Sphingosine 1-phosphate, lysophosphatidic acid and growth factor signaling and termination

Sphingosine 1 phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lipid phosphates that bind to cell surface G-protein coupled receptors (GPCR) and, in addition, exhibit intracellular actions. We have summarised herein, an important functional interaction between lipid phosphate GPCR and receptor tyrosine kinases (RTK) that enables growth factors to spatially regulate effectors, thereby governing the nature of the biological response. For instance, we describe how the formation of functional complexes between the S1P1 receptor and PDGFβ receptor may effectively re-programme platelet-derived growth factor from a mitogenic to a migratory stimulus. This is achieved by integration of RTK- and GPCR-specific signals that results in spatial regulation of a cytoplasmic retained pool of extracellular signal regulated kinase-1/2 linked to myosin light chain kinase, myosin light chain phosphorylation and migration. We therefore suggest that the lipid phosphate receptor is a major determinant in regulating growth factor-dependent biology. Growth factors can also increase S1P inside cells, and we discuss the concept of spatial/temporal aspects of compartmentalised intracellular signaling of S1P in relation to defined interactions between, for instance, sphingosine kinase, phospholipase D1 and lipid phosphate phosphatases and regulation of cell survival

The effect of hypoxia on lipid phosphate receptor and sphingosine kinase expression and mitogen-activated protein kinase signaling in human pulmonary smooth muscle cells

Both acute and chronic hypoxia had no effect on S1P1, S1P3 or LPA1 receptor transcript expression in human pulmonary smooth muscle cells. However, acute hypoxia increased sphingosine kinase SK1/2 and LPP1 mRNA transcript levels, while chronic hypoxia increased SK1 mRNA transcript alone. Acute hypoxia had no effect on S1P-, PDGF- or phorbol ester (PMA)-stimulated activation of ERK-1/2, but increased the ability of S1P to activate p38 MAPK. Chronic hypoxia increased the ability of S1P to stimulate the phosphorylation of ERK-1/2. Therefore, we have demonstrated for the first time that hypoxia can lead to marked changes in the expression of genes involved in S1P production and may modify post S1P receptor signal transduction pathways

Nerve growth factor stimulation of p42/p44 mitogen-activated protein kinase in PC12 cells: role of G(i/o), G protein-coupled receptor kinase 2, beta-arrestin I, and endocytic processing

In this study, we have shown that nerve growth factor (NGF)-dependent activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in PC12 cells can be partially blocked by pertussis toxin (which inactivates the G proteins Gi/o). This suggests that the Trk A receptor may use a G protein-coupled receptor pathway to signal to p42/p44 MAPK. This was supported by data showing that the NGF-dependent activation of p42/p44 MAPK is potentiated in cells transfected with G protein-coupled receptor kinase 2 (GRK2) or beta -arrestin I. Moreover, GRK2 is constitutively bound with the Trk A receptor, whereas NGF stimulates the pertussis toxin-sensitive binding of beta -arrestin I to the TrkA receptor-GRK2 complex. Both GRK2 and beta -arrestin I are involved in clathrin-mediated endocytic signaling to p42/p44 MAPK. Indeed, inhibitors of clathrin-mediated endocytosis (e.g., monodansylcadaverine, concanavalin A, and hyperosmolar sucrose) reduced the NGF-dependent activation of p42/p44 MAPK. Finally, we have found that the G protein-coupled receptor-dependent component regulating p42/p44 MAPK is required for NGF-induced differentiation of PC12 cells. Thus, NGF-dependent inhibition of DNA synthesis was partially blocked by PD098059 (inhibitor of MAPK kinase-1 activation) and pertussis toxin. Our findings are the first to show that the Trk A receptor uses a classic G protein-coupled receptor-signaling pathway to promote differentiation of PC12 cells

Modulation of cyclic nucleotides and cyclic nucleotide phosphodiesterases in pancreatic islet beta-cells and intestinal l-cells as targets for treating diabetes mellitus

Cyclic 3'5'-AMP (cAMP) is an important physiological amplifier of glucose-induced insulin secretion by the pancreatic islet beta-cell. In the beta-cell, cAMP is formed by the activity of adenylyl cyclase, especially in response to the incretin hormones glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide. cAMP may also play a similar role in regulating GLP-1 secretion from intestinal L-cells. cAMP influences many steps involved in glucose-induced insulin secretion and may be important in regulating pancreatic islet beta-cell differentiation, growth and survival. cAMP itself is rapidly degraded in the pancreatic islet beta-cell by cyclic nucleotide phosphodiesterase enzymes. This review will discuss the possibility of targeting cAMP mechanisms in the treatment of type 2 diabetes mellitus, in which insulin release in response to glucose is impaired

