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BBA - Molecular Cell Research (v.1803, #7)
Herpesviruses: Hijacking the Ras signaling pathway by Harilaos Filippakis; Demetrios A. Spandidos; George Sourvinos (pp. 777-785).
Cancer is the final result of the accumulation of several genetic alterations occurring in a cell. Several herpesviruses and especially gamma-herpesviruses have played an important role in Cancer Biology, contributing significantly to our comprehension of cell signaling and growth control pathways which lead to malignancy. Unlike other infectious agents, herpesviruses persist in the host by establishing a latent infection, so that they can reactivate periodically. Interestingly, some herpesviruses are able to either deliver or induce the expression of cellular oncogenes. Such alterations can result in the derailment of the normal cell cycle and ultimately shift the balance between continuous proliferation and programmed cell death. Herpesvirus infection employs key molecules of cellular signaling cascades mostly to enhance viral replication. However, most of these molecules are also involved in essential cellular functions, such as proliferation, cellular differentiation and migration, as well as in DNA repair mechanisms. Ras proteins are key molecules that regulate a wide range of cellular functions, including differentiation, proliferation and cell survival. A broad field of medical research is currently focused on elucidating the role of ras oncogenes in human tumor initiation as well as tumor progression and metastasis. Upon activation, Ras proteins employ several downstream effector molecules such as phosphatidylinositol 3-kinase (PI3-K) and Raf and Ral guanine nucleotide-dissociation stimulators (RALGDS) to regulate a cascade of events ranging from cell proliferation and survival to apoptosis and cellular death. In this review, we give an overview of the impact that herpesvirus infection has on the host-cell Ras signaling pathway, providing an outline of their interactions with the key cascade molecules with which they associate. Several of these interactions of viral proteins with member of the Ras signaling pathway may be crucial in determining herpesviruses' oncogenic potential or their oncomodulatory behavior. The questions that emerge concern the potential role of these molecules as therapeutic targets both for viral infections and cancer. Understanding the means by which viruses may cause oncogenesis would therefore provide a deeper knowledge of the overall oncogenic process.
Keywords: Abbreviations; BL; Burkitt's lymphoma; COX-2; cytochrome; c; oxidase subunit II; E; Early; EBNA; Epstein–Barr nuclear antigen; EBV; Epstein–Barr virus; EGF; Epidermal Growth Factor; EGFR; Epidermal Growth Factor Receptor; Elk; Ets-related transcription factor; ERK; extracellular regulated kinase; GDP; guanosine diphosphate; GTP; guanosine triphosphate; HCMV; Human Cytomegalovirus; HHV; Human Herpes Virus; HL; Hodgkin's lymphoma; HSV; Herpes Simplex Virus; IE; Immediate Early; IL; interleukin; JNK; c-Jun amino-terminal kinases; KSHV; Kaposi Sarcoma-associated Herpes Virus; L; Late; LAT; latency-associated transcript; LNA; latent nuclear antigen 1; MAPK; Mitogen-Activated Protein Kinases; MAPKK; MAPK kinase; MAPKKK; MAPK kinase kinase; MCD; multicentric variant of Castleman Disease; NF-κB; Nuclear Factor kappa-light-chain-enhancer of activated B cells; NGF; Neuronal Growth Factor; NPC; Nasopharyngeal Carcinoma; ORF; Open Reading Frame; PEL; Primary Effusion Lymphoma; PI3-K; phosphatidylinositol 3-kinase; PKR; RNA-activated protein kinase; RA; retinoic acid; RALGDS; Ral guanine nucleotide-dissociation stimulators; RTA; viral replication and transcriptional activation protein; TNF-α; tumor necrosis factor; TPA; 12-; O; -tetradecanoyl-phorbol-13-acetate; VZV; Varicella Zoster VirusHerpesvirus; Ras; HSV-1; HSV-2; VZV; EBV; HCMV; HHV6; HHV7; HHV8; Cancer
Herpesviruses: Hijacking the Ras signaling pathway by Harilaos Filippakis; Demetrios A. Spandidos; George Sourvinos (pp. 777-785).
