Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1

The activity of integrin LFA-1 (α_Lβ₂) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of α_L chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57''s strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57''s antagonistic mimicry of LFA-1''s natural ligands, the ICAM molecules.     

Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence.

Insulin-like growth factor (IGF)-binding protein 1 (IGFBP-1) contains an Arg-Gly-Asp (RGD) integrin recognition sequence. In vitro mutagenesis was used to alter this RGD sequence to Trp-Gly-Asp (WGD). Migration of Chinese hamster ovary (CHO) cells expressing the wild-type protein was more than 3-fold greater in 48 hr compared with cells expressing the WGD mutant form of IGFBP-1. Similarly, wild-type IGFBP-1 added to the media of control CHO cells stimulated migration 2-fold compared with the WGD protein. A synthetic RGD-containing peptide, when added to the medium with wild-type IGFBP-1, blocked the effect of IGFBP-1 on cell migration. The addition of IGF-I to the culture medium had no effect on the migration of cells expressing IGFBP-1 or vector alone. Affinity chromatography of 125I-labeled CHO cell membrane proteins, using IGFBP-1 coupled to agarose, identified the alpha 5 beta 1 integrin (fibronectin receptor) as the only cell surface molecule capable of binding IGFBP-1 in an RGD-dependent manner. Furthermore, wild-type IGFBP-1, but not the WGD mutant form, could be coprecipitated from CHO cells with an antibody directed against the alpha 5 integrin subunit. These studies demonstrate that IGFBP-1 stimulates CHO cell migration and binds to the alpha 5 beta 1 integrin receptor, both by an RGD-dependent mechanism. The effect of IGFBP-1 on migration is independent of IGF-I and is probably mediated through the alpha 5 beta 1 integrin.     

An atomic resolution view of ICAM recognition in a complex between the binding domains of ICAM-3 and integrin alphaLbeta2

Within the Ig superfamily (IgSF), intercellular adhesion molecules (ICAMs) form a subfamily that binds the leukocyte integrin alphaLbeta2. We report a 1.65-A-resolution crystal structure of the ICAM-3 N-terminal domain (D1) in complex with the inserted domain, the ligand-binding domain of alphaLbeta2. This high-resolution structure and comparisons among ICAM subfamily members establish that the binding of ICAM-3 D1 onto the inserted domain represents a common docking mode for ICAM subfamily members. The markedly different off-rates of ICAM-1, -2, and -3 appear to be determined by the hydrophobicity of residues that surround a metal coordination bond in the alphaLbeta2-binding interfaces. Variation in composition of glycans on the periphery of the interfaces influences on-rate.     

EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt

The cadherin-catenin complex is the major machinery for cell-cell adhesion in many animal species. This complex in general associates with actin fibers at its cytoplasmic side, organizing the adherens junction (AJ). In epithelial cells, the AJ encircles the cells near their apical surface and forms the "zonula adherens" or "adhesion belt." The mechanism as to how the cadherin-catenin complex and F-actin cooperate to generate these junctional structures, however, remains unknown. Here, we show that EPLIN (epithelial protein lost in neoplasm; also known as Lima-1), an actin-binding protein, couples with alpha-catenin and, in turn, links the cadherin-catenin complex to F-actin. Without EPLIN, this linkage was unable to form. When EPLIN had been depleted in epithelial cells, the adhesion belt was disorganized and converted into zipper-like junctions in which actin fibers were radially arranged. However, nonjunctional actin fibers were not particularly affected by EPLIN depletion. As EPLIN is known to have the ability to suppress actin depolymerization, our results suggest that EPLIN functions to link the cadherin-catenin complex to F-actin and simultaneously stabilizes this population of actin fibers, resulting in the establishment of the adhesion belt.     

Adhesion characteristics of porcine pancreatic islets and exocrine tissue to coating materials

Since the report of the Edmonton protocol in 2000, islet transplantation has been implemented worldwide, and xenotransplantation using porcine islets has also been reported. In addition, many basic experiments using pancreatic islets and exocrine tissue after isolation have been reported. Recently, exocrine cells have been found to be essential for inducing the differentiation of pancreatic islets. Therefore, the importance of the culture conditions for pancreatic tissue when conducting experiments using pancreatic tissue is also increasing. In this study, we focused on the coat material and examined the adhesive properties of porcine pancreatic islets and exocrine tissue after isolation. Porcine islet isolation was performed, and isolated islets (purity ≥95%) and exocrine tissue (purity ≥99%) were used to achieve adhesion to several extracellular matrixes, fibronectin, collagen type I, collagen type IV, laminin I, fibrinogen, and bovine serum albumin (BSA). DMEM with 0.5% FBS was used as the assay medium. For exocrine tissue, the adhesion was promoted in fibronectin, collagen type I, laminin I, and fibrinogen. The adhesive ability to fibronectin was more than twice that to BSA, while the adhesive ability to collagen type I, laminin I, and fibrinogen was less than twice that to BSA. For islets, the adhesive ability to fibronectin was weaker than that of exocrine tissue. Furthermore, the adhesion effect in fibronectin was obtained within 30 minutes and in medium containing little serum for both islets and exocrine tissues. These data suggest that fibronectin may be useful for the adhesion of pancreatic tissue.     

