Distinct roles of DKK1 and DKK2 in tumor angiogenesis.

Tumor angiogenesis is essential for tumor invasive growth and metastasis, and generates abnormal vascular structures unlike developmental neovessel formation. To reduce tumor vascular abnormalities such as leakage and perivascular cell coverage deficiency that limit cancer therapy effectiveness, novel therapeutic approaches focus on vessel normalization. We have previously shown that Dickkopf-1 (DKK1), a Wnt antagonist, inhibits and its homolog DKK2 enhances, angiogenesis in normal tissues. In the present study, we investigated the effects of DKK1 and DKK2 on tumor growth and angiogenesis. Treatment of B16F10 melanoma-bearing mice with adenovirus expressing DKK1 significantly reduced tumor growth but DKK2 increased growth compared with controls. Similar pattern of tumor growth was observed in endothelial-specific DKK1 and DKK2 transgenic mice. Interestingly, tumor vascular density and perfusion were significantly decreased by DKK1 but increased by DKK2. Moreover, coverage of blood vessels by pericytes was reduced by DKK1, while DKK2 increased it. We further observed that DKK1 diminished retinal vessel density and increased avascular area in an in vivo murine model of oxygen-induced retinopathy, whereas DKK2 showed opposite results. These findings demonstrate that DKK1 and DKK2 have differential roles in normalization and functionality of tumor blood vessels, in addition to angiogenesis.

Rk1, a ginsenoside, is a new blocker of vascular leakage acting through actin structure remodeling.

Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage.

The WNT antagonist Dickkopf2 promotes angiogenesis in rodent and human endothelial cells.

Neovessel formation is a complex process governed by the orchestrated action of multiple factors that regulate EC specification and dynamics within a growing vascular tree. These factors have been widely exploited to develop therapies for angiogenesis-related diseases such as diabetic retinopathy and tumor growth and metastasis. WNT signaling has been implicated in the regulation and development of the vascular system, but the detailed mechanism of this process remains unclear. Here, we report that Dickkopf1 (DKK1) and Dickkopf2 (DKK2), originally known as WNT antagonists, play opposite functional roles in regulating angiogenesis. DKK2 induced during EC morphogenesis promoted angiogenesis in cultured human endothelial cells and in in vivo assays using mice. Its structural homolog, DKK1, suppressed angiogenesis and was repressed upon induction of morphogenesis. Importantly, local injection of DKK2 protein significantly improved tissue repair, with enhanced neovascularization in animal models of both hind limb ischemia and myocardial infarction. We further showed that DKK2 stimulated filopodial dynamics and angiogenic sprouting of ECs via a signaling cascade involving LRP6-mediated APC/Asef2/Cdc42 activation. Thus, our findings demonstrate the distinct functions of DKK1 and DKK2 in controlling angiogenesis and suggest that DKK2 may be a viable therapeutic target in the treatment of ischemic vascular diseases.

Hypnotic effects and binding studies for GABA(A) and 5-HT(2C) receptors of traditional medicinal plants used in Asia for insomnia.

AIM OF THE STUDY: Many medicinal plants have been used for treatment of insomnia in Asia. However, scientific evidence and precise mechanism for their sedative-hypnotic activity have not been fully investigated. Thus, we investigated the binding activity of the oriental plant extracts (mainly from Korea and Japan) to the well-known molecular targets for sleep regulation, GABA(A) and 5-HT(2C) receptors. Following the binding assay, sedative-hypnotic effects of the extracts with high affinity were examined in an animal model of sleep. MATERIALS AND METHODS: Aqueous and ethanol extracts of 15 medicinal plants were tested for binding at the benzodiazepine site of GABA(A) receptor and 5-HT site of 5-HT(2C) receptor. The sedative-hypnotic effects of selected extracts were evaluated by measuring the sleep latency and sleep duration during pentobarbital-induced sleep in mice after oral administration of extracts. RESULTS: In the GABA(A) assay, the ethanol extracts of licorice and danshen displayed concentration-dependent, high affinity binding, whereas in the 5-HT(2C) assay, the ethanol extracts of ginseng and silk tree showed high affinity. Among these extracts we tested previously uncharacterized licorice and silk tree for hypnotic effects. We found the ethanol extracts of licorice and silk tree significantly decreased sleep latency and increased sleep duration in pentobarbital-induced sleep. CONCLUSIONS: We demonstrate for the first time that licorice and silk tree have the sedative-hypnotic activity possibly by modulating GABA(A) and 5-HT(2C) receptors. We propose that licorice and silk tree might be effective candidates for treatment of insomnia.