Cancer is the final result of the accumulation of several genetic alterations occurring in a cell. Several herpesviruses and especially gamma-herpesviruses have played an important role in Cancer Biology, contributing significantly to our comprehension of cell signaling and growth control pathways which lead to malignancy. Unlike other infectious agents, herpesviruses persist in the host by establishing a latent infection, so that they can reactivate periodically. Interestingly, some herpesviruses are able to either deliver or induce the expression of cellular oncogenes. Such alterations can result in the derailment of the normal cell cycle and ultimately shift the balance between continuous proliferation and programmed cell death. Herpesvirus infection employs key molecules of cellular signaling cascades mostly to enhance viral replication. However, most of these molecules are also involved in essential cellular functions, such as proliferation, cellular differentiation and migration, as well as in DNA repair mechanisms. Ras proteins are key molecules that regulate a wide range of cellular functions, including differentiation, proliferation and cell survival. A broad field of medical research is currently focused on elucidating the role of ras oncogenes in human tumor initiation as well as tumor progression and metastasis. Upon activation, Ras proteins employ several downstream effector molecules such as phosphatidylinositol 3-kinase (PI3-K) and Raf and Ral guanine nucleotide-dissociation stimulators (RALGDS) to regulate a cascade of events ranging from cell proliferation and survival to apoptosis and cellular death. In this review, we give an overview of the impact that herpesvirus infection has on the host-cell Ras signaling pathway, providing an outline of their interactions with the key cascade molecules with which they associate. Several of these interactions of viral proteins with member of the Ras signaling pathway may be crucial in determining herpesviruses' oncogenic potential or their oncomodulatory behavior. The questions that emerge concern the potential role of these molecules as therapeutic targets both for viral infections and cancer. Understanding the means by which viruses may cause oncogenesis would therefore provide a deeper knowledge of the overall oncogenic process.
Keywords: Abbreviations; BL; Burkitt's lymphoma; COX-2; cytochrome; c; oxidase subunit II; E; Early; EBNA; Epstein–Barr nuclear antigen; EBV; Epstein–Barr virus; EGF; Epidermal Growth Factor; EGFR; Epidermal Growth Factor Receptor; Elk; Ets-related transcription factor; ERK; extracellular regulated kinase; GDP; guanosine diphosphate; GTP; guanosine triphosphate; HCMV; Human Cytomegalovirus; HHV; Human Herpes Virus; HL; Hodgkin's lymphoma; HSV; Herpes Simplex Virus; IE; Immediate Early; IL; interleukin; JNK; c-Jun amino-terminal kinases; KSHV; Kaposi Sarcoma-associated Herpes Virus; L; Late; LAT; latency-associated transcript; LNA; latent nuclear antigen 1; MAPK; Mitogen-Activated Protein Kinases; MAPKK; MAPK kinase; MAPKKK; MAPK kinase kinase; MCD; multicentric variant of Castleman Disease; NF-κB; Nuclear Factor kappa-light-chain-enhancer of activated B cells; NGF; Neuronal Growth Factor; NPC; Nasopharyngeal Carcinoma; ORF; Open Reading Frame; PEL; Primary Effusion Lymphoma; PI3-K; phosphatidylinositol 3-kinase; PKR; RNA-activated protein kinase; RA; retinoic acid; RALGDS; Ral guanine nucleotide-dissociation stimulators; RTA; viral replication and transcriptional activation protein; TNF-α; tumor necrosis factor; TPA; 12-; O; -tetradecanoyl-phorbol-13-acetate; VZV; Varicella Zoster VirusHerpesvirus; Ras; HSV-1; HSV-2; VZV; EBV; HCMV; HHV6; HHV7; HHV8; Cancer
Hedgehog signaling and gastrointestinal cancer by Milena Saqui-Salces; Juanita L. Merchant (pp. 786-795).
Hedgehog (Hh) signaling is critical for embryonic development and in differentiation, proliferation, and maintenance of multiple adult tissues. De-regulation of the Hh pathway is associated with birth defects and cancer. In the gastrointestinal tract, Hh ligands Sonic (Shh) and Indian (Ihh), as well as the receptor Patched (Ptch1), and transcription factors of Glioblastoma family (Gli) are all expressed during development. In the adult, Shh expression is restricted to the stomach and colon, while Ihh expression occurs throughout the luminal gastrointestinal tract, its expression being highest in the proximal duodenum. Several studies have demonstrated a requirement for Hh signaling during gastrointestinal tract development. However to date, the specific role of the Hh pathway in the adult stomach and intestine is not completely understood. The current review will place into context the implications of recent published data related to the biochemistry and cell biology of Hh signaling on the luminal gastrointestinal tract during development, normal physiology and subsequently carcinogenesis.
Keywords: Hedgehog signaling; Gastric epithelium; Intestine; Colon cancer; Gastric cancer; Gastrointestinal development
Hedgehog signaling and gastrointestinal cancer by Milena Saqui-Salces; Juanita L. Merchant (pp. 786-795).
Hedgehog (Hh) signaling is critical for embryonic development and in differentiation, proliferation, and maintenance of multiple adult tissues. De-regulation of the Hh pathway is associated with birth defects and cancer. In the gastrointestinal tract, Hh ligands Sonic (Shh) and Indian (Ihh), as well as the receptor Patched (Ptch1), and transcription factors of Glioblastoma family (Gli) are all expressed during development. In the adult, Shh expression is restricted to the stomach and colon, while Ihh expression occurs throughout the luminal gastrointestinal tract, its expression being highest in the proximal duodenum. Several studies have demonstrated a requirement for Hh signaling during gastrointestinal tract development. However to date, the specific role of the Hh pathway in the adult stomach and intestine is not completely understood. The current review will place into context the implications of recent published data related to the biochemistry and cell biology of Hh signaling on the luminal gastrointestinal tract during development, normal physiology and subsequently carcinogenesis.
