Domain-and species-specific monoclonal antibodies recognize the Von Willebrand Factor-C domain of CCN5

The CCN family of proteins typically consists of four distinct peptide domains: an insulin-like growth factor binding protein-type (IGFBP) domain, a Von Willebrand Factor C (VWC) domain, a thrombospondin type 1 repeat (TSP1) domain, and a carboxy-terminal (CT) domain. The six family members participate in many processes, including proliferation, motility, cell-matrix signaling, angiogenesis, and wound healing. Accumulating evidence suggests that truncated and alternatively spliced isoforms are responsible for the diverse functions of CCN proteins in both normal and pathophysiologic states. Analysis of the properties and functions of individual CCN domains further corroborates this idea. CCN5 is unique among the CCN family members because it lacks the CT-domain. To dissect the domain functions of CCN5, we are developing domain-specific mouse monoclonal antibodies. Monoclonal antibodies have the advantages of great specificity, reproducibility, and ease of long-term storage and production. In this communication, we injected mixtures of GST-fused rat CCN5 domains into mice to generate monoclonal antibodies. To identify the domains recognized by the antibodies, we constructed serial expression plasmids that express dual-tagged rat CCN5 domains. All of the monoclonal antibodies generated to date recognize the VWC domain, indicating it is the most highly immunogenic of the CCN5 domains. We characterized one particular clone, 22H10, and found that it recognizes mouse and rat CCN5, but not human recombinant CCN5. Purified 22H10 was successfully applied in Western Blot analysis, immunofluorescence of cultured cells and tissues, and immunoprecipitation, indicating that it will be a useful tool for domain analysis and studies of mouse-human tumor models

The Extracellular Matrix and Blood Vessel Formation: Not Just a Scaffold

The extracellular matrix plays a number of important roles, among them providing structural support and information to cellular structures such as blood vessels imbedded within it. As more complex organisms have evolved, the matrix ability to direct signalling towards the vasculature and remodel in response to signalling from the vasculature has assumed progressively greater importance. This review will focus on the molecules of the extracellular matrix, specifically relating to vessel formation and their ability to signal to the surrounding cells to initiate or terminate processes involved in blood vessel formation

CCN5: biology and pathophysiology

CCN5 is one of six proteins in the CCN family. This family of proteins has been shown to play important roles in many processes, including proliferation, migration, adhesion, extracellular matrix regulation, angiogenesis, tumorigenesis, fibrosis, and implantation. In this review, we focus on the biological and putative pathophysiological roles of CCN5. This intriguing protein is structurally unique among the CCN family members, and has a unique biological activity profile as well

CCN5, a secreted protein, localizes to the nucleus

CCN5, a member of the CCN family of growth factors, inhibits the proliferation and migration of smooth muscle cells in cell culture and animal models. Expressed in both embryonic and adult tissues, CCN5 exhibits a matricellular localization pattern characteristic of secreted proteins that are closely associated with the cell surface. In addition to this observed expression pattern, immunohistochemical evidence suggests the presence of nuclear CCN5 in some cells. To determine if CCN5 localizes to the nucleus we performed immunofluorescence, confocal imaging, and cell fractionation to corroborate the immunohistochemical observations. After confirming the presence of nuclear CCN5 using four independent experimental methods, we identified a single putative nuclear localization signal in the von Willebrand factor C domain of mouse and rat CCN5. Site directed mutagenesis of the three basic amino acids in the putative nuclear localization sequence did not prevent nuclear localization of CCN5 in four different cell types, suggesting that CCN5 nuclear transport is not mediated by the only canonical nuclear localization signal present in the primary amino acid sequence. Future work will address the mechanism of nuclear localization and the function of nuclear versus secreted CCN5

CCN5 expression in mammals: I. Embryonic and fetal tissues of mouse and human

The six proteins of the CCN family have important roles in development, angiogenesis, cell motility, proliferation, and other fundamental cell processes. To date, CCN5 distribution in developing rodents and humans has not been mapped comprehensively. CCN5 strongly inhibits adult smooth muscle cell proliferation and motility. Its anti-proliferative action predicts that CCN5 would not be present in developing tissues until the proliferation phase of tissue morphogenesis is complete. However, estrogen induces CCN5 expression in epithelial and smooth muscle cells, suggesting that CCN5 might be widely expressed in embryonic tissues exposed to high levels of estrogen. 9–16 day murine embryos and fetuses and 3–7 month human fetal tissues were analyzed by immunohistochemistry. CCN5 was detected in nearly all developing tissues. CCN5 protein expression was initially present in most tissues, and at later times in development tissue-specific expression differences were observed. CCN5 expression was particularly strong in vascular tissues, cardiac muscle, bronchioles, myotendinous junctions, and intestinal smooth muscle and epithelium. CCN5 expression was initially absent in bone cartilaginous forms but was increasingly expressed during bone endochondral ossification. Widespread CCN5 mRNA expression was detected in GD14.5 mice. Although CCN2 and CCN5 protein expression patterns in some adult pathologic conditions are inversely expressed, this expression pattern was not found in developing mouse and human tissues. The widespread expression pattern of CCN5 in most embryonic and fetal tissues suggests a diverse range of functions for CCN5