KITENIN functions as a fine regulator of ErbB4 expression level in colorectal cancer via protection of ErbB4 from E3-ligase Nrdp1-mediated degradation.

Understanding the complex biological functions of E3-ubiquitin ligases may facilitate the development of mechanism-based anti-cancer drugs. We recently identified that the KITENIN/ErbB4-Dvl2-c-Jun axis works as a novel unconventional downstream signal of epidermal growth factor (EGF) in colorectal cancer (CRC) tissues. Here we addressed whether E3-ubiquitin ligases are required for operation of this axis. We found that Nrdp1, an E3-ligase for ErbB3/ErbB4, interacted with KITENIN (KAI1 C-terminal interacting tetraspanin) to form a functional KITENIN/ErbB4/Nrdp1 complex and is responsible for down-regulating Dvl2 within this complex. Interestingly, ErbB4 was resistant to degradation by Nrdp1 in KITENIN/Nrdp1-co-transfected CRC cells, and KITENIN bound to the C-terminal coiled-coil domain of Nrdp1. Chemical blockade of ErbB kinase did not block the action of EGF to increase in total/phospho-ErbB4 and phospho-ERK in KITENIN/ErbB4-cotransfected cells, whereas it blocked the action of EGF in ErbB4 alone-transfected CRC cells. In human CRC tissues, higher expressions of ErbB4 and KITENIN and lower expression of Dvl2 was observed in stage IV samples than in stage I, but a low level of Nrdp1 was expressed in both stages and it did not differ significantly by stage. These results indicated that Nrdp1 is necessary for the reduction in Dvl2 to generate c-Jun in the EGF-KITENIN/ErbB4-c-Jun axis, but more importantly, elevated KITENIN protects KITENIN-bound ErbB4 from Nrdp1-mediated degradation via physical collaboration between the KITENIN/ErbB4 complex and Nrdp1, but not via modulation of ErbB kinase activity. Thus, KITENIN functions in the maintenance of a higher expression level of ErbB4 in advanced CRC tissues, independent of ubiquitin-mediated degradation via Nrdp1. © 2016 Wiley Periodicals, Inc.

Proteolytic activity of Porphyromonas gingivalis attenuates MCP-1 mRNA expression in LPS-stimulated THP-1 cells.

Bacteria can modulate cytokine production of host cells. In this study, we examined effects of Porphyromonas gingivalis, an important periodontal pathogen, on the cytokine production in lipopolysaccharide (LPS)-stimulated THP-1 macrophagic cells. A wide range of doses of P. gingivalis increased the tumor necrosis factor-α (TNF-α) production. However, monocyte chemoattractant protein-1 (MCP-1) production was substantially suppressed by high doses of P. gingivalis and this effect was demonstrated at the mRNA level. Challenges with a congenic protease mutant strain did not significantly attenuate the MCP-1 mRNA expression and addition of leupeptin, a protease inhibitor, to the cultures largely prevented the inhibition of MCP-1 expression by P. gingivalis. Transwell experiments showed that direct contact of P. gingivalis with THP-1 cells was not required for the MCP-1 inhibition. Furthermore, blockade of internalization of P. gingivalis into THP-1 cells had no effect on the MCP-1 inhibition by P. gingivalis. Finally, degradation of MCP-1 mRNA in LPS-stimulated THP-1 cells was accelerated in the presence of P. gingivalis. These results suggest that the proteolytic activity of P. gingivalis attenuate MCP-1 mRNA expression by promoting the decay of MCP-1 mRNA in LPS-stimulated THP-1 cells.

Enhancement of cytokine-mediated NF-kappaB activation by phosphatidylinositol 3-kinase inhibitors in monocytic cells.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays crucial roles in inflammation and immunity. Understanding the positive and negative regulation of NF-kappaB activity is therefore of fundamental importance. A few previous studies reported that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway enhances lipopolysaccharide (LPS)-induced activation of NF-kappaB. However, many aspects of the PI3K negative regulation of NF-kappaB activation remain to be clarified. The present study was conducted to shed light on cell-type specificity, stimulus specificity, and upstream mechanisms of the enhanced NF-kappaB activation by PI3K inhibitors. Gel shift assays showed that LY294002 (LY29) potently increased interleukin (IL)-1-induced NF-kappaB DNA binding in human monocytic THP-1 cells. Moreover, another PI3K inhibitor 3-methyladenine also strongly enhanced IL-1-induced NF-kappaB DNA binding, while LY303511, an inactive analogue of LY29, did not increase the NF-kappaB DNA binding. Compared with LY29, wortmannin (WM) effected only a marginal enhancement of NF-kappaB DNA binding. LY29 treatment also augmented tumor necrosis factor (TNF)-mediated NF-kappaB DNA binding. Furthermore, LY29, but not WM, increased cyclooxygenase (COX)-2 mRNA expression by IL-1 or TNF in THP-1 cells. Likewise, prostaglandin E2 production by IL-1 was increased by LY29, but not by WM. Western blot analysis demonstrated that IkappaB kinase (IKK) activation as well as IkappaB-alpha degradation and NF-kappaB nuclear translocation was elevated by LY29 and WM. Among the tested cell lines (HL-60, ECV304, Hep-2, and Molt-4), only HL-60, a promyelocytic cell line, showed enhanced NF-kappaB DNA binding by LY29. These results suggest that pharmacological inhibition of PI3K enhances the NF-kappaB-activating pathways by IL-1 through augmentation of IKK activation in myeloid/monocytic cells and the NF-kappaB enhancement is more robustly achieved by LY29 than by WM.

Expression of Nm23 in gliomas and its effect on migration and invasion in vitro.

BACKGROUND: Despite the well-documented importance of Nm23 in the control of metastasis, there is currently a paucity of data regarding the role of this gene family in the control of glioma invasion. MATERIALS AND METHODS: Nm23-H1 expression in gliomas was assessed via immunohistochemistry, Western blot, RT-PCR and Northern blot analyses. The migration and invasion ability were also investigated in primary glioma culture cells, human glioma cell lines and nm23-H1 transfectant, using an in vitro brain slice invasion model and a simple scratch technique. RESULTS: Although no significant correlations were detected between nm23-H1 expression and pathological grade, the endogenous nm23-H1 expression in gliomas was found to be inversely correlated with their migratory abilities. Additionally, the nm23-H1 transfectant resulted in a reduction of approximately 45% of the migratory ability and suppressed the invasiveness of the parental cell line. CONCLUSION: Our overall findings suggest that nm23-H1 may play an important role in the suppression of glioma invasion and migration.