Mutations in the human endoglin gene result in hereditary hemorrhagic telangiectasia type 1, a vascular disorder characterized by multisystemic vascular dysplasia, arteriovenous malformations, and focal dilatation of postcapillary venules. Previous studies have implicated endoglin in the inhibition of cell migration in vivo and in vitro. In the course of studies to address the relationship of the conserved cytosolic domain to endoglin function, we identified zyxin, a LIM domain protein that is concentrated at focal adhesions, as an interactor with endoglin in human umbilical vein vascular endothelial cells. This interaction is localized within the 47-amino acid carboxyl-terminal cytosolic domain of endoglin, and maps within zyxin residues 326 -572. The endoglin-zyxin interaction was found to be largely mediated by the third LIM domain of zyxin, and is specific for endoglin because the homologous cytosolic domain of the transforming growth factor-␤ type III receptor, betaglycan, fails to interact with zyxin. Expression of endoglin is associated with reduction of zyxin, as well as its interacting proteins p130 cas and CrkII, from a focal adhesion protein fraction, and this reduction is correlated with inhibition of cell migration. We also show that endoglin-dependent: (i) inhibition of cell migration, (ii) reduction of focal adhesion-associated p130 cas /CrkII protein levels, (iii) tyrosine phosphorylation of p130 cas , and (iv) focal adhesion-associated endoglin levels are mediated by the cytosolic domain of endoglin. These results suggest a novel mechanism of endoglin function involving its interaction with LIM domain-containing proteins, and associated adapter proteins, affecting sites of focal adhesion.Endoglin is a 190-kDa homodimeric transmembrane glycoprotein composed of 95-kDa disulfide-linked subunits (1). Mutations in the gene encoding endoglin have been linked to the human disease: hereditary hemorrhagic telangiectasia type 1 (HHT1) 1 (2, 3), an autosomal dominant inherited vascular disorder characterized by localized vascular dysplasia and a tendency toward arteriovenous malformations. Arteriovenous malformations occur in ϳ20% of patients and are associated with life-threatening complications including stroke and brain abscess. Pathological features found in HHT telangiectases include focal dilatation of postcapillary venules and a prominence of actin stress fibers in pericytes (4 -6). Mice lacking endoglin die from defective angiogenesis characterized by failure of vascular smooth muscle investment of embryonic blood vessels, suggesting a defect in vascular smooth muscle cell development (7). The molecular mechanism underlying the angiogenic phenotype in the endoglin null mouse remains unclear.In vivo data points to a role for endoglin in the vascular response to injury. Findings from this laboratory showed that endoglin was expressed in human aortic smooth muscle cells in atherosclerotic plaques but was absent from normal smooth muscle (8). Ma et al. (9) found that expression of endoglin in normal p...

We have previously demonstrated that the expression of the soluble extracellular domain of the transmembrane ligand for Notch receptors, Jagged 1 (sJ1), in NIH 3T3 cells results in the formation of a matrix-dependent chord-like phenotype, the loss of contact inhibition of growth, and an inhibition of pro-␣1(I) collagen expression. In an effort to define the mechanism by which sJ1 induces this phenotype, we report that sJ1 transfectants display biochemical and cytoskeletal alterations consistent with the activation of Src. Indeed, cotransfection of sJ1 transfectants with a dominant-negative mutant of Src resulted in the loss of matrix-dependent chord formation and correlated with the restoration of type I collagen expression and contact inhibition of growth. We also report that the sJ1-mediated induction of Src activity and related phenotypes, including chord formation, may result from the inhibition of endogenous Jagged 1-mediated Notch signaling since it was not possible to detect an sJ1-dependent induction of CSL-dependent transcription in these cells. Interestingly, NIH 3T3 cells transfected with dominant-negative (but not constitutively active) mutants of either Notch 1 or Notch 2 displayed a similar Src-related phenotype as the sJ1 transfectants. These data suggest that the ability of sJ1 to mediate chord formation is Src-dependent and requires the repression of endogenous Jagged 1-mediated Notch signaling, which is tolerant to the destabilization of the actin cytoskeleton, a mediator of cell migration.

