In-silico assessment of high-risk non-synonymous SNPs in ADAMTS3 gene associated with Hennekam syndrome and their impact on protein stability and function.

Hennekam Lymphangiectasia-Lymphedema Syndrome 3 (HKLLS3) is a rare genetical disorder caused by mutations in a few genes including ADAMTS3. It is characterized by lymphatic dysplasia, intestinal lymphangiectasia, severe lymphedema and distinctive facial appearance. Up till now, no extensive studies have been conducted to elucidate the mechanism of the disease caused by various mutations. As a preliminary investigation of HKLLS3, we sorted out the most deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs) that might affect the structure and function of ADAMTS3 protein by using a variety of in silico tools. A total of 919 nsSNPs in the ADAMTS3 gene were identified. 50 nsSNPs were predicted to be deleterious by multiple computational tools. 5 nsSNPs (G298R, C567Y, A370T, C567R and G374S) were found to be the most dangerous and can be associated with the disease as predicted by different bioinformatics tools. Modelling of the protein shows it can be divided into segments 1, 2 and 3, which are connected by short loops. Segment 3 mainly consists of loops without substantial secondary structures. With prediction tools and molecular dynamics simulation, some SNPs were found to significantly destabilize the protein structure and disrupt the secondary structures, especially in segment 2. The deleterious effects of mutations in segment 1 are possibly not from destabilization but from other factors such as the change in phosphorylation as suggested by post-translational modification (PTM) studies. This is the first-ever study of ADAMTS3 gene polymorphism, and the predicted nsSNPs in ADAMST3, some of which have not been reported yet in patients, will serve for diagnostic purposes and further therapeutic implications in Hennekam syndrome, contributing to better diagnosis and treatment.

Hypoxic regulation of ADAMTS-2 and -3 (a disintegrin and matrix metalloproteinase with thrombospondin motifs 2 and 3) procollagen N proteinases by HIF-1α in endothelial cells.

ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.

New Strategies for the Treatment of Neuropsychiatric Disorders Based on Reelin Dysfunction.

Reelin is an extracellular matrix protein that is mainly produced in Cajal-Retzius cells and controls neuronal migration, which is important for the proper formation of cortical layers in the developmental stage of the brain. In the adult brain, Reelin plays a crucial role in the regulation of N-methyl-D-aspartate receptor-dependent synaptic function, and its expression decreases postnatally. Clinical studies showed reductions in Reelin protein and mRNA expression levels in patients with psychiatric disorders; however, the causal relationship remains unclear. Reelin-deficient mice exhibit an abnormal neuronal morphology and behavior, while Reelin supplementation ameliorates learning deficits, synaptic dysfunctions, and spine loss in animal models with Reelin deficiency. These findings suggest that the neuronal deficits and brain dysfunctions associated with the down-regulated expression of Reelin are attenuated by enhancements in its expression and functions in the brain. In this review, we summarize findings on the role of Reelin in neuropsychiatric disorders and discuss potential therapeutic approaches for neuropsychiatric disorders associated with Reelin dysfunctions.

[USF1 Suppresses Expression of Fibrillar Type I, II, and III Collagen and pNP Adamts-3 in Osteosarcoma Cells].

Collagens are the main components of human tissues. Various regulatory factors and cytokines may influence expression levels for collagen-encoding genes, and, therefore, contrubite to some collagen-associated pathologies. In this study, we demonstrate regulatory effects of USF1 on expression of genes encoding fibrillar collagen types I, II, and III in osteoblastic Saos-2 and MG-63 cells. An ectopic expression of the human USF1 led to a decrease in both mRNA and protein expression levels of the collagen-encoding genes mentioned above. ADAMTS-3 is a proteinase primarily responsible for the amino-terminal cleavage of type I and type II collagen precursors. The ADAMTS-3 promoter region contains potential binding sites for USF1. Here we show that an overexpression of USF1 lead to a decrease in ADAMTS-3 mRNA and protein expression levels. In co-transfection studies, USF1 negatively regulated ADAMTS-3 promoter activity. Further, in EMSA studies, we showed that USF1 binds to the ADAMTS-3 promoter region. In conclusion, it seems that ADAMTS-3 and USF1 contribute to the regulation of collagen encoding genes in osteosarcoma.

