Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

In neural stem cells, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote cell proliferation and self-renewal. In the bFGF- and EGF-responsive neural stem cells, β1-integrin also plays important roles in crucial cellular processes, including proliferation, migration, and apoptosis. The cross-talk of the signaling pathways mediated by these growth factors and β1-integrin, however, has not been fully elucidated. Here we report a novel molecular mechanism through which bFGF or EGF promotes the proliferation of mouse neuroepithelial cells (NECs). In the NECs, total β1-integrin expression levels and proliferation were dose-dependently increased by bFGF but not by EGF. EGF rather than bFGF strongly induced the increase of β1-integrin localization on the NEC surface. bFGF- and EGF-induced β1-integrin up-regulation and proliferation were inhibited after treatment with a mitogen-activated protein kinase kinase inhibitor, U0126, which indicates the dependence on the mitogen-activated protein kinase pathway. Involvement of β1-integrin in bFGF- and EGF-induced proliferation was confirmed by the finding that NEC proliferation and adhesion to fibronectin-coated dishes were inhibited by knockdown of β1-integrin using small interfering RNA. On the other hand, apoptosis was induced in NECs treated with RGD peptide, a small β1-integrin inhibitor peptide with the Arg-Gly-Asp motif, but it was independent of β1-integrin expression levels. Those results suggest that regulation of β1-integrin expression/localization is involved in cellular processes, such as proliferation, induced by bFGF and EGF in NECs. The mechanism underlying the proliferation through β1-integrin would not be expected to be completely identical, however, for bFGF and EGF.     

The αvβ6 Integrin Is Transferred Intercellularly via Exosomes

Exosomes, cell-derived vesicles of endosomal origin, are continuously released in the extracellular environment and play a key role in intercellular crosstalk. In this study, we have investigated whether transfer of integrins through exosomes between prostate cancer (PrCa) cells occurs and whether transferred integrins promote cell adhesion and migration. Among others, we have focused on the α_vβ₆ integrin, which is not detectable in normal human prostate but is highly expressed in human primary PrCa as well as murine PrCa in Pten^pc−/− mice. After confirming the fidelity of the exosome preparations by electron microscopy, density gradient, and immunoblotting, we determined that the α_vβ₆ integrin is actively packaged into exosomes isolated from PC3 and RWPE PrCa cell lines. We also demonstrate that α_vβ₆ is efficiently transferred via exosomes from a donor cell to an α_vβ₆-negative recipient cell and localizes to the cell surface. De novo α_vβ₆ expression in an α_vβ₆-negative recipient cell is not a result of a change in mRNA levels but is a consequence of exosome-mediated transfer of this integrin between different PrCa cells. Recipient cells incubated with exosomes containing α_vβ₆ migrate on an α_vβ₆ specific substrate, latency-associated peptide-TGFβ, to a greater extent than cells treated with exosomes in which α_vβ₆ is stably or transiently down-regulated by shRNA or siRNA, respectively. Overall, this study shows that exosomes from PrCa cells may contribute to a horizontal propagation of integrin-associated phenotypes, which would promote cell migration, and consequently, metastasis in a paracrine fashion.     

Gap Junction–mediated Cell–Cell Communication Modulates Mouse Neural Crest Migration

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in α₁ connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for α₁ connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing α₁ connexins, and from α₁ connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative α₁ connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and α₁ connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous α₁ connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and α₁ connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the α₁ connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium— an elevation in the CMV43 mice vs. a reduction in the α₁ connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non–neural crest– derived tissues. Overall, these findings suggest that gap junction communication mediated by α₁ connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of α₁ connexin function.     

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin α_vβ₅-dependent latent transforming growth factor (TGF)-β1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin α_vβ₅, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin α_vβ₅ interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin α_vβ₅-dependent latent TGF-β1 activation and TGF-β1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-α_vβ₅ interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-β1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin α_vβ₅ interactions inhibit contraction-induced latent TGF-β1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-β1 with integrin α_vβ₅. Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.     

Interplay between αvβ3 Integrin and Nucleolin Regulates Human Endothelial and Glioma Cell Migration

The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because α_vβ₃ integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and α_vβ₃ by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing α_vβ₃ and depended on the phosphorylation of β₃ at Tyr⁷⁷³ through receptor protein-tyrosine phosphatase β/ζ (RPTPβ/ζ) and c-Src activation. Downstream of α_vβ_3, PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both α_vβ₃ and RPTPβ/ζ. Positive correlation of cell surface NCL and α_vβ₃ expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing α_vβ₃. Collectively, these data suggest that both expression and β₃ integrin phosphorylation at Tyr⁷⁷³ determine the cell surface localization of NCL downstream of the RPTPβ/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.     

