CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5beta.

CLASPs are mammalian microtubule-stabilizing proteins that can mediate the interaction between distal microtubule ends and the cell cortex. Using mass spectrometry-based assays, we have identified two CLASP partners, LL5beta and ELKS. LL5beta and ELKS form a complex that colocalizes with CLASPs at the cortex of HeLa cells as well as at the leading edge of motile fibroblasts. LL5beta is required for cortical CLASP accumulation and microtubule stabilization in HeLa cells, while ELKS plays an accessory role in these processes. LL5beta is a phosphatidylinositol-3,4,5-triphosphate (PIP3) binding protein, and its recruitment to the cell cortex is influenced by PI3 kinase activity but does not require intact microtubules. Cortical clusters of LL5beta and ELKS do not overlap with focal adhesions but often form in their vicinity and can affect their size. We propose that LL5beta and ELKS can form a PIP3-regulated cortical platform to which CLASPs attach distal microtubule ends.

Anti-inflammatory effect of pitavastatin on NF-kappaB activated by TNF-alpha in hepatocellular carcinoma cells.

As nuclear factor-kappa B (NF-kappaB) is essential for promoting inflammation-associated cancer, it is a potential target for cancer prevention in chronic inflammatory diseases. Here we examined the anti-inflammatory effect of pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on NF-kappaB activated by TNF-alpha in hepatocellular carcinoma (HCC) cells. Western blot revealed that the treatment of Huh 7 cells with pitavastatin at 0.1 microM inhibited the nuclear expression of NF-kappaB p65 induced by TNF-alpha. Furthermore, electrophoretic mobility shift assay showed that after the cells were incubated with pitavastatin alone or with pitavastatin and TNF-alpha for 24 h, pitavastatin significantly decreased the DNA binding activity of NF-kappaB induced by TNF-alpha. Subsequently, luciferase assay revealed that pitavastatin suppressed the transcriptional activity of the NF-kappaB promoter, which was clearly related to the HMG-CoA reductase activity because the addition of mevalonic acid (MEV) elevated the TNF-alpha activity. Moreover, the Rho kinase inhibitor Y27632 had no major effect on the NF-kappaB inhibitory activity of pitavastatin. The inhibitory effect of pitavastatin is possibly independent of the Rho kinase pathway in inflammation-associated HCC cells is. Finally, the addition of TNF-alpha significantly increased IL-6 protein production, which was suppressed by the addition of pitavastatin. These results suggest that pitavastatin at a low dose (0.1 microM) inhibits NF-kappaB activation and decreases IL-6 production induced by TNF-alpha, and is therefore expected to be a new strategy for treating HCC.

BMP-2 prevents apoptosis of the N1511 chondrocytic cell line through PI3K/Akt-mediated NF-kappaB activation.

The signal transduction pathway by which bone morphogenetic protein-2 (BMP-2) regulates apoptosis in chondrocytes remains largely unknown. We investigated the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt-mediated NF-kappaB activation by BMP-2 stimulation in the modulation of this antiapoptotic process in a chondrocytic cell line, N1511. BMP-2 prevented apoptosis through the inhibition of caspase-3 and -9 and an increase in Bcl-xL expression, and this antiapoptotic effect was inhibited by Noggin. Not only was NF-kappaB p65 activated transiently in the early phase (5-15 min) after treatment with BMP-2 but p65 at serine 536 was phosphorylated from 5 min as well. Akt was rapidly phosphorylated in response to BMP-2 treatment; however, the inhibition of PI3K by Wortmannin markedly reduced the phosphorylation of Akt by BMP-2. Wortmannin also decreased the NF-kappaB transcriptional activity that was up-regulated by BMP-2. Thus, BMP-2-induced NF-kappaB activation is mediated by PI3K/Akt signaling. Wortmannin treatment inhibited the antiapoptotic effect of BMP-2. These data indicate that BMP-2 can utilize a new signal transduction pathway in the NF-kappaB activation system, which plays a crucial role in the survival of the N1511 chondrocytic cell line.

Pitavastatin at low dose activates endothelial nitric oxide synthase through PI3K-AKT pathway in endothelial cells.

Pitavastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor that is used for suppressing cholesterol biosynthesis. Previously, we have reported that pitavastatin induces the activation of endothelial nitric oxide synthase (eNOS) and increases nitric oxide (NO) production in vascular endothelial cells (EC). However, the mechanism of eNOS activation by pitavastatin remains unknown. Here, we examined the implications of pitavastatin-induced signaling in eNOS phosphorylation in EC. We found that treatment of EC with a low dose of pitavastatin induced eNOS phosphorylation at Ser-1177, activated Akt phosphorylation at Ser-473 in a time-and dose-dependent manner, and increased NO production. These processes were suppressed by the addition of either mevalonic acid (MEV) or geranylgeranyl pyrophosphate (GGPP). In addition, northern blot analysis revealed that pitavastatin did not increase eNOS mRNA expression level in EC. These results suggest that the activation of eNOS with a low dose of pitavastatin (0.1 microM) involves phosphoinositide 3-kinase and the Akt pathway and produces NO in EC, which is dependent on post-transcriptional regulation. This pathway is critical for cellular responses that contribute to EC function.