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Oroxylin A (Oro-A), the main bioactive flavonoid extracted from Scutellaria radix , has been reported to inhibit migration in various human cancer cell models. In this study, we further explored the anti-migration effects of Oro-A on oral squamous cell carcinoma (OSCC) cells and investigated the underlying mechanisms. A 24-h (short-term) exposure of OSCC cells to non-cytotoxic concentrations (5⁻20 μM) of Oro-A significantly suppressed cell migration according to a wound-healing assay. Furthermore, a 30-day exposure (long-term) to Oro-A (20 μM), which did not exhibit a cytotoxic effect on OSCC cells, significantly suppressed cell migration more than short-term Oro-A exposure. To uncover the molecular mechanisms underlying the inhibitory effect of long-term Oro-A exposure on OSCC migration, a cDNA microarray and the Ingenuity software were used. Overall, 112 upregulated and 356 downregulated genes were identified in long-term Oro-A-exposed cells compared with untreated OSCC cells. Among them, 75 genes were reported to be associated with cancer cell migration. Consistent with the cDNA microarray results, we found that the expression levels of several cell migration-related genes, such as LCN2, ID-1, MDK, S100A9 and CCL2, were significantly decreased in long-term Oro-A-exposed OSCC cells using a quantitative real-time polymerase chain reaction (Q-PCR) assay. The Western blotting and enzyme-linked immunosorbent assay (ELISA) results also demonstrated that CCL2 expression at the mRNA and protein levels was significantly decreased in long-term Oro-A-exposed OSCC cells compared with untreated OSCC cells. Moreover, the expression levels of downstream CCL2 targets, including p-ERK1/2, NFκB, MMP2, and MMP9, were also decreased in long-term Oro-A-exposed OSCC cells. Further, Oro-A treatment suppressed in vivo metastasis. These results suggest that long-term Oro-A treatment inhibits metastasis via CCL2 signaling in OSCC cells., Competing Interests: available online at www.mdpi.com/xxx/s1: Table S1: Q-PCR primer; Figure S1: The effect of 24-h Oro-A exposure on LCN2, ID-1, MDK, S100A9, and CCL2 mRNA expression was determined by quantitative real-time polymerase chain reaction (Q-PCR); Figure S2: The effect of 24-h Oro-A exposure of on CCL2, CCR2, phosphorylated MER, IKK and P65 expression was determined by western blotting.

Angiogenesis is the process of developing vascular sprouts from existing blood vessels. Luminal endothelial cells convert into "tip" cells that contribute to the development of a multicellular stalk, which then undergoes lumen formation. In this review, we consider a variety of cellular and molecular pathways that mediate these transitions. We focus first on Notch signaling in cell fate determination as a mechanism to define tip and stalk cells. We next discuss the current models of lumen formation and describe new players in this process, such as chloride intracellular channel proteins. Finally, we consider the possible medical therapeutic benefits of understanding these processes and acknowledge potential obstacles in drug development.

Background: Little is known about the role of CLIC1 in endothelium. These studies investigate CLIC1 as a regulator of angiogenesis by in vitro techniques that mimic individual steps in the angiogenic process., Methods: Using shRNA against clic1, we determined the role of CLIC1 in primary human endothelial cell behavior., Results: Here, we report that reduced CLIC1 expression caused a reduction in endothelial migration, cell growth, branching morphogenesis, capillary-like network formation, and capillary-like sprouting. FACS analysis showed that CLIC1 plays a role in regulating the cell surface expression of various integrins that function in angiogenesis including β1 and α3 subunits, as well as αVβ3 and αVβ5., Conclusions: Together, these results indicate that CLIC1 is required for multiple steps of in vitro angiogenesis and plays a role in regulating integrin cell surface expression.

Visual perception of the environment plays an important role in many mosquito behaviors. Characterization of the cellular and molecular components of mosquito vision will provide a basis for understanding these behaviors. A unique feature of the R7 photoreceptors in Aedes aegypti and Anopheles gambiae is the extreme apical projection of their rhabdomeric membrane. We show here that the compound eye of both mosquitoes is divided into specific regions based on nonoverlapping expression of specific rhodopsins in these R7 cells. The R7 cells of the upper dorsal region of both mosquitoes express a long wavelength op2 rhodopsin family member. The lower dorsal hemisphere and upper ventral hemisphere of both mosquitoes express the UV-sensitive op8 rhodopsin. At the lower boundary of this second region, the R7 cells again express the op2 family rhodopsin. In Ae. aegypti, this third region is a horizontal stripe of one to three rows of ommatidia, and op8 is expressed in a fourth region in the lower ventral hemisphere. However, in An. gambiae the op2 family member expression is expanded throughout the lower region in the ventral hemisphere. The overall conserved ommatidial organization and R7 retinal patterning show these two species retain similar visual capabilities. However, the differences within the ventral domain may facilitate species-specific visual behaviors.

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1(-/-) mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1(-/-); Ccm2(lacZ/+) and Ccm2(lacZ/lacZ) mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity.

