological analysis and genome-wide screening revealed that loss of Dscr-1 in mice triggers increased immune cell infiltration and upregulation of SDF (stromal derived factor)-1 and its receptor, CXCR4 (C-X-C motif chemokine ligand receptor-4), potentiating this signaling axis in the cornea, thereby contributing to pathological corneal angiogenesis and opacity. CONCLUSIONS: This study is the first demonstration of the critical role for the endogenous inhibitor of calcineurin, DSCR-1, and pathological corneal angiogenesis in hypercholesterolemia induced corneal opacity.

Organ/Tissue-Specific Vascular Endothelial Cell Heterogeneity in Health and Disease.

The vascular system forms the largest surface in our body, serving as a critical interface between blood circulation and our diverse organ/tissue environments. Thus, the vascular system performs a gatekeeper function for organ/tissue homeostasis and the body's adjustment to pathological challenges. The endothelium, as the most inner layer of the vasculature, regulates the tissue microenvironment, which is critical for development, hemostatic balance, inflammation, and angiogenesis, with a role as well in tumor malignancy and metastasis. These multitudinous functions are primarily mediated by organ/tissue-specifically differentiated endothelial cells, in which heterogeneity has long been recognized at the molecular and histological level. Based on these general principles of vascular-bed heterogeneity and characterization, this review largely covers landmark discoveries regarding organ/tissue microenvironment-governed endothelial cell phenotypic changes. These involve the physical features of continuous, discontinuous, fenestrated, and sinusoidal endothelial cells, in addition to the more specialized endothelial cell layers of the lymphatic system, glomerulus, tumors, and the blood brain barrier (BBB). Major signal pathways of endothelial specification are outlined, including Notch as a key factor of tip/stalk- and arterial-endothelial cell differentiation. We also denote the shear stress sensing machinery used to convey blood flow-mediated biophysical forces that are indispensable to maintaining inert and mature endothelial phenotypes. Since our circulatory system is among the most fundamental and emergent targets of study in pharmacology from the viewpoint of drug metabolism and delivery, a better molecular understanding of organ vasculature-bed heterogeneity may lead to better strategies for novel vascular-targeted treatments to fight against hitherto intractable diseases.

Loss of Endogenous HMGB2 Promotes Cardiac Dysfunction and Pressure Overload-Induced Heart Failure in Mice.

BACKGROUND: The rapid increase in the number of heart failure (HF) patients in parallel with the increase in the number of older people is receiving attention worldwide. HF not only increases mortality but decreases quality of life, creating medical and social problems. Thus, it is necessary to define molecular mechanisms underlying HF development and progression. HMGB2 is a member of the high-mobility group superfamily characterized as nuclear proteins that bind DNA to stabilize nucleosomes and promote transcription. A recent in vitro study revealed that HMGB2 loss in cardiomyocytes causes hypertrophy and increases HF-associated gene expression. However, it's in vivo function in the heart has not been assessed. Methods and Results: Western blotting analysis revealed increased HMGB2 expression in heart tissues undergoing pressure overload by transverse aorta constriction (TAC) in mice. Hmgb2 homozygous knockout (Hmgb2-/-) mice showed cardiac dysfunction due to AKT inactivation and decreased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a activity. Compared to wild-type mice, Hmgb2-/- mice had worsened cardiac dysfunction after TAC surgery, predisposing mice to HF development and progression. CONCLUSIONS: This study demonstrates that upregulation of cardiac HMGB2 is an adaptive response to cardiac stress, and that loss of this response could accelerate cardiac dysfunction, suggesting that HMGB2 plays a cardioprotective role.

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[Braf V600E] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.

Defensive effect of microRNA-200b/c against amyloid-beta peptide-induced toxicity in Alzheimer's disease models.

