Extrinsic sphingosine 1-phosphate activates S1P5 and induces autophagy through generating endoplasmic reticulum stress in human prostate cancer PC-3 cells.

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that binds to a family of G protein-coupled receptors (GPCRs), termed S1P1-S1P5. Our previous study has reported that S1P induces autophagy in human prostate cancer PC-3 cell. In addition, S1P-induced autophagy plays a prosurvival role in PC-3 cells. Accumulating evidence has shown that the autophagy responses triggered by ER stress signaling have cytoprotective effects. Thus, we attempted to investigate whether S1P-induced autophagy is a result of triggering ER stress in PC-3 cells. By monitoring XBP-1 mRNA splicing, a characteristic of ER stress, we demonstrate that S1P triggers ER stress in a concentration-dependent and time-dependent manner. Moreover, DiH S1P, a membrane-nonpermeable S1P analog without intracellular effects also enhances ER stress. Meanwhile, we also show that S1P5 is required for S1P-induced ER stress by using RNA interference experiments. Furthermore, signaling analyses revealed that PI3K, PLC, and ROS production were involved in S1P's effects on ER stress induction. On the other hand, knockdown of XBP-1 abolished S1P-induced autophagy. In summary, our results demonstrate for the first time that the extracellular S1P-triggered ER stress is responsible for autophagy induction in PC-3 cells.

Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells.

Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA1 and LPA3 siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.

Fibroblast growth factor-2 up-regulates the expression of nestin through the Ras-Raf-ERK-Sp1 signaling axis in C6 glioma cells.

Nestin is a 240-kDa intermediate filament protein expressed mainly in neural and myogenic stem cells. Although a substantial number of studies have focused on the expression of nestin during development of the central nervous system, little is known about the factors that induce and regulate its expression. Fibroblast growth factor-2 (FGF-2) is an effective mitogen and stimulates the proliferation and differentiation of a subset of nestin-expressing cells, including neural progenitor cells, glial precursor cells, and smooth muscle cells. To assess whether FGF-2 is a potent factor that induces the expression of nestin, C6 glioma cells were used. The results showed that nestin expression was up-regulated by FGF-2 via de novo RNA and protein synthesis. Our RT-PCR results showed that C6 glioma cells express FGFR1/3, and FGFRs is required for FGF-2-induced nestin expression. Further signaling analysis also revealed that FGF-2-induced nestin expression is mediated through FGFR-MAPK-ERK signaling axis and the transcriptional factor Sp1. These findings provide new insight into the regulation of nestin in glial system and enable the further studies on the function of nestin in glial cells.

Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro.

AIM: To investigate whether sphingosine-1-phosphate (S1P), a potent angiogenic factor, induced vascular endothelial growth factor-C (VEGF-C) expression in endothelial cells in vitro and to examine its underlying mechanisms. METHODS: Human umbilical vein endothelial cells (HUVECs) were examined. VEGF-C mRNA expression in the cells was assessed using real-time PCR. VEGF-C protein and FGFR-1 phosphorylation in the cells were measured with ELISA. RNA interference was used to downregulate the expression of matrix metalloproteinase-2 (MMP-2), fibroblast growth factor-1 (FGF-1) and FGF receptor-1 (FGFR-1). RESULTS: Incubation of HUVECs with S1P (1, 5, and 10 µmol/L) significantly increased VEGF-C expression. The effect was blocked by pretreatment with the MMP inhibitor GM6001 or the FGFR inhibitor SU5402, but not the EGFR inhibitor AG1478. The effect was also blocked in HUVECs that were transfected with FGFR-1 or MMP-2 siRNA. Furthermore, incubation of HUVECs with S1P (5 µmol/L) significantly increased FGFR-1 phosphorylation, which was blocked by GM6001. Moreover, knockdown of FGF-1, not FGF-2, in HUVECs with siRNAs, blocked S1P-induced VEGF-C expression. CONCLUSION: S1P induces VEGF-C expression through a MMP-2/ FGF-1/FGFR-1-dependent pathway in HUVECs.

Phosphorylation of CBP by IKKalpha promotes cell growth by switching the binding preference of CBP from p53 to NF-kappaB.

CBP plays a central role in coordinating and integrating multiple signaling pathways. Competition between NF-kappaB and p53 for CBP is a crucial determinant of whether a cell proliferates or undergoes apoptosis. However, how the CBP-dependent crosstalk between these two transcription factors is regulated remains unclear. Here, we show that IKKalpha phosphorylates CBP at serine 1382 and serine 1386 and consequently increases CBP's HAT and transcriptional activities. Importantly, such phosphorylation enhances NF-kappaB-mediated gene expression and suppresses p53-mediated gene expression by switching the binding preference of CBP from p53 to NF-kappaB, thus promoting cell growth. The CBP phosphorylation also correlates with constitutive IKKalpha activation in human lung tumor tissue compared with matched nontumor lung tissue. Our results suggest that phosphorylation of CBP by IKKalpha regulates the CBP-mediated crosstalk between NF-kappaB and p53 and thus may be a critical factor in the promotion of cell proliferation and tumor growth.

MSAP, the meichroacidin homolog of carp (Cyprinus carpio), differs from the rodent counterpart in germline expression and involves flagellar differentiation.

To gain access to the molecular mechanisms of spermatogenesis, the genes from a subtractive screen of the carp testis cDNA library were investigated. In this study, a male-specific homolog of the meichroacidin gene, called MSAP (MORN motif-containing sperm-specific axonemal protein), was isolated and further characterized. Database search and zoo-Western blot analyses revealed that MSAP homologs might be widespread in a variety of phyla but divergent in their C-terminal length and sequences. Carp MSAP is exclusively transcribed in testis, while mouse meichroacidin message is present in gonads of both sexes, although especially enriched in testis. In mouse, meichroacidin is expressed in male germ cells of meiotic stages, while carp MSAP is expressed during late spermiogenesis and accumulated in mature spermatozoa, in which MSAP is localized to the basal body and flagellum. Contrary to mouse meichroacidin revealed previously, existence of multiple pI variants of MSAP in two-dimensional electrophoresis suggested regulatory differences of the homologous molecules between mammal and teleost. These results indicate that MSAP homologs may play different roles in male germline development between vertebrates. Proteomic analysis and immunolocalization disclosed that MSAP is associated with septin7, a conserved GTPase that may participate in cellular morphogenesis, in the basal body of carp sperm. These findings suggest the involvement of carp MSAP in flagellar differentiation during spermiogenesis.