A synthetic method to assay adhesion-family G-protein coupled receptors. Determination of the G-protein coupling profile of ADGRG6(GPR126).

G-protein coupled receptors (GPCRs) are the largest family of membrane-spanning receptors in metazoans and mediate diverse biological processes such as chemotaxis, vision, and neurotransmission. Adhesion GPCRs represent an understudied class of GPCRs. Adhesion GPCRs (ADGRs) are activated by an intrinsic proteolytic mechanism executed by the G-protein autoproteolysis inducing domain that defines this class of GPCRs. It is hypothesized that agonist ligands modulate the proteolyzed receptor to regulate the activity of a tethered agonist peptide that is an intramolecular activator of ADGRs. The mechanism of activation of ADGRs in physiological settings is unclear and the toolbox for interrogating ADGR physiology in cellular models is limited. Therefore, we generated a novel enterokinase-activated tethered ligand system for ADGRG6(GPR126). Enterokinase addition to cells expressing a synthetic ADGRG6 protein induced potent and efficacious signal transduction through heterotrimeric G-protein coupled second messenger pathways including cyclic nucleotide production, intracellular calcium mobilization, and GPCR-pathway linked reporter gene induction. These studies support the hypothesis that ADGRG6(GPR126) is coupled to multiple heterotrimeric G-proteins: including Gαs, Gαq, and Gα12. This novel assay method is robust, specific, and compatible with numerous cell pharmacology approaches. We present a new tool for determination of the biological function of ADGRs and the identification of ligands that engage these receptors.

The Impact of Spaceflight and Simulated Microgravity on Cell Adhesion.

Microgravity induces a number of significant physiological changes in the cardiovascular, nervous, immune systems, as well as the bone tissue of astronauts. Changes in cell adhesion properties are one aspect affected during long-term spaceflights in mammalian cells. Cellular adhesion behaviors can be divided into cell-cell and cell-matrix adhesion. These behaviors trigger cell-cell recognition, conjugation, migration, cytoskeletal rearrangement, and signal transduction. Cellular adhesion molecule (CAM) is a general term for macromolecules that mediate the contact and binding between cells or between cells and the extracellular matrix (ECM). In this review, we summarize the four major classes of adhesion molecules that regulate cell adhesion, including integrins, immunoglobulin superfamily (Ig-SF), cadherins, and selectin. Moreover, we discuss the effects of spaceflight and simulated microgravity on the adhesion of endothelial cells, immune cells, tumor cells, stem cells, osteoblasts, muscle cells, and other types of cells. Further studies on the effects of microgravity on cell adhesion and the corresponding physiological behaviors may help increase the safety and improve the health of astronauts in space.

The semaphorins and their receptors as modulators of tumor progression.

The semaphorins were initially characterized as repulsive axon guidance factors. However, they are currently also recognized as important regulators of diverse biological processes which include regulation of immune responses, angiogenesis, organogenesis, and a variety of additional physiological and developmental functions. The semaphorin family consists of more than 20 genes divided into seven subfamilies, all of which contain the sema domain signature. They usually transduce signals by activation of receptors belonging to the plexin family, either directly, or indirectly following the binding of some semaphorins to receptors of the neuropilin family which subsequently associate with plexins. Additional receptors which form complexes with these primary semaphorin receptors are also frequently involved in semaphorin signalling, and can strongly influence the nature of the biological responses of cells to semaphorins. Recent evidence suggests that semaphorins play important roles in the etiology of multiple forms of cancer. Some semaphorins such as some semaphorins belonging to the class-3 semaphorin subfamily, have been found to function as bona fide tumor suppressors and to inhibit tumor progression by various mechanisms. Because these class-3 semaphorins are secreted proteins, these semaphorins may potentially be used as anti-tumorigenic drugs. Other semaphorins, such as semaphorin-4D, function as inducers of tumor progression and represent targets for the development of novel anti-tumorigenic drugs. The mechanisms by which semaphorins affect tumor progression are diverse, ranging from direct effects on tumor cells to modulation of accessory processes such as modulation of immune responses and inhibition or promotion of tumor angiogenesis and tumor lymphangiogenesis. This review focuses on the diverse mechanisms by which semaphorins affect tumor progression.

Coevolution of paired receptors in Xenopus carcinoembryonic antigen-related cell adhesion molecule families suggests appropriation as pathogen receptors.

