Jun Ichi Abe

Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase.

Mitogen-activated protein (MAP) kinases are a multigene family activated by many extracellular stimuli. There are three groups of MAP kinases based on their dual phosphorylation motifs, TEY, TPY, and TGY, which are termed extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinases, and p38, respectively. A new MAP kinase family member termed Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. BMK1 has a TEY sequence similar to ERK1/2 but has unique COOH-terminal and loop-12 domains. To define BMK1 regulation, its activation in cultured rat vascular smooth muscle cells was characterized. Angiotensin II, phorbol ester, platelet-derived growth factor, and tumor necrosis factor-α were the strongest stimuli for ERK1/2 but were weak activators of BMK1. In contrast, H2O2 caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and ERK1/2. BMK1 activation by H2O2 was calcium-dependent and appeared ubiquitous as shown by stimulation in human skin fibroblasts, human vascular smooth muscle cells, and human umbilical vein endothelial cells. These findings demonstrate that activation of BMK1 is different from ERK1/2 and suggest an important role for BMK1 as a redox-sensitive kinase.

p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1

The Na+/H+exchanger isoform-1 (NHE-1) is the key member of a family of exchangers that regulates intracellular pH and cell volume. Activation of NHE-1 by growth factors is rapid, correlates with increased NHE-1 phosphorylation and cell alkalinization, and plays a role in cell cycle progression. By two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identify serine 703 as the major serum-stimulated amino acid. Mutation of serine 703 to alanine had no effect on acid-stimulated Na+/H+ exchange but completely prevented the growth factor-mediated increase in NHE-1 affinity for H+. In addition, we show that p90 ribosomal S6 kinase (p90RSK) is a key NHE-1 kinase since p90RSK phosphorylates NHE-1 serine 703 stoichiometricallyin vitro, and transfection with kinase-inactive p90RSK inhibits serum-induced phosphorylation of NHE-1 serine 703 in transfected 293 cells. These findings establish p90RSK as a serum-stimulated NHE-1 kinase and a mediator of increased Na+/H+ exchange in vivo.

Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II–induced aortic aneurysms

Inflammation and oxidative stress are pathogenic mediators of many diseases, but molecules that could be therapeutic targets remain elusive. Inflammation and matrix degradation in the vasculature are crucial for abdominal aortic aneurysm (AAA) formation. Cyclophilin A (CypA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMCs), is secreted in response to reactive oxygen species (ROS) and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe−/− mice, we show that Apoe−/−Ppia−/− mice are completely protected from AngII–induced AAA formation, in contrast to Apoe−/−Ppia+/+ mice. Apoe−/−Ppia−/− mice show decreased inflammatory cytokine expression, elastic lamina degradation and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia+/+ mice. Mechanistic studies showed that VSMC-derived intracellular and extracellular CypA are required for ROS generation and matrix metalloproteinase-2 activation. These data define a previously undescribed role for CypA in AAA formation and suggest CypA as a new target for treating cardiovascular disease.

Endothelial atheroprotective and anti-inflammatory mechanisms

Abstract: Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor‐α (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF‐mediated EC activation. EC apoptosis is likely important in the process termed “plaque erosion” that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide‐dependent protective pathways that involve enzymes PI3‐kinase and Akt. Conversely, our laboratory has identified nitric oxide‐independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen‐activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF‐mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm2) on TNF‐stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c‐Jun N‐terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre‐exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen‐activated protein kinase kinase inhibitor, blocked the flow‐mediated inhibition of TNF activation of JNK. Flow‐mediated inhibition of JNK was unaffected by 0.1 mM L‐nitroarginine, 100 μM 8‐bromo‐cyclic GMP, or 100 μM 8‐bromo‐cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow‐mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.

Fyn and JAK2 mediate Ras activation by reactive oxygen species.

