Endothelial activation: intracellular signaling pathways

Tumor necrosis factor (TNF) is the prototypic proinflammatory cytokine and endothelial cells are the principal cellular targets of its actions. Here I review the responses of endothelial cells to TNF, with emphasis on the induction of endothelial leukocyte adhesion molecules. I focus on the biochemistry and cell biology of signal transduction in TNF-treated endothelial cells that lead to the expression of adhesion molecules.     

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-κB activity with and without lipopolysacharide (LPS) stimulation.     

Neuroprotective effects and intracellular signaling pathways of erythropoietin in a rat model of multiple sclerosis

In multiple sclerosis (MS), long-term disability is primarily caused by axonal and neuronal damage. We demonstrated in a previous study that neuronal apoptosis occurs early during experimental autoimmune encephalomyelitis, a common animal model of MS. In the present study, we show that, in rats suffering from myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis, systemic application of erythropoietin (Epo) significantly increased survival and function of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. We identified three independent intracellular signaling pathways involved in Epo-induced neuroprotection in vivo: Protein levels of phospho-Akt, phospho-MAPK 1 and 2, and Bcl-2 were increased under Epo application. Using a combined treatment of Epo together with a selective inhibitor of phosphatidylinositol 3-kinase (PI3-K) prevented upregulation of phospho-Akt and consecutive RGC rescue. We conclude that in MOG-EAE the PI3-K/Akt pathway has an important influence on RGC survival under systemic treatment with Epo.     

Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways

CCR5 is a CC chemokine receptor expressed on memory lymphocytes, macrophages, and dendritic cells and also constitutes the main coreceptor for macrophage-tropic (or R5) strains of human immunodeficiency viruses. In the present study, we investigated whether CCR5 was palmitoylated in its carboxyl-terminal domain by generating alanine substitution mutants for the three cysteine residues present in this region, individually or in combination. We found that wild-type CCR5 was palmitoylated, but a mutant lacking all three Cys residues was not. Through the use of green fluorescent fusion proteins and immunofluorescence studies, we found that the absence of receptor palmitoylation resulted in sequestration of CCR5 in intracellular biosynthetic compartments. By using the fluorescence recovery after photobleaching technique, we showed that the non-palmitoylated mutant had impaired diffusion properties within the endoplasmic reticulum. We next studied the ability of the mutants to bind and signal in response to chemokines. Chemokines binding and activation of G(i)-mediated signaling pathways, such as calcium mobilization and inhibition of adenylate cyclase, were not affected. However, the duration of the functional response, as measured by a microphysiometer, and the ability to increase [(35)S]guanosine 5'-3-O-(thio)triphosphate binding to membranes were severely affected for the non-palmitoylated mutant. The ability of RANTES (regulated on activation normal T cell expressed and secreted) and aminooxypentane-RANTES to promote CCR5 endocytosis was not altered by cysteine replacements. Finally, we found that the absence of receptor palmitoylation reduced the human immunodeficiency viruses coreceptor function of CCR5, but this effect was secondary to the reduction in surface expression. In conclusion, we found that palmitoylated cysteines play an important role in the intracellular trafficking of CCR5 and are likely necessary for efficient coupling of the receptor to part of its repertoire of signaling cascades.     

Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways

Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-alpha (mERalpha). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E2-P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E2 and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E2-P induced a monophasic ERK activation. E2 also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10(-14) and 10(-8)M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca2+) involvement and timing of pathway activations; all affected ERK activation as early as 3-6 min, suggesting simultaneous, not sequential, activation. Therefore, E2 and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation.     

Huntington's disease: intracellular signaling pathways and neuronal death

Huntington's disease (HD) is a mid-life onset neurodegenerative disorder characterized by unvoluntary movements (chorea), personality changes and dementia that progress to death within 10-20 years of onset. There are currently no treatment to delay or prevent appearance of the symptoms in the patients. The defective gene in HD contains a trinucleotide CAG repeat expansion within its coding region that is expressed as a polyglutamine (polyQ) repeat in the protein huntingtin. The exact molecular mechanims by which mutant huntingtin induces cell death as well as the function of huntingtin are not totally understood. Studying mechanisms by which polyQ-huntingtin induces neurodegeneration has shown that phosphorylation plays a key role in HD. The IGF-1/Akt/SGK pathway reduces polyQ-huntingtin induced toxicity. This anti-apopototic effect is mediated via the phosphorylation of serine 421 of huntingtin. Moreover, components of this pathway are altered in disease. What is the function of huntingtin? Several studies indicate that huntingtin is an anti-apoptotic protein that could regulate intracellular dynamic. We recently demonstrated, that huntingtin specifically enhances vesicular transport of brain-derived neurotrophic factor (BDNF) along microtubules. Huntingtin-mediated transport involves Huntingtin-Associated Protein-1 (HAP1) and the p150(Glued) subunit of dynactin, an essential component of molecular motors. BDNF transport is attenuated both in the disease context and by reducing the levels of wild-type huntingtin. The alteration of the huntingtin/HAP1/ p150(Glued) complex correlates with reduced association of motor proteins with microtubules. Finally, polyQ-huntingtin-induced transport deficit results in the loss of neurotrophic support and neuronal toxicity.     

