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Cellular fibronectin enhances hepatic stellate cell migration
S46
Abstracts
II collagen was present on porcine ACVs. Human ACVs contained easily measurable quantities of type II collagen which was of higher molecular weight in old human ACVs. Conclusions ACVs contain crucial collagen receptors. Increased quantities of type II collagen in human ACVs may explain the seemingly paradoxical mineralization pattern in isolated ACVs from human cartilage. doi:10.1016/j.matbio.2008.09.364
ylation and an overall reduction in cellular phosphotyrosine levels consistent with the loss of FAK activity. FAK-related Pyk2 expression and phosphorylation is similar in KD FAK and WT MEFs. Time-lapse and indirect fluorescent microscopy revealed that KD FAK MEFs initially spread normally on fibronectin, but form an over-abundant focal adhesions and exhibit elevated numbers of membrane protrusions within 1–2 h. These morphological alterations of KD FAK MEFs are associated with severe motility defects. Together, our results support the notion that KD FAK embryo lethality is associated with defects in vascular cell morphogenesis and that FAK activity is not essential for cell survival. Our studies highlight a differential role for FAK in cell migration-survival. doi:10.1016/j.matbio.2008.09.366
150 Cellular fibronectin enhances hepatic stellate cell migration Abby L. Olsena,b, Rebecca G. Wellsa,c a University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States b Cellular and Molecular Biology Graduate Program, United States c Department of Medicine, Division of Gastroenterology, United States Fibronectins regulate diverse cell behaviors via integrin signaling. Cellular fibronectin (cFN) can contain two alternatively spliced domains, extra domain A (EDA) and B (EDB), which are excluded from plasma fibronectin (pFN). EDA- and EDB-containing isoforms of cFN are upregulated during liver fibrosis, in which EDA has been reported to promote myofibroblastic differentiation of hepatic stellate cells (HSC). We hypothesized that EDA and EDB influence HSC behavior by altering integrin signaling. To model myofibroblastic differentiation, we cultured HSC on plastic or polyacrylamide supports coated with pFN or cFN. HSC grown on cFN had decreased organization of αSMA into stress fibers and increased lamellipodia, suggestive of enhanced cell motility. This motility difference was confirmed by transwell migration assays, in which HSC plated on cFN-coated inserts had increased baseline chemotaxis toward serum as well as a greater enhancement of migration in response to treatment with PDGF. HSC grown on cFN also demonstrated greater expression of the integrin signaling mediators phosphoFAK and phosphopaxillin, and smaller but more numerous focal adhesions. These effects were further augmented by treatment with PDGF. Collectively, these data demonstrate a clear role for cFN in promoting HSC motility and suggest that this effect may be due to changes in integrin signaling that occur via synergy with PDGF. Thus, in concert with PDGF, cFN may contribute to the pathology of liver fibrosis by promoting HSC migration to areas of injury within the liver.
152 Extracellular ligands involved in learning and memory Richard G. LeBarona, Amber M. Ashera, David A. Medinaa, Mary M. Navarroa, Maria V. Tejada-Simonb a Department of Biology, University of Texas, San Antonio, TX 78249, United States b Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, United States Models describing mechanisms of CNS synaptic plasticity increasingly include cell-surface adhesion receptors as important molecules in long-term potentiation (LTP). LTP is an activity-dependent prolonged increase in synaptic transmission that represents a cellular substrate for some forms of learning and memory. In rat CA1 excitatory synapses, integrin receptors are crucial for the maintenance of LTP, thereby implicating integrin ligands in important complement roles. However, integrin ligands that are crucial for hippocampal LTP have not been definitively identified. To test for ligands, substrata derived from preparations of mouse hippocampal neurosynaptosomes were probed with cells that express the integrins that play roles in hippocampal LTP. Proteins within the molecular mass ranges of 50–60 kDa and 70–80 kDa sustained cell attachment. Peptide mass fingerprints of the regions revealed candidate integrin ligands. In a complement approach, the chemical Bis(Sulfosuccinimidyl) suberate was used in rat hippocampal slices to crosslink integrin β1 subunits to their near-neighbor proteins. Peptide mass fingerprints of crosslinked β1 pull-downs identified proteins that were detected by cell probe analysis. Candidate ligands include molecules of the ECM and cell surface proteins that will be applied to rat hippocampal slices to determine whether field excitatory post-synaptic potentials are diminished, thereby showing functionally that the candidate ligands operate in slice preparations of CA1 LTP.
