Cell migration in response to the amino-terminal fragment of urokinase requires epidermal growth factor receptor activation through an ADAM-mediated mechanism.

BACKGROUND: Cell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of amino-terminal fragment (ATF) of urokinase on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. We hypothesize that A Disintegrin and Metalloproteinase Domains (ADAM) allows the transactivation of EGFR by ATF. OBJECTIVE: To determine the role of ADAM in EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC) during cell migration. METHODS: Human coronary VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to ATF (10 nM) in the presence and absence of the matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, epidermal growth factor (EGF) inhibitory antibodies, and the EGFR inhibitor AG1478. The small interference ribonucleic acid (siRNA) against EGFR and ADAM-9, ADAM-10, ADAM-12, and adenoviral delivered Gbg inhibitor, betaARK(CT) were also used. RESULTS: ATF produced concentration-dependent VSMC migration (by wound assay and Boyden chamber), which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at fourfold greater than control. Pre-incubation with the Gbetagamma inhibitor betaARK(CT) inhibited EGFR activation by ATF. This migratory and EGFR response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF-mediated migratory and EGFR responses. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of MMP, ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA to ADAM-9 and -10 significantly reduced HB-EGF release from VSMC and EGFR activation in response to ATF. The siRNA against ADAM-12 had no effect. CONCLUSION: ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR is an attractive molecular target to inhibit cell migration.

Mechanisms of sphingosine-1-phosphate-induced akt-dependent smooth muscle cell migration.

BACKGROUND: Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets that stimulates migration of vascular smooth muscle cells (VSMC) in vitro. S-1-P will activate akt, which can regulate multiple cellular functions including cell migration. Akt activation is downstream of phosphatidylinositol 3'-kinase (PI3-K) and phosphoinositide-dependent protein kinase-1 (PDK1). The objective of this study was to examine the regulation of akt signaling during smooth muscle cell (SMC) migration in response to S-1-P. METHODS: Murine arterial SMC were cultured in vitro. Linear wound and microchemotaxis assays of migration in Boyden chambers were performed in the presence of S-1-P with and without an akt inhibitor (aktI). Assays were performed for PI3-K, PDK1, akt, and GSK3beta in the presence of various inhibitors and after transfection with the G beta gamma inhibitor beta ARK(CT). RESULTS: S-1-P induced time-dependent PI3-K, PDK1, and akt activation. The migratory responses in both assays to S-1-P were blocked by aktI. Activation of akt and dephosphorylation of its downstream kinase, GSK3 beta, were inhibited by aktI. Inhibition of PI3-K with LY294002 significantly decreased activation of both PI3-K and akt. Inhibition of G beta gamma inhibited akt activation through a decrease in activation of both PI3-K and PDK1. Although inhibition of the ras with manumycin A had no effect, inhibition of rho with C3 limited activation of both PI3K and akt. PDK1 responses were unchanged by inhibition of GTPases. Inhibiting the generation of reactive oxygen species with N-acetylcysteine and of epidermal growth factor receptor with AG1478 inhibited PDK1 activation in response to S-1-P. CONCLUSION: S-1-P-mediated migration is akt-dependent. S-1-P-mediated akt phosphorylation is controlled by a G beta gamma-dependent PI3-K activation, which requires the GTPase rho and G beta gamma. PDK1 activation requires G beta gamma-dependent generation of reactive oxygen species and epidermal growth factor receptor activation.

Insulin-induced epidermal growth factor activation in vascular smooth muscle cells is ADAM-dependent.

BACKGROUND: With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. This study examines the role of insulin actions on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor-linked tyrosine kinases and is key to many of their responses. OBJECTIVE: To determine the pathway of EGFR transactivation by insulin in human vascular smooth muscle cells (VSMC). METHODS: VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the A disintegrin and metalloproteinase domain (ADAM) inhibitors tumor necrosis factor-alpha protease inhibitor (TAPI)-0 and TAPI-1, heparin-binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. RESULTS: Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration-dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. CONCLUSION: Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. This is the first description of cross-talk via ADAM between insulin and EGFR in VSMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target.

Sphingosine-1-phosphate-induced oxygen free radical generation in smooth muscle cell migration requires Galpha12/13 protein-mediated phospholipase C activation.