BACKGROUND: In mammals, CEACAM1 and closely related members represent paired receptors with similar extracellular ligand-binding regions and cytoplasmic domains with opposing functions. Human CEACAM1 and CEACAM3 which have inhibitory ITIM/ITSM and activating ITAM-like motifs, respectively, in their cytoplasmic regions are such paired receptors. Various bacterial pathogens bind to CEACAM1 on epithelial and immune cells facilitating both entry into the host and down-regulation of the immune response whereas interaction with granulocyte-specific CEACAM3 leads to their uptake and destruction. It is unclear whether paired CEACAM receptors also exist in other vertebrate clades. RESULTS: We identified more than 80 ceacam genes in Xenopus tropicalis and X. laevis. They consist of two subgroups containing one or two putative paired receptor pairs each. Analysis of genomic sequences of paired receptors provide evidence that their highly similar ligand binding domains were adjusted by recent gene conversion events. In contrast, selection for diversification is observed among inhibitory receptor orthologs of the two frogs which split some 60 million years ago. The allotetraploid X. laevis arose later by hybridization of two closely related species. Interestingly, despite the conservation of the genomic landscape surrounding the homeologous ceacam loci only one locus resembles the one found in X. tropicalis. From the second X. laevis locus more than 80 % of the ceacam genes were lost including 5 of the 6 paired receptor genes. This suggests that once the gene for one of the paired receptors is lost the remaining gene cluster degrades rapidly probably due to lack of selection pressure exerted by pathogens. CONCLUSIONS: The presence of paired receptors and selection for diversification suggests that also in amphibians CEACAM1-related inhibitory proteins are or were used as pathogen receptors.

Discordant immunophenotypic profiles of adhesion molecules and cytokines in acute myeloid leukemia involving bone marrow and skin.

We investigated the role of adhesion molecules in skin involvement by acute myeloid leukemia (AML) using immunohistochemical analysis. Ten paired cases of skin and bone marrow biopsy specimens from patients with myeloid leukemia cutis (MLC) and 15 bone marrow biopsy specimens from patients without MLC were studied with antibodies directed against CD29, CD34, CD54, CD62-L, CD183, and cutaneous lymphocyte antigen (CLA). CLA was expressed in all cases of leukemia whereas CD54 was negative within blasts. CD62-L was expressed in 4 of 10 specimens of marrow infiltrates with MLC and 6 of 10 specimens of matching skin infiltrates; in marrows without MLC, only 2 of 15 were positive. CD29 was expressed in 1 of 10 marrow infiltrate specimens with MLC and 4 of 10 matching skin infiltrate specimens; in marrows without MLC, only 1 of 15 were positive. CD183 was expressed in 1 of 10 marrow infiltrate specimens with MLC and 4 of 10 matching skin infiltrate specimens; in marrows without MLC, CD183 was negative. The gain of CD62-L, CD29, and CD183 expression in bone marrow and skin infiltrates in leukemia cutis, relative to bone marrow infiltrates of cases without MLC, suggests a role for these markers in AML homing to skin.

Effects of ozone on nasal mucosa (endothelial cells).

After exposure to ozone, humans develop neutrophil infiltration of the nasal mucosa. To investigate the events contributing to inflammatory cell recruitment in the nasal mucosa we exposed 10 healthy nonsmoking volunteers to 400 ppb ozone or filtered air for 2h at rest on two separate occasions. Nasal biopsies were performed 6h after ozone/filtered air exposure. The nasal biopsies were embedded in glycol mathacrylate and immunostained for inflammatory cells, including neutrophils, mast cells, total T-cells (CD3), T-cell subsets CD8 and CD4, macrophages, eosinophils adhesion molecules (P-selectin, E-selectin, ICAM-1, VCAM-1), cytokines (TNF-α, IL-1ß, GM-CSF, IL-6), chemokines (IL-8 and RANTES), and nuclear factor NF-κB. No significant changes were seen in the number of T-cells, and T-cell subsets, eosinophils, macrophages, or percentages of vessels expressing P-selectin, VCAM-1, GM-CSF, IL-6 and RANTES in the biopsies. The number of neutrophils and mast cells in the submucosa was significantly higher after ozone exposure (p=0.009 and p=0.005 respectively). The percentage of vessels expressing E-selectin (p=0.01), ICAM-1 (p=0.005), IL-8 (p=0.02), TNF-α (p=0.02), IL-1ß (p=0.009), and NF-κB (p=0.05) increased significantly after ozone exposure as compared to filtered air exposure. Exposure of normal subjects to ozone increases the expression of proinflammatory cytokines resulting in upregulation of IL-8 and adhesion molecules via activation of NF-κB, leading to neutrophil inflitration in the nasal mucosa.

Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic antigen families.

