Genetic profiling：from bench to bedside
Choong-Chin Liew
The Cardiovascular Genome Unit, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA
From discovery of glycoproteins to identification of the receptors for Chlamydiae
Cho-Chou Kuo
Department of Pathobiology, University of Washington, Seattle, USA
The role of thrombomodulin in atherosclerosis
Yi-Heng Li
Department of Internal Medicine,National Cheng KungUniversity Hospital,Tainan, Taiwan
Thrombomodulin (TM) is a cell membrane-bound glycoprotein. Its main physiological function is to bind thrombin. Thrombin-TM complex catalyzes the activation of protein C which may proteolytically inactivates factor Va and VIIIa, thereby effectively inhibiting propagation of the coagulant response. The TM structure exhibits a N-terminal lectin-like domain, six homologous epidermal growth factor (EGF) repeats, a serine-threonine-rich region, a transmembrance domain and a short cytoplasmic tail. In addition to the presence on endothelium, TM was also found on smooth muscle cell, platelet, monocyteand some tumor cells. Although originally discovered as an anticoagulant protein, TM itself has received much attention in recent years because of its direct cellular effects. The first one is its directeffect on cellular proliferation. Previous studies demonstrated that TM expression level in tumor cells inversely correlates with tumor cell proliferation and invasion. However, the effect of TM on vascular smooth muscle cell proliferation is still controversial. Recombinant TM inhibits in vitro smooth muscle cell proliferation induced by thrombin. In contrast, recombinant soluble TM consisting only of the six EGF domains enhances proliferation of smooth muscle cells. TM has also anti-inflammatory effect. Gene-engineered mice lacking lectin-domain of TM have more leukocytes accumulation in the lung after lipopolysaccharide inhalation, increased mortality after endotoxin-induced sepsis and larger infarction size after coronary artery ligation.From the effects on thrombosis, cellular proliferation andinflammation, it seems that an important role of TM in the pathogenesis of atherosclerosis can be expected. We have searched for polymorphisms in the TM gene in large patient groups. The TM G-33A variants that we discovered in patients and normals are produced via site-directed mutagenesis and expressed in endothelial cell culture systems. We found that this genetic variant could reduce luciferase reporter gene expression and soluble TM level. The genetic variant constitutes a risk factor to coronary and carotid atherosclerosis. For further elucidating the role of TM in the pathogenesis of atherosclerosis, mouse atherosclerotic model produced by carotid artery ligation was established. In this model the common carotid artery was disrupted by ligating the vessel near the distal bifurcation. The ligated vessel does not contain too much thrombus, and arterial pulsation is present at all times. The cessation of blood flow causes carotid luminal narrowing by formation of an extensive smooth muscle cell-rich neointima and a reduction in vessel diameter. There was TM expression in the medial and neointimal smooth muscle cells in this mouse atherosclerosis model. These study results may elucidate the role of TM in the process of atherosclerosis and add our knowledge about the pathogenesis of atherosclerosis.
A Novel Family of Cell-Surface ProteinsExpressed in Human Vascular Endothelium
Ruey-Bing Yang
Institute of Biomedical Sciences, Academia Sinica, Taipei,Taiwan
We have identified a novel family of cell-surface proteins expressed in human vascular endothelium by a combination of high throughput sequencing and genome-wide microarray profiling analyses. The first member of this protein family was termed SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1. All family members share a similar domain structure composed of one amino-terminal signal peptide followed by nine EGF-like repeats and one CUB domain at the carboxyl terminus. When overexpressed in human embryonic kidney 293T cells, SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. Interestingly, the secreted SCUBE1 can be further proteolytically processed by a serum-associated protease to release the EGF-like repeats from the CUB domain. To further investigate its protein expression profile, we have raised antibodies specifically against the EGF-like or CUB domain of human SCUBE1, respectively. Immunohistochemical analysis using the anti-EGF-like domain-specific antibody confirmed the endothelial origin of SCUBE1 protein, whereas the anti-CUB domain-specific antibody revealed the immunostaining exclusively associated with arterial or venous thrombi in a variety of monkey tissue sections. Together, these data suggest that this novel cell-surface endothelial protein, SCUBE1, may be modulated by a proteolytic mechansim and may be involved in the thrombus formation in vivo.
The role of p38 MAPK in T cell activation and T cell positive selection
Chia-Cheng Wu‡, Shu-Ching Hsu‡, Hsiu-ming Shih†, Jiahuai Han¶,and Ming-Zong Lai‡
‡Institute of Molecular Biology, Academia Sinica, Taipei; †Division of Molecular & Genomic Medicine, National Health Research Institute, and ¶Department of Immunology, Scripps Research Institute, La Jolla, California, U.S.A.
p38 mitogen activated protein kinase (MAPK) is essential for T cell activation. Here we demonstrated that nuclear factor of activated T cell (NFAT) is a direct target of p38 MAPK. Inhibition of p38 MAPK led to selective inactivation of NFAT in T cells. We further linked a strict requirement of p38 MAPK to activation of NFATc. Stimulatory effect of p38 MAPK was found on at least four other stages of NFATc activation. First, the p38 MAPK cascade activated the NFATc promoter and induced transcription of NFATc mRNA. Second, p38 MAPK mildly increased the mRNA stability of NFATc. Third, p38 MAPK enhanced translation of NFATc mRNA. Fourth, p38 MAPK promoted the interaction of NFATc with coactivator CREB-binding protein (CBP). In contrast, p38 MAPK moderately enhanced expulsion of NFATc from nucleus in T cells. Therefore, p38 MAPK displays opposite effect on the different stages of NFATc activation. All together, the overall effect of p38 MAPK on NFATc in T cells is a clear activation. Positive selection of thymocytes during T cell development is mediated by T cell receptor (TCR)-activated signals. For different mitogen activated protein kinases (MAPKs) activated by TCR complex, a selective involvement of extracellular signal-regulated kinase (ERK) but not p38 MAPK in positive selection has been suggested. Using transgenic mice with dominant negative mutant of both MAP kinase kinase 3 (MMK3) and MKK6, we obtained mice with different extents of inhibition on p38 MAPK activation. Partial inhibition of p38 MAPK impaired CD4-CD8- thymocyte development and T cell proliferation, but not positive selection. Interference of thymocyte positive selection was observed in mice with effective suppression of p38 MAPK. Our resultssuggest that, in addition to early thymocyte development, p38 is involved in positive selection.
Salicylate suppresses COX-2 transcription by targeting P90 ribosomal S-6 kinase
Kenneth K. Wu
Vascular Biology Research Center, Institute of Molecular Medicine and Division of Hematology, University of Texas at Houston, Houston, Texas, USA
We have shown that aspirin and sodium salicylate equipotently suppress cyclooxygense-2 (COX-2) and nitric oxide synthase –2 (NOS-2) in expressions human and murine cells by blocking binding of CCAAT/enhancer binding protein (C/EBP) to a C/EBP response element. C/EBP contains intramolecular inhibitory elements that hinder its DNA binding site. Phosphorylation of C/EBP at Thr-235 by ERK1/2, Thr-266 by RSK (p90 ribosomal S-6 kinase), Ser-288 hy PKA (protein kinase A), or Ser-325 hy CaMKIV (calmodulin-dependent kinase IV) has been shown to increase its DNA binding activity in vitro. We tested the hypothesis that phorbal 12-myristate 13-acetate (PMA) induces C/EBP binding to COX-2 promoter by phosphorylating C/EBP via a kinase signaling pathway and aspirin/salicylate inhibits the kinase activity. We constructed FLAG-tagged wild type (WT) C/EBP and mutants T235A, T266A, S288A and S325A in pCMV-Tag 2B and transfected in human foreskin fibroblasts with these vectors. Binding of nuclear extract proteins from these transfected cells to a COX-2 promoter probe was analyzed by electrophoretic mobility shift assay. T266A had a significant reduction in binding activity whereas other mutants had a normal activity when compared to WT. These results suggest that phosphorylation of Thr-266 hy RSK is required for PMA-induced C/EBP binding. To confirm the requirement of RSK for PMA-induced COX-2 promoter activity, we transfected cells with WT RSK-2, a RSK-2 dominant negative (DN) mutant or constitutively active (CA) mutant and determined COX-2 promoter activity in response to PMA stimulation. PMA-induced COX-2 promoter activity was abrogated by DN mutant and enhanced by CA mutant. We next transfected cells with HA-RSK-2 and treated the transfected cells with PMA with or without salicylate. RSK was immunoprecipated with a HA antibody and RSK activity was assayed using a synthetic peptide as a substrate. RSK activity was increased by PMA which was abrogated by salicylate. These results indicate that PMA induces C/EBP binding via the RSK pathway and aspirin/salicylate inhibit C/EBP binding by blocking RSK activity.