BACKGROUND: Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid that stimulates the migration of vascular smooth muscle cell (VSMC) through G-protein coupled receptors; it has been shown to activate reduced nicotinamide dinucleotide phosphate hydrogen (NAD[P]H) oxidase. The role of phospholipase C (PLC) in oxygen free radical generation, and the regulation of VSMC migration in response to S-1-P, are poorly understood. METHODS: Rat arterial VSMC were cultured in vitro. Oxygen free radical generation was measured by fluorescent redox indicator assays in response to S-1-P (0.1microM) in the presence and absence of the active PLC inhibitor (U73122; U7, 10nM) or its inactive analog U73343 (InactiveU7, 10nM). Activation of PLC was assessed by immunoprecipitation and Western blotting for the phosphorylated isozymes (beta and gamma). Small interfering (si) RNA to the G-proteins Galphai, Galphaq, and Galpha12/13 was used to downregulate specific proteins. Statistics were by one-way analysis of variance (n = 6). RESULTS: S-1-P induced time-dependent activation of PLC-beta and PLC-gamma; PLC-beta but not PLC-gamma activation was blocked by U7 but not by InactiveU7. PLC-beta activation was Galphai-independent (not blocked by pertussis toxin, a Galphai inhibitor, or Galphai2 and Galphai3 siRNA) and Galphaq-independent (not blocked by glycoprotein [GP] 2A, a Galphaq inhibitor, or Galphaq siRNA). PLC-beta activation and cell migration was blocked by siRNA to Galpha12/13. Oxygen free radical generation induced by S-1-P, as measured by dihydroethidium staining, was significantly inhibited by U7 but not by InactiveU7. Inhibition of oxygen free radicals with the inhibitor diphenyleneiodonium resulted in decreased cell migration to S-1-P. VSMC mitogen-activated protein kinase activation and VSMC migration in response to S-1-P was inhibited by PLC- inhibition. CONCLUSION: S-1-P induces oxygen free radical generation through a Galpha12/13, PLC-beta-mediated mechanism that facilitates VSMC migration. To our knowledge, this is the first description of PLC-mediated oxygen free radical generation as a mediator of S-1-P VSMC migration and illustrates the need for the definition of cell signaling to allow targeted strategies in molecular therapeutics for restenosis.

Patterns of kinase activation induced by injury in the murine femoral artery.

BACKGROUND: Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. Cell signaling in vascular smooth muscle cells remains a potential molecular target to modulate the development of intimal hyperplasia. The aim of this study was to define a baseline pattern of histological changes and kinase activation in a murine model. METHODS: The murine femoral wire injury model was used in which a microwire was passed through a branch of the femoral artery and used to denude the common femoral artery. Pluronic gel was used to apply mitogen-activated protein kinases (MAPK) inhibitors (PD98059, SB230580, and SP600125) on the exterior of the vessels. Specimens were perfusion-fixed and sections were stained for morphometry using an ImagePro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting and zymography to allow for the study of kinase and protease activation. Contralateral vessels were used as controls. RESULTS: The injured femoral arteries developed intimal hyperplasia, which is maximal at 28 days and does not change substantially between day 28 and day 56. Sham-operated vessels did not produce such a response. Cell apoptosis peaked within 3 days and cell proliferation peaked at 7 days after injury. There is a time-dependent increase in kinase activity immediately after injury. MEK1/2 activation peaks at 20 min after injury and is followed by a peak in extracellular signal-regulated kinase-1/2 activation at 45 min. The stress kinases p38(MAPK) and JNK peak between 10 and 20 min. Activation of akt is later at 45 min and 120 min and activation of p70S6K was biphasic. There was a time-dependent increase in uPA/PAI-1 expression and activity after injury. Local application of MAPK inhibitors (PD98059, SB230580, and SP600125) within a pluronic gel reduced respective MAPK activity, decreased cell proliferation and enhanced cell apoptosis, increased PAI-1, and decreased uPA expression and activity; at 14 days there was a decrease in intimal hyperplasia. CONCLUSIONS: These data demonstrate that femoral wire injury in the mouse induces a consistent model of intimal hyperplasia and that it is associated with a time dependent increase in signaling kinase activity. Interruption of these pathways will interrupt the uPA/PAI-1 pathway and decrease intimal hyperplasia development. Accurate characterization of cell signaling is a necessary step in the development of molecular therapeutics.

