A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1): a novel modulator of mitochondrial autophagy.

A20 binding inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1), a polyubiquitin-binding protein, is a signal-induced autophagy receptor that attenuates NF-κB-mediated inflammation and cell death. The present study aimed to elucidate the potential role of ABIN-1 in mitophagy, a biological process whose outcome is decisive in diverse physiological and pathological settings. Microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) was found to be in complex with ectopically expressed hemagglutinin (HA)-tagged-full length (FL)-ABIN-1. Bacterial expression of ABIN-1 and LC3A and LC3B showed direct binding of ABIN-1 to LC3 proteins, whereas mutations in the LC3-interacting region (LIR) 1 and 2 motifs of ABIN-1 abrogated ABIN-1/LC3B-II complex formation. Importantly, induction of autophagy in HeLa cells resulted in colocalization of ABIN-1 with LC3B-II in autophagosomes and with lysosomal-associated membrane protein 1 (LAMP-1) in autophagolysosomes, leading to degradation of ABIN-1 with p62. Interestingly, ABIN-1 was found to translocate to damaged mitochondria in HeLa-mCherry-Parkin transfected cells. In line with this observation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated deletion of ABIN-1 significantly inhibited the degradation of the mitochondrial outer membrane proteins voltage-dependent anion-selective channel 1 (VDAC-1), mitofusin-2 (MFN2), and translocase of outer mitochondrial membrane (TOM)20. In addition, short interfering RNA (siRNA)-mediated knockdown of ABIN-1 significantly decreased lysosomal uptake of mitochondria in HeLa cells expressing mCherry-Parkin and the fluorescence reporter mt-mKEIMA. Collectively, our results identify ABIN-1 as a novel and selective mitochondrial autophagy regulator that promotes mitophagy, thereby adding a new player to the complex cellular machinery regulating mitochondrial homeostasis.

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44-/- mice but not Cd14-/- mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

Biglycan is a new high-affinity ligand for CD14 in macrophages.

Sterile inflammation is a therapeutic target in many diseases where it represents an important initiator of disease progression. However, the detailed mechanisms underlying its evolution and biological relevance are not yet completely elucidated. Biglycan, a prototype extracellular matrix-derived damage-associated molecular pattern, mediates sterile inflammation in macrophages through Toll-like receptor (TLR) 2 and/or TLR4-dependent signaling pathways. Here we discovered that soluble biglycan is a novel high-affinity ligand for CD14, a well-known GPI-anchored co-receptor for TLRs. CD14 is required for all biglycan-mediated TLR2/4 dependent inflammatory signaling pathways in macrophages. By binding to CD14 and choosing different TLR signaling branches, biglycan induced TNF-α and CCL2 via TLR2/4, HSP70 through TLR2, and CCL5 via TLR4. Mechanistically, biglycan evoked phosphorylation and subsequent nuclear translocation of p38, p44/42, and NF-κB, and these effects were due to a specific, high-affinity interaction between biglycan protein core and CD14. Finally, we provide proof-of-principle for the requirement of CD14, by transiently overexpressing biglycan in a mouse model of renal ischemia/reperfusion injury performed in Cd14-/- mice. Lack of Cd14 prevented biglycan-mediated cytokine expression, recruitment of macrophages, M1 macrophage polarization as well as mitigated the tubular damage and serum creatinine levels, thereby improving renal function. Thus, CD14 inhibition could lead to the reduction in the activation of biglycan-TLR2/4 signaling pathways and could be a novel therapeutic approach in inflammatory kidney diseases.

Analysis of Cell Type-Specific Effects of MicroRNA-92a Provides Novel Insights Into Target Regulation and Mechanism of Action.

