Leslie V. Parise

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.

Identification of a Novel Calcium-binding Protein That Interacts with the Integrin αIIb Cytoplasmic Domain

The mechanism by which platelets regulate the function of integrin αIIbβ3 (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 αIIb 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 αIIb, since it does not interact with the αv, α2, α5, β1, or β3 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 (∼25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured αIIbβ3 indicates a specific interaction between CIB and intact αIIbβ3. These results suggest that CIB is a candidate regulatory molecule for integrin αIIbβ3.

Integrins and GTPases in tumour cell growth, motility and invasion

One of the most exciting aspects of recent research on cell-adhesion receptors is the realization that these molecules can participate in bidirectional signal-transduction processes. Integrins can convey signals from the extracellular matrix to the cell interior, and intracellular events can influence the affinity of integrins for their ligands. Another important emerging concept is that integrins and small GTPases of the Rho family work together to coordinate both cytoskeletal organization and signalling processes. In this review, the authors examine integrin signalling and integrin-GTPase interplay in the context of cancer cell growth and behaviour.

Coagulation activation and inflammation in sickle cell disease-associated pulmonary hypertension.

Patients with sickle cell disease-associated pulmonary hypertension have increased endothelial dysfunction, coagulation activation and inflammation compared with patients without pulmonary hypertension. Endothelial dysfunction and coagulation activation appear to be the result of chronic hemolysis. See related perspective on page 1. Background Pulmonary hypertension (PHT) is common in sickle cell disease (SCD). The purpose of this study was to determine whether markers of coagulation activation and inflammation are associated with PHT in SCD. Design and Methods This cross-sectional study was performed using a cohort of patients followed at an adult Sickle Cell Clinic. Pulmonary artery systolic pressure was determined by Doppler echocardiography, and the diagnosis of PHT was defined using age, sex and body mass index-adjusted reference ranges. Clinical laboratory examinations, including hematologic studies and biochemical tests, as well as various measures of coagulation activation, endothelial activation and inflammation, were conducted on SCD subjects and on healthy, race-matched control subjects without SCD. Results Patients with SCD (n=76) had higher plasma levels of markers of coagulation (thrombin-antithrombin complex, prothrombin fragment F1+2, D-dimer) and endothelial (soluble vascular endothelial cell adhesion molecule, sVCAM) activation compared with control subjects (n=6). SCD patients with PHT (n=26) had significantly higher levels of sVCAM compared with those patients without PHT (n=50). Although PHT patients showed increased plasma measures of coagulation activation, the differences were not statistically significant when compared to those of patients without PHT. HbSS patients with PHT also had a trend towards higher levels of other inflammatory cytokines (interleukins 6, 8 and 10) than HbSS patients without PHT. There was a modest negative correlation between hemoglobin and plasma measures of coagulation and endothelial activation, and modest positive correlations between markers of hemolysis and plasma measures of coagulation and endothelial activation. Conclusions SCD patients with PHT have higher levels of markers of endothelial activation and other inflammatory markers than patients without PHT. A trend towards an increased level of markers of coagulation activation was observed in SCD patients with PHT compared with that in patients without PHT. Markers of hemolysis are associated with coagulation activation and endothelial dysfunction in SCD patients. Clinical trials of anticoagulants and anti-inflammatory agents are warranted in SCD patients with PHT.

Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48

Sensorineural hearing loss is genetically heterogeneous. Here, we report that mutations in CIB2, which encodes a calcium- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). One mutation in CIB2 is a prevalent cause of deafness DFNB48 in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In mice, CIB2 is localized to the mechanosensory stereocilia of inner ear hair cells and to retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of calcium binding, CIB2 significantly decreased the ATP-induced calcium responses in heterologous cells, whereas mutations in deafness DFNB48 altered CIB2 effects on calcium responses. Furthermore, in zebrafish and Drosophila melanogaster, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We also show that CIB2 is a new member of the vertebrate Usher interactome.

