BBA - Molecular Cell Research (v.1783, #9)

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE2 release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-δ, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-κB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-κB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-κB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(δ)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-κB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-κB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE2 release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.
Keywords: Thrombin; EGF receptor; ERK1/2; COX-2; Vascular smooth muscle cell;

Nitrotyrosine promotes human aortic smooth muscle cell migration through oxidative stress and ERK1/2 activation by Hong Mu; Xinwen Wang; Peter Lin; Qizhi Yao; Changyi Chen (1576-1584).
Nitrotyrosine is a new biomarker of atherosclerosis and inflammation. The objective of this study was to determine the direct effects of free nitrotyrosine on human aortic smooth muscle cell (AoSMC) migration and molecular mechanisms. By a modified Boyden chamber assay, nitrotyrosine significantly increased AoSMC migration in a concentration-dependent manner. For example, nitrotyrosine at 300 nM increased AoSMC migration up to 152% compared with l-tyrosine-treated control cells (P  < 0.01). Cell wound healing assay confirmed this effect. Nitrotyrosine significantly increased the expression of some key cell migration-related molecules including PDGF receptor-B, matrix metalloproteinase 2 (MMP2) and integrins αV and β3 at both mRNA and protein levels in AoSMC (P  < 0.01). In addition, nitrotyrosine increased reactive oxygen species (ROS) production in AoSMC by staining with fluorescent dye DCFHDA. Furthermore, nitrotyrosine induced transient phosphorylation of ERK2 by Bio-Plex luminex immunoassay and western blot analysis. AoSMC were able to uptake nitrotyrosine. Antioxidants including seleno-l-methionine and superoxide dismutase mimetic (MnTBAP) as well as ERK1/2 inhibitor PD98059 effectively blocked the promoting effect of nitrotyrosine on AoSMC migration and the mRNA expression of above cell migration-related molecules. Thus, nitrotyrosine directly increases AoSMC migration in vitro and the expression of migration-related molecules through overproduction of ROS and activation of ERK1/2 pathway. Nitrotyrosine may contribute to cardiovascular pathogenesis.
Keywords: Nitrotyrosine; Smooth muscle cell migration; Oxidative stress; Antioxidant; Atherosclerosis;

Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and ΔF508 CFTR by Tsukasa Okiyoneda; Akiko Niibori; Kazutsune Harada; Taijun Kohno; Marek Michalak; Marek Duszyk; Ikuo Wada; Masahito Ikawa; Tsuyoshi Shuto; Mary Ann Suico; Hirofumi Kai (1585-1594).
Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl channel at the plasma membrane of epithelium. The most common mutant, ΔF508 CFTR, has competent Cl channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of ΔF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl channel function of ΔF508 CFTR. Importantly, cAMP-induced Cl current in colonic epithelium from CNX KO/ΔF508 CFTR mice was comparable with that of ΔF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of ΔF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not ΔF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not ΔF508 CFTR, which has folding defects.
Keywords: Calnexin; Calnexin knockout; CFTR; ER quality control;

Crosstalk between Nap1 protein and Cds1 checkpoint kinase to maintain chromatin integrity by Maribel Grande; Eva Lambea; Alba Fajardo; Sandra López-Avilés; Douglas Kellogg; Rosa Aligue (1595-1604).
The nucleosome assembly protein Nap1 has been implicated in various cellular functions such as histone shuttling into the nucleus, nucleosome assembly, chromatin remodelling, transcriptional control and cell-cycle regulation in Saccharomyces cerevisiae. In Schizosaccharomyces pombe nap1 null mutant cells are viable but they showed a delay in the onset of mitosis which is rescued by the absence of the replication Cds1 checkpoint kinase. In contrast, the absence of the DNA-damage Chk1 checkpoint kinase is unable to rescue the delay. Moreover, the double nap1 cds1 mutant cells lose viability and cells show positive H2AX phosphorylation, suggesting that the viability of nap1-deleted cells is due to the Cds1 kinase. We also show that overexpression of Nap1 protein blocks the cell cycle in G1 phase.
Keywords: Cell cycle; Checkpoint; Chromatin; Nap1; Fission yeast;

Transforming growth factor beta (TGF-β1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-α-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-β1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-β1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-β1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-β induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-β. TIMP-3 promoter deletion analysis revealed that − 828 deletion retains the original promoter activity while − 333 and − 167 deletions display somewhat reduced activity suggesting that most of the TGF-β-responsive, cis-acting elements are found in the − 333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the − 940 and − 333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-β-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.
Keywords: Articular chondrocyte; Transforming growth factor beta; Tissue inhibitor of metalloproteinases-3; Smad; RNA interference; Promoter analysis;

