In response to agonist stimulation, the αIIbβ3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of αIIbβ3 is believed to occur in part via engagement of the β3 cytoplasmic tail with talin; however, the role of the αIIb tail and its potential binding partners in regulating αIIbβ3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the αIIb tail, is an endogenous inhibitor of αIIbβ3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced αIIbβ3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to αIIbβ3, thus providing a model for tightly controlled regulation of αIIbβ3 activation.

CIB1 is a 22-kDa calcium binding, regulatory protein with ϳ50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin ␣IIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1 ؊/؊ mice. Although Cib1 ؊/؊ mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1؊/؊ testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1 ؊/؊ mice exhibit a much slower growth rate compared to Cib1 ؉/؉ MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.

Objective-Reactive oxygen species (ROS) integrate cellular signaling pathways involved in aortic smooth muscle cell (SMC) proliferation and migration associated with atherosclerosis. However, the effect of subcellular localization of ROS on SMC mitogenic signaling is not yet fully understood. Methods and Results-We used superoxide dismutase (SOD)-deficient mouse aortic SMCs to address the role of subcellular ROS localization on SMC phenotype and mitogenic signaling. Compared with wild-type, a 54% decrease in total SOD activity (Ϸ50% decrease in SOD1 protein levels) and a 42% reduction in SOD2 activity (Ϸ50% decrease in SOD2 protein levels) were observed in SOD1 ϩ/Ϫ and SOD2 ϩ/Ϫ SMCs, respectively. Consistent with this, basal and thrombin-induced superoxide levels increased in these SMCs. SOD1ϩ/Ϫ and SOD2 ϩ/Ϫ SMCs exhibit increased basal proliferation and enhanced [3 H]-thymidine and [ 3 H]-leucine incorporation in basal and thrombin-stimulated conditions. Our results indicate preferential activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases in SOD1 ϩ/Ϫ and janus kinase/signal transducer and activator of transcriptase (JAK/STAT) pathway in SOD2 ϩ/Ϫ SMCs. Pharmacological inhibitors of ERK1/2 p38 and JAK2 confirm the SOD genotypedependent SMC proliferation. Conclusions-Our results suggest that SOD1 and SOD2 regulate SMC quiescence by suppressing divergent mitogenic signaling pathways, and dysregulation of these enzymes under pathophysiological conditions may lead to SMC hyperplasia and hypertrophy. Key Words: ROS Ⅲ SMC Ⅲ thrombin Ⅲ SOD Ⅲ cell signaling S mooth muscle cells (SMCs) are characterized by marked plasticity in their proliferative potential and differentiation status. As the primary constituents of the arterial media, they provide mechanical support for blood vessels and participate in regulation of vasomotor tone. In addition, SMCs contribute to neointimal formation after vascular injury and during atherosclerotic lesion progression in humans. In advanced atherosclerotic lesions, SMCs are prone to apoptosis and release proteolytic enzymes such as matrix metalloproteinases that may contribute to plaque instability. The remarkable diversity of SMC phenotype and function is determined in large part through regulatory cues in the extracellular microenvironment and interactions among intracellular signaling pathways. In addition, SMC phenotypes are modified over time by the accumulation of injuries to cellular macromolecules. See page 887Reactive oxygen species (ROS) are key components for integration of SMC signaling events, whereas at the same time, they are major contributors to the degradation of cellular function through their interactions with proteins and structural components of the cell. Exogenous ROS are potent stimuli for activation of SMC signaling and mitogenesis, 1 although in high (but physiological) concentrations, they may induce DNA damage and mitochondrial dysfunction. 2 SMCs have several intrinsic mechanisms for generating ROS, the majo...

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.

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