Mechanisms of kringle fragment of urokinase-induced vascular smooth muscle cell migration.

BACKGROUND: Urokinase plasminogen activator (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to uPA is dependent on both the growth factor binding domain at the aminoterminal end and the kringle (K) domain of the molecule. uPA is readily degraded in vivo into these constitutive domains. The aim of this study was to examine cell signaling during the migration of smooth muscle cell in response to the kringle domain of urokinase. MATERIALS AND METHODS: Murine arterial smooth muscle cells were cultured in vitro. Migration assays were performed in the presence of K with and without the plasmin inhibitors (aprotinin and -aminocaproic acid), the Galphai inhibitor Pertussis toxin, the MMP inhibitor (GM6001), the PI3-K inhibitors, Wortmannin and LY294002, and the MAPK inhibitors PD98089 (MEK1 inhibitor) and SB203580 (p38(MAPK) inhibitor). Western blotting was performed for ERK 1/2 and p38(MAPK) phosphorylation after stimulation with K in the presence and absence of the inhibitors. Statistics were analyzed by one-way ANOVA (n = 6). RESULTS: The kringle domain (K) induced a plasmin-independent, MMP-dependent increase in cell migration (2-fold, P < 0.05) compared to control. This migratory response to K was Galphai mediated and dependent on both ERK 1/2 and p38(MAPK) activation. K induced time-dependent increases in the phosphorylation of ERK 1/2 (3-fold, P < 0.05) and p38(MAPK) (3-fold, P < 0.05). Activation of p38(MAPK) and ERK 1/2 was completely inhibited by the PI3-K inhibitors. We explored a potential role for the epidermal growth factor receptor (EGFR). K induced EGFR phosphorylation and the presence of AG1478, the EGFR inhibitor, inhibited both cell migration and akt activation in response to K. CONCLUSION: Kringle domain of uPA induces smooth muscle cell migration through a G-protein-coupled PI3-K-dependent process involving both ERK 1/2 and p38(MAPK) and is mediated in part through EGFR. Defining the differences in response to key molecular domains of uPA is vital to understand its role in vessel remodeling.

Plasminogen activator system and vascular disease.

Vascular diseases, such as atherosclerosis, thromboembolic disorders and stroke, in addition to surgical procedures such as restenosis, all share the plasminogen activator system as a central component in the pathogenesis of vascular injury. Since the development of plasminogen deficient mice our knowledge of the effects of this proteolytic system in cardiovascular disease has vastly increased. The plasminogen activator system plays a key role in vascular homeostasis and constitutes a critical response mechanism to cardiovascular injury. The central components of the PA system are the proteolytic activators, urokinase-plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), plasminogen (plg) and its degradation product, plasmin, together with the major inhibitors of this system, plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2). An extensive network of additional proteases, inhibitors, receptors and modulators directly associate and are influenced by the PA system, the largest group being the Matrix Metalloproteinases (MMPs) and their respective inhibitors the Tissue inhibitors of MMPs (TIMPS).

Plasmin-induced smooth muscle cell proliferation requires epidermal growth factor activation through an extracellular pathway.

BACKGROUND: Plasminogen activators are used routinely for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. We have shown recently that plasmin induces extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated cell proliferation. Plasmin can also activate metalloproteinases on the cell surface, which can release the tethered ligand heparin-binding epidermal growth factor (HB-EGF), which can in turn activate the epidermal growth factor receptor (EGFR). METHODS: Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis and cell proliferation, EGFR phosphorylation, and ERK1/2 activation were examined in response to plasmin in the presence and absence of the plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), matrix metalloproteinase (MMP) inhibitor GM6001, HB-EGF inhibitor CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. RESULTS: Plasmin-induced smooth muscle cell DNA synthesis, which was blocked by EGFR and HB-EGF inhibition. Plasmin-induced time-dependent EGFR phosphorylation and ERK1/2 activation, which were inhibited by AG1478. This response was dependent on the proteolytic activity of plasmin since both plasmin inhibitors blocked the response. EGFR phosphorylation by plasmin was blocked by inhibition of MMP activity and the ligand HB-EGF. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs, or HB-EGF. Direct blockade of the EGFR prevented activation by both plasmin and EGF. CONCLUSIONS: Plasmin can induce smooth muscle cell proliferation through activation of EGFR by an extracellular MMP-mediated, HB-EGF-dependent process.

