Agrin has a pathological role in the progression of oral cancer.

BACKGROUND: The extracellular matrix modulates the hallmarks of cancer. Here we examined the role of agrin-a member of this matrix-in progression of oral squamous cell carcinoma (OSCC). METHODS: We evaluated the immunohistochemical expression of agrin in OSCC and dysplasias. Benign lesions were used as control. In subsequent experiments, we investigated whether the silencing of agrin interferes with tumour expansion both in vitro as well as in vivo. To gain insights into the role of agrin, we identified its protein network (interactome) using mass spectrometry-based proteomics and bioinformatics. Finally, we evaluated the clinical relevance of agrin interactome. RESULTS: Agrin was elevated in malignant and premalignant lesions. Further, we show that agrin silencing interferes with cancer cell motility, proliferation, invasion, colony and tumour spheroid formation, and it also reduces the phosphorylation of FAK, ERK and cyclin D1 proteins in OSCC cells. In orthotopic model, agrin silencing reduces tumour aggressiveness, like vascular and neural invasion. From a clinical perspective, agrin contextual hubs predict a poor clinical prognosis related with overall survival. CONCLUSIONS: Altogether, our results demonstrate that agrin is a histological marker for the progression of oral cancer and is a strong therapeutic target candidate for both premalignant and OSCC lesions.

Engineered agrin attenuates the severity of experimental autoimmune myasthenia gravis.

INTRODUCTION: Agrin is essential for the formation and maintenance of neuromuscular junctions (NMJs). NT-1654 is a C-terminal fragment of mouse neural agrin. In this study, we determined the effects of NT-1654 on the severity of experimental autoimmune myasthenia gravis (EAMG). METHODS: EAMG was induced in female Lewis rats by immunization with the Torpedo acetylcholine receptor (tAChR) and complete Freund's adjuvant (CFA). NT-1654 was dissolved in phosphate-buffered saline (PBS) and injected daily subcutaneously into tAChR immunized rats during the first 10 days after immunization, and then every other day for the following 20 days. RESULTS: We showed that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. DISCUSSION: We demonstrated that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. Muscle Nerve 57: 814-820, 2018.

Agrin mediates chondrocyte homeostasis and requires both LRP4 and α-dystroglycan to enhance cartilage formation in vitro and in vivo.

OBJECTIVES: Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. In other tissues, it connects the cytoskeleton to the basement membrane through binding to α-dystroglycan. Prompted by an unexpected expression pattern, we investigated the role and receptor usage of agrin in cartilage. METHODS: Agrin expression pattern was investigated in human osteoarthritic cartilage and following destabilisation of the medial meniscus in mice. Extracellular matrix (ECM) formation and chondrocyte differentiation was studied in gain and loss of function experiments in vitro in three-dimensional cultures and gain of function in vivo, using an ectopic cartilage formation assay in nude mice. Receptor usage was investigated by disrupting LRP4 and α-dystroglycan by siRNA and blocking antibodies respectively. RESULTS: Agrin was detected in normal cartilage but was progressively lost in OA. In vitro, agrin knockdown resulted in reduced glycosaminoglycan content, downregulation of the cartilage transcription factor SOX9 and other cartilage-specific ECM molecules. Conversely, exogenous agrin supported cartilage differentiation in vitro and ectopic cartilage formation in vivo. In the context of cartilage differentiation, agrin used an unusual receptor repertoire requiring both LRP4 and α-dystroglycan. CONCLUSIONS: We have discovered that agrin strongly promotes chondrocyte differentiation and cartilage formation in vivo. Our results identify agrin as a novel potent anabolic growth factor with strong therapeutic potential in cartilage regeneration.

Agrin-signaling is necessary for the integration of newly generated neurons in the adult olfactory bulb.

In the adult forebrain, new interneurons are continuously generated and integrated into the existing circuitry of the olfactory bulb (OB). In an attempt to identify signals that regulate this synaptic integration process, we found strong expression of agrin in adult generated neuronal precursors that arrive in the olfactory bulb after their generation in the subventricular zone. While the agrin receptor components MuSK and Lrp4 were below detection level in neuron populations that represent synaptic targets for the new interneurons, the alternative receptor α3-Na(+)K(+)-ATPase was strongly expressed in mitral cells. Using a transplantation approach, we demonstrate that agrin-deficient interneuron precursors migrate correctly into the OB. However, in contrast to wild-type neurons, which form synapses and survive for prolonged periods, mutant neurons do not mature and are rapidly eliminated. Using in vivo brain electroporation of the olfactory system, we show that the transmembrane form of agrin alone is sufficient to mediate integration and demonstrate that excess transmembrane agrin increases the number of dendritic spines. Last, we provide in vivo evidence that an interaction between agrin and α3-Na(+)K(+)-ATPase is of functional importance in this system.

