Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.     

Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress

Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0 +/- 3.0% (n = 3; P < 0.001) and primary cancer cells by 41.0 +/- 3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5 +/- 0.13 fold) and Western analysis (1.34 +/- 0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 microM), PP2 (20 microM), and actin-cytoskeleton stabilizer phalloidin (10 microM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0 +/- 2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection.     

Regulation of biological activity of laminin-5 by proteolytic processing of gamma2 chain

Laminin-5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino-terminal region of the gamma2 chain as well as at a carboxyl-terminal region of the alpha3 chain. To clarify the biological effect of the gamma2 chain processing, we prepared a human recombinant LN5 with the 150-kDa, non-processed gamma2 chain (GAA-LN5) and natural LN5 with the 105-kDa, processed gamma2 chain (Nat-LN5). Comparison of their biological activities demonstrated that GAA-LN5 had an about five-times higher cell adhesion activity but an about two-times lower cell migration activity than Nat-LN5. This implies that the proteolytic processing of LN5 gamma2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non-processed gamma2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed gamma2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the gamma2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion.     

Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences

The major surface protein of the genus Pseudomonas, OprF, is a non-specific porin that plays an important role in maintenance of cell shape, in growth in a low osmolarity environment, and in adhesion to various supports. The objectives of our study were (i) to carry out a comparative analysis of phylogenies obtained from the OprF protein and from the 16S rRNA gene in 41 isolates from various sources (water, soil, milk and the hospital) and (ii) to investigate the physiological characteristics correlated with the phylogeny of OprF. We report here an important incongruence between the phylogenies of the 16S rRNA gene and the OprF protein. Phylogenetic analysis of 16S rRNA genes grouped Pseudomonas fluorescens isolates into one cluster (termed fluorescens r-cluster) whilst the phylogeny of the OprF protein divided Pseudomonas fluorescens isolates into two quite distinct clusters (termed fluorescens 1 o-cluster and fluorescens 2 o-cluster) that may be related to the original habitat of the strain. The fluorescens 1 o-cluster contained the majority of non-rhizospheric soil isolates, while the fluorescens 2 o-cluster contained all our clinical isolates and most of the rhizospheric isolates (which are fixed to the roots). In order to check this correlation, we studied two physiological characteristics: the range of growth temperature and the capacity for non-specific adhesion to polystyrene. The temperature range study for strains did not explain the existence of the two o-clusters but it did confirm the capacity of certain P. fluorescens strains to grow at 37 degrees C. The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche.     

TGF-beta1 enhances betaig-h3-mediated keratinocyte cell migration through the alpha3beta1 integrin and PI3K

betaig-h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF-beta1). We previously demonstrated that betaig-h3 has two alpha3beta1 integrin-interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both betaig-h3 and TGF-beta1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF-beta1 enhances keratinocyte adhesion and migration toward betaig-h3 through the alpha3beta1 integrin. TGF-beta1 did not increase the amount of the alpha3beta1 integrin on the cell surface, but rather increased its affinity for betaig-h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF-beta1-enhanced cell migration but not adhesion to betaig-h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to betaig-h3. The PI3K pathway is also not associated with the affinity of the alpha3beta1 integrin to betaig-h3. TGF-beta1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF-beta1 increases affinity of the alpha3beta1 integrin to betaig-h3, resulting in enhanced adhesion and migration of keratinocytes toward betaig-h3. TGF-beta1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the alpha3beta1 integrin or its adhesion to betaig-h3.     