Urokinase-induced smooth muscle cell migration requires PI3-K and Akt activation.

OBJECTIVE: To examine the role of the phospho-inositol-3'-kinase (PI3-K)-akt signaling axis during smooth muscle cell (SMC) migration in response to the aminoterminal fragment of urokinase (ATF). BACKGROUND: Urokinase (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to urokinase is dependent on ATF. The role of PI3-K/akt signaling during migration in response to the uPA fragments is not understood. METHODS: Murine arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of ATF with and without the PI3-K inhibitors (Wortmannin, Wn [10 nm] and LY294002, LY [10 microm]) and an akt inhibitor (aktI, [10 microm]). Western blotting was performed for akt, ERK1/2, and GSK3beta phosphorylation after cells were stimulated with ATF in the presence and absence of the inhibitors. Statistics were analyzed by one-way ANOVA. RESULTS: Both PI3-K and akt inhibitors blocked the migratory response to ATF in both assays. ATF induced time-dependent increases in akt phosphorylation at both S472 and T308 sites and ERK1/2 phosphorylation. Activation of akt and ERK1/2 was inhibited by Wn and LY. Manumycin A, a ras inhibitor, did not inhibit activation of akt but did inhibit ERK1/2 activation. Activation of akt and the dephosphorylation of its downstream kinase GSK3beta were inhibited by the akt inhibitor. Direct inhibition of akt did not influence ERK1/2 activation and inhibition of ERK1/2 did not influence akt activation. CONCLUSION: ATF mediated migration is PI3-K dependent and activates two separate pathways: ERK1/2 and akt. ATF induces akt phosphorylation through a PI3K-mediated but ras-independent mechanism while both ras and PI3K are required for ERK1/2 activation. Defining key signaling pathways is vital to regulate vessel remodeling.

Urokinase-induced smooth muscle cell responses require distinct signaling pathways: a role for the epidermal growth factor receptor.

OBJECTIVE: Urokinase plasminogen activator (uPA) a key serine protease during remodeling, is capable of inducing both smooth muscle cell migration and proliferation. However, the signals that produce these responses are poorly understood. METHODS: Early passage rat aortic arterial smooth muscle cells were cultured in vitro and standard assays of DNA synthesis ([ 3 H]thymidine incorporation), cell proliferation (manual cell counting), and migration (linear wound assay and Boyden chamber) were used to study the cells responses to uPA. Activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK , Akt, MAP kinase/ERK kinase (MEK1/2), MAP kinase kinase (MKK)3/6, and epidermal growth factor receptor (EGFR) in response to uPA was assayed by Western blot analysis for the phosphorylated form of each kinase. These assays were repeated in the presence of the Galphai inhibitor pertussis toxin (PTx, 100 ng/mL), the Ras inhibitor manumycin A (MA, 10 microM), the phosphatidyl-inositol 3' kinase (PI3K) inhibitor wortmannin (WN, 1 microM), the EGFR inhibitor AG1478 (AG, 10 nM), the MEK1 inhibitor PD98059 (PD, 10 microM), the p38 MAPK inhibitor SB203580 (SB, 10 microM), and the plasmin inhibitors aprotinin and epsilon-aminocaproic acid. RESULTS: uPA induced a twofold increase in smooth muscle cell migration and increased smooth muscle cell DNA synthesis and proliferation. The ERK1/2 and p38 MAPK inhibitors PD98059 (PD) and SB203580 (SB) blocked cell proliferation, but only PD blocked cell migration. Although uPA-induced phosphorylation of both ERK1/2 and p38 MAPK was blocked by Galphai inhibition, inhibition of PI3K and Ras decreased the uPA-induced phosphorylation of ERK1/2 but not p38 MAPK . Activation of MEK1/2 was abrogated by inhibitors of Galphai and Ras, but not by PI3K inhibition. In contrast, activation of MKK3/6 was abrogated by inhibition of Galphai, but not by Ras or PI3K inhibition. uPA induced time-dependent phosphorylation of EGFR, which was dependent on plasmin activity. Inhibition of EGFR reduced both ERK1/2 and p38 MAPK activation. uPA activation of PI3K and MKK3/6 was EGFR-dependent and that of MEK1 was EGFR-independent. CONCLUSION: uPA induces smooth muscle cell proliferation through ERK1/2- and p38 MAPK -mediated pathways. Migration appears to be dependent on ERK1/2 activity alone. Activation of EGFR appears to be required. The differential activation of pathways for ERK1/2 and p38 MAPK by uPA allows for two distinct biologic responses that both require tyrosine kinase receptor transactivation. CLINICAL RELEVANCE: Elevated urokinase-like plasminogen activator (uPA) and decreased plasminogen activator inhibitor-1 (PAI-1) levels are predictors for restenosis. Matrix remodeling and smooth muscle cell responses are integrally linked. Changes in smooth muscle cell migration and proliferation are dependent on the extracellular matrix environment in which they are encased. Proteases such as uPA can effect smooth muscle cells and alter the matrix; their activity is controlled by a series of inhibitors (eg, PAI-1). The balance of activation and inhibition forms the basis of the proteolytic thermostat in the vessel wall. Understanding the biology of the proteolytic thermostat will allow for structured therapeutic interventions to control restenosis and thus improve patient care and avoid secondary interventions. Our study demonstrates that uPA is capable of inducing separate responses through more than one signaling pathway, in part, by transactivation of a nearby receptor for the unrelated ligand epidermal growth factor receptor (EGFR). Blockade of EGFR can inhibit both cell migration and proliferation induced by uPA. This is the first description of cross talk between uPA and EGFR in vascular smooth muscle cells. Targeting a pivotal receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases, is an attractive molecular target to control restenosis.

