Alleviation of Diabetic Retinopathy by Glucose-Triggered Delivery of Vitamin D via Dextran-Gated Functionalized Mesoporous Silica Nanoparticles.

Diabetic retinopathy (DR) is the most common retinal disorder, developed in 35% of patients with diabetes mellitus. Lower serum levels of 25-hydroxyvitamin D are associated with the increased risk of developing DR. High doses of the active form of vitamin D (VD), on the contrary, for a long period of time may lead to hypercalcemia and an imbalance in the regulation of bone metabolism. Herein, we studied the efficacy of dextran-gated carboxyphenylboronic acid (CPBA)-functionalized mesoporous silica nanoparticles (MSNs) for glucose-sensitive delivery of 1,25-dihydroxyvitamin D3 to modulate cellular oxidative stress and inflammation for managing DR. The physical adsorption technique was employed to load VD onto nanoparticles (263.63 µg/mg (w/w)). In the presence of glucose, the dextran molecules detach from pores, allowing VD to release since glucose has 1,2-cis diol groups which have very high affinity to CPBA. Approximately 75% of VD was released upon exposure to 25 mM glucose at a time point of 10 h, demonstrating glucose-responsive delivery. Furthermore, MSN-CPBA was able to deliver VD in a glucose-dependent manner and improve the bioavailability of VD. In high-glucose-supplemented human retinal cells, MSN-CPBA increased the bioavailability of VD and reduced cellular oxidative stress and inflammation. The results suggested that the VD-loaded nanocarrier exerted remarkable therapeutic capacity in reducing the risk of developing DR. By using MSN-CPBA as a delivery platform with dextran gating, the research proposes an effective treatment approach for improving the bioavailability and effectiveness of a hydrophobic molecule in the treatment of DR.

Gαq Is the Specific Mediator of PAR-1 Transactivation of Kinase Receptors in Vascular Smooth Muscle Cells.

AIMS: G protein-coupled receptor (GPCR) transactivation of kinase receptors greatly expands the actions attributable to GPCRs. Thrombin, via its cognate GPCR, protease-activated receptor (PAR)-1, transactivates tyrosine and serine/threonine kinase receptors, specifically the epidermal growth factor receptor and transforming growth factor-ß receptor, respectively. PAR-1 transactivation-dependent signalling leads to the modification of lipid-binding proteoglycans involved in the retention of lipids and the development of atherosclerosis. The mechanisms of GPCR transactivation of kinase receptors are distinct. We aimed to investigate the role of proximal G proteins in transactivation-dependent signalling. MAIN METHODS: Using pharmacological and molecular approaches, we studied the role of the G⍺ subunits, G⍺q and G⍺11, in the context of PAR-1 transactivation-dependent signalling leading to proteoglycan modifications. KEY FINDINGS: Pan G⍺q subunit inhibitor UBO-QIC/FR900359 inhibited PAR-1 transactivation of kinase receptors and proteoglycans modification. The G⍺q/11 inhibitor YM254890 did not affect PAR-1 transactivation pathways. Molecular approaches revealed that of the two highly homogenous G⍺q members, G⍺q and G⍺11, only the G⍺q was involved in regulating PAR-1 mediated proteoglycan modification. Although G⍺q and G⍺11 share approximately 90% homology at the protein level, we show that the two isoforms exhibit different functional roles. SIGNIFICANCE: Our findings may be extrapolated to other GPCRs involved in vascular pathology and highlight the need for novel pharmacological tools to assess the role of G proteins in GPCR signalling to expand the preeminent position of GPCRs in human therapeutics.

Smad linker region phosphorylation is a signalling pathway in its own right and not only a modulator of canonical TGF-ß signalling.

Transforming growth factor (TGF)-ß signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-ß signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-ß receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-ß can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-ß signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-ß signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.

Thyroid Hormone Distributor Proteins During Development in Vertebrates.