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue–ECM boundary, rather than by differential homo- and heterotypic energies of cell–cell interaction. Surprisingly, interactions with the tissue–ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell–cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell–cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell–ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.Self-organization is a process that contributes to pattern formation and repair at all scales of biological complexity. At the tissue scale, defining robust strategies of self-organization is critical for engineering functional tissues, as well as for understanding development and the breakdown of tissue structure during diseases such as cancer (1). During development, two or more populations of motile cells can self-organize into spatially ordered tissues by a process referred to as cell sorting (2–4). The outcome of cell sorting can be rationalized using physical models that invoke cell-type-specific differences in interfacial energies. Interfacial energies arise through the action of a contractile cell cortex coupled to adhesion molecules (e.g., cadherins) that link the cortices of neighboring cells and signal to modulate cortical tension at specific cellular interfaces (5). In general, the organization of a tissue after cell sorting corresponds to a configuration that maximizes the formation of the most energetically favorable (hereafter referred to as most cell–cell cohesive)^† cellular interfaces (6). For example, with an intermediate level of heterotypic cell–cell cohesion the most self-cohesive cell type is typically found in the tissue core, with the less cohesive cell type spread around the tissue surface (Fig. 1A). In order for self-organization to proceed robustly by this strategy, a tissue requires and must maintain a clearly delineated hierarchy of homo- and heterotypic cell–cell cohesive interactions.Open in a separate windowFig. 1.A self-generated and binary adhesive interaction directs cell positioning in the mammary epithelium. (A) Self-organization of two initially disordered populations of cells (Center) into spatially ordered tissues. In the mammary gland, the correct architecture (Right) can go on to polarize and form a lumen. (B) Self-organization of fourth-passage primary human mammary epithelial cells in agarose (Left) and Matrigel (Right) after 24 h. In Matrigel, the reconstituted microtissue can also polarize and form a lumen over an additional 72 h (MEP, red, keratin-14/K14; LEP, green, keratin-19/K19; blue, DAPI/nuclei). (C) Experiments as in B but with MEP and LEP stained before self-organization with CellTracker Red (CTR) and CellTracker Green (CTG), respectively. (Insets) Average intensity profiles under each condition (n = 30). (D) Frequency of indicated tissue architectures for experiments in C (n > 235). (E) Representative images and average intensity plots of CellTracker-labeled MEP and LEP self-organized in Col1-functionalized agarose (n = 30) and unfunctionalized PDMS microwells (n = 20). (F) Conceptual model for self-organization by a self-generated adhesive interaction at the tissue–ECM boundary. (Inset) An image of MEP on an unfunctionalized PDMS surface (dotted line, PDMS; yellow, fibronectin-1; red, actin; blue, nuclei). (G) Representative images of cell doublets and XZ sections of single cells after 4 h on Matrigel-coated substrate (green, CTG; red, CTR; purple, QD605). (H) Distribution of measured contact angles at all interfaces (n > 42). (I) Representative images of aggregates of homogeneous MEP and LEP after 12 h in agarose wells. (J) Aggregates prepared as in G but subsequently transferred to Matrigel-coated glass for 12 h (green, K19; red, K14). Error bars are SD. (Scale bars, 10 µm.)Regulated differences in cell–cell cohesion are also thought to contribute to the self-organization and repair of adult human secretory organs such as the mammary gland (7, 8). The mammary gland, along with the prostate, salivary, lacrimal, and sweat glands, has an architecture comprising two concentrically arranged epithelial cell types as shown in Fig. 1A. However, the cells in the mammary gland dynamically regulate their cohesivity and motility to serve specialized roles at different locations and at different times. For example, the inner luminal (LEP) and outer myoepithelial (MEP) cells are known to undergo physical and chemical changes throughout development, menstrual cycles, pregnancy, involution, and the early stages of malignant disease. However, experiments using the mouse as a model system indicate that at the terminal end bud, where the lumen is filled and cell motility and rearrangements are elevated, cell positioning is rarely lost (9, 10). Moreover, deletion of key cell–cell adhesion proteins such as E- and P-cadherin has no gross effect on MEP or LEP cell positioning in the developing mouse mammary gland (11, 12). This is surprising given the established role of cadherins in guiding cell positioning through cell sorting. How self-organization remains robust to such severe changes to cell–cell cohesion remains unclear.Adult tissues also comprise populations of cells that can be heterogeneous in their molecular and physical properties (13), and the mammary gland is a prototypical example of a heterogeneous tissue, possessing considerable spatial and temporal variability within both the inner luminal and outer myoepithelial populations. For example, neighboring cells in healthy tissue can differ markedly with respect to their expression of adhesion molecules, cytoskeletal proteins, hormone receptors, and the activation of specific signaling pathways (14–17). Such heterogeneity can affect the distribution of cell–cell cohesive properties among different cell types, thus confounding the ordered hierarchy of interactions necessary to drive self-organization robustly (6). Nevertheless, normal levels of tissue heterogeneity do not affect cell positioning in the gland. Even when heterogeneity in cell–cell cohesion is artificially elevated by mosaic deletion of E-cadherin, LEP and MEP retain their relative positions efficiently (18). How self-organization remains robust among these and other heterogeneous populations of cells is poorly understood.The robustness exhibited by the mammary gland during self-organization could derive from a variety of mechanisms, including the action of intercellular regulatory networks or microenvironmental cues that fine-tune cell–cell cohesion. Here, we investigate the hypothesis that spatially restricted interfacial interactions unique to the tissue–ECM boundary are sufficient to direct robust self-organization, even among heterogeneous or changing populations of cells. To test this hypothesis, we reconstitute the self-organization of the mammary and prostate glands in vitro from aggregates of primary human cells. We then estimate the hierarchy of cell–cell and cell–ECM cohesive energies in the mammary gland to reveal that only the MEP, and not the LEP cells, adhere and spread onto the tissue–ECM boundary. Using mathematical modeling and cell-type-specific knockdown of key adhesion proteins, we show that binary (i.e., MEP adhere and LEP do not) cell–ECM cohesion dominates self-organization and is robust to changes to the hierarchy of cell–cell cohesive interactions. Our results provide a conceptual framework for understanding robust tissue formation in vivo and in vitro but also suggest several potential mechanisms through which tissue structure might break down during the progression of malignant disease.     

