Exostosin-1 Research Articles

AEG-1 as a Novel Therapeutic Target in Colon Cancer: A Study from Silencing AEG-1 in BALB/c Mice to Large Data Analysis.

Astrocyte elevated gene-1 (AEG-1) is overexpressed in various malignancies. Exostosin-1 (EXT-1), a tumor suppressor, is an intermediate for malignant tumors. Understanding the mechanism behind the interaction between AEG-1 and EXT-1 may provide insights into colon cancer metastasis. AOM/DSS was used to induce tumor in BALB/c mice. Using an in vivo-jetPEI transfection reagent, transient transfection of AEG-1 and EXT-1 siRNAs were achieved. Histological scoring, immunohistochemical staining, and gene expression studies were performed from excised tissues. Data from the Cancer Genomic Atlas and GEO databases were obtained to identify the expression status of AEG-1 and itsassociation with the survival. In BALB/c mice, the AOM+DSS treated mice developed necrotic, inflammatory and dysplastic changes in the colon with definite clinical symptoms such as loss of goblet cells, colon shortening, and collagen deposition. Administration of AEG-1 siRNA resulted in a substantial decrease in the disease activity index. Mice treated with EXT-1 siRNA showed diffusely reduced goblet cells. In vivo investigations revealed that PTCH-1 activity was influenced by upstream gene AEG-1, which in turn may affect EXT-1 activity. Data from The Cancer Genomic Atlas and GEO databases confirmed the upregulation of AEG-1 and downregulation of EXT-1 in cancer patients. This study revealed that AEG-1 silencing might alter EXT-1 expression indirectly through PTCH-1, influencing cell-ECM interactions, and decreasing dysplastic changes, proliferation and invasion.

Endothelial dysfunction is dampened by early administration of fresh frozen plasma in a rodent burn shock model.

Endothelial dysfunction has been implicated in the pathogenesis of burn shock affecting patients with large thermal injury. In response to injury, glycocalyx components like Syndecan-1 (SDC-1) are shed into circulation and have been used as markers of endothelial damage. Previous work in our laboratory has shown that plasma inclusive resuscitation (PIR) with fresh frozen plasma (FFP) ameliorates endothelial damage. However, there remains a paucity of information regarding optimal timing and dosing of PIR as well as organ-specific endothelial responses to shock. We aimed to examine the impact of PIR on endothelial dysfunction using clinically translatable timing and dosing. Sprague-Dawley rats were used to create thermal burns. Rats were subjected to 40% total body surface area scald burns and were resuscitated with lactated Ringer's (LR) only, LR plus albumin, and LR plus early 1 mL boluses of FFP at 0, 2, 4, and 8 hours postinjury. A late group also received LR plus FFP starting at hour 10 postinjury. Syndecan-1 levels were quantified by enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction analysis characterized transcription of glycocalyx components and inflammatory cytokines in the lung and spleen. Evan's blue dye was used to quantify amount of vascular leakage. Lactated Ringer's plus early FFP reduced Evan's blue dye extravasation when compared with LR only groups, while late FFP did not. When comparing LR only versus LR plus early FFP, SDC-1 levels were reduced in the LR plus early FFP group at hours 8, 12, and 24 (5.23 vs. 2.07, p < 0.001; 4.49 vs. 2.05, p < 0.01; and 3.82 vs. 2.08, p < 0.05, respectively). Lactated Ringer's only groups had upregulation of Exostosin-1 and SDC-1 in the lung compared with LR plus early FFP groups ( p < 0.01 and p < 0.05) and upregulation of cytokines interluekin-10 and interferon γ ( p < 0.001 and p < 0.001). Early administration of LR plus FFP reduces the magnitude of SDC-1 shedding and dampens the cytokine response to injury. The upregulation of glycocalyx components as a response to endothelial injury is also decreased in the lung and spleen by LR plus early FFP administration.