An additional case of Hennekam lymphangiectasia-lymphedema syndrome caused by loss-of-function mutation in ADAMTS3.

Hennekam lymphangiectasia-lymphedema syndrome (HKLLS) is a genetically heterogeneous lymphatic dysplasia with characteristic of facial dysmorphism, neurocognitive impairments, and abnormalities of the pericardium, intestinal tract, and extremities. It is an autosomal recessive condition caused by biallelic mutations in CCBE1 (collagen- and calcium-binding epidermal growth factor domain-containing protein 1) (HKLLS1; OMIM 235510) or FAT4 (HKLLS2; OMIM 616006). CCBE1 acts via ADAMTS3 (a disintegrin and metalloprotease with thrombospondin motifs-3 protease) to enhance vascular endothelial growth factor C signaling. There is report of one family supporting mutations in ADAMTS3 as causative for the phenotype labeled as HKLLS3. Here, we report an additional case of HKLLS that appears to be associated with homozygous nonsense mutation of ADAMTS3.

Van Maldergem syndrome and Hennekam syndrome: Further delineation of allelic phenotypes.

Biallelic variants in FAT4 are associated with the two disorders, Van Maldergem syndrome (VMS) (n = 11) and Hennekam syndrome (HS) (n= 40). Both conditions are characterized by a typical facial gestalt and mild to moderate intellectual disability, but differ in the occurrence of neonatal hypotonia and feeding problems, hearing loss, tracheal anomalies, and osteopenia in VMS, and lymphedema in HS. VMS can be caused by autosomal recessive variants in DCHS1 as well, and HS can also be caused by autosomal recessive variants in CCBE1 and ADAMTS3. Here we report two siblings with VMS and one girl with HS, all with FAT4 variants, and provide an overview of the clinical findings in all patients reported with FAT4 variants. Our comparison of the complete phenotypes of patients with VMS and HS indicates a resemblance of several signs, but differences in several other main signs and symptoms, each of marked importance for affected individuals.

Downregulation of ADAMTS3 Suppresses Stemness and Tumorigenicity in Glioma Stem Cell.

AIMS: Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models. METHODS: ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets. RESULTS: ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM. CONCLUSION: We identified ADAMTS3 as a potential therapeutic target for GBM.

ADAMTS3 and FLT4 gene mutations result in congenital lymphangiectasia in newborns: A case report.

BACKGROUND: Congenital lymphangiectasia is a rare disease characterized by dilated interstitial lymphatic vessels and cystic expansion of the lymphatic vessels. Congenital lymphangiectasia can affect various organ systems; however, it frequently occurs in the lungs accompanied with unexplained pleural effusion. Further, it might not be diagnosed during prenatal examination owing to the absence of pronounced abnormalities. However, after birth the newborn rapidly develops respiratory distress that quickly deteriorates. Genetic variations in proteins controlling the development of lymphatic vessels contribute to the pathophysiology of this disease. We report a rare case of heterozygous mutation of ADAMTS3 and FLT4 genes, which have not been reported previously. CASE SUMMARY: We analysed the case of a neonate who had presented with only pleural effusion at a late gestational age and eventually died due to its inability to establish spontaneous breathing after birth. An autopsy revealed lymphangiectasia of the organ systems. Further, whole exome sequencing revealed heterozygous mutations of the lymphangiogenesis-controlling genes, ADAMTS3 and FLT4, and Sanger verification revealed similar lesions in the mother with no symptoms. CONCLUSION: Considering the presented case, obstetricians should observe unexplained foetal pleural effusion, and perform pathology analysis and whole exome sequencing for a conclusive diagnosis and prompt treatment.

