Barry L. Fanburg

Activation of the JAK-STAT pathway by reactive oxygen species

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinsons disease, pulmonary fibrosis, and Alzheimers disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c- fos and c- myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-l-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinsons disease, pulmonary fibrosis, and Alzheimers disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c-fos and c-myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2 stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-L-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.

Mechanical Properties of Rat Cardiac Muscle during Experimental Hypertrophy

The mechanical properties of trabecular muscles from the hearts of 77 rats subjected to aortic arch constriction were compared with those from 77 unoperated and sham-operated control animals at 1, 3, 7, 14, and 28 days after operation. Significant hypertrophy, as evidenced by an increase in left ventricle to body weight ratio, was first seen at three days (P < 0.02), reached a maximum of 30 to 40% by seven days (P < 0.001), and remained relatively constant throughout the remainder of the experiment. Depression of isotonic shortening velocity and maximum isometric force of trabecular muscles from hypertrophied hearts was first seen at seven days. These changes persisted at 14 and 28 days. When alterations in muscle mechanics due to changes in muscle thickness were taken into consideration, muscles from hypertrophied hearts demonstrated a depressed maximum velocity of shortening (P < 0.001), while development of isometric tension was unaltered. The latter appeared to be maintained at least in part by a prolonged contraction time, as reflected by increases in the time to peak isometric tension (P < 0.05) and the time to peak “unloaded” isotonic shortening (P < 0.001). Resting tension was increased in trabecular muscles from hypertrophied hearts. Tissue hydroxyproline concentration was elevated with hypertrophy. The observed depression in muscle shortening velocity at light loads may be explained by altered contractile state or by increased stiffness of the parallel elastic element.

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.

Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the interaction of lipoprotein lipase, purified from bovine skim milk with monolayer cultures of endothelial cells, isolated from bovine pulmonary artery. Endothelial cells in primary culture had no intrinsic lipoprotein lipase activity but were able to bind lipoprotein lipase quantitatively. The binding reached equilibrium and was saturable at 0.24 nmol of lipoprotein lipase/mg of cell protein. The concentration of lipoprotein lipase at half-maximal binding was 0.52 microM. Bound lipoprotein lipase could be detached from cultured cells by increasing concentrations of heparin, and at and above 0.6 microgram/ml of heparin, 90% of the cell-bound lipoprotein lipase activity was released. Heparan sulfate and dermatan sulfate released the enzyme to a lesser extent and chondroitin sulfate caused little, if any, release of lipoprotein lipase. The release of lipoprotein lipase with heparin was not associated with a release of [3S]glycosaminoglycans from 35S-prelabeled cells. Reductions of lipoprotein lipase binding to endothelial cells and of cell surface-associated [3S]glycosaminoglycans in 35S-prelabeled cells occurred in parallel both when cells were pretreated with crude Flavobacterium heparinum enzyme before lipoprotein lipase binding and when cells were treated with this enzyme after lipoprotein lipase binding. The removal of heparan sulfate from the cell surface by purified heparinase totally inhibited the binding of lipoprotein lipase by endothelial cells, but the removal of chondroitin sulfate by chondroitin ABC lyase had no effect on this binding. These results provide direct evidence for lipoprotein lipase attachment to endothelial cells through heparan sulfate on the cell surface, and provide evidence for the release of lipoprotein lipase by heparin through a detachment from this binding site.

Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture.

