Yinglin Liu

Rho Kinase–Induced Nuclear Translocation of ERK1/ERK2 in Smooth Muscle Cell Mitogenesis Caused by Serotonin

There is now considerable evidence supporting a mitogenic action of serotonin (5-HT) on vascular smooth muscle cells (SMC) that might participate in pulmonary hypertension (PH). Our previous studies have demonstrated that 5-HT–induced proliferation depends on the generation of reactive oxygen species and activation of extracellular signal-regulated kinase (ERK) 1/ERK2. Activation of Rho kinase (ROCK) in SMC also may be important in PH. We undertook the present study to assess the role of Rho A/ROCK and its possible relation to ERK1/ERK2 in 5-HT–induced pulmonary artery SMC proliferation. We found that this stimulation of SMC proliferation requires Rho A/ROCK as inhibition with Y27632, a ROCK inhibitor, or dominant negative (DN) mutant Rho A blocks 5-HT–induced proliferation, cyclin D1 expression, phosphorylation of Elk, and the DNA binding of transcription factors, Egr-1 and GATA-4. 5-HT activated ROCK, and the activation was blocked by GR 55562 and GR127935, 5-HT 1B/1D receptor antagonists, but not by serotonin transport (SERT) inhibitors. Activation of Rho kinase by 5-HT was independent of activation of ERK1/ERK2, and 5-HT activated ERK1/ERK2 independently of ROCK. Treatment of SMC with Y27632 and expression of DNRho A in cells blocked translocation of ERK1/ERK2 to the cellular nucleus. Depolymerization of actin with cytochalasin D (CD) and latrunculin B (latB) failed to block the translocation of ERK, suggesting that the actin cytoskeleton does not participate in the translocation. The studies show for the first time to our knowledge combinational action of SERT and a 5-HT receptor in SMC growth and Rho A/ROCK participation in 5-HT receptor 1B/1D-mediated mitogenesis of vascular SMCs through an effect on cytoplasmic to nuclear translocation of ERK1/ERK2.

The 5-HT transporter transactivates the PDGFβ receptor in pulmonary artery smooth muscle cells

Serotonin (5‐HT) stimulates smooth muscle cell growth through 5‐HT receptors and the 5‐HT transporter (5‐HTT), and has been associated with pulmonary hypertension (PH). Platelet‐derived growth factor receptors (PDGFR) have also been associated with PH. We present evidence for the first time that 5‐HT transactivates PDGFRβ through the 5‐HTT in pulmonary artery (PA) SMCs. Inhibition of PDGFR kinase with imatinib or AG1296 blocks 5‐HT‐stimulated PDGFRβ phosphorylation. 5‐HTT inhibitors and the Na+/K+‐ATPase inhibitor ouabain, but not 5‐HT2 and 5‐HT1B/1D receptor inhibitors, block PDGFRβ activation by 5‐HT. Notably, 5‐HTT binds the PDGFRβ upon 5‐HT stimulation and the 5‐HTT inhibitor fluoxetine blocks both the binding and PDGDRβ activation. Activation of PDGFRβ may occur through oxidation of a catalytic cysteine of tyrosine phosphatase. 5‐HT‐acti‐vated PDGFRβ phosphorylation is blocked by the anti‐oxidant N‐acetyl‐L‐cysteine and the NADPH oxidase inhibitor, DPI. Inhibition of PDGFR kinase with ima‐tinib or AG1296 significantly inhibits SMC proliferation and migration induced by 5‐HT in vitro. Infusion of 5‐HT by miniosmotic pumps enhances PDGFRβ activation in mouse lung in vivo. In summary, these results demonstrate that 5‐HT transactivates PDGFRβ in PASMCs leading to SMC proliferation and migration, and may be an important signaling pathway in the production of PH in vivo..—Liu, Y., Li, M., Warburton, R. R., Hill, N. S., Fanburg, B. L. The 5‐HT transporter transactivates the PDGFβ receptor in pulmonary artery smooth muscle cells. FASEB J. 21, 2725–2734 (2007)