The effect of elevated extracellular glucose on adherens junction proteins in cultured rat heart endothelial cells

Vascular permeability is a proof of vascular endothelial cell dysfunction induced by diabetes. Vascular permeability is directly related to the width of intercellular endothelial cells junctions, which may become permeable to macromolecules as a result of a change in endothelial cell shape. To determine the role of hyperglycemia in endothelial cell shape, the study examined the effect of high concentrations of glucose on the shape of cultured rat heart endothelial cells. This result indicated that the high-glucose-induced changes in the morphology of endothelial cells, via the glucose-mediated reorganization of F-actin. In endothelial cells, the actin cytoskeleton is tethered to the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix interactions respectively. The present study demonstrated that the high-glucose-induced changes in the actin-binding protein such as filamin, zonula adherens proteins such as alpha-, beta-, and gamma-catenin, focal adhesions proteins such as focal adhesion kinase, paxillin, and tyrosine phosphorylation of paxillin. It appears that differences in expression of adherens junctions molecules on rat heart endothelial cells in response to high glucose reflect endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.     

Involvement of NAD(P)H oxidase in the enhanced expression of cell adhesion molecules in the aorta of diabetic mice

The increased levels of cell adhesion molecules (CAM) have been identified in diabetic vasculatures, but the underlying mechanisms remain unclear. To determine the relationship among vascular production of superoxide, expression of CAM and diabetes, superoxide generation and expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E- and P-selectin in the aorta from control (C57BL/6J) and diabetic mice (ob/ob) were measured. In situ staining for superoxide using dihydroethidium showed an increased superoxide production in diabetic aorta in association with an enhanced NAD(P)H oxidase activity. Immunohistochemical analysis revealed that the endothelial expression of ICAM-1 (3.5 +/- 0.4) and VCAM-1 (3.8 +/- 0.3) in diabetic aorta was significantly higher than that in control aorta (0.9 +/- 0.5 and 1.6 +/- 0.3, respectively). Furthermore, there was a strong positive correlation (r = 0.89, p < 0.01 in ICAM-1 and r = 0.88, p < 0.01 in VCAM-1) between ICAM-1/VCAM-1 expression and vascular production of superoxide. The present data indicate that the increased production of superoxide via NAD(P)H oxidase may explain the enhanced expression of CAM in diabetic vasculatures.     

Transforming growth factor-beta1 stimulated protein kinase B serine-473 and focal adhesion kinase tyrosine phosphorylation dependent on cell adhesion in human hepatocellular carcinoma SMMC-7721 cells

Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor and apoptosis inducer for most normal cells. However, tumor cells are commonly nevertheless sensitive to the tumor-suppressing effects of TGF-beta1. In this paper, we focus on the effects of TGF-beta1 on two important anti-apoptotic protein kinases, protein kinase B (PKB), and focal adhesion kinase (FAK), in SMMC-7721 cells. We found that PKB-Ser-473 phosphorylation was apparently up-regulated by TGF-beta1. In the meantime, PKB-Thr-308 phosphorylation was slightly up-regulated by TGF-beta1. TGF-beta1 could also enhance FAK-Tyr phosphorylation. We observed that integrin-linked kinase (ILK) was also up-regulated by TGF-beta1 in good accordance with PKB-Ser-473 phosphorylation. We first found that TGF-beta1 could stimulate PKB-Ser-473 phosphorylation possibly via up-regulating ILK expression. Furthermore, we also failed to detect PKB-Ser-473 and FAK-Tyr phosphorylation with various concentrations of TGF-beta1 treatment when cells were kept in suspension. The above results indicate that PKB-Ser-473 and FAK-Tyr phosphorylation stimulated by TGF-beta1 are both dependent on cell adhesion.     

Tumor angiogenesis inhibitors

Formation of the blood supply system is a critical step in malignant transformation of neoplasms which results in the penetration of tumor cells into neighboring tissues and metastatic growth. Significant progress in the elucidation of mechanisms underlying tumor angiogenesis and the discovery of a great diversity of biomolecules involved in its regulation have culminated in the development of a radically new approach to antitumor therapy based on the search for efficient inhibitors of tumor angiogenesis. This review is devoted to the analysis of action mechanisms and expression of the major endogenous inhibitors involved in regulation of tumor and physiological angiogenesis. The antiangiogenic effects of the majority of currently known synthetic inhibitors are considered in the context of their roles in the main steps of tumor angiogenesis. Possible applications of antiangiogenic therapy in the chemotherapy of cancer diseases are discussed.