Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Proteomic study of DBA/2J mice retina: Down‐regulation of Integrin β7 correlated with retinal ganglion cell death

To identify and determine the function of the proteins associated with the death of retinal ganglion cells (RGCs) in DBA/2J mice, an animal model of glaucoma, retinas of DBA/2J mice, were analyzed by proteomics at 5‐, 7‐, and 11‐months‐of‐age. The proteins showing significant alterations were selected for identification by MS and 18 proteins were differentially expressed and the identified proteins included cell membrane receptors and proteins associated with intracellular signaling pathways. Among of identified proteins, the expression of Integrin β7 at 7‐months‐of‐age was decreased by about 89% of that at 5‐months‐of‐age. Integrin β7 was expressed in the RGCs. The effect of glutamate toxicity on the expression pattern of Integrin β7 in a RGC line was also investigated and the glutamate‐induced death of RGC was inhibited by the RNA knockdown of Integrin β7. Our data showed also that the expression of 18 proteins in the DBA/2J was significantly altered in DBA2 mice and down‐regulation of Integrin β7 may have a protective effect on glutamate‐induced death of RGCs.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

α6β2*‐subtype nicotinic acetylcholine receptors are more sensitive than α4β2*‐subtype receptors to regulation by chronic nicotine administration

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*‐nAChR are down‐regulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose–responses and quantitative ligand‐binding autoradiography were used to define nicotine sensitivity of changes in α4β2*‐nAChR and α6β2*‐nAChR expression. α6β2*‐nAChR down‐regulation by chronic nicotine exposure in dopaminergic and optic‐tract nuclei was ≈three‐fold more sensitive than up‐regulation of α4β2*‐nAChR. In contrast, nAChR‐mediated [³H]‐dopamine release from dopamine‐terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR‐mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [³H]‐DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function.

     

LPS antagonism of TGF‐β signaling results in prolonged survival and activation of rat primary microglia

Accumulating evidence indicates that activated microglia contribute to the neuropathology involved in many neurodegenerative diseases and after traumatic injury to the CNS. The cytokine transforming growth factor‐beta 1 (TGF‐β1), a potent deactivator of microglia, should have the potential to reduce microglial‐mediated neurodegeneration. It is therefore perplexing that high levels of TGF‐β1 are found in conditions where microglia are chronically activated. We hypothesized that TGF‐β1 signaling is suppressed in activated microglia. We therefore activated primary rat microglia with lipopolysaccharide (LPS) and determined the expression of proteins important to TGF‐β1 signaling. We found that LPS treatment decreased the expression of the TGF‐β receptors, TβR1 and TβR2, and reduced protein levels of Smad2, a key mediator of TGF‐β signaling. LPS treatment also antagonized the ability of TGF‐β to suppress expression of pro‐inflammatory cytokines and to induce microglial cell death. LPS treatment similarly inhibited the ability of the TGF‐β related cytokine, Activin‐A, to down‐regulate expression of pro‐inflammatory cytokines and to induce microglial cell death. Together, these data suggest that microglial activators may oppose the actions of TGF‐β1, ensuring continued microglial activation and survival that eventually may contribute to the neurodegeneration prevalent in chronic neuroinflammatory conditions.

     

β‐catenin regulates IRF3‐mediated innate immune signalling in colorectal cancer

Objective

β‐catenin is one of the most critical oncogenes associated with many kinds of human cancers, especially in the human CRC. Innate immunity recognizes tumour derived damage‐associated molecular patterns (DAMPs) and primes the anti‐tumour adaptive responses. While the function of β‐catenin in CRC tumourigenesis is well established, its impact on innate immune evasion is largely unknown. The aim of this study is to characterize the role of β‐catenin in inhibiting RIG‐I‐like receptor (RLR)‐mediated IFN‐β signalling in colorectal cancer.

