α6 integrin subunit regulates cerebellar development

Mutations in genes encoding several basal lamina components as well as their cellular receptors disrupt normal deposition and remodeling of the cortical basement membrane resulting in a disorganized cerebral and cerebellar cortex. The α6 integrin was the first α subunit associated with cortical lamination defects and formation of neural ectopias. In order to understand the precise role of α6 integrin in the central nervous system (CNS), we have generated mutant mice carrying specific deletion of α6 integrin in neuronal and glia precursors by crossing α6 conditional knockout mice with Nestin-Cre line. Cerebral cortex development occurred properly in the resulting α6^{fl/fl;nestin-Cre} mutant animals. Interestingly, however, cerebellum displayed foliation pattern defects although granule cell (GC) proliferation and migration were not affected. Intriguingly, analysis of Bergmann glial (BG) scaffold revealed abnormalities in fibers morphology associated with reduced processes outgrowth and altered actin cytoskeleton. Overall, these data show that α6 integrin receptors are required in BG cells to provide a proper fissure formation during cerebellum morphogenesis.     

Will the real integrin please stand up?

Recent structure-function studies of integrins are beginning to unravel the mechanism of signal transduction across the plasma membrane, but controversy exists over the nature of the conformational changes underlying this process.     

Targeted inactivation of β1 integrin induces β3 integrin switching,which drives breast cancer metastasis by TGF-β

Mammary tumorigenesis and epithelial–mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin–deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin–specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.     

Multiple non-catalytic ADAMs are novel integrin α4 ligands

The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM–integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these “dead” enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.     

Pro-apoptotic role of integrin β3 in glioma cells

Malignant gliomas are the most destructive type of brain cancer. In order to gain a better understanding of the molecular mechanisms of glioma cell death and survival, we previously established an alkylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant variant of C6 rat glioma cells. Proteomic analysis indicated a significant down-regulation of integrin beta 3 (ITGB3) in the BCNU-resistant C6R cells. Re-expression of ITGB3 in C6R cells restored the BCNU sensitivity. In U87MG, U373MG, and T98G human glioma cells, there was a positive correlation between ITGB3 expression and the sensitivity to BCNU and etoposide, suggesting an important role of ITGB3 in glioma cell death. Over-expression of ITGB3 cDNA significantly increased the sensitivity of the human glioma cells to the anticancer drug-induced apoptosis. Nitric oxide showed an additive effect on the anticancer drug-induced glioma cell death by increasing ITGB3 expression. Subsequent dissection of signaling pathways indicated that extracellular signal-regulated kinase and unligated integrin-mediated cell death pathway may be involved in the pro-apoptotic role of ITGB3 in glioma cells. These results implicate ITGB3 in glioma cell death/survival and drug resistance.     

The roles of integrin β4 in vascular endothelial cells

Integrin heterodimers play diverse and important roles in physiological and pathological processes, such as cell adhesion, migration, proliferation, differentiation, angiogenesis, and tumor progression, via the outside-in and/or inside-out signaling pathways. Aberrant functions of integrins have been implicated in the causation and intervention of multiple diseases. Integrin β(4), a laminin-5 (LN5) receptor, mainly locates in the adhesion structure of hemidesmosome (HD). Most of the previous researches concentrated on the role of integrin β(4) in cancer and cancer therapy, and a few focused on the physiological roles of normal mammalian cells. Recently, accumulating data reveal that integrin β(4) participates in cell death, macroautophagy (hereafter autophagy), senescence, and differentiation regulations in various cell types including human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells, and mouse neural cells, implying the key roles of integrin β(4) in the physiological alteration of mammalian cells. Thus, the elucidation of integrin β(4)-mediated signaling may undoubtedly contribute to novel therapeutic strategies for various human diseases, such as vascular and neural disorders. We have reviewed the roles of integrin β(4) in neural cells. In the present review we will discuss the recent research progress in the inherent functions and pharmacological modulation of integrin β(4) in vascular endothelial cells.     

Discovery of orally available integrin α5β1 antagonists

Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin α5β1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin α5β1 inhibitor scaffolds for potential systemic treatment is described.     

Autophagy modulates cell migration and β1 integrin membrane recycling

Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3^?/? and atg5^?/? MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of β1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized β1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of β1 integrins.     

