Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells

A precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet-derived growth factor (PDGF)-mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin-binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an Α v -containing integrin receptor, but not Α v Β 1 or Α v Β 3 , appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50–100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation. © 1993 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49886/1/1041570104_ftp.pd

A Role for Syndecan-1 in Coupling Fascin Spike Formation by Thrombospondin-1

An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure

Regulation of sea urchin egg myosin by 53K, a myosin-binding protein

A myosin-binding protein is identified that mediates the low ionic strength solubility of myosin in extracts of unfertilized sea urchin eggs. The protein, termed 53K, possesses a subunit molecular weight on SDS-PAGE of 53 kD and an S value of 7. The association between myosin and 53K is demonstrated by their co-precipitation from extracts upon the addition of nucleotides and co-sedimention at 24S by sedimentation velocity centrifugation. Further, myosin is immunoprecipitated from extract with antibody to 53K, and 53K binds to a myosin affinity column. When extract is depleted of 53K, myosin precipitates out of extract in a nucleotide-independent manner. Reconstituting 53K and myosin partially restores the low ionic strength solubility demonstrated by myosin in fresh egg extracts.The binding site of 53K on myosin was examined to determine how the interaction between these two proteins might influence myosin\u27s solubility. Egg myosin digested with papain yields discrete fragments representing head and rod portions. Blot overlay techniques established that 53K binds to the head-rod junction on the myosin molecule. Additionally, both cross-linking studies using myosin fragments and 53K, and immunogold labelling of 53K bound to myosin indicated binding of 53K to the head-rod junction of myosin. Therefore, 53K may exert an effect on myosin solubility dependent, in part, on is binding site on myosin.It was demonstrated that 53K possesses myosin light chain kinase activity. 53K phosphorylates the regulatory light chains of egg and chicken gizzard myosin and increases the actin-activated ATPase activity of both myosins by approximately three fold. Autophosphorylation of 53K occurs, and may indicate that phosphorylation regulates 53K activity. 53K kinase activity is independent of Ca\sp{2+}/CaM although a second kinase activity exists in extracts that is Ca\sp{2+}/CaM-dependent. Contraction of reconstituted egg actin gels is enhanced by myosin light chain phosphorylation which is likely mediated by 53K. The myosin-binding protein 53K appears to be bifunctional in nature. Apart from the ability of 53K to mediate the low ionic strength solubility of myosin, it is also capable of myosin light chain phosphorylation. These two functional aspects of 53K may be closely linked in regulating myosin in the egg cytoplasm