Chondroitin sulfate proteoglycan promotes APRIL‐induced tumor cell proliferation

AbstractA proliferation‐inducing ligand (APRIL), a member of the tumor necrosis factor superfamily, affects the survival and proliferation of tumor cells. Understanding the mechanism of action of APRIL in tumor cells, including intracellular signaling, is important for its potential use in diagnostics and prognosis. It has been shown that APRIL‐induced tumor proliferation requires heparan sulfate (HS) proteoglycans to mediate the binding of APRIL to tumor cells. Here, we show that chondroitin sulfate (CS) proteoglycan mainly contributes to the APRIL‐stimulated proliferation of triple‐negative breast cancer BT‐549 cells. Knockout of chondroitin 4‐O‐sulfotransferase‐1 (C4ST‐1), a key CS biosynthetic enzyme, suppressed APRIL‐induced tumor proliferation, whereas deficiency of exostosin 1 (EXT1), a key HS biosynthetic enzyme, had only weak effects. Molecular interaction analyses using Biacore revealed that although CS did not directly bind to tumor growth through Ca2+ modulator interactor (TACI), it enhanced the binding of APRIL to the APRIL receptor, TACI. The small leucine‐rich proteoglycan, biglycan, plays a pivotal role in tumor growth and progression. Biglycan knockdown inhibited BT‐549 cell proliferation. These results suggest that CS synthesized by C4ST‐1 participates in APRIL signaling and modulates pathological events in tumors.

Clinical significance of exostosin 1 in confirmed and suspected lupus membranous nephropathy

ObjectiveThis study aimed to investigate the clinical significance of exostosin 1 (EXT1) in confirmed and suspected lupus membranous nephropathy (LMN).MethodsEXT1 was detected in 67 renal tissues of M-type phospholipase A2...

A Diagnostic and Therapeutic Dilemma Concerning Exostosin 1/Exostosin 2-associated Lupus-like Membranous Nephropathy with Positive Antinuclear Antibody in an Elderly Man with Various Immune Abnormalities.

Exostosin 1 (EXT1) and exostosin 2 (EXT2)-associated membranous nephropathy (MN) may be associated with active autoimmune disease. We encountered an elderly man who presented with EXT1/EXT2-associated lupus-like MN with full house immune deposits, monoclonal gammopathy of uncertain significance and Sjögren's syndrome. The patient exhibited various other immune abnormalities. Although he did not fulfill the criteria of clinical systemic lupus erythematosus (SLE), he met a stand-alone renal criterion of the Systemic Lupus International Collaborating Clinics (SLICC) 2012. Whether or not a stand-alone renal criterion with EXT1/EXT2 positivity, as in the present patient, can efficiently guide decisions regarding the diagnosis and treatment of SLE remains a clinical dilemma.

Clinical and Histopathologic Characteristics of Pediatric Patients With Primary Membranous Nephropathy

Primary membranous nephropathy (PMN) is uncommon in children. Therefore, data on the clinical course of affected children are scarce. In recent years, several novel antigens have been implicated in the pathogenesis of PMN. However, the histopathologic characteristics of pediatric patients with PMN remain poorly represented in the literature. We have retrospectively analyzed the clinical presentation and outcomes data of 21 children with PMN from 3 centers in the United States. In addition, we have identified novel antigens in biopsy specimens from these patients and correlated their presence or absence to clinical outcomes. Finally, we compared the results of the novel antigen staining from our clinical cohort to a validation cohort of 127 biopsy specimens from children with PMN at Arkana Laboratories. The data from the 2 cohorts demonstrated similar overall antigen positivity rates of 62% to 63%, with phospholipase A2 receptor (PLA2R) and exostosin 1 (EXT1) being the most commonly found antigens. Results from the clinical cohort showed that overall, the kidney prognosis for children with PMN was good, with 17 of 21 patients entering a complete or partial remission. Children who were positive for PLA2R or EXT1 were significantly more likely to enter remission than those in the antigen negative group. Approximately 60% of pediatric membranous cases are positive for a novel antigen on kidney biopsy and the clinical prognosis is generally favorable. More studies are needed to understand the clinical implications of each specific novel antigen.

Exostosin 1 Knockdown Induces Chemoresistance in MV3 Melanoma Cells by Upregulating JNK and MEK/ERK Signaling.

Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC50 values two- or fourfold, respectively. Resistance formation was scarcely related to HSPG deficiency, suggested by enzymatic cleavage of HSPG in control cells. Notably, EXT1kd induced an upregulation of EGFR signaling via JNK and MEK/ERK, and hence blocking these kinases returned resistance to a sensitive level. JNK appeared as a key signal component, also inducing higher migratory activity of EXT1kd cells. Furthermore, EXT1kd upregulated thrombotic properties of MV3 cells, indicated by tissue factor and PAR-1 expression, functionally reflected by a stronger activation of platelet aggregation. EXT1 was confirmed to act as a tumor suppressor, shown here for the first time to affect chemosensitivity of melanoma cells.

Role of Heparan Sulfate in Vasculogenesis of Dental Pulp Stem Cells

Dental pulp stem cells (DPSCs) can differentiate into vascular endothelial cells and display sprouting ability. During this process, DPSC responses to the extracellular microenvironment and cell–extracellular matrix interactions are critical in regulating their ultimate cell fate. Heparan sulfate (HS) glycosaminoglycan, a major component of extracellular matrix, plays important roles in various biological cell activities by interacting with growth factors and relative receptors. However, the regulatory function of HS on vasculogenesis of mesenchymal stem cells remains unclear. The objective of this study was to investigate the role of HS in endothelial differentiation and vasculogenesis of DPSCs. Our results show that an HS antagonist suppressed the proliferation and sprouting ability of DPSCs undergoing endothelial differentiation. Furthermore, expression of proangiogenic markers significantly declined with increasing dosages of the HS antagonist; in contrast, expression of stemness marker increased. Silencing of exostosin 1 (EXT1), a crucial glycosyltransferase for HS biosynthesis, in DPSCs using a short hairpin RNA significantly altered their gene expression profile. In addition, EXT1-silenced DPSCs expressed lower levels of endothelial differentiation markers and displayed a reduced vascular formation capacity compared with control DPSCs transduced with scrambled sequences. The sprouting ability of EXT1-silenced DPSCs was rescued by the addition of exogenous HS in vitro. Next, we subcutaneously transplanted biodegradable scaffolds seeded with EXT1-silenced or control DPSCs into immunodeficient mice. Lumen-like structures positive for human CD31 and von Willebrand factor were formed by green fluorescent protein–transduced DPSCs. Numbers of blood-containing vessels were significantly lower in scaffolds loaded with EXT1-silenced DPSCs than specimens implanted with control DPSCs. Collectively, our findings unveil the crucial role of HS on endothelial differentiation and vasculogenesis of DPSCs, opening new perspectives for the application of HS to tissue engineering and dental pulp regeneration.

Heparan sulfate is necessary for the early formation of nascent fibronectin and collagen I fibrils at matrix assembly sites

Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.

Protectin conjugates in tissue regeneration 1 restores lipopolysaccharide-induced pulmonary endothelial glycocalyx loss via ALX/SIRT1/NF-kappa B axis

BackgroundEndothelial glycocalyx loss is integral to increased pulmonary vascular permeability in sepsis-related acute lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel macrophage-derived lipid mediator exhibiting potential anti-inflammatory and pro-resolving benefits.MethodsPCTR1 was administrated intraperitoneally with 100 ng/mouse after lipopolysaccharide (LPS) challenged. Survival rate and lung function were used to evaluate the protective effects of PCTR1. Lung inflammation response was observed by morphology and inflammatory cytokines level. Endothelial glycocalyx and its related key enzymes were measured by immunofluorescence, ELISA, and Western blot. Afterward, related-pathways inhibitors were used to identify the mechanism of endothelial glycocalyx response to PCTR1 in mice and human umbilical vein endothelial cells (HUVECs) after LPS administration.ResultsIn vivo, we show that PCTR1 protects mice against lipopolysaccharide (LPS)-induced sepsis, as shown by enhanced the survival and pulmonary function, decreased the inflammatory response in lungs and peripheral levels of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1β. Moreover, PCTR1 restored lung vascular glycocalyx and reduced serum heparin sulphate (HS), syndecan-1 (SDC-1), and hyaluronic acid (HA) levels. Furthermore, we found that PCTR1 downregulated heparanase (HPA) expression to inhibit glycocalyx degradation and upregulated exostosin-1 (EXT-1) protein expression to promote glycocalyx reconstitution. Besides, we observed that BAY11-7082 blocked glycocalyx loss induced by LPS in vivo and in vitro, and BOC-2 (ALX antagonist) or EX527 (SIRT1 inhibitor) abolished the restoration of HS in response to PCTR1.ConclusionPCTR1 protects endothelial glycocalyx via ALX receptor by regulating SIRT1/NF-κB pathway, suggesting PCTR1 may be a significant therapeutic target for sepsis-related acute lung injury.

Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells.

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.

Mechanisms of Primary Membranous Nephropathy.

Membranous nephropathy (MN) is an autoimmune disease of the kidney glomerulus and one of the leading causes of nephrotic syndrome. The disease exhibits heterogenous outcomes with approximately 30% of cases progressing to end-stage renal disease. The clinical management of MN has steadily advanced owing to the identification of autoantibodies to the phospholipase A2 receptor (PLA2R) in 2009 and thrombospondin domain-containing 7A (THSD7A) in 2014 on the podocyte surface. Approximately 50–80% and 3–5% of primary MN (PMN) cases are associated with either anti-PLA2R or anti-THSD7A antibodies, respectively. The presence of these autoantibodies is used for MN diagnosis; antibody levels correlate with disease severity and possess significant biomarker values in monitoring disease progression and treatment response. Importantly, both autoantibodies are causative to MN. Additionally, evidence is emerging that NELL-1 is associated with 5–10% of PMN cases that are PLA2R- and THSD7A-negative, which moves us one step closer to mapping out the full spectrum of PMN antigens. Recent developments suggest exostosin 1 (EXT1), EXT2, NELL-1, and contactin 1 (CNTN1) are associated with MN. Genetic factors and other mechanisms are in place to regulate these factors and may contribute to MN pathogenesis. This review will discuss recent developments over the past 5 years.

Silencing of Astrocyte Elevated Gene-1 (AEG-1) inhibits the proliferative and invasive potential through interaction with Exostosin-1 (EXT-1) in primary and metastatic colon cancer cells

Silencing of Astrocyte Elevated Gene-1 (AEG-1) inhibits the proliferative and invasive potential through interaction with Exostosin-1 (EXT-1) in primary and metastatic colon cancer cells

Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling

Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. Hypertrophic chondrocytes were significantly more abundant (P=0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P=0.036 for femur; and P<0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P=0.007). HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

Correlation between mutated genes and forearm deformity in patients with multiple osteochondroma

BackgroundsExostosin-1 (EXT1) and exostosin-2 (EXT2) cause multiple osteochondromas (MO). In this study, we investigated the correlation between forearm deformity and mutant EXTs in Japanese families with MO. MethodsWe evaluated 112 patients in 71 families with MO. Genomic DNA was isolated from peripheral blood leucocytes. Of these, 28 patients were selected and underwent radiography for their forearms since they had gross forearm deformities. We measured the radial articular angle (RAA), ulna variance (UV), carpal slip (CS), and percentage of radial bowing (%RB) to compare between patients with mutant EXT1 or EXT2 and those with missense or other mutations using Student's t-test. ResultsTwenty-two (78.6%) and 6 (11.4%) out of 28 patients had mutations in EXT1 and EXT2, respectively. Nine (32.1%) and 19 (67.9%) of the 28 patients had missense and other mutations, respectively. The mean age of patients with EXT1 and EXT2 were 25.9 ± 20.3 and 33.5 ± 25.4 years, respectively and those with missense mutation and other mutations were 28.7 ± 27.0 and 24.6 ± 17.0 years, respectively. There were no significant differences in RAA, UV, and RB between patients harbouring mutant EXT1 or EXT2 (RAA, 40.1 ± 8.7 and 31.5 ± 13.9°; UV, −2.7 ± 5.7 and −3.1 ± 3.7 mm; %RB, 8.6 ± 1.5 and 8.3 ± 2.0%). CS was significantly greater in patients with mutant EXT1 than that in those with mutant EXT2 (EXT1, 44.1 ± 16.8%; EXT2, 18.6 ± 14.0%). There were no significant differences in RAA, UV, CS and %RB between patients with missense and other mutations. ConclusionsPatients with mutant EXT1 displayed greater CS than patients with mutant EXT2, indicating that patients with MO harbouring EXT1 mutations sustain more severe ulnar drift deformities than those with EXT2 mutations.

