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AbstractIt is well accepted that atherosclerosis occurs in a site-specific manner especially at branch points where disturbed blood flow (d-flow) predisposes to the development of plaques. Investigations both in vivoand in vitrohave shown that d-flow is pro-atherogenic by promoting oxidative and inflammatory states in the artery wall. In contrast, steady laminar blood flow (s-flow) is atheroprotective by inhibition of oxidative stress and inflammation in the vessel wall. The mechanism for inflammation in endothelial cells (ECs) exposed to d-flow has been well studied and includes redox-dependent activation of apoptosis signal-regulating kinase 1 (ASK1) and Jun NH2-terminal kinase (JNK) that ultimately lead to the expression of adhesive molecules. In contrast, s-flow leads to the activation of the mitogen extracellular-signal-regulated kinase kinase 5/extracellular signal-regulated kinase-5 (MEK5/ERK5) pathway that prevents pro-inflammatory signaling. Important transcriptional events that reflect the pro-oxidant and pro-inflammatory condition of ECs in d-flow include the activation of activator protein 1 (AP-1) and nuclear factor kappaB (NFκB), whereas in s-flow, activation of Krüppel-like factor 2 (KLF2) and nuclear factor erythroid 2-like 2 (Nrf2) are dominant. Recent studies have shown that protein kinase c zeta (PKCζ) is highly activated under d-flow conditions and may represent a molecular switch for EC signaling and gene expression. The targeted modulation of proteins activated in a site-specific manner holds the promise for a new approach to limit atherosclerosis. Antioxid. Redox Signal. 15, 1405–1414. [ABSTRACT FROM AUTHOR]

Osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor or tumor necrosis factor receptor superfamily member 11B, is well known as a modulator of bone remodeling. The contribution of OPG to cardiovascular disease (CVD) has been suggested, but its molecular mechanism is complex and remains unclear. In the present study, Alves-Lopes et al. (Clin. Sci. (Lond.) (2021) 135(20): https://doi.org/10.1042/CS20210643) reported the critical role of syndecan-1 (SDC-1, also known as CD138), a surface protein part of the endothelial glycocalyx, in OPG-induced vascular dysfunction. The authors found that in endothelial cells (ECs), through SDC-1, OPG increased eNOS Thr495 phosphorylation, thereby inhibiting eNOS activity. Furthermore, the OPG--SDC-1 interaction increased reactive oxygen species (ROS) production through NOX1/4 activation. Both the reduced eNOS activity and induced ROS production inhibited NO production and impaired EC function. In vascular smooth muscle cells (VSMCs), the OPG--SDC-1 interaction increased ROS production through NOX1/4 activation, subsequently increased MLC phosphorylation-mediated Rho kinase-MYPT1 regulation, leading to increased vascular contraction. Ultilizing wire myography and mechanistic studies, the authors nicely provide the evidence that SDC-1 plays a crucial role in OPG-induced vascular dysfunction. As we mentioned above, the molecular mechanism and roles of OPG in cardiovascular system are complex and somewhat confusing. In this commentary, we briefly summarize the OPG-mediated signaling pathways in cardiovascular system. [ABSTRACT FROM AUTHOR]

Significance: Hemodynamic shear stress, the frictional force exerted onto the vascular endothelial cell (EC) surface, influences vascular EC functions. Atherosclerotic plaque formation in the endothelium is known to be site specific: disturbed blood flow (d-flow) formed at the lesser curvature of the aortic arch and branch points promotes plaque formation, and steady laminar flow (s-flow) at the greater curvature is atheroprotective. Recent Advances: Post-translational modifications (PTMs), including phosphorylation and SUMOylation, and epigenetic events, including DNA methylation and histone modifications, provide a new perspective on the pathogenesis of atherosclerosis, elucidating how gene expression is altered by d-flow. Activation of PKCζ and p90RSK, SUMOylation of ERK5 and p53, and DNA hypermethylation are uniquely induced by d-flow, but not by s-flow. Critical Issues: Extensive cross talk has been observed among the phosphorylation, SUMOylation, acetylation, and methylation PTMs, as well as among epigenetic events along the cascade of d-flow-induced signaling, from the top (mechanosensory systems) to the bottom (epigenetic events). In addition, PKCζ activation plays a role in regulating SUMOylation-related enzymes of PIAS4, p90RSK activation plays a role in regulating SUMOylation-related enzymes of Sentrin/SUMO-specific protease (SENP)2, and DNA methyltransferase SUMOylation may play a role in d-flow signaling. Future Directions: Although possible contributions of DNA events such as histone modification and the epigenetic and cytosolic events of PTMs in d-flow signaling have become clearer, determining the interplay of each PTM and epigenetic event will provide a new paradigm to elucidate the difference between d-flow and s-flow and lead to novel therapeutic interventions to inhibit plaque formation. [ABSTRACT FROM AUTHOR]

