Peter A. Wilden

ATP-stimulated smooth muscle cell proliferation requires independent ERK and PI3K signaling pathways

Vascular smooth muscle cells respond to the purinergic agonist ATP by increasing intracellular calcium concentration and increasing the rate of cell proliferation. In many cells the extracellular signal-regulated kinase (ERK) cascade plays an important role in cellular proliferation. We have studied the effect of extracellular ATP on ERK activation and cell proliferation. ATP binding to a UTP-sensitive P2Y nucleotide receptor activates ERK1/ERK2 in a time- and dose-dependent manner in coronary artery smooth muscle cells (CASMC). ATP-induced activation of ERK1/ERK2 is dependent on the dual-specificity kinase mitogen-activated protein kinase/ERK kinase (i.e., MEK) but independent of phosphatidylinositol 3-kinase (PI3K) activity. We provide evidence that both ERK1/ERK2 and PI3K activities are required for CASMC proliferation. Thus ATP-stimulation of CASMC proliferation requires independent activation of both the ERK and PI3K signaling pathways.Vascular smooth muscle cells respond to the purinergic agonist ATP by increasing intracellular calcium concentration and increasing the rate of cell proliferation. In many cells the extracellular signal-regulated kinase (ERK) cascade plays an important role in cellular proliferation. We have studied the effect of extracellular ATP on ERK activation and cell proliferation. ATP binding to a UTP-sensitive P2Y nucleotide receptor activates ERK1/ERK2 in a time- and dose-dependent manner in coronary artery smooth muscle cells (CASMC). ATP-induced activation of ERK1/ERK2 is dependent on the dual-specificity kinase mitogen-activated protein kinase/ERK kinase (i.e., MEK) but independent of phosphatidylinositol 3-kinase (PI3K) activity. We provide evidence that both ERK1/ERK2 and PI3K activities are required for CASMC proliferation. Thus ATP-stimulation of CASMC proliferation requires independent activation of both the ERK and PI3K signaling pathways.

APS, AN ADAPTER PROTEIN WITH A PH AND SH2 DOMAIN, IS A SUBSTRATE FOR THE INSULIN RECEPTOR KINASE

APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk. We previously identified SH2-B as an insulin-receptor-binding protein and substrate [Kotani, Wilden and Pillay (1998) Biochem J. 335, 103-109]. Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS. Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction.

Cell-Signaling Evidence for Adenosine Stimulation of Coronary Smooth Muscle Proliferation via the A1 Adenosine Receptor

For decades, it has been thought that adenosine is exclusively antimitogenic on vascular smooth muscles via the A2-type adenosine receptor. Recently, we have demonstrated that adenosine stimulates proliferation of porcine coronary artery smooth muscle cells (CASMC) through the A1 adenosine receptor. However, the cell-signaling mechanisms underlying A1 receptor–mediated CASMC proliferation in response to adenosine have not been defined. Here, we show that in cultured CASMC, adenosine stimulates phosphorylation of extracellular signal–regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT in a concentration- and time-dependent manner. This effect is fully mimicked by NECA (nonselective agonist), largely mimicked by CCPA (A1-selective agonist), weakly mimicked by 2-Cl-IB-MECA (A3-selective agonist), but not by CGS21680 (A2A-selective agonist), indicating that adenosine signals strongly via the A1 receptor to these mitogenic signaling pathways. This interpretation is supported by the finding that adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT are inhibited by pertussis toxin (inactivator of Gi proteins) and by DPCPX (A1-selective antagonist), but not by SCH58261, MRS1706, and VUF5574 (A2A-, A2B-, and A3-selective antagonists, respectively). In addition, adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT is inhibited, respectively, by U0126, PD98059 (mitogen-activated protein kinase kinase inhibitors), SP600125 (JNK kinase inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor). Furthermore, these kinase inhibitors abolish or diminish adenosine- and CCPA-induced increases in the rate of cellular DNA synthesis, bromodeoxyuridine incorporation, protein synthesis, and cell number. We conclude that adenosine activates the ERK, JNK, and phosphatidylinositol 3-kinase/AKT pathways primarily through the A1 receptor, leading to CASMC mitogenesis.

Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells Role for the A1 Adenosine Receptor

Adenosine is a vascular endothelial cell mitogen, but anti-mitogenic for aortic smooth muscle cells and fibroblasts when acting via the A2B adenosine receptor. However, we show that adenosine increases porcine coronary artery smooth muscle cell (CASMC) number, cellular DNA content, protein synthesis, and PCNA staining. RT-PCR analysis indicates that porcine CASMC express A1, A2A, A3, and barely detectable levels of A2B receptor mRNAs. The mitogenic effect of adenosine is mimicked by NECA, CCPA, and R-PIA, but not by CGS21680 and 2-Cl-IB-MECA, and is inhibited by DPCPX, indicating a prominent role for the A1 receptor. This interpretation is supported by the finding that adenosine- and CCPA-induced DNA synthesis is significantly inhibited by pertussis toxin, but substantially potentiated by PD81723, an allosteric enhancer of the A1 receptor. When a cDNA encoding the porcine A1 receptor was cloned and expressed in COS-1 cells, A1 receptor pharmacology is confirmed. Anti-sense oligonucleotides to the cloned sequence dramatically suppress the mitogenic effect of adenosine and CCPA. Conversely, over-expression of the cloned A1 receptor in CASMC increases adenosine- and CCPA-induced DNA synthesis. Furthermore, stimulation with adenosine or CCPA of intact coronary arteries in an organ culture model of vascular disease increases cellular DNA synthesis, which was abolished by DPCPX. We conclude that adenosine acts as a novel mitogen in porcine CASMC that express the A1 adenosine receptor, possibly contributing to the development of coronary artery disease.

P2Y Receptor Regulation of PAI-1 Expression in Vascular Smooth Muscle Cells

P2Y-type purine and pyrimidine nucleotide receptors play important roles in the regulation of vascular hemostasis. In this article, the regulation of plasminogen activator inhibitor-1 (PAI-1) expression in rat aortic smooth muscle cells (RASMCs) by adenine and uridine nucleotides was examined and compared. Northern analysis revealed that RASMCs express multiple P2Y receptor subtypes, including P2Y(1), P2Y(2), and P2Y(6). Treatment of RASMCs with UTP increased PAI-1 mRNA expression and extracellular PAI-1 protein levels by 21-fold (P<0.001) and 7-fold (P<0.001), respectively. The ED(50) for the effect of UTP on PAI-1 expression was approximately 1 micromol/L, and its maximal effect occurred at 3 hours. UDP stimulated a 5-fold increase (P<0.005) in PAI-1 expression. In contrast to these potent stimulatory effects of uridine nucleotides, ATP and 2-methylthioadenosine triphosphate (2-MeSATP) caused a small and transient increase in PAI-1 mRNA at 1 hour, followed by a rapid decrease to baseline levels. ADP produced only an inhibitory effect, reducing PAI-1 mRNA levels by 63% (P<0.05) at 3 hours. The relative nucleotide potency in stimulating PAI-1 expression is UTP>UDP>ATP=2-MeSATP, consistent with a predominant role of the P2Y(6) receptor. Further studies revealed that exposure of RASMCs to either ATP or ADP for 3 hours inhibited both UTP- and angiotensin II-stimulated PAI-1 expression by up to 90% (P<0.001). Thus, ATP induced a small and transient upregulation of PAI-1 that was followed by a strong inhibition of PAI-1 expression. These results show that extracellular adenine and uridine nucleotides exert potent and opposing effects on vascular PAI-1 expression.

Insulin receptor structural requirements for the formation of a ternary complex with IRS-1 and PI 3-kinase.

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor tyrosine kinase and is an intermediate in insulin signaling. Phosphotyrosyl-IRS-1 binds to other signaling proteins including phosphatidylinositol 3-kinase (PI 3-kinase). We examined the role of three insulin receptor tyrosine autophosphorylation domains in association of the receptor with IRS-1. Our data support the idea that tyrosine phosphorylation of the insulin receptor juxtamembrane domain is necessary for receptor association with IRS-1. We provide evidence that the kinase regulatory domain, which is part of a loop structure at the mouth of the catalytic cleft, when mutated to replace Tyr1146, Tyr1150, and Tyr1151 with phenylalanine can bind receptor substrates without tyrosine phosphorylation of residues in the receptor juxtamembrane region. In addition, our data show that the amount of PI 3-kinase directly associated with the insulin receptor C-terminus is low when compared to the PI 3-kinase associating with IRS-1. We also demonstrate that a substantial amount (approximately 25%) of the IRS-1 associated PI 3-kinase is associated with the insulin receptor in a ternary complex of insulin receptor/IRS-1/PI 3-kinase.