Beta1 integrin mediated adhesion increases Bim protein degradation and contributes to drug resistance in leukaemia cells

It has been shown that the tumour microenvironment confers resistance to chemotherapy. Specifically, it was previously reported that adhesion of haematopoietic tumour cells to fibronectin (FN) via beta1 integrins confers a multi-drug resistance phenotype commonly referred to as cell adhesion mediated drug resistance. The present study showed that the pro-apoptotic BCL-2 family member Bim was reduced when leukaemia cells were adherent to FN via beta1 integrins. beta1 integrin-mediated regulation of Bim in K562 cells was demonstrated to be partly a result of increased proteasomal-mediated degradation of Bim protein levels, and proteasome inhibitors prevent Bim degradation. Increased degradation of Bim was not related to activation of the mitogen-activated protein kinase pathway, as adhesion of K562 cells caused a reduction in phospho-extracellular signal-related kinase (ERK)1/2 levels. In addition, pharmacological inhibition of MAP/ERK (MEK) with PD98059 did not increase Bim levels. Reducing Bim levels by short hairpin RNA targeting inhibited imatinib and mitoxantrone-induced cell death. These results showed that beta1 integrin-mediated adhesion regulates Bim degradation and may contribute to the minimal residual disease associated with many haematopoietic malignancies. Together our data indicate that disrupting beta1 integrin-mediated regulation of Bim degradation may increase the efficacy of drugs, including imatinib, used to treat haematopoietic malignancies.     

B-CAM/LU expression and the role of B-CAM/LU activation in binding of low- and high-density red cells to laminin in sickle cell disease

