Lyna Thach

Structure, Function, Pharmacology, and Therapeutic Potential of the G Protein, Gα/q,11.

G protein coupled receptors (GPCRs) are one of the major classes of cell surface receptors and are associated with a group of G proteins consisting of three subunits termed alpha, beta, and gamma. G proteins are classified into four families according to their α subunit; Gαi, Gαs, Gα12/13, and Gαq. There are several downstream pathways of Gαq of which the best known is upon activation via guanosine triphosphate (GTP), Gαq activates phospholipase Cβ, hydrolyzing phosphatidylinositol 4,5-biphosphate into diacylglycerol and inositol triphosphate and activating protein kinase C and increasing calcium efflux from the endoplasmic reticulum. Although G proteins, in particular, the Gαq/11 are central elements in GPCR signaling, their actual roles have not yet been thoroughly investigated. The lack of research of the role on Gαq/11 in cell biology is partially due to the obscure nature of the available pharmacological agents. YM-254890 is the most useful Gαq-selective inhibitor with antiplatelet, antithrombotic, and thrombolytic effects. YM-254890 inhibits Gαq signaling pathways by preventing the exchange of guanosine diphosphate for GTP. UBO-QIC is a structurally similar compound to YM-254890, which can inhibit platelet aggregation and cause vasorelaxation in rats. Many agents are available for the study of signaling downstream of Gαq/11. The role of G proteins could potentially represent a novel therapeutic target. This review will explore the range of pharmacological and molecular tools available for the study of the role of Gαq/11 in GPCR signaling.

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

Transforming growth factor (TGF)‐β regulates the function of vascular endothelial cells and may be involved in endothelial dysfunction. The canonical TGF‐β pathway involves TGF‐β receptor‐mediated carboxy‐terminal phosphorylation of Smad2; however, TGF‐β 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‐β stimulation in the absence and presence of different serine/threonine kinase inhibitors in vascular endothelial cells.

Protease activated receptor-1 mediated dual kinase receptor transactivation stimulates the expression of glycosaminoglycan synthesizing genes.

G protein-coupled receptors (GPCR) are one of the most important targets for therapeutics due to their abundance and diversity. The G protein-coupled receptor for thrombin can transactivate protein tyrosine kinase receptors (PTKR) and we have recently established that it can also transactivate serine/threonine kinase receptors (S/TKR). A comprehensive knowledge of the signalling pathways that GPCR transactivation elicits is necessary to fully understand the implications of both GPCR activation and the impact of target drugs. Here, we demonstrate that thrombin elicits dual transactivation-dependent signalling pathways to stimulate mRNA expression of glycosaminoglycan synthesizing enzymes chondroitin 4-O-sulfotransferase 1 and chondroitin sulfate synthase 1. The PTKR mediated response involves matrix metalloproteinases and the phosphorylation of the MAP kinase Erk. The S/TKR mediated response differs markedly and involves the phosphorylation of Smad2 carboxy terminal serine residues and does not involve matrix metalloproteinases. This work shows that all of the thrombin mediated signalling to glycosaminoglycan synthesizing enzyme gene expression occurs via transactivation-dependent pathways and does not involve transactivation-independent signalling. These findings highlight the complexity of thrombin-mediated transactivation signalling and the broader implications of GPCR targeted therapeutics.

(S)-[6]-Gingerol inhibits TGF-β-stimulated biglycan synthesis but not glycosaminoglycan hyperelongation in human vascular smooth muscle cells

(S)‐[6]‐Gingerol is under investigation for a variety of therapeutic uses. Transforming growth factor (TGF)‐β stimulates proteoglycan synthesis, leading to increased binding of low‐density lipoproteins, which is the initiating step in atherosclerosis. We evaluated the effects of (S)‐[6]‐gingerol on these TGF‐β‐mediated proteoglycan changes to explore its potential as an anti‐atherosclerotic agent.

Platelet-derived growth factor-stimulated versican synthesis but not glycosaminoglycan elongation in vascular smooth muscle is mediated via Akt phosphorylation

Proteoglycans are associated with the initiation of atherosclerosis due to their binding of apolipoproteins on lipid particles leading to retention in the vessel wall. The signaling pathways through which growth factors regulate the synthesis and structure of proteoglycans are potential therapeutic targets. Platelet-derived growth factor (PDGF) is present in atherosclerotic plaques and activates phosphorylation of the serine/threonine kinase Akt. We have investigated the role of Akt in the signaling pathways for proteoglycan core protein expression and elongation of glycosaminoglycan chains on proteoglycans secreted by human vascular smooth muscle cells. The pharmacological inhibitor of Akt phosphorylation, SN30978, blocked PDGF stimulated phosphorylation of Akt. SN30978 caused concentration dependent inhibition of PDGF stimulated radiosulfate incorporation into secreted proteoglycans and the response was blocked by the PDGF receptor antagonists Ki11502 and imatinib. Analysis of the size of the biglycan molecules by SDS-PAGE showed that PDGF increased the apparent size of biglycan but this effect on glycosaminoglycan chain elongation was blocked by Ki11502 but not by SN30978. PDGF also stimulated total protein core protein synthesis assessed as (35)S-methionine/cysteine incorporation and specifically the expression of versican mRNA. Both of these responses were blocked by SN30978. This data shows that PDGF-stimulated proteoglycan core protein synthesis but not glycosaminoglycan chain elongation is mediated via Akt phosphorylation. These data identify potential pathways for the development of agents which can pharmacologically regulate individual components of the synthesis of proteoglycans.