Mechanical force regulates integrin turnover in Drosophila in vivo

Regulated assembly and disassembly,?or?turnover,?of integrin-mediated cell-extracellular matrix?(ECM)?adhesions is essential for dynamic cell movements?and?long-term tissue maintenance. For example, in Drosophila,?misregulation of integrin turnover disrupts muscle-tendon?attachment at myotendinous junctions (MTJs). We demonstrate that?mechanical force, which modulates integrin activity, also regulates integrin and intracellular adhesion complex (IAC) turnover in vivo. Using conditional mutants to alter the tensile force on MTJs, we found that the proportion of IAC components undergoing turnover inversely correlated with the force applied on MTJs. This effect was disrupted by point mutations in β-integrin that interfere with ECM-induced conformational changes and activation of β-integrin or integrin-mediated cytoplasmic signalling. These mutants also disrupted integrin dynamics at MTJs during larval development. Together, these data suggest that specific β-integrin-mediated signals regulate adhesion turnover in response to tension during tissue?formation. We propose that integrin-ECM adhesive stability is continuously controlled by force in vivo through integrin-dependent auto-regulatory feedback mechanisms so that tissues can quickly adapt to and withstand mechanical stresses.     

Lumican inhibits cell migration through α2β1 integrin

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K_d ≥ 200 nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.     

小菜蛾整合素integrin β1基因的克隆及其功能分析

整合素(Integrin)是一种由α和β亚基组成的跨膜异二聚体蛋白,在昆虫血细胞对外物的包囊过程中起着重要的作用。为阐明integrinβ1参与小菜蛾Plutella xylostella体内细胞免疫中的功能,本研究利用RT-PCR结合RACE技术克隆了小菜蛾integrinβ1基因的cDNA全长序列,命名为PxIntβ1(Gen Bank登录号GQ178290)。小菜蛾PxIntβ1的cDNA全长序列为2 832 bp,开放阅读框为2 487 bp,编码828个氨基酸,成熟的氨基酸序列中有一个跨膜区和一个integrin亚基。系统进化树显示小菜蛾PxIntβ1与亚洲玉米螟Ostrinia furnacalis整合素的同源性最高,两者同源性达到78%。利用半定量RT-PCR技术检测PxIntβ1基因在小菜蛾不同发育历期(卵、1-4龄幼虫、蛹、成虫)、不同组织(血细胞、体壁、脂肪体、中肠、马氏管)和细菌(金黄色葡萄球菌和大肠杆菌混合菌液)诱导下的表达模式。半定量结果表明小菜蛾PxIntβ1基因在小菜蛾整个发育历期除了卵之外都有表达,4龄幼虫转录水平最高,在血细胞中特异性的高表达,混合细菌诱导2-48 h后,PxIntβ1基因在小菜蛾诱导后24 h达到诱导表达高峰。为了进一步获得重组蛋白PxIntβ1,本研究构建了原核表达质粒pET-32a-PxIntβ1,融合蛋白在大肠杆菌Eschericha coli BL21中获得高效表达,Ni-NTA一步纯化了融合蛋白,并免疫新西兰大白兔,获得了多克隆抗体anti-PxIntβ1。SDS-PAGE电泳和Western blot分析结果表明,His抗体和多克隆抗体anti-PxIntβ1能特异性识别融合蛋白PxIntβ1;进一步利用显微镜观察了小菜蛾血细胞对凝胶珠Sephadex-A25的包囊情况,结果表明抗血清anti-PxIntβ1处理过的小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用受到明显抑制,重组蛋白PxIntβ1可以提高小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用;以上研究结果表明小菜蛾PxIntβ1在小菜蛾细胞免疫中起着重要的作用。     

Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin α_IIbβ₃, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin α_IIbβ₃ by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin α_IIbβ₃. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin α_IIbβ₃ and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin α_IIbβ₃ is important to understand the role of platelets in amyloid pathology.     