Analysis of Reelin signaling and neurodevelopmental trajectory in primary cultured cortical neurons with RELN deletion identified in schizophrenia.

Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca2+/calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain.

Case Report: Systemic lupus erythematous associated with thrombotic thrombocytopenic purpura, a diagnostic challenge.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon microangiopathic disease and sometimes is associated with systemic lupus erythematous (SLE). However, this probable causal relationship has not been completely proven. The diagnostic differentiation of both diseases is difficult in the first instance because they share similar characteristics that may overlap. We present a case of a 32-year-old woman with antecedents of epileptic seizures since she was 12 years old. The patient was admitted to the emergency room with a clinical picture of headaches, fever, paleness in the skin and mucosa, confused state, paresthesia, and transient spasticity of the extremities. The laboratory results revealed direct Coombs negative hemolytic anemia, severe thrombocytopenia, significant elevation of lactate dehydrogenase, and presence of schistocytes ++ in the peripheral film. In addition, positive antinuclear antibodies and positive anti-native DNA in titers of 1/320 and 1/160, respectively, were found. Urinalysis showed that serum creatinine was in normal range. Because of limited hospital resources, ADAMTS13 was not evaluated. However, based on clinical, hematological, and biochemical findings, we concluded that it was a case of TTP associated with SLE and indicated treatment with plasmapheresis and methylprednisolone pulses, obtaining a satisfactory response (normalization of biomarker levels, health condition) after the second session of plasmapheresis. Diagnosis of both SLE and TTP is often difficult to achieve; however, adequate correlation of clinical manifestations and laboratory tests, along with the help of partial therapeutic interventions, may lead to good clinical response.

Proteolytic Cleavages in the VEGF Family: Generating Diversity among Angiogenic VEGFs, Essential for the Activation of Lymphangiogenic VEGFs.

Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymph-angiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. At least five different proteases mediate the activating cleavage of VEGF-C: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the "mature" growth factors, which differ in affinity and receptor activation potential. The "default" VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D, and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by distinct proteases. During embryonic development, ADAMTS3 activates VEGF-C. The other activating proteases are likely important for non-developmental lymphangiogenesis during, e.g., tissue regeneration, inflammation, immune response, and pathological tumor-associated lymphangiogenesis. The better we understand these events at the molecular level, the greater our chances of developing successful therapies targeting VEGF-C and VEGF-D for diseases involving the lymphatics such as lymphedema or cancer.

Assay for Reelin-Cleaving Activity of ADAMTS and Detection of Reelin and Its Fragments in the Brain.

Proteolytic cleavage of the secreted signaling protein Reelin has been suggested to play causative roles in many neuropsychiatric and neurodegenerative disorders. Therefore, characterization of the proteolytic activity against Reelin is important not only for understanding how the brain works but also for the development of novel therapy for these disorders. Notably, ADAMTS family proteases are the primary suspects of Reelin-cleaving proteases under many, though not all, circumstances. Here we describe how to measure the Reelin-cleaving activity of ADAMTS (or of any other protease that may cleave Reelin), how to purify the Reelin-cleaving protease ADAMTS-3 from the culture supernatant of cortical neurons, and how to detect endogenous Reelin protein and its fragments in the brain.

A case report of familial 4q13.3 microdeletion in three individuals with syndromic intellectual disability.

