CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca2+-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

CIB1 prevents nuclear GAPDH accumulation and non-apoptotic tumor cell death via AKT and ERK signaling.

CIB1 is a 22-kDa regulatory protein previously implicated in cell survival and proliferation. However, the mechanism by which CIB1 regulates these processes is poorly defined. Here, we report that CIB1 depletion in SK-N-SH neuroblastoma and MDA-MB-468 breast cancer cells promotes non-apoptotic, caspase-independent cell death that is not initiated by increased outer mitochondrial membrane permeability or translocation of apoptosis-inducing factor to the nucleus. Instead, cell death requires nuclear GAPDH accumulation. Furthermore, CIB1 depletion disrupts two commonly dysregulated, oncogenic pathways-PI3K/AKT and Ras/MEK/ERK, resulting in a synergistic mechanism of cell death, which was mimicked by simultaneous pharmacological inhibition of both pathways, but not either pathway alone. In defining each pathway's contributions, we found that AKT inhibition alone maximally induced GAPDH nuclear accumulation, whereas MEK/ERK inhibition alone had no effect on GAPDH localization. Concurrent GAPDH nuclear accumulation and ERK inhibition were required, however, to induce a significant DNA damage response, which was critical to subsequent cell death. Collectively, our results indicate that CIB1 is uniquely positioned to regulate PI3K/AKT and MEK/ERK signaling and that simultaneous disruption of these pathways synergistically induces a nuclear GAPDH-dependent cell death. The mechanistic insights into cell death induced by CIB1 interference suggest novel molecular targets for cancer therapy.

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated alpha2beta1 activation.

BACKGROUND: The platelet alpha2beta1 integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that alpha2beta1 function can be activated via inside-out signaling, similar to the prototypical platelet integrin alphaIIbbeta3. However, signaling molecules that regulate alpha2beta1 activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates alphaIIbbeta3 activation. OBJECTIVES: We hypothesized that Rap1b positively regulates alpha2beta1 during agonist-induced platelet activation. METHODS: To test whether Rap1b activates alpha2beta1 downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b-/- or wild-type mice with diverse agonists and measured alpha2beta1 activation using fluorescein isothiocyanate-labeled monomeric collagen. We also examined the role of Rap1b in outside-in signaling pathways by analyzing adhesion and spreading of Rap1b-/- or wild-type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b-mediated events. RESULTS: Rap1b-/- platelets displayed comparable ADP-induced or thrombin-induced alpha2beta1 activation as wild-type platelets, but reduced convulxin-dependent alpha2beta1 activation. Rap1b-/- platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild-type platelets. Rap1b-/- platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. CONCLUSIONS: Rap1b is required for maximal GPVI-induced but not ADP-induced activation of alpha2beta1 in murine platelets.

The alpha4beta1 integrin in sickle cell disease.

The alpha4beta1 integrin is an adhesion receptor expressed on reticulocytes in sickle cell disease (SCD) and mediates the adhesion of these cells to sub-endothelial matrix proteins and the endothelium. In this review, we describe the mechanism of activation of the alpha4beta1 integrin on sickle reticulocytes and discuss novel roles for this integrin in SCD as a result of this activation. We also illustrate novel therapies in SCD that may target the integrin and alleviate vaso-occlusion.

Activation of sickle red blood cell adhesion via integrin-associated protein/CD47-induced signal transduction.

Peripheral human red blood cells (RBCs) are not generally known to become activated and adhesive in response to cell signaling. We show, however, that soluble thrombospondin via integrin-associated protein (IAP; CD47) increases the adhesiveness of sickle RBCs (SS RBCs) by activating signal transduction in the SS RBC. This stimulated adhesion requires occupancy of IAP and shear stress and is mediated by the activation of large G proteins and tyrosine kinases. Reticulocyte-enriched RBCs derived from sickle-cell disease (SCD) patients are most responsive to IAP-induced activation. These studies therefore establish peripheral SS RBCs as signaling cells that respond to a novel synergy between IAP-induced signal transduction and shear stress, suggesting new therapeutic targets in SCD.

Integrin-associated protein is an adhesion receptor on sickle red blood cells for immobilized thrombospondin.

