Effects Of Soluble Fibronection On RhoA And CAMP/PKA-Mediated Signal Transduction Pathways

Integrin is an important receptor of cellular adhesion molecules and mediates the adhesion between cell and cell or cell and extra-cellular matrix. It is also involved in intracellular and extracellular information transmission. Integrin consists of one a and oneβsubunit. So far,8 (3 subunits and 18 a subunits have been found and the subunits may assemble into 25 instinct integrins. Among them, integrinα5β1 is the receptor of fibronectin. When cells attach to extracellular matrix containing fibronectin, integrinα5β1 is activated. However, a large portion of fibronectin is soluble within the body. Soluble fibronectin exists mainly in the blood plasma and reaches each part of the body through the blood stream. There is no research data addressing whether soluble fibronectin activates integrin-mediated signal transduction.RhoA is a key member of Rho family of small GTP-binding proteins and mediates signaling relating to cytoskeleton arrangement, migration, proliferation and gene expression. Study found that there was a variety of cancer cells over-expressing RhoA and invasive growth and metastasis of cancer cell were inhibited when RhoA activity was inhibited. A lot of research data have addressed the significance of the association between integrin and RhoA. However, the exact mechanism is still unclear. PKA is a protein kinase related to the cAMP signaling pathway. PKA is an isozyme existing as a tetramer of two R-and two C-subunits. Occupancy of the R-subunits by cAMP subsequently results in the rapid dissociation of the holoenzyme, allowing the catalytically active c-subunit to phosphorylate substrates.Research data suggested that PKA could inhibit the activity and the function of RhoA. The possible interaction among integrin, RhoA and PKA is unknown.ObjectivesThis study was designed to investigate the relationship among soluble fibronectin/integrin-, cAMP/PKA-, and RhoA-mediated intracellular signal transduction pathways. This thesis was focused on investigating the effect of soluble fibronectin on cytoskeleton, RhoA activity, cAMP content and PKA activity.Methods(1) To examine cytoskeletal changes, we stained F-actin with rhodamine-conjugated phalloidin.(2) The morphological dynamic changes of the PC-3 cells treated with drug were visualized by a reverse microscope.(3) Rho activity was detected by pull down assay.(4) cAMP concentration was measured by Radioimmunoassay (RIA).(5) The distribution of PKA catalytic subunit was detected by fluorescent microscopy.(6) The amount of the phosphorylation of VASP and PKA catalytic subunit were detected by Western blotting. (7) The reporter gene plasmid pcDNACRE-Luc and plasmid of referrence geneβ-galactosidase were co-transfected into SGC-7901 cells. The reporter gene expression activity of the cells treated with drug was analyzed by luciferase reporter gene assay.(8) The SGC-7901 cells transfected with wild type pcDNA HT31 and pGFP were selected with G418. The cells stably expressing the peptide Ht31 were obtained by Limited-Dilution Method.(9) The SGC-7901/HT31wt cells which stably expressed the peptide Ht31 were grown on 24-well plate. The cells treated with drug were stained with phalloidin and the F-actin was visualized under fluorescent microscope.Results(1) Soluble fibronectin caused obviously morphological changes of PC-3 cells. Y27632, an inhibitor of RhoA related protein kinase Rock, and antibody against integrinα5β1 partially blocked the effect of soluble fibronectin.(2) Soluble fibronectin dose and time-dependently increased RhoA activity. Pre-incubating the cells with the antibody against integrinα5β1 partially inhibited the RhoA activation induced by soluble fibronectin. PKA could inhibit RhoA activity and fibronectin could reduce this effect.(3) The elevation of cAMP level induced by forskolin or PTX could be inhibited by soluble fibronectin.(4) The increase of PKA catalytic subunit indeuced by forskolin or PTX could be inhibited by soluble fibronectin.(5) The increases of VASP phosphorylation and CRE-dependent transcription activity induced by forskolin could be inhibited by soluble fibronectin. However, soluble fibronectin could not inhibite these increase induced by cAMP.(6) The cell clone which stably expressed the peptide Ht31 selected out was identified with SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and two specific protein bands, respectively at 37KD and 27KD, were proved.(7) Soluble fibronectin dose-dependently increased the formation of F-actin. PKA was able to antagonize the soluble fibronectin-induced F-actin formation.This effect was partly impaired upon the disruption of PKA anchorage.Conclusions(1) Soluble fibronectin also binds integrinα5β1 and activates consequent signaling events.(2) Soluble fibronectin increased the formation of F-actin and caused obviously morphological changes. These effects were related to RhoA and PKA.(3) Soluble fibronectin could increase RhoA activity and antagonize the inhibition of PKA on RhoA activity.(4) Soluble fibronectin could inhibit cAMP/PKA mediated signal transduction pathway. The possible acting poin was adenylate cyclase.Our results not only help us to understand the relationship among fibronectin/integrin-, cAMP/PKA-and RhoA-mediated signal transduction pathways, but also provides novel target for strategy of cancer therapy.