BBA - Molecular Cell Research (v.1783, #3)

Cross-talk between PDGF and S1P signalling elucidates the inhibitory effect and potential antifibrotic action of the immunomodulator FTY720 in activated HSC-cultures by Anna Maria Brunati; Elena Tibaldi; Amedeo Carraro; Enrico Gringeri; Francesco D’Amico; Antonio Toninello; Maria Lina Massimino; Mario Angelo Pagano; Giovanna Nalesso; Umberto Cillo (347-359).
Platelet-derived growth factor (PDGF) has been shown to be essential in the activation of hepatic stellate cells (HSCs), contributing to the onset and development of hepatic fibrosis. Recently, sphingosine-1-phosphate (S1P) has been shown to be a mitogen and stimulator of chemotaxis also for HSCs. Since it has been demonstrated in several cell types that cross-talk between PDGF and S1P signalling pathways occurs, our aim was to investigate the potential antifibrotic effect of FTY720, whose phosphorylated form acts as a potent S1P receptor (S1PR) modulator, on HSCs. FTY720 inhibits cell proliferation and migration after PDGF stimulation on HSCs in a concentration range between 0.1 and 1 μM. By using compounds that block S1P signalling (PTX and VPC23019), we assessed that FTY720 also acts in an S1P receptor-independent way by decreasing the level of tyrosine phosphorylation of PDGF receptor, with subsequent inhibition of the PDGF signalling pathway. In addition, inhibition of sphingosine kinase2 (SphK2), which is responsible for FTY720 phosphorylation, by DMS/siRNA unveils a mechanism of action irrespective of its phosphorylation, in particular decreasing the level of S1P1 on the plasma membrane. These findings led us to hypothesize a potential use of FTY720 as a potential antifibrotic drug for further clinical application.
Keywords: Integrative signalling; S1P receptor; PDGF receptor; PTX;

Rac1 GTPase is activated by hepatitis B virus replication — involvement of HBX by Tuan Lin Tan; Ning Fang; Tuan Ling Neo; Pritpal Singh; Jianhua Zhang; Ruijie Zhou; Cheng-Gee Koh; Vincent Chan; Seng Gee Lim; Wei Ning Chen (360-374).
Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (βPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.
Keywords: Hepatitis B virus replication; Rac1 activation; Cell morphology; HBX; βPIX;

Cotinine-induced convergence of the cholinergic and PI3 kinase-dependent anti-inflammatory pathways in innate immune cells by Kunal Rehani; David A. Scott; Diane Renaud; Hashir Hamza; Lisa R. Williams; Huizhi Wang; Michael Martin (375-382).
Nicotine [(S)-3-(1-methyl-2-pyrrolidinyl)pyridine] is a major component of tobacco and a highly efficient acetylcholine receptor (nAChR) agonist that triggers the cholinergic anti-inflammatory pathway. We demonstrate that pre-treatment of monocytes with the stable nicotine catabolite, cotinine [(S)-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone], dramatically alters the nature of the inflammatory response to Gram negative bacteria by abrogating the production of cytokines that are under the transcriptional control of the NF-κB system (TNF-α, IL-1β, IL-6, IL-12/IL-23 p40) and shifting the response towards an IL-10-dominated anti-inflammatory profile. This anti-inflammatory phenomenon is initiated specifically by engagement of the monocytic α7 nAChR; and is PI3K/GSK-3β-dependent; but NF-κB-independent. These mechanistic insights suggest an ability to exploit convergent, endogenous anti-inflammatory pathway(s) to either up-regulate or down-regulate the production of specific cytokine groups (pro- or anti-inflammatory cytokines) depending on the clinical necessity.
Keywords: Cholinergic anti-inflammatory pathway; Cotinine; Cytokine; Inflammation; Monocyte; PI3K-dependent anti-inflammatory pathway; Tobacco smoking;

KIBRA interacts with discoidin domain receptor 1 to modulate collagen-induced signalling by Heidi N. Hilton; Prudence M. Stanford; Jessica Harris; Samantha R. Oakes; Warren Kaplan; Roger J. Daly; Christopher J. Ormandy (383-393).
Mammary gland development is coupled to reproductive events by hormonal cues of ovarian and pituitary origin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. In this study KIBRA was identified as a transcript showing decreased expression associated with failed mammary gland development in Prlr knockout mammary epithelium. It is strongly up-regulated during pregnancy, falls during lactation and is again up-regulated during involution of the gland at weaning. A bioinformatic approach was undertaken to identify potential binding partners which interact with the WW domains of KIBRA. We show that KIBRA binds to a WW domain binding motif, PPxY, in the tyrosine kinase receptor DDR1, and dissociates upon treatment with the DDR1 ligands collagen type I or IV. In addition we show that KIBRA and DDR1 also interact with PKCz to form a trimeric complex. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodeling events of mammary gland development.
Keywords: Mammary; Progesterone; Cell matrix; PKCζ, ERK MAPK;

