Tumor samples were collected from primary lesion of metastatic ccRCC patients

ent by Necl4-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. P<0.01 vs. FLAG. K, Reduced VEGF-induced Digitoxin 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. P<0.05 vs. FLAG. L and M, 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. P<0.01 vs. control siRNA. doi:10.1371/journal.pone.0124259.g004 Necl-4 strongly interacts with VEGFR2 with no effect on VEGFR2 activation and enhancement of VEGFR2 signaling and cellular responses in sparsely cultured ECs Because Necl-4 was localized at the leading edges of sparsely cultured ECs, we examined whether Necl-4 coordinated VEGFR2 activation, signaling, and cellular responses under sparse conditions. Although Necl-4 strongly interacted with VEGFR2, Necl-4-knockdown did not influence the VEGF-induced phosphorylation of VEGFR2. Necl-4-knockdown decreased the basal and VEGF-stimulated activities of Rac1. Moreover, Necl4-knockdown did not influence the basal phosphorylation levels of ERK1/2, but decreased the VEGF-induced increase in the phosphorylation of ERK1/2. These results indicate that Necl-4 increases the basal activity of Rac1 and the VEGF-induced activation of Rac1 and ERK1/2 under sparse conditions. Because there is a crosstalk between Rho-associated protein kinase and Rac1 whereby PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768747 ROCK inhibits the activity of Rac1, we examined whether ROCK was activated by Necl-4-knockdown. The phosphorylation of Thr853 residues of the MBS of myosin light chain phosphatase was increased by Necl-4-knockdown, indicating the activation of ROCK. The decreased activation of Rac1 in Necl-4-knockdown cells was restored by specific ROCK inhibitors, Y-27632 and fasudil. Consistent with this, Necl-4-knockdown increased formation of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768500 stress fibers and focal adhesions, which were regulated by ROCK, and the increased formation of stress fibers and focal adhesions was restored by Y-27632 and fasudil. However, the decreased phosphorylation of ERK1/2 was not restored by Y-27632 or fasudil. These results indicate that the increased activity of ROCK inhibits the activation of Rac1, but is not involved in the decreased activation of ERK1/2 by Necl-4-knockdown. In Necl-4-knockdown cells, additional knockdown of PTPN13 restored the increased phosphorylation of MBS and the decreased activation of Rac1. These results indicate that PTPN13 mediates the Necl-4-knockdown-induced activation of ROCK. We next investigated the role of Necl-4 in cellular responses under sparse conditions. In the presence or absence of VEGF, closure of scraped areas was delayed in Necl-4-knockdown ECs. Similarly, cell proliferation and tubulogenesis in the presence or absence of VEGF were significantly reduced in Necl-4-knockdown ECs. The reduced movement and tubulogenesis were partly restored by Y-27632 and fasudil, whereas the reduced proliferation was not affected by these inhibitors. These results in