Expression of angiopoietin-like 3 associated with puromycin-induced podocyte damage.

Angiopoietin-like 3 (ANGPTL3) is a secreted protein of the angiopoietin family and is involved in angiogenesis and lipid metabolism regulation. However, there is little data regarding the role of ANGPTL3 in kidney injury. We recently reported the glomerular distribution of ANGPTL3 in Adriamycin nephropathy in rats. In the present paper, we report expression of ANGPTL3 by murine podocytes in vitro. Puromycin-induced injury of cultured podocytes showed a time-dependent upregulation of ANGPTL3 accompanied by a time-dependent downregulation of perlecan and agrin by Western blot and RT-PCR analysis. In addition, the increased expression of ANGPTL3 following gene transfection upregulated the expression of perlecan and agrin in podocytes. Double immunolabeling demonstrated colocalization of perlecan and ANGPTL3 on podocytes following pcDNA3.1-ANGPTL3 transfection. To explore how ANGPTL3 transfection modulates the effect of puromycin on podocytes, we compared cell adhesion in untreated podocytes and ANGPTL3-transfected podocytes. ANGPTL3 gene transfection significantly ameliorated puromycin-induced podocyte detachment. In conclusion, ANGPTL3 expression is upregulated in puromycin-induced podocyte damage and is associated with the reduction of perlecan and agrin expression.

Distinctive peri-luminal presence of agrin in murine and human carotid atherosclerotic plaques.

The clinical consequences of arterial atherosclerotic lesions depend, apart from their size, on their composition of cellular and extracellular components. While an intact endothelium at the interface of atherosclerotic plaques towards the blood can prevent its erosion, underlying smooth muscle cells within the plaque can reduce the risk of plaque ruptures, due to the deposition of stabilizing extracellular matrix. Basement membranes underlay and support the function of endothelial cells, and embed smooth muscle cells in the media, the source of most smooth muscle cells within atherosclerotic plaques. In the present study mouse atherosclerotic plaques were comparatively analyzed for the basement membrane components laminin, type IV collagen, perlecan, and agrin. Distinct agrin immunofluorescence was found in the peri-luminal area in mouse carotid atherosclerotic plaques. Agrin was also clearly present in the media, with a significant increase in regions directly associated with plaque tissue. In addition, ten human endarterectomy specimens were investigated for this heparan sulfate proteoglycan. No statistically significant differences in agrin immunofluorescence were noticed between five specimens from symptomatic and five from asymptomatic patients. In all these plaques agrin was present in a distinctive manner in a narrow zone partially or almost completely surrounding the lumen. Additionally it was also present around the small lumina of the CD31-positive neovessels. The presence of agrin at locations with particular importance for the growth and stability of atherosclerotic plaques renders this molecule strategically positioned to influence plaque development and vulnerability.

Comparison of the expression of agrin, a basement membrane heparan sulfate proteoglycan, in cholangiocarcinoma and hepatocellular carcinoma.

Heparan sulfate proteoglycans mediate cell adhesion and control the activities of numerous growth and motility factors. They play a critical role in carcinogenesis and tumor progression. Agrin is a large multidomain heparan sulfate proteoglycan associated with basement membranes in several tissues. The expression of agrin in the liver has recently been described under physiologic and pathologic conditions. However, little is known about its role in malignancies. We aimed to study the mRNA and protein expression of agrin in cholangiocarcinoma (CC) and focused on the differences between CC and hepatocellular carcinoma (HCC). Eighty surgically removed liver specimens were studied by immunohistochemistry. Representative samples were used for immunoblotting. mRNA expression was measured in 32 samples by real-time polymerase chain reaction. By immunohistochemistry, agrin was seen around bile ducts and blood vessels within the portal areas in the normal liver. Although no expression was found within the hepatic lobules, agrin was deposited in the neovascular basement membrane in HCCs. Agrin was abundant in the tumor-specific basement membrane in well-differentiated areas of CCs, whereas with immunostaining, it was fragmented, decreased, or it even disappeared in less differentiated areas and sites of infiltration. By real-time polymerase chain reaction, up-regulation of agrin expression was measured in HCCs compared with that in the normal liver. CC samples showed an even higher expression of agrin. Immunoblotting confirmed these findings. Our results indicate that agrin might play an important role in neoangiogenesis in human HCC, being a part of the newly formed vasculature. In CC, however, agrin might be involved in tumor progression.