We have previously reported that serotonin (5-hydroxytryptamine [5HT]) alters cultured bovine pulmonary artery smooth muscle cell (SMC) configuration through two different regulatory mechanisms. We now report that 5HT also regulates SMC growth through these same two mechanisms--a stimulatory event initiated intracellularly and inhibition of growth resulting from a cell surface action. 5HT (1 microM) plus 0.1 mM iproniazid (a 5HT metabolic inhibitor) produced a severalfold stimulation of DNA synthesis (as measured by [3H]thymidine incorporation) of SMCs after a 17-24-hour incubation with only a slight elevation of cellular cAMP. This stimulatory effect responded synergistically with other growth factors including platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor and was effectively reversed by 5HT uptake inhibition. It was not produced by 5-hydroxyindoleacetic acid, a metabolite of 5HT. In the presence of 1 microM 5HT plus 0.1 mM isobutylmethylxanthine (IBMX), cAMP was elevated eightfold, dendritic formation occurred, and [3H]thymidine labeling of SMCs was inhibited. Inhibition of labeling by [3H]thymidine was mimicked by other agents that elevated cellular cAMP (10 microM histamine, 1 microM isoproterenol plus 0.1 mM IBMX, and 10 microM forskolin) and by 1 mM dibutyryl cAMP. This inhibitory effect was not blocked by either inhibition of 5HT uptake or 5HT-receptor antagonists ketanserin (5HT2); methiothepin, spiperone, and mianserin (5HT1/5HT2); and 3-tropanyl-indole-3-carboxylate and 3-tropanyl-3,5-dichlorobenzoate (5HT3). However, similar to 5HT, the 5HT1A agonist, (+/-)-8-hydroxy-(+/-)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalenehydrobromide, in association with IBMX, produced an elevation in cAMP and inhibition of labeling by [3H]thymidine. 5HT, in the presence of either iproniazid or IBMX, did not alter [Ca2+]i, indicating that [Ca2+]i was not a signal for either of these actions.(ABSTRACT TRUNCATED AT 250 WORDS)

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-β1

Mitogenic growth factors and transforming growth factor β1 (TGF‐β1) induce the generation of reactive oxygen species (ROS) in non‐phagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF‐β1 to activate a cell surface‐associated NADH:flavin: O2 oxidoreductase (NADH oxidase) that generates extracellular H2O2. In this study, we compared the ROS‐generating enzymatic systems activated by mitogenic growth factors and TGF‐β1 with respect to the primary reactive species produced (O2 vs. H2O2), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF‐BB, FGF‐2, and TGF‐α (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O2 without detectable NADH oxidase activity or extracellular H2O2 release. In contrast, TGF‐β1 does not stimulate intracellular O2 production and the delayed induction of extracellular H2O2 release is not associated with O2 production. Expression of dominant‐negative Ras (N17Ras) protein by herpes simplex virus‐mediated gene transfer blocks mitogen‐stimulated intracellular O2 generation but has no effect on TGF‐β1‐induced NADH oxidase activation/H2O2 production. These results demonstrate that there are at least two distinctly different ROS‐generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF‐β1 via Ras‐dependent and ‐independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/ cytokines may have different biological effects depending on the primary reactive species generated and site of production.—Thannickal, V. J., Day, R. M., Klinz, S. G., Bastien, M. C., Larios, J. M., Fanburg, B. L. Ras‐dependent and ‐independent regulation of reactive oxygen species by mitogenic growth factors and TGF‐β1. FASEB J. 14, 1741–1748 (2000)

Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion

Our previous studies have shown that, through an active transport process, serotonin (5-HT) rapidly elevates[Formula: see text] formation, stimulates protein phosphorylation, and enhances proliferation of bovine pulmonary artery smooth muscle cells (SMCs). We presently show that 1 μM 5-HT also rapidly elevates phosphorylation and activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) 1 and ERK2 of SMCs, and the enhanced phosphorylation is blocked by the antioxidants Tiron, N-acetyl-l-cysteine (NAC), and Ginkgo biloba extract. Inhibition of MAP kinase with PD-98059 failed to block enhanced[Formula: see text] formation by 5-HT. Chinese hamster lung fibroblasts (CCL-39 cells), which demonstrate both 5-HT transporter and receptor activity, showed a similar response to 5-HT (i.e., enhanced mitogenesis, [Formula: see text]formation, and ERK1 and ERK2 phosphorylation and activation). Unlike SMCs, they also responded to 5-HT receptor agonists. We conclude that downstream signaling of MAP kinase is a generalized cellular response to 5-HT that occurs secondary to[Formula: see text] formation and may be initiated by either the 5-HT transporter or receptor depending on the cell type.

