Signalling pathways regulating galactosaminoglycan synthesis and structure in vascular smooth muscle: Implications for lipoprotein binding and atherosclerosis.

Atherosclerosis commences with the trapping of low density lipoproteins (LDLs) in blood vessels by modified proteoglycans (PGs) with hyperelongated glycosaminoglycan (GAG) chains. GAG chain synthesis and growth factor mediated hyperelongation regulates the composition and size of PGs in a manner that would cause low density lipoprotein (LDLs) retention in vessel wall. Galactosaminoglycans are a class of GAGs, commonly observed on PGs. Multiple enzymes are involved in galactosaminoglycan biosynthesis. Galactosaminoglycan synthesis is regulated by various signalling pathways which are amenable to pharmacological manipulation to treat atherosclerosis. Receptor mediated signalling pathways including protein tyrosine kinase receptors (PTKRs), serine/threonine kinase receptors (S/TKRs) and G-protein coupled receptors (GPCRs) pathways regulate galactosaminoglycan synthesizing enzyme expression. Increased expression of these enzymes modify galactosaminoglycan chain structure by making them hyperelongated. This review focuses on the signalling pathways regulating the expression of genes involved in galactosaminoglycan synthesis and modification. Furthermore, there are multiple other processes for inhibiting the interactions between LDL and galactosaminoglycans such as peptide mimetics of ApoB100 and anti-galactosaminoglycan antibodies and the therapeutic potential of these strategies is also addressed.

The expansion of GPCR transactivation-dependent signalling to include serine/threonine kinase receptors represents a new cell signalling frontier.

G protein-coupled receptor (GPCR) signalling is mediated through transactivation-independent signalling pathways or the transactivation of protein tyrosine kinase receptors and the recently reported activation of the serine/threonine kinase receptors, most notably the transforming growth factor-ß receptor family. Since the original observation of GPCR transactivation of protein tyrosine kinase receptors, there has been considerable work on the mechanism of transactivation and several pathways are prominent. These pathways include the "triple membrane bypass" pathway and the generation of reactive oxygen species. The recent recognition of GPCR transactivation of serine/threonine kinase receptors enormously broadens the GPCR signalling paradigm. It may be predicted that the transactivation of serine/threonine kinase receptors would have mechanistic similarities with transactivation of tyrosine kinase pathways; however, initial studies suggest that these two transactivation pathways are mechanistically distinct. Important questions are the relative importance of tyrosine and serine/threonine transactivation pathways, the contribution of transactivation to overall GPCR signalling, mechanisms of transactivation and the range of cell types in which this phenomenon occurs. The ultimate significance of transactivation-dependent signalling remains to be defined but it appears to be prominent and if so will represent a new cell signalling frontier.

Transforming growth factor ß-mediated site-specific Smad linker region phosphorylation in vascular endothelial cells.

OBJECTIVES: Transforming growth factor (TGF)-ß regulates the function of vascular endothelial cells and may be involved in endothelial dysfunction. The canonical TGF-ß pathway involves TGF-ß receptor-mediated carboxy-terminal phosphorylation of Smad2; however, TGF-ß signalling also activates numerous serine/threonine kinases that phosphorylate Smad2 in its linker region. The expression of phosphorylated Smad linker proteins were determined following TGF-ß stimulation in the absence and presence of different serine/threonine kinase inhibitors in vascular endothelial cells. METHODS: Proteins were quantified by Western blotting using specific antibodies to individual phosphorylated Smad2 linker region residues. KEY FINDINGS: TGF-ß mediated the phosphorylation of all four Smad2 linker region residues of interest. Erk and Jnk specifically phosphorylate Ser245 while all mitogen-activated protein kinases phosphorylate Ser250 and Ser255. Thr220 and Ser245 are phosphorylated by phosphoinositide 3 kinase (PI3K), while Ser255 was phosphorylated by the PI3K/Akt pathway. CDK and GSK-3 were shown to phosphorylate Thr220 and Ser245. TGF-ß also mediated plasminogen activator inhibitor-1 gene expression that was attenuated by p38 and CDK inhibitors. CONCLUSIONS: TGF-ß-mediated phosphorylation of individual serine/threonine sites in the linker region of Smad2 occurs in a highly specific manner by kinases. These phosphorylations provide an opportunity to further understand a therapeutically targeted and very specific signalling pathway in vascular endothelial cells.

