Actomyosin organization at adherens junctions

Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with β-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with β-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with β-catenin. Although a “catch-bond model” partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with β-catenin and enhances its F-actin–binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with β-catenin and E-cadherin at adherens junctions

The Cell Adhesion Molecule Necl-4/CADM4 Serves as a Novel Regulator for Contact Inhibition of Cell Movement and Proliferation

Contact inhibition of cell movement and proliferation is critical for proper organogenesis and tissue remodeling. We show here a novel regulatory mechanism for this contact inhibition using cultured vascular endothelial cells. When the cells were confluently cultured, Necl-4 was up-regulated and localized at cell-cell contact sites where it cis-interacted with the vascular endothelial growth factor (VEGF) receptor. This interaction inhibited the tyrosine-phosphorylation of the VEGF receptor through protein-tyrosine phosphatase, non-receptor type 13 (PTPN13), eventually reducing cell movement and proliferation. When the cells were sparsely cultured, Necl-4 was down-regulated but accumulated at leading edges where it inhibited the activation of Rho-associated protein kinase through PTPN13, eventually facilitating the VEGF-induced activation of Rac1 and enhancing cell movement. Necl-4 further facilitated the activation of extracellular signal-regulated kinase 1/2, eventually enhancing cell proliferation. Thus, Necl-4 serves as a novel regulator for contact inhibition of cell movement and proliferation cooperatively with the VEGF receptor and PTPN13

Increased Insulin Action in SKIP Heterozygous Knockout Mice▿ †

Insulin controls glucose homeostasis and lipid metabolism, and insulin impairment plays a critical role in the pathogenesis of diabetes mellitus. Human skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) is a member of the phosphatidylinositol 3,4,5-trisphosphate phosphatase family (T. Ijuin et al. J. Biol. Chem. 275:10870-10875, 2000; T. Ijuin and T. Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). Previous studies showed that SKIP negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling (Ijuin and Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). We now have generated mice with a targeted mutation of the mouse ortholog of the human SKIP gene, Pps. Adult heterozygous Pps mutant mice show increased insulin sensitivity and reduced diet-induced obesity with increased Akt/protein kinase B (PKB) phosphorylation in skeletal muscle but not in adipose tissue. The insulin-induced uptake of 2-deoxyglucose into the isolated soleus muscle was significantly enhanced in Pps mutant mice. A hyperinsulinemic-euglycemic clamp study also revealed a significant increase in the rate of systemic glucose disposal in Pps mutant mice without any abnormalities in hepatic glucose production. Furthermore, in vitro knockdown studies in L6 myoblast cells revealed that reduction of SKIP expression level increased insulin-stimulated Akt/PKB phosphorylation and 2-deoxyglucose uptake. These results imply that SKIP regulates insulin signaling in skeletal muscle. Thus, SKIP may be a promising pharmacologic target for the treatment of insulin resistance and diabetes

Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions.

<p><b>A</b>, Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. <b>B</b>, Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. <b>C and D</b>, Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 (<b>C</b>) or VEGFR2 (<b>D</b>) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.</p

Necl-4 enhances cellular responses in sparsely cultured ECs.

<p><b>A–C</b>, Reduced movement by Necl-4-knockdown. HUVECs transfected with control or Necl-4 siRNAs were subjected to wound-healing assays in the presence or absence of 50 ng/ml VEGF. Culture dishes were coated with collagen (<b>A and B</b>) or vitronectin (<b>C</b>) (<i>n</i> = 3). †<i>P</i><0.01 vs. control siRNA. <b>D</b>, Reduced proliferation by Necl-4-knockdown. HUVECs transfected with control or Necl-4 siRNAs were cultured on 96-well plates coated with type I collagen in EBM-2 plus 2% FBS in the absence or presence of 50 ng/ml VEGF. At the indicated time points, the numbers of the cells were quantified by crystal violet staining (<i>n</i> = 3). (<b>E and F</b>, Reduced tubulogenesis by Necl-4-knockdown. HUVECs, transfected with control or Necl-4 siRNAs were subjected to Matrigel network formation assays in the presence or absence of 50 ng/ml VEGF (<i>n</i> = 4). †<i>P</i><0.01 vs. control siRNA. <b>G, H, J, and K</b>, Restoration of the reduced movement and tubulogenesis of Necl-4-knockdown HUVECs by ROCK inhibitors. HUVECs, transfected with control or Necl-4 siRNAs and incubated with or without 10 μM Y-27632 or fasudil, were subjected to wound-healing assays (<b>G and H</b>) (<i>n</i> = 3) or Matrigel network formation assays (<b>J and K</b>) (<i>n</i> = 4) in the presence of 50 ng/ml VEGF. †<i>P</i><0.01 vs. VEGF. <b>I</b>, No effects of ROCK inhibitors on the reduced proliferation of Necl-4-knockdown HUVECs. HUVECs, transfected with control or Necl-4 siRNAs and incubated with or without 10μM Y-27632 or fasudil, were cultured on 24-well plates coated with collagen in EBM-2 plus 2% FBS in the presence of 50 ng/ml VEGF. After 48 h, the numbers of the cells were quantified by crystal violet staining (<i>n</i> = 3). <b>L–O</b>, Restoration of the reduced tubulogenesis and movement of Necl-4-knockdown HUVECs by additional knockdown of PTPN13. HUVECs, transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs, were subjected to Matrigel network formation assays (<b>L and M</b>) (<i>n</i> = 3) or wound-healing assays (<b>N and O</b>) (<i>n</i> = 3) in the presence of 50 ng/ml VEGF. *<i>P</i><0.05; †<i>P</i><0.01; ns, not significant.</p