Thyroid hormones (THs) are ancient hormones that not only influence the growth, development and metabolism of vertebrates but also affect the metabolism of (at least some) bacteria. Synthesized in the thyroid gland (or follicular cells in fish not having a discrete thyroid gland), THs can act on target cells by genomic or non-genomic mechanisms. Either way, THs need to get from their site of synthesis to their target cells throughout the body. Despite being amphipathic in structure, THs are lipophilic and hence do not freely diffuse in the aqueous environments of blood or cerebrospinal fluid (in contrast to hydrophilic hormones). TH Distributor Proteins (THDPs) have evolved to enable the efficient distribution of THs in the blood and cerebrospinal fluid. In humans, the THDPs are albumin, transthyretin (TTR), and thyroxine-binding globulin (TBG). These three proteins have distinct patterns of regulation in both ontogeny and phylogeny. During development, an additional THDP with higher affinity than those in the adult, is present during the stage of peak TH concentrations in blood. Although TTR is the only THDP synthesized in the central nervous system (CNS), all THDPs from blood are present in the CSF (for each species). However, the ratio of albumin to TTR differs in the CSF compared to the blood. Humans lacking albumin or TBG have been reported and can be asymptomatic, however a human lacking TTR has not been documented. Conversely, there are many diseases either caused by TTR or that have altered levels of TTR in the blood or CSF associated with them. The first world-wide RNAi therapy has just been approved for TTR amyloidosis.

Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation.

Protease activated receptors (PARs) transactivate both epidermal growth factor receptors (EGFR) and transforming growth factor (TGF)-ß receptors (TGFBR1) in vascular smooth muscle leading to the increased expression of genes (CHST11 and CHSY1) which are rate limiting for the enzymes that mediate hyperelongation of glycosaminoglycan (GAG) chains on the lipid-binding proteoglycan, biglycan. This is an excellent model to investigate mechanisms of transactivation as the processes are biochemically distinct. EGFR transactivation is dependent on the classical matrix metalloprotease (MMP) based triple membrane bypass mechanism and TGFBR1 transactivation is dependent on Rho/ROCK signalling and integrins. We have shown that all kinase receptor signalling is targeted towards phosphorylation of the linker region of the transcription factor, Smad2. We investigated the mechanisms of thrombin mediated kinase receptor transactivation signalling using anti-phospho antibodies and Western blotting and gene expression by RT-PCR. Thrombin stimulation of phospho-Smad2 (Ser 245/250/255) and of phospho-Smad2(Thr220) via EGFR transactivation commences quickly and extends out to at least 4 h whereas transactivation via TGFBR1 is delayed for 120 min but also persists for at least 4 h. Signalling of thrombin stimulated Smad linker region phosphorylation is approximately equally inhibited by the MMP inhibitor, GM6001 and the ROCK inhibitor, Y27632, and similarly expression of CHST11 and CHSY1 is approximately equally inhibited by GM6001 and Y27632. The data establishes Smad linker region phosphorylation as a central target of all transactivation signalling of GAG gene expression and thus an upstream kinase may be a target to prevent all transactivation signalling and its pathophysiological consequences.

Recent advances in the management of diabetic retinopathy.

Diabetic retinopathy (DR) is a microvascular complication of diabetes and is the leading cause of vision loss in people with diabetes. The current treatments do not target early stages of disease or impede disease progression. Therefore, the identification of new therapeutic targets, the development of novel therapies targeting early stages of the disease and accurate models that simulate pathological characteristics of this disorder are crucial. This review provides an overview of the pathological mechanisms underlying the development of DR, highlighting the recent advances in current and emerging treatments for DR.

Individual Smad2 linker region phosphorylation sites determine the expression of proteoglycan and glycosaminoglycan synthesizing genes.

Growth factors such as thrombin and transforming growth factor (TGF)-ß facilitate glycosaminoglycan (GAG) chain hyperelongation on proteoglycans, a phenomenon that increases lipoprotein binding in the vessel wall and the development of atherosclerosis. TGF-ß signals via canonical carboxy terminal phosphorylation of R-Smads and also non-canonical linker region phosphorylation of R-Smads. The G protein coupled receptor agonist, thrombin, can transactivate the TGF-ß receptor leading to both canonical and non-canonical Smad signalling. Linker region phosphorylation drives the expression of genes for the synthesis of the proteoglycan, biglycan. Proteoglycan synthesis involves core protein synthesis, the initiation of GAG chains and the subsequent elongation of GAG chains. We have explored the relationship between the thrombin stimulated phosphorylation of individual serine and threonine sites in the linker region of Smad2 and the expression of GAG initiation xylosyltransferase-1 (XT-1) and GAG elongation chondroitin 4-sulfotransferase-1 (C4ST-1) and chondroitin synthase-1 (CHSY-1) genes. Thrombin stimulated the phosphorylation of all four target residues (Thr220, Ser245, Ser250 and Ser255 residues) with a similar temporal pattern - phosphorylation was maximal at 15 min (the earliest time point studied) and the level of the phospho-proteins declined thereafter over the following 4 h. Jnk, p38 and PI3K, selectively mediated the phosphorylation of the Thr220 residue whereas the serine residues were variously phosphorylated by multiple kinases. Thrombin stimulated the expression of all three genes - XT-1, C4ST-1 and CHSY-1. The three pathways mediating Thr220 phosphorylation were also involved in the expression of XT-1. The target pathways (excluding Jnk) were involved in the expression of the GAG elongation genes (C4ST-1 and CHSY-1). These findings support the contention that individual Smad linker region phosphorylation sites are linked to the expression of genes for the initiation and elongation of GAG chains on proteoglycans. The context of this work is that a specific inhibitor of GAG elongation represents a potential therapeutic agent for preventing GAG elongation and lipid binding and the results indicate that the specificity of the pathways is such that it might be therapeutically feasible to specifically target GAG elongation without interfering with other physiological processes with which proteoglycans are involved.