Plasmin induces smooth muscle cell proliferation.

BACKGROUND: Plasminogen activators are routinely used for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. The signaling pathways used by plasmin are not understood. METHODS: Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis, cell proliferation, MAPKK and MAPK activation were examined in response to plasmin alone and in the presence of plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), pertussis toxin (Galphai inhibitor, PTx), GP-2A (Galphaq inhibitor), wortmannin (PI3-K inhibitor, Wn), LY294002, (PI3-K inhibitor, LY), PD98059 (MEK inhibitor, PD), and SB203580 (p38MAPK inhibitor, SB). RESULTS: Plasmin produced concentration dependent smooth muscle cells DNA synthesis and proliferation and induced ERK1/2 and p38MAPK phosphorylation. Inhibition of the proteolytic activity of plasmin prevented these responses. The ERK1/2 inhibitor, PD, but not the p38MAPK inhibitors, SB, blocked cell proliferation. The activation of the MEK1/2 and ERK1/2 pathway was both Galphai dependent (PTx-sensitive) and Galphaq dependent (GP-2A-sensitive). It was blocked by the PI3-K inhibitors, Wn and LY. PI3-K activation as measured by akt phosphorylation was dependent on Galphai, but was independent of Galphaq. CONCLUSION: Plasmin induces smooth muscle cell proliferation. Plasmin induced ERK1/2 phosphorylation occurs through two pathways: one which is Galphai mediated/PI3-K dependent and a second which is Galphaq mediated/PI3K independent. p38MAPK appears not to be involved in plasmin-mediated cell proliferation. This pattern of activation is distinct from that seen with urokinase plasminogen activator.

Sphingosine-1-phosphate-induced smooth muscle cell migration involves the mammalian target of rapamycin.