TONAL SCHEMATA ARE SHAPED BY CULTURE-SPECIFIC music exposure. The acquisition process of tonal schemata has been delineated in Western mono-musical children, but cross-cultural variations have not been explored. We examined how Japanese children acquire tonal schemata in a bi-musical culture characterized by the simultaneous, and unbalanced, appearances of Western (dominant) music along with traditional Japanese (non-dominant) music. Progress of this acquisition was indexed by gauging children's sensitivities to musical scale membership (differentiating scale-tones from non-scale-tones) and differences in tonal stability among scale tones (differentiating the tonic from another scale tone). Children (7-, 9-, 11-, 13-, and 14-year-olds) and adults judged how well two types of target tones (scale tone vs. non-scale tone; tonic vs. non-tonic) fit a preceding Western or traditional Japanese tonal context. Results showed that even 7-year-olds showed sensitivity to Western scale membership while sensitivity to Japanese scale membership did not appear until age nine. Also, sensitivity to the tonic emerged at age 13 for both types of melodies. These results suggest that even though they are exposed to both types of music simultaneously from birth, Japanese children begin by acquiring the tonal schema of the dominantWestern music and this age of acquisition is not delayed relative to Western monomusical peers. [ABSTRACT FROM AUTHOR]

BACKGROUND: Radiation exposure increases the risk of coronary artery disease (CAD). We explored the association of CAD with coronary artery dose-volume parameters in patients treated with 3D-planned radiation therapy (RT). METHODS: Patients who received thoracic RT and were evaluated by cardiac computed tomography = 1 year later were included. Demographic data and cardiac risk factors were retrospectively collected. Dosimetric data (mean heart dose, dmax, dmean, V50 - V5) were collected for the whole heart and for each coronary artery. A coronary artery calcium (CAC) Agatston score was calculated on a per-coronary basis and as a total score. Multivariable generalized linear mixed models were generated. The predicted probabilities were used for receiver operating characteristic analyses. RESULTS: Twenty patients with a median age of 53 years at the time of RT were included. Nine patients (45%) had = 3/6 conventional cardiac risk factors. Patients received RT for breast cancer (10, 50%), lung cancer (6, 30%), or lymphoma/myeloma (4, 20%) with a median dose of 60 Gy. CAC scans were performed a median of 32 months after RT. CAC score was significantly associated with radiation dose and presence of diabetes. In a multivariable model adjusted for diabetes, segmental coronary artery dosimetric parameters (dmax, dmean, V50, V40 V30, V20, V10, and V5) were significantly associated with CAC score > 0. V50 had the highest area under the ROC curve (0.89, 95% confidence interval, 0.80-0.97). J Cardiovasc Imaging. 2019 Oct;27(4):268-279. CONCLUSIONS: Coronary artery radiation exposure is strongly correlated with subsequent segmental CAC score. Coronary calcification may occur soon after RT and in individuals with conventional cardiac risk factors. [ABSTRACT FROM AUTHOR]