Cellular and Molecular Pathology of Age-Related Macular Degeneration: Potential Role for Proteoglycans

Age-related macular degeneration (AMD) is a retinal disease evident after the age of 50 that damages the macula in the centre of retina. It leads to a loss of central vision with retained peripheral vision but eventual blindness occurs in many cases. The initiation site of AMD development is Bruchs membrane (BM) where multiple changes occur including the deposition of plasma derived lipids, accumulation of extracellular debris, changes in cell morphology, and viability and the formation of drusen. AMD manifests as early and late stage; the latter involves cell proliferation and neovascularization in wet AMD. Current therapies target the later hyperproliferative and invasive wet stage whilst none target early developmental stages of AMD. In the lipid deposition disease atherosclerosis modified proteoglycans bind and retain apolipoproteins in the artery wall. Chemically modified trapped lipids are immunogenic and can initiate a chronic inflammatory process manifesting as atherosclerotic plaques and subsequent artery blockages, heart attacks, or strokes. As plasma derived lipoprotein deposits are found in BM in early AMD, it is possible that they arise by a similar process within the macula. In this review we consider aspects of the pathological processes underlying AMD with a focus on the potential role of modifications to secreted proteoglycans being a cause and therefore a target for the treatment of early AMD.

Evaluation of the potential synergism of imatinib-related poly kinase inhibitors using growth factor stimulated proteoglycan synthesis as a model response.

Tyrosine kinase inhibitors were the first class of smart drugs being specifically designed to inhibit a disease causing target. There is a very important but unresolved question as whether or not the overall therapeutic role of an individual tinib results from an action at its primary target, a single most likely, tyrosine kinase, or from the combined or aggregate action at the multiple targets which each tinib addresses.

Multiple growth factors, but not VEGF, stimulate glycosaminoglycan hyperelongation in retinal choroidal endothelial cells

A major feature of early age-related macular degeneration (AMD) is the thickening of Bruchs membrane in the retina and an alteration in its composition with increased lipid deposition. In certain pathological conditions proteoglycans are responsible for lipid retention in tissues. Growth factors are known to increase the length of glycosaminoglycan chains and this can lead to a large increase in the interaction between proteoglycans and lipids. Using choroidal endothelial cells, we investigated the effects of a number of AMD relevant growth factors TGFβ, thrombin, PDGF, IGF and VEGF on proteoglycan synthesis. Cells were characterized as of endothelial origin using the specific cell markers endothelial nitric oxide synthesis and von Willebrand factor and imaged using confocal microscopy. Cells were treated with growth factors in the presence and absence of the appropriate inhibitors and were radiolabeled with [35S]-SO4. Proteoglycans were isolated by ion exchange chromatography and sized using SDS-PAGE. Radiosulfate incorporation was determined by the cetylpyridinium chloride (CPC) precipitation technique. To measure cellular glycosaminoglycan synthesizing capacity we added xyloside and assessed the xyloside-GAGs by SDS-PAGE. TGFβ, thrombin, PDGF & IGF dose-dependently stimulated radiosulfate incorporation and GAG elongation as well as xyloside-GAG synthesis, however VEGF treatment did not stimulate any changes in proteoglycan synthesis. VEGF did not increase pAKT but caused a large increase in pERK relative to the response to PDGF. Thus, AMD relevant agonists cause glycosaminoglycan hyperelongation of proteoglycans synthesised and secreted by retinal choroidal endothelial cells. The absence of a response to VEGF is intriguing and identifies proteoglycans as a novel potential target in AMD. Future studies will examine the relevance of these changes to enhanced lipid binding and the development of AMD.

Assessing the role of Gαq/11 in cellular responses: an analysis of investigative tools

Seven transmembrane G Protein Coupled Receptors (GPCRs) are one of the major classes of cell surface receptors and play a major role through agonists and antagonists in human therapeutics [1]. GPCRs are associated with a group of G proteins which consist of 3 subunits termed alpha, beta and gamma. G proteins may be classified according to their effector molecules of the alpha subunit, which in mammals falls into several subtypes, Gαs, Gαi, Gα12 and Gαq. The Gαq family consists of four subunits Gαq, Gα11, Gα14 and Gα15/16. In contrast to the protein kinase receptors which have intrinsic (kinase) enzymatic activity, GPCRs do not have enzymatic activity-enzymatic activity mediating signal transduction resides in the Gα proteins which have GTPase activity [2]. Gα proteins exist in the GTP bound form. Ligand initiated conformational changes in the GPCR causes the release of bound Gα proteins. This dissociation initiates the GTPase activity, hydrolyzing GTP to GDP which is released from the proteins and allows alterative interactions leading to downstream signal transduction.

Insights into cellular signalling by G protein coupled receptor transactivation of cell surface protein kinase receptors

G protein coupled receptor (GPCR) signalling is mediated by transactivation independent and transactivation dependent pathways. GPCRs transactivate protein tyrosine kinase receptors (PTKRs) and protein serine/threonine kinase receptors (PS/TKR). Since the initial observations of transactivation dependent signalling, there has been an effort to understand the mechanisms behind this phenomena. GPCR signalling has evolved to include biased signalling. Biased signalling, whereby selected ligands can activate the same GPCR that can generate multiple signals, but drive only a unique response. To date, there has been no focus on the ability of biased agonists to activate the PTKR and PS/TKR transactivation pathways differentially. As such, this represents a novel direction for future research. This review will discuss the main mechanisms of GPCR mediated receptor transactivation and the pathways involved in intracellular responses.