Schwann cells express active agrin and enhance aggregation of acetylcholine receptors on muscle fibers.

To explore novel roles of glial cells in synaptic function and formation, we examined the expression of agrin in frog Schwann cells and tested their role in the aggregation of acetylcholine receptors (AChRs). Using reverse transcription-PCR, we found that Schwann cells along nerve fibers in tadpoles expressed only the inactive agrin isoform B0 but began to also express active agrin isoforms B11 and B19 at approximately metamorphosis. During nerve regeneration in the adult, the expression of these active agrin isoforms in Schwann cells was upregulated, including the appearance of the most potent isoform, B8. This upregulation was induced by regenerating axons but not by nerve injury per se. In muscle cultures, the presence of adult Schwann cells enhanced the number and the total area of AChR aggregates 2.2- and 4.5-fold, respectively, and this enhancement was eliminated by heparin treatment. Furthermore, adult Schwann cells in culture expressed active agrin isoforms and produced agrin protein. Using a novel technique to selectively ablate perisynaptic Schwann cells (PSCs) at the neuromuscular junction, we found that PSCs also expressed active agrin isoforms B11 and B19, and these active isoforms were upregulated, including the appearance of B8, during reinnervation. Observation in vivo showed that extrajunctional AChR aggregates were associated with PSC sprouts after nerve injury and subsequent reinnervation. These results suggest that, contrary to the prevailing view that only neurons express active agrin, glial cells also express active agrin and play a role in the aggregation of AChRs both in vitro and in vivo.

Synapse-forming axons and recombinant agrin induce microprocess formation on myotubes.

We examined cell-surface behavior at nerve-muscle contacts during synaptogenesis in cocultures of rat ventral spinal cord (VSC) neurons and myotubes. Developing synapses in 1-d-old cocultures were identified by the presence of axon-induced acetylcholine receptor (AChR) aggregation. Identified regions were then examined by transmission and scanning electron microscopy. The myotube surface near contacts with axons that induced AChR aggregation typically displayed ruffles, microvilli, and filopodia (microprocesses), indicating motility of the myotube surface. At some of these contact sites microprocesses were wrapped around the axon, resulting in the partial or total "submersion" of the axon within the myotube contours. Sites of myotube contact with somata and dendrites of the same neurons showed much less evidence of motility and surface interaction than sites of contact with axons. Moreover, the distance between opposed membranes of axons and myotubes was smaller than between dendrites or somata and myotubes, suggesting stronger adhesion of axons. These results suggest polarized expression of molecules involved in the induction of microprocess formation and adhesion in developing VSC neurons. We therefore tested the ability of agrin, which is preferentially secreted by axons, to induce microprocess formation in myotubes. Addition of recombinant C-terminal agrin to culture medium resulted in formation of microprocesses within 3 hr. Myotubes transfected with full-length rat agrin constructs displayed numerous filopodia, as revealed by fluorescence microscopy. The results suggest that the induction of muscle cell surface motility may be linked to the signaling processes that trigger the initial formation of the neuromuscular junction.

Expression, identification and biological effects of the novel recombination protein, PACAP38-NtA, with high bioactivity.

Pituitary adenylate cyclase­activating polypeptide (PACAP) is a type of neuropeptide with multiple biological functions. However, it has a short half­life period in the body, ~3 to 5 min, restricting its further development as a drug that can promote the recovery of nerve injury. In vitro and in vivo experiments have shown that PACAP can repair the epithelial cell on the surface of the injured cornea, as PACAP can act on the trigeminal nerve cell to secrete other active neurotransmitters, which can promote corneal epithelial cell proliferation and differentiation. In the present study, PACAP is connected to the N­terminal agrin domain (NtA) with a genetic engineering method, which allows the function of repairing the injured nerve. Notably, the recombinant polypeptide can interact with laminin, improving the biological effect of PACAP in repairing the injured nerve. In the study, the recombinant protein was constructed by combining PACAP38 and NtA by genetic engineering, and it is expressed in the pronucleus escherichia coli. The recombinant protein, PACAP38­NtA, is obtained with a two­step purification method, including anion­exchange chromatography and Ni­affinity chromatography, with the purity reaching >90%. The in vitro experiment has shown that this recombinant protein not only has the neurotrophy and neural restoration function of PACAP, but also has the function of an anchoring protein as laminin interacts with NtA. According to the in vitro anti­apoptosis, PC12 axon growth and ELISA experiments, this protein has the biological activity of a recombinant protein. PACAP38­NtA also has an anchoring function as NtA and laminin interact with good biological activity.