TDAG8介导的细胞内信号传送通路及其功能

T细胞死亡相关基因8编码的受体蛋白/G蛋白偶联受体65(TDAG8)是G蛋白偶联受体家族成员之一,起初它被鉴定为脂质分子(鞘氨醇半乳糖苷)的受体;后来的研究证明TDAG8也具有感知细胞外的质子或pH的功能。因此,TDAG8是一种特殊的双配体受体。TDAG8受体广泛表达在免疫组织等正常组织和肿瘤组织中,具有潜在的重要生理作用。本文对TDAG8介导的细胞内信号通路及TDAG8功能研究进展进行综述。     

Designing cell-permeant phosphopeptides to modulate intracellular signaling pathways

A central theme in intracellular signaling is the regulatable interaction of proteins via the binding of specialized domains on one protein to short linear sequences on other molecules. The capability of these short sequences to mediate the required specificity and affinity for signal transduction allows for the rational design of peptide-based modulators of specific protein-protein interactions. Such inhibitors are valuable tools for elucidating the role of these interactions in cellular physiology and in targeting such interactions for potential therapeutic intervention. This approach is exemplified by the study of the role of phosphorylation of specific sites on signaling proteins. However, the difficulty of introducing large hydrophilic molecules such as phosphopeptides into cells has been a major drawback in this area. This review describes the application of recently developed cell-permeant peptide vectors in the introduction of biologically active peptides into cells, with particular emphasis on the antennapedia/penetratin, TAT, and signal-peptide based sequences. In addition, the modification of such peptides to increase uptake efficiency and affinity for their targets is discussed. Finally, the use of cell-permeant phosphopeptides to both inhibit and stimulate intracellular signaling mechanisms is described, by reference to the PLCgamma, Grb2, and PI-3 kinase pathways.     

On the problem of identification of intracellular signaling pathways

A hypothesis is proposed about the involvement of mitochondria in transduction of intracellular signals. In addition to the endoplasmic reticulum responsible for fluctuations of intracellular concentration of Ca²⁺, mitochondria are supposed to determine changes in the concentration of the secondary messenger cAMP. This hypothesis extends our ideas concerning mechanisms of intracellular signal transduction.     

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.     

Fatty acylated proteins as components of intracellular signaling pathways

From the studies presented above, it is obvious that fatty acylation is a common modification among proteins involved in cellular regulatory pathways, and in certain cases mutational analyses have demonstrated the importance of covalent fatty acids in the functioning of these proteins. Indeed, certain properties provided by fatty acylation make it an attractive modification for regulatory proteins that might interact with many different substrates, particularly those found at or near the plasma membrane/cytosol interface. In the case of intracellular fatty acylated proteins, the fatty acyl moiety allows tight binding to the plasma membrane without the need for cotranslational insertion through the bilayer. For example, consider the tight, salt-resistant interaction of myristoylated SRC with the membrane, whereas its nonmyristoylated counterpart is completely soluble. Likewise for the RAS proteins, which associate weakly with the membrane in the absence of fatty acylation, while palmitoylation increases their affinity for the plasma membrane and their biological activity. Fatty acylation also permits reversible membrane association in some cases, particularly for several myristoylated proteins, thus conferring plasticity on their interactions with various signaling pathway components. Finally, although this has not been demonstrated, it is conceivable that covalent fatty acid may allow for rapid mobility of proteins within the membrane. Several questions remain to be answered concerning requirements for fatty acylation by regulatory proteins. The identity of the putative SRC "receptor" will provide important clues as to the pathways in which normal SRC functions, as well as into the process of transformation by oncogenic tyrosine kinases. The possibility that other fatty acylated proteins associate with the plasma membrane in an analogous manner also needs to be investigated. An intriguing observation that can be made from the information presented here is that at least three different families of proteins involved in growth factor signaling pathways encode both acylated and nonacylated members, suggesting that selective fatty acylation may provide a means of determining the specificity of their interactions with other regulatory molecules. Further studies of fatty acylated proteins should yield important information concerning the regulation of intracellular signaling pathways utilized during growth and differentiation.     

DNA damage-induced activation of ATM and ATM-dependent signaling pathways

Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to ionizing radiation. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage response pathways, most notably cell cycle checkpoints. In this review, we describe recent developments in our understanding of the mechanism of activation of ATM and its downstream signaling pathways, and explore whether DNA double-strand breaks are the sole activators of ATM and ATM-dependent signaling pathways.     

Lysophospholipid receptor activation of RhoA and lipid signaling pathways

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) signal through G-protein coupled receptors (GPCRs) which couple to multiple G-proteins and their effectors. These GPCRs are quite efficacious in coupling to the Gα_12/13 family of G-proteins, which stimulate guanine nucleotide exchange factors (GEFs) for RhoA. Activated RhoA subsequently regulates downstream enzymes that transduce signals which affect the actin cytoskeleton, gene expression, cell proliferation and cell survival. Remarkably many of the enzymes regulated downstream of RhoA either use phospholipids as substrates (e.g. phospholipase D, phospholipase C-epsilon, PTEN, PI3 kinase) or are regulated by phospholipid products (e.g. protein kinase D, Akt). Thus lysophospholipids signal from outside of the cell and control phospholipid signaling processes within the cell that they target. Here we review evidence suggesting an integrative role for RhoA in responding to lysophospholipids upregulated in the pathophysiological environment, and in transducing this signal to cellular responses through effects on phospholipid regulatory or phospholipid regulated enzymes. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.