The α7β1 integrin in muscle development and disease

The alpha7beta1 integrin is a laminin receptor on the surface of skeletal myoblasts and myofibers. Alternative forms of both the alpha7 and beta1 chains are expressed in a developmentally regulated fashion during myogenesis. These different alpha7beta1 isoforms localize at specific sites on myofibers and appear to have distinct functions in skeletal muscle. These functions include the migration and proliferation of developing myoblasts, the formation and integrity of neuromuscular and myotendinous junctions, and the "gluing" together of muscle fibers that is essential to the generation of contractile force. The alpha7beta1 integrin appears to be both directly and indirectly causally related to several muscle diseases. Enhanced expression of alpha7beta1-mediated linkage of the extracellular matrix is seen in Duchenne muscular dystrophy and may compensate for the absence of the dystrophin-mediated linkage. Downregulation of expression of the integrin may contribute to the development of pathology in congenital laminin deficiencies. Mutations in the alpha7 integrin gene underlie additional congenital muscle diseases. The functional roles of this integrin in the formation and stability of the neuromuscular and myotendinous junctions and its localization between fibers suggest that altered expression or function of this integrin may have widespread involvement in other myopathies. The localization of the alpha7 gene at human chromosome 12q13 is a useful clue for focusing such studies.     

Modeling the molecular basis for α4β1 integrin antagonism

We report a 3D QSAR study of almost 300 structurally diverse small molecule antagonists of the integrin α4β1 whose biological activity spans six orders of magnitude. The alignment of the molecules was based on the conformation of a structurally related ligand bound to the αIIBβ3 and αvβ3 integrins in X-ray crystallographic studies. The molecular field method, CoMSIA, was used to generate the 3D QSAR models. The resulting models showed that the lipophilic properties were the most important, with hydrogen bond donor and steric properties less relevant. The models were highly significant (r(2)=0.89, q2(LOO)=0.67, r(2) (test set)=0.76), and could make robust predictions of the data (SEE=0.46, SEP=0.78, SEP (test set)=0.66). We predicted the antagonist activities of a further ten compounds with useful accuracy. The model appears capable of predicting α4β1 integrin antagonist activity to within a factor of five for compounds within its domain of applicability. The implications for design of improved integrin antagonists will be discussed.     

The α6 integrin subunit in the developing mouse olfactory bulb

Integrins are heterodimeric cell surface receptors that mediate developmental events by binding extracellular matrix ligands. Several lines of evidence suggest a role for integrins, specifically the α 6 subunit, in neuronal migration, neurite outgrowth, and axon guidance during olfactory development. Therefore, we undertook an analysis of the expression of the α 6 subunit in the olfactory system of the embryonic and early postnatal mouse to understand the role it may play during neural development. In addition, as a functional assay we examined the developmental effects of the loss of this subunit on olfactory development by analyzing an α 6 knockout (α 6^?/?). Immunohistochemical analyses and confocal microscopy were used to examine α 6 expression in the CD-1 embryonic and early postnatal olfactory system and also to examine the organization of the olfactory system in the α 6^?/? mouse. In CD-1 mice from E13 to E17, α 6 localizes in radial patterns extending from the core of the olfactory bulb to the nerve layer and colocalizes with RC2, an antibody specific for radial glia. By the day of birth (P0; ～E19), expression is limited to the external plexiform layer and the olfactory nerve layer, where it colocalizes with laminin and p75. In the α 6^?/? mouse, areas of ectopic granule cells were observed in the mitral cell layer of the olfactory bulb. These ectopias coincided with areas of disorganization of the radial glial processes and breaks in the mitral cell layer. These observations suggest a role for α 6 integrin in neural migration during olfactory development, likely secondary to organization of the radial glial scaffold.     

Neuritogenesis: the prion protein controls β1 integrin signaling activity

Cytoskeleton modifications are required for neuronal stem cells to acquire neuronal polarization. Little is known, however, about mechanisms that orchestrate cytoskeleton remodeling along neuritogenesis. Here, we show that the silencing of the cellular prion protein (PrP(C)) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrP(C) is necessary to neuritogenesis. Such PrP(C) function relies on its capacity to negatively regulate the clustering, activation, and signaling activity of β1 integrins at the plasma membrane. β1 Integrin aggregation caused by PrP(C) depletion triggers overactivation of the RhoA-Rho kinase-LIMK-cofilin pathway, which, in turn, alters the turnover of focal adhesions, increases the stability of actin microfilaments, and in fine impairs neurite formation. Inhibition of Rho kinases is sufficient to compensate for the lack of PrP(C) and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrP(C)-depleted 1C11 cells, which likely self-sustains β1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP(C) contributes to the acquisition of neuronal polarization by modulating β1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.     

Diacylglycerol kinase α controls RCP-dependent integrin trafficking to promote invasive migration

Inhibition of αvβ3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of α5β1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase α (DGK-α), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent α5β1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-α drove RCP-dependent invasion in the absence of mutant p53 expression or αvβ3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-α is essential to connect expression of mutant p53s or inhibition of αvβ3 to RCP and for this Rab11 effector to drive the trafficking of α5β1 that is required for tumor cell invasion through three-dimensional matrices.     