Expression of agrin in the developing and adult rat brain.

Agrin, a synaptic basal lamina protein, is essential for the formation of the vertebrate neuromuscular junction. Agrin's role in synaptogenesis in the central nervous system has, however, not been elucidated. Therefore, we performed immunohistochemical analysis of agrin localization in adult rat brain using agrin-specific polyclonal antibodies. Our results show that agrin immunoreactivity is detected in neuronal cells throughout the brain, and that agrin is expressed in many morphologically and neurochemically distinct neuronal populations. Within neurons, agrin-immunoreactive material is present in dendrites. To determine agrin isoform expression in the central nervous system, we analysed the pattern of expression of several isoforms during development of the rat brain. Our results indicate that alternative splicing of agrin is specifically regulated in the nervous system; isoforms of the Y=4 (i.e. Ag x,4,0, Ag x,4,8 and Ag x,4,19), Z=8 and Z=19 type are expressed exclusively in the nervous system. Agrin expression precedes synaptogenesis and is developmentally regulated in neural tissues. To evaluate stimuli that may be involved in the regulation of agrin expression, we monitored the patterns of isoform expression following a depolarizing stimulus. Our results show that agrin expression in the adult hippocampus is regulated in an activity-dependent manner, with kinetics of induction resembling a delayed early response gene.

Agrin gene expression in mouse somatosensory cortical neurons during development in vivo and in cell culture.

Agrin is an extracellular matrix protein involved in the formation of the postsynaptic apparatus of the neuromuscular junction. In addition to spinal motor neurons, agrin is expressed by many other neuronal populations throughout the nervous system. Agrin's role outside of the neuromuscular junction, however, is poorly understood. Here we use the polymerase chain reaction to examine expression and alternative splicing of agrin in mouse somatosensory cortex during early postnatal development in vivo and in dissociated cell culture. Peak levels of agrin gene expression in developing cortex coincide with ingrowth of thalamic afferent fibres and formation of thalamocortical and intracortical synapses. Analysis of alternatively spliced agrin messenger RNA variants shows that greater than 95% of all agrin in developing and adult somatosensory cortex originates in neurons, including isoforms that have little or no activity in acetylcholine receptor aggregation assays. The levels of expression of "active" and "inactive" isoforms, however, are regulated during development. A similar pattern of agrin gene expression is also observed during a period when new synapses are being formed between somatosensory neurons growing in dissociated cell culture. Changes in agrin gene expression, observed both in vivo and in vitro, are consistent with a role for agrin in synapse formation in the central nervous system.

Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)

Expression of agrin mRNA is altered following seizures in adult rat brain.

Agrin mRNA is broadly distributed throughout the adult rat brain, consistent with its proposed role in synaptogenesis and the organization of synaptic proteins in the central nervous system. The present study examined the effect of neuronal activity on agrin mRNA expression in adult rat forebrain using the hilus lesion paradigm for seizure induction and in situ hybridization and polymerase chain reaction techniques for quantification and characterization of agrin mRNA content. Seizures induced rapid, prolonged, and region-specific changes in agrin mRNA expression with the most prominent alterations occurring in hippocampal and cortical neurons. However, there were no detectable perturbations in the relative abundance of alternatively spliced agrin transcripts in affected brain regions. Activity-dependent changes in agrin expression suggest a role for this protein in modifications of synaptic structure associated with functional synaptic plasticity.

Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

Over-expression of acetylcholinesterase stimulates the expression of agrin in NG108-15 cells.

Several lines of evidence suggest the non-cholinergic functions of acetylcholinesterase (AChE) in promoting neurite outgrowth of cultured neurons and in inducing the postsynaptic specializations of developing neuromuscular junctions. In order to support the hypothesis, a cholinergic synapse-forming cell line NG108-15 was over-expressed with chick AChE by cDNA transfection. The transfected NG108-15 cells secreted a approximately 105-kDa protein, recognized by anti-AChE antibody in Western blot analysis, corresponding to the chick AChE catalytic subunit. Over 80% of the recombinant enzyme were secreted into the conditioned medium and they were enzymatically active. In the NG108-15 cell-muscle co-cultures, the AChR-aggregating activity of NG108-15 cells was increased by the over-expression of AChE. The increase in AChR-aggregating activity of the transfected NG108-15 cells paralleled with the increase in agrin and neurofilament expression of the transfected cells as determined by their corresponding antibodies. However, the intracellular cAMP level remained unchanged in the AChE over-expressed NG108-15 cells. These results support the hypothesis that AChE could play a role in promoting neuron differentiation.

