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The synthesis and secretion of versican isoform V3 by mammalian cells: A role for N-linked glycosylation.

ABSTRACT: Versican is a large extracellular matrix (ECM) chondroitin sulfate (CS) proteoglycan found in most soft tissues, which is encoded by the VCAN gene. At least four major isoforms (V0, V1, V2, and V3) are generated via alternative splicing. The isoforms of versican are expressed and accumulate in various tissues during development and disease, where they contribute to ECM structure, cell growth and migration, and immune regulation, among their many functions. While several studies have identified the mRNA transcript for the V3 isoform in a number of tissues, little is known about the synthesis, secretion, and targeting of the V3 protein. In this study, we used lentiviral generation of doxycycline-inducible rat V3 with a C-terminal tag in stable NIH 3T3 cell lines and demonstrated that V3 is processed through the classical secretory pathway. We further show that N-linked glycosylation is required for efficient secretion and solubility of the protein. By site-directed mutagenesis, we identified amino acids 57 and 330 as the active N-linked glycosylation sites on V3 when expressed in this cell type. Furthermore, exon deletion constructs of V3 revealed that exons 11-13, which code for portions of the carboxy region of the protein (G3 domain), are essential for V3 processing and secretion. Once secreted, the V3 protein associates with hyaluronan along the cell surface and within the surrounding ECM. These results establish critical parameters for the processing, solubility, and targeting of the V3 isoform by mammalian cells and establishes a role for V3 in the organization of hyaluronan.

SUBMITTER: Harten IA

PROVIDER: S-EPMC7282976 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Arterial smooth muscle cells (ASMCs) undergo phenotypic changes during development and pathological processes in vivo and during cell culture in vitro. Our previous studies demonstrated that retrovirally-mediated expression of the versican V3 splice variant (V3) that lacks glycosaminoglycan chains by ASMCs retards cell proliferation and migration in vitro and reduces neointimal thickening, macrophage and lipid accumulation in animal models of vascular injury and atherosclerosis. However, the molecular pathways induced by V3 expression that are responsible for these changes are not yet clear. In the present study, we employed a microarray approach to examine how expression of V3 induced changes in gene expression and the molecular pathways in ASMCs. We found that forced expression of V3 by ASMCs affected expression of 521 genes by more than 1.5 fold. Gene ontology (GO) analysis shows that components of extracellular matrix were the most significantly affected by V3 expression, indicating that V3 expression elicits profound remodeling of extracellular matrix. In addition, genes regulating the formation of the cytoskeleton which also serve as markers of contractile smooth muscle cells were significantly upregulated. On the other hand, components of the complement system, chemokines, chemokine receptors, and transcription factors crucial for regulating inflammatory processes were among the genes most downregulated. Consistently, we found that the level of myocardin, a key transcription factor promoting contractile ASMC phenotype, was greatly increased while proinflammatory transcription factors NFkappaB1 and C/EBPβ were significantly attenuated in V3-expressing SMCs. Such results indicate that V3 expression reprograms ASMC into differentiated and anti-inflammatory phenotypes. Overall, these findings demonstrate that expression of V3 reprograms ASMCs promoting anti-inflammatory and differentiated smooth muscle cell phenotypes potentially by altering cell-ECM interaction and focal adhesion signaling pathways.

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The synthesis and secretion of versican isoform V3 by mammalian cells: A role for N-linked glycosylation.

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