A p38–p65 transcription complex induced by endothelin-1 mediates signal transduction in cancer cells by Melanie Gerstung von Brandenstein; Anna Ngum Abety; Reinhard Depping; Tanja Roth; Matthias Koehler; Hans-Peter Dienes; Jochen W.U. Fries (1613-1622).
Endothelin-1 is a powerful mitogen for various tumor and non-tumor cells. Its signaling cascade induces the inflammatory NF-κB complex, leading to expression of a number of target genes. In this context, MAPK p38 has been regarded as a potential phosphate donor for the p65 subunit of NF-κB. In the present study in HeLa cells, we have found that ET-1 induced signalling activates the NF-κB transcription complex (TC) in the nucleus at 6 h specifically via ET-A — but not ET-B receptor. The TC contains p65, p38 (α and β) – binding to the NLS of p65 in the cytoplasm – as well as p50, but no IκBα. Specific p38 inhibition by SB203580 or by siRNA interferes markedly with gene expression of several target genes. Complex formation occurs in the cytoplasm, and both transcription factors transmigrate as a complex in the nucleus. Overexpression of p38, treatment with Chrysin, MG132, or dimethylformamide shows dependence of TC on p38 as partner. In other tumor cells lines studied, ET-1 activates TC, with p38 as an important complex partner of p65. TC-induction by ET-1 contains about twice the amount of p38 than by TNFα. Thus, p38 may be an additional therapeutic target to control inflammatory gene expression in tumor cells.
Keywords: Endothelin-1; MAPK p38; NF-κB; MG 132; Chrysin; Immunoprecipitation;

The tumor necrosis factor-alpha (TNF)-induced inflammatory response in human lung microvascular endothelial cells (MVECs) is an early event in acute lung injury. Studies have shown that p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase 2 (MK2) and heat shock protein 27 (HSP27) are involved in the expression of pro-inflammatory mediators in other cell types. However, their role in the TNF-induced inflammatory response in lung MVECs has not been determined. We evaluated the role of p38 MAPK, MK2 and HSP27 in regulating the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs. Inhibition of p38 MAPK reduced ICAM-1 and IL-8 expression without influencing NF-κB activation or ICAM-1 and IL-8 mRNA levels. TNF stimulation induced p38 MAPK-dependent phosphorylation of MK2 and HSP27. MK2 silencing reduced ICAM-1 and IL-8 expression without influencing NF-κB activation or ICAM-1 and IL-8 mRNA levels. HSP27 silencing reduced cellular HSP27 levels and HSP27 phosphorylation following TNF stimulation but had no effect on ICAM-1 and IL-8 expression. Our study demonstrates for the first time that MK2 mediates post-transcriptional regulation by p38 MAPK of the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs, and that this regulation by the p38 MAPK/MK2 pathway is dissociated from HSP27 phosphorylation.
Keywords: Human lung microvascular endothelial cells; MK2; HSP27; siRNA; ICAM-1; IL-8;

Regulation of TM4SF5-mediated tumorigenesis through induction of cell detachment and death by tiarellic acid by Suyong Choi; Sei-Ryang Oh; Sin-Ae Lee; Sung-Yul Lee; Kyungseop Ahn; Hyeong-Kyu Lee; Jung Weon Lee (1632-1641).
mRNA for four-transmembrane L6 family member 5 (TM4SF5), a homolog of tumor antigen L6, was previously shown to be highly expressed in diverse tumors. We recently found that human hepatocarcinoma tissues also overexpressed TM4SF5 protein, in comparison to normal liver tissues. We also found that tiarellic acid (TA) caused cell detachment-related apoptosis in cells expressing endogenous or stably-overexpressing TM4SF5. When cells expressing TM4SF5 were treated with TA, we observed reduced phosphorylation of focal adhesion kinase, paxillin, and p130Cas, but not c-Src. TA treatment also caused focal adhesion loss and reduced cell adhesion, and increased the numbers of floating cells and apoptotic cells. These effects were blocked by overexpression of focal adhesion molecules, suggesting that treatment with TA mediates anoikis of TM4SF5-expressing cells. However, TM4SF5-null cells were not affected by TA, indicating that these effects occur specifically in TM4SF5-positive cells. TA administration reduced tumor formation in nude mice injected with TM4SF5-expressing cells, presumably through increased apoptosis in TM4SF5-positive tumors. These observations indicate that TM4SF5-positive tumorigenesis can be inhibited by TA via induction of cell detachment-related apoptosis, and suggest that TA may be developed as a putative therapeutic reagent against TM4SF5-positive tumorigenesis.
Keywords: TM4SF5; Apoptosis; Focal adhesion; Tiarellic acid; Tumorigenesis;