BACKGROUND: Most rapidly evolving gene families are involved in immune responses and reproduction, two biological functions which have been assigned to the carcinoembryonic antigen (CEA) gene family. To gain insights into evolutionary forces shaping the CEA gene family we have analysed this gene family in 27 mammalian species including monotreme and marsupial lineages. RESULTS: Phylogenetic analysis provided convincing evidence that the primordial CEA gene family in mammals consisted of five genes, including the immune inhibitory receptor-encoding CEACAM1 (CEA-related cell adhesion molecule) ancestor. Our analysis of the substitution rates within the nucleotide sequence which codes for the ligand binding domain of CEACAM1 indicates that the selection for diversification is, perhaps, a consequence of the exploitation of CEACAM1 by a variety of viral and bacterial pathogens as their cellular receptor. Depending on the extent of the amplification of an ancestral CEACAM1, the number of CEACAM1-related genes varies considerably between mammalian species from less than five in lagomorphs to more than 100 in bats. In most analysed species, ITAM (immunoreceptor tyrosine-based activation motifs) or ITAM-like motif-containing proteins exist which contain Ig-V-like, ligand binding domains closely related to that of CEACAM1. Human CEACAM3 is one such protein which can function as a CEACAM1 decoy receptor in granulocytes by mediating the uptake and destruction of specific bacterial pathogens via its ITAM-like motif. The close relationship between CEACAM1 and its ITAM-encoding relatives appears to be maintained by gene conversion and reciprocal recombination. Surprisingly, secreted CEACAMs resembling immunomodulatory CEACAM1-related trophoblast-specific pregnancy-specific glycoproteins (PSGs) found in humans and rodents evolved only in a limited set of mammals. The appearance of PSG-like genes correlates with invasive trophoblast growth in these species. CONCLUSIONS: These phylogenetic studies provide evidence that pathogen/host coevolution and a possible participation in fetal-maternal conflict processes led to a highly species-specific diversity of mammalian CEA gene families.

Genistein, a soybean isoflavone, reduces the production of pro-inflammatory and adhesion molecules induced by hemolysate in brain microvascular endothelial cells.

Genistein (4',5,7-trihydroxyisoflavone) is the most abundant isoflavone found in the soybean that exhibits an anti-inflammatory effect. The present study was designed to examine the effects of genistein on expression levels of hemolysate-induced proinflammatory and adhesion molecules in SD rat brain microvascular endothelial cells (BMECs). Genistein treatment attenuated hemolysate-induced nuclear factor-kappa B (NF-kappaB) p65 translocation in BMECs. In addition, genistein suppressed the expression levels of tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein 1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1), but not vascular cell adhesion molecule-1 (VCAM-1). The inhibitory rate of 50 pM genistein for TNF-alpha, MCP-1 and ICAM-1 was 65.4%, 60.5% and 54.9% respectively. These inhibitory effects of genistein on proinflammatory and adhesion molecules were not due to decreased BMEC viability as assessed by MTT test. Taken together the present study suggests that genistein suppresses expression levels of hemolysate-induced pro-inflammatory and adhesion molecules in cerebral endothelial cells.

Prognostic significance of cadherin-based adhesion molecules in cutaneous malignant melanoma.

BACKGROUND: The need for novel molecular prognostic markers that can supplement validated clinicopathologic correlates for cutaneous malignant melanoma is well recognized. Proteins that mediate the epithelial-mesenchymal transition, the process by which a cancer cell disengages from its parent tumor, are important candidates. METHODS: The prognostic relevance of E-cadherin, N-cadherin, and P-cadherin, calcium-dependent transmembrane glycoproteins that regulate cell-cell adhesion, and their adaptors, alpha-catenin, beta-catenin, and p120-catenin, was evaluated on a cohort of 201 primary and 274 metastatic melanoma tumors using fluorescence-based immunohistochemical methods and Automated Quantitative Analysis of protein expression on digitally captured photomicrographs. RESULTS: Increasing levels of N-cadherin expression improved overall survival (log-rank = 7.31; P = 0.03) but did not retain significance following adjustment for established clinicopathologic correlates (P = 0.50). Higher levels of E-cadherin approached significance for favorable prognosis on both univariate (P = 0.13) and multivariable (P = 0.10) analyses. Hierarchical clustering of the composite profiles for all six markers identified four unique clusters that yielded differential overall survival (log-rank = 10.54; P = 0.01). Cluster 4, expressing high E-cadherin and N-cadherin levels, possessed the most favorable outcome and cluster 2, featuring low E-cadherin and alpha-catenin but modest N-cadherin, showed least favorable outcomes. Cluster 2 remained significant on multivariable analysis (hazard ratio, 3.29; 95% confidence interval, 1.50-7.19; P = 0.003). CONCLUSIONS: Although none of the cadherin-based adhesion molecules were independently prognostic, multimarker profiles were significant. Similar to epithelial-derived tumors, loss of E-cadherin correlates with poor outcome. In contrast, for neural crest-derived cutaneous malignant melanoma, N-cadherin overexpression can be associated with either a successful epithelial-mesenchymal transition or a favorably differentiated tumor. Additional cadherin profiles are needed to discriminate these distinctive phenotypes.

