No abstract

Necl-5/Tage4/poliovirus receptor/CD155 is the poliovirus receptor and upregulated in rodent and human carcinoma. We have recently shown that mouse Necl-5 is upregulated by an oncogenic Ki-Ras (V12Ki-Ras) in NIH3T3 cells and enhances cell movement induced by growth factors, including platelet-derived growth factor and fibroblast growth factor (FGF), in an integrin a v b 3 -dependent manner in wild type and V12Ki-Ras-transformed NIH3T3 cells. In addition, it enhances the growth factor-induced cell proliferation. We examined here how mouse Necl-5 was upregulated by V12Ki-Ras in NIH3T3 cells. Expression of the luciferase reporter gene fused to the Necl-5 promoter was induced by V12Ki-Ras in NIH3T3 cells. This induction was mediated through the Raf-MEK-ERK pathway. The Necl-5 promoter has an AP-1-binding site and this site was required for the V12Ki-Ras-induced activation of the Necl-5 promoter. Expression of the luciferase reporter gene fused to the Necl-5 promoter was also induced by FGF through the Raf-MEK-ERK-AP-1 pathway in NIH3T3 cells. These results indicate that the expression of mouse Necl-5 is induced by FGF or V12Ki-Ras through the Raf-MEK-ERK-AP-1 pathway.

E-cadherin and nectins are major cell-cell adhesion molecules at adherens junctions (AJs) in epithelial cells. When Madin-Darby canine kidney (MDCK) cells stably expressing nectin-1 (nectin-1-MDCK cells) are cultured at normal Ca(2+), E-cadherin and nectin-1 are concentrated at the cell-cell contact sites. When these cells are cultured at low Ca(2+), E-cadherin disappears from the cell-cell contact sites, but nectin-1 persists there. When these cells are re-cultured at normal Ca(2+), E-cadherin is recruited to the nectin-based cell-cell contact sites. We found here that this recruitment was dependent on protein synthesis, because a protein synthesis inhibitor, cycloheximide, prevented the accumulation of E-cadherin. When nectin-1-MDCK cells, precultured at low Ca(2+) in the presence of a proteasome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal), were re-cultured at normal Ca(2+), E-cadherin was recruited to the nectin-based cell-cell contact sites but the level of E-cadherin was reduced. Similar results were obtained when wild-type MDCK cells were used instead of nectin-1-MDCK cells. These results suggest that degradation of one or more protein factors and de novo synthesis of the same or different protein factor(s) are needed for the formation of the E-cadherin-based AJs. We biochemically identified the annexin II-S100A10 complex as such a candidate. Depletion of plasma membrane cholesterol, which abolished the localization of the annexin II-S100A10 complex at the plasma membrane, inhibited the re-concentration of E-cadherin at the nectin-based cell-cell contact sites in the Ca(2+) switch experiment. Knockdown of annexin II by RNA interference also inhibited the re-concentration of E-cadherin. These results indicate that the annexin II-S100A10 complex is involved in the formation of the E-cadherin-based AJs in MDCK cells.

Milk casein-derived tripeptides, valyl prolyl proline (VPP), and isoleucyl prolyl proline (IPP) inhibit angiotensin-converting enzyme (ACE) and both fermented milk and proteolytic hydrolysates of milk casein containing these peptides exert blood pressure-lowering effects in animals and humans. On the top of these results, we have recently reported that the hydrolysate of milk casein containing both VPP and IPP improved the vascular endothelial function of subjects with stage I hypertension, enforcing us to elucidate the mechanism of the improvement of endothelial dysfunction by these peptides. For this purpose, we examined the effect of VPP and IPP on induction of nitric oxide (NO) production using cultured vascular endothelial cells and isolated arterial vessels. When both VPP and IPP were added to the medium of cultured endothelial cells at final concentrations of more than 100 nmol/l, the NO(x) (NO(2) and NO(3)) concentration in the medium was significantly higher than that of the control. Moreover, both VPP and IPP induced endothelium-dependent relaxation of isolated aortic rings, and these effects were inhibited by NO synthase inhibitors, K channel inhibitors, and bradykinin B2 receptor antagonists. These lines of results suggested that both VPP and IPP induced production of vasodilative substances including NO.

The antiviral effects of both a live and non-live Lactobacillus acidophilus strain L-92 (L-92) were investigated by oral administration (10 mg/mouse per d) daily for 21 d in a mouse model infected intranasally with influenza virus (H1N1). Virus titres in the lung of mice administered either live or non-live L-92 cells daily for 15 d were repressed 6 d after virus infection compared with the control group. Natural killer (NK) activity in the orally administered non-live L-92 group was higher compared with that of the control group before virus infection and on day 6. In contrast, NK activity in the live L-92 group compared with the control group was not significantly changed on both days, but was significantly higher on day 1. In contrast, live L-92 showed a greater repression of virus proliferation compared with non-live L-92, 6 d after the infection. Live L-92 decreased the number of neutrophils in the lung and suppressed lung weight, leading to the consequent deterioration of consolidation scores of the lung. These results indicated that pretreatment of live or non-live L-92 cells had protective effects against influenza virus infection. Among the measured cytokines and chemokines, eotaxin, macrophage colony-stimulating factor, IL-1b, RANTES (regulated on activation, normal T cell expressed and secreted) and interferon-a were significantly increased in the lung: IL-17 was significantly increased in Peyer's patch of the live L-92 group compared with the control group. A mechanistic study suggested that the enhancement of NK activity in the lung caused by stimulating various antiviral cytokines and chemokines after the oral administration of L-92 cells might be important in protecting against virus infection.