Healthy kidney structure and environment rely on epithelial integrity and interactions between epithelial cells and other kidney cells. The Ser/Thr kinase 90 kDa ribosomal protein S6 kinase 1 (p90RSK) belongs to a protein family that regulates many cellular processes, including cell motility and survival. p90RSK is predominantly expressed in the kidney, but its possible role in chronic kidney disease (CKD) remains largely unknown. Here, we found that p90RSK expression is dramatically activated in a classic mouse obstructive chronic kidney disease model, largely in the interstitial FSP-1-positive fibroblasts. We generated FSP-1-specific p90RSK transgenic mouse (RSK-Tg) and discovered that these mice, after obstructive injury, display significantly increased fibrosis and enhanced tubular epithelial damage compared with their wt littermates (RSK-wt), indicating a role of p90RSK in fibroblast-epithelial communication. We established an in vitro fibroblast-epithelial coculture system with primary kidney fibroblasts from RSK-Tg and RSK-wt mice and found that RSK-Tg fibroblasts consistently produce excessive H2O2 causing epithelial oxidative stress and inducing nuclear translocation of the signaling protein β-catenin. Epithelial accumulation of β-catenin, in turn, promoted epithelial apoptosis by activating the transcription factor forkhead box class O1 (FOXO1). Of note, blockade of reactive oxygen species (ROS) or β-catenin or FOXO1 activity abolished fibroblast p90RSK-mediated epithelial apoptosis. These results make it clear that p90RSK promotes kidney fibrosis by inducing fibroblast-mediated epithelial apoptosis through ROS-mediated activation of β-catenin/FOXO1 signaling pathway. [ABSTRACT FROM AUTHOR]

We investigated tonal perception of melodies from 2 cultures (Western and traditional Japanese) by 5 different cultural groups (44 Japanese, 25 Chinese, 16 Vietnamese, 18 Indonesians, and 25 U.S. citizens). Listeners rated the degree of "melodic completeness" of the final tone (a tonic vs. a nontonic) and "happiness-sadness" in the mode (major vs. minor, YOH vs. IN) of each melody. When Western melodies were presented, American and Japanese listeners responded similarly, such that they reflected implicit tonal knowledge of Western music. By contrast, the responses of Chinese, Vietnamese, and Indonesian listeners were different from those of American and Japanese listeners. When traditional Japanese melodies were presented, Japanese listeners exhibited responses that reflected implicit tonal knowledge of traditional Japanese music. American listeners also showed responses that were like the Japanese; however, the pattern of responses differed between the 2 groups. Alternatively, Chinese, Vietnamese, and Indonesian listeners exhibited different responses from the Japanese. These results show large differences between the Chinese/Vietnamese/Indonesian group and the American/Japanese group. Furthermore, the differences in responses to Western melodies between Americans and Japanese were less pronounced than that between Chinese, Vietnamese, and Indonesians. These findings imply that cultural differences in tonal perception are more diverse and distinctive than previously believed. [ABSTRACT FROM AUTHOR]

A major advantage of satellite communications is the ability to implement communication networks virtually anywhere in Japan very simply. The NTT Group has been utilizing satellite communications to provide services in situations where optical fiber, mobile telephony, and other terrestrial facilities are impractical: remote islands, offshore areas, and stricken regions where people have been forced to temporarily flee in the face of earthquakes and other natural disasters. The Group continues to pursue research and development on satellite systems to make them more efficient and advanced. Here we present an overview of NTT's new satellite communications modem unit called COM-U (satellite circuit-terminating common unit), that markedly improves the spectrum utilization efficiency of satellite transponders and the maintenance and operations of satellite communications used for remote island and disaster relief satellite communications. [ABSTRACT FROM AUTHOR]

The pathophysiological mechanisms of the immune activation of smooth muscle cells are not well understood. Increased expression of Axl, a receptor tyrosine kinase, was recently found in arteries from patients after coronary bypass grafts. In the present study, we hypothesized that Axl-dependent immune activation of smooth muscle cells regulates vein graft remodeling. We observed a twofold decrease in intimal thickening after vascular and systemic depletion of Axl in vein grafts. Local depletion of Axl had the greatest effect on immune activation, whereas systemic deletion of Axl reduced intima due to an increase in apoptosis in vein grafts. Primary smooth muscle cells isolated from Axl knockout mice had reduced proinflammatory responses by prevention of the STAT1 pathway. The absence of Axl increased suppressor of cytokine signaling (SOCS)1 expression in smooth muscle cells, a major inhibitory protein for STAT1. Ultrasound imaging suggested that vascular depletion of Axl reduced vein graft stiffness. Axl expression determined the STAT1-SOCS1 balance in vein graft intima and progression of the remodeling. The results of this investigation demonstrate that Axl promotes STAT1 signaling via inhibition of SOCS1 in activated smooth muscle cells in vein graft remodeling. [ABSTRACT FROM AUTHOR]