Reactive oxygen species (ROS) activate Ras and the extracellular signal-regulated kinase (ERK) cascade. Because JAK2 is a critical mediator for Ras/Raf/ERK activation by several hormones, we examined the role of JAK2 in ROS signal events. H2O2 stimulated JAK2 activity in fibroblasts with peak at 2–5 min. To determine the specific role of Src and Fyn as mediators of JAK2 activation and its downstream events, we used fibroblasts derived from transgenic mice deficient in Src (Src−/−) or Fyn (Fyn−/−). H2O2-stimulated JAK2 activity was completely inhibited in Fyn−/− cells. Shc tyrosine phosphorylation and Ras activation by H2O2 were also significantly reduced in Fyn−/− cells, but not altered in Src−/− cells. Activation of JAK2 was restored when Fyn−/− cells were transfected with B-Fyn but not with Src. Inhibiting JAK2 activity with the specific inhibitor AG-490 prevented H2O2 stimulated Shc and Ras activation. H2O2-mediated ERK1/2 activation in Fyn−/− cells and AG-490 treated cells was completely inhibited at an early time (5 min), but not at late times (20–40 min) after stimulation. These results define a new redox-sensitive pathway for Ras activation and rapid ERK1/2 activation, which is mediated by Fyn and JAK2.

Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: Role of Gab1 and MEKK3 in TNF-α-induced c-Jun and NF-κB activation and adhesion molecule expression

Insulin-like growth factor (IGF)-1 and the type I IGF-1 receptor are important regulators of vascular function that may contribute to cardiovascular disease. We hypothesized that IGF-1 causes endothelial cell dysfunction and expression of neutrophil and monocyte adhesion molecules by enhancing pro-inflammatory cytokine signal transduction. Long-term IGF-1 treatment of endothelial cells potentiated c-Jun and nuclear factor NF-kappaB activation by tumor necrosis factor (TNF)-alpha and enhanced TNF-alpha-mediated adhesion molecule expression. In response to IGF-1 treatment, the expression of kinases in the c-Jun/c-Jun NH(2)-terminal kinase signaling pathway (MEKK1, MEK4, and JNK1/2) was unchanged, but expressions of insulin receptor substrate-1 and Grb2-associated binder-1 (Gab1) were significantly decreased. Because Gab1 is involved in both c-Jun and NF-kappaB activation by TNF-alpha, we focused on Gab1-dependent signaling. Gab1 inhibited c-Jun and NF-kappaB transcriptional activation by TNF-alpha. Interestingly, Gab1 inhibited c-Jun transcriptional activity induced by MEKK3 but not MEKK1 and MEK4. Gab1 associated with MEKK3, and a catalytically inactive form of MEKK3 inhibited TNF-alpha-induced c-Jun and NF-kappaB transcriptional activation, suggesting a critical role for Gab1 and MEKK3 in TNF-alpha signaling. These data demonstrate that Gab1 and MEKK3 play important roles in endothelial cell inflammation via regulating the activation of c-Jun and NF-kappaB. Furthermore, the IGF-1-mediated downregulation of Gab1 expression represents a novel mechanism to promote vascular inflammation and atherosclerosis.

Functional Role of Phosphodiesterase 3 in Cardiomyocyte Apoptosis Implication in Heart Failure

Background—Myocyte apoptosis plays an important role in pathological cardiac remodeling and the progression of heart failure. cAMP signaling is crucial in the regulation of myocyte apoptosis and cardiac remodeling. Multiple cAMP-hydrolyzing phosphodiesterases (PDEs), such as PDE3 and PDE4, coexist in cardiomyocytes and elicit differential temporal/spatial regulation of cAMP signaling. However, the role of PDE3 and PDE4 in the regulation of cardiomyocyte apoptosis remains unclear. Although chronic treatment with PDE3 inhibitors increases mortality in patients with heart failure, the contribution of PDE3 expression/activity in heart failure is not well known. Methods and Results—In this study we report that PDE3A expression and activity were significantly reduced in human failing hearts as well as mouse hearts with chronic pressure overload. In primary cultured cardiomyocytes, chronic inhibition of PDE3 but not PDE4 activity by pharmacological agents or adenovirus-delivered antisense PDE3A promoted cardiomyocyte apoptosis. Both angiotensin II (Ang II) and the β-adrenergic receptor agonist isoproterenol selectively induced a sustained downregulation of PDE3A expression and induced cardiomyocyte apoptosis. Restoring PDE3A via adenovirus-delivered expression of wild-type PDE3A1 completely blocked Ang II– and isoproterenol-induced cardiomyocyte apoptosis, suggesting the critical role of PDE3A reduction in cardiomyocyte apoptosis. Moreover, we defined a crucial role for inducible cAMP early repressor expression in PDE3A reduction–mediated cardiomyocyte apoptosis. Conclusions—Our results suggest that PDE3A reduction and consequent inducible cAMP early repressor induction are critical events in Ang II– and isoproterenol-induced cardiomyocyte apoptosis and may contribute to the development of heart failure. Drugs that maintain PDE3A function may represent an attractive therapeutic approach to treat heart failure.