Biologically Active CD40 Ligand Is Elevated in Sickle Cell Anemia. Potential Role for Platelet-Mediated Inflammation

Objective—After activation, platelets expose CD40 ligand (CD40L) on their surface, then subsequently release the inflammatory mediator as a soluble fragment (sCD40L). Because sickle cell anemia (SCA) is noted for both platelet activation and chronic inflammation, we asked whether platelet-released CD40L potentially plays a role in SCA. Methods and Results—ELISAs demonstrate that SCA patient plasma contains 30-fold more sCD40L than control plasma. Correspondingly, platelets from these patients contain less than half the CD40L found in control platelets. Platelets from patients in painful crises are further depleted of CD40L, with even higher plasma levels, suggesting a correlation to the patient’s clinical state. In addition, elevated sCD40L correlates with increased tissue factor in SCA plasma. Blockage of the CD40L receptor CD40 reduces SCA plasma-induced production of tissue factor and endothelial intercellular adhesion molecule-1 (ICAM-1). Finally, sCD40L activity in SCA plasma is confirmed by its induction of B-cell proliferation. Conclusions—Platelet-derived sCD40L is elevated in SCA, further elevated in crises, and biologically active. The participation of sCD40L in SCA plasma-induced production of B cells, tissue factor, and ICAM-1 suggests that CD40L may contribute to the chronic inflammation and increased thrombotic activity known to occur in SCA.

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.

Dissecting clot retraction and platelet aggregation. Clot retraction does not require an intact fibrinogen gamma chain C terminus.

Fibrinogen mediates the processes of platelet aggregation and clot retraction. Previous studies have demonstrated that fibrinogen binding to the platelet receptor αβ requires the C-terminal residues of the fibrinogen chain. We made a recombinant human fibrinogen that lacks the chain C-terminal four residues (AGDV). As expected this fibrinogen did not support platelet aggregation. Unexpectedly, this variant did support clot retraction that was indistinguishable from retraction with normal recombinant or plasma fibrinogen. These results suggest that the site on fibrinogen that is required for platelet aggregation differs from the site on fibrin that is required for clot retraction.

Integrin αIIbβ3 signaling in platelet adhesion and aggregation

Abstract Intracellular signals are received and generated by the α IIb β 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 α IIb β 3 that cause further signaling and clot retraction.

CIB1 is a Regulator of Pathological Cardiac Hypertrophy

Hypertrophic heart disease is a leading health problem in Western countries. Here we identified the small EF hand domain–containing protein Ca2+ and integrin–binding protein-1 (CIB1) in a screen for previously unknown regulators of cardiomyocyte hypertrophy. Yeast two-hybrid screening for CIB1-interacting partners identified a related EF hand domain–containing protein, calcineurin B, the regulatory subunit of the prohypertrophic protein phosphatase calcineurin. CIB1 localizes primarily to the sarcolemma in mouse and human myocardium, where it anchors calcineurin to control its activation in coordination with the L-type Ca2+ channel. CIB1 protein amounts and membrane association were enhanced in cardiac pathological hypertrophy, but not in physiological hypertrophy. Consistent with these observations, Cib1-deleted mice showed a marked reduction in myocardial hypertrophy, fibrosis, cardiac dysfunction and calcineurin–nuclear factor of activated T cells (NFAT) activity after pressure overload, whereas the degree of physiologic hypertrophy after swimming exercise was not altered. Transgenic mice with inducible and cardiac-specific overexpression of CIB1 showed enhanced cardiac hypertrophy in response to pressure overload or calcineurin signaling. Moreover, mice lacking Ppp3cb (encoding calcineurin A, β isozyme) showed no enhancement in cardiac hypertrophy associated with CIB1 overexpression. Thus, CIB1 functions as a previously undescribed regulator of cardiac hypertrophy through its ability to regulate the association of calcineurin with the sarcolemma and its activation.