The Lbc Rho Guanine Nucleotide Exchange Factor/α-Catulin Axis Functions in Serotonin-induced Vascular Smooth Muscle Cell Mitogenesis and RhoA/ROCK Activation

Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling that occurs in pulmonary arterial hypertension. RhoA/Rho kinase (ROCK) function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT. Lbc knockdown by siRNA attenuated 5-HT-induced thymidine uptake in PASMC, indicating a role in PASMC mitogenesis. 5-HT triggered Rho-dependent serum response factor-mediated reporter activation in PASMC, and this was reduced by Lbc depletion. Lbc knockdown reduced 5-HT-induced RhoA/ROCK activation, but not p42/44 ERK MAP kinase activation, suggesting that Lbc is an intermediary between 5-HT and RhoA/ROCK, but not ERK. 5-HT stimulation of PASMC led to increased association between Lbc, RhoA, and the α-catulin scaffold. Furthermore, α-catulin knockdown attenuated 5-HT-induced PASMC thymidine uptake. 5-HT-induced PASMC mitogenesis was reduced by dominant-negative G_q protein, suggesting cooperation with Lbc/α-catulin. These results for the first time define a Rho GEF involved in vascular smooth muscle cell growth and serotonin signaling, and suggest that Lbc Rho GEF family members play distinct roles. Thus, the Lbc/α-catulin axis participates in 5-HT-induced PASMC mitogenesis and RhoA/ROCK signaling, and may be an interventional target in diseases involving vascular smooth muscle remodeling.     

Tissue transglutaminase promotes serotonin-induced AKT signaling and mitogenesis in pulmonary vascular smooth muscle cells

Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that cross-links proteins with monoamines such as serotonin (5-hydroxytryptamine, 5-HT) via a transglutamidation reaction, and is associated with pathophysiologic vascular responses. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PASMCs) that has been linked to pulmonary vascular remodeling underlying pulmonary hypertension development. We previously reported that 5-HT-induced PASMC proliferation is inhibited by the TG2 inhibitor monodansylcadaverine (MDC); however, the mechanisms are poorly understood. In the present study we hypothesized that TG2 contributes to 5-HT-induced signaling pathways of PASMCs. Pre-treatment of bovine distal PASMCs with varying concentrations of the inhibitor MDC led to differential inhibition of 5-HT-stimulated AKT and ROCK activation, while p-P38 was unaffected. Concentration response studies showed significant inhibition of AKT activation at 50 μM MDC, along with inhibition of the AKT downstream targets mTOR, p-S6 kinase and p-S6. Furthermore, TG2 depletion by siRNA led to reduced 5-HT-induced AKT activation. Immunoprecipitation studies showed that 5-HT treatment led to increased levels of serotonylated AKT and increased TG2-AKT complex formations which were inhibited by MDC. Overexpression of TG2 point mutant cDNAs in PASMCs showed that the TG2 C277V transamidation mutant blunted 5-HT-induced AKT activation and 5-HT-induced PASMC mitogenesis. Finally, 5-HT-induced AKT activation was blunted in SERT genetic knock-out rat cells, but not in their wild-type counterpart. The SERT inhibitor imipramine similarly blocked AKT activation. These results indicate that TG2 contributes to 5-HT-induced distal PASMC proliferation via promotion of AKT signaling, likely via its serotonylation. Taken together, these results provide new insight into how TG2 may participate in vascular smooth muscle remodeling.     

H(2)O(2) signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells

Our previous studies have shown that 5-hydroxytryptamine (5-HT) induces cellular hyperplasia/hypertrophy through protein tyrosine phosphorylation, rapid formation of superoxide (O(2)(-)), and extracellular signal-regulated kinase (ERK)1/ERK2 mitogen-activated protein (MAP) kinase activation. Intracellularly released O(2)(-) is rapidly dismuted by superoxide dismutase (SOD) to H(2)O(2), another possible cellular growth mediator. In the present study, we assessed whether H(2)O(2) participates in 5-HT-induced mitogenic signaling. Inactivation of cellular Cu/Zn SOD by copper-chelating agents inhibited 5-HT-induced DNA synthesis of bovine pulmonary artery smooth muscle cells (BPASMCs). Infection of BPASMCs with an adenovirus containing catalase inhibited both ERK1/ERK2 MAP kinase activation and DNA synthesis induced by 5-HT. Although we could not find evidence of p38 MAP kinase activation by 5-HT, SB-203580 and SB-202190, reported inhibitors of p38 MAP kinase, inhibited the 5-HT-induced growth of BPASMCs. However, these inhibitors also inhibited 5-HT-induced O(2)(-) release. Thus quenching of O(2)(-) may be their mechanism for inhibition of cellular growth unrelated to p38 MAP kinase inhibition. These data indicate that generation of O(2)(-) in BPASMCs in response to 5-HT is followed by an increase in intracellular H(2)O(2) that mediates 5-HT-induced mitogenesis through activation of ERK1/ERK2 but not of p38 MAP kinase.     

