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Proliferation of aortic smooth muscle cells contributes to atherogenesis and neointima formation. Sublytic activation of complement, particularly C5b-9, induces cell cycle progression in aortic smooth muscle cells. RGC-32 is a novel protein that may promote cell cycle progression in response to complement activation. We cloned human RGC-32 cDNA from a human fetal brain cDNA library. The human RGC-32 cDNA encodes a 117-amino acid protein with 92% similarity to the rat and mouse protein. Human RGC-32 maps to chromosome 13 and is expressed in most tissues. Sublytic complement activation enhanced RGC-32 mRNA expression in human aortic smooth muscle cells and induced nuclear translocation of the protein. RGC-32 was physically associated with cyclin-dependent kinase p34 CDC2 and increased the kinase activity in vivo and in vitro. In addition, RGC-32 was phosphorylated by p34 CDC2 -cyclin B1 in vitro. Mutation of RGC-32 protein at Thr-91 prevented the p34 CDC2 -mediated phosphorylation and resulted in loss of p34 CDC2 kinase enhancing activity. Overexpression of RGC-32 induced quiescent aortic smooth muscle cells to enter S-phase. These data indicate that cell cycle activation by C5b-9 may involve p34 CDC2 activity through RGC-32. RGC-32 appears to be a cell cycle regulatory factor that mediates cell proliferation, both as an activator and substrate of p34 CDC2 .C5b-9, the membrane attack complex of complement, causes cell death by forming transmembrane pores (1). When the number of C5b-9 molecules is limited to a sublytic level, nucleated cells are able to escape cell death by eliminating membrane-inserted terminal complement complexes (TCC 1 ; C5b-7, C5b-8, and C5b-9) by endocytosis and/or membrane shedding (2-4). Among these complexes, C5b-9 is most potent in activating target cells. C5b-9 causes a Ca 2ϩ influx and generates intracellular second messengers, including phosphatidylinositol triphosphates, diacylglycerol, and ceramide (5-8). Membrane-inserted TCC activates the G i /G o family of G proteins (9). Activation of G i /G o by TCC is responsible for the G␤␥-mediated activation of cell cycle through activation of Ras, Raf-1, extracellular signal regulated kinase-1 (10), and activation of phosphatidylinositol 3-phosphate kinase (10, 11).Cell cycle activation by C5b-9 is associated with an increase in CDK4, CDK2, and p34 CDC2 activities, and this is followed by an increase in DNA synthesis and cell proliferation (11, 12, 24 -26). The C5b-9-induced DNA synthesis is abolished by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 and phosphatidylinositol 3-phosphate kinase (11). Cell cycle activation by C5b-9 in postmitotic cells, such as oligodendrocytes (OLG) and myotubes, is associated with expression of c-JUN and c-FOS protooncogenes and loss of differentiation (12)(13)(14).In an effort to find novel C5b-9-induced genes involved in cell cycle regulation, we cloned the rat Response Gene to Complement (RGC-32) using mRNA differential display PCR in OLG (15, 16). C5b-9 en...