Interleukin-1-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKs, and NF-B pathways in human tracheal smooth muscle cells
Chuen-Mao Yang, Chih-Chung Lin,Chin-Sung Chien, and Li-Der Hsiao
Department of Pharmacology,College of Medicine, Chang Gung University, Tao-Yuan, Taiwan
Interleukin-1 (IL-1) was exerted as a potent stimulus for synthesis of prostaglandin (PG) implicated in inflammatory responses of the airways. However, the mechanisms underlying IL-1 action on cyclooxygenase (COX) expression and PGE2synthesis in human tracheal smooth muscle cells (HTSMCs) were not completely understood.IL-1 markedly increased COX-2 expression and PGE2 formationin a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE2 formationin response to IL-1 were attenuated by a tyrosine kinase inhibitor genistein, a phosphatidyl choline-phospholipase C inhibitor D609, a phosphatidyl inositol-phospholipase C inhibitor U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca2+ by addition of BAPTA/AM plus EGTA, and phosphatidyl inositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitorsof p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by the results that IL-1 induced a transient activation of p42/p44 and p38 MAPKs. Furthermore, IL-1-induced activation of NF-B reversely correlated with the degradation of IB-. IL-1-induced COX-2 expression and PGE2 synthesis mediated through NF-B activationwas inhibited by NF-B inhibitor pyrrolidinedithiocarbamate (PDTC), but not by PD98059 and SB203580, suggesting that activation of p42/p44 and p38 MAPKs and NF-B signaling pathways were independently required for these responses. To further identify the upstream components of NF-B cascade, IL-1-inducedNF-B translocation, COX-2 expression and PGE2 synthesis were inhibited by transfection with dominant negative mutants of NIK and IKK-, but not with IKK-. These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from IL-1-challenged TSMCs, at least in part, independently mediated through p42/p44 and p38 MAPKs and NF-B signaling pathways in canine TSMCs. IL-1-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in these cells.
c-Jun C-terminus but not N-terminal phosphorylation is required for EGF-induced expression of cyclooxygenase-2 in human epidermoid carcinoma A431 cells
Lei-Chin Chen, Ben-Kuen Chen, and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high level prostaglandin production during inflammation and carcinogenesis.In this study, the transcriptional mechanism of COX-2 expression induced by epidermal growth factor (EGF) in human epidermoid carcinoma A431 cells was studied. Transient transfection with a series of 5’-deletion constructs and site-directed mutagenesis mutants showed that the CREelement on COX-2 promoter region ranging from -57 to -53 bp played an important role in the EGF response. Analysis of the transcription factors binding to CRE element by gel mobility shift assay, DNA affinity precipitation assay and chromatin immunoprecipitation assay revealed that c-Jun binding to CRE site was responsible for the EGF-induced COX-2 gene transcription. SP600125, a pharmacological inhibitor of JNK, efficiently inhibited JNK kinase activity and c-Jun N-terminal Ser-63 and Ser-73 phosphorylation levels, but could not inhibit COX-2 expression induced by EGF. Overexpression of c-Jun N-terminal Ser-63 and Ser-73 mutant (c-JunS63/73A) had the same stimulatory effects on COX-2 promoter activity and protein expression as wild type c-Jun. TAM-67, a mutant of c-Jun which lacking N-terminal transactivation domain of c-Jun could also enhance COX-2 promoter activity in cells treated with EGF.Furthermore, DNA affinity precipitation assay revealed that c-Jun binding to CRE element on COX-2 promoter region could be enhanced by EGF treatment. Mutation of c-Jun at Thr-231, Ser-243, and Ser-249 to alanine could significantly increase the COX-2 promoter activity as that of wild type c-Jun. c-Fos, that can form heterodimer with c-Jun, also could bind to CRE element on COX-2 promoter. Overexpression of c-Fos could increase COX-2 promoter activity which effect could be enhanced by co-expression of wild type c-Jun or c-JunS63/73A mutant. Fusion of c-Fos C-terminal activation domain to GAL4 DNA binding domain could activate pXC44-G, on which CRE/E-box element on COX-2 promoter was replaced by GAL4 binding element. These results indicated c-Jun N-terminal Ser-63 and Ser-73 phosphorylation was not required for EGF-induced expression of COX-2. Regulation of c-Jun C-terminus domain and activation of c-Fos by EGF were required at least in part for EGF-induced expression of COX-2 in A431 cells.
