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.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

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.     

Functional α1‐ and β2‐adrenergic receptors in human osteoblasts

Central (hypothalamic) control of bone mass is proposed to be mediated through β2‐adrenergic receptors (β2‐ARs). While investigations in mouse bone cells suggest that epinephrine enhances both RANKL and OPG mRNA via both β‐ARs and α‐ARs, whether α‐ARs are expressed in human bone cells is controversial. The current study investigated the expression of α1‐AR and β2‐AR mRNA and protein and the functional role of adrenergic stimulation in human osteoblasts (HOBs). Expression of α1B‐ and β2‐ARs was examined by RT‐PCR, immunofluorescence microscopy and Western blot (for α1B‐ARs). Proliferation in HOBs was assessed by ³H‐thymidine incorporation and expression of RANKL and OPG was determined by quantitative RT‐PCR. RNA message for α1B‐ and β2‐ARs was expressed in HOBs and MG63 human osteosarcoma cells. α1B‐ and β2‐AR immunofluorescent localization in HOBs was shown for the first time by deconvolution microscopy. α1B‐AR protein was identified in HOBs by Western blot. Both α1‐agonists and propranolol (β‐blocker) increased HOB replication but fenoterol, a β2‐agonist, inhibited it. Fenoterol nearly doubled RANKL mRNA and this was inhibited by propranolol. The α1‐agonist cirazoline increased OPG mRNA and this increase was abolished by siRNA knockdown of α1B‐ARs in HOBs. These data indicate that both α1‐ARs and β2‐ARs are present and functional in HOBs. In addition to β2‐ARs, α1‐ARs in human bone cells may play a role in modulation of bone turnover by the sympathetic nervous system. J. Cell. Physiol. 220: 267–275, 2009. © 2009 Wiley‐Liss, Inc.     

β-Turn conformations in crystal structures of model peptides containing α,α-Di-n-propylglycine and α,α-Di-n-butylglycine

The crystal state conformations of three peptides containing the α,α-dialkylated residues. α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Alu-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II β-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: ? = 66.2°, ψ = 19.3°; III: ? = 66.5°. ψ = 21.1°) deviate appreciably from ideal values for the i + 2 residue in a type II β-turn. In both peptides the observed (N…O) distances between the Boc CO and Ala (3) NH groups are far too long (1: 3.44 Å: III: 3.63 Å) for an intramolecular 4 → 1 hydrogen bond. Boc-Ala-Dpg-Ata-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules HA and HB adopt consecutive β-turn (type III-III in HA and type III-I in IIB) or incipient 3₁₀-helical structures, stabilized by two intramolecular 4 → 1 hydrogen bonds. In all four molecules the bond angle N-C^α-C′ (τ) at the Dxg residues are ≥ 110°. The observation of conformational angles in the helical region of ?,ψ space at these residues is consistent with theoretical predictions. © 1995 John Wiley & Sons, Inc.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

Exploration of the active site of Escherichia coli cystathionine γ‐synthase

Cystathionine γ‐synthase (CGS) catalyzes the condensation of O‐succinyl‐L ‐homoserine (L ‐OSHS) and L ‐cysteine (L ‐Cys), to produce L ‐cystathionine (L ‐Cth) and succinate, in the first step of the bacterial transsulfuration pathway. In the absence of L ‐Cys, the enzyme catalyzes the futile α,γ‐elimination of L ‐OSHS, yielding succinate, α‐ketobutyrate, and ammonia. A series of 16 site‐directed variants of Escherichia coli CGS (eCGS) was constructed to probe the roles of active‐site residues D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361. The effects of these substitutions on the catalytic efficiency of the α,γ‐elimination reaction range from a reduction of only ～2‐fold for R49K and the E325A,Q variants to 310‐ and 760‐fold for R361K and R48K, respectively. A similar trend is observed for the k_cat/K of the physiological, α,γ‐replacement reaction. The results of this study suggest that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in bacterial CGS sequences, tether the distal and α‐carboxylate moieties, respectively, of the L ‐OSHS substrate. In contrast, with the exception of the 13‐fold increase observed for R106A, the K is not markedly affected by the site‐directed replacement of the residues investigated. The decrease in k_cat observed for the S326A variant reflects the role of this residue in tethering the side chain of K198, the catalytic base. Although no structures exist of eCGS bound to active‐site ligands, the roles of individual residues is consistent with the structures inhibitor complexes of related enzymes. Substitution of D45, E325, or Y101 enables a minor transamination activity for the substrate L ‐Ala.     