Role of Protein Transamidation in Serotonin-Induced Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells

Pulmonary hypertension is characterized by elevated pulmonary artery pressure and pulmonary artery smooth muscle cell (SMC) proliferation and migration. Clinical and experimental evidence suggests that serotonin (5-HT) is important in these responses. We previously demonstrated the participation of the 5-HT transporter and intracellular 5-HT (5-HTi) in the pulmonary vascular SMC-proliferative response to 5-HT. However, the mechanism underlying the intracellular actions of 5-HT is unknown. We speculated that 5-HTi activates SMC growth by post-translational transamidation of proteins via transglutaminase (TGase) activity, a process referred to as serotonylation. To test this hypothesis, serotonylation of pulmonary artery SMC proteins, and their role in 5-HT-induced proliferative and migratory responses, were assessed. 5-HT caused dose- and time-dependent increase in serotonylation of multiple proteins in both bovine and rat pulmonary artery SMCs. Inhibition of TGase with dansylcadaverin blocked this activity, as well as SMC-proliferative and migratory responses to 5-HT. Serotonylation of proteins also was blocked by 5-HT transporter inhibitors, and was enhanced by inhibition of monoamine oxidase, an enzyme known to degrade 5-HTi, indicating that 5-HTi levels regulate serotonylation. Immunoprecipitation assays and HPLC-mass spectral peptide sequencing revealed that a major protein serotonylated by TGase was fibronectin (FN). 5-HT-stimulated SMC serotonylation and proliferation were blocked by FN small interfering (si) RNA. These findings, together with previous observations that FN expression in the lung strongly correlates with the progression of pulmonary hypertension in both experimental animals and humans, suggest an important role of FN serotonylation in the pathogenesis of this disease.

Serotonin induces Rho/ROCK-dependent activation of Smads 1/5/8 in pulmonary artery smooth muscle cells

Serotonin (5‐HT) stimulates pulmonary artery smooth muscle cell proliferation and has been associated with pulmonary arterial hypertension (PAH). Bone morphogenetic protein receptor 2 (BMPR2) mutations similarly have been linked to PAH. However, possible crosstalk between 5‐HT and BMPR signaling remains poorly characterized. We report here that 5‐HT activates Smads 1/5/8 in bovine and human pulmonary artery smooth muscle cells (SMCs) and causes translocation of these Smads from cytoplasm to the nucleus. DN BMPR1A blocked 5‐HT activation of Smads 1/5/8 by 5‐HT and BMPR1A overexpression enhanced it. Activation of Smads by 5‐HT occurred through the 5‐HT 1B/1D receptor as it was blocked with the inhibitor GR 55562 but unaffected by inhibitors of the 5‐HT transporter and a variety of 5‐HT receptors. Activation of the Smads by 5‐HT depended on Rho/Rho kinase signaling as it was blocked by Y27632, but unaffected by inhibitors of PI3K or MAPK. Transfection of cells with BMPR1A and ligation of the BMP receptor with BMP‐2 also activated GTP‐Rho A of these SMCs, while DN BMPR1A blocked the activation. 5‐HT stimulated an increase in serine/threonine phosphorylation of BMPR1A, supporting the activation of BMPR1A by 5‐HT in SMCs. Infusion of 5‐HT into mice with miniosmotic infusion pumps caused activation of Smads 1/5/8 in lung tissue, demonstrating the effect in vivo. The studies support a unique concept that 5‐HT transactivates the serine kinase receptor, BMPR 1A, to activate Smads 1/5/8 via Rho and Rho kinase in pulmonary artery SMCs. Rho and Rho kinase also participate in the activation of Smads by BMP.—Liu, Y.,Ren, W., Warburton, R., Toksoz, D., Fanburg, B. L. Serotonin induces Rho/ROCK‐dependent activation of Smads 1/5/8 in pulmonary artery smooth muscle cells. FASEB J. 23, 2299–2306 (2009)

