Agrin Mediates Angiogenesis in the Tumor Microenvironment.

Angiogenesis represents a hallmark of cancer. Several proteoglycans associate with cell surface receptors and regulate angiogenesis within the tumor microenvironment (TME). We highlight the recent discovery that the proteoglycan Agrin cross talks between the tumor and the endothelium to promote an angiogenesis privileged niche during cancer progression.

JQ1 is a potential therapeutic option for COPD patients with agrin overexpression.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and death worldwide. It is characterized by chronic pulmonary inflammation and obstructed airflow from the lungs. To date, there is no effective treatment for COPD. The activation of the agrin (AGRN-YAP pathway can promote heart regeneration. Because agrin can induce only mild cardiomyocyte proliferation compared with ERBB2 pathway activation, it might exert pleiotropic effects, such as mitigation of innate inflammation, immune response, and fibrosis. Previously, we demonstrated that several common pathological gene regulatory programs such as innate inflammatory and profibrotic transcriptional networks were shared by COPD and heart failure. In this study, we show that agrin is inversely correlated with COPD development and progression and may exert its effects by suppressing innate inflammation and profibrotic signaling pathways. BET inhibitor JQ1, in line with our previous findings, is a promising therapeutic option in the treatment of patients with COPD. Nevertheless, wet laboratory experiments and clinical trials are needed before its application in clinical practice.

Sodium nitrate decreases agrin-induced acetylcholine receptor clustering.

BACKGROUND: Humans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined. METHODS: C2C12 skeletal muscle cell cultures were maintained, myoblasts were fused into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance acetylcholine receptor (AChR) clustering. Untreated cultures were compared with cultures exposed to sodium nitrate at concentrations ranging from 10 ng/mL-100 µg/mL. RESULTS: The results reported here demonstrate that 1 µg/mL sodium nitrate was sufficient to decrease the frequency of agrin-induced AChR clustering without affecting myotube formation. In addition, concentrations of sodium nitrate of 1 µg/mL or 100 µg/mL decreased gene expression of the myogenic transcription factor myogenin and AChR in correlation with the agrin-induced AChR clustering data. CONCLUSIONS: These results reveal that sodium nitrate decreases the frequency of agrin-induced AChR clustering by a mechanism that includes myogenin and AChR gene expression. As a consequence sodium nitrate may pose a risk for skeletal muscle development and subsequent neuromuscular synapse formation in humans.

Clustering transmembrane-agrin induces filopodia-like processes on axons and dendrites.

The transmembrane form of agrin (TM-agrin) is primarily expressed in the CNS, particularly on neurites. To analyze its function, we clustered TM-agrin on neurons using anti-agrin antibodies. On axons from the chick CNS and PNS as well as on axons and dendrites from mouse hippocampal neurons anti-agrin antibodies induced the dose- and time-dependent formation of numerous filopodia-like processes. The processes appeared within minutes after antibody addition and contained a complex cytoskeleton. Formation of processes required calcium, could be inhibited by cytochalasine D, but was not influenced by staurosporine, heparin or pervanadate. Time-lapse video microscopy revealed that the processes were dynamic and extended laterally along the entire length of the neuron. The lateral processes had growth cones at their tips that initially adhered to the substrate, but subsequently collapsed and were retracted. These data provide the first evidence for a specific role of TM-agrin in shaping the cytoskeleton of neurites in the developing nervous system.

Functional innervation of cultured human skeletal muscle proceeds by two modes with regard to agrin effects.

Nerve-derived agrin is a specific isoform of agrin that promotes clustering of nicotinic acetylcholine receptors (AChR) and other components of the neuromuscular junction (NMJ). We investigated the effects of agrin on functional maturation of NMJs at the early stages of synaptogenesis in human muscle. Specifically, we assessed the importance of agrin for the differentiation of developing NMJs to the stage where they are able to transmit signals that result in contractions of myotubes. We utilized an in vitro model in which human myotubes are innervated by neurons extending from spinal cord explants of fetal rat. This model is suitable for functional studies because all muscle contractions are the result of neuromuscular transmission and can be quantitated. An anti-agrin antibody, Agr 33, was applied to co-cultures during de novo NMJ formation. Quantitative analyses demonstrated that Agr 33 reduced the number of AChR clusters to 20% and their long axes to 50% of control, yet still permitted early, NMJ-mediated muscle contractions that are normally observed in 7-10-day-old co-cultures. However, at later times of development, the same treatment completely prevented the increase in the number of contracting units as compared with untreated co-cultures. It is concluded that there are two modes of functional maturation of NMJs with regard to agrin effects: one that is insensitive and the other that is sensitive to agrin blockade. Agrin-insensitive mode is limited to the small population of NMJs that become functional at the earlier stages of functional innervation. However, most of the NMJs become contraction-competent at the later stages of the innervation process. These NMJs become functional only if agrin action is uncompromised. This is the first characterization of the contribution of agrin to NMJ development on human muscle.

Agrin differentially regulates the rates of axonal and dendritic elongation in cultured hippocampal neurons.

