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Scientific Reports Jan 2021ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Dysregulated aggrecanase activity of ADAMTS-5 has been directly...
ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Dysregulated aggrecanase activity of ADAMTS-5 has been directly linked to the etiology of osteoarthritis (OA). For this reason, ADAMTS-5 is a pharmaceutical target for the treatment of OA. ADAMTS-5 shares high structural and functional similarities with ADAMTS-4, which makes the design of selective inhibitors particularly challenging. Here we exploited the ADAMTS-5 binding capacity of β-N-acetyl-D-glucosamine to design a new class of sugar-based arylsulfonamides. Our most promising compound, 4b, is a non-zinc binding ADAMTS-5 inhibitor which showed high selectivity over ADAMTS-4. Docking calculations combined with molecular dynamics simulations demonstrated that 4b is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Furthermore, the interaction between 4b and the ADAMTS-5 Dis domain is mediated by hydrogen bonds between the sugar moiety and two lysine residues (K532 and K533). Targeted mutagenesis of these two residues confirmed their importance both for versicanase activity and inhibitor binding. This positively-charged cluster of ADAMTS-5 represents a previously unknown substrate-binding site (exosite) which is critical for substrate recognition and can therefore be targeted for the development of selective ADAMTS-5 inhibitors.
Topics: ADAMTS5 Protein; Aggrecans; Amino Acid Sequence; Binding Sites; Disintegrins; Endopeptidases; Glycoconjugates; Humans; Lysine; Metalloproteases; Mutagenesis; Osteoarthritis; Protein Binding; Protein Domains; Sequence Alignment; Sulfonamides; Versicans
PubMed: 33441904
DOI: 10.1038/s41598-020-80294-1 -
Redox Biology Aug 2023Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis....
Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO/ONOOH. Both reagent ONOO/ONOOH and SIN-1 (a thermal source of ONOO/ONOOH) modified Tyr, Trp and Met residues. ONOO/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.
Topics: Humans; Oxidants; Peroxynitrous Acid; Versicans; Hyaluronic Acid; Plaque, Atherosclerotic; Extracellular Matrix; Atherosclerosis; Protein Isoforms; Inflammation
PubMed: 37402332
DOI: 10.1016/j.redox.2023.102794 -
Connective Tissue Research Jan 2022: The proper function of the tenocyte network depends on cell-matrix as well as intercellular communication that is mechanosensitive. Building on the concept that the...
: The proper function of the tenocyte network depends on cell-matrix as well as intercellular communication that is mechanosensitive. Building on the concept that the etiopathogenic stimulus for tendon degeneration is the catabolic response of tendon cells to mechanobiologic under-stimulation, we studied the pericellular matrix rich in versican and its predominant proteolytic enzyme ADAMTS-1, as well as Connexin-43 (Cx43), a major gap junction forming protein in tendons, in stress-deprived rat tail tendon fascicles (RTTfs).: RTTfs were stress-deprived for up to 7 days under tissue culture conditions. RT-qPCR was used to measure mRNA expression of versican, ADAMTS-1, and Cx43. Protein synthesis was determined using Western blotting and immunohistochemistry.: Stress-deprivation (SD) caused a statistically significant up-regulation of versican, ADAMTS-1, and Cx43 mRNA expression that was persistent over the 7-day test period. Western blot analysis and immunohistochemical assessment of protein synthesis revealed a marked increase of the respective proteins with SD. Inhibition of proteolytic enzyme activity with ilomastat prevented the increased versican degradation and Cx43 synthesis in 3 days stress-deprived tendons when compared with non-treated, stress-deprived tendons.: In the absence of mechanobiological signaling the immediate pericellular matrix is modulated as tendon cells up-regulate their production of ADAMTS-1, and versican with subsequent proteoglycan degradation potentially leading to cell signaling cues increasing Cx43 gap junctional protein. The results also provide further support for the hypothesis that the cellular changes associated with tendinopathy are a result of decreased mechanobiological signaling and a loss of homeostatic cytoskeletal tension.
Topics: Animals; Connexin 43; Connexins; RNA, Messenger; Rats; Tendons; Up-Regulation; Versicans
PubMed: 33467936
DOI: 10.1080/03008207.2021.1873302 -
The Journal of Histochemistry and... Nov 2020Hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilizes interactions between two important extracellular matrix (ECM) macromolecules, versican and hyaluronan,...
Hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilizes interactions between two important extracellular matrix (ECM) macromolecules, versican and hyaluronan, which facilitate proliferation of fibroblasts and their conversion to myofibroblasts. However, the role of HAPLN1 in these events has not been studied. Using immunocytochemistry, cellular and ECM locations of HAPLN1 were evaluated in cultured human lung fibroblasts during proliferation and conversion to myofibroblasts. HAPLN1 localized to pericellular matrices, associating with both versican and hyaluronan in the ECM and on the cell surface. Nuclear and total HAPLN1 immunostaining increased after myofibroblast induction. Confocal microscopy showed HAPLN1 predominant in the ECM under cells while versican predominated above cells. Versican and HAPLN1 were also juxtaposed in columnar inclusions in the cytoplasm and nucleus. Nuclear HAPLN1 staining in interphase cells redistributed to the cytosol during mitosis. In the absence of TGF-β1, addition of exogenous bovine HAPLN1 (together with aggrecan G1) facilitated myofibroblast formation, as seen by significant upregulation of α-smooth muscle actin (SMA) staining, while adding full-length bovine versican had no effect. Increased compaction of hyaluronan-rich ECM suggests that HAPLN1 plus G1 addition affects hyaluronan networks and myofibroblast formation. These observations demonstrate changes in both extracellular and intracellular localization of HAPLN1 during fibroblast proliferation and myofibroblast conversion suggesting a possible role in fibrotic remodeling.
Topics: Cell Proliferation; Cells, Cultured; Extracellular Matrix Proteins; Fibroblasts; Humans; Lung; Phenotype; Proteoglycans; Versicans
PubMed: 33064036
DOI: 10.1369/0022155420966663 -
JCI Insight Apr 2019Chronic tubulointerstitial injury impacts the prognosis of focal segmental glomerulosclerosis (FSGS). We found that the level of versican V1 was increased in tubular...
Chronic tubulointerstitial injury impacts the prognosis of focal segmental glomerulosclerosis (FSGS). We found that the level of versican V1 was increased in tubular cells of FSGS patients. Tubular cell-derived versican V1 induced proliferation and collagen synthesis by activating the CD44/Smad3 pathway in fibroblasts. Both urine C3a and suPAR were increased and bound to the tubular cells in FSGS patients. C3a promoted the transcription of versican by activating the AKT/β-catenin pathway. C3aR knockout decreased the expression of versican in Adriamycin-treated (ADR-treated) mice. On the other hand, suPAR bound to integrin β6 and activated Rac1, which bound to SRp40 at the 5' end of exon 7 in versican pre-mRNA. This binding inhibited the 3'-end splicing of intron 6 and the base-pair interactions between intron 6 and intron 8, leading to the formation of versican V1. Cotreatment with ADR and suPAR specifically increased the level of versican V1 in tubulointerstitial tissues and caused more obvious interstitial fibrosis in mice than treatment with only ADR. Altogether, our results show that C3a and suPAR drive versican V1 expression in tubular cells by promoting transcription and splicing, respectively, and the increases in tubular cell-derived versican V1 induce interstitial fibrosis by activating fibroblasts in FSGS.
Topics: Adult; Animals; Biopsy; Case-Control Studies; Cell Line; Complement C3a; Disease Models, Animal; Doxorubicin; Epithelial Cells; Female; Fibroblasts; Fibrosis; Gene Expression Profiling; Glomerulosclerosis, Focal Segmental; Humans; Kidney Tubules; Male; Mice; Mice, Knockout; Middle Aged; Protein Isoforms; Receptors, Complement; Receptors, Urokinase Plasminogen Activator; Versicans; Young Adult
PubMed: 30944246
DOI: 10.1172/jci.insight.122912 -
Matrix Biology : Journal of the... Jan 2023The extracellular matrix (ECM) in the endometrium plays a crucial role in mammalian pregnancy. We have shown that versican secreted from the endometrial epithelium...
The extracellular matrix (ECM) in the endometrium plays a crucial role in mammalian pregnancy. We have shown that versican secreted from the endometrial epithelium promotes embryo implantation. Versican is a proteoglycan, a major player in the provisional matrix, and versikine, its N-terminal fragment cleaved by ADAMTS proteinases, serves as a bioactive molecule. Here, since versican expression in the placenta was dynamically altered in humans and mice, we investigated the role of versican in pregnancy using uterine-specific Vcan deletion mice (uKO mice) and ADAMTS-resistant versican expressing mice (V1R mice). uKO mice exhibited insufficient spiral artery dilation, followed by fetal growth restriction and maternal hypertension. Further analysis revealed impaired proliferation of tissue-resident natural killer cells required for spiral artery dilation. V1R mice showed the same results as the control, eliminating the involvement of versikine. Our results provide a new concept that versican, one factor of ECM, contributes to placentation and following fetal growth.