BACKGROUND: MicroRNAs (miRs) regulate nearly all biological pathways. Because the dysregulation of miRs can lead to disease progression, they are being explored as novel therapeutic targets. However, the cell type-specific effects of miRs in the heart are poorly understood. Thus, we assessed miR target regulation using miR-92a-3p as an example. Inhibition of miR-92a is known to improve endothelial cell function and recovery after acute myocardial infarction. METHODS: miR-92a-3p was inhibited by locked nucleic acid (LNA)-based antimiR (LNA-92a) in mice after myocardial infarction. Expression of regulated genes was evaluated 3 days after myocardial infarction by RNA sequencing of isolated endothelial cells, cardiomyocytes, fibroblasts, and CD45+ hematopoietic cells. RESULTS: LNA-92a depleted miR-92a-3p expression in all cell types and derepressed predicted miR-92a-3p targets in a cell type-specific manner. RNAseq showed endothelial cell-specific regulation of autophagy-related genes. Imaging confirmed increased endothelial cell autophagy in LNA-92a treated relative to control animals. In vitro inhibition of miR-92a-3p augmented EC autophagy, derepressed autophagy-related gene 4a, and increased luciferase activity in autophagy-related gene 4a 3'UTR containing reporters, whereas miR-92a-3p overexpression had the opposite effect. In cardiomyocytes, LNA-92a derepressed metabolism-related genes, notably, the high-density lipoprotein transporter Abca8b. LNA-92a further increased fatty acid uptake and mitochondrial function in cardiomyocytes in vitro. CONCLUSIONS: Our data show that miRs have cell type-specific effects in vivo. Analysis of miR targets in cell subsets disclosed a novel function of miR-92a-3p in endothelial cell autophagy and cardiomyocyte metabolism. Because autophagy is upregulated during ischemia to supply nutrients and cardiomyocyte metabolic-switching improves available substrate utilization, these prosurvival mechanisms may diminish tissue damage.

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants.

In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-ß (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.

An Essential Role for SHARPIN in the Regulation of Caspase 1 Activity in Sepsis.

Sepsis is burdened by high mortality due to uncontrolled inflammatory response to pathogens. Increased caspase 1 activation causing maturation of IL1ß/18 remains a therapeutic challenge in sepsis. SHARPIN (shank-associated regulator of G-protein signaling homology domain-interacting protein), a component of the LUBAC (linear ubiquitin chain-assembly complex), regulates inflammation, with unknown effects on caspase 1 activation. Mice lacking Casp1, Casp11, or both in a Sharpin-deficient background were generated, exposed to lipopolysaccharide-induced endotoxemia, and injected with caspase 1 inhibitor. We monitored survival, Il1ß/18, and caspase 1/11 levels in plasma and organs and deciphered mechanisms of SHARPIN-dependent caspase 1 inhibition. A correlation between LUBAC and active caspase 1 was found in blood mononuclear cells from septic patients. SHARPIN bound caspase 1 and disrupted p20/p10 dimer formation, the last step of caspase 1 processing, thereby inhibiting enzyme activation and maturation of IL1ß/18 in a LUBAC-independent manner. In septic patients, LUBAC-independent decline in SHARPIN correlated with enhancement of active caspase 1 in circulating mononuclear cells. Septic Sharpin-deficient mice displayed enrichment in mature Il1ß/18 and active caspase 1, and shortened survival. Inhibition of caspase 1 reduced levels of Il1ß/18 and splenic cell death, and prolonged survival in septic Sharpin-deficient mice. Our findings identify SHARPIN as a potent in vivo caspase 1 inhibitor and propose the caspase 1-SHARPIN interaction as a target in sepsis.

Endostatin and endorepellin: A common route of action for similar angiostatic cancer avengers.

Traditional cancer therapy typically targets the tumor proper. However, newly-formed vasculature exerts a major role in cancer development and progression. Autophagy, as a biological mechanism for clearing damaged proteins and oxidative stress products released in the tumor milieu, could help in tumor resolution by rescuing cells undergoing modifications or inducing autophagic-cell death of tumor blood vessels. Cleaved fragments of extracellular matrix proteoglycans are emerging as key players in the modulation of angiogenesis and endothelial cell autophagy. An essential characteristic of cancer progression is the remodeling of the basement membrane and the release of processed forms of its constituents. Endostatin, generated from collagen XVIII, and endorepellin, the C-terminal segment of the large proteoglycan perlecan, possess a dual activity as modifiers of both angiogenesis and endothelial cell autophagy. Manipulation of these endogenously-processed forms, located in the basement membrane within tumors, could represent new therapeutic approaches for cancer eradication.

Bimodal role of NADPH oxidases in the regulation of biglycan-triggered IL-1ß synthesis.

