Sanja Arandjelovic

Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion

Skeletal muscle arises from the fusion of precursor myoblasts into multinucleated myofibres. Although conserved transcription factors and signalling proteins involved in myogenesis have been identified, upstream regulators are less well understood. Here we report an unexpected discovery that the membrane protein BAI1, previously linked to recognition of apoptotic cells by phagocytes, promotes myoblast fusion. Endogenous BAI1 expression increased during myoblast fusion, and BAI1 overexpression enhanced myoblast fusion by means of signalling through ELMO/Dock180/Rac1 proteins. During myoblast fusion, a fraction of myoblasts within the population underwent apoptosis and exposed phosphatidylserine, an established ligand for BAI1 (ref. 3). Blocking apoptosis potently impaired myoblast fusion, and adding back apoptotic myoblasts restored fusion. Furthermore, primary human myoblasts could be induced to form myotubes by adding apoptotic myoblasts, even under normal growth conditions. Mechanistically, apoptotic cells did not directly fuse with the healthy myoblasts, rather the apoptotic cells induced a contact-dependent signalling with neighbours to promote fusion among the healthy myoblasts. In vivo, myofibres from Bai1−/− mice are smaller than those from wild-type littermates. Muscle regeneration after injury was also impaired in Bai1−/−mice, highlighting a role for BAI1 in mammalian myogenesis. Collectively, these data identify apoptotic cells as a new type of cue that induces signalling via the phosphatidylserine receptor BAI1 to promote fusion of healthy myoblasts, with important implications for muscle development and repair.

PAD4-Mediated Neutrophil Extracellular Trap Formation Is Not Required for Immunity against Influenza Infection

During an inflammatory response, neutrophils migrate to the site of infection where they can kill invading pathogens by phagocytosis, secretion of anti-microbicidal mediators or the release of neutrophil extracellular traps (NETs). NETs are specialized anti-microbial structures comprised of decondensed chromatin decorated with microbicidal agents. Increased amount of NETs have been found in patients suffering from the chronic lung inflammatory disease cystic fibrosis, correlating with increased severity of pulmonary obstruction. Furthermore, acute lung inflammation during influenza A infection is characterized by a massive influx of neutrophils into the lung. The role of NETs during virus-mediated lung inflammation is unknown. Peptidylarginine deiminase 4 (PAD4)-mediated deimination of histone H3 and H4 is required for NET formation. Therefore, we generated a PAD4-deficient mouse strain that has a striking inability to form NETs. These mice were infected with influenza A/WSN, and the disease was monitored at the level of leukocytic lung infiltration, lung pathology, viral replication, weight loss and mortality. PAD4 KO fared comparable to WT mice in all the parameters tested, but they displayed slight but statistically different weight loss kinetics during infection that was not reflected in enhanced survival. Overall, we conclude that PAD4-mediated NET formation is dispensable in a mouse model of influenza A infection.

Regulation of tumor necrosis factor receptor-1 and the IKK-NF-κB pathway by LDL receptor-related protein explains the antiinflammatory activity of this receptor

Low-density lipoprotein receptor-related protein (LRP-1) functions in endocytosis and in cell signaling directly (by binding signaling adaptor proteins) or indirectly (by regulating levels of other cell-surface receptors). Because recent studies in rodents suggest that LRP-1 inhibits inflammation, we conducted activity-based protein profiling experiments to discover novel proteases, involved in inflammation, that are regulated by LRP-1. We found that activated complement proteases accumulate at increased levels when LRP-1 is absent. Although LRP-1 functions as an endocytic receptor for C1r and C1s, complement protease mRNA expression was increased in LRP-1-deficient cells, as was expression of inducible nitric oxide synthase (iNOS) and interleukin-6. Regulation of expression of inflammatory mediators was explained by the ability of LRP-1 to suppress basal cell signaling through the I kappaB kinase-nuclear factor-kappaB (NF-kappaB) pathway. LRP-1-deficient macrophages, isolated from mice, demonstrated increased expression of iNOS, C1r, and monocyte chemoattractant protein-1 (MCP-1); MCP-1 expression was inhibited by NF-kappaB antagonism. The mechanism by which LRP-1 inhibits NF-kappaB activity involves down-regulating cell-surface tumor necrosis factor receptor-1 (TNFR1) and thus, inhibition of autocrine TNFR1-initiated cell signaling. TNF-alpha-neutralizing antibody inhibited NF-kappaB activity selectively in LRP-1-deficient cells. We propose that LRP-1 suppresses expression of inflammatory mediators indirectly, by regulating TNFR1-dependent cell signaling through the I kappaB kinase-NF-kappaB pathway.

PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis.