Thrombin promotes PAI-1 expression and migration in keratinocytes via ERK dependent Smad linker region phosphorylation.

Keratinocyte proliferation and migration is essential during re-epithelialisation for the restoration of the epithelial barrier during skin wound healing. Numerous growth factors are involved in the stimulation of keratinocyte proliferation and migration. The signalling pathways that drive these processes during wound healing are not well defined. This study investigated thrombin-mediated signalling in keratinocytes. The thrombin receptor, protease-activated receptor 1 (PAR-1) is a seven transmembrane G-protein coupled receptor that is known to transactivate the epidermal growth factor receptor (EGFR). Immortalized human keratinocytes (HaCaT cells) were treated with thrombin and selective inhibitors to EGFR and MAP kinases. Whole cell lysates were separated on SDS-PAGE and analysed by Western blot using antibodies against transcription factor Smad2. Quantitative real-time polymerase chain reaction was used to measure the mRNA expression of PAI-1 while scratch wound assays were used to measure keratinocyte migration. Western blot data showed that thrombin mediates PAR-1 transactivation of EGFR and the downstream phosphorylation of the transcription factor Smad2 linker (Smad2L) region. ERK1/2 inhibition by UO126 caused a decrease in Smad2L phosphorylation while the p38 inhibitor SB202190 and JNK inhibitor SP600125 did not. Smad2L Ser250 was specifically phosphorylated by this thrombin mediated pathway while Ser245 and Ser255 were not. Thrombin increased PAI-1 mRNA expression and keratinocyte migration and this was reduced when either EGFR or ERK1/2 were blocked. Taken together these results show that thrombin mediated mRNA expression of PAI-1 in keratinocytes and migration occurs via EGFR transactivation and involves signalling intermediates ERK1/2 and Smad2 and may be a key pathway in skin wound healing.

Flavopiridol Inhibits TGF-ß-Stimulated Biglycan Synthesis by Blocking Linker Region Phosphorylation and Nuclear Translocation of Smad2.

Transforming growth factor-ß (TGF-ß) is a pleiotropic growth factor implicated in the development of atherosclerosis for its role in mediating glycosaminoglycan (GAG) chain hyperelongation on the proteoglycan biglycan, a phenomenon that increases the binding of atherogenic lipoproteins in the vessel wall. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signaling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have used flavopiridol, a well-known cyclin-dependent kinase inhibitor, to study the role of linker region phosphorylation in the TGF-ß-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS-PAGE to assess proteoglycan synthesis, real-time polymerase chain reaction to assess gene expression, and chromatin immunoprecipitation to assess the binding of Smads to the promoter region of GAG Synthesizing genes. Flavopiridol blocked TGF-ß-stimulated synthesis of mRNA for the GAG synthesizing enzymes, and chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1) and TGF-ß-mediated proteoglycans synthesis as well as GAG hyperelongation. Flavopiridol blocked TGF-ß-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-ß, and this response was blocked by flavopiridol, demonstrating that linker region phosphorylated Smad can pass to the nucleus and positively regulate transcription. These results demonstrate the validity of the kinases, which phosphorylate the Smad linker region as potential therapeutic target(s) for the development of an agent to prevent atherosclerosis.

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.

RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells.

G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the ß-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

Role Played by Signalling Pathways in Overcoming BRAF Inhibitor Resistance in Melanoma.

The discovery of the BRAFV600E mutation led to the development of vemurafenib (PLX4032), a selective BRAF inhibitor specific to the kinase, for the treatment of metastatic melanomas. However, initial success of the drug was dampened by the development of acquired resistance. Melanoma was shown to relapse in patients following treatment with vemurafenib which eventually led to patients' deaths. It has been proposed that mechanisms of resistance can be due to (1) reactivation of the mitogen-activated protein kinase (MAPK) signalling pathway via secondary mutations, amplification or activation of target kinase(s), (2) the bypass of oncogenic pathway via activation of alternative signalling pathways, (3) other uncharacterized mechanisms. Studies showed that receptor tyrosine kinases (RTK) such as PDGFRß, IGF1R, EGFR and c-Met were overexpressed in melanoma cells. Along with increased secretion of growth factors such as HGF and TGF-α, this will trigger intracellular signalling cascades. This review discusses the role MAPK and Phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) pathways play in the mechanism of resistance of melanomas.