In the present study, we examined the role of agrin in axonal and dendritic elongation in central neurons. Dissociated hippocampal neurons were grown in the presence of either recombinant agrin or antisense oligonucleotides designed to block agrin expression. Our results indicate that agrin differentially regulates axonal and dendritic growth. Recombinant agrin decreased the rate of elongation of main axons but induced the formation of axonal branches. On the other hand, agrin induced both dendritic elongation and dendritic branching. Conversely, cultured hippocampal neurons depleted of agrin extended longer, nonbranched axons and shorter dendrites when compared with controls. These changes in the rates of neurite elongation and branching were paralleled by changes in the composition of the cytoskeleton. In the presence of agrin, there was an upregulation of the expression of microtubule-associated proteins MAP1B, MAP2, and tau. In contrast, a downregulation of the expression of these MAPs was detected in agrin-depleted cells. Taken collectively, these results suggest an important role for agrin as a trigger of the transcription of neuro-specific genes involved in neurite elongation and branching in central neurons.

Sialic acid inhibits agrin signaling in C2 myotubes.

Acetylcholine receptor (AChR) clustering is an early event in neuromuscular synapse formation that is commonly studied using muscle cell culture. Motor neuron-derived agrin induces the postsynaptic tyrosine phosphorylation of both a muscle-specific kinase (MuSK) and the AChR beta-subunit. These phosphorylation events are required for AChR clustering, suggesting an agrin-driven signaling pathway. Both the phosphorylation events and AChR clustering can also be induced by neuraminidase, an enzyme that cleaves sialic acid from glycoconjugates, suggesting that neuraminidase is able to activate the agrin signaling pathway. A postulated signal for postsynaptic differentiation at sites of nerve-muscle contact during vertebrate development is the enzymatic removal of basal lamina components. We show here that bath-applied sialic acid has an effect directly opposite that of agrin or neuraminidase. Sialic acid not only decreases AChR clustering but also diminishes the tyrosine phosphorylation of MuSK and the AChR beta-subunit signal-transduction events normally driven by agrin. However, sialic acid does not prevent agrin-binding molecules from colocalizing with the decreased number of AChR clusters that do form, suggesting that sialic acid is acting to inhibit the agrin signaling pathway downstream of agrin binding to the muscle cell membrane. We propose a regulatory role for sialic acid in the signal transduction events of neuromuscular synapse formation, in which agrin or neuraminidase can overcome this sialic acid repression, resulting in the clustering of AChRs and other postsynaptic molecules.

Heparin blocks functional innervation of cultured human muscle by rat motor nerve.

In vitro innervated human muscle is the only experimental model to study synaptogenesis of the neuromuscular junction in humans. Cultured human muscle never contracts spontaneously but will if innervated and therefore is a suitable model to study the effects of specific neural factors on the formation of functional neuromuscular contacts. Here, we tested the hypothesis that nerve derived factor agrin is essential for the formation of functional synapses between human myotubes and motoneurons growing from the explant of embryonic rat spinal cord. Agrin actions were blocked by heparin and the formation of functional neuromuscular contacts was quantitated. At a heparin concentration of 25 microg/ml, the number of functional contacts was significantly reduced. At higher concentrations, formation of such contacts was blocked completely. Except at the highest heparin concentrations (150 microg/ml) neuronal outgrowth was normal indicating that blockade of neuromuscular junction formation was not due to neuronal dysfunction. Our results are in accord with the concept that binding of neural agrin to the synaptic basal lamina is essential for the formation of functional neuromuscular junctions in the human muscle.

Heparin inhibits acetylcholine receptor aggregation at two distinct steps in the agrin-induced pathway.

Muscle cells depend on motoneurons for the initiation of postsynaptic differentiation during early development of the neuromuscular junction. Motoneurons secrete specific isoforms of the extracellular matrix protein agrin which trigger the aggregation of acetylcholine receptors (AChRs) on the muscle surface. Both motoneuron- and agrin-induced AChR aggregation are inhibited by heparin. Here we show that this inhibition is due to two separate and distinguishable mechanisms. At high concentrations, heparin directly binds to agrin isoforms which contain the peptide KSRK, resulting in a virtually complete inhibition of AChR clustering. Heparin and other polyanions do not bind to agrin splicing variants without KSRK insert. Isoforms containing or lacking the KSRK insert have a high potency to induce AChR aggregation in the presence of an activating eight-amino-acid insert. This activity is inhibited by low concentrations of heparin even in the absence of any binding of heparin to agrin. Therefore, this second type of inhibition is due to the interaction of heparin with a downstream component of the agrin-induced clustering pathway. Binding of heparin to this yet unidentified component substantially decreases, but does not completely abolish AChR aggregation. The inhibition is particularly strong on myotubes which have not completely matured in culture.

Inhibition of agrin-mediated acetylcholine receptor clustering by utrophin C-terminal peptides.

BACKGROUND: Agrin is an extracellular matrix protein that is required for neuromuscular synaptogenesis and is particularly important in the clustering of acetylcholine receptors at post-synaptic sites. Little is known about the signal transduction pathway of agrin-mediated receptor clustering, although cytoskeletal elements and a dystrophin associated glycoprotein complex (DGC) have been implicated. Because agrin binds to alpha-dystroglycan, a member of the DGC, and the DGC is linked to actin through utrophin at postsynaptic sites, it has been suggested that binding of utrophin to the DGC plays a central role in agrin mediated receptor clustering. RESULTS: To test this hypothesis, we expressed at high levels the DGC binding domains of utrophin in cultured myotubes using recombinant Semliki Forest Virus. Myotubes expressing the utrophin and dystrophin DGC binding domain formed significantly fewer acetylcholine receptor clusters in response to agrin than myotubes expressing other proteins. CONCLUSIONS: These results suggest involvement of the DGC and utrophin in the signal transduction pathway of agrin-mediated acetylcholine receptor cluster formation or stabilization.