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

Thermotolerant genes essential for survival at a critical high temperature in thermotolerant ethanologenic Zymomonas mobilis TISTR 548

Abstract Background High-temperature fermentation (HTF) technology is expected to reduce the cost of bioconversion of biomass to fuels or chemicals. For stable HTF, the development of a thermotolerant microbe is indispensable. Elucidation of the molecular mechanism of thermotolerance would enable the thermal stability of microbes to be improved. Results Thermotolerant genes that are essential for survival at a critical high temperature (CHT) were identified via transposon mutagenesis in ethanologenic, thermotolerant Zymomonas mobilis TISTR 548. Surprisingly, no genes for general heat shock proteins except for degP were included. Cells with transposon insertion in these genes showed a defect in growth at around 39 °C but grew normally at 30 °C. Of those, more than 60% were found to be sensitive to ethanol at 30 °C, indicating that the mechanism of thermotolerance partially overlaps with that of ethanol tolerance in the organism. Products of these genes were classified into nine categories of metabolism, membrane stabilization, transporter, DNA repair, tRNA modification, protein quality control, translation control, cell division, and transcriptional regulation. Conclusions The thermotolerant genes of Escherichia coli and Acetobacter tropicalis that had been identified can be functionally classified into 9 categories according to the classification of those of Z. mobilis, and the ratio of thermotolerant genes to total genomic genes in Z. mobilis is nearly the same as that in E. coli, though the ratio in A. tropicalis is relatively low. There are 7 conserved thermotolerant genes that are shared by these three or two microbes. These findings suggest that Z. mobilis possesses molecular mechanisms for its survival at a CHT that are similar to those in E. coli and A. tropicalis. The mechanisms may mainly contribute to membrane stabilization, protection and repair of damage of macromolecules and maintenance of cellular metabolism at a CHT. Notably, the contribution of heat shock proteins to such survival seems to be very low

Necl-4 enhances the VEGFR2 signaling in sparsely cultured ECs.

<p><b>A</b>, No effect of Necl-4-knockdown on the phosphorylation of VEGFR2 under sparse conditions. Lysates of HUVECs, transfected with control or Necl-4 siRNAs and cultured in the presence or absence of 50 ng/ml VEGF for 1 min, were subjected to Western blotting using the indicated antibodies. <b>B and C</b>, Reduced activation of Rac1 by Necl-4-knockdown. Lysates of HUVECs, transfected with control or Necl-4 siRNAs and cultured in the presence or absence of 50 ng/ml VEGF for the indicated periods of time, were subjected to pull-down assays using GST-PAK-CRIB (<i>n</i> = 4). †<i>P</i><0.01 vs. control siRNA. <b>D–F</b>, Reduced phosphorylation of ERK by Necl-4-knockdown. Lysates of HUVECs, transfected with FLAG or FLAG-Necl-4 and cultured in the presence or absence of 50 ng/ml VEGF for the indicated periods of time, were subjected to Western blotting using the indicated antibodies (<i>n</i> = 4). *<i>P</i><0.05; †<i>P</i><0.01 vs. FLAG. <b>G and H</b>, Activation of ROCK by Necl-4-knockdown. Lysates of HUVECs transfected with control or Necl-4 siRNAs were subjected to Western blotting using the indicated antibodies. *<i>P</i><0.05; †<i>P</i><0.01 vs. control siRNA. <b>I and J</b>, Restoration of the reduced activation of Rac1 in Necl-4-knockdown HUVECs by ROCK inhibitors. Lysates of HUVECs, transfected with control or Necl-4 siRNAs, incubated with or without 10 μM Y-27632 or fasudil, and cultured in the presence or absence of 50 ng/ml VEGF for 5 min, were subjected to Western blotting using the indicated antibodies or pull-down assays using GST-PAK-CRIB (<i>n</i> = 3). *<i>P</i><0.05; †<i>P</i><0.01. <b>K and L</b>, Restoration of the activity of ROCK by additional knockdown of PTPN13. Lysates of HUVECs transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs were subjected to Western blotting using the indicated antibodies (<i>n</i> = 3). †<i>P</i><0.01; ns, not significant. <b>M and N</b>, Restoration of the activity of Rac1 by additional knockdown of PTPN13. Lysates of HUVECs, transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs and cultured in presence of 50 ng/ml VEGF for 5 min, were subjected to pull-down assays using GST-PAK-CRIB (<i>n</i> = 3). *<i>P</i><0.05; †<i>P</i><0.01; ns, not significant.</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

