Effects of the acid polysaccharide fraction isolated from a cultivated Cordyceps sinensis on macrophages in vitro

The acid polysaccharide fraction (APSF) extracted from the mycelia of cultivated Cordyceps sinensis is water-soluble polysaccharide. In this study we evaluated the modulating effects of APSF on murine macrophage cell line RAW264.7. Phagocytotic assay by neutral red and FITC-dextran internalization showed that APSF stimulated the phagocytosis of macrophages. The nitrite levels in the culture supernatant determined using Griess reagent revealed the elevation of NO production after treatment with APSF. RT-PCR and immunocytochemistry assay indicated that APSF promoted both the mRNA and protein expressions of inducible nitric oxide synthase (iNOS). Furthermore, Western blotting demonstrated that NF-κB levels in nucleuses increased after APSF treatment, suggesting that APSF probably stimulated macrophage activities by activating the IκB-NF-κB pathway.     

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.     

The flexible evolutionary anchorage-dependent Pardee's restriction point of mammalian cells. How its deregulation may lead to cancer

Living cells oscillate between the two states of quiescence and division that stand poles apart in terms of energy requirements, macromolecular composition and structural organization and in which they fulfill dichotomous activities. Division is a highly dynamic and energy-consuming process that needs be carefully orchestrated to ensure the faithful transmission of the mother genotype to daughter cells. Quiescence is a low-energy state in which a cell may still have to struggle hard to maintain its homeostasis in the face of adversity while waiting sometimes for long periods before finding a propitious niche to reproduce. Thus, the perpetuation of single cells rests upon their ability to elaborate robust quiescent and dividing states. This led yeast and mammalian cells to evolve rigorous Start [L.H. Hartwell, J. Culotti, J. Pringle, B.J. Reid, Genetic control of the cell division cycle in yeast, Science 183 (1974) 46–51] and restriction (R) points [A.B. Pardee, A restriction point for control of normal animal cell proliferation, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 1286–1290], respectively, that reduce deadly interferences between the two states by enforcing their temporal insulation though still enabling a rapid transition from one to the other upon an unpredictable change in their environment. The constitutive cells of multicelled organisms are extremely sensitive in addition to the nature of their adhering support that fluctuates depending on developmental stage and tissue specificity. Metazoan evolution has entailed, therefore, the need for exceedingly flexible anchorage-dependent R points empowered to assist cells in switching between quiescence and division at various times, places and conditions in the same organism. Programmed cell death may have evolved concurrently in specific contexts unfit for the operation of a stringent R point that increase the risk of deadly interferences between the two states (as it happens notably during development). But, because of their innate flexibility, anchorage-dependent R points have also the ability to readily adjust to a changing structural context so as to give mutated cells a chance to reproduce, thereby encouraging tumor genesis. The Rb and p53 proteins, which are regulated by the two products of the Ink4a-Arf locus [C.J. Sherr, The INK4a/ARF network in tumor suppression, Nat. Rev., Mol. Cell Biol. 2 (2001) 731–737], govern separable though interconnected pathways that cooperate to restrain cyclin D- and cyclin E-dependent kinases from precipitating untimely R point transit. The expression levels of the Ink4a and Arf proteins are especially sensitive to changes in cellular shape and adhesion that entirely remodel at the time when cells shift between quiescence and division. The Arf proteins further display an extremely high translational sensitivity and can activate the p53 pathway to delay R point transit, but, only when released from the nucleolus, ‘an organelle formed by the act of building a ribosome’ [T. Mélèse, Z. Xue, The nucleolus: an organelle formed by the act of building a ribosome, Curr. Opin. Cell Biol. 7 (1995) 319–324]. In this way, the Ink4a/Rb and Arf/p53 pathways emerge as key regulators of anchorage-dependent R point transit in mammalian cells and their deregulation is, indeed, a rule in human cancers. Thus, by selecting the nucleolus to mitigate cell cycle control by the Arf proteins, mammalian cells succeeded in forging a highly flexible R point enabling them to match cell division with a growth rate imposed by factors controlling nucleolar assembling, such as nutrients and adhesion. It is noteworthy that nutrient control of critical size at Start in budding yeast has been shown recently to be governed by a nucleolar protein interaction network [P. Jorgensen, J.L. Nishikawa, B.-J. Breitkreutz, M. Tyers, Systematic identification of pathways that couple cell growth and division in yeast, Science 297 (2002) 395–400].     

Internalization of CD40 regulates its signal transduction in vascular endothelial cells

The CD40 ligand (CD40L)-CD40 dyad can ignite proinflammatory and procoagulatory activities of the vascular endothelium in the pathogenesis and progression of atherosclerosis. Besides being expressed on the activated CD4(+) T cell surface (mCD40L), the majority of circulating CD40L reservoir (sCD40L) in plasma is released from stimulated platelets. It remains debatable which form of CD40L triggers endothelial inflammation. Here, we demonstrate that the agonistic antibody of CD40 (G28.5), which mimics the action of sCD40L, induces rapid endocytosis of CD40 independent of TRAF2/3/6 binding while CD40L expressed on the surface of HEK293A cells captures CD40 at the cell conjunction. Forced internalization of CD40 by constitutively active mutant of Rab5 preemptively activates NF-kappaB pathway, suggesting that CD40 was able to form an intracellular signal complex in the early endosomes. Internalized CD40 exhibits different patterns of TRAF2/3/6 recruitment and Akt phosphorylation from the membrane anchored CD40 complex. Finally, mCD40L but not sCD40L induces the upregulation of proinflammatory cytokines and cell adhesion factors in the primary human vascular endothelial cells in vitro, although both forms of CD40L activate NF-kappaB pathway. These results therefore may help understand the molecular mechanism of CD40L signaling that contributes to the pathophysiology of atherosclerosis.     

Dioscorin isolated from Dioscorea alata activates TLR4-signaling pathways and induces cytokine expression in macrophages

The Toll-like receptor 4 (TLR4)-signaling pathway is crucial for activating both innate and adaptive immunity. TLR4 is a promising molecular target for immune-modulating drugs, and TLR4 agonists are of therapeutic potential for treating immune diseases and cancers. Several medicinal herb-derived components have recently been reported to act via TLR4-dependent pathways, suggesting that medicinal plants are potential resources for identifying TLR4 activators. We have applied a screening procedure to systematically identify herbal constituents that activate TLR4. To exclude possible LPS contamination in these plant-derived components, a LPS inhibitor, polymyxin B, was added during screening. One of the plant components we identified from the screening was dioscorin, the glycoprotein isolated from Dioscorea alata. It induced TLR4-downstream cytokine expression in bone marrow cells isolated from TLR4-functional C3H/HeN mice but not from TLR4-defective C3H/HeJ mice. Dioscorin also stimulated multiple signaling molecules (NF-kappaB, ERK, JNK, and p38) and induced the expression of cytokines (TNF-alpha, IL-1beta, and IL-6) in murine RAW 264.7 macrophages. Furthermore, the ERK, p38, JNK, and NF-kappaB-mediated pathways are all involved in dioscorin-mediated TNF-alpha production. In summary, our results demonstrate that dioscorin is a novel TLR4 activator and induces macrophage activation via typical TLR4-signaling pathways.