Materials and Methods

Immunohistochemical staining and western blotting were conducted to study the expression of β‐catenin, IRF3 and phospho‐IRF3 (p‐IRF3) in CRC samples and cell lines. Plaque assay determining virus replication was performed to assess the regulation of β‐catenin on IFN‐β signalling. The inhibition of β‐catenin on RLR‐mediated IFN‐β signalling was further studied by real‐time analyses and reporter assays in the context of lentiviral‐mediated β‐catenin stably knocking down. Lastly, co‐immunoprecipitation and nuclear fractionation assay were conducted to monitor the interaction between β‐catenin and IRF3.

Results

We found that high expression of β‐catenin positively correlated with the expression of IRF3 in CRC cells. Overexpression of β‐catenin increased the viral replication. Conversely knocking down of β‐catenin inhibited viral replication. Furthermore, our data demonstrated that β‐catenin could inhibit the expression of IFN‐β and interferon‐stimulated gene 56 (ISG56). Mechanistically, we found that β‐catenin interacted with IRF3 and blocked its nuclear translocation.

Conclusion

Our study reveals an unprecedented role of β‐catenin in enabling innate immune evasion in CRC.

     

Mannose 6‐phosphate‐independent Lysosomal Sorting of LIMP‐2

The lysosomal integral membrane protein type 2 (LIMP‐2/SCARB2) has been described as a mannose 6‐phosphate (M6P)‐independent trafficking receptor for β‐glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP‐2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP‐2 with the cation‐independent M6P receptor (MPR) results in M6P‐dependent targeting of LIMP‐2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P‐dependent delivery of LIMP‐2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP‐2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P‐forming N‐acetylglucosamine (GlcNAc)‐1‐phosphotransferase, LIMP‐2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP‐2 levels within lysosomes purified from liver of wild‐type (wt) and GlcNAc‐1‐phosphotransferase‐defective mice. Heterologous expression of the luminal domain of LIMP‐2 in wild‐type, LIMP‐2‐deficient and GlcNAc‐1‐phosphotransferase‐defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP‐2. Finally, cathepsin Z, a known GlcNAc‐1‐phosphotransferase substrate, but not LIMP‐2, could be precipitated with M6P‐specific antibodies. These data prove M6P‐independent lysosomal sorting of LIMP‐2 and subsequently GC in vivo.      

Identification of P‐glycoprotein co‐fractionating proteins and specific binding partners in rat brain microvessels

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P‐glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood‐brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP‐containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post‐translationally regulated at the BBB. The goal of the current study was to identify proteins that co‐localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co‐localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co‐fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post‐translational regulation of PgP activity at the BBB.

     

The role of β‐adrenergic receptors and p38MAPK signaling pathways in physiological processes of cardiosphere‐derived cells

The effects of β adrenergic receptors (β‐ARs) and p38 mitogen‐activated protein kinases (MAPK) pathways on cardiosphere‐derived cells (CDCs) are largely unknown. This study aimed to investigate the roles of β‐ARs and p38MAPK pathways on the proliferation, apoptosis, and differentiation capacity of CDCs. The CDCs were treated with β1‐AR blocker (Met group), β2‐AR antagonist (ICI group), and p38MAPK inhibitor (SB group), non‐selective β‐AR blocker (PRO group), and β‐AR agonist (ISO group). The viability, apoptotic rate and differentiation status of CDCs were determined by MST‐1 assay, flow cytometery, and Western blot, respectively. The CDCs viability significantly reduced in ICI group (all P < 0.05), and SB group had a significant high viability after 48 h treatment (P < 0.05). Compared with control group, all treated groups had a low apoptotic rate. After treatment for 72 h, ISO treatment elevated the expression of Nkx2.5, and could partially or fully attenuate the inhibitory effects of β‐AR antagonists and/or p38MAPK inhibitor. A similar overall trend of protein expression levels among all groups could be observed between protein pairs of cTnT and β1‐AR as well as c‐Kit and β2‐AR, respectively. These results suggested that β‐ARs and p38MAPK signaling pathways play crucial roles in the proliferation and differentiation of CDCs. Our findings should be helpful for better understanding the molecular mechanism underlying the physiological processes of CDCs.     

