BBA - Molecular Cell Research (v.1763, #8)

FGF6 in myogenesis by Anne-Sophie Armand; Iman Laziz; Christophe Chanoine (773-778).
Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage. However, the analyses of Fgf6 (−/−) mutant mice gave contradictory results and the role of FGF6 during myogenesis remained largely unclear. Recent reports support the concept that FGF6 has a dual function in muscle regeneration, stimulating myoblast proliferation/migration and muscle differentiation/hypertrophy in a dose-dependent manner. The alternative use of distinct signaling pathways recruiting either FGFR1 or FGFR4 might explain the dual role of FGF6 in myogenesis. A role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle has been also strongly suggested. The aim of this review is to summarize our knowledge on the involvement of FGF6 in myogenesis.
Keywords: FGF6; Myogenesis; Growth factor; Muscle regeneration; Development;

Integrins play a key role in cellular immune responses in a variety of organisms; however, knowledge of integrins and their effects on cell signalling and functional responses in molluscan defence reactions is poor. Using integrin-mediated cell adhesion kits, αVβ3 and β1 integrin-like subunits were identified on the surface of Lymnaea stagnalis haemocytes. Haemocyte binding via these integrins was found to be dependent on Ca2+/Mg2+. Western blotting with an anti-phospho (anti-active) focal adhesion kinase (FAK) antibody revealed a 120–125 kDa FAK-like protein in these cells; this protein was transiently phosphorylated upon haemocyte adhesion over 90 min, with maximal phosphorylation occurring after 30 min binding. Also, integrin engagement with the tetrapeptide Arg–Gly–Asp–Ser (RGDS) resulted in a rapid increase in phosphorylation of the FAK-like protein; however, RGDS did not affect the phosphorylation of extracellular signal-regulated kinase. Treatment of haemocytes with RGDS (2 mM) inhibited phagocytosis of E. coli bioparticles by 88%. Moreover, at this concentration, RGDS reduced cell spreading by 61%; stress fiber formation was also impaired. Taken together, these results demonstrate a role for integrins in L. stagnalis haemocyte adhesion and defence reactions and, for the first time, link integrin engagement to FAK activation in molluscs.
Keywords: Mollusc; Invertebrate defence; Haemocytes; FAK; ERK; RGDS;

p14ARF inhibits the growth of p53 deficient cells in a cell-specific manner by Yanxia Li; Lizhi He; Anthony Bruce; Kavita Parihar; Alistair Ingram; Lieqi Liu; Damu Tang (787-796).
While p14ARF suppression of tumorigenesis in a p53-dependent manner is well studied, the mechanism by which p14ARF inhibits tumorigenesis independently of p53 remains elusive. A variety of factors have been reported to play a role in this latter process. We report here that p14ARF displays different effects on the anchorage-dependent and -independent growth of p53-null/Mdm2 wild type cells. p14ARF blocks both the anchorage-dependent and-independent (soft agar) proliferation of 293T and p53−/− HCT116, but not p53-null H1299 lung carcinoma cells. While p14ARF had no effect on the anchorage-dependent proliferation of p53−/− MEFs and Ras12V-transformed p53−/− MEFs, it inhibited the growth of Ras12V-transformed p53−/− MEFs in soft agar. Furthermore, ectopic expression of p14ARF did not lead to degradation of the E2F1 protein and did not result in the reduction of E2F1 activity detected by two E2F1 responsible promoters, Apaf1 and p14ARF promoter, in 293T, p53−/− HCT116, and H1299 cells. This is consistent with our observations that p14ARF did not result in G1 arrest, but induced apoptosis via Bax up-regulation. Taken together, our data demonstrate that the response of p53-null cells to ARF is cell type dependent and involves factors other than Mdm2 and E2F1.
Keywords: p14ARF; p53; Cell cycle; Tumor suppression;

Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor β in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal by Satoshi Akiba; Satomi Kumazawa; Hidenori Yamaguchi; Naoya Hontani; Takeyoshi Matsumoto; Takako Ikeda; Mayuko Oka; Takashi Sato (797-804).
Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor β (PDGFR-β) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-β and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-β and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-β and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-β and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.
Keywords: Matrix metalloproteinase; Oxidized LDL; Platelet-derived growth factor receptor; Smooth muscle cell;