Biglycan, a ubiquitous proteoglycan, acts as a danger signal when released from the extracellular matrix. As such, biglycan triggers the synthesis and maturation of interleukin-1ß (IL-1ß) in a Toll-like receptor (TLR) 2-, TLR4-, and reactive oxygen species (ROS)-dependent manner. Here, we discovered that biglycan autonomously regulates the balance in IL-1ß production in vitro and in vivo by modulating expression, activity and stability of NADPH oxidase (NOX) 1, 2 and 4 enzymes via different TLR pathways. In primary murine macrophages, biglycan triggered NOX1/4-mediated ROS generation, thereby enhancing IL-1ß expression. Surprisingly, biglycan inhibited IL-1ß due to enhancement of NOX2 synthesis and activation, by selectively interacting with TLR4. Synthesis of NOX2 was mediated by adaptor molecule Toll/IL-1R domain-containing adaptor inducing IFN-ß (TRIF). Via myeloid differentiation primary response protein (MyD88) as well as Rac1 activation and Erk phosphorylation, biglycan triggered translocation of the cytosolic NOX2 subunit p47(phox) to the plasma membrane, an obligatory step for NOX2 activation. In contrast, by engaging TLR2, soluble biglycan stimulated the expression of heat shock protein (HSP) 70, which bound to NOX2, and consequently impaired the inhibitory function of NOX2 on IL-1ß expression. Notably, a genetic background lacking biglycan reduced HSP70 expression, rescued the enhanced renal IL-1ß production and improved kidney function of Nox2(-/y) mice in a model of renal ischemia reperfusion injury. Here, we provide a novel mechanism where the danger molecule biglycan influences NOX2 synthesis and activation via different TLR pathways, thereby regulating inflammation severity. Thus, selective inhibition of biglycan-TLR2 or biglycan-TLR4 signaling could be a novel therapeutic approach in ROS-mediated inflammatory diseases.

Endorepellin evokes autophagy in endothelial cells.

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity via dual receptor antagonism, through concurrent binding to the α2ß1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2). Here, we discovered that soluble endorepellin induced autophagy in endothelial cells by modulating the expression of Beclin 1, LC3, and p62, three established autophagic markers. Moreover, endorepellin evoked expression of the imprinted tumor suppressor gene Peg3 and its co-localization with Beclin 1 and LC3 in autophagosomes, suggesting a major role for this gene in endothelial cell autophagy. Mechanistically, endorepellin induced autophagy by down-regulating VEGFR2 via the two LG1/2 domains, whereas the C-terminal LG3 domain, the portion responsible for binding the α2ß1 integrin, was ineffective. Endorepellin also induced transcriptional activity of the BECN1 promoter in endothelial cells, and the VEGFR2-specific tyrosine kinase inhibitor, SU5416, blocked this effect. Finally, we found a correlation between endorepellin-evoked inhibition of capillary morphogenesis and enhanced autophagy. Thus, we have identified a new role for this endogenous angiostatic fragment in inducing autophagy through a VEGFR2-dependent but α2ß1 integrin-independent pathway. This novel mechanism specifically targets endothelial cells and could represent a promising new strategy to potentiate the angiostatic effect of endorepellin and perhaps other angiostatic matrix proteins.

Decorin causes autophagy in endothelial cells via Peg3.

Soluble decorin affects the biology of several receptor tyrosine kinases by triggering receptor internalization and degradation. We found that decorin induced paternally expressed gene 3 (Peg3), an imprinted tumor suppressor gene, and that Peg3 relocated into autophagosomes labeled by Beclin 1 and microtubule-associated light chain 3. Decorin evoked Peg3-dependent autophagy in both microvascular and macrovascular endothelial cells leading to suppression of angiogenesis. Peg3 coimmunoprecipitated with Beclin 1 and LC3 and was required for maintaining basal levels of Beclin 1. Decorin, via Peg3, induced transcription of Beclin 1 and microtubule-associated protein 1 light chain 3 alpha genes, thereby leading to a protracted autophagic program. Mechanistically, decorin interacted with VEGF receptor 2 (VEGFR2) in a region overlapping with its natural ligand VEGFA, and VEGFR2 was required for decorin-evoked Beclin 1 and microtubule-associated protein 1 light chain 3 alpha expression as well as for Peg3 induction in endothelial cells. Moreover, decorin induced VEGFR2-dependent mitochondrial fragmentation and loss of mitochondrial membrane potential. Thus, we have unveiled a mechanism for a secreted proteoglycan in inducing Peg3, a master regulator of macroautophagy in endothelial cells.

Endorepellin laminin-like globular 1/2 domains bind Ig3-5 of vascular endothelial growth factor (VEGF) receptor 2 and block pro-angiogenic signaling by VEGFA in endothelial cells.