Differential regulation of the high‐affinity choline transporter by wild‐type and Swedish mutant amyloid precursor protein

The high‐affinity choline transporter (CHT) is responsible for choline uptake into cholinergic neurons, with this being the rate‐limiting step for acetylcholine production. Altering CHT protein disposition directly impacts choline uptake activity and cholinergic neurotransmission. Amyloid precursor protein (APP) interacts with CHT proteins and increases their endocytosis from the cell surface. The goal of this study was to examine regulation of CHT trafficking and activity by wild‐type APP (APP_wt) and determine if this differs with Swedish mutant APP (APP_Swe) in SH‐SY5Y human neuroblastoma cells. APP_Swe differs from APP_wt in its trafficking from the cell surface through endosomes. We report for the first time that CHT interacts significantly less with APP_Swe than with APP_wt. Surprisingly, however, CHT cell surface levels and choline uptake activity are decreased to the same extent and CHT co‐localization to early endosomes increased similarly in cells expressing either APP_wt or APP_Swe. A critical observation is that CHT co‐immunoprecipitates with βCTF from APP_Swe‐expressing cells. We propose that decreased CHT function is mediated differently by APP_wt and APP_Swe; APP_wt interaction with CHT facilitates its endocytosis from the cell surface, whereas the effect of APP_Swe on CHT is mediated indirectly potentially by binding to the βCTF fragment or by Aβ released from cells.

     

Functional analysis and purification of a Pen‐2 fusion protein for γ‐secretase structural studies

The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.

     

Myofibroblasts protect myoblasts from intrinsic apoptosis associated with differentiation via β1 integrin‐PI3K/Akt pathway

Skeletal myoblasts withdrawing from cell cycle is a prerequisite for myodifferentiation, while upon proliferation/differentiation transformation, a large portion of myoblasts will undergo apoptosis. Skeletal fibroblasts, residing in muscle tissue both during and post myogenesis, have been proofed to play pivotal roles in muscle development, while their effect on myoblast apoptosis being coincident with differentiation has not been reported. Using a membrane insert co‐culture system, we studied it and found that the mitochondrial pathway played a crucial role in myoblast apoptosis during differentiation, and fibroblasts promoted not only cell cycle withdrawal but also myoblast survival in a paracrine fashion, which was coupled with upregulations of β1 integrin, phosphorylated Akt and anti‐apoptotic protein Bcl2. To determine the effect of β1 integrin in the process, we transfected myoblasts with siRNA specific for β1 integrin before co‐culture and found that β1 integrin knockdown abolished anti‐apoptotic ability of myoblasts and inhibited Akt activation and Bcl2 expression. Blockage of PI3K/Akt pathway with wortmannin also seriously impaired the protective effect of fibroblasts on myoblasts and fibroblast‐induced Bcl2 expression. The data demonstrated that fibroblasts protected myoblasts from intrinsic apoptosis associated with differentiation, and β1 integrin‐PI3K/Akt pathway activation was required for the process.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Low levels of methyl β‐cyclodextrin disrupt GluA1‐dependent synaptic potentiation but not synaptic depression

Methyl‐β‐cyclodextrin (MβCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol‐enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MβCD. However, how MβCD changes synaptic function, such as N‐methyl‐d ‐aspartate receptor (NMDA‐R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MβCD. At low concentrations (0.5 mg/mL), MβCD decreased basal synaptic transmission and miniature excitatory post‐synaptic current without changing NMDA‐R‐mediated synaptic transmission and the paired‐pulse facilitation ratio. Interestingly, low doses of MβCD failed to deplete cholesterol or affect α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA‐R) and NMDA‐R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MβCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2‐lacking AMPA‐R. MβCD successfully decreased NMDA‐R‐mediated long‐term potentiation but did not affect the formation of either NMDA‐R‐mediated or group I metabotropic glutamate receptor‐dependent long‐term depression. MβCD inhibited de‐depression without affecting de‐potentiation. These results suggest that MβCD regulates GluA1‐dependent synaptic potentiation but not synaptic depression in a cholesterol‐independent manner.