JNK regulates serotonin-mediated proliferation and migration of pulmonary artery smooth muscle cells

JNK is a member of the MAPK family and has essential roles in inflammation and cell differentiation and apoptosis. In recent years, there have been accumulating data indicating a novel role for JNK in cell growth and migration. In this report, we demonstrate that JNK activity is necessary for serotonin (5-HT)-induced proliferation and migration of bovine pulmonary artery smooth muscle cells (PASMCs). Stimulation with 5-HT was found to lead to activation of JNK with a maximal activation at 10 min. Inhibition of JNK with its specific inhibitor, SP-600125, or its dominant-negative form, DN-JNK, significantly reduced 5-HT-stimulated [(3)H]thymidine incorporation and cyclin D1 expression. A similar inhibitory effect on SMC migration produced by 5-HT, as detected by a wound healing assay, was observed with inhibition of JNK. Furthermore, inhibition of 5-HT receptors (1B) and (2A), but not inhibition of the 5-HT transporter, blocked 5-HT-induced JNK activation. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY-294002 and wortmannin had little or no effect on 5-HT-induced JNK phosphorylation, but JNK inhibitor SP-600125 and DN-JNK blocked 5-HT-stimulated phosphorylation of Akt and its downstream effectors, p70S6K1 and S6, indicating that Akt is a downstream effector of JNK. Activation of Akt by 5-HT was blocked only minimally, if at all, by inhibitors of ERK and p38 MAPK, indicating a uniqueness of JNK MAPK in this activation of Akt. Coimmunoprecipitation showed binding of Akt to JNK, further supporting the interaction of JNK and Akt. Thus JNK is a critical molecule in 5-HT-induced PASMC proliferation and migration and may act at an important point for cross talk of the MAPK and PI3K pathways. Its activation by 5-HT is initiated through 5-HT (1B) and (2A) receptors, and its stimulation of SMC proliferation and migration occurs through the Akt pathway.

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 Gq 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.

Classical and alternative activation of rat hepatic sinusoidal endothelial cells by inflammatory stimuli

The ability of rat hepatic sinusoidal endothelial cells (HSEC) to become activated in response to diverse inflammatory stimuli was analyzed. Whereas the classical macrophage activators, IFNγ and/or LPS upregulated expression of iNOS in HSEC, the alternative macrophage activators, IL-10 or IL-4+IL-13 upregulated arginase-1 and mannose receptor. Similar upregulation of iNOS and arginase-1 was observed in classically and alternatively activated Kupffer cells, respectively. Removal of inducing stimuli from the cells had no effect on expression of these markers, demonstrating that activation is persistent. Washing and incubation of IFNγ treated cells with IL-4+IL-13 resulted in decreased iNOS and increased arginase-1 expression, while washing and incubation of IL-4+IL-13 treated cells with IFNγ resulted in decreased arginase-1 and increased iNOS, indicating that classical and alternative activation of the cells is reversible. HSEC were more sensitive to phenotypic switching than Kupffer cells, suggesting greater functional plasticity. Hepatocyte viability and expression of PCNA, β-catenin and MMP-9 increased in the presence of alternatively activated HSEC. In contrast, the viability of hepatocytes pretreated for 2 h with 5 mM acetaminophen decreased in the presence of classically activated HSEC. These data demonstrate that activated HSEC can modulate hepatocyte responses following injury. The ability of hepatocytes to activate HSEC was also investigated. Co-culture of HSEC with acetaminophen-injured hepatocytes, but not control hepatocytes, increased the sensitivity of HSEC to classical and alternative activating stimuli. The capacity of HSEC to respond to phenotypic activators may represent an important mechanism by which they participate in inflammatory responses associated with hepatotoxicity.

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.