Regulation of ICAM-1 transcription by c-Src and PI3K/Akt
Wei-Chien Huang and Ching-Chow Chen*
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Our previous studies have shown the involvement of PKC/tyrosine kinase in the TNF-α-induced ICAM-1 expression in alveolar epithelial cells, we further identified the tyrosine kinase and examined its role in this regulation. In A549 cells, TNF-α- or TPA-induced IkB kinase (IKK) activation was blocked by a protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src-specific kinase inhibitor (PP2). Co-immunoprecipitation and immunoblot experiments showed that IKKβ was associated with c-Src and phosphorylated on tyrosine residues after TNF-α or TPA treatment. Tyr188 and Tyr199, near the activation loop of IKKβ, were identified as being important for NF-κB activation. Substitution of these residues with phenylalanines abolished ICAM-1 promoter activity and c-Src-dependent phosphorylation of IKKβ. c-Src-induced NF-κB promoter activity was attenuated by IKKβ tyrosine (FF) mutant but not serine (AA) mutant. These data suggest that, in addition to activating NIK, TNF-α also activates PKC-dependent c-Src. These two pathways converge at IKKβ and go on to activate NF-κB, via serine phosphorylation and degradation of IκB-α, and, finally, to initiate ICAM-1 expression.
The PI3K/Akt pathway has been reported to play a critical role in the regulation of gene expression induced by numerous stimuli. We show that the TNF-α-induced PI3K/Akt activation also participates in the regulation of ICAM-1 expression. Inhibition of PI3K/Akt pathway by Ly294002 (a PI3-K inhibitor) or kinase-dead Akt attenuates TNF-α-induced ICAM-1 expression and promoter activity without affecting IB degradation and NF-κB activation. TNF-α activates Akt and increases its nuclear localization. Nuclear Akt associates with transcriptional co-activator p300 and phosphorylates its Ser-1834. Mutation of Ser-1834to alanine inhibited the TNF-α-induced ICAM-1 promoter activity and decreased the recruitment of p300 to the ICAM-1 promoter. The phosphorylation of p300 at Ser-1834 increasedits intrinsic histone acetyltransferase (HAT) activity. Furthermore, GST pull-down assay showed that interaction of p300 with C/EBPβ and P/CAFrequired Akt phosphorylation of its Ser-1834.These results demonstrated thatAkt phosphorylation of p300 at Ser-1834 has two effects. One isenhancing its recruitment to the ICAM-1 promoter and the other is increasing its HAT activity to promote the ICAM-1 gene expression.
Lysophospholipids Enhance IL-8 and MCP-1 Expression in Human Endothelial Cells
Hsinyu Lee, Chi Iou Lin, Yu Wei Lee, Jia Jun Liao, Wei Feng Yen and Jiun Hong Chen
Department of Life Science and Institute of Zoology, National Taiwan University, Taipei, Taiwan
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low molecular weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors. Both ligands activate multiple signaling pathways in a variety of cell types. In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. IL-8 is a cysteine-x-cysteine (C-X-C) chemokine and acts as a chemoattractant of neutrophils, whereas MCP-1 is a cysteine-cysteine (C-C) chemokine and functions mainly as achemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. In the present study, we investigated the effects of LPA and S1P on IL-8 and MCP-1 synthesis in endothelial cells. Using RT-PCR and ELISA, we have demonstrated that LPA and S1Penhanced IL-8 and MCP-1 RNA expression and subsequent protein expression in human umbilical cord vein endothelial cells (HUVEC). The effects of LPA and S1P on IL-8 and MCP-1 expression appear to be time and concentration dependent. Further elucidations of the mechanisms involving the action of LPL’s on endothelial cells were investigated by prior treatment of cells with 15 ng/ml of pertussis toxin, a specific inhibitor of Gi and 1g/ml of Clostridium botulinum C3 exoenzyme, an inhibitor of rho, showed suppression of the enhancement effects elicited by LPA and S1P on IL-8 and MCP-1 expressions in HUVEC. This suggests that LPLs released by activated platelets might chemmoattract leukocytes to the endothelium through a Gi-dependent and rho-dependent mechanism, thus facilitating the wound healing and inflammation process. These results might enable us to elucidate the mechanisms of LPLs in modulating wound healing, inflammation and possibly atherosclerosis.