| Hyperproliferation of smooth muscle cells is the hallmark of several important diseases, including restenosis following vascular surgery, hypertension, fibroids, asthma, and persistent pulmonary hypertension of the newborn. Vascular smooth muscle cells (VSMC) go through a phenotypic switch from the quiescent/contractile state to a proliferative/synthetic state during vascular injury and remodeling. Many factors are thought to participate and regulate this process. Heparin, which is a specific inhibitor of VSMC, induces expression of CCN5. CCN5, also called WISP2, is a member of the CCN family of proteins. CCN5 has been shown to inhibit VSMC proliferation and migration in vitro and in vivo, but the mechanism is not clear. CCN proteins regulate many cellular processes including adhesion, proliferation, differentiation, migration, apoptosis, and ECM remodeling. As a result, they play essential roles in development, wound healing, angiogenesis, chondrogenesis, tumorigenesis, and several disease states. CCN5 is unique among the CCN family members in that it lacks the CT domain present in the other five CCN proteins. The four domains present in CCN 1-4 and CCN6 are: 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. Accumulating evidence suggests that domains of CCN family proteins may have separate functions. Furthermore, various truncated isoforms exist and appear to be responsible for some of the diverse functions of CCN proteins in both normal and pathophysiological states.;With the clues from other CCN family members and previous work on CCN5, my research is based on the hypothesis that multifunctional isoforms of CCN5 exist in VSMC. Currently, no study has been done to explore the isoforms of CCN5. By examining endogenous isoforms and constructing recombinant CCN5 domains, my goal is to better understand the mechanism and regulation of CCN5 function. This will hopefully provide therapeutic insights into the treatment of smooth muscle cell diseases characterized by hyperproliferation, including restenosis and uterine fibroids. To test my hypothesis, my research was designed to answer three important questions: (1) Do endogenous CCN5 splicing variants exist, and if so, what is their function? I explored the alternative RNA splicing variants of CCN5 in VSMC. In this dissertation, I report that alterative RNA splicing generates two variants in rat VSMC, named CCN5-tr1 and CCN5-tr2, in addition to the canonical full-length CCN5 (CCN5-f). By testing lentiviral expression and lentiviral sh-RNA specific for CCN5-f and CCN5-tr1 constructs in VSMC, I found that full-length CCN5 seemly promotes a highly organized cytoskeleton, mimicking the contractile phenotype of VSMC, while CCN5-tr1 seems promotes a diffused cytoskeleton, mimicking the synthetic phenotype of VSMC. (2) Can domain-specific monoclonal antibodies provide a useful tool for understanding the function of individual CCN5 domains? I tried to make domain-specific mouse monoclonal antibodies to examine endogenous protein isoforms expression. However, all the identified hybridoma is against VWC domain of CCN5. These studies indicate that the VWC domain is the most antigenic region of CCN5, and provides a highly useful tool for measuring and localizing CCN5. However, the lack of apparent antigenicity of the other two domains limits the utility of this approach. (3) Which isoforms of CCN5 localize to the nucleus and how do they get there? I explored some of the mechanisms by which CCN5 might localize to the cell nucleus by testing recombinant CCN5 domains. One putative but weak nuclear localization signal (NLS) was found in the amino acid sequence of the rodent forms of CCN5. However, mutagenesis of the basic amino acids within this sequence did not cause a loss of nuclear localization, thus it appears that CCN5 is not imported into the nucleus via a canonical NLS. Further studies showed that the non-secreted CCN5 and some of its truncated forms containing VWC domain were localized in the cell nucleus. These results strongly suggest that the VWC domain is required for nuclear import.;In summary, my work provides a model of how CCN5 might regulate the phenotypic switch of VSMC, and hence proliferation and migration. It also suggests that targeting the regulation of CCN5 alternative splicing may be useful in controlling VSMC proliferation, which could have important clinical implications. Finally, the reagents generated in these studies provide a useful toolkit for future research on CCN5. |