The adhesive protein thrombospondin (TSP) potentially mediates sickle (SS) red blood cell (RBC) adhesion to the blood vessel wall, thereby contributing to vaso-occlusive crises in sickle cell disease. We previously reported that SS RBCs bind to immobilized TSP under flow conditions, whereas normal (AA) red cells do not. However, the SS RBC receptors that mediate this interaction are largely unknown. Here it is reported that integrin-associated protein (IAP), or CD47, mediates the adhesion of these cells to immobilized TSP under both flow and static conditions. A peptide derived from the C-terminal IAP binding site of TSP also supports sickle cell adhesion; adhesion to this peptide or to TSP is inhibited specifically by the anti-IAP monoclonal antibody, 1F7. Furthermore, these data suggest that IAP on SS RBCs is structurally different from that expressed on AA RBCs but that IAP expression levels do not vary between AA and SS RBCs. This structural difference may contribute to the enhanced adhesion of SS RBCs to immobilized TSP. These results identify IAP as a TSP receptor on SS RBCs and suggest that this receptor and its binding site within TSP represent potential therapeutic targets to decrease vaso-occlusion. (Blood. 2001;97:2159-2164)

Multiple roles of integrins in cell motility.

Motility is essential for many important biological events, including embryonic development, inflammatory responses, wound healing, and tumor metastasis. During these events cells are in dynamic contact with the extracellular matrix through integrins. Integrins are the primary receptors for extracellular matrix proteins and consequently are required for cell motility. Cells have evolved multiple mechanisms to modulate integrin adhesive functions, which impact cell migration. In addition to providing a mechanism that allows cells to contact the extracellular matrix, integrins also promote intracellular signals that stimulate and regulate cell movement. Here we discuss the role of integrins during the multiple steps of cell migration.

New aspects of integrin signaling in cancer.

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behavior, including motility and invasiveness, cell growth, and cell survival. Engagement of integrins with extracellular matrix (ECM) proteins can activate members of the Rho-family of small GTPases; conversely, Rho- and Ras-family proteins can influence the ability of integrins to bind their ligands. These events impinge on the control of cell motility, and ultimately on invasive and metastatic behavior. Integrin engagement with ECM also has important effects on cell survival, particularly for cells of epithelial origin. In some cases, specific integrins have selective effects on the efficiency of signal transduction in cell survival pathways.

Integrin alpha(IIb)beta(3) signaling in platelet adhesion and aggregation.

Intracellular signals are received and generated by the alpha(IIb)beta(3) integrin on platelets. Recent advances have been made in the areas of agonist receptors that initiate platelet activation, downstream signaling molecules (e.g. small G-proteins and kinases) and changes in ligand-occupied alpha(IIb)beta(3) that cause further signaling and clot retraction.

Calcium-dependent properties of CIB binding to the integrin alphaIIb cytoplasmic domain and translocation to the platelet cytoskeleton.

The alphaIIbbeta3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to alphaIIbbeta3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional alphaIIbbeta3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the alphaIIb cytoplasmic domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed. In addition, isothermal titration calorimetry indicated that CIB binds to an alphaIIb cytoplasmic-domain peptide in a Ca(2+)-dependent manner, with moderate affinity (K(d), 700 nM) and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and alphaIIbbeta3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by alphaIIbbeta3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca(2+)-modulated binding to the alphaIIb cytoplasmic domain and changes in intracellular distribution.

R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells.

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the alpha2, but not the alpha5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin beta cytoplasmic domains was observed for the alpha2 cytoplasmic domain; alpha2beta1-mediated migration was inhibited by the expression of excess alpha2 but not alpha5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin alpha subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin alpha cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.

Sickle cell adhesion to laminin: potential role for the alpha5 chain.

Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the alpha1, alpha2, beta1, and gamma1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin alpha5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin alpha5 chain.

Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K.

Transformation of mammary epithelial cells into invasive carcinoma results in alterations in their integrin-mediated responses to the extracellular matrix, including a loss of normal epithelial polarization and differentiation, and a switch to a more motile, invasive phenotype. Changes in the actin cytoskeleton associated with this switch suggest that the small GTPases Cdc42 and Rac, which regulate actin organization, might modulate motility and invasion. However, the role of Cdc42 and Rac1 in epithelial cells, especially with respect to integrin-mediated events, has not been well characterized. Here we show that activation of Cdc42 and Rac1 disrupts the normal polarization of mammary epithelial cells in a collagenous matrix, and promotes motility and invasion. This motility does not require the activation of PAK, JNK, p70 S6 kinase, or Rho, but instead requires phosphatidylinositol-3-OH kinase (PI(3)K). Further, direct PI(3)K activation is sufficient to disrupt epithelial polarization and induce cell motility and invasion. PI(3)K inhibition also disrupts actin structures, suggesting that activation of PI(3)K by Cdc42 and Rac1 alters actin organization, leading to increased motility and invasiveness.

Structure and function of platelet alpha IIb beta 3.