An investigation of genetic polymorphisms in heparan sulfate proteoglycan core proteins and key modification enzymes in an Australian Caucasian multiple sclerosis population

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system in young adults. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to the cell surface and the extracellular matrix. HSPG biosynthesis is a complex process involving enzymatic attachment of heparan sulfate (HS) chains to a core protein. HS side chains mediate specific ligand and growth factor interactions directing cellular processes including cell adhesion, migration and differentiation. Two main families of HSPGs exist, the syndecans (SDC1-4) and glypicans (GPC1-6). The SDCs are transmembrane proteins, while the GPC family are GPI linked to the cell surface. SDC1 has well-documented interactions with numerous signalling pathways. Genome-wide association studies (GWAS) have identified regions of the genome associated with MS including a region on chromosome 13 containing GPC5 and GPC6. International studies have revealed significant associations between this region and disease development. The exostosin-1 (EXT1) and sulfatase-1 (SULF1) are key enzymes contributing to the generation of HS chains. EXT1, with documented tumour suppressor properties, is involved in the initiation and polymerisation of the growing HS chain. SULF1 removes 6-O-sulfate groups from HS chains, affecting protein-ligand interactions and subsequent downstream signalling with HS modification potentially having significant effects on MS progression. In this study, we identified significant associations between single nucleotide polymorphisms in SDC1, GPC5 and GPC6 and MS in an Australian Caucasian case-control population. Further significant associations in these genes were identified when the population was stratified by sex and disease subtype. No association was found for EXT1 or SULF1.

Drug Discovery for Multiple Hereditary Exostoses

Multiple hereditary exostoses (MHE) is an autosomal dominant disorder that results from heterozygous loss‐of‐function genetic mutations in exostosin 1 (EXT1) or exostosin 2 (EXT2). One in 50,000 children worldwide are affected by this relatively rare pediatric disease, which is characterized by osteochondromas that form next to the growth plates of skeletal elements. EXT1 and EXT2 encode the copolymerase responsible for biosynthesis of the backbone of heparan sulfate (HS), a cell surface polysaccharide. Heterozygous mutations in EXT1 or EXT2 lead to shortened HS chains in plasma and various tissues. Currently, there are no approved therapies for this disease; therefore, there is much interest in preventing and/or slowing the growth of exostoses through the development of small molecules that enhance HS levels on the cell surface. In this study, a high‐throughput phenotypic screen was developed to discover potential small molecule drug candidates that augment HS expression in mammalian cells. Screening of a compound library of ~57,000 molecules resulted in the discovery of several N‐aryl‐2‐aminothiazoles that displayed favorable “drug‐like” physicochemical properties, exhibited minimal cytotoxicity, and enhanced HS expression up to 3‐fold on the cell surface. Preliminary studies of structure‐activity relationships identified active analogs of the primary hit and key physicochemical properties required for their biological activity. Ongoing studies aim to expand the SAR studies, identification of a preferred candidate for in vivo studies, and finding the biological target for these compounds. Overall, these studies provide a promising new avenue for the development of novel therapies for MHE and other disorders involving glycosaminoglycan deficiencies.Support or Funding InformationR01AR064202‐03 (to J.E.), K12HL141956 (to R.W.)

