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Ras, protein kinase C zeta, and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation of NF-kappa B by hyaluronic acid fragments in T-24 carcinoma cellsWe have investigated the ability of hyaluronic acid (HA) fragments to activate the transcription factor NF-kappa B. HA fragments activated NF-kappa B in the cell lines T-24, HeLa, MCF7, and J774. Further studies in T-24 cells demonstrated that HA fragments also induced I kappa B alpha phosphorylation and degradation, kappa B-linked reporter gene expression, and ICAM-1 promoter activity in an NF-kappa B-dependent manner. The effect of HA was size dependent as neither disaccharide nor native HA were active. CD44, the principal cellular receptor for HA, was critical for the response because the anti-CD44 Ab IM7.8.1 blocked the effect on NF-kappa B. HA fragments activated the I kappa B kinase complex, and the effect on a kappa B-linked reporter gene was blocked in T-24 cells expressing dominant negative I kappa B kinases 1 or 2. Activation of protein kinase C (PKC) was required because calphostin C inhibited NF-kappa B activation and I kappa B alpha phosphorylation. In particular, PKC zeta was required because transfection of cells with dominant negative PKC zeta blocked the effect of HA fragments on kappa B-linked gene expression and HA fragments increased PKC zeta activity. Furthermore, damnacanthal and manumycin A, two mechanistically distinct inhibitors of Ras, blocked NF-kappa B activation. Transfection of T-24 cells with dominant negative Ras (RasN17) blocked HA fragment-induced kappa B-linked reporter gene expression, and HA fragments activated Ras activity within 5 min. Taken together, these studies establish a novel signal transduction cascade emanating from CD44 to Ras, PKC zeta, and I kappa B kinase 1 and 2.
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NF-kappaB activation in premalignant mouse tal-1/scl thymocytes and tumorsTAL-1/SCL activation is a common genetic event in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Expression of tal-1/scl or a DNA binding mutant of tal-1/scl induces arrest of thymocyte development, resulting in decreases in double-positive and single-positive CD4 thymocytes. Moreover, nuclear p65/p50 heterodimers are detected in premalignant tal-1/scl and mut tal-1/scl thymocytes, suggesting that E2A depletion may induce developmental arrest and stimulate NF-kappaB activation. Increased NF-kappaB activity is also observed in tal-1/scl tumors and bcl-2 is overexpressed. To examine the contribution of NF-kappaB to tal-1/scl tumor growth in vivo, we expressed a mutant form of IkappaBalpha in tal-1/scl tumor cells. Although expression of mutant IkappaBalpha inhibited the tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB response, it had no effect on tumor growth in mice. These data suggest that NF-kappaB activation is an early event in tal-1/scl-induced leukemogenesis, associated with arrest of thymocyte development, and does not appear to contribute to tal-1/scl-induced tumor growth.
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Flagellin Acting Via TLR5 is the Major Activator of Key Signaling Pathways Leading to NF-kappa B and Proinflammatory Gene Program activation in intestinal epithelial cellsBACKGROUND: Infection of intestinal epithelial cells by pathogenic Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-kappa B is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-kappa B during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-kappa B activation in all the cells; invasion of cells by the bacteria is not required to activate NF-kappa B. RESULTS: Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and I kappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-kappa B via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-kappa B activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-kappa B activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both. CONCLUSION: These collective results provide the evidence that flagellin acts as the main determinant of Salmonella mediated NF-kappa B and proinflammatory signaling and gene activation by this flagellated pathogen. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Lastly, TLR5 expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition.