A splice variant of CD99 increases motility and MMP-9 expression of human breast cancer cells through the AKT-, ERK-, and JNK-dependent AP-1 activation signaling pathways.

The CD99 gene encodes two distinct transmembrane proteins by alternative splicing of its transcript. To examine the effects of two CD99 isoforms on the invasive phenotypes of breast cancer cells, MDA-MB-231 and MCF-7 human breast cancer cell lines were stably transfected with CD99 cDNAs encoding the major wild-type form (type I) or a minor splice variant (type II). As a result, expression of CD99 type II, but not type I, markedly elevated the motility, binding to fibronectin, MMP-9 expression, and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. In MDA-MB-435 breast cancer cells expressing both CD99 type I and type II, invasion-related cellular activities were inhibited by the transfection of small interfering RNA (siRNA) targeted to CD99 type II. Meanwhile, CD99 type II-induced MMP-9 expression in MDA-MB-231 cells was shown to be mediated by the binding of AP-1 factors to the MMP-9 gene promoter. Gel shift assay revealed that ligation of CD99 type II with antibody resulted in the binding of JunD to the AP-1 site of the MMP-9 promoter region. Initiation of CD99 type II signaling by antibody ligation increased expression of JunD and FosB AP-1 factors, along with phosphorylation of Src, Akt, p38 MAPK, ERK, and JNK. Knockdown of JunD and FosB by siRNA transfection abolished the positive effects of CD99 type II on the motility and MMP-9 expression of MDA-MB-231 cells. Increased expression of JunD and FosB as well as elevated cell motility and MMP-9 expression by CD99 type II ligation were also abrogated by inhibitors, dominant-negative forms, and siRNAs for Akt1, ERK1/2, and JNK1 but not for p38 MAPK. These results suggest that expression of a splice variant of CD99 contributes to the invasive ability of human breast cancer cells by up-regulating AP-1-mediated gene expression through the Akt-dependent ERK and JNK signaling pathways.

PNUTS, a protein phosphatase 1 (PP1) nuclear targeting subunit. Characterization of its PP1- and RNA-binding domains and regulation by phosphorylation.

PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a recently described protein that targets protein phosphatase 1 (PP1) to the nucleus. In the present study, we characterized the biochemical properties of PNUTS. A variety of truncation and site-directed mutants of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia coli or as FLAG-tagged proteins in 293T cells. A 50-amino acid domain in the center of PNUTS mediated both high affinity PP1 binding and inhibition of PP1 activity. The PP1-binding domain is related to a motif found in several other PP1-binding proteins but is distinct in that Trp replaces Phe. Mutation of the Trp residue essentially abolished the ability of PNUTS to bind to and inhibit PP1. The central PP1-binding domain of PNUTS was an effective substrate for protein kinase A in vitro, and phosphorylation substantially reduced the ability of PNUTS to bind to PP1 in vitro and following stimulation of protein kinase A in intact cells. In vitro RNA binding experiments showed that a C-terminal region including several RGG motifs and a novel repeat domain rich in His and Gly interacted with mRNA and single-stranded DNA. PNUTS exhibited selective binding for poly(A) and poly(G) compared with poly(U) or poly(C) ribonucleotide homopolymers, with specificity being mediated by distinct regions within the domain rich in His and Gly and the domain containing the RGG motifs. Finally, a PNUTS-PP1 complex was isolated from mammalian cell lysates using RNA-conjugated beads. Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.

TNF-related activation-induced cytokine (TRANCE) induces angiogenesis through the activation of Src and phospholipase C (PLC) in human endothelial cells.

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.