Platelet aggregation is mediated by the platelet fibrinogen receptor, the alpha IIb beta 3 integrin (glycoprotein IIb-IIIa). This integrin has served as a prototype for studies probing structure-function relationships, cellular modulation of integrin function, and integrin-mediated signaling. Use of new model systems and molecular approaches have helped investigators to make rapid progress in our understanding of these areas, as reviewed here.

Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain.

The mechanism by which platelets regulate the function of integrin alphaIIbbeta3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, we screened a human fetal liver cDNA library in the yeast two-hybrid system, using the alphaIIb cytoplasmic domain as "bait," and isolated a novel 855-base pair clone. The open reading frame encodes a novel 191-amino acid polypeptide (termed CIB for calcium- and integrin-binding protein) that appears to be specific for the cytoplasmic domain of alphaIIb, since it does not interact with the alphav, alpha2, alpha5, beta1, or beta3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has sequence homology to two known Ca2+-binding regulatory proteins, calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs corresponding to the two C-terminal Ca2+ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45Ca2+ in blot overlay assays. Using reverse transcriptase-polymerase chain reaction and Western blot analysis, we detected CIB mRNA and protein ( approximately 25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured alphaIIbbeta3 indicates a specific interaction between CIB and intact alphaIIbbeta3. These results suggest that CIB is a candidate regulatory molecule for integrin alphaIIbbeta3.

Ras activation in platelets after stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C.

Several reports have indicated that the small G-protein Ras is not present immunologically in platelets. However, here we report the identification of Ras in platelets by immunoprecipitation with the Ras-specific monoclonal antibodies Y13-259 or Y13-238, followed by Western blotting. The presence of Ras was not due to contamination of samples with erythrocytes or leucocytes. Immunofluorescence studies indicated that Ras was present in a peripheral rim pattern in fixed, permeabilized platelets, suggesting an intracellular, plasma membrane location. Activation of platelets with the thrombin receptor peptide42-50, the prostaglandin H2/thromboxane A2 mimetic U46619 or phorbol 12-myristate 13-acetate induced a rapid increase in GTP-bound, activated Ras. In each case, this increase was inhibited by the protein kinase C (PKC) inhibitor bisindolylmaleimide GF 109203X, suggesting that Ras is activated downstream of PKC in platelets. Thus the activation of Ras in platelets by agonists will now allow consideration of multiple potential Ras-dependent signal transduction pathways in platelet activation processes.

Characterization of a new hereditary thrombopathy in a closed colony of Wistar rats.

A new hereditary thrombopathy has been identified in a closed colony of Wistar rats. A simple and reproducible cuticle bleeding time test was developed as a rapid screening procedure for the bleeding diathesis. Affected animals exhibit markedly prolonged bleeding times and complete absence of platelet aggregation either with adenosine diphosphate (ADP) or with thrombin. Inheritance data suggest an autosomal dominant inheritance pattern with variable penetrance. Coagulation tests, platelet counts, plasma von Willebrand factor (vWF) activity, and clot retraction are within normal limits in thrombopathic animals. GPIb-dependent botrocetin-induced platelet agglutination was present in washed thrombopathic rat platelets. No discernible abnormality of intraplatelet organelles or granules was seen by transmission electron microscopy of thrombopathic platelets. A qualitative morphologic assessment of intraplatelet fibrinogen in thrombopathic rat platelets showed no discernible difference as compared with control rat platelets. Thrombopathic rat platelets exhibit decreased glycoprotein IIb/IIIa (GPIIb/IIIa) antigen by flow cytometric analysis and markedly decreased iodine 125-labeled fibrinogen binding to platelet GPIIb/IIIa after ADP activation. This rat colony demonstrates a unique thrombopathy, distinct from previously described animal thrombopathies, with some characteristics of variant Glanzmann's thrombasthenia. This animal model may provide further insight into the regulatory mechanisms and pathophysiology of platelet GPIIb/IIIa.

The alpha2beta1 integrin is a necessary co-receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase Cgamma2 in platelets.