Endorepellin, a processed fragment of perlecan protein core, possesses anti-angiogenic activity by antagonizing endothelial cells. Endorepellin contains three laminin G-like (LG) domains and binds simultaneously to vascular endothelial growth factor receptor 2 (VEGFR2) and α2ß1 integrin, resulting in dual receptor antagonism. Treatment of endothelial cells with endorepellin inhibits transcription of VEGFA, the natural ligand for VEGFR2, attenuating the pro-survival and migratory activities of VEGFA/VEGFR2 signaling cascade. Here, we investigated the specific binding site of endorepellin within the ectodomain of VEGFR2. Full-length endorepellin was not capable of displacing VEGFA binding from VEGFR2 and LG3 domain alone did not bind VEGFR2. This suggested different binding mechanisms of the extracellular Ig domains of VEGFR2. Therefore, we hypothesized that endorepellin would bind through its proximal LG1/2 domains to VEGFR2 in a different region than VEGFA. Indeed, we found that LG1/2 did not bind Ig1-3, but did bind with high affinity to Ig3-5, distal to the known VEGFA binding site, i.e. Ig2-3. These results support a role for endorepellin as an allosteric inhibitor of VEGFR2. Moreover, we found that LG1/2 blocked the rapid VEGFA activation of VEGFR2 at Tyr1175 in endothelial cells. In contrast, LG1/2 did not result in actin cytoskeletal disassembly in endothelial cells whereas LG3 alone did induce cytoskeletal collapse. However, LG1/2 did inhibit VEGFA-dependent endothelial migration through fibrillar collagen I. These studies provide a mechanistic understanding of how the different LG domains of endorepellin signal in endothelial cells while serving as a template for protein design of receptor tyrosine kinase antagonists.

Endorepellin affects angiogenesis by antagonizing diverse vascular endothelial growth factor receptor 2 (VEGFR2)-evoked signaling pathways: transcriptional repression of hypoxia-inducible factor 1α and VEGFA and concurrent inhibition of nuclear factor of activated T cell 1 (NFAT1) activation.

Endorepellin, the angiostatic C-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits angiogenesis by simultaneously binding to the α2ß1 integrin and the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) on endothelial cells. This interaction triggers the down-regulation of both receptors and the concurrent activation of the tyrosine phosphatase SHP-1, which leads to a signaling cascade resulting in angiostasis. Here, we provide evidence that endorepellin is capable of attenuating both the PI3K/PDK1/Akt/mTOR and the PKC/JNK/AP1 pathways. We show that hypoxia-inducible factor 1α (HIF-1α) transcriptional activity induced by VEGFA was inhibited by endorepellin independent of oxygen concentration and that only a combination of both PI3K and calcineurin inhibitors completely blocked the suppressive activity evoked by endorepellin on HIF1A and VEGFA promoter activity. Moreover, endorepellin inhibited the PKC/JNK/AP1 axis induced by the recruitment of phospholipase γ and attenuated the VEGFA-induced activation of NFAT1, a process dependent on calcineurin activity. Finally, endorepellin inhibited VEGFA-evoked nuclear translocation of NFAT1 and promoted NFAT1 stability. Thus, we provide evidence for a novel downstream signaling axis for an angiostatic fragment and for the key components involved in the dual antagonistic activity of endorepellin, highlighting its potential use as a therapeutic agent.

Endorepellin, the angiostatic module of perlecan, interacts with both the α2ß1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2): a dual receptor antagonism.

Endorepellin, the C-terminal module of perlecan, negatively regulates angiogenesis counter to its proangiogenic parental molecule. Endorepellin (the C-terminal domain V of perlecan) binds the α2ß1 integrin on endothelial cells and triggers a signaling cascade that leads to disruption of the actin cytoskeleton. Here, we show that both perlecan and endorepellin bind directly and with high affinity to both VEGF receptors 1 and 2, in a region that differs from VEGFA-binding site. In both human and porcine endothelial cells, this interaction evokes a physical down-regulation of both the α2ß1 integrin and VEGFR2, with concurrent activation of the tyrosine phosphatase SHP-1 and downstream attenuation of VEGFA transcription. We demonstrate that endorepellin requires both the α2ß1 integrin and VEGFR2 for its angiostatic activity. Endothelial cells that express α2ß1 integrin but lack VEGFR2, do not respond to endorepellin treatment. Thus, we provide a new paradigm for the activity of an antiangiogenic protein and mechanistically explain the specificity of endorepellin for endothelial cells, the only cells that simultaneously express both receptors. We hypothesize that a mechanism such as dual receptor antagonism could operate for other angiostatic fragments.