Proteomic Analysis of Complement Proteins in Membranous Nephropathy

IntroductionMembranous nephropathy (MN) is the most common cause of nephrotic syndrome in Caucasian adults. Phospholipase A2 receptor (PLA2R)– and exostosin 1 (EXT1)/exostosin 2 (EXT2)–associated MN represent the most common primary and secondary forms of MN. The complement profile using a proteomics approach has not been studied in these 2 common forms of MN.MethodsWe used laser microdissection and mass spectrometry (MS/MS) to dissect glomeruli and identify glomerular complement proteins in PLA2R-associated (n = 7), EXT1/EXT2-associated MN (n = 21), and 11 control cases (time 0 transplant biopsies).ResultsMS/MS identified high total spectral counts for PLA2R and EXT1/EXT2 in corresponding cases of PLA2R- and EXT1/EXT2-positive MN. Both PLA2R- and EXT1/EXT2-associated MN had high spectral counts of complement proteins C3, C4, C5, C6, C7, C8, and C9. Complement protein C1 was present in low spectral counts in EXT1/EXT2-associated MN. Regulators of complement activation that were detected in MN included higher spectral counts of FH, FHR-1, FHR-5, clusterin, vitronectin and lower spectral counts of FHR-3, FHR-4, and CD59. Low spectral counts of FB and properdin, key components of the alternative pathway, also were detected. IgG4 and IgG1 were the most abundant IgG subclasses in PLA2R- and EXT1/EXT2-associated MN. Lower spectral counts for C3, C4, and C5 were detected in control cases when compared with MN.ConclusionSignificant complement activation is present in MN as evidenced by large spectral counts of complement proteins from C3- and C4-based pathways, including regulatory proteins of complement pathways. These data suggest that anticomplement drugs may be effective in treatment for MN.

Insights into the molecular regulatory network of pathomechanisms in osteochondroma.

Osteochondroma is a benign autosomal dominant hereditary disease characterized by abnormal proliferation of cartilage in the long bone. It is divided into solitary osteochondroma and hereditary multiple exostoses (HMEs). The exostosin-1 (EXT-1) and exostosin-2 (EXT-2) gene mutations are well-defined molecular mechanisms in the pathogenesis of HME. EXT-1 and EXT-2 encode glycosyltransferases that are necessary for the synthesis of heparin sulfate. Accumulating evidence suggests that mutations in the EXT family induce changes in isolated hypogonadotropic hypogonadism-parathyroid hormone-related protein, bone morphogenetic protein, and fibroblast growth factor signaling pathways. Studies have also found that a large number of microRNAs (miRNAs) are abnormally expressed in osteochondroma tissues, and some of them also participate in several major signaling pathways. The regulation of miRNA expression could be another breakthrough in the treatment of osteochondroma. Although the pathogenesis of osteochondroma is very complicated, significant progress has been made in recent years. It is hoped that the pathogenesis of osteochondroma will be clearly understood and the most effective methods for the prevention and treatment of osteochondroma will be determined. This review provides an update on the recent progress in the interpretation of the underlying molecular mechanisms of osteochondroma.

Exostosin 1/Exostosin 2-Associated Membranous Nephropathy.

In membranous nephropathy (MN), which is characterized by deposition of immune complexes along the glomerular basement membrane (GBM), phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A are target antigens in approximately 70% and 1%-5% of cases of primary MN, respectively. In other cases of primary MN and in secondary MN, the target antigens are unknown. We studied 224 cases of biopsy-proven PLA2R-negative MN and 102 controls (including 47 cases of PLA2R-associated MN) in pilot and discovery cohorts. We also evaluated 48 cases of PLA2R-negative presumed primary MN and lupus MN in a validation cohort. We used laser microdissection and mass spectrometry to identify new antigens, which were localized by immunohistochemistry. Mass spectrometry detected exostosin 1 (EXT1) and exostosin 2 (EXT2) in 21 cases of PLA2R-negative MN, but not in PLA2R-associated MN and control cases. Immunohistochemistry staining revealed bright granular GBM staining for EXT1 and EXT2. Clinical and biopsy findings showed features of autoimmune disease, including lupus, in 80.7% of the 26 EXT1/EXT2-associated MN cases we identified. In the validation cohort, we confirmed that EXT1/EXT2 staining was detected in pure class 5 lupus nephritis (eight of 18 patients) and in presumed primary MN associated with signs of autoimmunity (three of 16 patients); only one of the 14 cases of mixed class 5 and 3/4 lupus nephritis was positive for EXT1/EXT2. Tests in seven patients with EXT1/EXT2-associated MN found no circulating anti-exostosin antibodies. A subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Autoimmune disease is common in this group of patients.