BACKGROUND: Interstitial 4q deletions are rare chromosomal alterations. Most of the previously reported deletions involving the 4q13.3 region are large chromosomal alterations with a common loss of band 4q21 resulting in marked growth restriction, severe intellectual disability, and absent or severely delayed speech. A microdeletion of 4q13.3 hasn't been previously reported. We discuss the involvement of genes and the observed phenotype, comparing it with that of previously reported patients. CASE PRESENTATION: We report on a 4q13.3 microdeletion detected in three affected individuals of a Lithuanian family. The clinical features of two affected children and their affected mother are very similar and include short stature, congenital heart defect, skeletal anomalies, minor facial anomalies, delayed puberty, and intellectual disability. Whole genome SNP microarray analysis of one child revealed an interstitial 4q13.3 microdeletion, 1.56 Mb in size. FISH analysis confirmed the deletion in the proband and identified the same deletion in her affected sib and mother, while it was not detected in a healthy sib. Deletion includes ADAMTS3, ANKRD17, COX18, GC, and NPFFR2 protein-coding genes. CONCLUSIONS: Our findings suggest that 4q13.3 microdeletion is a cause of a recognizable phenotype of three affected individuals. The detected microdeletion is the smallest interstitial deletion in 4q13. We highlight ADAMTS3, ANKRD17 and RNU4ATAC9P as candidate genes for intellectual disability, growth retardation and congenital heart defect.

SP1-mediated downregulation of ADAMTS3 gene expression in osteosarcoma models.

ADAMTS3 is a member of procollagen N-proteinase subfamily of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family. It has an important function in the procollagen maturation process. The removal of N-peptidases is required for the accurate processing of fibrillar collagens. Otherwise, several disorders can occur that is related with the collagenous tissues. ADAMTS3 mainly maturates type II collagen molecule which is the main component of the bone and cartilage. There are several expression studies about ADAMTS3 gene however its transcriptional regulation has not been lightened up, yet. Here we first time cloned and functionally analyzed the promoter region of ADAMTS3 gene, approximately 1380 bp upstream of the transcription start site. Transient transfection experiments showed that all truncated promoter constructs are active and 171 bp fragment is sufficient to activate gene expression in both Saos-2 and MG63 cells. In silico analysis showed that ADAMTS3 has a TATA-less promoter and contains several SP1/GC box binding motifs and a CpG island. Therefore we mainly investigated the SP1 dependent regulation of ADAMTS3 promoter. SP1 downregulated ADAMTS3 transcriptional activity. As consistent with the transcriptional activity, mRNA, and protein expression levels were also decreased by SP1. On the other hand, functional binding of the SP1 on multiple regions of ADAMTS3 promoter was confirmed by EMSA studies. As ADAMTS3 is responsible for the collagen maturation and biosynthesis, further we investigated the effect of SP1 on type I-II and III collagen gene expressions. We point out that SP1 increased type II and III collagen expression and in contrast decreased type I collagen expression levels in Saos-2 cells. mRNA expression level was decreased for all collagen types in MG63 model. Decrease in the type II collagen expression was also demonstrated at the protein level by SP1. Collectively these results provide first findings for the SP1-related transcriptional regulation of ADAMTS3 and collagen genes in osteosarcoma cell lines.

Effect of insulin on the mRNA expression of procollagen N-proteinases in chondrosarcoma OUMS-27 cells.

Chondrosarcoma is one of the most common bone tumors, and at present, there is no non-invasive treatment option for this cancer. The chondrosarcoma OUMS-27 cell line produces proteoglycan and type II, IX, and XI collagens, which constitutes cartilage tissue. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases are a group of secreted proteases, which include the procollagen N-proteinases ADAMTS-2, -3 and -14. These procollagen N-proteinases perform a role in the processing of procollagens to collagen and the maturation of type I collagen. The present study aimed to improve the understanding of the causes of metastasis, local invasion and resistance to chemo- and radiotherapy in chondrosarcoma, as well as the effect of insulin on cancer cells. The present study was designed to reveal the effects of insulin on procollagen N-proteinases in chondrosarcoma OUMS-27 cells. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) alone or in DMEM containing 10 µg/ml insulin. The medium was changed every other day for 11 days. The cells were harvested on days 1, 3, 7 and 11, and total RNA isolation was performed immediately following harvesting. The expression levels of ADAMTS2, ADAMTS3 and ADAMTS14 mRNA were estimated by reverse transcription-quantitative polymerase chain reaction using appropriate primers. ADAMTS2 mRNA expression was found to be decreased on day 7 (P=0.028) and increased at day 11 compared with the control group (P=0.016). The increase in mRNA concentration at day 11 was significantly different compared to the concentrations on days 3 (P=0.047) and 7 (P=0.008). The expression of ADAMTS3 mRNA decreased immediately subsequent to insulin induction on day 1 compared with the control group (P=0.008). The most evident decrease in mRNA concentration was seen at day 7 subsequent to insulin induction (P=0.008). The present results demonstrated that ADAMTS2 and ADAMTS3 may perform a role in the invasion and metastasis of tumors, and may also possess proteolytic activity that results in the breakdown of the extracellular matrix (ECM). Insulin itself can modulate the biosynthesis of ECM macromolecules that are altered in diabetes through various pathways.