MiRNA molecules are important post-transcriptional regulators of gene expression in the brain function. Altered miRNA profiles could represent a defensive response against the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD). Endogenous miRNAs have lower toxic effects than other gene silencing methods, thus enhancing the expression of defensive miRNA could be an effective therapy. However, little is known about the potential of targeting miRNAs for the treatment of AD. Here, we examined the function of the miR-200 family (miR-200a, -141, -429, -200b, -200c), identified using miRNA microarray analysis of cortical tissue from Tg2576 transgenic mice. In murine primary neurons, we found that upregulation of miR-200b or -200c was induced by the addition of amyloid beta (Aß). Neurons transfected with miR-200b or -200c reduced secretion of Aß in conditioned medium. Moreover, mice infused with miR-200b/c into the brain were relieved of memory impairments induced by intracerebroventricular injection of oligomeric Aß, and demonstrated proper spatial learning in the Barnes maze. To gain further understanding of the relationship between miR-200b/c and Aß, we identified target mRNAs via an RNA-binding protein immunoprecipitation-microarray assay. Western blot analysis showed that expression of ribosomal protein S6 kinase B1 (S6K1), a candidate target, was inhibited by miR-200c. S6K1, a downstream effector of mammalian target of rapamycin (mTOR), serves as a negative feedback mediator that phosphorylates insulin receptor substrate 1 at serine residues (IRS-1pSer). S6K1-dependent IRS-1pSer suppresses insulin signaling leading to insulin resistance, which is frequently observed in AD brains. Notably, miR-200b/c transfection of SH-SY5Y cells reduced the levels of IRS-1pSer. This finding indicates that miR-200b/c has the potential to alleviate insulin resistance via modulation of S6K1. Taken together, miR-200b/c may contribute to reduce Aß secretion and Aß-induced cognitive impairment by promoting insulin signaling.

SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts.

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [1]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[BrafV600E] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naïve WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naïve KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-ß-galactosidase, p21waf1 and p16ink4a, and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.

A subset of cerebrovascular pericytes originates from mature macrophages in the very early phase of vascular development in CNS.

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31+F4/80+ hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31+F4/80+ cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31+F4/80+ cells transdifferentiated into NG2/PDGFRß/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.

Significance of Extracellular Vesicles: Pathobiological Roles in Disease.

Over the past decade, many studies have been conducted on extracellular vesicles (EVs) in the fields of basic and clinical research. EVs are small sized membranous vesicles generated from many type of cells upon activation by environmental stresses such as heat, hypoxia, and irradiation. EVs theoretically consist of microparticles/microvesicles, exosomes, and apoptotic bodies by different productive mechanisms. Clinically, EVs are observed in the blood stream of patients suffering from acute and chronic inflammation evoked by various diseases, and number of EVs in blood flow is often dependent on the inflammatory status and severity of the diseases. To date, it has been reported that small molecules such as RNAs and proteins are encapsulated in EVs; however, the functions of EVs are still unclear in the biological, pathological, and clinical aspects. In this review, we summarize and discuss the biogenesis-based classification, expected function, and pathobiological activities of EVs.

Inflammation-induced endothelial cell-derived extracellular vesicles modulate the cellular status of pericytes.

Emerging lines of evidence have shown that extracellular vesicles (EVs) mediate cell-to-cell communication by exporting encapsulated materials, such as microRNAs (miRNAs), to target cells. Endothelial cell-derived EVs (E-EVs) are upregulated in circulating blood in different pathological conditions; however, the characteristics and the role of these E-EVs are not yet well understood. In vitro studies were conducted to determine the role of inflammation-induced E-EVs in the cell-to-cell communication between vascular endothelial cells and pericytes/vSMCs. Stimulation with inflammatory cytokines and endotoxin immediately induced release of shedding type E-EVs from the vascular endothelial cells, and flow cytometry showed that the induction was dose dependent. MiRNA array analyses revealed that group of miRNAs were specifically increased in the inflammation-induced E-EVs. E-EVs added to the culture media of cerebrovascular pericytes were incorporated into the cells. The E-EV-supplemented cells showed highly induced mRNA and protein expression of VEGF-B, which was assumed to be a downstream target of the miRNA that was increased within the E-EVs after inflammatory stimulation. The results suggest that E-EVs mediate inflammation-induced endothelial cell-pericyte/vSMC communication, and the miRNAs encapsulated within the E-EVs may play a role in regulating target cell function. E-EVs may be new therapeutic targets for the treatment of inflammatory diseases.

Delphinidin, one of the major anthocyanidins, prevents bone loss through the inhibition of excessive osteoclastogenesis in osteoporosis model mice.