Lamin B1, a major component of the nuclear lamina, anchors the nucleus to the cytoskeletal cage, and controls nuclear orientation, chromosome positioning and, alongside several enzymes, fundamental nuclear functions. Exposing polyomavirus-transformed rat pyF111 fibroblasts and human cervical carcinoma (HCC) C4-I cells for 30 min to photoexcited perylenequinone calphostin C, i.e. Cal CφE, an established reactive oxygen species (ROS)-generator and protein kinase C (PKC) inhibitor, caused the cells to selectively oxidize and then totally destroy their nuclear lamin B1 by only 60 min after starting the treatment, i.e. when apoptotic caspases' activities had not yet increased. However, while the oxidized lamin B1 was being destroyed, lamins A/C, the lamin A-associated nuclear envelope protein emerin, and the nucleoplasmic protein cyclin E were neither oxidized nor destroyed. The oxidized lamin B was ubiquitinated and demolished in the proteasome probably by an enhanced peptidyl-glutaminase-like activity. Hence, the Cal CφE-induced rapid and selective lamin B1 oxidation and proteasomal destruction ahead of the activation of apoptotic caspases was by itself a most severe molecular lesion impairing vital nuclear functions. Conversely, Cal C directly added to the cells kept in the dark damaged neither nuclear lamin B1 nor cell viability. Thus, our findings reveal a novel cell-damaging mechanism of a photodynamic tumor therapeutic agent.
Keywords: Calphostin C; H cervical carcinoma C4-I cell; Rat pyF111 fibroblast; Lamin; Ubiquitin proteasome system;

Interferon regulatory factor-1 (IRF-1) regulates VEGF-induced angiogenesis in HUVECs by Jeong Heon Lee; Taehoon Chun; Sang-Yoon Park; Seung Bae Rho (1654-1662).
Interferon regulatory factor-1 (IRF-1) is a tumor suppressor and transcriptional modulator that can regulate gene expression involved in cell growth control, induction of apoptosis, and post-translation modification. In this study, we found that IRF-1 inhibits endothelial cell angiogenesis using human umbilical vein endothelial cell (HUVECs) culture system. In addition, IRF-1 directly inhibited the tube formation of endothelial cells on Matrigel and reduced the expression of p-Akt, and p-eNOS, which play a significant role in angiogenesis when stimulated by VEGF. We also demonstrate that C-terminal region including transactivation domain (TA) of IRF-1 functions as a signal for its angiostatic activity, and is spliced in human tumor tissues. These findings indicate that splicing variant involving exons 7 of IRF-1 could potentially modulate anti-angiogenic effect of IRF-1. In overall, this study provides the first evidence for anti-angiogenic role of IRF-1, which may have therapeutic values for cancer and angiogenesis-associated diseases.
Keywords: Interferon regulatory factor; Angiogenesis; Alternative splicing; Endothelial cell; Vessel sprouting; CAM;

Cell surface targeting of VPAC1 receptors: Evidence for implication of a quality control system and the proteasome by Ingrid Langer; Karelle Leroy; Nathalie Gaspard; Jean-Pierre Brion; Patrick Robberecht (1663-1672).
Like for most transmembrane proteins, translation of G protein-coupled receptors (GPCRs) mRNA takes place at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. The molecular mechanisms involved in the transport process of GPCRs from ER to the plasma membrane are poorly investigated. Here we studied the mechanisms involved in glycosylation-dependent cell surface expression and quality control of the receptor for Vasoactive Intestinal Polypeptide (VIP) VPAC1, a member of the B family of GPCRs. Using biochemical and pharmacological techniques and fluorescence microscopy, we have shown that only a fraction of newly synthesized VPAC1 attains properly conformation that allows their cell surface targeting. Misfolded or immature VPAC1 are taken in charge by co- and post-translational quality control that involves: 1) calnexin-dependent folding strictly through a glycan-dependent mechanism, 2) BiP-dependant folding, 3) translocation to the cytoplasm and proteasome-dependent degradation of improper proteins, and 4) post-ER quality control check points. Our data suggest that VPAC1 expression/trafficking pathways are under the control of complex and precise molecular mechanisms to ensure that only proper VPAC1 reaches the cell surface.
Keywords: Cell surface expression; Glycosylation; GPCR; Proteasome; Trafficking; VIP receptor; VPAC1;