Intracellular localization of RORα is isoform and cell line-dependent by Angelique Aschrafi; Nadine Meindl; Beate Firla; Ralf P. Brandes; Dieter Steinhilber (805-814).
The retinoid-related orphan receptor α (RORα) belongs to the nuclear receptor superfamily and comprises four isoforms generated by different promotor usage and alternative splicing. To better understand its function, the subcellular distribution of RORα was investigated. We could show that subcellular distribution of RORα is cell line and isoform-dependent. Isoform specific differences were mediated by the A/B domains which with the exception of RORα1 contain a signal that mediates cytoplasmic localization. The lack of this signal in RORα1 results in a complete nuclear localization and prevents cell membrane association observed for RORα2, 3, and 4. The region responsible for membrane association was identified as the C-terminal α-helix 12. Furthermore, the hinge region/ligand binding domain mediates nuclear localization. Our results show that isoform specific activity of RORα is not only regulated by different expression and DNA binding affinities but also by different subcellular distribution. Different access to the nucleus reveals an important mechanism regulating the activity of this constitutively active nuclear receptor.
Keywords: RORα; Cholesterol; 293 HEK cell; HeLa cell; Cellular distribution; Nuclear receptor;

Induction of H-ferritin synthesis by oxalomalate is regulated at both the transcriptional and post-transcriptional levels by Rita Santamaria; Maria Assunta Bevilacqua; Carmen Maffettone; Carlo Irace; Barbara Iovine; Alfredo Colonna (815-822).
Ferritin gene expression is complex and is controlled at transcriptional level in response to a variety of stimuli such as hormones, cytokines and cAMP. Iron, hemin and several compounds, chemically different, also activate the transcription of the ferritin gene. Ferritin biosynthesis is mainly regulated at post-transcriptional level by iron regulatory proteins (IRP1 and IRP2). We previously reported that oxalomalate, a competitive inhibitor of aconitase, remarkably decreases the IRP1 RNA-binding activity and induces a significant increase of ferritin expression. Here, we examined in cells cultured in presence of OMA the IRP1 intracellular content, ferritin biosynthesis and the transcriptional efficiency of H-ferritin gene promoter. Our results demonstrate a peculiar role of OMA that rapidly inactivates IRP1 without affecting IRP1 protein content and subsequently activates H-ferritin gene transcription leading to an overall increase of ferritin biosynthesis. We conclude that OMA regulates H-ferritin biosynthesis acting early at the post-transcriptional level and later on at transcriptional level.
Keywords: Oxalomalate; Iron metabolism; Ferritin; Iron Regulatory Protein; Transcription; H-ferritin promoter;

RpS3 is a component of the 40S ribosomal subunit of eukaryotes and also plays a role as a base damage endonuclease. Nm23-H1 encodes nucleoside diphosphate kinase A and acts as a suppressor of metastasis in certain human tumors. RpS3 interacted with nm23-H1, and the two proteins were colocalized in the cell periphery and cytoplasm. The 190th leucine of rpS3, and the 118th histidine and the 120th serine of nm23-H1 play key roles in the interaction of two proteins, respectively. The expression of rpS3 reduced the secretion of MMP-9 and the invasive potential in HT1080 cells. Additionally, the phosphorylated ERK was reduced by the expression of rpS3. In MCF7 cells, where the ERK pathway is inactivated and MMPs are not secreted and the ERK pathway can be activated by PMA, the PMA-induced ERK phosphorylation was reduced by the expression of rpS3. However, the L190A mutant of rpS3, which did not interact with nm23-H1, did not inhibit the invasive potential, the secretion of MMP-9, and the activation of the ERK pathway in HT1080 cells and PMA-activated MCF7 cells. These results suggest that rpS3 inhibits invasion via blocking the ERK pathway and MMP-9 secretion; the results also suggest that the interaction of rpS3 and nm23-H1 appears to be critical in this inhibition.
Keywords: rpS3; nm23-H1; Invasion; Metastasis suppressor;

The 3′-untranslated region directs ribosomal protein-encoding mRNAs to specific cytoplasmic regions by Annapina Russo; Giulia Russo; Monica Cuccurese; Corrado Garbi; Concetta Pietropaolo (833-843).
mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. We have analyzed the subcellular distribution of mRNAs encoding human cytosolic and mitochondrial ribosomal proteins. Biochemical fractionation experiments showed that the transcripts for cytosolic ribosomal proteins associate preferentially with the cytoskeleton via actin microfilaments. Transfection in HeLa cells of a GFP reporter construct containing the cytosolic ribosomal protein L4 3′-UTR showed that the 3′-UTR is necessary for the association of the transcript to the cytoskeleton. Using confocal analysis we demonstrate that the chimeric transcript is specifically associated with the perinuclear cytoskeleton. We also show that mRNA for mitochondrial ribosomal protein S12 is asymmetrically distributed in the cytoplasm. In fact, this transcript was localized mainly in the proximity of mitochondria, and the localization was 3′-UTR-dependent. In summary, ribosomal protein mRNAs constitute a new class of localized transcripts that share a common localization mechanism.
Keywords: mRNA localization; Ribosomal protein; Cytoskeleton; 3′-UTR localization signal; In situ hybridization;