Six cadm/SynCAM genes are expressed in the nervous system of developing zebrafish.

The Cadm (cell adhesion molecule) family of cell adhesion molecules (also known as IGSF4, SynCAM, Necl and TSLC) has been implicated in a multitude of physiological and pathological processes, such as spermatogenesis, synapse formation and lung cancer. The precise mechanisms by which these adhesion molecules mediate these diverse functions remain unknown. To investigate mechanisms of action of these molecules during development, we have identified zebrafish orthologs of Cadm family members and have examined their expression patterns during development and in the adult. Zebrafish possess six cadm genes. Sequence comparisons and phylogenetic analysis suggest that four of the zebrafish cadm genes represent duplicates of two tetrapod Cadm genes, whereas the other two cadm genes are single orthologs of tetrapod Cadm genes. All six zebrafish cadms are expressed throughout the nervous system both during development and in the adult. The spatial and temporal patterns of expression suggest multiple roles for Cadms during nervous system development.

[The role of integrins in the physiologic and pathogenic processes]. / Udzial integryn w procesach fizjo- i patologicznych.

Cell adhesion molecules (CAM) are a numerous, diverse group of cell surface proteins, which are both receptors and ligands for receptors. Their functions include adhesion, recognition, cell-cell interaction, and communication between mediate cells and extracellular matrix. The following groups of CAM can be distinguished: seletins, integrins, cadherins and other isoforms, including CD 44. Integrins are heterodimers formed from the alpha and beta chains. The a subclass is responsible for a specific bond with ligands. It defines the specificity of integrins. The 8 chain participates in the integration with cytoskeleton ptoteins. It determines the functions of the integrin receptor. The best recognized integrins include: integrin beta1, beta2 and beta3. The expression and activity of integrins have been found to be affected by a variety of factors being either activators or inhibitors. Adhesion molecules (including integrins) play a significant role in both physiological processes (embryogenesis, organogenesis, the normal growth and tissue development) and pathogenic ones. In the latter case, they are particularly involved in inflammatory, allergic and neoplastic diseases. The role of integrins is also emphasized in organ response to trauma and in skin lesion redevelopment. The knowlegde of the integrin molecular basis and that of other adhesion molecules can contribute significantly to the creation of new diagnostic and therapeutic perspectives. An adequate modification of cellular adhesion constitutes a promising way of the pathogenic processes control.

BT-IgSF, a novel immunoglobulin superfamily protein, functions as a cell adhesion molecule.

BT-IgSF is a newly identified cell surface glycoprotein belonging to the immunoglobulin superfamily (IgSF). We have previously shown that the expression of the BT-IgSF gene was highly restricted to brain and testis, and its transcript was detected in both neurons and glial cells. In this study, to explore its function, we generated cells overexpressing BT-IgSF proteins and analyzed their phenotypes. We found that the constitutive expression of BT-IgSF in the myeloid leukemia cell line TF-1-fms did not alter the growth rates, but caused the formation of large cell aggregates. The cell aggregates were also observed with mutant BT-IgSF lacking its cytoplasmic tail, the amino acid sequences of which were highly conserved among the BT-IgSF subgroup proteins. The neutralizing antibody to beta(1) integrin did not diminish the cell aggregate formation. These results indicate that BT-IgSF functions as a cell adhesion molecule, that its cytoplasmic tail is not essential for the function, and that beta(1) integrin is not involved in the function. We confirmed the cell adhesion function using NIH/3T3 fibroblastic cells expressing BT-IgSF in an inducible system. Flow cytometric analyses with the cells demonstrated that the cell aggregation mediated by BT-IgSF was through homophilic molecular interaction, and in a Ca(2+)/Mg(2+)-independent manner. Coupled with its restricted pattern of the expression, the cell adhesion-inducing function of BT-IgSF suggests a role of the cell surface molecule in the development/function of the central nervous system and spermatogenesis.

The trophinin gene encodes a novel group of MAGE proteins, magphinins, and regulates cell proliferation during gametogenesis in the mouse.