Transforming growth factor-ß signalling: role and consequences of Smad linker region phosphorylation.

Transforming growth factor-ß (TGF-ß) is a secreted homodimeric protein that plays an important role in regulating various cellular responses including cell proliferation and differentiation, extracellular matrix production, embryonic development and apoptosis. Disruption of the TGF-ß signalling pathway is associated with diverse disease states including cancer, renal and cardiac fibrosis and atherosclerosis. At the cell surface TGF-ß complex consists of two type I and two type II transmembrane receptors (TßRI and TßRII respectively) which have serine/threonine kinase activity. Upon TGF-ß engagement TßRII phosphorylates TßRI which in turn phosphorylates Smad2/3 on two serine residues at their C-terminus which enables binding to Smad4 to form heteromeric Smad complexes that enter the nucleus to initiate gene transcription including for extracellular matrix proteins. TGF-ß signalling is also known to activate other serine/threonine kinase signalling that results in the phosphorylation of the linker region of Smad2. The Smad linker region is defined as the domain which lies between the MH1 and MH2 domains of a Smad protein. Serine/threonine kinases that are known to phosphorylate the Smad linker region include mitogen-activated protein kinases, extracellular-signal regulated kinase, Jun N-terminal kinase and p38 kinase, the tyrosine kinase Src, phosphatidylinositol 3'-kinase, cyclin-dependent kinases, rho-associated protein kinase, calcium calmodulin-dependent kinase and glycogen synthase kinase-3. This review will cover the role of Smad linker region phosphorylation downstream of TGF-ß signalling in vascular cells. Key factors including the identification of the kinases that phosphorylate individual Smad residues, the upstream agents that activate these kinases, the cellular location of the phosphorylation event and the importance of the linker region in regulation and expression of genes induced by TGF-ß are covered.

Suramin inhibits PDGF-stimulated receptor phosphorylation, proteoglycan synthesis and glycosaminoglycan hyperelongation in human vascular smooth muscle cells.

OBJECTIVES: Suramin is a polysulfonated naphthylurea with antiparasitic and potential antineoplastic activity. Suramin's pharmacological actions, which have not yet been fully elucidated, include antagonism of the action of platelet-derived growth factor (PDGF) at its receptor. We investigated the effects of suramin on PDGF-stimulated proteoglycan synthesis. METHODS: Human vascular smooth muscle cells (VSMCs) were incubated in the presence and absence of PDGF and suramin with [(3) H]thymidine or (35) SO4 as radiolabels. Mitogenic response was determined by [(3) H]thymidine incorporation. PDGFß receptor phosphorylation was assessed by western blotting. Proteoglycan size and glycosaminoglycan chain synthesis and size were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Alphascreen phosphotyrosine assay kit was used to investigate PDGFß receptor tyrosine kinase inhibition by suramin. KEY FINDINGS: Suramin decreased PDGF-stimulated proliferation, proteoglycan synthesis and GAG chain hyperelongation. Suramin also directly inhibited PDGFß receptor kinase activity as well as PDGFß receptor phosphorylation in intact VSMCs. CONCLUSIONS: These data show that inhibition of PDGFß receptor phosphorylation in intact cells is necessary to define a fully active PDGF antagonist. They also confirm that PDGFß receptor kinase activity is necessary for PDGF-mediated atherogenic changes in proteoglycan synthesis and support efforts to develop PDGFß receptor antagonists as potential anti-atherosclerotic agents.