Anthocyanins, one of the flavonoid subtypes, are a large family of water-soluble phytopigments and have a wide range of health-promoting benefits. Recently, an anthocyanin-rich compound from blueberries was reported to possess protective property against bone loss in ovariectomized (OVX) animal models. However, the active ingredients in the anthocyanin compound have not been identified. Here we show that delphinidin, one of the major anthocyanidins in berries, is a potent active ingredient in anti-osteoporotic bone resorption through the suppression of osteoclast formation. In vitro examinations revealed that delphinidin treatment markedly inhibited the differentiation of RAW264.7 cells into osteoclasts compared with other anthocyanidins, cyanidin and peonidin. Oral administration of delphinidin significantly prevented bone loss in both RANKL-induced osteoporosis model mice and OVX model mice. We further provide evidence that delphinidin suppressed the activity of NF-κB, c-fos, and Nfatc1, master transcriptional factors for osteoclastogenesis. These results strongly suggest that delphinidin is the most potent inhibitor of osteoclast differentiation and will be an effective agent for preventing bone loss in postmenopausal osteoporosis.

Inhibition of histone demethylase JMJD1A improves anti-angiogenic therapy and reduces tumor-associated macrophages.

Antiangiogenic strategies can be effective for cancer therapy, but like all therapies resistance poses a major clinical challenge. Hypoxia and nutrient starvation select for aggressive qualities that may render tumors resistant to antiangiogenic attack. Here, we show that hypoxia and nutrient starvation cooperate to drive tumor aggressiveness through epigenetic regulation of the histone demethylase JMJD1A (JHDM2A; KDM3A). In cancer cells rendered resistant to long-term hypoxia and nutrient starvation, we documented a stimulation of AKT phosphorylation, cell morphologic changes, cell migration, invasion, and anchorage-independent growth in culture. These qualities associated in vivo with increased angiogenesis and infiltration of macrophages into tumor tissues. Through expression microarray analysis, we identified a cluster of functional drivers such as VEGFA, FGF18, and JMJD1A, the latter which was upregulated in vitro under conditions of hypoxia and nutrient starvation and in vivo before activation of the angiogenic switch or the prerefractory phase of antiangiogenic therapy. JMJD1A inhibition suppressed tumor growth by downregulating angiogenesis and macrophage infiltration, by suppressing expression of FGF2, HGF, and ANG2. Notably, JMJD1A inhibition enhanced the antitumor effects of the anti-VEGF compound bevacizumab and the VEGFR/KDR inhibitor sunitinib. Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation-resistant cancer cells as an approach to leverage antiangiogenic treatments and limit resistance to them.

Increased expression of histone demethylase JHDM1D under nutrient starvation suppresses tumor growth via down-regulating angiogenesis.

Histone demethylase JHDM1D (also known as KDM7A) modifies the level of methylation in histone and participates in epigenetic gene regulation; however, the role of JHDM1D in tumor progression is unknown. Here, we show that JHDM1D plays a tumor-suppressive role by regulating angiogenesis. Expression of JHDM1D was increased in mouse and human cancer cells under long-term nutrient starvation in vitro. Expression of JHDM1D mRNA was increased within avascular tumor tissue at the preangiogenic switch, along with increased expression of angiogenesis-regulating genes such as Vegf-A. Stable expression of JHDM1D cDNA or siRNA silencing of JHDM1D in cancer cells did not affect cell proliferation, anchorage-independent cell growth, or cell cycle progression in vitro. Notably, JHDM1D-expressing mouse melanoma (B16) and human cervical carcinoma (HeLa) cells exhibited significantly slower tumor growth in vivo compared with the original cells. This reduction in tumor growth was associated with decreased formation of CD31(+) blood vessels and reduced infiltration of CD11b(+) macrophage linage cells into tumor tissues. Expression of multiple angiogenic factors such as VEGF-B and angiopoietins was decreased in tumor xenografts of JHDM1D-expressing B16 and HeLa cells. Our results provide evidence that increased JHDM1D expression suppressed tumor growth by down-regulating angiogenesis under nutrient starvation.

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway.

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.