c-Abl Kinase Is a Regulator of αvβ3 Integrin Mediated Melanoma A375 Cell Migration

Integrins are heterodimeric transmembrane receptors that physically link the extracellular matrix (ECM) to the intracellular actin cytoskeleton, and are also signaling molecules that transduce signals bi-directionally across the plasma membrane. Integrin regulation is essential for tumor cell migration in response to growth factors. c-Abl kinase is a nonreceptor tyrosine kinase and is critical for signaling transduction from various receptors. Here we show that c-Abl kinase is involved in A375 cell migration mediated by α_vβ₃ integrin in response to PDGF stimulation. c-Abl kinase colocalizes with α_vβ₃ integrin dynamically and affects α_vβ₃ integrin affinity by regulating its cluster. The interaction between c-Abl kinase and α_vβ₃ integrin was dependent on the activity of c-Abl kinase induced by PDGF stimulation, but was not dependent on the binding of α_vβ₃ integrin with its ligands, suggesting that c-Abl kinase is not involved in the outside-in signaling of α_vβ₃ integrin. Talin head domain was required for the interaction between c-Abl kinase and α_vβ₃ integrin, and the SH3 domain of c-Abl kinase was involved in its interaction with talin and α_vβ₃ integrin. Taken together, we have uncovered a novel and critical role of c-Abl kinase in α_vβ₃ integrin mediated melanoma cell migration.     

The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells

The α_vβ₃ integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the α_vβ₃ integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to α_vβ₃ requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances α_vβ₃ recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt α_vβ₃-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated α_vβ₃ integrins.     

Type IIB Procollagen NH2-propeptide Induces Death of Tumor Cells via Interaction with Integrins αVβ3 and αVβ5

Cartilage is resistant to tumor invasion. In the present study, we found that the NH₂-propeptide of the cartilage-characteristic collagen, type IIB, PIIBNP, is capable of killing tumor cells. The NH₂-propeptide is liberated into the extracellular matrix prior to deposition of the collagen fibrils. This peptide adheres to and kills cells from chondrosarcoma and cervical and breast cancer cell lines via the integrins α_vβ₅ and α_vβ₃. Adhesion is abrogated by blocking with anti α_vβ₅ and α_vβ₃ antibodies. When α_v is suppressed by small intefering RNA, adhesion and cell killing are blocked. Normal chondrocytes from developing cartilage do not express α_vβ₃ and α_vβ₅ integrins and are thus protected from cell death. Morphological, DNA, and biochemical evidence indicates that the cell death is not by apoptosis but probably by necrosis. In an assay for invasion, PIIBNP reduced the number of cells crossing the membrane. In vivo, in a tumor model for breast cancer, PIIBNP was consistently able to reduce the size of the tumor.     

Protogenin prevents premature apoptosis of rostral cephalic neural crest cells by activating the α5β1-integrin

The bones and connective tissues of the murine jaws and skull are partly derived from cephalic neural crest cells (CNCCs). Here, we report that mice deficient of protogenin (Prtg) protein, an immunoglobulin domain-containing receptor expressed in the developing nervous system, have impairments of the palatine and skull. Data from lineage tracing experiments, expression patterns of neural crest cell (NCC) marker genes and detection of apoptotic cells indicate that the malformation of bones in Prtg-deficient mice is due to increased apoptosis of rostral CNCCs (R-CNCCs). Using a yeast two-hybrid screening, we found that Prtg interacts with Radil, a protein previously shown to affect the migration and survival of NCCs in zebrafish with unknown mechanism. Overexpression of Prtg induces translocation of Radil from cytoplasm to cell membrane in cultured AD293 cells. In addition, overexpression of Prtg and Radil activates α5β1-integrins to high-affinity conformational forms, which is further enhanced by the addition of Prtg ligand ERdj3 into cultured cells. Blockage of Radil by RNA interference abolishes the effect of ERdj3 and Prtg on the α5β1-integrin, suggesting that Radil acts downstream of Prtg. Prtg-deficient R-CNCCs display fewer activated α5β1-integrins in embryos, and these cells show reduced migratory ability in in vitro transwell assay. These results suggest that the inside-out activation of the α5β1-integrin mediated by ERdj3/Prtg/Radil signaling is crucial for proper functions of R-CNCCs, and the deficiency of this pathway causes premature apoptosis of a subset of R-CNCCs and malformation of craniofacial structures.     

Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

Background

Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β₂-integrin activation and function in neutrophilic transmigration through endothelium.

Methods and results

Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β₂-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β₂-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.

Conclusion

This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β₂-integrins on the cell surface. Blocking this activation of β₂-integrins might be an important target in cigarette smoke induced neutrophilic diseases.     