BACKGROUND: Vascular smooth muscle cell (SMC) migration is an important component of the development of intimal hyperplasia. Sphingosine-1-phosphate (S-1-P) is a lipid released from activated platelets with numerous cellular effects including the stimulation of SMC migration in vitro. We examined the role of the mammalian target of rapamycin and ribosomal p70S6 kinase (p70S6K) in S-1-P-induced SMC migration . METHODS: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P (0.01 to 100 micromol/L) with and without rapamycin (10 nmol/L). Western blotting was performed for phosphorylated and total p70S6K, ERK1/2, and p38(MAPK) after stimulation with S-1-P (0.1 micromol/L), with and without rapamycin pretreatment. Phosphorylation of p70S6K was also assayed after S-1-P treatment in the presence and absence of inhibitors of PI3 kinase (wortmannin, WN, and LY294002, LY), Akt (AktI), p38(MAPK) (SB203580), and MEK1 (PD98059). RESULTS: S-1-P stimulated migration of SMCs in both linear wound and Boyden chamber assays compared to control (P < .05); these responses were inhibited by rapamycin to below the level of control (P < .05 vs S-1-P alone for both assays) in a dose-dependent manner (inhibitory concentration of 50%, 10 nmol/L). S-1-P stimulated phosphorylation of ERK1/2, p38(MAPK), and p70S6K, which peaked at 5 minutes for ERK1/2 and p38(MAPK) and10 minutes for p70S6K (2-fold increase over control for each, P < .05). Rapamycin prevented the phosphorylation of p70S6K at the Thr 389 site (which correlates with enzyme activity), reduced ERK1/2 phosphorylation, but had no effect on the Thr 421/Ser 424 site or on p38(MAPK) phosphorylation. Wortmannin and LY294002 inhibited phosphorylation of the Thr 389 site of p70S6K. AktI and SB203580 had no effect on p70S6K, whereas PD98059 had a marginal effect. CONCLUSIONS: S-1-P-induced SMC migration was completely inhibited by rapamycin, indicating that the p70S6K pathway is involved. This mechanism likely involves modulation of the ERK1/2 pathway. S-1-P stimulates phosphorylation of p70S6K in a MEK1-dependent, PI3 kinase-dependent, but Akt-independent manner.

Differential regulation of ERK1/2 and p38(MAPK) by components of the Rho signaling pathway during sphingosine-1-phosphate-induced smooth muscle cell migration.

OBJECTIVE: To determine the role of rhosignaling in sphingosine-1-phosphate (S-1-P)-induced smooth muscle cell migration. BACKGROUND: S-1-P is a bioactive sphingolipid released from activated platelets stimulating migration of smooth muscle cells (SMC) in vitro through Galphai G-proteins and MAPK activation. Rho is one of the key small GTPases required for cytoskeletal reorganization and MAPK activation during migration. We hypothesized that S-1-P-stimulated migration is regulated by the rho-signaling pathway. METHODS: Rat arterial SMCs were cultured in vitro. Linear wound assays of migration were performed in the presence of S-1-P with and without C3 (a rho antagonist) and Y (Y27632, a Rho kinase inhibitor). Western blotting was performed for MEK1-ERK1/2 and MMK3/MKK6-p38(MAPK) phosphorylation after stimulation with S-1-P with and without pre-incubation with the inhibitors. Statistics were analyzed by one-way ANOVA. RESULTS: S-1-P stimulated migration of SMCs in a wound assay (2-fold over control; P < 0.01), which was blocked by Rho inhibition (P < 0.05). S-1-P activated rho and induced a time-dependent increase in ERK1/2 and p38(MAPK) activation. In the presence of C3, MEK1 and ERK1/2 phosphorylation were significantly decreased, while MKK3/6 and p38(MAPK) phosphorylation were unchanged. In contrast, when rho kinase was inhibited, there was an increase in ERK1/2 and a decrease in p38(MAPK) phosphorylation. Rho kinase inhibition resulted in a decrease in MEK1/2 and MKK3/6 phosphorylation. CONCLUSIONS: S-1-P differentially regulates the MAPK pathway through components of the rho pathway. Rho regulates ERK1/2 activation through MEK1/2, while Rho kinase negatively modulates ERK1/2 in a MEK1/2-independent manner and regulates p38(MAPK) through MKK3/6. This is the first description of differential MAPK regulation by a G-protein-coupled receptor through the rho pathway. Understanding signal transduction in SMCs will contribute to the development of molecular therapeutics for intimal hyperplasia.

Plasminogen activator inhibitor-1 deficiency enhances flow-induced smooth muscle cell migration.