IntroductionBoth murine and human genome-wide association studies have implicated peptidyl arginine deiminase (PAD4) as a susceptibility gene in rheumatoid arthritis (RA). In addition, patients with RA commonly have autoantibodies which recognize PAD4 or and/or citrullinated peptides. This study aims to evaluate the role of PAD4 in the effector phase of arthritis.MethodsPAD4 knock out (KO) and wild type (WT) C57BL/6J mice were injected with K/BxN sera to induce disease. Progression of disease was monitored by measuring paw and ankle swelling and clinical indexes of disease, and pathogenesis was assessed by indexing of clinical progression on paws collected from WT and PAD4 KO mice injected with K/BxN serum. PAD4 activity was determined by visualization of neutrophil extracellular traps (NETs) and immunohistological analysis of histone citrullination.ResultsPAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion.ConclusionsPAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease.

A shed form of LDL receptor–related protein–1 regulates peripheral nerve injury and neuropathic pain in rodents

Injury to the peripheral nervous system (PNS) initiates a response controlled by multiple extracellular mediators, many of which contribute to the development of neuropathic pain. Schwann cells in an injured nerve demonstrate increased expression of LDL receptor-related protein-1 (LRP1), an endocytic receptor for diverse ligands and a cell survival factor. Here we report that a fragment of LRP1, in which a soluble or shed form of LRP1 with an intact alpha-chain (sLRP-alpha), was shed by Schwann cells in vitro and in the PNS after injury. Injection of purified sLRP-alpha into mouse sciatic nerves prior to chronic constriction injury (CCI) inhibited p38 MAPK activation (P-p38) and decreased expression of TNF-alpha and IL-1beta locally. sLRP-alpha also inhibited CCI-induced spontaneous neuropathic pain and decreased inflammatory cytokine expression in the spinal dorsal horn, where neuropathic pain processing occurs. In cultures of Schwann cells, astrocytes, and microglia, sLRP-alpha inhibited TNF-alpha-induced activation of p38 MAPK and ERK/MAPK. The activity of sLRP-alpha did not involve TNF-alpha binding, but rather glial cell preconditioning, so that the subsequent response to TNF-alpha was inhibited. Our results show that sLRP-alpha is biologically active and may attenuate neuropathic pain. In the PNS, the function of LRP1 may reflect the integrated activities of the membrane-anchored and shed forms of LRP1.

Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris

Background: LRP1 is a scavenger receptor involved in the clearance of apoptotic cells and myelin vesicles. Results: Novel ligands for LRP1 were discovered in CNS myelin by affinity purification combined with proteomics. Conclusion: Some ligands are intracellular proteins, suggesting a function for LRP1 in the clearance of necrotic debris. Significance: LRP1 mediates the removal of cellular waste and could maintain homeostasis. In the central nervous system (CNS), fast neuronal signals are facilitated by the oligodendrocyte-produced myelin sheath. Oligodendrocyte turnover or injury generates myelin debris that is usually promptly cleared by phagocytic cells. Failure to remove dying oligodendrocytes leads to accumulation of degraded myelin, which, if recognized by the immune system, may contribute to the development of autoimmunity in diseases such as multiple sclerosis. We recently identified low density lipoprotein receptor-related protein-1 (LRP1) as a novel phagocytic receptor for myelin debris. Here, we report characterization of the LRP1 interactome in CNS myelin. Fusion proteins were designed corresponding to the extracellular ligand-binding domains of LRP1. LRP1 partners were isolated by affinity purification and characterized by mass spectrometry. We report that LRP1 binds intracellular proteins via its extracellular domain and functions as a receptor for necrotic cells. Peptidyl arginine deiminase-2 and cyclic nucleotide phosphodiesterase are novel LRP1 ligands identified in our screen, which interact with full-length LRP1. Furthermore, the extracellular domain of LRP1 is a target of peptidyl arginine deiminase-2-mediated deimination in vitro. We propose that LRP1 functions as a receptor for endocytosis of intracellular components released during cellular damage and necrosis.

Regulation of the Composition of the Extracellular Matrix by Low Density Lipoprotein Receptor-related Protein-1 ACTIVITIES BASED ON REGULATION OF mRNA EXPRESSION

Low density lipoprotein receptor-related protein-1 (LRP-1) is a catabolic receptor for extracellular matrix (ECM) structural proteins and for proteins that bind to ECM. LRP-1 also is implicated in integrin maturation. In this study, we applied a proteomics strategy to identify novel proteins involved in ECM modeling that are regulated by LRP-1. We show that LRP-1 deficiency in murine embryonic fibroblasts (MEFs) is associated with increased levels of type III collagen and pigment epithelium-derived factor, which accumulate in the substratum surrounding cells. The collagen receptor, uPAR-AP/Endo-180, is also increased in LRP-1-deficient MEFs. Human LRP-1 reversed the changes in protein expression associated with LRP-1 deficiency; however, the endocytic activity of LRP-1 was not involved. Instead, regulation occurred at the mRNA level. Inhibition of c-Jun amino-terminal kinase (JNK) blocked type III collagen expression in LRP-1-deficient MEFs, suggesting regulation of JNK activity as a mechanism by which LRP-1 controls mRNA expression. The ability of LRP-1 to regulate expression of the factors identified here suggests a role for LRP-1 in determining blood vessel structure and in angiogenesis.