Expression of α4 and β2 nicotinic acetylcholine receptor subunit mRNA and localization of α-bungarotoxin binding proteins in the rat vestibular periphery

In situ hybridization histochemistry was used to map the distribution of α2, α3, α4, and β2 nAChR subunit mRNAs throughout the peripheral vestibular system of the rat. The α4 and β2 nAChR subunit genes were co-expressed by populations of primary afferent neurons within Scarpa's ganglion, while there was no expression of the α2, α3, α4, or β2 nAChR subunit genes by type I or type II vestibular hair cells. α-bungarotoxin binding to nAChRs in the vestibular end-organs was primarily limited to the afferent chalices surrounding type I hair cells and the basal aspect of type II hair cells. These data suggest that nAChRs composed of α4 and β2 subunits are localized on afferent chalices innervating the type I vestibular hair cells and that the direct cholinergic efferent innervation of the type II vestibular hair cells utilizes nAChR composed of other subunits.     

β-hexosaminidase immunolocalization and α- and β-subunit gene expression in the rat testis and epididymis

β-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G_M2 gangliosidoses. β-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for β-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the α- and β-subunits of β-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. β-hexosaminidase α-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, β-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7–91 postnatal days of age, testis levels of α-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, β-subunit mRNA was expressed at low levels throughout testis development. In isolated male germ cells, β-hexosaminidase α-subunit expression was most abundant in haploid round spermatids, whereas the β-subunit mRNA was not detected in germ cells. Within the epididymis both α- and β-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that β-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, β-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput, and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that β-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of β-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions. Mol. Reprod. Dev. 46:227–242, 1997. © 1997 Wiley-Liss, Inc.     

Activation mechanisms of αVβ3 integrin by binding to fibronectin: A computational study

Integrin αVβ3 plays an important role in regulating cellular activities and in human diseases. Although the structure of αVβ3 has been studied by crystallography and electron microscopy, the detailed activation mechanism of integrin αVβ3 induced by fibronectin remains unclear. In this study, we investigated the conformational and dynamical motion changes of Mn²⁺‐bound integrin αVβ3 by binding to fibronectin with molecular dynamics simulations. Results showed that fibronectin binding to integrin αVβ3 caused the changes of the conformational flexibility of αVβ3 domains, the essential mode of motion for the domains of αV subunit and β3 subunit and the degrees of correlated motion of residues between the domains of αV subunit and β3 subunit of integrin αVβ3. The angle of Propeller domain with respect to the Calf‐2 domain of αV subunit and the angle of Hybrid domain with respect to βA domain of β3 subunit significantly increased when integrin αVβ3 was bound to fibronectin. These changes could result in the conformational change tendency of αVβ3 from a bend conformation to an extended conformation and lead to the open swing of Hybrid domain relative to βA domain of β3 subunit, which have demonstrated their importance for αVβ3 activation. Fibronectin binding to integrin αVβ3 significantly decreased the relative position of α1 helix to βA domain and that to metal ion‐dependent adhesion site, stabilized Mn²⁺ ions binding in integrin αVβ3 and changed fibronectin conformation, which are important for αVβ3 activation. Results from this study provide important molecular insight into the “outside‐in” activation mechanism of integrin αVβ3 by binding to fibronectin.     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.     

Phorbol myristate acetate transactivates the avian β3 integrin gene and induces αvβ3 integrin expression