Induction of human ADAMTS-2 gene expression by IL-1α is mediated by a multiple crosstalk of MEK/JNK and PI3K pathways in osteoblast like cells.

Up-regulation of ADAMTS genes with proinflammatory cytokines is important for some pathological conditions such as osteoarthritis (OA) that is a disease based on ECM degradation in cartilage. IL-1α is a proinflammatory cytokine and important both to normal and pathophysiologic conditions in cartilage and bone. Effects of some proinflammatory cytokines such as TNF-α and IL-1ß on the some members of ADAMTS family have been investigated in some chondrocyte tissues or cell lines. However the effect of the IL-1α on the expression of ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cell lines, remains unclear. Therefore, the aim of this study is to investigate the effect of IL-1α on ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cells, Saos-2 and MG-63. The present study, for the first time, demonstrated that IL-1α increases ADAMTS-2 and ADAMTS-3 gene expressions in both Saos-2 and MG-63 cells. Having correlation to mRNA induction, the upregulation of ADAMTS-2,-3 protein levels by IL-1α stimulation is also observed. The inhibition studies showed that this upregulation occurred at the level of transcription, and there was no effect of IL-1α on ADAMTS-2 mRNA half-life in Saos-2 cells. Transactivation potential of IL-1α on ADAMTS-2 promoter was investigated by transient transfection assay. Specifically, IL-1α strongly increased -658/+112 and -530/+112 ADAMTS-2 promoter constructs. Further, we analyzed signaling pathways involved in ADAMTS-2 induction. Pathway inhibition studies revealed that this upregulation depends on the activation of MEK, JNK and PI3K pathways. These findings suggested that IL-1α is a strong positive regulator of ADAMTS-2 and ADAMTS-3 expression. These findings would provide novel insight into the pathophysiology of OA.

Cellular dissection of the spinal cord motor column by BAC transgenesis and gene trapping in zebrafish.

Bacterial artificial chromosome (BAC) transgenesis and gene/enhancer trapping are effective approaches for identification of genetically defined neuronal populations in the central nervous system (CNS). Here, we applied these techniques to zebrafish (Danio rerio) in order to obtain insights into the cellular architecture of the axial motor column in vertebrates. First, by using the BAC for the Mnx class homeodomain protein gene mnr2b/mnx2b, we established the mnGFF7 transgenic line expressing the Gal4FF transcriptional activator in a large part of the motor column. Single cell labeling of Gal4FF-expressing cells in the mnGFF7 line enabled a detailed investigation of the morphological characteristics of individual spinal motoneurons, as well as the overall organization of the motor column in a spinal segment. Secondly, from a large-scale gene trap screen, we identified transgenic lines that marked discrete subpopulations of spinal motoneurons with Gal4FF. Molecular characterization of these lines led to the identification of the ADAMTS3 gene, which encodes an evolutionarily conserved ADAMTS family of peptidases and is dynamically expressed in the ventral spinal cord. The transgenic fish established here, along with the identified gene, should facilitate an understanding of the cellular and molecular architecture of the spinal cord motor column and its connection to muscles in vertebrates.