Keywords: Hedgehog signaling; Gastric epithelium; Intestine; Colon cancer; Gastric cancer; Gastrointestinal development
Interaction between the motor protein prestin and the transporter protein VAPA by Soma Sengupta; Katharine K. Miller; Kazuaki Homma; Roxanne Edge; Mary Ann Cheatham; Peter Dallos; Jing Zheng (pp. 796-804).
Prestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify prestin-associated proteins. Several proteins were recognized as potential prestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and prestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and prestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from prestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of prestin. In other words, less VAPA protein is found in OHCs lacking prestin. Thus, prestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more prestin protein appears at the plasma membrane when VAPA is co-expressed with prestin. These data suggest that VAPA could be involved in prestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.
Keywords: Prestin; VAPA; Outer hair cell; Protein trafficking
Interaction between the motor protein prestin and the transporter protein VAPA by Soma Sengupta; Katharine K. Miller; Kazuaki Homma; Roxanne Edge; Mary Ann Cheatham; Peter Dallos; Jing Zheng (pp. 796-804).
Prestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify prestin-associated proteins. Several proteins were recognized as potential prestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and prestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and prestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from prestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of prestin. In other words, less VAPA protein is found in OHCs lacking prestin. Thus, prestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more prestin protein appears at the plasma membrane when VAPA is co-expressed with prestin. These data suggest that VAPA could be involved in prestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.
Keywords: Prestin; VAPA; Outer hair cell; Protein trafficking
Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6 by Sylvie Cayouette; Simon M. Bousquet; Nancy Francoeur; Dupre Émilie Dupré; Michaël Monet; Hugo Gagnon; Youssef B. Guedri; Christine Lavoie; Guylain Boulay (pp. 805-812).
TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9S21N, a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11S25N abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface.
Keywords: Abbreviations; Ca; 2+; i; intracellular Ca; 2+; concentration; BSA; bovine serum albumin; CCh; carbachol; CFTR; cystic fibrosis transmembrane conductance regulator; EGFP; enhanced green fluorescent protein; ENaC; epithelial sodium channel; GST; glutathione S-transferase; HA; hemagglutinin antigen; HBSS; Hepes buffered salt solution; IP; 3; inositol 1, 4, 5-trisphosphate; PBS; phosphate buffered saline; GqPCR; Gq-protein coupled receptor; TRP; transient receptor potential; TRPC; TRP canonical; TRPV; TRP vanilloidTRPC; Ca2+ entry; Rab GTPase; Trafficking; trans; -Golgi network; Endosome
Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6 by Sylvie Cayouette; Simon M. Bousquet; Nancy Francoeur; Dupre Émilie Dupré; Michaël Monet; Hugo Gagnon; Youssef B. Guedri; Christine Lavoie; Guylain Boulay (pp. 805-812).
TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9S21N, a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11S25N abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface.
Keywords: Abbreviations; Ca; 2+; i; intracellular Ca; 2+; concentration; BSA; bovine serum albumin; CCh; carbachol; CFTR; cystic fibrosis transmembrane conductance regulator; EGFP; enhanced green fluorescent protein; ENaC; epithelial sodium channel; GST; glutathione S-transferase; HA; hemagglutinin antigen; HBSS; Hepes buffered salt solution; IP; 3; inositol 1, 4, 5-trisphosphate; PBS; phosphate buffered saline; GqPCR; Gq-protein coupled receptor; TRP; transient receptor potential; TRPC; TRP canonical; TRPV; TRP vanilloidTRPC; Ca2+ entry; Rab GTPase; Trafficking; trans; -Golgi network; Endosome
The M5 muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization by Dasiel O. Borroto-Escuela; Garcia-Negredo Gloria García-Negredo; Pere Garriga; Kjell Fuxe; Francisco Ciruela (pp. 813-825).
Besides some pharmacological, biochemical and biophysical evidences support the contention that muscarinic acetylcholine receptors can form homo- and heterodimers, the existence of specific M3 and M5 muscarinic receptors oligomers in living cells is a new concept. Interestingly, this phenomenon might have relevance in lymphocytic cholinergic function since both T- and B-cells naturally express high levels of these two receptor subtypes. Here, by means of co-immunoprecipitation and bioluminescence resonance energy transfer methods we demonstrated that M3 and M5 muscarinic receptors could form constitutive homo- and heterodimers in transiently transfected HEK-293T cells. Interestingly, this receptor–receptor interaction was unaltered by carbachol treatment but it was affected by the expression of a peptide corresponding to a portion of the third intracellular loop of the M5 muscarinic receptor. In addition, the same peptide was able to abrogate the carbachol-induced mitogen-activated protein kinase phosphorylation and the carbachol-enhanced PHA-induced IL-2 production in derived lymphocytic T cells. Overall, these results suggest that the third intracellular loop of the M5 muscarinic receptor might play a regulatory role in receptor function and heteromerization, thus providing the molecular framework for a potential cholinergic-based therapeutic intervention of the immune system.