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) transactivates the avian β₃ integrin gene whose promoter contains at least two vitamin D response elements, one of which is in close proximity to a candidate AP1 site (TGACTCA). Since fos/jun and steroid hormones interact to regulate gene expression, we asked whether phorbol-12-myristate-13-acetate (PMA), which stimulates binding of fos/jun to AP1 sites, transactivates the avian β₃ integrin gene and, if so, does the phorbol ester modulate 1,25(OH)₂D₃ induction of the gene. We find the candidate AP1 sequence comigrates with the consensus AP1 sequence on electromobility shift assay when incubated with recombinant c-jun protein. Furthermore, PMA prompts expression of β₃ integrin mRNA in the avian monocytic line, HD11. The increase in message reflects transactivation of the β₃ gene and is mirrored by plasma membrane appearance of the integrin heterodimer α_vβ₃. Moreover, attesting to the functional significance of PMA-enhanced α_vβ₃ expression, cells treated with concentrations of the phorbol ester that induce the β₃ gene, spread extensively on plastic, an event blocked by an anti-α_v antibody and a peptide mimetic known to inhibit α_vβ₃-mediated cell attachment. Interestingly, co-addition of 1.25(OH)₂D₃ and PMA prompts greater expression of α_vβ₃ than when the cells are exposed to either agent alone and PMA enhances 1,25(OH)₂D₃-induced β₃ integrin mRNA expression. Thus, PMA and 1,25(OH)₂D₃ impact on the avian β₃ integrin gene independently and in combination. © 1996 Wiley-Liss, Inc.     

CHARACTERIZATION OF THE Na+K+-ATPase IN ISOLATED BOVINE ARTICULAR CHONDROCYTES; MOLECULAR EVIDENCE FOR MULTIPLE α AND β ISOFORMS

We have used isoform-specific antibodies against the Na⁺K⁺-ATPase αα1, α2 and α3) and ββ1 and β2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the α1 and α3 isoforms, although α1 expression was significantly greater than α3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the β1 and β2 isoforms in chondrocytes, although β2 immunostaining on the plasma membrane was more punctate than β1 which in contrast predominated in a subcellular compartment. The plasma membrane abundance of the Na⁺K⁺-ATPase was found to be sensitive to the extracellular ionic concentration and long-term elevation of extracellular Na⁺concentration significantly upregulated Na⁺K⁺-ATPase density as measured by specific³H-ouabain binding. Our observations suggest that the expression of α3 and β2 is not restricted to excitable tissues as previously reported. The physiological relevance of α3 expression in chondrocytes may be related to its low affinity for intracellular Na⁺in an extracellular environment where Na⁺concentration is unusually high (260–350mm) compared to other cell types (140mm). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the β2 subunit glycoprotein may allow the chondrocyte to detect changes in its extracellular environment by physically interacting with components of the cellular cytoskeleton and matrix macromolecules.     

Characterization of a Cerebellar Granule Cell-Specific Gene Encoding the γ-Aminobutyric Acid Type A Receptor α6 Subunit

Abstract: The α6 subunit of γ-aminobutyric type A receptors is a marker for cerebellar granule cells and is an attractive candidate to study cell-specific gene expression in the brain. The mouse α6 subunit gene has nine exons and spans ～14 kb. The largest intron (intron 8) is ～7 kb. For a minority of mRNAs, a missplice of the first exon was identified that disrupts the signal peptide and most likely results in the production of nonfunctional protein. The gene is transcribed from a TATA-less promoter that uses multiple start sites. Using transgenic mice, it was found that the proximal 0.5 kb of the rat α6 gene upstream region confers expression on a β-galactosidase reporter gene. One founder gave rise to a line with cerebellar granule cell-specific expression, although expression varied with lobule region. Other founders had ectopic but neuron-specific expression, with β-galactosidase found in cerebellar Purkinje cells, neocortex, thalamus, hippocampus, caudate-putamen, and inferior colliculi. Thus, we have defined a region containing the basal promoter of the α6 subunit gene and that confers neuron-specific expression.     

Impaired integrin α5/β1‐mediated hepatocyte growth factor release by stellate cells of the aged liver

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α₅/β₁ is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α₅ and β₁ decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α₅ and β₁ knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α₅ and laminin gene expression whereas integrin β₁ remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α₅ knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α₅/β₁ in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.     

β-Bungarotoxin antagonizes the effect of α-latrotoxin from black widow spider venom on the neuromuscular junction

Sustained contraction of the chick biventer cervicis nerve-muscle preparations evoked by α-latrotoxin was antagonized quickly by β-bungarotoxin. This effect of β-bungarotoxin was dependent on its phospholipase A₂ activity. In contrast, pancreatic phospholipase A₂ was ineffective even at a much higher dose. It is concluded that α-latrotoxin needs intact presynaptic membrane to exert its effect.