New capillaries are formed through angiogenesis and an integral step in this process is endothelial tubulogenesis. The molecular mechanisms driving tube formation during angiogenesis are not yet delineated. Recently, the chloride intracellular channel 4 (CLIC4)-orthologue EXC-4 was found to be necessary for proper development and maintenance of the Caenorhabditis elegans excretory canal, implicating CLIC4 as a regulator of tubulogenesis. Here, we studied the role of CLIC4 in angiogenesis and endothelial tubulogenesis. We report the effects of inhibiting or inducing CLIC4 expression on distinct aspects of endothelial cell behavior in vitro. Our experiments utilized RNA interference to establish cultured human endothelial cell lines with significant reduction of CLIC4 expression, and a CLIC4-expressing lentiviral plasmid was used to establish CLIC4 overexpression in endothelial cells. We observed no effect on cell migration and a modest effect on cell survival. Reduced CLIC4 expression decreased cell proliferation, capillary network formation, capillary-like sprouting, and lumen formation. This suggests that normal endogenous CLIC4 expression is required for angiogenesis and tubulogenesis. Accordingly, increased CLIC4 expression promoted proliferation, network formation, capillary-like sprouting, and lumen formation. We conclude that CLIC4 functions to promote endothelial cell proliferation and to regulate endothelial morphogenesis, and is thus involved in multiple steps of in vitro angiogenesis.

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. Here we describe a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction.

Cytostatic factor (CSF) arrests unfertilized vertebrate eggs in metaphase of meiosis II by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from mediating cyclin destruction. The APC/C inhibitor Emi2/XErp1 satisfies a number of historical criteria for the molecular identification of CSF, but the mechanism by which CSF is activated selectively in meiosis II is the remaining unexplained criterion. Here we provide an explanation by showing that Emi2 is expressed specifically in meiosis II through translational de-repression or "unmasking" of its mRNA. We find that Emi2 protein is undetectable in immature, G2/prophase-arrested Xenopus oocytes and accumulates approximately 90 minutes after germinal vesicle breakdown. The 3' untranslated region of Emi2 mRNA contains cytoplasmic polyadenylation elements that directly bind the CPEB protein and confer temporal regulation of Emi2 polyadenylation and translation. Our results demonstrate that cytoplasmic polyadenylation and translational unmasking of Emi2 directs meiosis II-specific CSF arrest.

Background: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood., Results: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions., Conclusions: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

In vertebrate meiosis, unfertilized eggs are arrested in metaphase II by cytostatic factor (CSF), which is required to maintain mitotic cyclin-dependent kinase activity. Fertilization triggers a transient increase in cytosolic free Ca(2+), which leads to CSF inactivation and ubiquitin-dependent cyclin destruction through the anaphase promoting complex or cyclosome (APC/C). The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the Polo-like kinase Plx1 are essential factors for Ca(2+)-induced meiotic exit, but the critical targets of these kinases were unknown. The APC/C inhibitor Emi2 or XErp1 has recently been characterized as a pivotal CSF component, required to maintain metaphase II arrest and rapidly destroyed in response to Ca(2+) signaling through phosphorylation by Plx1 and ubiquitination by the SCF(betaTrCP) complex. An important question is how the increase in free Ca(2+) targets Plx1 activity toward Emi2. Here, we demonstrate that CaMKII is required for Ca(2+)-induced Emi2 destruction, and that CaMKII functions as a "priming kinase," directly phosphorylating Emi2 at a specific motif to induce a strong interaction with the Polo Box domain of Plx1. We show that the strict requirement for CaMKII to phosphorylate Emi2 is a specific feature of CSF arrest, and we also use phosphatase inhibitors to demonstrate an additional mode of Emi2 inactivation independent of its destruction. We firmly establish the CSF component Emi2 as the first-known critical and direct target of CaMKII in CSF release, providing a detailed molecular mechanism explaining how CaMKII and Plx1 coordinately direct APC/C activation and meiotic exit upon fertilization.

Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCF(betaTrCP) ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified Delta90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of nondestructible Delta90 cyclin B rescues the MI-MII transition in Emi1-inhibited oocytes.

Mutations in ATM, the gene defective in the human genetic disorder ataxia-telangiectasia (A-T), have been described in A-T patients and in a variety of tumor samples. Most of these arise due to exon skipping. We developed an RT-PCR based protein truncation test (PTT) to screen for ATM mutations in breast cancer patients showing adverse response to radiotherapy. An additional PTT product was evident in the ATM gene in peripheral blood mononuclear cells (PBMCs) from blood samples that were collected 2 days or more prior to RNA extraction. Lymphoblastoid cell lines established from the same blood samples showed no evidence of the additional band. Cloning and sequencing of the additional RT-PCR product revealed an exact deletion of exon 20 (2639 del 200), pointing to exon skipping. RT-PCR analysis of RNA extracted from freshly prepared PBMCs from 3 normal individuals showed no evidence of the additional RT-PCR product but when the blood was stored for 2-3 days prior to RNA extraction the lower molecular weight band was evident in every sample. DNA sequencing confirmed this to be due to loss of exon 20. These data suggest that mRNA-based mutation analysis on ATM should be carried out on unstored blood samples to avoid artificial loss of exons that give rise to apparent mutations., (Copyright 2001 Wiley-Liss, Inc.)