信号转导和转录活化因子3通过趋化因子CX3C配体1促进血管内皮细胞增殖迁移

信号转导和转录活化因子3 (STAT3)与趋化因子CX3C配体1 (Fractalkine/CX3CL1)在血管炎症和损伤中起重要作用,为了探讨STAT3是否通过CX3CL1促进血管内皮细胞增殖和迁移,在血管内皮细胞(HUVEC)中过表达或敲降STAT3,通过quantitative real-time PCR、Western blotting实验确定STAT3对CX3CL1表达的影响。构建含有STAT3结合位点及突变STAT3结合位点的CX3CL1启动子荧光素酶报告基因质粒,利用荧光素酶活性分析实验研究STAT3对CX3CL1启动子转录活性的作用。利用MTT实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞增殖率的影响。利用划痕实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞迁移率的影响。结果显示,过表达STAT3可以促进CX3CL1表达,敲降STAT3可以使CX3CL1表达下调。STAT3可以直接结合到CX3CL1的启动子促进其转录激活,其促进作用依赖于CX3CL1启动子上的GAS位点。敲降STAT3可以抑制血管内皮细胞的迁移,过表达CX3CL1拮抗该抑制作用。总结得出,STAT3通过结合到CXCL1启动子促进CX3CL1转录与表达进而促进血管内皮的增殖与迁移。     

The role of sphingomyelin and sphingomyelin synthases in cell death,proliferation and migration—from cell and animal models to human disorders

Sphingomyelin constitutes membrane microdomains such as lipid raft, caveolae, and clathrin-coated pits and implicates in the regulation of trans-membrane signaling. On the other hand, sphingomyelin emerges as an important molecule to generate bioactive sphingolipids through ceramide. Sphingomyelin synthase is an enzyme that generates sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. Although ceramide has a well-known role as a lipid mediator to regulate cell death and survival, the only known biological role of sphingomyelin regulated by sphingomyelin synthases was limited to being a source of bioactive lipids. Here, we describe the basic characters of sphingomyelin synthases and discuss additional roles for sphingomyelin and sphingomyelin synthase in biological functions including cell migration, apoptosis, autophagy, and cell survival/proliferation as well as in human disorders such as cancer and cardiovascular disorders. It is expected that a better understanding of the role of sphingomyelin regulated by sphingomyelin synthase will shed light on new mechanisms in cell biology, physiology and pathology. In the future, novel therapeutic procedures for currently incurable diseases could be developed through modifying the function of not only sphingolipids, such as sphingomyelin and ceramide, but also of their regulatory enzymes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Sphingomyelin synthase 1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation

Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.     

Expression of Nanog gene promotes NIH3T3 cell proliferation

Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion.     

Reduction of airway anion secretion via CFTR in sphingomyelin pathway

The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (Isc) measurement revealed that isoproterenol (ISO, 0.1 microM)-induced anion secretion was prevented by pretreatment with SMase (0.3 U/ml, for 30 min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolinone (1-EBIO, a Ca2+-activated K+ channel opener)-induced Isc were unaffected. The effects of SMase were reproduced in responses to forskolin (20 microM) or 8-bromo-cAMP (2 mM). C2-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C2-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K+ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion.