Identification of Osteopontin as a Novel Ligand for the Integrin α8β1 and Potential Roles for This Integrin–Ligand Interaction in Kidney Morphogenesis

Epithelio–mesenchymal interactions during kidney organogenesis are disrupted in integrin α8β1-deficient mice. However, the known ligands for integrin α8β1—fibronectin, vitronectin, and tenascin-C—are not appropriately localized to mediate all α8β1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for α8β1. We have coexpressed the extracellular domains of the mouse α8 and β1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the α8β1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with α8β1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In “far Western” blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to α8β1-AP. Cell adhesion assays using K562 cells expressing α8β1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin α8β1 may help regulate kidney development and other morphogenetic processes.

The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway

The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.

Delayed Activation of the Store-operated Calcium Current Induced by Calreticulin Overexpression in RBL-1 Cells

Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.

The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ∼100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.

Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid–cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4°C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid–cholesterol microdomains in living cells.

α5β1 Integrin Controls Cyclin D1 Expression by Sustaining Mitogen-activated Protein Kinase Activity in Growth Factor-treated Cells

Cyclin D1 expression is jointly regulated by growth factors and cell adhesion to the extracellular matrix in many cell types. Growth factors are thought to regulate cyclin D1 expression because they stimulate sustained extracellular signal-regulated kinase (ERK) activity. However, we show here that growth factors induce transient ERK activity when added to suspended fibroblasts and sustained ERK activity only when added to adherent fibroblasts. Cell attachment to fibronectin or anti-α5β1 integrin is sufficient to sustain the ERK signal and to induce cyclin D1 in growth factor-treated cells. Moreover, when we force the sustained activation of ERK, by conditional expression of a constitutively active MAP kinase/ERK kinase, we overcome the adhesion requirement for expression of cyclin D1. Thus, at least in part, fibroblasts are mitogen and anchorage dependent, because integrin action allows for a sustained ERK signal and the expression of cyclin D1 in growth factor-treated cells.

Complex Patterns of Alternative Splicing Mediate the Spatial and Temporal Distribution of Perlecan/UNC-52 in Caenorhabditis elegans

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan.  Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear.

On the Role of Myosin-II in Cytokinesis: Division of Dictyostelium Cells under Adhesive and Nonadhesive Conditions

We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (ΔBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing ΔBLCBS-myosin were previously shown to divide in suspension (Uyeda et al., 1996). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, ΔBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a ΔBLCBS-myosin is similar to that in wild-type cells.

Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator

Nitric oxide (NO) has diverse roles in intercellular communication and (at higher levels) in immune-mediated cell killing. NO reacts with many cellular targets, with cell-killing effects correlated to inactivation of key enzymes through nitrosylation of their iron-sulfur centers. SoxR protein, a redox-sensitive transcription activator dependent on the oxidation state of its binuclear iron-sulfur ([2Fe-2S]) centers, is also activated in Escherichia coli on exposure to macrophage-generated NO. We show here that SoxR activation by NO occurs through direct modification of the [2Fe-2S] centers to form protein-bound dinitrosyl-iron-dithiol adducts, which we have observed both in intact bacterial cells and in purified SoxR after NO treatment. Functional activation through nitrosylation of iron-sulfur centers contrasts with the inactivation typically caused by this modification. Purified, nitrosylated SoxR has transcriptional activity similar to that of oxidized SoxR and is relatively stable. In contrast, nitrosylated SoxR is short-lived in intact cells, indicative of mechanisms that actively dispose of nitrosylated iron-sulfur centers.

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal α-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal α-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

Expression of αv and β3 integrin chains on murine lymphocytes

The vitronectin receptor is a member of the integrin family of adhesion protein receptors and binds a broad spectrum of ligands, including fibronectin and fibrinogen in addition to vitronectin. We have generated four mAbs that recognize the murine αvβ3 vitronectin receptor. Biochemical and expression analyses showed that two of the mAbs are specific for the αv chain, and two are specific for the β3 chain. The mAbs are effective blocking reagents and inhibited cell adhesion to vitronectin, fibrinogen, and fibronectin. Staining analysis revealed expression of αv and β3 on certain populations of murine thymocytes, splenocytes, and bone marrow cells. The expression of αv and β3 appeared to be modulated at specific stages of thymocyte development, suggesting a possible function for the αvβ3 vitronectin receptor in T cell development.

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic–nuclear transport

The product of the c-abl protooncogene is a nonreceptor tyrosine kinase found in both the cytoplasm and the nucleus. We report herein that cell adhesion regulates the kinase activity and subcellular localization of c-Abl. When fibroblastic cells are detached from the extracellular matrix, kinase activity of both cytoplasmic and nuclear c-Abl decreases, but there is no detectable alteration in the subcellular distribution. Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of a subset of c-Abl to early focal contacts is observed coincident with the export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating closely with its return to the nucleus and suggesting that the active nuclear c-Abl originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not activated upon cell attachment. These results show that c-Abl activation requires cell adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus where it may function to integrate adhesion and cell cycle signals.

Importance of newly generated neurons in the adult olfactory bulb for odor discrimination

In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively γ-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.