Agrin requires specific proteins to selectively activate γ-aminobutyric acid neurons for pain suppression.

Agrin, a heparan sulfate proteoglycan functioning as a neuro-muscular junction inducer, has been shown to inhibit neuropathic pain in sciatic nerve injury rat models, via phosphorylation of N-Methyl-d-aspartate receptor NR1 subunits in gamma-aminobutyric acid neurons. However, its effects on spinal cord injury-induced neuropathic pain, a debilitating syndrome frequently encountered after various spine traumas, are unknown. In the present investigation, we studied the 50kDa agrin isoform effects in a quisqualic acid dorsal horn injection rat model mimicking spinal cord injury-induced neuropathic pain. Our results indicate that 50kDa agrin decreased only in the dorsal horn of neuropathic animals and increased 50kDa agrin expression in the dorsal horn, via intra-spinal injection of adeno-associated virus serum type two, suppressed spinal cord injury-induced neuropathic pain. Also, the reason why 50kDa agrin only activates the N-Methyl-d-aspartate receptor NR1 subunits in the GABA neurons, but not in sensory neurons, is unknown. Using immunoprecipitation and Western-blot analysis, two dimensional gel separation, and mass spectrometry, we identified several specific proteins in the reaction protein complex, such as neurofilament 200 and mitofusin 2, that are required for the activation of the NR1 subunits of gamma-aminobutyric acid inhibitory neurons by 50kDa agrin. These findings indicate that 50kDa agrin is a promising agent for neuropathic pain treatment.

Apoptosis inhibitors and mini-agrin have additive benefits in congenital muscular dystrophy mice.

Mutations in LAMA2 cause a severe form of congenital muscular dystrophy, called MDC1A. Studies in mouse models have shown that transgenic expression of a designed, miniaturized form of the extracellular matrix molecule agrin ('mini-agrin') or apoptosis inhibition by either overexpression of Bcl2 or application of the pharmacological substance omigapil can ameliorate the disease. Here, we tested whether mini-agrin and anti-apoptotic agents act on different pathways and thus exert additive benefits in MDC1A mouse models. By combining mini-agrin with either transgenic Bcl2 expression or oral omigapil application, we show that the ameliorating effect of mini-agrin, which acts by restoring the mechanical stability of muscle fibres and, thereby, reduces muscle fibre breakdown and concomitant fibrosis, is complemented by apoptosis inhibitors, which prevent the loss of muscle fibres. Treatment of mice with both agents results in improved muscle regeneration and increased force. Our results show that the combination of mini-agrin and anti-apoptosis treatment has beneficial effects that are significantly bigger than the individual treatments and suggest that such a strategy might also be applicable to MDC1A patients.

Quantitative and qualitative alterations of heparan sulfate in fibrogenic liver diseases and hepatocellular cancer.

Heparan sulfate (HS), due to its ability to interact with a multitude of HS-binding factors, is involved in a variety of physiological and pathological processes. Remarkably diverse fine structure of HS, shaped by non-exhaustive enzymatic modifications, influences the interaction of HS with its partners. Here we characterized the HS profile of normal human and rat liver, as well as alterations of HS related to liver fibrogenesis and carcinogenesis, by using sulfation-specific antibodies. The HS immunopattern was compared with the immunolocalization of selected HS proteoglycans. HS samples from normal liver and hepatocellular carcinoma (HCC) were subjected to disaccharide analysis. Expression changes of nine HS-modifying enzymes in human fibrogenic diseases and HCC were measured by quantitative RT-PCR. Increased abundance and altered immunolocalization of HS was paralleled by elevated mRNA levels of HS-modifying enzymes in the diseased liver. The strong immunoreactivity of the normal liver for 3-O-sulfated epitope further increased with disease, along with upregulation of 3-OST-1. Modest 6-O-undersulfation of HCC HS is probably explained by Sulf overexpression. Our results may prompt further investigation of the role of highly 3-O-sulfated and partially 6-O-desulfated HS in pathological processes such as hepatitis virus entry and aberrant growth factor signaling in fibrogenic liver diseases and HCC.