Red blood cells from patients with sickle cell disease (SS RBC) adhere to laminin and over-express the high-affinity laminin receptor basal cell adhesion molecule/Lutheran protein (B-CAM/LU). This receptor has recently been shown to undergo activation in vitro through a protein kinase A-dependent mechanism. Low-density SS RBC express two-thirds more B-CAM/LU than high-density SS RBC. However, high-density SS RBC have been identified as most adherent to laminin under flow conditions. We investigated the ability of low- and high-density SS RBC to interact with laminin under various conditions and explored factors that might be responsible for the differences in B-CAM/LU-laminin interaction between high- and low-density SS RBC. We confirmed that high-density SS RBC adhere to laminin more strongly than low-density SS RBC under flow conditions. However, low-density SS RBC bind soluble laminin most strongly and are the most adherent to laminin under static conditions. Soluble recombinant Lutheran extracellular domain protein completely blocked SS RBC adhesion to laminin under both static and flow conditions. The protein kinase A inhibitor 14-22 amide inhibited adhesion to laminin during flow by high-density SS RBC from patients with strongly adherent cells but had no effect on adhesion observed after a static phase. Deletion of the cytoplasmic domain of B-CAM as well as mutation of the juxtamembranous tyrosine residue failed to reduce B-CAM-mediated adhesion to laminin by transfected MEL cells. These studies confirm that B-CAM/LU is the most critical receptor mediating adhesion to laminin under both static and flow conditions. Dense SS RBC are most adherent to laminin despite bearing fewer laminin receptors, apparently due to a reversible protein kinase A-dependent process that is unlikely to involve direct phosphorylation of B-CAM/LU. Our results also suggest that the nature of the interaction of B-CAM/LU with laminin may be different under static and flow conditions.     

整合素与钙通道协同调节人肾小管上皮细胞转分化过程及其机制

目的 用转化生长因子-β1（TGF-β1）诱导人肾小管上皮细胞（HK-2）转分化,探讨转分化过程中可能的细胞信号转导机制。方法 25ml TGF-β1（5ng／ml）刺激HK-2,用特异性细胞外信号调节激酶（ERK）抑制剂PD98059（25μmol／L）和（或）钙通道拮抗剂Nifedipine（10μmol／L）处理;于不同时间段分别用免疫荧光、S-P法和免疫印迹法检测黏着斑激酶（FAK）、整合素β1（β1-Integrin）。结果 相比空白组TGF-β1成功诱导HK-2转分化;TGF-β1刺激15min后FAK-Tyr397磷酸化增强,60min达顶峰。相比TGF-β1对照组,PD98059使FAK-Tyr397的磷酸化减弱（P〈0．05）,而Nifedipine无明显抑制作用（P〉0．05）,两者协同作用明显（P〈0．01）;PD98059使FAK蛋白表达呈时间依赖性减弱（P〈0．05）,PD98059及Nifedipine均呈时间依赖性抑制β1-Integrin表达（P〈0．05）。二者协同作用明显（P〈0．01）。结论 TGF-β1与Integrin信号通路之间通过ERK／FAK相互作用;钙通道参与调节FAK活化及FAK与β1-Integrin的表达。     

Crypt-villus differentiation reflected by lectin and protein binding to rat small intestinal brush border membranes

To compare differentiation along the crypt-villus axis in adult rats with changes observed in postnatal maturation with respect to binding capacities for lectins and food proteins, crypts and villi were isolated byin vivo perfusion andin vitro incubation. Brush border membranes were prepared from adults and newborns, and binding of¹²⁵I-labeled lectins and food proteins was assessed by airfuge ultracentrifugation. Crypt and villus membrane protein patterns looked almost identical, unlike newborn membranes. Considerable shifts in lectin binding to membranes were observed during postnatal maturation, but not in crypt-villus differentiation. For instance, fucose-specific lectin binding patterns in both preparations resembled the general adult mode. Contrary to differences in food protein binding between newborn and adult membranes, food protein binding did not show a consistent significant difference between membranes of crypt and villus origin in adult animals. In conclusion, membrane differentiation along the crypt-villus axis was found to follow a pattern dissimilar from neonatal maturation as far as protein and carbohydrate composition and food protein binding were concerned.Supported by Deutsche Forschungsgemeinschaft DFG Ste 305.     

血管活性肠肽对体外大肠癌 HT29细胞粘附和侵袭作用的影响

目的观察血管活性肠肽(VIP)和双丁酰环磷酸腺苷(db-cAMP)对体外大肠癌 HT29细胞对大鼠血管内皮的粘附和侵袭的影响。方法取出大鼠主动脉,建立体外癌细胞—血管内皮粘附试验模型。用光镜和扫描电镜观察 HT29细胞对大鼠血管内皮粘附作用和其形态学变化,以及 VIP 和 db-cAMP 对细胞粘附的影响并在扫描电镜上计数量化分析。结果在血管内皮上可清晰辨认癌细胞并可观察其对血管内皮的粘附和侵袭的形态学改变.VIP 组的粘附细胞和高活性粘附细胞显著多于对照组(P<0.05),相反 db-cAMP 则无此作用.结论 VIP可促进体外 HT29细胞对血管内皮的粘附和侵袭,其机制可能与激活癌细胞 A 激酶系统有关。