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Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation has been associated with tumor progression, poor prognosis and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavourable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells, in vitro. Results exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent down regulation of Notch-mediated signalling, as demonstrated by reduced levels of the Notch target genes, Hes1 and Hey1. Neuroendocrine differentiation was promoted by attenuation of Hes1 transcription, as cells expressing a dominant negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia down regulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen independent cell lines, PC3 and Du145, it did not change the extent of NE differentiation in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Conclusions hypoxia induces neuroendocrine differentiation of LNCaP cells in vitro, which appears to be driven by the inhibition of Notch signalling with subsequent down-regulation of Hes1 transcription.

Edited by Ned ManteiThe article is dedicated to the memory of Tom Maciag, scientist, friend, and mentor.Abstract Notch signaling involves proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands, Delta or Jagged; and the resultant soluble intracellular domain of Notch stimulates a cascade of transcriptional events. The Delta1 ligand also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. We demonstrate that the expression of the intracellular domain of Delta1 results in a non-proliferating senescent-like cell phenotype which is dependent on the expression of the cell cycle inhibitor, p21, and is abolished by coexpression of constitutively active Notch1. These data suggest a new intracellular role for Delta1.

Sphingosine kinase 1 catalyzes the formation of sphingosine-1-phosphate, a lipid mediator involved in the regulation of angiogenesis. Sphingosine kinase 1 is constitutively released from cells, even though it lacks a classical signal peptide sequence. Because copper-dependent non-classical stressinduced release of FGF1 also regulates angiogenesis, we questioned whether sphingosine kinase 1 is involved in the FGF1 release pathway. We report that (i) the coexpression of sphingosine kinase 1 with FGF1 inhibited the release of sphingosine kinase 1 at 37°C; (ii) sphingosine kinase 1 was released at 42°C in complex with FGF1; (iii) sphingosine kinase 1 null cells failed to release FGF1 at stress; (iv) sphingosine kinase 1 is a high affinity copper-binding protein which formed a complex with FGF1 in a cell-free system, and (v) sphingosine kinase 1 over expression rescued the release of FGF1 from inhibition by the copper chelator, tetrathiomolybdate. We propose that sphingosine kinase 1 is a component of the copper-dependent FGF1 release pathway.

Introduction Severe burn injuries are known to initiate a profound systemic inflammatory response (SIRS) that may lead to burn shock and other SIRS related complications. Damage associated molecular patterns (DAMPs) are important early signaling molecules that initiate SIRS after burn injury. Mitochondrial DAMPs (mtDAMPs) – such as mitochondrial DNA (mtDNA) - are thought to be the most critical of these early signaling molecules. Previous work in a rodent model has shown that application of a topical immune modulator (p38 MAPK inhibitor) applied directly to the burn wound decreases cytokine expression, reduces pulmonary inflammation and edema and improves survival. Our group has demonstrated that tranexamic acid (TXA) – in addition to its use as an anti-fibrinolytic – has cell protective in vitro effects. We hypothesized that administration of TXA after burn injury would attenuate mtDAMP release and reduce lung inflammation. Methods C57/BL6 male mice were subjected to a 40% TBSA scald burn by immersion in an 80°C water bath. Sham animals underwent the same procedure, but were immersed in room temperature water. All animals were resuscitated according to the Parkland formula (3cc × %TBSA × Weight [Kg]) by intraperitoneal injection (IP). One treatment group received the topical application of 1mM solution of p38 MAPK inhibitor after burn injury. The other treatment group received an IP administration of TXA (100 mg TXA per 1 kg weight) after burn injury. Animals were sacrificed at 5 hours after injury or sham treatment. Plasma was collected by cardiac puncture. MtDNA levels in plasma were determined by qPCR. Syndecan-1 levels in plasma were determined by ELISA. Lungs were harvested, formalin fixed and paraffin embedded. Sections of lungs were deparaffinized and stained for Mac1 antigen to detect macrophages. Numbers of macrophages per a standard square unit of lung section were calculated. Results Topical p38 MAPK inhibitor and TXA significantly attenuated mtDNA release. Both TXA and the topical p38 MAPK inhibitor reduced lung inflammation as represented by decreased macrophage infiltration. Circulating syndecan-1 levels showed no difference between the untreated burn group and either treatment group. Conclusion Both p38 MAPK inhibitor and TXA demonstrated the ability to attenuate burn induced DAMP release and lung inflammation. Beyond its role as an anti-fibrinolytic, TXA may have significant anti-inflammatory effects pertinent to burn resuscitation. Further study is required; however, TXA may be a useful adjunct in burn resuscitation and other non-hemorrhagic shock states.

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