ATP Induces Protein Arginine Deiminase 2-Dependent Citrullination in Mast Cells through the P2X7 Purinergic Receptor

Posttranslational modifications regulate physiology either by directly modulating protein function or by impacting immune recognition of self-proteins. Citrullination is a posttranslational modification formed by the conversion of arginine residues into the citrulline amino acid by protein arginine deiminase (PAD) family members. We have identified mast cells as a major source of the PAD2 enzyme. Activation of the P2X7 purinergic receptor (P2X7) by the inflammatory “danger” signal ATP induces PAD2 activity and robust protein citrullination. P2X7-mediated activation of PAD2 is sensitive to p38 MAPK and protein kinase C inhibitors, and PAD2 regulates the expression of the TNFR2, Adamts-9, and Rab6b transcripts in mast cells. Further, the PAD2 enzyme and its citrullinated substrate proteins are released from mast cells on activation with ATP. PAD2 expression is closely linked with inflammation in rheumatoid arthritis (RA) synovial tissue, and PAD2 and citrullinated proteins are found in the synovial fluid of RA patients. In addition, RA is associated with the development of autoantibodies to citrullinated self-proteins. Our results suggest that P2X7 activation of mast cells may play a role in inflammation by providing PAD2 and PAD2 substrates access to the extracellular space.

Characterization of the interaction between alpha2-macroglobulin and fibroblast growth factor-2: the role of hydrophobic interactions.

Basic fibroblast growth factor (FGF-2) is important in development, wound healing and angiogenesis. The human plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) binds to and regulates the biological activity of various growth factors, including FGF-2. FGF-2 binds specifically and saturably to native alpha2M and conformationally modified alpha2M (alpha2M*); however, the KD for FGF-2 binding to alpha2M* is 10-fold lower. This study investigates the biochemical nature of the interaction between FGF-2 and alpha2M* and localizes a possible FGF-2 binding site in the alpha2M subunit. FGF-2 binding to alpha2M* was not affected by shifts in pH between 6.5 and 10; however, increasing temperature decreased the KD for this interaction. The binding affinity of FGF-2 for alpha2M* also increased with increasing ionic strength. These results are consistent with the hypothesis that hydrophobic interactions predominate in promoting FGF-2 association with alpha2M*. Consistent with this hypothesis, FGF-2 bound to a glutathione S-transferase fusion protein containing amino acids 591-774 of the alpha2M subunit (FP3) and to a hydrophobic 16-amino-acid peptide (amino acids 718-733) within FP3. Specific binding of FGF-2 to the 16-amino-acid peptide was inhibited by excess transforming growth factor-beta1. When the 16-amino-acid peptide was chemically modified to neutralize the only two charged amino acids, FGF-2-binding activity was unaffected, supporting the predominant role of hydrophobic interactions. FGF-2 presentation to signalling receptors is influenced by growth factor binding to heparan sulphate proteoglycans (HSPGs), which is electrostatic in nature. Our results demonstrate that the interactions of FGF-2 with alpha2M* and HSPGs are biochemically distinct, suggesting that different FGF-2 sequences are involved.

Effects of low density lipoprotein receptor‐related protein‐1 on the expression of platelet‐derived growth factor β‐receptor in vitro

The low density lipoprotein receptor related protein‐1 (LRP‐1) is a cargo transport receptor that undergoes constitutive endocytosis and recycling. Platelet‐derived growth factor‐BB (PDGF‐BB) binds to LRP‐1 and may bridge LRP‐1 to PDGF receptors. Bridging of LRP‐1 to other receptors by bifunctional ligands may represent a general mechanism whereby LRP‐1 facilitates internalization of membrane proteins. The goal of this study was to determine whether LRP‐1 regulates cell‐surface levels of PDGF β‐receptor or PDGF β‐receptor degradation following treatment with PDGF‐BB. Unexpectedly, in both murine embryonic fibroblasts (MEFs) and HT 1080 fibrosarcoma cells, LRP‐1 expression was associated with increased levels of PDGF β‐receptor. In MEFs, the mechanism involved increased PDGF β‐receptor transcription and/or RNA stabilization. LRP‐1 expression was not associated with increased levels of PDGF β‐receptor in Chinese hamster ovary (CHO) cells, suggesting that cell context is important. The kinetics of PDGF β‐receptor phosphorylation, in response to PDGF‐BB, and the extent of degradation of PDGF β‐receptor were equivalent in LRP‐1‐expressing and ‐deficient MEFs. We conclude that PDGF β‐receptor expression and cell surface levels may be regulated by LRP‐1; however, this activity is cell type‐specific. LRP‐1 does not directly regulate PDGF β‐receptor phosphorylation or degradation in PDGF‐BB‐treated cells.