Keywords: G protein-coupled receptor; Muscarinic acetylcholine receptor; Receptor oligomerization
The M5 muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization by Dasiel O. Borroto-Escuela; Garcia-Negredo Gloria García-Negredo; Pere Garriga; Kjell Fuxe; Francisco Ciruela (pp. 813-825).
Besides some pharmacological, biochemical and biophysical evidences support the contention that muscarinic acetylcholine receptors can form homo- and heterodimers, the existence of specific M3 and M5 muscarinic receptors oligomers in living cells is a new concept. Interestingly, this phenomenon might have relevance in lymphocytic cholinergic function since both T- and B-cells naturally express high levels of these two receptor subtypes. Here, by means of co-immunoprecipitation and bioluminescence resonance energy transfer methods we demonstrated that M3 and M5 muscarinic receptors could form constitutive homo- and heterodimers in transiently transfected HEK-293T cells. Interestingly, this receptor–receptor interaction was unaltered by carbachol treatment but it was affected by the expression of a peptide corresponding to a portion of the third intracellular loop of the M5 muscarinic receptor. In addition, the same peptide was able to abrogate the carbachol-induced mitogen-activated protein kinase phosphorylation and the carbachol-enhanced PHA-induced IL-2 production in derived lymphocytic T cells. Overall, these results suggest that the third intracellular loop of the M5 muscarinic receptor might play a regulatory role in receptor function and heteromerization, thus providing the molecular framework for a potential cholinergic-based therapeutic intervention of the immune system.
Keywords: G protein-coupled receptor; Muscarinic acetylcholine receptor; Receptor oligomerization
JNK inhibition arrests cotranslational degradation by Valentina Gandin; Daniela Brina; Pier Carlo Marchisio; Stefano Biffo (pp. 826-831).
Adhesion to fibronectin stimulates protein synthesis (translation) of fibroblasts. Protein synthesis stimulation is dependent from the activation of β1-integrin. β1-Integrin elicits a PI3K cascade that modulates eIF4F (eukaryotic initiation factor 4F) complex formation. In the attempt to further dissect elements of the PI3K cascade that might be responsible for fibronectin-stimulated translation, we used pharmacological inhibitors of known kinases. We found that JNK inhibition, by SP600125 treatment, increased35S-methionine incorporation. Paradoxically, the increase in methionine incorporation was associated to a reduction of initiation of translation. These data imply that, during the adhesion of fibroblasts to fibronectin, conspicuous protein degradation occurs. Indeed, we found that inhibition of the proteasome by MG132 also increased methionine incorporation. Cotranslational degradation depended on PI3K activation. In spite of this, serum promoted translation, but not cotranslational degradation. The crosstalk between translation and degradation was further analyzed by studying the phosphorylation of initiation factors. Briefly, inhibition of JNK leads to eIF2α phosphorylation, which may account for the decrease in initiation of translation. In conclusion, β1-integrin-activated translation causes the synthesis of short-lived proteins, whose degradation is controlled by the JNK pathway. We hypothesize that JNK is a general regulator of cotranslational degradation.
Keywords: eIF2; eIF4E; Fibronectin; Spreading; Proteasome; Polysomes
JNK inhibition arrests cotranslational degradation by Valentina Gandin; Daniela Brina; Pier Carlo Marchisio; Stefano Biffo (pp. 826-831).
Adhesion to fibronectin stimulates protein synthesis (translation) of fibroblasts. Protein synthesis stimulation is dependent from the activation of β1-integrin. β1-Integrin elicits a PI3K cascade that modulates eIF4F (eukaryotic initiation factor 4F) complex formation. In the attempt to further dissect elements of the PI3K cascade that might be responsible for fibronectin-stimulated translation, we used pharmacological inhibitors of known kinases. We found that JNK inhibition, by SP600125 treatment, increased35S-methionine incorporation. Paradoxically, the increase in methionine incorporation was associated to a reduction of initiation of translation. These data imply that, during the adhesion of fibroblasts to fibronectin, conspicuous protein degradation occurs. Indeed, we found that inhibition of the proteasome by MG132 also increased methionine incorporation. Cotranslational degradation depended on PI3K activation. In spite of this, serum promoted translation, but not cotranslational degradation. The crosstalk between translation and degradation was further analyzed by studying the phosphorylation of initiation factors. Briefly, inhibition of JNK leads to eIF2α phosphorylation, which may account for the decrease in initiation of translation. In conclusion, β1-integrin-activated translation causes the synthesis of short-lived proteins, whose degradation is controlled by the JNK pathway. We hypothesize that JNK is a general regulator of cotranslational degradation.
