G-Proteins and Intimal Hyperplasia

Mark G. Davies, MD

Intimal hyperplasia is the universal response of a vessel to injury. It involves the coordinated stimulation of smooth muscle cells by mechanical, cellular and humoral factors to induce a program of cellular activation that leads to proliferation, migration and extracellular matrix deposition. Signal transduction is the mechanism by which a cell responds to these extracellular signals and converts them to intracellular instructions that co-ordinate cell responses. There are two basic ligand operated receptor mechanisms responsible for signal transduction: ligand-modulated release of a secondary intracellular mediator (G-protein coupled receptors) and ligand-regulated enzyme activity (tyrosine kinase coupled receptors). Targeting signal transduction pathways is a broad and novel avenue for molecular therapeutics. There are over 1000 G-protein coupled receptors (GPCRs) and 400 of these are considered non-sensory GPCRs; 41 are targets of commercial drugs and 48 are targeted for drug development.

G-Proteins Classification and Action

G-proteins are intrinsic membrane-bound proteins which act as transmembrane signal transducers in cells. They consist of three distinct subunits: a, b, and g; the bg subunits form a tightly associated dimer. G-protein hetero-trimers are classified according to differences in their Ga subunits (G,ai, Gas, Gaq, Ga11/12). Both Ga and Gbg subunits have different cellular targets. and their activation is intimately associated with receptor activation. Receptor-G-protein activity is tightly regulated by a series of G-protein related kinases, which dephosphorylate the receptors, and a second family of proteins, regulators of G-protein signaling (RGS), which regulate the GTP activity of the heterotrimeric units. Thereafter, the process of receptor internalization, which involves the key proteins dynein, b-arrestin and caveolin, regulates downstream secondary mediator activation. In general, Gai protein subtypes (ai1, ai2, and ai3) inhibit adenylate cyclase and decrease intracellular cAMP levels. Gas proteins can activate adenylate cyclase and increase intracellular cAMP, and can also regulate calcium channels. Gaq subunits have been shown to activate phospholipase Cb. The traditional role of bg subunits, released following activation of Gai proteins, is to bind active as-GTP species, reducing their activity and leading to decreased cAMP levels. However, Gbg subunits of the pertussis toxin sensitive Gai proteins can enhance the effect of Gas on type II and IV adenylate cyclase but can inhibit Gas-stimulated type I adenylate cyclase. The other three Gas-stimulated adenylate cyclases (type III, V and VI) appear not to be modulated by the Gbg subunits. bg subunits have also been shown to activate phospholipase C isozymes (PLC-b, g and d), to activate small GTPases, and to transactivate the epidermal growth factor receptor.

G-Proteins and Proliferation

The role of G-protein signal transduction in cellular proliferation is being defined. In mesenchymal cells, Gai G-proteins enhance ERK activity, DNA synthesis and cell proliferation. Furthermore, a Gai-regulated role in mitosis has been identified in fibroblast cell lines. In BALB/c3T3 cells, Gai2 and Gbg are responsible for ERK activation and DNA synthesis, while Gai3 is responsible for cell transformation. Gbg subunit activation results in proliferation through the activation of receptor tyrosine kinase pathways, involving Src-like kinases that activate Shc-Grb2-Sos and ras pathways. Ras activates raf directly or acts on PI3-Kinase to induce raf, protein kinase B (akt) and rac phosphorylation. These various pathways lead to ERK / p38 /JNK activation and subsequent transcription. Gbg-mediated ERK activation has recently been shown to involve transactivation of the epidermal growth factor receptor in smooth muscle cells.

G-Proteins and Migration

The role of G-protein signal transduction in the cellular migration is less well defined. Migration of inflammatory cells is driven in part by families of chemokines, whose receptors are G-protein coupled and pertussis toxin sensitive suggesting a role for Gai. Further recent data has demonstrated roles for ras, rac, rho and ERK1/2 in chemokine linked Gai induced cascades. It appears that receptors coupled to Gai can mediate chemotaxis, even when they lack the distinguishing features of chemokine receptors and when their agonist ligands are not classical chemokines. Activation of Gai is required but probably not sufficient for chemotaxis. Although Gai coupled receptors undergo internalization during many cell-induced responses (such as mitogenesis), similar Gai receptors can mediate chemotaxis without undergoing agonist-induced internalization. Irrespective Gai, Gbg is an essential mediator of chemotaxis. Integrin signaling in cells is mediated through Focal Adhesion Kinase but also involves an integrin-associated protein, which is G-protein coupled and pertussis toxin sensitive. Thrombin induces migration that is pertussis toxin sensitive and also involves transactivation of the epidermal growth factor receptor by Gbg. PDGF and IGF both have pertussis toxin sensitive elements when they are used as chemoattractants in migration assays. PDGF activation induces formation of sphingosine-1-phosphate which in turn binds to its receptors and is responsible for the migration to PDGF.

G-Protein Expression and Response to Injury

In vein grafts, there is enhanced expression of Gai2 and Gbg subunits and de novo expression of Gai3. These dramatic changes in G-protein expression are associated with a change in the functional coupling of serotonergic receptors to G-proteins; vein grafts develop pertussis toxin sensitive responses, while control veins have pertussis toxin insensitive response. The changes in the expression of G-proteins in vein grafts, particularly the Gai3 and Gbg subunits, parallel the formation of intimal hyperplasia. A similar pattern of changes occurs during the development of intimal hyperplasia after arterial injury. These results suggest that the smooth muscle cell phenotype within the intimal hyperplasia response undergoes changes in membrane transducion systems with the expression of new components and changes in the coupling of these components to surface receptors.

Given the changes in G-proteins, modulating G-protein expression or function should affect the intimal hyperplastic response. Gbg signaling is common to all G-proteins and was targeted by a gene encoding a specific Gbg inhibitor, the carboxyl terminus of the b-adrenergic receptor kinase (bARKCT). Intimal hyperplasia, both in vein graft and arterial injury models was significantly reduced by a single application of this inhibitor. The altered G protein-coupling after vein grafting (i.e. development of pertussis toxin sensitivity) was blocked by bARKCT treatment. It was recently demonstrated that one time exposure to pertussis toxin, a Gai inhibitor, reduces intimal hyperplasia in vivo. Thus, Gai/Gbg-mediated pathways appear to play major roles in intimal hyperplasia development.

Conclusion

G-proteins represent a significant, little explored signal transduction system in the development of vascular disease. Targeting G-protein coupled responses, instead of individual receptors, appears to offer a novel therapeutic strategy.

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