RUNX2 is a member of the runt family of DNA-binding transcription factors. RUNX2 mediates endothelial cell migration and invasion during tumor angiogenesis and is expressed in metastatic breast and prostate tumors. Our published studies showed that RUNX2 DNA-binding activity is low during growth arrest, but elevated in proliferating endothelial cells. To investigate its role in cell proliferation and cell cycle regulation, RUNX2 was depleted in human bone marrow endothelial cells using RNA interference. Specific RUNX2 depletion inhibited DNA-binding activity as measured by electrophoretic mobility shift assay resulting in inhibition of cell proliferation. Cells were synchronized at the G 1 /S boundary with excess thymidine or in mitosis (M phase) with nocodazole. Endogenous or ectopic RUNX2 activity was maximal at late G 2 and during M phase. Inhibition of RUNX2 expression by RNA interference delayed entry into and exit out of the G 2 /M phases of the cell cycle. RUNX2 was coimmunoprecipitated with cyclin B1 in mitotic cells, which further supported a role for RUNX2 in cell cycle progression. Moreover, in vitro kinase assays using recombinant cdc2 kinase showed that RUNX2 was phosphorylated at Ser 451 . The cdc2 inhibitor roscovitine dose dependently inhibited in vivo RUNX2 DNAbinding activity during mitosis and the RUNX2 mutant S451A exhibited lower DNA-binding activity and reduced stimulation of anchorage-independent growth relative to wild type RUNX2. These results suggest for the first time that RUNX2 phosphorylation by cdc2 may facilitate cell cycle progression possibly through regulation of G 2 and M phases, thus promoting endothelial cell proliferation required for tumor angiogenesis.The RUNX gene family of transcriptional regulators is the mammalian homologue of the Drosophila transcription factor runt. The three RUNX factors contain an evolutionarily conserved 128-amino acid runt domain, which is responsible for DNA binding and heterodimerization with the cofactor CBF␤ (1-3). RUNX factors are essential regulators of hematopoietic (4 -6), osteogenic (7-10), and gastric development (11-13). RUNX2 promotes preosteoblast growth and osteoblast lineage commitment (14 -16). Mice nullizygous for RUNX2 lack bone matrix gene expression and exhibit no bone formation, whereas heterozygous mice display skeletal abnormalities similar to cleidocranial dysplasia in humans (17)(18)(19)(20). Increasing evidence suggests that RUNX2 exhibits oncogenic potential in a wide variety of cells (21). Expression of RUNX2 is higher in H-Ras-transformed NIH3T3 fibroblasts compared with normal cells (22) and, after retroviral integration in T-cells, RUNX2 expression cooperates with c-Myc to stimulate cell growth (23). Forced expression of Runx2 in transgenic mice was found to interfere with T-cell development and to predispose mice to lymphoma (23). Endogenous RUNX2 is expressed in malignant breast cancer cells and prostate tumors, regulates bone sialoprotein expression, and promotes osteolytic lesions in breast cancer metastases (24...

The signaling pathway mediated by JNK transduces different types of signals, such as stress stimuli and cytokines, into functional responses that mediate apoptosis, as well as proliferation, differentiation, and inflammation. To better characterize the dynamic information flow and signal processing of this pathway in the cellular context, a genetically encoded, fluorescent protein-based biosensor was engineered to detect endogenous JNK activity. This biosensor, named JNKAR1 (for JNK activity reporter), specifically detects stress-(ribotoxic and osmotic) and cytokine-(TNF-α) induced JNK activity in living cells with a 15 to 30% increase in the yellow-tocyan emission ratio because of a phosphorylation-dependent increase in FRET between two fluorescent proteins. JNK activity was detected not only in the cytoplasm, but also in the nucleus, mitochondria, and plasma membrane with similar kinetics after induction of ribotoxic stress by anisomycin, suggesting relatively rapid signal propagation to the nuclear, mitochondrial, and plasma membrane compartments. Furthermore, quantitative single-cell analysis revealed that anisomycin-induced JNK activity exhibited ultrasensitivity, sustainability, and bimodality, features that are consistent with behaviors of bistable systems. The development of JNKAR1, therefore, laid a foundation for evaluating the signaling properties and behaviors of the JNK cascade in single living cells.

Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.

Migration and proliferation of aortic endothelial cells (AEC)are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/ Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1.

Activation of the complement system plays an important role in innate and acquired immunity. Activation of complement and subsequent formation of C5b-9 channels on the surface of cellular membranes leads to cell lysis. When the number of channels assembled on the surface of nucleated cells is limited, C5b-9 does not cause lysis, but instead can induce cell-cycle progression by activating signal transduction pathways, transcription factors, and key components of the cell-cycle machinery. Cell-cycle induction by C5b-9 is dependent on the activation of phosphatidylinositol 3-kinase and the ERK1 pathway in a Gi protein-dependent manner. Cell-cycle activation is regulated, in part, by activation of proto-oncogene c-jun and AP1 DNA binding activity. C5b-9 induces sequential activation of CDK4 and CDK2, leading to G1/S-phase transition and cellular proliferation. RGC-32 is a novel gene whose expression is induced by C5b-9. RGC-32 may play a key role in cell-cycle activation by increasing cyclin B1-CDC2 activity. C5b-9-mediated cell-cycle activation plays an important role in cellular proliferation and protection from apoptosis.

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