Necl-4 enhances the PLCγ-c-Raf-MEK-ERK pathway without affecting internalization of VEGFR2.

We have reported that knockdown of Necl-4 decreases vascular endothelial growth factor (VEGF)-induced phosphorylation of extracellular signal-regulated kinase (ERK) without affecting phosphorylation of VEGF receptor 2 (VEGFR2) in sparsely cultured human umbilical vein endothelial cells (HUVECs). However, the underlying molecular mechanism is unknown. Compared with control HUVECs, VEGF-induced phosphorylation of phospholipase Cγ (PLCγ), c-Raf, mitogen-activated protein kinase/ERK kinase (MEK) and ERK were all inhibited in Necl-4-knockdown HUVECs. However, VEGF-induced internalization of VEGFR2 was not different between control and Necl-4-knockdown HUVECs. We have reported that protein-tyrosine phosphatase, non-receptor type 13 (PTPN13) and Rho-associated kinase (ROCK) are involved in the Necl-4-knockdown-induced inhibition of the VEGF-induced activation of Rac1. However, the effects of Necl-4-knockdown on VEGF-induced phosphorylation of VEGFR2 and ERK were not affected either by knockdown of PTPN13 or by ROCK inhibitors. These results suggest that Necl-4 enhances VEGF-induced activation of PLCγ-c-Raf-MEK-ERK pathway without affecting the phosphorylation and internalization of VEGFR2.

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.

Successful steroid therapy for heart failure due to myocarditis associated with primary biliary cirrhosis.

This report concerns a 65-year-old woman with primary biliary cirrhosis (PBC) featuring high titers of M2 antimitochondrial antibody who developed intractable heart failure (HF). Although the etiology is unclear, involvement of skeletal muscles has been reported with PBC as a result of mitochondrial damage from the autoimmune process. In this case, histopathological examination and (18)F-fluorodeoxy glucose-positron emission tomography imaging revealed myocarditis concomitant with myositis. The patient showed favourable response to high-dose corticosteroid administration. To the best of our knowledge, this is the first case of HF with myocarditis and polymyositis associated with asymptomatic PBC who was successfully treated with prednisolone.

Necl-5/poliovirus receptor interacts with VEGFR2 and regulates VEGF-induced angiogenesis.

RATIONALE: Vascular endothelial growth factor (VEGF), a major proangiogenic agent, exerts its proangiogenic action by binding to VEGF receptor 2 (VEGFR2), the activity of which is regulated by direct interactions with other cell surface proteins, including integrin α(V)ß(3). However, how the interaction between VEGFR2 and integrin α(V)ß(3) is regulated is not clear. OBJECTIVE: To investigate whether Necl-5/poliovirus receptor, an immunoglobulin-like molecule that is known to bind integrin α(V)ß(3), regulates the interaction between VEGFR2 and integrin α(V)ß(3), and to clarify the role of Necl-5 in the VEGF-induced angiogenesis. METHODS AND RESULTS: Necl-5-knockout mice displayed no obvious defect in vascular development; however, recovery of blood flow after hindlimb ischemia and the VEGF-induced neovascularization in implanted Matrigel plugs were impaired in Necl-5-knockout mice. To clarify the mechanism of the regulation of angiogenesis by Necl-5, we investigated the roles of Necl-5 in the VEGF-induced angiogenic responses in vitro. Knockdown of Necl-5 by siRNAs in human umbilical vein endothelial cells (HUVECs) inhibited the VEGF-induced capillary-like network formation on Matrigel, migration, and proliferation, and conversely, enhanced apoptosis. Coimmunoprecipitation assays showed the interaction of Necl-5 with VEGFR2, and knockdown of Necl-5 prevented the VEGF-induced interaction of integrin α(V)ß(3) with VEGFR2. Knockdown of Necl-5 suppressed the VEGFR2-mediated activation of downstream proangiogenic and survival signals, including Rap1, Akt, and endothelial nitric oxide synthase. CONCLUSIONS: These results demonstrate the critical role of Necl-5 in angiogenesis and suggest that Necl-5 may regulate the VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin α(v)ß(3), and the VEGFR2-mediated Rap1-Akt signaling pathway.