Keywords: eIF2; eIF4E; Fibronectin; Spreading; Proteasome; Polysomes
ErbB4 regulates the timely progression of late fetal lung development by Washa Liu; Erkhembulgan Purevdorj; Katja Zscheppang; Dietlinde von Mayersbach; Jan Behrens; Maria-Jantje Brinkhaus; Heber C. Nielsen; Andreas Schmiedl; Christiane E.L. Dammann (pp. 832-839).
The ErbB4 receptor has an important function in fetal lung maturation. Deletion of ErbB4 leads to alveolar hypoplasia and hyperreactive airways similar to the changes in bronchopulmonary dysplasia (BPD). BPD is a chronic pulmonary disorder affecting premature infants as a consequence of lung immaturity, lung damage, and abnormal repair. We hypothesized that proper ErbB4 function is needed for the timely progression of fetal lung development. An ErbB4 transgenic cardiac rescue mouse model was used to study the effect of ErbB4 deletion on fetal lung structure, surfactant protein (SP) expression, and synthesis, and inflammation. Morphometric analyses revealed a delayed structural development with a significant decrease in saccular size at E18 and more pronounced changes at E17, keeping these lungs in the canalicular stage. SP-B mRNA expression was significantly down regulated at E17 with a subsequent decrease in SP-B protein expression at E18. SP-D protein expression was significantly decreased at E18. Surfactant phospholipid synthesis was significantly decreased on both days, and secretion was down regulated at E18. We conclude that pulmonary ErbB4 deletion results in a structural and functional delay in fetal lung development, indicating a crucial regulatory role of ErbB4 in the timely progression of fetal lung development.
Keywords: Abbreviations; BPD; Bronchopulmonary dysplasia; DCt; Difference in threshold cycle; DSPC; Disaturated phosphatidylcholine; E; Embryonic day; SP; Surfactant protein; HER4; heart−/−; Homozygote ErbB negative transgene; HER4; heart+/−; Heterozygote transgeneTransgenic mouse; Surfactant; Lung structure; Inflammation
ErbB4 regulates the timely progression of late fetal lung development by Washa Liu; Erkhembulgan Purevdorj; Katja Zscheppang; Dietlinde von Mayersbach; Jan Behrens; Maria-Jantje Brinkhaus; Heber C. Nielsen; Andreas Schmiedl; Christiane E.L. Dammann (pp. 832-839).
The ErbB4 receptor has an important function in fetal lung maturation. Deletion of ErbB4 leads to alveolar hypoplasia and hyperreactive airways similar to the changes in bronchopulmonary dysplasia (BPD). BPD is a chronic pulmonary disorder affecting premature infants as a consequence of lung immaturity, lung damage, and abnormal repair. We hypothesized that proper ErbB4 function is needed for the timely progression of fetal lung development. An ErbB4 transgenic cardiac rescue mouse model was used to study the effect of ErbB4 deletion on fetal lung structure, surfactant protein (SP) expression, and synthesis, and inflammation. Morphometric analyses revealed a delayed structural development with a significant decrease in saccular size at E18 and more pronounced changes at E17, keeping these lungs in the canalicular stage. SP-B mRNA expression was significantly down regulated at E17 with a subsequent decrease in SP-B protein expression at E18. SP-D protein expression was significantly decreased at E18. Surfactant phospholipid synthesis was significantly decreased on both days, and secretion was down regulated at E18. We conclude that pulmonary ErbB4 deletion results in a structural and functional delay in fetal lung development, indicating a crucial regulatory role of ErbB4 in the timely progression of fetal lung development.
Keywords: Abbreviations; BPD; Bronchopulmonary dysplasia; DCt; Difference in threshold cycle; DSPC; Disaturated phosphatidylcholine; E; Embryonic day; SP; Surfactant protein; HER4; heart−/−; Homozygote ErbB negative transgene; HER4; heart+/−; Heterozygote transgeneTransgenic mouse; Surfactant; Lung structure; Inflammation
iPLA2, a novel determinant in Ca2+- and phosphorylation-dependent S100A8/A9 regulated NOX2 activity by Véronique Schenten; Brechard Sabrina Bréchard; Plancon Sébastien Plançon; Chantal Melchior; Jean-Pol Frippiat; Eric J. Tschirhart (pp. 840-847).
The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to inflammatory processes, which could lead to the development of diseases such as atherosclerosis. In this paper, we characterize a pathway leading to NOX2 activation in which iPLA2-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. Studies in cell-free or recombinant systems involved two Ca2+-binding proteins of the S100 family, namely S100A8 and S100A9, in NOX2 activation dependent on intracellular Ca2+ concentration ([Ca2+]i) elevation. Using differentiated HL-60 cells as a model of neutrophils, we provide evidence that [Ca2+]i-regulated S100A8/A9 translocation is mediated by an increase in [Ca2+]i through intracellular Ca2+ store depletion. Moreover, we confirm that p38 MAPK induces S100A9 phosphorylation, a mandatory precondition for S100 translocation. Based on a pharmacological approach and an siRNA strategy, we identify iPLA2 as a new molecular player aiding S100 translocation and NOX2 activity. Inhibition of p38 MAPK activity and S100A9 phosphorylation by bromoenol lactone, a selective inhibitor of iPLA2, indicated that p38 MAPK-mediated S100A9 phosphorylation is dependent on iPLA2. In conclusion, we have characterized a pathway leading to NOX2 activation in which iPLA2-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation.
Keywords: Abbreviations; ATF-2; activating transcription factor-2; BAPTA; 1,2-bis(o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid; BEL; bromoenol lactone; [Ca; 2+; ]; i; intracellular Ca; 2+; concentration; DMSO; dimethylsulfoxide; fMLF; N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine; HRP; horseradish peroxidase; iPLA; 2; Ca; 2+; -independent phospholipase A; 2; NOX2; NADPH oxidase; p38 MAPK; p38 mitogen-activated protein kinaseNOX2; S100A8/A9 translocation; p38 MAPK; iPLA; 2; Intracellular Ca; 2+; Neutrophil-like HL-60 cells
iPLA2, a novel determinant in Ca2+- and phosphorylation-dependent S100A8/A9 regulated NOX2 activity by Véronique Schenten; Brechard Sabrina Bréchard; Plancon Sébastien Plançon; Chantal Melchior; Jean-Pol Frippiat; Eric J. Tschirhart (pp. 840-847).
The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to inflammatory processes, which could lead to the development of diseases such as atherosclerosis. In this paper, we characterize a pathway leading to NOX2 activation in which iPLA2-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. Studies in cell-free or recombinant systems involved two Ca2+-binding proteins of the S100 family, namely S100A8 and S100A9, in NOX2 activation dependent on intracellular Ca2+ concentration ([Ca2+]i) elevation. Using differentiated HL-60 cells as a model of neutrophils, we provide evidence that [Ca2+]i-regulated S100A8/A9 translocation is mediated by an increase in [Ca2+]i through intracellular Ca2+ store depletion. Moreover, we confirm that p38 MAPK induces S100A9 phosphorylation, a mandatory precondition for S100 translocation. Based on a pharmacological approach and an siRNA strategy, we identify iPLA2 as a new molecular player aiding S100 translocation and NOX2 activity. Inhibition of p38 MAPK activity and S100A9 phosphorylation by bromoenol lactone, a selective inhibitor of iPLA2, indicated that p38 MAPK-mediated S100A9 phosphorylation is dependent on iPLA2. In conclusion, we have characterized a pathway leading to NOX2 activation in which iPLA2-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation.
Keywords: Abbreviations; ATF-2; activating transcription factor-2; BAPTA; 1,2-bis(o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid; BEL; bromoenol lactone; [Ca; 2+; ]; i; intracellular Ca; 2+; concentration; DMSO; dimethylsulfoxide; fMLF; N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine; HRP; horseradish peroxidase; iPLA; 2; Ca; 2+; -independent phospholipase A; 2; NOX2; NADPH oxidase; p38 MAPK; p38 mitogen-activated protein kinaseNOX2; S100A8/A9 translocation; p38 MAPK; iPLA; 2; Intracellular Ca; 2+; Neutrophil-like HL-60 cells
α9β1 integrin engagement inhibits neutrophil spontaneous apoptosis: Involvement of Bcl-2 family members by Roberta F. Saldanha-Gama; João A. Moraes; Andrea Mariano-Oliveira; Ana Lucia Coelho; Erin M. Walsh; Cezary Marcinkiewicz; Christina Barja-Fidalgo (pp. 848-857).
Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. α9β1 integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how α9β1 integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by α9β1 integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to α9β1 integrin in neutrophils, we demonstrate that α9β1 integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/α9β1 interaction. Moreover we show that VLO5/α9β1 engagement induces NF-κB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-xL. VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of α9β1 integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role.