Nuclear translocation of hypoxia-inducible factors (HIFs): Involvement of the classical importin α/β pathway by Reinhard Depping; Amrei Steinhoff; Susann G. Schindler; Beate Friedrich; Riku Fagerlund; Eric Metzen; Enno Hartmann; Matthias Köhler (394-404).
Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-α subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1α into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors importin α/β. HIF-1α, -1β, and HIF-2α are binding to importin α1, α3, α5, and α7. The direct interaction of HIF-1α to α importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.
Keywords: Hypoxia-inducible factor; HIF; Importin; Nuclear transport; NLS; Oxygen homeostasis;

Oxidative stress mislocalizes and retains transport factor importin-α and nucleoporins Nup153 and Nup88 in nuclei where they generate high molecular mass complexes by Mohamed Kodiha; Dan Tran; Cynthia Qian; Andreea Morogan; John F. Presley; Claire M. Brown; Ursula Stochaj (405-418).
Nuclear trafficking of proteins requires the cooperation between soluble transport components and nucleoporins. As such, classical nuclear import depends on the dimeric carrier importin-α/β1, and CAS, a member of the importin-β family, which exports importin-α to the cytoplasm. Here we analyzed the effect of oxidative stress elicited by diethyl maleate (DEM) on classical nuclear transport. Under conditions that do not induce death in the majority of cells, DEM has little effect on the nucleocytoplasmic concentration gradient of Ran, but interferes with the nuclear accumulation of several reporter proteins. Moreover, DEM treatment alters the distribution of soluble transport factors and several nucleoporins in growing cells. We identified nuclear retention of importin-α, CAS as well as nucleoporins Nup153 and Nup88 as a mechanism that contributes to the nuclear concentration of these proteins. Both nucleoporins, but not CAS, associate with importin-α in the nuclei of growing cells and in vitro. Importin-α generates high molecular mass complexes in the nucleus that contain Nup153 and Nup88, whereas CAS was not detected. The formation of high molecular mass complexes containing importin-α, Nup153 and Nup88 is increased upon oxidant treatment, suggesting that complex formation contributes to the anchoring of importin-α in nuclei. Taken together, our studies link oxidative stress to the proper localization of soluble transport factors and nucleoporins and to changes in the interactions between these proteins.
Keywords: Stress; Oxidant; Nucleus; Nuclear transport;

Crossregulation of β-catenin/Tcf pathway by NF-κB is mediated by lzts2 in human adipose tissue-derived mesenchymal stem cells by Hyun Hwa Cho; Hye Joon Joo; Ji Sun Song; Yong Chan Bae; Jin Sup Jung (419-428).
β-catenin/Tcf and NF-κB signaling pathways play an important role in biological functions and crosstalk between these pathways has been reported.  We found that the modulation of NF-κB activity showed a direct correlation with β-catein/Tcf pathway in human adipose tissue (hASCs) and bone marrow (hBMSCs)-derived mesenchymal stem cells. Expression of lzts2, which inhibits nuclear translocation of β-catenin and its transactivation activity, was regulated by NF-κB activity. Downregulation of lzts2 by RNA interference increased the nuclear translocation of β-catenin and NF-κB activity in hASCs. NF-κB activation by the downregulation of lzts2 was accompanied by the increase of β-TrCP1 expression and the decrease of IκB level. Downregulation of lzts2 increased the proliferation of hASCs and hBMSC, and blocked the NF-κB inhibitor-induced inhibitory effect on their proliferation and Tcf promoter activation. These findings provide the first evidence that the reciprocal crosstalk between β-catenin/Tcf pathway and NF-κB signaling in hMSCs is mediated through the regulation of lzts2 expression.
Keywords: hASCs; β-Catenin/Tcf; NF-κB; lzts2; β-TrCP;