p38 Kinase Is a Negative Regulator of Angiotensin II Signal Transduction in Vascular Smooth Muscle Cells: Effects on Na+/H+ Exchange and ERK1/2

Activation of the Na+/H+ exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II-mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na+/H+ exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na+/H+ exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na+/H+ exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na+/H+ exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II-stimulated Na+/H+ exchange. First, angiotensin II activation of ERK1/2 was increased 1. 5- to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II-stimulated MAP kinases to phosphorylate a glutathione S-transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II-mediated regulation of the Na+/H+ exchanger in VSMC.

Reactive Oxygen Species Activate p90 Ribosomal S6 Kinase via Fyn and Ras

Reactive oxygen species and growth factors stimulate similar intracellular signal transduction events including activation of Src kinase family members and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of Src and ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth by activating several transcription factors as well as the Na+/H+ exchanger. In the present study, we determined whether H2O2 activates p90RSK to gain insight into signal transduction mechanisms activated by reactive oxygen species. H2O2 (200 μm) stimulated ERK1/2 and p90RSK activity in lymphocytes, endothelial cells, and fibroblasts. The MEK-1 inhibitor, PD98059 (30 μm), inhibited H2O2-mediated activation of ERK1/2 but not of p90RSK. An essential role for Fyn and Ras in p90RSK activation was suggested by five findings. 1) The tyrosine kinase inhibitor, herbimycin A, and the specific Src kinase family inhibitor, PP1, blocked p90RSK activation by H2O2 in a concentration-dependent manner. 2) p90RSK activation by H2O2 was significantly reduced in fibroblasts derived from transgenic mice deficient in Fyn, but not c-Src. 3) H2O2rapidly activated Ras (peak at 2–5 min), which preceded p90RSK activation (peak at 20 min). 4) Dominant negative Ras completely blocked H2O2-induced activation of p90RSK. 5) In Fyn−/− fibroblasts, activation of Ras by H2O2 was significantly attenuated. These results show essential roles for Fyn and Ras in H2O2-mediated activation of p90RSK and establish redox-sensitive regulation of Ras and p90RSK as a new function for Fyn.

Cyclophilin A Mediates Vascular Remodeling by Promoting Inflammation and Vascular Smooth Muscle Cell Proliferation

Background— Oxidative stress, generated by excessive reactive oxygen species, promotes cardiovascular disease. Cyclophilin A (CyPA) is a 20-kDa chaperone protein secreted from vascular smooth muscle cells (VSMCs) in response to reactive oxygen species that stimulates VSMC proliferation and inflammatory cell migration in vitro; however, the role CyPA plays in vascular function in vivo remains unknown. Methods and Results— We tested the hypothesis that CyPA contributes to vascular remodeling by analyzing the response to complete carotid ligation in CyPA knockout mice, wild-type mice, and mice that overexpress CyPA in VSMC (VSMC-Tg). After carotid ligation, CyPA expression in vessels of wild-type mice increased dramatically and was significantly greater in VSMC-Tg mice. Reactive oxygen species–induced secretion of CyPA from mouse VSMCs correlated significantly with intracellular CyPA expression. Intimal and medial hyperplasia correlated significantly with CyPA expression after 2 weeks of carotid ligation, with marked decreases in CyPA knockout mice and increases in VSMC-Tg mice. Inflammatory cell migration into the intima was significantly reduced in CyPA knockout mice and increased in VSMC-Tg mice. Additionally, VSMC proliferation assessed by Ki67+ cells was significantly less in CyPA knockout mice and was increased in VSMC-Tg mice. The importance of CyPA for intimal and medial thickening was shown by strong correlations between CyPA expression and the number of both inflammatory cells and proliferating VSMCs in vivo and in vitro. Conclusions— In response to low flow, CyPA plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating flow-mediated vascular remodeling and intima formation.