Keywords: Abbreviations; ADAM; a disintegrin and metalloprotease; ECM; extracellular matrix; Erk; extracellular signal-regulated kinase; FAK; focal adhesion kinase; JNK; c-Jun kinase; NF-κB; nuclear factor-κB; PI3K; phosphatidylinositol-3-kinase; PMN; polymorphonuclear leukocyte; VCAM-1; vascular cell adhesion molecule-1Neutrophil; Apoptosis; Integrin; Disintegrin; Signaling; Bcl-xL
α9β1 integrin engagement inhibits neutrophil spontaneous apoptosis: Involvement of Bcl-2 family members by Roberta F. Saldanha-Gama; João A. Moraes; Andrea Mariano-Oliveira; Ana Lucia Coelho; Erin M. Walsh; Cezary Marcinkiewicz; Christina Barja-Fidalgo (pp. 848-857).
Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. α9β1 integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how α9β1 integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by α9β1 integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to α9β1 integrin in neutrophils, we demonstrate that α9β1 integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/α9β1 interaction. Moreover we show that VLO5/α9β1 engagement induces NF-κB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-xL. VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of α9β1 integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role.
Keywords: Abbreviations; ADAM; a disintegrin and metalloprotease; ECM; extracellular matrix; Erk; extracellular signal-regulated kinase; FAK; focal adhesion kinase; JNK; c-Jun kinase; NF-κB; nuclear factor-κB; PI3K; phosphatidylinositol-3-kinase; PMN; polymorphonuclear leukocyte; VCAM-1; vascular cell adhesion molecule-1Neutrophil; Apoptosis; Integrin; Disintegrin; Signaling; Bcl-xL
A cancer-derived mutation in the PSTAIRE helix of cyclin-dependent kinase 2 alters the stability of cyclin binding by Emma S. Child; Hendrychova Tereza Hendrychová; Karen McCague; Andy Futreal; Michal Otyepka; David J. Mann (pp. 858-864).
Cyclin-dependent kinase 2 (cdk2) is a central regulator of the mammalian cell cycle. Here we describe the properties of a mutant form of cdk2 identified during large-scale sequencing of protein kinases from cancerous tissue. The mutation substituted a leucine for a proline in the PSTAIRE helix, the central motif in the interaction of the cdk with its regulatory cyclin subunit. We demonstrate that whilst the mutant cdk2 is considerably impaired in stable cyclin association, it is still able to generate an active kinase that can functionally complement defective cdks in vivo. Molecular dynamic simulations and biophysical measurements indicate that the observed biochemical properties likely stem from increased flexibility within the cyclin-binding helix.
Keywords: Cyclin-dependent kinase; cdk; PSTAIRE; p21Cip1; p27Kip1; cdc28
A cancer-derived mutation in the PSTAIRE helix of cyclin-dependent kinase 2 alters the stability of cyclin binding by Emma S. Child; Hendrychova Tereza Hendrychová; Karen McCague; Andy Futreal; Michal Otyepka; David J. Mann (pp. 858-864).
Cyclin-dependent kinase 2 (cdk2) is a central regulator of the mammalian cell cycle. Here we describe the properties of a mutant form of cdk2 identified during large-scale sequencing of protein kinases from cancerous tissue. The mutation substituted a leucine for a proline in the PSTAIRE helix, the central motif in the interaction of the cdk with its regulatory cyclin subunit. We demonstrate that whilst the mutant cdk2 is considerably impaired in stable cyclin association, it is still able to generate an active kinase that can functionally complement defective cdks in vivo. Molecular dynamic simulations and biophysical measurements indicate that the observed biochemical properties likely stem from increased flexibility within the cyclin-binding helix.
Keywords: Cyclin-dependent kinase; cdk; PSTAIRE; p21Cip1; p27Kip1; cdc28
Identification of importin α1 as a novel constituent of RNA stress granules by Ken Fujimura; Tomonori Suzuki; Yoshinari Yasuda; Masayuki Murata; Jun Katahira; Yoshihiro Yoneda (pp. 865-871).
Importin α is a nuclear transport receptor well established for its ability to mediate importin β-mediated nuclear import of proteins that possess classical nuclear localization signal (cNLS). Previously, we reported that importin α rapidly accumulates to the nucleus in response to H2O2-induced oxidative stress, which implies a role for this protein in stress response. In this study, we show that importin α1 (also known as KPNA2 or Rch1), a major subtype of the importin α family, localizes to RNA stress granules (SGs), large cytoplasmic bodies that are thought to function as RNA triage sites during stress response. The recruitment of importin α1 to SGs was compatible with its nuclear accumulation during heat shock. Depletion of endogenous importin α1 using siRNA showed that importin α1 regulates the dynamics of SG assembly, and that it promotes cell survival in arsenite-treated cells. These data revealed, for the first time, the involvement of importin α in the assembly of RNA granules and its pro-survival role during stress response.
Keywords: Importin alpha; Stress granule; Stress response
Identification of importin α1 as a novel constituent of RNA stress granules by Ken Fujimura; Tomonori Suzuki; Yoshinari Yasuda; Masayuki Murata; Jun Katahira; Yoshihiro Yoneda (pp. 865-871).
