Endoglin Requirement for BMP9 Signaling in Endothelial Cells Reveals New Mechanism of Action for Selective Anti-Endoglin Antibodies

Endoglin (ENG), a co-receptor for several TGFβ-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFβ and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFβ1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFβ only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFβ1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.     

MdmX promotes bipolar mitosis to suppress transformation and tumorigenesis in p53-deficient cells and mice

Mdm2 and MdmX are structurally related p53-binding proteins that function as critical negative regulators of p53 activity in embryonic and adult tissue. The overexpression of Mdm2 or MdmX inhibits p53 tumor suppressor functions in vitro, and the amplification of Mdm2 or MdmX is observed in human cancers retaining wild-type p53. We now demonstrate a surprising role for MdmX in suppressing tumorigenesis that is distinct from its oncogenic ability to inhibit p53. The deletion of MdmX induces multipolar mitotic spindle formation and the loss of chromosomes from hyperploid p53-null cells. This reduction in chromosome number, not observed in p53-null cells with Mdm2 deleted, correlates with increased cell proliferation and the spontaneous transformation of MdmX/p53-null mouse embryonic fibroblasts in vitro and with an increased rate of spontaneous tumorigenesis in MdmX/p53-null mice in vivo. These results indicate that MdmX has a p53-independent role in suppressing oncogenic cell transformation, proliferation, and tumorigenesis by promoting centrosome clustering and bipolar mitosis.     

Multiple effects of brood parasitism reduce the reproductive success of prothonotary warblers, Protonotaria citrea

Avian brood parasitism often has multiple negative effects on the reproductive success of the host. Most studies have focused on one or two of these effects, but rarely have they all been studied simultaneously for one species. I studied prothonotary warblers to quantify the effects of different intensities of (i.e. multiple) brood parasitism by brown-headed cowbirds, Molothrus ater, on the production of host and cowbird young and on the between-year returns of adult warblers. Host clutch size decreased with an increase in the number of cowbird eggs laid in nests. The hatching success of warbler and cowbird eggs decreased with increased cowbird eggs in nests, but was always higher for cowbird eggs than warbler eggs. The survival of warbler nestlings, but not cowbird nestlings, decreased with increased cowbird nestlings in the brood. An increase in the number of cowbird nestlings in broods resulted in a reduction in the average mass of warbler nestlings but not cowbird nestlings. The number of cowbird eggs or nestlings present did not affect nest predation, and the fledging of cowbirds did not influence the renesting interval of female warblers. In addition, the between-year returns of adult warblers were not negatively affected by brood parasitism. Decreased hatching success and nestling survival reduced the reproductive output of the warblers the most. These effects were substantial and appear to favour the evolution of behavioural responses that reduce the effects of brood parasitism on prothonotary warblers. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour      

Glutamate transprot in rat kidney mitochondria

The quantitative characteristics of [U-14C]glutamate transport were determined in rotenone-inhibited energized rat kidney mitochondria at pH 7.0 and 28 degrees C. Glutamate efflux was observed to be first order with respect to matrix glutamate with a rate constant of 0.457 min-1. Uptake kinetic studies indicated that the Km of external glutamate was 1.4 mM and the Vmax 3.2 nmol/mg X min. These kinetic values were found to be unchanged at pH 6.6 or in mitochondria obtained from kidneys of chronically acidotic rats. Parallel studies of glutamate deamination were performed in which mitochondria were incubated in state 3, state 4, and with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, in the presence of malonate. The oxidative deamination of glutamate determined with 1 and 10 mM glutamate never exceeded the simultaneously measured rate of glutamate transport. No glutamate was detectable within the mitochondrial matrix under the conditions of these metabolic experiments. The studies indicate that the glutamate hydroxyl transporter is quite slow and rate limiting for the oxidative deamination of external glutamate in rat kidney mitochondria.     

Decrease in adhesion of cells cultured in polyunsaturated fatty acids

The addition of long chain unsaturated fatty acids (linoleic, linolenic and arachidonic acids) to BHK cells reduces the cell to substrate adhesion, causes morphological changes and alters the cellular growth properties. The new characteristics are similar to those of transformed cells. The data indicate that the effects are probably due to actual changes in the surface membrane lipids and not due to prostaglandin synthesis.     

Simultaneous Comparisons of Multiple Treatments to Two (or More) Control

DUNNETT (1955) developed a procedure simultaneously comparing k treatments to one control with an exact overall type I error of α when all sampling distributions are normal. Sometimes it is desirable to compare k treatments to m≧2 controls, in particular to two controls. For instance, several new therapies (e.g., pain relievers) could be compared to two standard therapies (e.g., Aspirin and Tylenol). Alternatively, a standard therapy could be very expensive, difficult to apply and/or have bad side effects, making it useful to compare each new therapy to both standard therapy and no therapy (Placebo). Dunnett's method is expanded here to give comparisons of mean values for k treatments to mean values for m≧2 controls at an exact overall type I error of α when all sampling distributions are normal. Tabled values needed to make exact simultaneous comparisons at α = .05 are given for m = 2. An application is made to an example from the literature.     

Changes in gene expression associated with IFN-beta and IL-2-induced augmentation of human natural killer cell function

NK function can be augmented by a variety of agents, including the cytokines IL-2 and IFN. The mechanisms associated with IL-2- and IFN-mediated augmentation of NK function are largely unknown. In order to learn more about the regulation of NK activity, we have studied changes in gene expression that occur upon treatment of a cloned line of NK cells (NK 3.3) with rIL-2 and rIFN-beta. Both IL-2 and IFN-beta induced rapid augmentation of lysis mediated by NK 3.3, which was significant within 1 h, peaked at 6 h of treatment, and declined by 12 h. This enhancement of lytic function was independent of proliferation and associated with a corresponding increase in steady state levels of RNA coding for both the nuclear proto-oncogene c-myb and for the IL-2R. These changes were specific in that RNA levels of another nuclear proto-oncogene, c-myc, were increased by IL-2 but not by IFN-beta, whereas HLA class I RNA levels were relatively unchanged by either IL-2 or IFN-beta treatment. Treatment of NK 3.3 with the combination of IL-2 and IFN enhanced both lysis and c-myb expression in an additive fashion. These findings suggest that c-myb may play a regulatory role in the cytolytic activity of NK cells.