Expression and functional characterization of a novel fibronectin in fish and mammals

Fibronectin (FN) is a highly conserved extracellular matrix (ECM) protein that plays a crucial role in many cellular processes. Previously, we identified a novel form of FN (FN2) in zebrafish that possesses a truncated structure and is generated by a unique RNA splicing pattern. In this study, it is shown that FN2 is evolutionarily conserved, found in multiple species of fish and in mammals. Unlike other FNs, FN2 exists as a monomer and localizes on the cell surface instead of in the ECM. Using a serum-free mouse embryo (SFME) cell line, factors that regulate FN2 expression were examined. TGF-β induces SFME cells to differentiate into astrocytes and also upregulates expression of FN2 in the cultures. BMP-4 induces the cells to differentiate into neuron and upregulates several FN isoforms, but has no effect on expression of FN2. Experiments conducted with recombinant zebrafish FN2 demonstrate that this protein has the ability to promote the attachment and spreading of Chinook salmon embryo (CHSE) and mouse NIH 3T3 cells, however it has minimal effect on SFME cells. RGD peptide can inhibit attachment of CHSE cells to FN2 substrate, indicating that FN2 may interact with a integrin receptor on the cell surface. Addition of soluble FN2 protein to the culture inhibits endogenous and exogenous FN assembly into the ECM. Also, transfected cells expressing zebrafish FN2 or FN2 fragment lacking the unique C-tail are less able to assemble FN matrix. These results indicate that FN2 has the potential to act as a negative regulator of FN matrix assembly. Cell culture studies have shown that FN2 is able to mediate infection by infectious hematopoietic necrosis virus (IHNV). CHSE cells expressing recombinant FN2 are more susceptible to IHNV infection, with a greater percentage of cells exhibiting cytopathic effect (CPE) compared to nontransfected control cells and cells expressing full length FN. Incubation of nontransfected cells with soluble recombinant FN2 also increases IHNV infection. Results demonstrating that FN2 possesses unique properties and it is evolutionarily conserved indicate that this protein may have an important and novel function in normal cellular processes