doi:10.1016/j.matbio.2008.09.365 doi:10.1016/j.matbio.2008.09.367
151 FAK activity is essential for vascular development Ssang-Taek Lim, Xiao Lei Chen, Alok Tomar, Nichol L. Miller, David D. Schlaepfer UCSD Moores Cancer Center, La Jolla, CA, United States Extracellular matrix regulates cellular architecture during development via integrin activation. Integrins transmit intracellular signals via activation of protein tyrosine kinases such as focal adhesion kinase (FAK), a key mediator of cell motility and survival. FAK deletion during development results in p53-dependent growth arrest, we found FAK promotes cell survival in a kinase-independent manner through FAK FERM-mediated p53 regulation. To test whether FAK activity is important during development, we created a kinase-dead (KD) FAK knockin mouse by homologous recombination. Homozygous KD FAK is lethal at E9.5 with irregular yolk sac and embryo vascular structures and associated hemorrhage. However, in contrast to FAK-null cells that exhibit p53dependent growth arrest, primary KD FAK cells grow in culture. KD FAK mouse embryonic fibroblasts (MEFs) exhibit reduced FAK Y397 phosphor-
153 Sarcoma MMP expression is influenced by integrin-ECM interactions Adriana Roopnarianeb, Rajeev Boregowdab, Brooke Krovicb, Timothy M. Rittya,b a Penn State Cancer Institute, United States b Orthopaedics, Penn State College of Medicine, Hershey, PA 17030, United States During metastasis, osteosarcoma cells encounter a variety of microenvironments. Matrix metalloproteinases (MMPs) are known to have multiple functions that include facilitation of cell migration. We hypothesize that metastasizing osteosarcoma cells alter MMP production in response to their integrin-perceived extracellular environment. To test this hypothesis, we have quantified the protein levels of seven secreted MMPs produced after human MG63 osteosarcoma cells attach to specific ECM proteins. MG63 cells specifically down-regulate production of MMP1 and MMP3 after attachment to type I collagen and fibronectin. Further, we have selectively down-regulated expression of integrin subunits alpha 2, 5, V and beta 3 using siRNA produced by stably transfected plasmids we
Abstracts
II collagen was present on porcine ACVs. Human ACVs contained easily measurable quantities of type II collagen which was of higher molecular weight in old human ACVs. Conclusions ACVs contain crucial collagen receptors. Increased quantities of type II collagen in human ACVs may explain the seemingly paradoxical mineralization pattern in isolated ACVs from human cartilage. doi:10.1016/j.matbio.2008.09.364
ylation and an overall reduction in cellular phosphotyrosine levels consistent with the loss of FAK activity. FAK-related Pyk2 expression and phosphorylation is similar in KD FAK and WT MEFs. Time-lapse and indirect fluorescent microscopy revealed that KD FAK MEFs initially spread normally on fibronectin, but form an over-abundant focal adhesions and exhibit elevated numbers of membrane protrusions within 1–2 h. These morphological alterations of KD FAK MEFs are associated with severe motility defects. Together, our results support the notion that KD FAK embryo lethality is associated with defects in vascular cell morphogenesis and that FAK activity is not essential for cell survival. Our studies highlight a differential role for FAK in cell migration-survival. doi:10.1016/j.matbio.2008.09.366
150 Cellular fibronectin enhances hepatic stellate cell migration Abby L. Olsena,b, Rebecca G. Wellsa,c a University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States b Cellular and Molecular Biology Graduate Program, United States c Department of Medicine, Division of Gastroenterology, United States Fibronectins regulate diverse cell behaviors via integrin signaling. Cellular fibronectin (cFN) can contain two alternatively spliced domains, extra domain A (EDA) and B (EDB), which are excluded from plasma fibronectin (pFN). EDA- and EDB-containing isoforms of cFN are upregulated during liver fibrosis, in which EDA has been reported to promote myofibroblastic differentiation of hepatic stellate cells (HSC). We hypothesized that EDA and EDB influence HSC behavior by altering integrin signaling. To model myofibroblastic differentiation, we cultured HSC on plastic or polyacrylamide supports coated with pFN or cFN. HSC grown on cFN had decreased organization of αSMA into stress fibers and increased lamellipodia, suggestive of enhanced cell motility. This motility difference was confirmed by transwell migration assays, in which HSC plated on cFN-coated inserts had increased baseline chemotaxis toward serum as well as a greater enhancement of migration in response to treatment with PDGF. HSC grown on cFN also demonstrated greater expression of the integrin signaling mediators phosphoFAK and phosphopaxillin, and smaller but more numerous focal adhesions. These effects were further augmented by treatment with PDGF. Collectively, these data demonstrate a clear role for cFN in promoting HSC motility and suggest that this effect may be due to changes in integrin signaling that occur via synergy with PDGF. Thus, in concert with PDGF, cFN may contribute to the pathology of liver fibrosis by promoting HSC migration to areas of injury within the liver.