Although there are multiple potential collagen-binding proteins on platelets, the contribution of each to collagen-induced signaling events and platelet activation is unclear. We investigated which early platelet signaling events, if any, could be attributed specifically to the binding of collagen to one of its receptors, the alpha2beta1 integrin. Treatment of platelets with collagen induced a rapid activation of the non-receptor tyrosine kinase, Syk, as measured by an increase in phosphorylation and kinase activity. Collagen also induced the rapid phosphorylation of phospholipase Cgamma2 (PLCgamma2). The phosphorylation of both Syk and PLCgamma2, as well as platelet aggregation, was blocked by an anti-alpha2beta1 integrin monoclonal antibody (P1E6), demonstrating that collagen binding to alpha2beta1 is necessary for signaling. Cross-linking of the alpha2beta1 integrin with stimulatory monoclonal antibody against either the beta1 or alpha2 subunit stimulated the phosphorylation of both Syk and PLCgamma2. However, antibody stimulation was dependent on co-stimulation of the FcgammaII receptor (CD32) since specific F(ab')2 fragments did not induce Syk and PLCgamma2 phosphorylation. Thus, these results suggest that occupancy of alpha2beta1 by collagen is necessary, but that a co-receptor, in addition to alpha2beta1, is required for these collagen-induced signaling events. Moreover, the P1E6 antibody did not inhibit all collagen-induced tyrosine phosphorylation events, demonstrating that collagen also induces phosphorylation events that are independent of the alpha2beta1 integrin. In addition to Syk and PLCgamma2, we identified the FcgammaII receptor (FcgammaRII) as being rapidly phosphorylated in response to collagen stimulation, even in the absence of antibodies. Finally, to determine if Syk activation precedes and directly contributes to the phosphorylation of PLCgamma2, platelets were preincubated with the Syk-selective kinase inhibitor, piceatannol. A concentration of piceatannol that inhibited the phosphorylation and kinase activity of Syk, but had no effect on Src kinase activity, blocked the collagen-induced phosphorylation of PLCgamma2 and also inhibited collagen-induced platelet aggregation. Our results begin to delineate a signaling pathway whereby occupancy of the alpha2beta1 integrin is required, but not sufficient, for collagen-induced activation of Syk and the subsequent phosphorylation of PLCgamma2. These events are necessary for platelet activation and aggregation in response to collagen.

Modulation of integrin expression in endometrial stromal cells in vitro.

Integrins, a class of cell adhesion molecules found on virtually all cells, display dynamic temporal and spatial patterns of expression in the endometrium during the menstrual cycle and in early pregnancy. To study integrin regulation, we measured the expression of eight different integrin subunits on cultured human endometrial stromal cells obtained from proliferative phase endometrium, using immunofluorescence and flow cytometry. Treatment with estrogen and progesterone induced hormonal changes of decidualization but did not alter the expression of any of the integrins. It is presently unknown whether steroid hormones other than estrogen or progesterone affect integrin expression. In contrast, treatment with several growth factors and cytokines resulted in specific alterations of integrin levels. Epidermal growth factor, transforming growth factor-alpha, and transforming growth factor-beta 1 induced expression of the alpha 1 beta 1 collagen/laminin receptor. There also was a trend towards decreased expression of the alpha 6 subunit in response to interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor-alpha. The expression of alpha 1 beta 1 was accompanied by increased adhesion to collagen but there was no change in the binding to fibronectin and vitronectin. Our findings suggest that some aspects of decidualization may be regulated by steroid hormones, whereas others, such as integrin expression, are regulated by cytokines or growth factors, possibly of trophoblast origin. Integrins are likely to play a role in the interaction between trophoblast and endometrium.

Glycoprotein IV-independent adhesion of sickle red blood cells to immobilized thrombospondin under flow conditions.

The abnormal adherence of red blood cells (RBC to the blood vessel wall is believed to contribute to the vascular occlusion observed in patients with sickle call anemia. The cell adhesion receptors GPIV (CD36) and integrin alpha 4 beta 1 (CD49d/CD29) were previously identified on circulating sickle reticulocytes, and shown to mediate sickle RBC adhesion to the endothelium. The presence of damaged endothelium in these patients suggests that exposed extracellular matrix proteins could provide a potential substrate for sickle RBC adhesion. To determine whether RBC adhesion receptors could mediate adhesion to extracellular matrix proteins, we tested their ability to adhere to a variety of immobilized, purified proteins under flow conditions. Neither sickle nor normal RBC adhered to fibronectin, vitronectin, fibrinogen, or collagen. In contrast, we observed substantial adhesion of sickle but not normal RBC to thrombospondin (TSP). The adhesion was not inhibited with known antagonists of the GPIV-TSP interaction, nor by inhibitors of several other known binding domains in TSP. Moreover, the adhesion was resistant to inhibition by soluble TSP, suggesting that immobilization of TSP exposes an adhesive site that is cryptic on TSP in solution. However, the glycosaminoglycans, chondroitin sulfate A, and dextran sulfate were potent inhibitors of this adhesion. These results suggest that a mechanism distinct from GPIV is responsible for sickle RBC adhesion to immobilized TSP under flow conditions.