INTRODUCTION: We determined the role of smooth muscle cell (SMC)-derived plasminogen activator inhibitor-1 (PAI-1) in the flow-induced SMC migratory response. MATERIALS AND METHODS: Wild type (wt) or PAI-1 knockout SMC were cultured in the absence or presence of endothelial cells (EC) under static or pulsatile flow conditions in a perfused culture system. SMC migration was then assessed by Transwell assay. RESULTS: Pulsatile flow significantly increased SMC PAI-1 mRNA and protein levels, approximately 4- and 3-fold respectively (n = 4, p < 0.05). In the absence, but not in the presence of EC, pulsatile flow significantly increased ( approximately 2.4-fold) the migration of wt SMC when compared to wt SMC cultured under static conditions. PAI-1 -/-SMC migration was significantly increased under flow conditions as compared to wild-type controls (334 +/- 22% vs. 237 +/- 11%, n = 6, p < 0.05). This flow-induced migration was significantly attenuated, but not completely inhibited, when PAI-1 -/-SMC were cultured in the presence of EC (147 +/- 13%, n = 6, p < 0.05). The flow-induced PAI-1 -/-SMC migratory response was partially inhibited by an anti-urokinase plasminogen activator (uPA) antibody (#1189), and completely inhibited by both 1189 and the matrix metalloproteinase (MMP) inhibitor BB3103. In parallel PAI-1 -/-SMC cells, there was a greater flow-induced increase in proMMP-2 activity as compared to wild-type control cells. Moreover, under both static and flow conditions, tissue inhibitors of matrix metalloproteinases (TIMP)-2 activity was reduced in these PAI-1-deficient cells as compared to wild-type controls. CONCLUSIONS: These results suggest that SMC PAI-1 plays a role in limiting flow-induced SMC migration and thus may be an important mechanism for controlling the process of vascular remodelling.

Neuropeptide Y Y(1) receptor regulates protein turnover and constitutive gene expression in hypertrophying cardiomyocytes.

Increased levels of neuropeptide Y correlate with severity of left ventricular hypertrophy in vivo. At cardiomyocyte level, hypertrophy is characterised by increased mass and altered phenotype. The aims were to determine the contributions of increased synthesis and reduced degradation of protein to neuropeptide Y-mediated increase in mass, assess effects on gene expression, and characterise neuropeptide Y Y receptor subtype involvement. Neuropeptide Y (10 nM) increased protein mass of adult rat ventricular cardiomyocytes maintained in culture (24 h) (16%>basal) and de novo protein synthesis (incorporation of [(14)C]phenylalanine) (18%>basal). Neuropeptide Y (100 nM) prevented degradation of existing protein at 8 h. Actinomycin D (5 microM) attenuated increases in protein mass to neuropeptide Y (< or = 1 nM) but not to neuropeptide Y (10 nM). [Leu(31), Pro(34)]neuropeptide Y (10 nM), an agonist at neuropeptide Y Y(1) receptors, increased protein mass (25%>basal) but did not stimulate protein synthesis. Neuropeptide Y-(3-36) (10 nM), an agonist at neuropeptide Y Y(2) receptors, increased protein mass (29%>basal) and increased protein synthesis (13%>basal), respectively. Actinomycin D (5 microM) abolished the increase in protein mass elicited by neuropeptide Y-(3-36) but not that by [Leu(31), Pro(34)]neuropeptide Y. BIBP3226 [(R)-N2-(diphenylacetyl)-N-(4-hydroxyphenylmethyl)-D-arginine amide] (1 microM), a neuropeptide Y Y(1) receptor subtype-selective antagonist, and T(4) [neuropeptide Y-(33-36)](4), a neuropeptide Y Y(2) receptor subtype-selective antagonist, attenuated the increase in protein mass to 100 nM neuropeptide Y by 68% and 59%, respectively. Neuropeptide Y increased expression of the constitutive gene, myosin light chain-2 (MLC-2), maximally at 12 h (4.7-fold>basal) but did not induce (t< or = 36 h) expression of foetal genes (atrial natriuretic peptide (ANP), skeletal-alpha-actin and myosin heavy chain-beta). This increase was attenuated by 86% and 51%, respectively, by BIBP3226 (1 microM) and T(4) [neuropeptide Y-(33-36)](4) (100 nM). [Leu(31), Pro(34)]neuropeptide Y (100 nM) (2.4-fold>basal) and peptide YY-(3-36) (100 nM) (2.3 fold>basal) increased expression of MLC-2 mRNA at 12 h. In conclusion, initiation of cardiomyocyte hypertrophy by neuropeptide Y requires activation of both neuropeptide Y Y(1) and neuropeptide Y Y(2) receptors and is associated with enhanced synthesis and attenuated degradation of protein together with increased expression of constitutive genes but not reinduction of foetal genes.