Endothelial-Rac1 Is Not Required for Tumor Angiogenesis unless αvβ3-Integrin Is Absent

Endothelial cell migration is an essential aspect of tumor angiogenesis. Rac1 activity is needed for cell migration in vitro implying a requirement for this molecule in angiogenesis in vivo. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. Here we show that depletion of endothelial Rac1 expression in adult mice, unexpectedly, has no effect on tumor growth or tumor angiogenesis. In addition, repression of Rac1 expression does not inhibit VEGF-mediated angiogenesis in vivo or ex vivo, nor does it affect chemotactic migratory responses to VEGF in 3-dimensions. In contrast, the requirement for Rac1 in tumor growth and angiogenesis becomes important when endothelial β3-integrin levels are reduced or absent: the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in β3-null mice are all Rac1-dependent. These data indicate that in the presence of αvβ3-integrin Rac1 is not required for tumor angiogenesis.     

Hypoxia-inducible factor-1α regulates the expression of L-type voltage-dependent Ca2+ channels in PC12 cells under hypoxia

Hypoxia is an important factor in regulation of cell behavior both under physiological and pathological conditions. The mechanisms of hypoxia-induced cell death have not been completely elucidated yet. It is well known that Ca²⁺ is critically related to cell survival. Hypoxia-inducible factor-1α (HIF-1α) is a core regulatory factor during hypoxia, and L-type voltage-dependent Ca²⁺ channels (L-VDCCs) have been reported to play a critical role in cell survival. This study was conducted to explore the relationship between L-VDCC expression and HIF-1α regulation in PC12 cells under hypoxia. PC12 cells were treated at 20 or 3 % O₂ to observe its proliferation and the intracellular calcium concentration. Then, we detected the protein expression of HIF-1α and L-VDCCs subtypes, Ca_v1.2 and Ca_v1.3. At last, to verify the relationship between HIF-1α and Ca_v1.2 and Ca_v1.3, we got the expression of Ca_v1.2 and Ca_v1.3 with Western blot and luciferase report gene assays after PC12 cells were treated by echinomycin, which is an HIF-1α inhibitor. Compared with 20 % O₂ (normoxia), 3 % O₂ (hypoxia) inhibited cell proliferation, increased the intracellular calcium concentration, and induced protein expression of HIF-1α. The protein expression of two L-VDCCs subtypes expressed in the nervous system, Ca_v1.2 and Ca_v1.3, was upregulated by hypoxia and reduced dose dependently by treatment with echinomycin, a HIF-1α inhibitor. Luciferase report gene assays showed that the expression of Ca_v1.2 and Ca_v1.3 genes was augmented under 3 % O₂. However, echinomycin only slightly and dose dependently decreased expression of the Ca_v1.2 gene, but not that of the Ca_v1.3 gene. These data indicated that Ca_v1.2 might be regulated by HIF-1α as one of its downstream target genes and involved in regulation of PC12 cells death under hypoxia.     

Foot-and-Mouth Disease Virus Virulent for Cattle Utilizes the Integrin αvβ3 as Its Receptor

Adsorption and plaque formation of foot-and-mouth disease virus (FMDV) serotype A₁₂ are inhibited by antibodies to the integrin α_vβ₃ (A. Berinstein et al., J. Virol. 69:2664–2666, 1995). A human cell line, K562, which does not normally express α_vβ₃ cannot replicate this serotype unless cells are transfected with cDNAs encoding this integrin (K562-α_vβ₃ cells). In contrast, we found that a tissue culture-propagated FMDV, type O₁BFS, was able to replicate in nontransfected K562 cells, and replication was not inhibited by antibodies to the endogenously expressed integrin α₅β₁. A recent report indicating that cell surface heparan sulfate (HS) was required for efficient infection of type O₁ (T. Jackson et al., J. Virol. 70:5282–5287, 1996) led us to examine the role of HS and α_vβ₃ in FMDV infection. We transfected normal CHO cells, which express HS but not α_vβ₃, and two HS-deficient CHO cell lines with cDNAs encoding human α_vβ₃, producing a panel of cells that expressed one or both receptors. In these cells, type A₁₂ replication was dependent on expression of α_vβ₃, whereas type O₁BFS replicated to high titer in normal CHO cells but could not replicate in HS-deficient cells even when they expressed α_vβ₃. We have also analyzed two genetically engineered variants of type O₁Campos, vCRM4, which has greatly reduced virulence in cattle and can bind to heparin-Sepharose columns, and vCRM8, which is highly virulent in cattle and cannot bind to heparin-Sepharose. vCRM4 replicated in wild-type K562 cells and normal, nontransfected CHO (HS⁺ α_vβ_3⁻) cells, whereas vCRM8 replicated only in K562 and CHO cells transfected with α_vβ₃ cDNAs. A similar result was also obtained in assays using a vCRM4 virus with an engineered RGD→KGE mutation. These results indicate that virulent FMDV utilizes the α_vβ₃ integrin as a primary receptor for infection and that adaptation of type O₁ virus to cell culture results in the ability of the virus to utilize HS as a receptor and a concomitant loss of virulence.     

NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival

The α_vβ₃ integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β₃ integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β₁-integrin ligand) did not induce NF-κB activity. The α_vβ₃ integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β₃ integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in α_vβ₃ integrin-mediated endothelial cell survival.     

Bromoenol Lactone Attenuates Nicotine-Induced Breast Cancer Cell Proliferation and Migration

Objectives

Calcium independent group VIA phospholipase A₂ (iPLA₂β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA₂β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined.

Method and Results

We found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA₂β dependent manner. These results suggest that iPLA₂β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis.

Conclusion

The present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA₂β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis.     

Gene Splicing of an Invertebrate Beta Subunit (LCavβ) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels

The accessory beta subunit (Ca_vβ) of calcium channels first appear in the same genome as Ca_v1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Ca_vβ subunits (β₁, β₂, β₃, β₄) which associate with four Ca_v1 channel isoforms (Ca_v1.1 to Ca_v1.4) and three Ca_v2 channel isoforms (Ca_v2.1 to Ca_v2.3). Here we assess the fundamentally-shared features of the Ca_vβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCa_v1, LCa_v2, and LCa_vβ). Invertebrate Ca_vβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Ca_v2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCa_vβ subunit. LCa_vβ will also slow the inactivation kinetics of LCa_v3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Ca_vβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa_vβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Ca_v2.2 and Ca_vβ₃ subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCa_v2 nor LCa_vβ) appears to be compatible with this observed property.     

Secreted Heat Shock Protein 90α Induces Colorectal Cancer Cell Invasion through CD91/LRP-1 and NF-κB-mediated Integrin αV Expression

HCT-8 colon cancer cells secreted heat shock protein 90α (HSP90α) and had increased invasiveness upon serum starvation. The concentrated conditioned medium of serum-starved HCT-8 cells was able to stimulate the migration and invasion of non-serum-starved cells, which could be prevented by treatment with an anti-HSP90α antibody. Recombinant HSP90α (rHSP90α) also enhanced HCT-8 cell migration and invasion, suggesting a stimulatory role of secreted HSP90α in cancer malignancy. HSP90α binding to CD91α and Neu was evidenced by the proximity ligation assay, and rHSP90α-induced HCT-8 cell invasion could be suppressed by the addition of anti-CD91α or anti-Neu antibodies. Via CD91α and Neu, rHSP90α selectively induced integrin α_V expression, and knockdown of integrin α_V efficiently blocked rHSP90α-induced HCT-8 cell invasion. rHSP90α induced the activities of ERK, PI3K/Akt, and NF-κB p65, but only NF-κB activation was involved in HSP90α-induced integrin α_V expression. Additionally, we investigated the serum levels of HSP90α and the expression status of tumor integrin α_V mRNA in colorectal cancer patients. Serum HSP90α levels of colorectal cancer patients were significantly higher than those of normal volunteers (p < 0.001). Patients with higher serum HSP90α levels significantly exhibited elevated levels of integrin α_V mRNA in tumor tissues as compared with adjacent non-tumor tissues (p < 0.001). Furthermore, tumor integrin α_V overexpression was significantly correlated with TNM (Tumor, Node, Metastasis) staging (p = 0.001).     

Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal

Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to ¹³⁷Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure.     

Bi-transgenic Mice Reveal that K-rasVal12 Augments a p53-independent Apoptosis When Small Intestinal Villus Enterocytes Reenter the Cell Cycle

Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in ~30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-ras^Val12 on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-ras^Val12, with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAg^Wt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-ras^Val12 × TAg^Wt bi-transgenic animals indicated that K-ras^Val12 can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-ras^Val12 is expressed alone or with a TAg containing Glu^107,108→ Lys^107,108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-ras^Val12 × TAg^Wt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAg^Wt mice), and is not associated with detectable changes in the crypt–villus patterns of expression of apoptotic regulators (Bcl-2, Bcl-x_L, Bak, and Bax) or mediators of epithelial cell–matrix interactions and survival (e.g., α₅β₁ integrin and its ligand, fibronectin). Coexpression of K-ras^Val12 and TAg^Wt produces dysplasia. The K-ras^Val12-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-ras^Val12 and a TAg with a COOH-terminal truncation. The dysplastic epithelium of K-ras^Val12 × TAg^Wt mice does not develop neoplasms. Our results are consistent with this finding: (a) When expressed in initiated enterocytes with a proliferative abnormality, K-ras^Val12 facilitates progression to a dysplastic phenotype; (b) by diminishing cell survival on the villus, the oncoprotein may impede further progression; and (c) additional mutations may be needed to suppress this proapoptotic response to K-ras^Val12.