Differential roles of p80- and p130-angiomotin in the switch between migration and stabilization of endothelial cells by Mira Ernkvist; Olivier Birot; Indranil Sinha; Niina Veitonmaki; Staffan Nyström; Karin Aase; Lars Holmgren (429-437).
We have previously shown that angiomotin (Amot) plays an important role in growth factor-induced migration of endothelial cells in vitro. Genetic knock-down of Amot in zebrafish also results in inhibition of migration of intersegmental vessels in vivo. Amot is expressed as two different isoforms, p80-Amot and p130-Amot. Here we have analyzed the expression of the two Amot isoforms during retinal angiogenesis in vivo and demonstrate that p80-Amot is expressed during the migratory phase. In contrast, p130-Amot is expressed during the period of blood vessel stabilization and maturation. We also show that the N-terminal domain of p130-Amot serves as a targeting domain responsible for localization of p130-Amot to actin and tight junctions. We further show that the relative expression levels of p80-Amot and p130-Amot regulate a switch between a migratory and a non-migratory cell phenotype where the migratory function of p80-Amot is dominant over the stabilization and maturation function of p130-Amot. Our data indicates that homo-oligomerization of p80-Amot and hetero-oligomerization of both isoforms are critical for this regulation.
Keywords: Actin; Angiogenesis; Calcium switch; Retina; Tight junction;

The functional implications of Akt activity and TGF-β signaling in tamoxifen-resistant breast cancer by Young A. Yoo; Yeul Hong Kim; Jun Suk Kim; Jae Hong Seo (438-447).
Keywords: Tamoxifen; Akt; Transforming growth factor-β; Breast cancer;

Analysis of the minimal specificity of CED-3 using a yeast transcriptional reporter system by Sarah J. Westein; Fiona L. Scott; Christine J. Hawkins (448-454).
Determination of the substrate specificity of site-specific proteases helps define their physiological roles. We developed a yeast-based system for defining the minimal substrate specificity of site-specific proteases, within the context of a protein. Using this system, we characterized the P4–P1 substrate specificity of the nematode apoptotic caspase CED-3. Apart from an absolute requirement for aspartate at the P1 position, CED-3 is a relatively promiscuous caspase capable of cleaving substrates bearing many amino acids at P4–P2 sites.
Keywords: Caspase; Protease; S. cerevisiae; C. elegans; Nematode; Substrate;

Cartilage–hair hypoplasia-associated mutations in the RNase MRP P3 domain affect RNA folding and ribonucleoprotein assembly by Tim J.M. Welting; Sandy Mattijssen; Florence M.A. Peters; Nienke L. van Doorn; Lianne Dekkers; Walther J. van Venrooij; Hans A. Heus; Luisa Bonafé; Ger J.M. Pruijn (455-466).
Cartilage–hair hypoplasia (CHH) is caused by mutations in the gene encoding the RNA component of RNase MRP. Currently it is unknown how these mutations affect the function of this endoribonuclease. In this study we investigated the effect of mutations in the P3 domain on protein binding and RNA folding. Our data demonstrate that a number of P3 nucleotide substitutions reduced the efficiency of its interaction with Rpp25 and Rpp20, two protein subunits binding as a heterodimer to this domain. The CHH-associated 40G>A substitution, as well as the replacement of residue 47, almost completely abrogated Rpp25 and Rpp20 binding in different assays. Also other CHH-associated P3 mutations reduced the efficiency by which the RNase MRP RNA is bound by Rpp25–Rpp20. These data demonstrate that the most important residues for binding of the Rpp25–Rpp20 dimer reside in the apical stem-loop of the P3 domain. Structural analyses by NMR not only showed that this loop may adopt a pseudo-triloop structure, but also demonstrated that the 40G>A substitution alters the folding of this part of the P3 domain. Our data are the first to provide insight into the molecular mechanism by which CHH-associated mutations affect the function of RNase MRP.
Keywords: Cartilage–hair hypoplasia; Endoribonuclease; RNA processing; RNase MRP; RNA-protein interaction;

Prokineticin-1 (Prok-1) works coordinately with glial cell line-derived neurotrophic factor (GDNF) to mediate proliferation and differentiation of enteric neural crest cells by Elly S.W. Ngan; Cathy K.Y. Shum; Hiu-Ching Poon; Mai-Har Sham; Maria-Mercè Garcia-Barcelo; Vincent C.H. Lui; Paul K.H. Tam (467-478).
Enteric neural crest cells (NCC) are multipotent progenitors which give rise to neurons and glia of the enteric nervous system (ENS) during fetal development. Glial cell line-derived neurotrophic factor (GDNF)/RET receptor tyrosine kinase (Ret) signaling is indispensable for their survival, migration and differentiation. Using microarray analysis and isolated NCCs, we found that 45 genes were differentially expressed after GDNF treatment (16 h), 29 of them were up-regulated including 8 previously undescribed genes. Prokineticin receptor 1 (PK-R1), a receptor for Prokineticins (Prok), was identified in our screen and shown to be consistently up-regulated by GDNF in enteric NCCs. Further, PK-R1 was persistently expressed at a lower level in the enteric ganglions of the c-Ret deficient mice when compared to that of the wild-type littermates. Subsequent functional analysis showed that GDNF potentiated the proliferative and differentiation effects of Prok-1 by up-regulating PK-R1 expression in enteric NCCs. In addition, expression analysis and gene knock-down experiments indicated that Prok-1 and GDNF signalings shared some common downstream targets. More importantly, Prok-1 could induce both proliferation and expression of differentiation markers of c-Ret deficient NCCs, suggesting that Prok-1 may also provide a complementary pathway to GDNF signaling. Taken together, these findings provide evidence that Prok-1 crosstalks with GDNF/Ret signaling and probably provides an additional layer of signaling refinement to maintain proliferation and differentiation of enteric NCCs.
Keywords: GDNF; Prok-1; Neural crest cell;