Trophinin is a membrane protein that mediates apical cell adhesion between trophoblastic cells and luminal epithelial cells of the endometrium and is implicated in the initial attachment during the process of human embryo implantation. The present study identified novel trophinin gene transcripts, which encode proteins structurally distinct from trophinin protein in the mouse. We designated these proteins "magphinins," because they share consensus amino acid sequences with MAGE (melanoma-associated antigen) superfamily proteins. Among many MAGE proteins, magphinins are closely related to NRAGE, which mediates p75 neurotrophin receptor-dependent apoptosis, and necdin, which is a strong suppressor of cell proliferation in post-mitotic neurons. There are three major forms of magphinins, i.e. magphinin-alpha, -beta, and -gamma, in the mouse, which are formed due to alternative usage of different exons. Northern blot analysis revealed that magphinins are expressed in brain, ovary, testis, and epididymis. In addition, Western blot analysis and in vitro translation experiments showed that magphinins expressed in the mouse ovary and testis are translation products utilizing the second initiation AUG codon and contain an active nuclear localization signal. Ectopic expression of magphinins in mammalian cells resulted in nuclear localization of magphinin and suppressed cell proliferation. Immunohistochemistry of the mouse ovary and testis showed that magphinin proteins are distributed in the cytoplasm of the male and female germ cells, whereas these proteins are translocated to the nucleus at a specific stage of gametogenesis. These results strongly suggest that magphinins regulate cell proliferation during gametogenesis in the mouse.

Analysis of cell-cell contact mediated by Ig superfamily cell adhesion molecules.

This unit provides protocols to assay cell-cell adhesion and adhesive-dependent cellular functions mediated by calcium-independent adhesion molecules. These protocols have been developed for neural cell adhesion molecules of the Ig superfamily. However, most of the protocols allow a more general application to other categories of adhesion molecules and non-neural cells.

EMILIN, a component of the elastic fiber and a new member of the C1q/tumor necrosis factor superfamily of proteins.

EMILIN (elastin microfibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil alpha helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of beta-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative.

Blockade of metastasis formation by CD44-receptor globulin.

CD44 standard as well as variant isoforms have been frequently reported to be involved in the process of metastasis formation. Whereas in the rat system, but also in some human tumours, the variant exon v6 is of importance in the lymphatic spread of carcinomas, in human malignant melanoma CD44s and, possibly, CD44v10 appear to facilitate local invasion and haematogenous spread. This has been tested in the B16F10 murine melanoma model by treating B16F10-bearing C57BL/6 mice either with a CD44s-/ CD44v10-specific antibody, or with receptor globulins (Rg) containing the extracellular part of CD44s or CD44v10 linked to the constant region of the immunoglobulin kappa light chain. Prior characterization of the CD44s and CD44v10 Rg had shown that both Rgs bound to components of the extracellular matrix, CD44s in particular to hyaluronic acid. Immunohistological screening of organ sections from adult C57BL/6 mice revealed additional evidence for both Rgs binding to elements of the extracellular matrix, particularly in bone marrow, intestine and lung. In the absence of any further treatment, the CD44s Rg reduced the number of lung colonies by 70%, while application of the CD44v10 Rg resulted in 60% reduction. CD44-specific antibodies were equally efficient with regard to B16F10 settlement in the lung. However, only the CD44 Rgs prevented spread and settlement of melanoma cells in distant organs. The finding confirms the involvement of both CD44s and CD44v10 in melanoma progression, and is suggestive for the use of Rgs as therapeutic reagents.

Cell adhesion molecules--update.

Cell adhesion molecules are glycoproteins expressed on the cell surface and play an important role in inflammatory as well as neoplastic diseases. There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins. The integrin family has eight subfamilies, designated as beta 1 through beta 8. The most widely studied subfamilies are beta 1 (CD29, very late activation [VLA] members), beta 2 (leukocyte integrins such as CD11a/CD18, CD11b/CD18, CD11c/CD18, and alpha d beta 2), beta 3 (CD61, cytoadhesions), and beta 7 (alpha 4 beta 7 and alpha E beta 7). The immunoglobulin superfamily includes leukocyte function antigen-2 (LFA-2 or CD2), leukocyte function antigen-3 (LFA-3 or CD58), intercellular adhesion molecules (ICAMs), vascular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The selectin family includes E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). Cadherins are major cell-cell adhesion molecules and include epithelial (E), placental (P), and neural (N) subclasses. The binding sites (ligands/receptors) are different for each of these cell adhesion molecules (e.g., ICAM binds to CD11/CD18; VCAM-1 binds to VLA-4). The specific cell adhesion molecules and their ligands that may be involved in pathologic conditions and potential therapeutic strategies by modulating the expression of these molecules will be discussed.