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。     

Signal transducer and activator of transcription 3 is the dominant mediator of the anti-inflammatory effects of IL-10 in human macrophages

The signaling mechanism by which the anti-inflammatory cytokine IL-10 mediates suppression of proinflammatory cytokine synthesis remains largely unknown. Macrophage-specific STAT3-null mice have demonstrated that STAT3 plays a critical role in the suppression of LPS-induced TNF-alpha release, although the mechanism by which STAT3 mediates this inhibition is still not clear. Using an adenoviral system, we have expressed a dominant negative (DN) STAT3 in human macrophages to broaden the investigation to determine the role of STAT3 in IL-10-mediated anti-inflammatory signaling and gene expression. Overexpression of STAT3 DN completely inhibited IL-10-induced suppressor of cytokine signaling 3, tissue inhibitor of MMP-1, TNF receptor expression, and the recently identified IL-10-inducible genes, T cell protein tyrosine phosphatase and signaling lymphocyte activation molecule. STAT3 DN also blocked IL-10-mediated inhibition of MHC class II and COX2 expression. In agreement with the studies in STAT3-null mice, overexpression of the STAT3 DN completely reversed the ability of IL-10 to inhibit LPS-mediated TNF-alpha and IL-6 production. However, real-time PCR analysis showed that STAT3 DN expression did not affect immediate suppression of TNF-alpha mRNA, but did reverse the suppression observed at later time points, suggesting a biphasic regulation of TNF-alpha mRNA levels by IL-10. In conclusion, although STAT3 does appear to be the dominant mediator of the majority of IL-10 functions, there are elements of its anti-inflammatory activity that are STAT3 independent.     

Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction

Human mesenchymal stem cells (hMSCs) can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1-8 μg/mL) negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 μg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr). Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical.     

miR-141-3p inhibits human stromal (mesenchymal) stem cell proliferation and differentiation

Wnt signaling determines human stromal (mesenchymal) stem cell (hMSC) differentiation fate into the osteoblast or adipocyte lineage. microRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate many aspects of osteoblast biology. Thus, we examined miRNAs regulated by Wnt signaling in hMSC. We identified miRNA (miR)-141-3p as a Wnt target which in turn inhibited Wnt signaling. Moreover, miR-141-3p inhibited hMSC proliferation by arresting cells at the G1 phase of the cell cycle. miR-141-3p inhibited osteoblast differentiation of hMSC as evidenced by reduced alkaline phosphatase activity, gene expression and in vitro mineralized matrix formation. Bioinformatic studies, Western blot analysis and 3’UTR reporter assay demonstrated that cell division cycle 25A (CDC25A) is a direct target of miR-141-3p. siRNA-mediated knock-down of CDC25A inhibited hMSC proliferation and osteoblast differentiation. In summary, miR-141-3p acts as a negative regulator of hMSC proliferation and osteoblast differentiation. Targeting miR-141-3p could be used as an anabolic therapy of low bone mass diseases, e.g. osteoporosis.     

Inhibition of lung cancer cell proliferation mediated by human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are mostly studied for their potential clinical use. Recently, much attention in the field of cancer research has been paid to hMSCs. In this study, we investigated the influence of hMSCs on the proliferation of lung cancer cell lines SK-MES-1 and A549 in vitro and in vivo by using a co-culture system and the hMSCs-conditioned medium. Our results demonstrated that hMSCs could inhibit the proliferation of SK-MES-1 and A549 cells, and induce the apoptosis of tumor cells in vitro via some soluble factors. Animal study showed that these soluble factors from hMSCs could suppress tumorigenesis and tumor angiogenesis by treating preliminarily tumor cells with the hMSCs-conditioned medium. The downregulated expression of vascular endothelial growth factor in tumor cells might be the mechanism of interference in tumor angiogenesis, which was verified by western blot analysis and immunohistochemistry assay. Taken together, our results suggested that the hMSCs could inhibit tumor cell growth by secreting some soluble factors.     

MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation

Mechanical signals are important regulators of skeletal homeostasis, and strain-induced oscillatory fluid flow is a potent mechanical stimulus. Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm² triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway. mechanotransduction; bone; marrow