N-glycosylation of ß4 integrin controls the adhesion and motility of keratinocytes

α6?4 integrin is an essential component of hemidesmosomes and modulates cell migration in wound healing and cancer invasion. To elucidate the role of N-glycosylation on ?4 integrin, we investigated keratinocyte adhesion and migration through the re-expression of wild-type or N-glycosylation-defective ?4 integrin (ΔN?4) in ?4 integrin null keratinocytes. N-glycosylation of ?4 integrin was not essential for the heterodimer formation of ?4 integrin with α6 integrin and its expression on a cell surface, but N-glycosylation was required for integrin-mediated cell adhesion and migration. Concomitantly with the reduction of ?4 integrin in the membrane microdomain, the intracellular signals of Akt and ERK activation were decreased in cells expressing ΔN?4 integrin. Forced cross-linking of ?4 integrin rescued the decreased ERK activation in ΔN?4 integrin-expressing cells to a similar extent in wild-type ?4 integrin-expressing cells. Surprisingly, compared with cells expressing wild-type ?4 integrin, an alternation in N-glycan structures expressed on epidermal growth factor receptor (EGFR), and the induction of a stronger association between EGFR and ?4 integrin were observed in ΔN?4 integrin-expressing cells. These results clearly demonstrated that N-glycosylation on ?4 integrin plays an essential role in keratinocyte cellular function by allowing the appropriate complex formation on cell surfaces.     

Structures and interaction analyses of integrin αMβ2 cytoplasmic tails

Integrins are heterodimeric (α and β subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-specific integrin αMβ2 is essential for myeloid cell adhesion, phagocytosis, and degranulation. In this work, we determined solution structures of the myristoylated cytosolic tail of αM and a Ser phosphorylated variant in dodecylphosphocholine micelles by NMR spectroscopy. Furthermore, the interactions between non-phosphorylated and phosphorylated αM tails with β2 tail were investigated by NMR and fluorescence resonance energy transfer (FRET). The three-dimensional structures of the 24-residue cytosolic tail of αM or phosphorylated αM are characterized by an N-terminal amphipathic helix and a loop at the C terminus. The residues at the loop are involved in packing interactions with the hydrophobic face of the helix. 15N-1H heteronuclear single quantum coherence experiments identified residues of αM and β2 tails that may be involved in the formation of a tail-tail heterocomplex. We further examined interactions between myristoylated β2 tail in dodecylphosphocholine micelles with dansylated αM tail peptides by FRET. These studies revealed enhanced interactions between αM or phosphorylated αM tails with β2 tail with Kd values ～5.2±0.6 and ～4.4±0.7 μm, respectively. Docked structures of tail-tail complexes delineated that the αM/β2 interface at the cytosolic region could be sustained by a network of polar interactions, ionic interactions, and/or hydrogen bonds.     

Distribution of the collagen-binding integrin α10β1 during mouse development

We have previously identified and characterised the collagen type II-binding integrin subunit alpha10, which is a member of the beta1 family and is expressed by chondrocytes. In the present study, we examined the expression of the alpha10 integrin in various mouse tissues. Immunohistochemical analysis of alpha10 on cryosections from 3-day-old mice demonstrated that alpha10beta1 was present in the hyaline cartilage of joints, vertebral column, trachea and bronchi. In addition, alpha10 was found in the ossification groove of Ranvier, in the aortic and atrioventricular valves of the heart and in the fibrous tissue lining skeletal muscle and ligaments. Overall, the distribution was distinct from that of the collagen-binding integrins alpha1beta1 and alpha2beta1. We also found that alpha10beta1was the dominating collagen-binding integrin during cartilage development. Expression of alpha10 appeared at embryonic day 11.5 (E11.5) at the same time as chondrogenesis started as judged by collagen type II expression. At E13.5, alpha10 was present throughout the anlage as well as in the perichondrium and in mesenchyme just outside the perichondrium, where it localised with collagen type I. Four weeks after birth, alpha10 was prominent both at the articular surface and in the growth plate. In conclusion, we found that integrin alpha10beta1 was a major collagen-binding integrin during cartilage development and in mature hyaline cartilage. In addition, we found that alpha10beta1 was present in some fibrous tissues.