Topics: Pregnancy; Humans; Female; Mice; Animals; Versicans; Dilatation; Uterus; Fetal Development; Arteries; Mammals
PubMed: 36423736
DOI: 10.1016/j.matbio.2022.11.004 -
BioRxiv : the Preprint Server For... Aug 2023The mechanisms regulating the cellular behavior and cardiomyocyte organization during ventricular wall morphogenesis are poorly understood. Cardiomyocytes are surrounded...
AIMS
The mechanisms regulating the cellular behavior and cardiomyocyte organization during ventricular wall morphogenesis are poorly understood. Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. This study aims to determine whether and how β1 integrins regulate cardiomyocyte behavior and organization during ventricular wall morphogenesis in the mouse.
METHODS AND RESULTS
We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/β integrins and their ligands in the embryonic heart. Integrin β1 subunit (β1) and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, while fibronectin surrounds cardiomyocytes, creating a network for them. , which encodes the β1 integrin subunit, was deleted via to generate myocardial-specific knockout (B1KO) mice. B1KO hearts display an absence of trabecular zone but a thicker compact zone. The abundances of hyaluronic acid and versican are not significantly different. Instead, fibronectin, a ligand of β1, was absent in B1KO. We examined cellular behaviors and organization via various tools. B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. The reduction of Notch1 activation was not the cause of the abnormal cellular organization in B1KO hearts. Mosaic clonal lineage tracing shows that regulates cardiomyocyte transmural migration and proliferation autonomously.
CONCLUSIONS
β1 is asymmetrically localized in the cardiomyocytes, and its ECM ligands are enriched in the luminal side of the myocardium and surrounding cardiomyocytes. β1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of , leading to ablation of β1 integrins, causes the dissociation of cardiomyocytes from the ECM network and failure to establish tissue architecture to form trabeculae.
PubMed: 37693495
DOI: 10.1101/2023.08.28.555112 -
Biology of Reproduction Aug 2022Hyaluronan is a structural component of the expanded cumulus matrix, and hyaluronan synthase 2 is the major enzyme for the synthesis of hyaluronan in humans. Versican...
Hyaluronan is a structural component of the expanded cumulus matrix, and hyaluronan synthase 2 is the major enzyme for the synthesis of hyaluronan in humans. Versican cross-links the hyaluronan-rich matrix to cumulus cells and is critical for successful ovulation. Activin A is a critical intrafollicular regulator of ovarian function. Although activin A has been shown to promote cumulus matrix expansion in mice, the functional role of activin A in the regulation of cumulus expansion in the human ovary remains to be elucidated. Using primary and immortalized human granulosa-lutein cells as study models, we provide the first data showing that activin A increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 in these cells. Additionally, activin A also promoted the expression of the hyaluronan-binding protein versican. Moreover, using inhibitor- and small interfering RNA-mediated inhibition approaches, we found that these stimulatory effects of activin A are most likely mediated through the type I receptor activin receptor-like kinase (ALK4)-mediated Sma- and Mad-related protein (SMAD2)/SMAD3-SMAD4 signaling pathway. Notably, the chromatin immunoprecipitation analyses demonstrated that SMAD4 could bind to human hyaluronan synthase 2 and VERSICAN promoters. The results obtained from this in vitro study suggest that locally produced activin A plays a functional role in the regulation of hyaluronan production and stabilization in human granulosa-lutein cells.
Topics: Activins; Cells, Cultured; Female; Granulosa Cells; Humans; Hyaluronan Synthases; Hyaluronic Acid; Versicans
PubMed: 35403677
DOI: 10.1093/biolre/ioac070 -
Connective Tissue Research Mar 2018Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We...
Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using Vcan mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, Vcan dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and Vcan mice died at the neonatal period (P0). At P0, Vcan dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in Vcan fibroblasts as compared with that in wild type (WT). Transforming growth factor β (TGFβ) signaling mediated through the Smad2/3-dependent pathway was down-regulated in Vcan fibroblasts and a reduced TGFβ storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFβ signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFβ signaling.
Topics: Animals; Cells, Cultured; Dermis; Extracellular Matrix; Fibroblasts; Mice; Mutation; Protein Domains; Signal Transduction; Versicans
PubMed: 28488903
DOI: 10.1080/03008207.2017.1324432