Endothelin 1: Mitogenic Activity on Pulmonary Artery Smooth Muscle Cells and Release from Hypoxic Endothelial Cells

Abstract Endothelin, a recently described vasoconstrictor, has been shown to be a mitogen for vascular smooth muscle cells from systemic arteries and might play a role in pulmonary vascular remodeling produced by chronic exposure to hypoxia. We examined the effects of endothelin on proliferation of pulmonary artery smooth muscle cells and the effects of hypoxia and normoxia on the synthesis and secretion of endothelin by endothelial cells. Our results indicate that endothelin significantly increased the incorporation of [3H]thymidine by porcine pulmonary artery smooth muscle cells (122 ± 4% to 168 ± 13% of controls, with concentrations of endothelin from 1 to 1000 ng/ml). When tested on bovine pulmonary artery smooth muscle cells, endothelin increased [3H]thymidine incorporation over controls by approximately 140%; cell counts were increased by 107 ± 4% and 122 ± 7% at doses of 100 ng/ml and 1000 ng/ml, respectively. The secretion of endothelin by porcine endothelial cells was not affected by hypoxia (3520 ± 138 pg/ml/106 cells in hypoxia vs 3770 ± 326 pg/ml/106 cells in normoxia). Transforming growth factor-β1 stimulated the release by normoxic, and to a lesser degree by hypoxic, porcine endothelial cells of endothelin (4716 ± 43 pg/ml/106 cells vs 4074 ± 106 pg/ml/106 cells). Taken together, our results indicate that endothelin is weakly mitogenic for pulmonary artery smooth muscle cells, but may not significantly contribute to the remodeling seen in hypoxic pulmonary hypertension.

SUPEROXIDE AS AN INTERMEDIATE SIGNAL FOR SEROTONIN-INDUCED MITOGENESIS

Serotonin (5-HT) stimulates tyrosine phosphorylation and proliferation of bovine pulmonary artery smooth muscle cells (SMC) through its active transport (Lee et al, 1991). The present studies show that 5-HT also rapidly elevates O2.- formation by these cells within 10 minutes as measured by a lucigenin-enhanced chemiluminescence assay. The O2.- free radical quencher, Tiron, and N-acetyl-cysteine, a substrate for glutathione, block both the 5-HT-induced formation of O2.- and cellular proliferation. Similarly, inhibition of 5-HT transport with imipramine or treatment of cells with diphenyliodonium, a NAD(P)H oxidase inhibitor, block both 5-HT-induced elevation of O2.- and cellular proliferation. Alpha-hydroxyfarnesylphosphonic acid, an inhibitor of p21ras, also blocks 5-HT-induced proliferation. Endothelial cells from the same vessel show neither 5-HT-induced proliferation nor stimulation of O2.- formation. We conclude that 5-HT induced cellular proliferation of SMC through signaling pathways that utilize its transport system and O2.- formation.

Pulmonary Arterial Hypertension Future Directions: Report of a National Heart, Lung and Blood Institute/Office of Rare Diseases Workshop

Pulmonary arterial hypertension (PAH) is characterized by vascular obstruction and the variable presence of vasoconstriction, leading to increased pulmonary vascular resistance and right-sided heart failure. PAH can present in an idiopathic form, usually called primary pulmonary hypertension (PPH), and PAH is also associated with the scleroderma spectrum of diseases, HIV infection, portal hypertension with or without cirrhosis, and anorectic drug ingestion. Idiopathic PAH occurs in women more often than men (>2:1), has a mean age at diagnosis of 36 years, and is usually fatal within 3 years if untreated. Modern treatment has markedly improved physical function and has extended survival, and the 5-year mortality rate is ≈50%. We still do not understand what initiates the disease or what allows it to progress. New studies of the pathogenetic basis of PAH will lead to targeted therapies for PAH. The National Heart, Lung and Blood Institute (NHLBI) and the Office of Rare Diseases (ORD), National Institutes of Health, convened a workshop to bring together investigators with various interests in vascular biology and pulmonary hypertension to identify new research directions. Discussion included genetics of PAH, receptor function, mediators, ion channels, extracellular matrix, signaling, and potential clinical approaches. Molecular genetic studies have demonstrated mutations in a receptor in the transforming growth factor (TGF-β) superfamily, called bone morphogenetic protein receptor 2 (BMPR2), in most cases of familial pulmonary hypertension.1,2 Less common mutations associated with PAH occur in Alk1, a TGF receptor that also causes hereditary hemorrhagic telangectasia.3 Because only ≈10% to 20% of persons with a BMPR2 mutation develop PAH, it is likely that other genes, genetic polymorphisms, and environmental factors are necessary to initiate the pathological sequence that leads to disease.4 Most cases of PAH are not associated with known inherited genetic mutations.5 Thus, external stimuli coupled with as-yet-undefined genetic …