Adenovirus-mediated transfection of caspase-8 sensitizes hepatocellular carcinoma to TRAIL- and chemotherapeutic agent-induced cell death by Yumi Yamaguchi; Katsuya Shiraki; Hiroyuki Fuke; Tomoko Inoue; Kazumi Miyashita; Yutaka Yamanaka; Takeshi Nakano (844-853).
Caspase-8 belongs to the cysteine protease family and is known to be activated at the initial step in the cascade of TRAIL-induced apoptosis. The activation of procaspase-8 can be blocked by a relatively large amount of c-FLIP, which renders resistance to death receptor-mediated apoptosis in many types of cancer cells. To ask if extrinsic over-expression of caspase-8 contributes to the induction of apoptosis, we introduced the caspase-8 gene into HCC cells using an adenoviral (Adv) vector (Adv-Casp8). We demonstrated that Adv-Casp8 increased expression of active forms of caspase-8 in MOI-dependent manner. A large amount of Adv-Casp8 (MOI of 50) induced apoptosis significantly in HCC cells and resulted in downregulation of c-FLIP (in SK-Hep1, HLE, and HepG2 cells), XIAP, survivin, and Bcl-xL (in HLE cells) and dynamic release of cytochrome c and Smac from the mitochondria into the cytosol. On the other hand, a small amount of Adv-Casp8 (MOI of 10) causes a slight but detectable increase in the level of apoptosis with only a small effect on anti-apoptotic proteins and mitochondrial activation. However, small amounts of Adv-Casp8 augmented TRAIL- or chemotherapeutic agent-induced cell death (with an MOI of 10 or 20, respectively). These results suggest both that exogenous over-expression of caspase-8 by Adv-Casp8 may be essential for induction of HCC cell death and that the combination of Adv-Casp8 and TRAIL or chemotherapeutic agents could provide a useful strategy for treatment of HCC.

Ferritin levels in microglia depend upon activation: Modulation by reactive oxygen species by Jana Mehlhase; Jeanette Gieche; Rebecca Widmer; Tilman Grune (854-859).
Iron is one of the trace elements playing a key role in the normal cellular metabolism. Since an excess of free iron is catalyzing the Fenton reaction, most of the intracellular iron is sequestered in the iron storage protein ferritin. The binding of iron into ferritin is well described for physiological conditions, however, under certain pathophysiological situations, the efficiency of this process is unknown. In the brain, microglial cells are among others the cell population most importantly responsible for the maintenance of the extracellular environment. These cells might undergo activation, and little is known about the expression of ferritin during activation of microglial cells. Therefore, we tested the microglial model cell line RAW264.7 for the expression of ferritin after LPS activation. A significant decrease in the levels of the ferritin H-chain during activation and a significant increase in the early recovery phase were found. We were able to demonstrate that reactive oxygen species are responsible for a suppression of the H-chain of ferritin, whereas iNOS expression and NO synthesis are counteracting the reactive oxygen species effect. The balance of reactive oxygen species and NO production are, therefore, determining expression levels of the ferritin H-chain during activation of microglial cells.
Keywords: Microglia; Activation; Ferritin; ROS; iNOS;

Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation by Valéry Daubie; Sandra Cauwenberghs; Nicole H.M. Senden; Roland Pochet; Theo Lindhout; Wim A. Buurman; Johan W.M. Heemskerk (860-869).
Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca2+ signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor VIIa, complexed with tissue factor, evoked a greatly delayed, oscillatory Ca2+ response, which relied on its catalytic activity and resembled that of SLIGRL, a peptide specifically activating the protease-activated receptor 2 (PAR2). Thrombin even at low concentrations evoked a rapid, mostly non-oscillating Ca2+ response through activation of PAR1, which reinforced the factor Xa response. The additive Ca2+ signals persisted, when factor X and prothrombin were activated in situ, or in the presence of plasma that was triggered to coagulate with tissue factor. Further, thrombin reinforced the factor Xa-induced production of IL-8, but not of IL-6. Both interleukins were produced in the presence of coagulating plasma. In conclusion, under coagulant conditions, factor Xa and thrombin appear to contribute in different and additive ways to the Ca2+-mobilizing and proinflammatory reactions of endothelial cells. These data provide first evidence that these serine proteases trigger distinct signaling modules in endothelium that is activated by plasma coagulation.
Keywords: Calcium; Coagulation; Endothelial cells; Factor Xa; Thrombin;