Targeting sphingosine-1-phosphate signalling for cardioprotection

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid generated by the sphingosine kinase (SK1 or SK2)-catalysed phosphorylation of sphingosine. Plasma S1P is carried in high-density lipoprotein (HDL) or bound to albumin and is reported to arise from activated platelets and erythrocytes. In addition, extracellular SK1 released from vascular endothelial cells may also contribute to plasma S1P levels. S1P exerts its effects through a family of five high affinity S1P-specific G protein-coupled receptors (GPCRs), S1P1-5. Various S1P receptors are present in the cardiovascular system, including cardiac tissue. Additionally, intracellular S1P may have a second messenger action. Since S1P is recognised as a survival factor in many tissues, there has been much interest in S1P as a cardioprotective agent. Recent evidence indicates that S1P can pre-condition and post-condition the heart and that the cardioprotective effect of HDL may be because of its S1P content. In addition, evidence is emerging that the cardioprotective effects of cannabinoids and S1P may be linked

Role of sphingosine kinases and lipid phosphate phosphatases in regulating spatial sphingosine 1-phosphate signalling in health and disease

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is produced by the sphingosine kinase-catalysed phosphorylation of sphingosine. S1P is an important regulator of cell function, mediating many of its effects through a family of five closely related G protein-coupled receptors (GPCR) termed S1P1-5 which exhibit high affinity for S1P. These receptors function to relay the effects of extracellular S1P via well-defined signal transduction networks linked to the regulation of cell proliferation, survival, migration etc. Diverse agonists (e.g. cytokines) also activate sphingosine kinase and the resulting S1P formed may bind to specific undefined intracellular targets to elicit cellular responses. The purpose of this review is to discuss some of the spatial/temporal aspects of intracellular S1P signalling and to define the function of sphingosine kinases and lipid phosphate phosphatases (which catalyse dephosphorylation of S1P) in terms of their regulation of cell function. Finally, we survey the function of S1P in relation to disease, where the major challenge is to dissect the role of intracellular versus extracellular actions of S1P in terms of association with defined diseased phenotypes

Assessment of agonism at g-protein coupled receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293 cells

Several different molecular species of phosphatidic acid (PA) bind to a G-protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from G(i/o)). The de-acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial differentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an ∼100 – 1000 fold lower potency compared with the different molecular species of PA. RT – PCR using gene-specific primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist

The functional pDGFβ receptor-S1P1 receptor signaling complex is involved in regulating migration of mouse embryonic fibroblasts in response to platelet derived growth factor

We report here that mouse embryonic fibroblasts (MEF) express a functional PDGFβ receptor–S1P1 receptor complex. The S1P1 receptor is constitutively active and functions to enhance PDGF-stimulated migration of MEF. This was based on three pieces of evidence. Firstly, the S1P1 receptor and PDGFβ receptor are co-immunoprecipitated from cell lysates using anti-PDGFβ receptor antibody. These findings suggest that the receptors form a complex in MEF. Secondly, inverse agonism of the S1P1 receptor with SB649146 to eliminate the constitutive activity of the S1P1 receptor reduced the PDGF-induced activation of p42/p44 MAPK in MEF. Thirdly, SB649146 inhibited the migration of MEF in response to the selective S1P1 receptor agonist, SEW2871 or PDGF. In contrast, S1P inhibited PDGF-stimulated MEF migration, possibly mediated by the inhibitory S1P2 receptor. These findings resolve an important issue regarding the functional role of the S1P1 receptor in regulating MEF migration and suggest an important role within the context of PDGFβ receptor–S1P1 receptor complex signaling

The identification of the inhibitory gamma-subunits of the type 6 retinal cyclic guanosine monophosphate phosphodiesterase in non-retinal tissues: differential processing of mRNA transcripts

Here, we report that mouse lung expresses γ-subunit (PDEγ) transcripts of the rod and cone photoreceptor cGMP phosphodiesterase genes (Pde6g and Pde6h, respectively). Moreover, a major 14-kDa protein (p14) in lung membranes was immunostained with antibodies that react with both rod and cone PDEγ. We show that p14 is, in fact, a mixture of rod and cone PDEγ, based on three additional lines of evidence. First, p14 was also immunostained with antibodies specific for the cone PDEγ isoform. Second, the expression of p14 immunostained with antibodies recognizing both rod and cone PDEγ was substantially reduced in lung membranes from Pde6g−/− mice. In contrast, the fraction of p14 stained with cone PDEγ-specific antibodies was not altered in the Pde6g−/− mice. Third, the absence of the Pde6g transcript was correlated with reduced levels of p14 in Pde6g−/− mice. We have also found that mouse lung contains a small Pde6h transcript that has a 41-bp deletion resulting in a frame change, derived by differential mRNA processing of exon 3 of Pde6h. BLAST searches also revealed a rat ovary EST that has the same 41-bp deletion causing the same frame change. However, the premature in-frame stop codon seen in the short Pde6h transcript is absent and the regular stop codon is out of frame leading to a predicted ORF extension into the 3′ UTR. These findings show that rod and cone PDEγ isoforms are expressed in lung and seem to have a critical role in regulating p42/p44 mitogen-activated protein kinase signaling