Gaq proteins: molecular pharmacology and therapeutic potential.

Seven transmembrane G protein-coupled receptors (GPCRs) have gained much interest in recent years as it is the largest class among cell surface receptors. G proteins lie in the heart of GPCRs signalling and therefore can be therapeutically targeted to overcome complexities in GPCR responses and signalling. G proteins are classified into four families (Gi, Gs, G12/13 and Gq); Gq is further subdivided into four classes. Among them Gαq and Gαq/11 isoforms are most crucial and ubiquitously expressed; these isoforms are almost 88% similar at their amino acid sequence but may exhibit functional divergences. However, uncertainties often arise about Gαq and Gαq/11 inhibitors, these G proteins might also have suitability to the invention of novel-specific inhibitors for each isoforms. YM-254890 and UBO-QIC are discovered as potent inhibitors of Gαq functions and also investigated in thrombin protease-activated receptor (PAR)-1 inhibitors and platelet aggregation inhibition. The most likely G protein involved in PAR-1 stimulates responses is one of the Gαq family isoforms. In this review, we highlight the molecular structures and pharmacological responses of Gαq family which may reflect the biochemical and molecular role of Gαq and Gαq/11. The advanced understanding of Gαq and Gαq/11 role in GPCR signalling may shed light on our understanding on cell biology, cellular physiology and pathophysiology and also lead to the development of novel therapeutic agents for a number of diseases.

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 Bruch's 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.

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 Bruch's 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 TGFß, 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. TGFß, 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.

An Investigation of Sodium Fusidate and Recombinant Cytochrome P450 Enzymes Inhibition In-Vitro.

BACKGROUND: Sodium fusidate (fusidic acid) is an antimicrobial agent that is used in the treatment of staphylococcal and streptococcal infections. Several case reports have noted a drug interaction between sodium fusidate and CYP3A4 metabolised statins, leading to statin toxicity. It is unclear whether sodium fusidate has the potential to cause interactions with other cytochrome P450 enzymes. OBJECTIVE: To investigate the effects of sodium fusidate on recombinant cytochrome P450 enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in-vitro. METHODS: A range of sodium fusidate concentrations (0.1µM, 1µM, 10µM, 100µM, 300µM, 1000µM and 10000µM) were tested to examine its activity on rCYP1A2, rCYP2C9, rCYP2C19, rCYP2D6 and rCYP3A4 using a luminescent assay with a luciferin substrate. RESULTS: Sodium fusidate inhibited all enzymes at tested concentrations which are relevant to those likely to be achieved in clinical practice. Further, sodium fusidate was found to be a time-dependent inhibitor of all the tested isoenzymes, with the exception of rCYP2C9. CONCLUSION: These findings suggest that there is a potential for sodium fusidate to cause drug interactions when used with other agents that are substrates for rCYP1A2, rCYP2C9, rCYP2C19, rCYP2D6 or rCYP3A4. Understanding the basis of this potential drug interaction will assist in safer use of sodium fusidate in clinical practice.

The role of specific Smad linker region phosphorylation in TGF-ß mediated expression of glycosaminoglycan synthesizing enzymes in vascular smooth muscle.

Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-ß (TGF-ß) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-ß receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-ß. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-ß receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis.

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

INTRODUCTION: 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. METHODS: We selected a series of ten tinibs (gefitinib, sunitinib, lapatinib, erlotinib, imatinib, sorafenib, axitinib, vanitinib, bosutinib, dasatinib) with various known targets and investigated their activities in the inhibition of proteoglycan synthesis and GAG hyperelongation stimulated by a tyrosine kinase receptor agonist, platelet derived growth factor (PDGF) and for contrast, a serine/threonine kinase receptor agonist, TGF ß and some downstream signalling pathways. RESULTS: The inhibitory activity varied from little to total inhibition. The actions of the tinibs were directed more towards inhibition of the tyrosine kinase, PDGF receptor signalling pathway compared to the TGF ß. CONCLUSION: There was no suggestion of any synergistic effect arising from inhibition of multiple kinases as the most potent compound, dasatinib, is known to inhibit the broadest spectrum of kinases.

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.

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 Cß, 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.