Hypoxia promotes rabbit pulmonary artery smooth muscle cells proliferation through a 15-LOX-2 product 15(S)-hydroxyeicosatetraenoic acid

The initial event of hypoxic pulmonary hypertension is acute hypoxic pulmonary vasoconstriction followed by remodeling of pulmonary arteries. Although 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] is found to be able to induce hypoxic pulmonary vasoconstriction, role of 15(S)-HETE in pulmonary artery smooth muscle cells (PASMCs) proliferation has been studied less. We sought evidence for a role of 15(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that hypoxia enhances 15-lipoxygenase-2 (15-LOX-2) expression and stimulates cultured rabbit PASMCs proliferation. 15(S)-HETE at concentration 0.1 μM stimulated proliferation of PASMCs and induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 15(S)-HETE-stimulated PASMC proliferation was blocked by the MEK inhibitors PD-98059. Hypoxia (3% O(2))-stimulated PASMC proliferation was blocked by U0126, a MEK inhibitor, as well as by NDGA and CDC, inhibitors of 15-LOX, but not by the p38 MAPK inhibitor SB-202190. We conclude that 15-LOX-2 and its product, 15(S)-HETE, are important intermediates in hypoxia-induced rabbit PASMC proliferation and may participate in hypoxia-induced pulmonary hypertension.     

Inhibition of serotonin-induced mitogenesis, migration, and ERK MAPK nuclear translocation in vascular smooth muscle cells by atorvastatin

The HMG-CoA reductase inhibitors, statins, have pleiotropic effects which may include interference with the isoprenylation of Ras and Rho small GTPases. Statins have beneficial effects in animal models of pulmonary hypertension, although their mechanisms of action remain to be determined. Serotonin [5-hydroxytryptamine (5-HT)] is implicated in the process of pulmonary artery smooth muscle (PASM) remodeling as part of the pathophysiology of pulmonary hypertension. We examined the effect of atorvastatin on 5-HT-induced PASM cell responses. Atorvastatin dose dependently inhibits 5-HT-induced mitogenesis and migration of cultured bovine PASM cells. Inhibition by atorvastatin was reversed by mevalonate and geranylgeranylpyrophosphate (GGPP) supplement, suggesting that the statin targets a geranylgeranylated protein such as Rho. Concordantly, atorvastatin inhibits 5-HT-induced cellular RhoA activation, membrane localization, and Rho kinase-mediated phosphorylation of myosin phosphatase-1 subunit. Atorvastatin reduced activated RhoA-induced serum response factor-mediated reporter activity in HEK293 cells, indicating that atorvastatin inhibits Rho signaling, and this was reversed by GGPP. While 5-HT-induced ERK MAP and Akt kinase activation were unaffected by atorvastatin, 5-HT-induced ERK nuclear translocation was attenuated in a GGPP-dependent fashion. These studies suggest that atorvastatin inhibits 5-HT-induced PASM cell mitogenesis and migration through targeting isoprenylation which may, in part, attenuate the Rho pathway, a mechanism that may apply to statin effects on in vivo models of pulmonary hypertension.     

Serotonin uptake and configurational change of bovine pulmonary artery smooth muscle cells in culture

Although it is well known that endothelial cells transport serotonin (5-HT) from extracellular to intracellular locations, it has been generally assumed that smooth muscle cells do not accumulate 5-HT but, rather, respond to 5-HT through a receptor activity unrelated to uptake of this amine or via stimulation of endothelial-derived relaxing factor. In the present study smooth muscle cells (PASMC), isolated and cultured from bovine pulmonary artery, were evaluated for 5-HT uptake under a variety of conditions. 5-HT uptake was linear up to 15 min and the rate was seven- to eightfold higher than that by bovine pulmonary artery endothelial cells. There was intracellular metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA). The uptake was inhibited by exposure to 4 degrees C, absence of Na+ from the medium, and agents such as imipramine, verapamil, ketanserin, and methiothepin. Like that of endothelial cells, 5-HT uptake by PASMC was stimulated by exposure of cells to anoxia for 24 hr. Unlike endothelial cells that showed no morphological changes, PASMC at early passage showed dendritic formation after 30-60 min exposure to 5-HT at a concentration as low as 10(-8) M. Although this configurational change in response to 5-HT was lost with passage of cells, transport of 5-HT by these cells was retained. The configurational change was blocked by agents that inhibited 5-HT uptake, such as imipramine, verapamil, ketanserin, and methiothepin; it was unaffected by inhibitors of protein kinase C, phospholipase C, and calmodulin or absence of Ca2+ from the medium. We conclude that PASMC, as well as endothelial cells, accumulate 5-HT; there appears to be a close relationship between 5-HT uptake and configurational change of early passaged PASMC in culture. The factor(s) required for the configurational change are absent in endothelial cells and lost during passage of PASMC.     

Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells

In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5-hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca(2+) fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 microM) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni(2+) and to ruthenium red, a TRPV channel blocker, and mimicked by 4alpha-phorbol-12,13-didecanoate (4alpha-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4alpha-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension.     

Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and 5-HT7(a)

Receptor tyrosine kinases activate mitogen-activated protein (MAP) kinases through Ras, Raf-1, and MEK. Receptor tyrosine kinases can be transactivated by G protein-coupled receptors coupling to G(i) and G(q). The human G protein-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) couple to G(s) and elevate intracellular cAMP. Certain G(s)-coupled receptors have been shown to activate MAP kinases through a protein kinase A- and Rap1-dependent pathway. We report the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 (p44 and p42 MAP kinase) through the human serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in COS-7 and human embryonic kidney HEK293 cells. In transfected HEK293 cells, 5-HT-induced activation of ERK1/2 is sensitive to H89, which indicates a role for protein kinase A. The observed activation of ERK1/2 does not require transactivation of epidermal growth factor receptors. Furthermore, 5-HT induced activation of both Ras and Rap1. Whereas the presence of Rap1GAP1 did not influence the 5-HT-mediated activation of ERK1/2, the activation of ERK1/2 was abolished in the presence of dominant negative Ras (RasN17). ERK1/2 activation was reduced in the presence of "dominant negative" Raf1 (RafS621A) and slightly reduced by dominant negative B-Raf, indicating the involvement of one or more Raf isoforms. These findings suggest that activation of ERK1/2 through the human G(s)-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in HEK293 cells is dependent on Ras, but independent of Rap1.     

5-Hydroxytryptamine augments migration of human aortic smooth muscle cells through activation of RhoA and ERK

The purpose of this study was to elucidate the mechanism of 5-hydroxytryptamine (5-HT, serotonin) action on migration of vascular smooth muscle cells. Migration of cultured human aortic smooth muscle cells (HASMCs), evaluated using time-lapse microscopy, was significantly enhanced by 5-HT at concentrations of 1-100 nM. The enhancing effect of 5-HT on cell migration was markedly inhibited in the presence of ketanserin, a 5-HT2 receptor antagonist, but not by GR 55562, a 5-HT1 receptor antagonist. Activities of RhoA and ERK were increased by 5-HT, and the increase in cell migration by 5-HT was abolished in the presence of U0126, a MEK1/2 inhibitor, or Y-27632, a Rho-kinase inhibitor. Activation of ERK was strongly inhibited by Y-27632. 5-HT-induced formation of stress fiber and detachment of uropod (trailing edge) were abolished by Y-27632. Thus, 5-HT has a potent enhancing action on migration of HASMCs due to an increase in stress fiber formation by 5-HT2 receptor stimulation followed by activation of the Rho-kinase and ERK pathways.     

Serotonin transporter interacts with the PDGFβ receptor in PDGF-BB-induced signaling and mitogenesis in pulmonary artery smooth muscle cells