Importin α is a nuclear transport receptor well established for its ability to mediate importin β-mediated nuclear import of proteins that possess classical nuclear localization signal (cNLS). Previously, we reported that importin α rapidly accumulates to the nucleus in response to H2O2-induced oxidative stress, which implies a role for this protein in stress response. In this study, we show that importin α1 (also known as KPNA2 or Rch1), a major subtype of the importin α family, localizes to RNA stress granules (SGs), large cytoplasmic bodies that are thought to function as RNA triage sites during stress response. The recruitment of importin α1 to SGs was compatible with its nuclear accumulation during heat shock. Depletion of endogenous importin α1 using siRNA showed that importin α1 regulates the dynamics of SG assembly, and that it promotes cell survival in arsenite-treated cells. These data revealed, for the first time, the involvement of importin α in the assembly of RNA granules and its pro-survival role during stress response.
Keywords: Importin alpha; Stress granule; Stress response
gp-91 mediates histone deacetylase inhibition-induced cardioprotection by Ting C. Zhao; Ling X. Zhang; Guangmao Cheng; Jun T. Liu (pp. 872-880).
We have recently shown that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia and reperfusion (I/R) injury. The mechanism by which HDAC inhibition induces cardioprotection remains unknown. We sought to investigate whether the genetic disruption of gp-91, a subunit of NADPH-oxidase, would mitigate cardioprotection of HDAC inhibition. Wild-type and gp-91−/− mice were treated with a potent inhibitor of HDACs, trichostatin A (TSA, 0.1mg/kg, i.p.). Twenty-four hours later, the perfused hearts were subjected to 30min of ischemia and 30min of reperfusion. HDAC inhibition in wild-type mice produced marked improvements in ventricular functional recovery and the reduction of infarct size. TSA-induced cardioprotection was eliminated with genetic deletion of gp91. Notably, Western blot and immunostaining displayed a significant increase in gp-91 in myocardium following HDAC inhibition, which resulted in a mildly subsequent increase in the production of reactive oxygen species (ROS). The pre-treatment of H9c2 cardiomyoblasts with TSA (50nmol/l) decreased cell necrosis and increased viability in response to simulated ischemia (SI), which was abrogated by the transfection of cells with gp-91 siRNA, but not by scrambled siRNA. Furthermore, treatment of PLB-985 gp91+/+ cells with TSA increased the resistance to SI, which also diminished with genetic disruption of gp91 in gp91 phox-deficient PLB-985 cells. TSA treatment inhibited the increased active caspase-3 in H9c2 cardiomyoblasts and PLB-985 gp91+/+ cells exposed to SI, which were prevented by knockdown of gp-91 by siRNA. These results suggest that a cascade consisting of gp-91 and HDAC inhibition plays an essential role in orchestrating the cardioprotective effect.
Keywords: Histone deacetylase; gp-91; NADPH-oxidase; Ischemia; Myocardial infarction
gp-91 mediates histone deacetylase inhibition-induced cardioprotection by Ting C. Zhao; Ling X. Zhang; Guangmao Cheng; Jun T. Liu (pp. 872-880).
We have recently shown that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia and reperfusion (I/R) injury. The mechanism by which HDAC inhibition induces cardioprotection remains unknown. We sought to investigate whether the genetic disruption of gp-91, a subunit of NADPH-oxidase, would mitigate cardioprotection of HDAC inhibition. Wild-type and gp-91−/− mice were treated with a potent inhibitor of HDACs, trichostatin A (TSA, 0.1mg/kg, i.p.). Twenty-four hours later, the perfused hearts were subjected to 30min of ischemia and 30min of reperfusion. HDAC inhibition in wild-type mice produced marked improvements in ventricular functional recovery and the reduction of infarct size. TSA-induced cardioprotection was eliminated with genetic deletion of gp91. Notably, Western blot and immunostaining displayed a significant increase in gp-91 in myocardium following HDAC inhibition, which resulted in a mildly subsequent increase in the production of reactive oxygen species (ROS). The pre-treatment of H9c2 cardiomyoblasts with TSA (50nmol/l) decreased cell necrosis and increased viability in response to simulated ischemia (SI), which was abrogated by the transfection of cells with gp-91 siRNA, but not by scrambled siRNA. Furthermore, treatment of PLB-985 gp91+/+ cells with TSA increased the resistance to SI, which also diminished with genetic disruption of gp91 in gp91 phox-deficient PLB-985 cells. TSA treatment inhibited the increased active caspase-3 in H9c2 cardiomyoblasts and PLB-985 gp91+/+ cells exposed to SI, which were prevented by knockdown of gp-91 by siRNA. These results suggest that a cascade consisting of gp-91 and HDAC inhibition plays an essential role in orchestrating the cardioprotective effect.
Keywords: Histone deacetylase; gp-91; NADPH-oxidase; Ischemia; Myocardial infarction