The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys → Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts.
Keywords: EGF receptor; Lipid raft; Cholesterol; Tyrosine kinase; Microdomain;

Lipidation and glycosylation of a T cell antigen receptor (TCR) transmembrane hydrophobic peptide dramatically enhances in vitro and in vivo function by Michael A. Amon; Marina Ali; Vera Bender; Yiu-Ngok Chan; Istvan Toth; Nicholas Manolios (879-888).
A T cell antigen receptor (TCR) transmembrane sequence derived peptide (CP) has been shown to inhibit T cell activation both in vitro and in vivo at the membrane level of the receptor signal transduction. To examine the effect of sugar or lipid conjugations on CP function, we linked CP to 1-aminoglucosesuccinate (GS), N-myristate (MYR), mono-di-tripalmitate (LP1, LP2, or LP3), and a lipoamino acid (LA) and examined the effects of these compounds on T cell activation in vitro and by using a rat model of adjuvant-induced arthritis, in vivo. In vitro, antigen presentation results demonstrated that lipid conjugation enhanced CP's ability to lower IL-2 production from 56.99% ± 15.69 S.D. observed with CP, to 12.08% ± 3.34 S.D. observed with LA. The sugar conjugate GS resulted in only a mild loss of in vitro activity compared to CP (82.95% ± 14.96 S.D.). In vivo, lipid conjugation retarded the progression of adjuvant-induced arthritis by approximately 50%, whereas the sugar conjugated CP, GS, almost completely inhibited the progression of arthritis. This study demonstrates that hydrophobic peptide activity is markedly enhanced in vitro and in vivo by conjugation to lipids or sugars. This may have practical applications in drug delivery and bioavailability of hydrophobic peptides.
Keywords: Peptides; Lipopeptides; Peptide conjugation; Transmembranes; T cell antigen receptor;

Insulin-stimulated glucose uptake through GLUT4 plays a pivotal role in maintaining normal blood glucose levels. Glucose transport through GLUT4 requires both GLUT4 translocation to the plasma membrane and GLUT4 activation at the plasma membrane. Here we report that a cell-permeable phosphoinositide-binding peptide, which induces GLUT4 translocation without activation, sequestered PI 4,5-P2 in the plasma membrane from its binding partners. Restoring PI 4,5-P2 to the plasma membrane after the peptide treatment increased glucose uptake. No additional glucose transporters were recruited to the plasma membrane, suggesting that the increased glucose uptake was attributable to GLUT4 activation. Cells overexpressing phosphatidylinositol-4-phosphate 5-kinase treated with the peptide followed by its removal exhibited a higher level of glucose transport than cells stimulated with a submaximal level of insulin. However, only cells treated with submaximal insulin exhibited translocation of the PH-domains of the general receptor for phosphoinositides (GRP1) to the plasma membrane. Thus, PI 4,5-P2, but not PI 3,4,5-P3 converted from PI 4,5-P2, induced GLUT4 activation. Inhibiting F-actin remodeling after the peptide treatment significantly impaired GLUT4 activation induced either by PI 4,5-P2 or by insulin. These results suggest that PI 4,5-P2 in the plasma membrane acts as a second messenger to activate GLUT4, possibly through F-actin remodeling.
Keywords: GLUT4; Glucose transport; PI 4,5-P2; F-actin;

Identification of tocopherol-associated protein as an activin/TGF-β-inducible gene in mast cells by Masayuki Funaba; Masaru Murakami; Teruo Ikeda; Kenji Ogawa; Kunihiro Tsuchida; Hiromu Sugino (900-906).
Previous studies have demonstrated that treatment with activin A and TGF-β1, members of the TGF-β family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-β1-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-β family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by real-time RT-PCR analyses. Treatment with TGF-β1 at 200 pM but not BMP-2 at 4 nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with > 1 μM SB431542, an inhibitor of activin and TGF-β type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-β1, whereas < 0.5 μM SB431542 effectively reduced TGF-β1-induced expression of mmcp-1 and mmcp-7. These results suggest that inhibitory effects of SB431542 are different between TGF-β-induced genes. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-β pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-β occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-β signaling.
Keywords: Activin; TGF-β; Tocopherol-associated protein; Mast cell;