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

Expression of Necl-4 is regulated by cell-density through Rap1 and afadin in ECs.

<p><b>A–H</b>, Comparison of the expression levels under sparse vs. confluent conditions. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to Western blotting using the indicated antibodies (<i>n</i> = 4). *<i>P</i><0.05; †<i>P</i><0.01 vs. 25% confluence. <b>I</b>, Up-regulation of Necl-4 protein by confluence. Lysates of HAECs, HBMECs, the human colon epithelial cancer cell line Caco-2, and HEK293 cells, which were cultured under sparse or confluent conditions and subjected to Western blotting using the indicated antibodies (<i>n</i> = 3). <b>J</b>, Up-regulation of Necl-4 mRNA expression by confluence. RNAs extracted from HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to qPCR (<i>n</i> = 4). *<i>P</i><0.05 vs. 25% confluence.</p

Model of the regulation of the phosphorylation and signaling of VEGFR2 by Necl-4 and PTPN13.

<p>The details are described in the Discussion. N13, PTPN13; P, phosphorylation.</p

Necl-4 inhibits VEGFR2 activation, signaling, and cellular responses in confluently cultured ECs.

<p><b>A and B</b>, Enhanced phosphorylation of VEGFR2 by Necl-4-knockdown. HUVECs transfected with control or Necl-4 siRNAs were cultured under confluent conditions in the presence or absence of 50 ng/ml VEGF for the indicated periods of time. Cell lysates were subjected to Western blotting using the indicated antibodies (<i>n</i> = 4). †<i>P</i><0.01 vs. control siRNA. <b>C–H</b>, Reduced phosphorylation and signaling of VEGFR2 by Necl-4-overexpression. HUVECs transfected with FLAG or FLAG-Necl-4 were cultured under sparse conditions in the presence or absence of 50 ng/ml VEGF for the indicated periods of time. Cell lysates were subjected to Western blotting using the indicated antibodies or pull-down assays using GST-PAK-CRIB (<i>n</i> = 4). *<i>P</i><0.05; †<i>P</i><0.01 vs. FLAG. <b>I and J</b>, Reduced movement by Necl-4-overexpression. HUVECs transfected with FLAG or FLAG-Necl-4 were plated onto collagen-coated culture dishes and subjected to wound-healing assays in the presence or absence of 50 ng/ml VEGF (<i>n</i> = 4). †<i>P</i><0.01 vs. FLAG. <b>K</b>, Reduced VEGF-induced proliferation by Necl-4-overexpression. HUVECs transfected with FLAG or FLAG-Necl-4 were cultured on 24-well plates coated with collagen in EBM-2 plus 2% FBS in the presence of 50 ng/ml VEGF. At the indicated time points, HUVECs were detached and the number of the cells was counted (<i>n</i> = 3). *<i>P</i><0.05 vs. FLAG. <b>L and M</b>, Involvement of PTPN13 in the enhanced phosphorylation of VEGFR2 by Necl-4-knockdown. HUVECs transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs were cultured under confluent conditions in the presence or absence of 50 ng/ml VEGF for 1 min and their lysates were subjected to Western blotting using the indicated antibodies (<i>n</i> = 3). †<i>P</i><0.01 vs. control siRNA.</p

MOESM1 of Thermotolerant genes essential for survival at a critical high temperature in thermotolerant ethanologenic Zymomonas mobilis TISTR 548

Additional file 1. Additional figures and tables