152 Extracellular ligands involved in learning and memory Richard G. LeBarona, Amber M. Ashera, David A. Medinaa, Mary M. Navarroa, Maria V. Tejada-Simonb a Department of Biology, University of Texas, San Antonio, TX 78249, United States b Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, United States Models describing mechanisms of CNS synaptic plasticity increasingly include cell-surface adhesion receptors as important molecules in long-term potentiation (LTP). LTP is an activity-dependent prolonged increase in synaptic transmission that represents a cellular substrate for some forms of learning and memory. In rat CA1 excitatory synapses, integrin receptors are crucial for the maintenance of LTP, thereby implicating integrin ligands in important complement roles. However, integrin ligands that are crucial for hippocampal LTP have not been definitively identified. To test for ligands, substrata derived from preparations of mouse hippocampal neurosynaptosomes were probed with cells that express the integrins that play roles in hippocampal LTP. Proteins within the molecular mass ranges of 50–60 kDa and 70–80 kDa sustained cell attachment. Peptide mass fingerprints of the regions revealed candidate integrin ligands. In a complement approach, the chemical Bis(Sulfosuccinimidyl) suberate was used in rat hippocampal slices to crosslink integrin β1 subunits to their near-neighbor proteins. Peptide mass fingerprints of crosslinked β1 pull-downs identified proteins that were detected by cell probe analysis. Candidate ligands include molecules of the ECM and cell surface proteins that will be applied to rat hippocampal slices to determine whether field excitatory post-synaptic potentials are diminished, thereby showing functionally that the candidate ligands operate in slice preparations of CA1 LTP.
doi:10.1016/j.matbio.2008.09.365 doi:10.1016/j.matbio.2008.09.367
151 FAK activity is essential for vascular development Ssang-Taek Lim, Xiao Lei Chen, Alok Tomar, Nichol L. Miller, David D. Schlaepfer UCSD Moores Cancer Center, La Jolla, CA, United States Extracellular matrix regulates cellular architecture during development via integrin activation. Integrins transmit intracellular signals via activation of protein tyrosine kinases such as focal adhesion kinase (FAK), a key mediator of cell motility and survival. FAK deletion during development results in p53-dependent growth arrest, we found FAK promotes cell survival in a kinase-independent manner through FAK FERM-mediated p53 regulation. To test whether FAK activity is important during development, we created a kinase-dead (KD) FAK knockin mouse by homologous recombination. Homozygous KD FAK is lethal at E9.5 with irregular yolk sac and embryo vascular structures and associated hemorrhage. However, in contrast to FAK-null cells that exhibit p53dependent growth arrest, primary KD FAK cells grow in culture. KD FAK mouse embryonic fibroblasts (MEFs) exhibit reduced FAK Y397 phosphor-
153 Sarcoma MMP expression is influenced by integrin-ECM interactions Adriana Roopnarianeb, Rajeev Boregowdab, Brooke Krovicb, Timothy M. Rittya,b a Penn State Cancer Institute, United States b Orthopaedics, Penn State College of Medicine, Hershey, PA 17030, United States During metastasis, osteosarcoma cells encounter a variety of microenvironments. Matrix metalloproteinases (MMPs) are known to have multiple functions that include facilitation of cell migration. We hypothesize that metastasizing osteosarcoma cells alter MMP production in response to their integrin-perceived extracellular environment. To test this hypothesis, we have quantified the protein levels of seven secreted MMPs produced after human MG63 osteosarcoma cells attach to specific ECM proteins. MG63 cells specifically down-regulate production of MMP1 and MMP3 after attachment to type I collagen and fibronectin. Further, we have selectively down-regulated expression of integrin subunits alpha 2, 5, V and beta 3 using siRNA produced by stably transfected plasmids we