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response by Kevin Goggin; Simon Beaudoin; Catherine Grenier; Andrée-Anne Brown; Xavier Roucou (479-491).
In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2α, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.
Keywords: Prion protein; Aggresome; Stress response; PKR; Stress granule;

Sequential activation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), critically regulated by extracellular signal-regulated kinase 1 and 2 (ERK1/2)-dependent phosphorylation, mediates U937 cell survival to peroxynitrite. In contrast, a limiting factor is represented by the parallel mitochondrial formation of H2O2 leading to suppression of the survival signaling. We now report that the inhibitory effects of H2O2 are at the level of ERK1/2 phosphorylation and involve activation of orthovanadate-sensitive phosphotyrosine protein phosphatase(s). Under these conditions, the otherwise stimulatory effects of peroxynitrite on ERK1/2 phosphorylation are concealed by phosphatase-dependent dephosphorylation and the activities of cPLA2 and 5-LO are significantly reduced or suppressed, respectively. The ensuing inhibition of downstream events preventing mitochondrial permeability transition rapidly leads these cells to death. Thus, endogenous H2O2 limits U937 cell survival to peroxynitrite via activation of phosphotyrosine protein phosphatase(s) promoting upstream inhibition of the survival signaling critically regulated by the extent of ERK1/2 phosphorylation.
Keywords: Peroxynitrite; ERK1/2; Protein tyrosine phosphatase(s); H2O2; Mitochondria; Survival signaling;

Lyl1 interacts with CREB1 and alters expression of CREB1 target genes by Serban San-Marina; YouQi Han; Fernando Suarez Saiz; Michael R. Trus; Mark D. Minden (503-517).
The basic helix-loop-helix (bHLH) transcription factor family contains key regulators of cellular proliferation and differentiation as well as the suspected oncoproteins Tal1 and Lyl1. Tal1 and Lyl1 are aberrantly over-expressed in leukemia as a result of chromosomal translocations, or other genetic or epigenetic events. Protein-protein and protein-DNA interactions described so far are mediated by their highly homologous bHLH domains, while little is known about the function of other protein domains. Hetero-dimers of Tal1 and Lyl1 with E2A or HEB, decrease the rate of E2A or HEB homo-dimer formation and are poor activators of transcription. In vitro, these hetero-dimers also recognize different binding sites from homo-dimer complexes, which may also lead to inappropriate activation or repression of promoters in vivo. Both mechanisms are thought to contribute to the oncogenic potential of Tal1 and Lyl1. Despite their bHLH structural similarity, accumulating evidence suggests that Tal1 and Lyl1 target different genes. This raises the possibility that domains flanking the bHLH region, which are distinct in the two proteins, may participate in target recognition. Here we report that CREB1, a widely-expressed transcription factor and a suspected oncogene in acute myelogenous leukemia (AML) was identified as a binding partner for Lyl1 but not for Tal1. The interaction between Lyl1 and CREB1 involves the N terminal domain of Lyl1 and the Q2 and KID domains of CREB1. The histone acetyl-transferases p300 and CBP are recruited to these complexes in the absence of CREB1 Ser 133 phosphorylation. In the Id1 promoter, Lyl1 complexes direct transcriptional activation. We also found that in addition to Id1, over-expressed Lyl1 can activate other CREB1 target promoters such as Id3, cyclin D3, Brca1, Btg2 and Egr1. Moreover, approximately 50% of all gene promoters identified by ChIP-chip experiments were jointly occupied by CREB1 and Lyl1, further strengthening the association of Lyl1 with Cre binding sites. Given the newly recognized importance of CREB1 in AML, the ability of Lyl1 to modulate promoter responses to CREB1 suggests that it plays a role in the malignant phenotype by occupying different promoters than Tal1.
Keywords: Acute myelogenous leukemia; Gene expression; Helix-loop-helix transcription factor; Lyl1; Tal1; CREB1;