Disturbed blood flow (d-flow) causes endothelial cell (EC) dysfunction, leading to atherosclerotic plaque formation. We have previously shown that d-flow increases SUMOylation of p53 and ERK5 through down regulation of sentrin/SUMO-specific protease 2 (SENP2) function; however, it is not known how SENP2 itself is regulated by d-flow. Here, we determined that d-flow activated the serine/threonine kinase p90RSK, which subsequently phosphorylated threonine 368 (T368) of SENP2. T368 phosphorylation promoted nuclear export of SENP2, leading to down regulation ofeNOS expression and upregulation of proinflammatory adhesion molecule expression and apoptosis. In an LDLR-deficient murine model of atherosclerosis, EC-specific overexpression of p90RSK increased EC dysfunction and lipid accumulation in the aorta compared with control animals; however, these pathologic changes were not observed in atherosclerotic mice overexpressing dominant negative p90RSK (DN-p90RSK). Moreover, depletion of SENP2 in these mice abolished the protective effect of DN-p90RSK overexpression. We propose that p90RSK-mediated SENP2-T368 phosphorylation is a master switch in d-flow-induced signaling, leadingto EC dysfunction and atherosclerosis. [ABSTRACT FROM AUTHOR]

Because ERK5 inhibits endothelial inflammation and dysfunction, activating ERK5 might be a novel approach to protecting vascular endothelial cells (ECs) against various pathological conditions of the blood vessel. We have identified small molecules that protect ECs via ERK5 activation and determined their contribution to preventing cardiac allograft rejection. Using high-throughput screening, we identified certain statins and antimalarial agents including chloroquine, hydroxychloroquine, and quinacrine as strong ERK5 activators. Pitavastatin enhanced ERK5 transcriptional activity and Kruppel-like factor-2 expression in cultured human and bovine ECs, but these effects were abolished by the depletion of ERK5. Chloroquine and hydroxychloroquine upregulated ERK5 kinase activity and inhibited VCAM-1 expression in an ERK5-dependent but MAPK/ERK kinase 5– and Kruppel-like factor 2/4–independent manner. Leukocyte rolling and vascular reactivity were used to evaluate endothelial function in vivo, and we found that EC-specific ERK5 knockout (ERK5-EKO) mice exhibited increased leukocyte rolling and impaired vascular reactivity, which could not be corrected by pitavastatin. The role of endothelial ERK5 in acute cardiac allograft rejection was also examined by heterotopic grafting of the heart obtained from either wild-type or ERK5-EKO mice into allomismatched recipient mice. A robust increase in both inflammatory gene expression and CD45-positive cell infiltration into the graft was observed. These tissue rejection responses were inhibited by pitavastatin in wild-type but not ERK5-EKO hearts. Our study has identified statins and antimalarial drugs as strong ERK5 activators and shown that ERK5 activation is preventive of endothelial inflammation and dysfunction and acute allograft rejection. [ABSTRACT FROM AUTHOR]

Background--Efferocytosis is a process by which dead and dying cells are removed by phagocytic cells. Efferocytosis by macrophages is thought to curb the progression of atherosclerosis, but the mechanistic insight of this process is lacking. Methods and Results--When macrophages were fed apoptotic cells or treated with pitavastatin in vitro, efferocytosisrelated signaling and phagocytic capacity were upregulated in an ERK5 activity--dependent manner. Macrophages isolated from macrophage-specific ERK5-null mice exhibited reduced efferocytosis and levels of gene and protein expression of efferocytosis-related molecules. When these mice were crossed with low-density lipoprotein receptor -/- mice and fed a high-cholesterol diet, atherosclerotic plaque formation was accelerated, and the plaques had more advanced and vulnerable morphology. Conclusions--Our results demonstrate that ERK5, which is robustly activated by statins, is a hub molecule that upregulates macrophage efferocytosis, thereby suppressing atherosclerotic plaque formation. Molecules that upregulate ERK5 and its signaling in macrophages may be good drug targets for suppressing cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow--mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow--mediated peroxynitrite (ONOO-) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53--CBcl-2 binding, and EC apoptosis. Both d-flow and ONOO- increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301--410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ--PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53-/- mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ--PIASy interaction during d-flow--mediated EC apoptosis, which has potential relevance to early events of atherosclerosis. [ABSTRACT FROM AUTHOR]

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-α-induced NF-κB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-α- or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, lL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α- or LPSinduced lung inflammation. Interestingly, vinpocetine inhibits NF-κB-dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca2+ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases. [ABSTRACT FROM AUTHOR]

Testicular orphan nuclear receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily with diverse physiological functions. Using TR4 knockout (TR4-/-) mice to study its function in cardiovascular diseases, we found reduced cluster of differentia- tion (CD)36 expression with reduced foam cell formation in TR4-/- mice. Mechanistic dissection suggests that TR4 induces CD36 pro- tein and mRNA expression via a transcriptional regulation. Interestingly, we found this TR4-mediated CD36 transactivation can be further enhanced by polyunsaturated fatty acids (PUFAs), such as omega-3 and -6 fatty acids, and their metabolites such as 15-hydroxyeico-satetraonic acid (15-HETE) and 13-hydroxy octa-deca dieonic acid (13-HODE) and thiazolidinedione (TZD)-rosiglitazone. Both electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that TR4 binds to the TR4 response element located on the CD36 5′-promoter region for the induction of CD36 expression. Stably transfected TR4-siRNA or functional TR4 cDNA in the RAW264.7 macrophage cells resulted in either decreased or increased CD36 expression with decreased or increased foam cell formation. Restoring functional CD36 cDNA in the TR4 knockdown macrophage cells reversed the decreased foam cell formation. Together, these results reveal an important signaling pathway controlling CD36-mediated foam cell formation/cardiovascular diseases, and findings that TR4 transactivation can be activated via its ligands/activators, such as PUFA metabolites and TZD, may provide a platform to screen new drug(s) to battle the metabolism syndrome, diabetes, and cardiovascular diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptors (PPAR) decrease the production of cytokine and inducible nitric-oxide synthase (iNOS) expression, which are associated with aging-related inflammation and insulin resistance. Recently, the involvement of the induction of hemeoxygenase-1 (HO-1) in regulating inflammation has been suggested, but the exact mechanisms for reducing inflammation by HO-1 remains unclear, We found that overexpression of HO-1 and [Ru(CO)3Cl2]2, a carbon monoxide (CO)-releasing compound, increased not only ERK5 kinase activity, but also its transcriptional activity measured by luciferase assay with the transfection of the Gal4-ERK5 reporter gene. This transcriptional activity is required for coactivation of PPARδ by ERK5 in C2C12 cells. [Ru(CO)3Cl2]2 activated PPARδ transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-κB activity by ERK5 activation was reversed by a dominant negative form of PPARδ suggesting that ERK5/PPAR6 activation is required for the anti-inflammatory effects of CO and HO-1. Based on these data, we propose a new mechanism by which CO and HO-1 mediate anti-inflammatory effects via activating ERK5/PPARδ, and ERK5 mediates CO and HO-1-induced PPARδ activation via its interaction with PPARδ. [ABSTRACT FROM AUTHOR]

Dysregulation of apoptosis in endothelial cells (EC) and fibroblasts contributes to fibrosis. We have shown previously that apoptosis of EC triggers the proteolysis of extracellular matrix components and the release of a C-terminal fragment of perlecan, which in turn inhibits apoptosis of fibroblasts. Here we have defined the receptors and pathways implicated in this anti-apoptotic response in fibroblasts. Neutralizing α2β1 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or a recombinant perlecan C-terminal fragment (LG3) prevented resistance to apoptosis and is associated with decreased levels of Akt phosphorylation. Co-incubation of fibroblasts for 24 h with SSC or LG3 in the presence of PP2 (AG1879), a biochemical inhibitor of Src family kinases (SFKs) and focal adhesion kinase, showed a significantly decreased anti-apoptotic response. However, focal adhesion kinase gene silencing with RNA interference did not inhibit the anti-apoptotic response in fibroblasts. Src phosphorylation was increased in fibroblasts exposed to SSC, and transfection of fibroblasts with constitutively active Src mutants induced an anti-apoptotic response that was not further increased by SSC. Also, Src-/- Fyn-/- fibroblasts failed to mount an anti-apoptotic response in presence of SSC for 24 h but developed a complete anti-apoptotic response when exposed to SSC for 7 days. These results suggest that extracellular matrix fragments produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an α2α1 integrin/SFK (Src and Fyn)/phosphatidylinositol 3-kinase (PI3K)-dependent pathway. In the long term, additional SFK members are recruited for sustaining the anti-apoptotic response, which could play crucial roles in abnormal fibrogenic healing. [ABSTRACT FROM AUTHOR]