The serotonin transporter (SERT) and the platelet-derived growth factor receptor (PDGFR) have been implicated in both clinical and experimental pulmonary hypertension (PH) and the facilitation of pulmonary artery smooth muscle cell (PASMC) growth. To gain a better understanding of the possible relationship of these two cell surface molecules we have explored interactions between SERT and PDGFR. We have previously demonstrated that SERT transactivates PDGFRβ in serotonin-stimulated PASMC proliferation. We now provide evidence for a role for SERT in PDGF-BB signaling and PASMC proliferation by using pharmacological inhibitors, genetic ablation, and construct overexpression of SERT. The results show that four tested SERT blockers dose dependently inhibit PDGF-stimulated human and bovine PASMC proliferation with comparable efficacy to that of PDGFR inhibitors, whereas 5-HT1B or 5-HT2A receptor inhibitors had no effect. Combinations of the SERT and PDGFR inhibitors led to synergistic/additive inhibition. Similarly, PDGF-induced PASMC proliferation was attenuated by small interfering RNA downregulation of SERT. Inhibition of SERT in PASMCs attenuated PDGF-induced phosphorylation of PDGFRβ, Akt, and p38 but not Erk. Overexpression of SERT in HEK293 cells led to enhanced Akt phosphorylation by PDGF, which was blunted by a SERT PDZ motif mutant, indicating the mechanistic need for the PDZ motif of SERT in PDGF signaling. Furthermore, coimmunoprecipitation experiments showed that SERT and PDGFRβ become physically associated upon PDGF stimulation. In total, the data show for the first time an important interactive relationship between SERT and the PDGFRβ in the production of PASMC proliferation triggered by PDGF that may be important in PH.     

Phospholipase D signaling in serotonin-induced mitogenesis of pulmonary artery smooth muscle cells

We have previously reported the participation of mitogen-activated protein, Rho, and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study, we investigated the possible participation of phospholipase D (PLD) and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [(3)H]phosphatidylbutanol and PA generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mammalian target of rapamycin (mTOR), by 80 and 72%, respectively, and partially blocked activation of extracellular signal-regulated kinase (ERK) by 30% but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1, and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by approximately 50%. Inhibition of 5-HT receptor 2A (R 2A) with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3-kinase inhibitor failed to block either activation of PLD by 5-HT or PA-dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT.     

Inhalation of the BKCa-Opener NS1619 Attenuates Right Ventricular Pressure and Improves Oxygenation in the Rat Monocrotaline Model of Pulmonary Hypertension

Background

Right heart failure is a fatal consequence of chronic pulmonary hypertension (PH). The development of PH is characterized by increased proliferation of vascular cells, in particular pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells. In the course of PH, an escalated right ventricular (RV) afterload occurs, which leads to increased perioperative morbidity and mortality. BK_Ca channels are ubiquitously expressed in vascular smooth muscle cells and their opening induces cell membrane hyperpolarization followed by vasodilation. Moreover, BK activation induces anti-proliferative effects in a multitude of cell types. On this basis, we hypothesized that treatment with the nebulized BK channel opener NS1619 might be a therapy option for pulmonary hypertension and tested this in rats.

Methods

(1) Rats received monocrotaline injection for PH induction. Twenty-four days later, rats were anesthetized and NS1619 or the solvent was administered by inhalation. Systemic hemodynamic parameters, RV hemodynamic parameters, and blood gas analyses were measured before as well as 30 and 120 minutes after inhalation. (2) Rat PASMCs were stimulated with PDGF-BB in the presence and absence of NS1619. AKT, ERK1 and ERK2 activation were investigated by western blot analyses, and relative cell number was determined 48 hours after stimulation.

Results

Inhalation of a 12 µM and 100 µM NS1619 solution significantly reduced RV pressure without affecting systemic arterial pressure. Blood gas analyses demonstrated significantly reduced carbon dioxide and improved oxygenation in NS1619-treated animals pointing towards a considerable pulmonary shunt-reducing effect. In PASMC’s, NS1619 (100 µM) significantly attenuated PASMC proliferation by a pathway independent of AKT and ERK1/2 activation.

Conclusion

NS1619 inhalation reduces RV pressure and improves oxygen supply and its application inhibits PASMC proliferation in vitro. Hence, BK opening might be a novel option for the treatment of pulmonary hypertension.     

Angiotensin II induces tyrosine nitration and activation of ERK1/2 in vascular smooth muscle cells

Angiotensin II (Ang II) induces a prominent and sustained nitration and activation of ERK1/2 in rat vascular smooth muscle cells, both mediated via AT1 receptor. Nitration and activation was also shown for recombinant non-activated extracellular signal-regulated kinase (ERK) and MEK. Nitration and phosphorylation of ERK1/2 by Ang II was significantly inhibited by NAD(P)H inhibitors and scavengers of oxygen and nitrogen reactive species and completely blocked by a selective inducible nitric-oxide synthase inhibitor. MEK inhibitor U0126 did not affect ERK nitration but completely blocked activation. These data indicate that Ang II nitrates and activates ERK1/2 via a reactive species-sensitive pathway.     

ERK is regulated by sodium-proton exchanger in rat aortic vascular smooth muscle cells

The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells.