GRK2 在细胞迁移中的调控作用

细胞迁移是免疫应答、损伤修复等正常生理功能的基础,也和癌细胞侵袭、转移等病理现象有关。G蛋白偶联受体激酶(GRKs)是一大类能够磷酸化G蛋白偶联受体(GPCRs)的激酶,共有7种亚型(GRK1~7),其中GRK2是体内分布最广、被研究最多的亚型。近年来的研究发现GRK2能够与多种蛋白底物相互作用,在细胞迁移活动中起到重要的调控作用。本篇综述将对GRK2调控细胞迁移的研究结果进行归纳梳理,为将来进一步的机制研究提供参考。     

Vascular endothelial growth factor--structure and functions

Vascular endothelial cell growth factor (VEGF), originally described as a vascular permeability factor, is currently known as one of the main factors which regulate angiogenesis. It plays an important role in the regulation of normal as well as pathological angiogenesis. In this paper we try to shortly review the actual knowledge on VEGF protein family, its expression, VEGF receptors and role of VEGF in signal transduction. The aim of this review is also to summarize recent achievements in research on biological functions of vascular endothelial growth factor and their clinical applications.     

Nitric oxide, a biological double-faced janus--is this good or bad?

Nitric oxide (NO) is a biological messenger molecule produced by one of the essential amino acids L-arginine by the catalytic action of the enzyme NO synthase (NOS). The dual role of NO as a protective or toxic molecule is due to several factors, such as; the isoform of NOS involved, concentration of NO and the type of cells in which it is synthesised, the availability of the substrate L-arginine, generation of guanosine 3,5'-cyclic monophosphate (cGMP) from soluble guanylate cyclase and the overall extra and intracellular environment in which NO is produced. NOS activation as a result of trauma (calcium influx) or infection leads to NO production, which activates its downstream receptor sGC to synthesise cGMP and/or leads to protein nitrosylation. This may lead to one or more systemic effects including altered neurotransmission which can be protective or toxic, vaso/bronchodilatation in the cardiovascular and respiratory systems and enhanced immune activity against invading pathogens. In addition to these major functions, NO plays important role in thermoregulation, renal function, gastrointestinal motility, endocrine function, and various functions of the urogenital system ranging from renin secretion to micturation; spermatogenesis to penile erection; and ovulation to implantation and parturition. A schematic summary of the functions of NO and the various isoforms of NOS expressed in body systems is shown in figure 1. In this review, the historical background, biochemistry and biosynthesis of NO and its enzymes together with the mechanism of NO actions in physiology and pathophysiology are discussed.     

Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells

Mitochondrial dysfunction and endoplasmic reticulum stress(ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide(NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca~(2+) flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstratedthe need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.     

EPHB4 Protein Expression in Vascular Smooth Muscle Cells Regulates Their Contractility,and EPHB4 Deletion Leads to Hypotension in Mice

EPH kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. This work presents evidence that EPHB4 on vascular smooth muscle cells (VSMCs) is involved in blood pressure regulation. We generated gene KO mice with smooth muscle cell-specific deletion of EPHB4. Male KO mice, but not female KO mice, were hypotensive. VSMCs from male KO mice showed reduced contractility when compared with their WT counterparts. Signaling both from EFNBs to EPHB4 (forward signaling) and from EPHB4 to EFNB2 (reverse signaling) modulated VSMC contractility. At the molecular level, the absence of EPHB4 in VSMCs resulted in compromised signaling from Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to myosin light chain kinase (MLCK) to myosin light chain, the last of which controls the contraction force of motor molecule myosin. Near the cell membrane, an adaptor protein GRIP1, which can associate with EFNB2, was found to be essential in mediating EPHB4-to-EFNB reverse signaling, which regulated VSMC contractility, based on siRNA gene knockdown studies. Our research indicates that EPHB4 plays an essential role in regulating small artery contractility and blood pressure.     

Nitric oxide in physiology and pathology

Summary Nitric oxide (NO) can exert a multitude of biological actions. NO, formed froml-arginine by a calcium-dependent enzyme (NO synthase) plays a key physiological role in regulating vascular tone and integrity. NO, formed by a constitutive neuronal isoform of NO synthase, likewise plays an important neuromodulator role. By contrast, high levels of NO can be generated following induction of a calcium-independent isoform of NO synthase. This excessive production of NO can provoke hypotension such as that observed in septic shock, and can exert cytotoxic actions leading to tissue injury and inflammation. Selective inhibitors of this inducible isoform thus have therapeutic potential in a number of disease states.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation

The formation of nitric oxide (NO) from L-arginine by vascular endothelial cells and its relationship to endothelium-dependent relaxation of vascular rings was studied. The release of NO, measured by bioassay or chemiluminescence, from porcine aortic endothelial cells stimulated with bradykinin was enhanced by infusions of L-, but not D-arginine. The release of 15NO, determined by high resolution mass spectrometry, from L-guanidino 15N (99%) arginine was also observed, indicating that NO is formed from the terminal guanidino nitrogen atom(s) of L-arginine. L-NG-monomethyl arginine (L-NMMA), but not D-NMMA, inhibited both the generation of NO by endothelial cells in culture and the endothelium-dependent relaxation of rabbit aortic rings. Both these effects were reversed by L-arginine. These data indicate that L-arginine is the physiological precursor for the formation of NO which mediates endothelium-dependent relaxation.     

Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for L-arginine is 3.14 microM with a Vmax of 0.14 micromol mg-1 min-1, whereas Ki values of 0.9 microM and 1.1 microM were determined for ADMA and L-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 microM level of L-arginine, MA levels of >10 microM were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 microm. Similar effects were observed in isolated vessels where 5 microm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and L-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. L-arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, approximately 4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.     

Localization and activity of iNOS in normal human lung tissue and lung cancer tissue

Inducible nitric oxide synthase (iNOS) is one of three enzymes generating nitric oxide (NO) from the amino acid L-arginine. iNOS-derived NO plays an important role in several physiological and pathophysiological conditions. NO is a free radical which produces many reactive intermediates that account for its bioactivity. In the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Moreover, high concentrations of NO have been shown to increase nuclear factor kappaB (NF-kB) activation. Recent investigations of NO expression in tumor tissue indicated that, at least for certain tumors, NO may mediate one or more roles during the growth of human cancer. We have studied iNOS in two tissue groups: normal human lung tissue and human lung cancer tissue. We localized iNOS in these tissues by immunohistochemistry and tested the mRNA expression by RT-PCR, the protein level by Western blot, and the protein activity by radiometric analysis. The results demonstrate different expression, localization and activity of iNOS in normal versus tumor tissue. This is suggestive of a role for NO production from iNOS in human lung cancer because high concentrations of this short molecule may transform to highly reactive compounds such as peroxynitrite (ONOO-); moreover, through the upregulator NF-kB, they can induce a chronic inflammatory state representing an elevated risk for cell transformation to cancer.     

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

Inflammation plays a critical role in promoting smooth muscle migration and proliferation during vascular diseases such as postangioplasty restenosis and atherosclerosis. Another common feature of many vascular diseases is the contribution of reactive oxygen (ROS) and reactive nitrogen (RNS) species to vascular injury. Primary sources of ROS and RNS in smooth muscle are several isoforms of NADPH oxidase (Nox) and the cytokine-regulated inducible nitric oxide (NO) synthase (iNOS). One important example of the interaction between NO and ROS is the reaction of NO with superoxide to yield peroxynitrite, which may contribute to the pathogenesis of hypertension. In this review, we discuss the literature that supports an alternate possibility: Nox-derived ROS modulate NO bioavailability by altering the expression of iNOS. We highlight data showing coexpression of iNOS and Nox in vascular smooth muscle demonstrating the functional consequences of iNOS and Nox during vascular injury. We describe the relevant literature demonstrating that the mitogen-activated protein kinases are important modulators of proinflammatory cytokine-dependent expression of iNOS. A central hypothesis discussed is that ROS-dependent regulation of the serine/threonine kinase protein kinase Cdelta is essential to understanding how Nox may regulate signaling pathways leading to iNOS expression. Overall, the integration of nonphagocytic NADPH oxidase with cytokine signaling in general and in vascular smooth muscle in particular is poorly understood and merits further investigation.     

The influence of nitric oxide on the regulation of plasminogen activator inhibitor type 1 and tissue-type plasminogen activator expression

Nitric oxide produced in various human tissues by nitric oxide synthase is involved in the regulation of many physiological processes. Mechanism of its action is diverse. The most important physiological activity of nitric oxide is guanylate cyclase activation and an increase of cGMP synthesis. At low concentrations NO plays a pivotal role in vessel relaxation and possesses antithrombotic, antiproliferative and anti-inflammatory features as well. An excessive production of nitric oxide can disturb vascular hemostasis and contribute to development of cardiovascular diseases. Studies provide that NO also participate in fibrynolysis regulation by the influence on the PAI-1 and t-PA expression, what may have important clinical implications. The aim of this review is to present current knowledge about the role of nitric oxide in the regulation of these plasminogen activation system factors.     

ERK5: Structure, regulation and function

Extracellular signal-regulated kinase 5 (ERK5), also termed big mitogen-activated protein kinase-1 (BMK1), is the most recently identified member of the mitogen-activated protein kinase (MAPK) family and consists of an amino-terminal kinase domain, with a relatively large carboxy-terminal of unique structure and function that makes it distinct from other MAPK members. It is ubiquitously expressed in numerous tissues and is activated by a variety of extracellular stimuli, such as cellular stresses and growth factors, to regulate processes such as cell proliferation and differentiation. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade plays a critical role in cardiovascular development and vascular integrity. Recent data points to a potential role in pathological conditions such as cancer and tumour angiogenesis. This review focuses on the physiological and pathological role of ERK5, the regulation of this kinase and the recent development of small molecule inhibitors of the ERK5 signalling cascade.     

Nitric oxide signalling functions in plant-pathogen interactions

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. During the last few years NO has been detected in several plant species, and the increasing number of reports on its function in plants have implicated NO as a key molecular signal that participates in the regulation of several physiological processes; in particular, it has a significant role in plant resistance to pathogens by triggering resistance-associated cell death and by contributing to the local and systemic induction of defence genes. NO stimulates signal transduction pathways through protein kinases, cytosolic Ca2+ mobilization and protein modification (i.e. nitrosylation and nitration). In this review we will examine the synthesis of NO, its effects, functions and signalling giving rise to the hypersensitive response and systemic acquired resistance during plant-pathogen interactions.     

Nitric oxide inhibits nociceptive transmission by differentially regulating glutamate and glycine release to spinal dorsal horn neurons

Nitric oxide (NO) is involved in many physiological functions, but its role in pain signaling remains uncertain. Surprisingly, little is known about how endogenous NO affects excitatory and inhibitory synaptic transmission at the spinal level. Here we determined how NO affects excitatory and inhibitory synaptic inputs to dorsal horn neurons using whole-cell recordings in rat spinal cord slices. The NO precursor L-arginine or the NO donor SNAP significantly increased the frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (IPSCs) of lamina II neurons. However, neither L-arginine nor SNAP had any effect on GABAergic IPSCs. L-arginine and SNAP significantly reduced the amplitude of monosynaptic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root with an increase in paired-pulse ratio. Inhibition of the soluble guanylyl cyclase abolished the effect of L-arginine on glycinergic IPSCs but not on evoked monosynaptic EPSCs. Also, inhibition of protein kinase G blocked the increase in glycinergic sIPSCs by the cGMP analog 8-bromo-cGMP. The inhibitory effects of L-arginine on evoked EPSCs and high voltage-activated Ca(2+) channels expressed in HEK293 cells and dorsal root ganglion neurons were abolished by blocking the S-nitrosylation reaction with N-ethylmaleimide. Intrathecal injection of L-arginine and SNAP significantly increased mechanical nociceptive thresholds. Our findings suggest that spinal endogenous NO enhances inhibitory glycinergic input to dorsal horn neurons through sGC-cGMP-protein kinase G. Furthermore, NO reduces glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated Ca(2+) channels. Both of these actions probably contribute to inhibition of nociceptive transmission by NO at the spinal level.