The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH2-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH2-terminal deletion mutant of Dok-4 (Dok-4-(ΔN11–29)), which lacks the mitechondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4-(ΔN1–99), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4-mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor-α (TNT-α)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4-Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF-α-mediated NF-κB activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-KB activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF-α-mediated ROS production and NF-κB activation. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC)-induced phosphorylation of cardiac troponin I (cTnI) depresses the acto-myosin interaction and may be important during the progression of heart failure. Although both PKCβII and PKC∊ can phosphorylate cTnI, only PKCβ expression and activity are elevated in failing human myocardium during end-stage heart failure. Furthermore, although increased cTnI phosphorylation was observed in mice with cardiac-specific PKCβ II overexpression, no differences were observed in cTnI phosphorylation status between wild type and cardiac-specific PKC∊ overexpression mice. A potentially important downstream effector of PKCs is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth by activating several transcription factors as well as Na+/H+ exchanger. Since both Ser23 and Ser24 of cTnI are contained in putative consensus sequences of p90RSK phosphorylation sites, we hypothesized that p90RSK is downstream from PKCβ II and can be a cTnI (Ser23/24) kinase, p90RSK, but not ERK½ activation, was increased in PKCβII overexpression mice but not in PKC∊ overexpression mice. p90RSK could phosphorylate cTnI in vitro with high substrate affinity but not cardiac troponin T (cTnT). To confirm the role of p90RSK in cTnI phosphorylation in vivo, we generated adenovirus containing a dominant negative form of p90RSK (Ad-DN-p90RSK). We found that the inhibition of p90RSK prevented H2O2-mediated cTnI (Ser23/24) phosphorylation but not ERK½ and PKCα/βII activation. Next, we generated cardiac-specific p90RSK transgenic mice and observed that cTnI (Ser23/24) phosphorylation was significantly increased. LY333,531, a specific PKCβ inhibitor, inhibited both p90RSK and cTnI (Ser23/24) phosphorylation by H2O2. Taken together, our data support a new redox-sensitive mechanism regulating cTnI phosphorylation in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Endothelial cell (EC) migration contributes to reendothelialization after angioplasty or rupture of atherosclerotic plaques. Extracellular signal-regulated kinase (ERK)½ translocates to the nucleus and activates transcription factors such as Ets-like transcription factor-1 and early growth response factor-1 (Egr-1) during reendothelialization. Because ERK½ does not possess a nuclear localization signal (NLS), its mechanism of translocation and accumulation in the nucleus remains unclear. Because Gab1 has a putative NLS in its N-terminal region, and Gab1 associates with phosphorylated ERK½, we hypothesized that Gab1 participates in ERK½ and Egr-1 nuclear accumulation. Using regenerating EC as a model system, we found that endogenous growth factor receptor-bound protein 2-associated binder-1 (Gab1) translocates into the nucleus in migrating EC. Wild-type red fluorescent protein-tagged Gab1 could be observed in both nucleus and cytoplasm, whereas the putative NLS deletion mutant (ΔNLS-Gab1) specifically localized in the cytoplasm. In addition, reduction of Gab1 expression by antisense Gab1 oligos or overexpression of ΔNLS-Gab1 inhibited serum-induced ERK½ and Egr-1 nuclear accumulation, suggesting a functional role for the NLS of Gab1 and a role for Gab1ERK½ interactions in ERK½-Egr-1 nuclear accumulation. To investigate whether Gab1-ERK½ interaction is critical for ERK½ and Egr-1 nuclear accumulation, we created a dominant-negative Gab1 construct that consisted of the c-Met binding domain (amino acids 442536) of Gab1. We found that overexpression of the c-Met binding domain of Gab1 disrupted serum-induced Gab1ERK1 interaction and inhibited ERK1 and Egr-1 nuclear accumulation. These data suggest that Gab1-ERK½ binding and their nuclear translocation play a crucial role in Egr-1 nuclear accumulation. [ABSTRACT FROM AUTHOR]