Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-κB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.

Inhibition of DNA synthesis induces transcription of DNA damage-inducible genes and prevents mitotic entry through the action of the S phase checkpoint. We have isolated a mutant, dun2, defective for both of these responses. DUN2 is identical to POL2, encoding DNA polymerase epsilon (pol epsilon). Unlike sad1 mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUN1 kinase necessary for the transcriptional response to damage. Interallelic complementation and mutation analysis indicate that pol epsilon contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to epsilon polymerases. We propose that DNA pol epsilon acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks.

RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that RIP mediates TNF-induced activation of the anti-apoptotic NF-B pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced NF-B activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding to large prodomain initiator caspases.Tumor necrosis factor receptor-1 mediates both the proinflammatory and pro-apoptotic effects of the pleiotropic cytokine TNF (1, 2). 1 The proinflammatory effects are mediated by activation of the transcription factor NF-B. Recently it has been shown that NF-B activation results in both the transcriptional activation of proinflammatory genes, including , and in activation of a cell survival pathway mediated at least in part by induction of anti-apoptotic IAP family members (6 -8). Therefore, quite paradoxically, two diametrically opposed pathways emanate from TNFR-1: a cell death pathway and a cell survival pathway mediated by activation of NF-B.The intracellular segment of TNFR-1 contains a 70-amino acid homophilic interaction domain, dubbed the "death domain," which is required for both signaling and NF-B activation. Upon activation of TNFR-1, a multi-component signaling complex is assembled by a series of homophilic interactions (1). Initially, the death domain-containing platform adapter molecule TRADD is recruited to TNFR-1 by virtue of a homophilic death domain interaction (9). TRADD in turn binds the death adapter molecule FADD (10, 11), which interacts with the zymogen form of the initiator death protease caspase-8 (12). Subsequent activation of caspase-8 by an induced proximity mechanism leads to amplification of the death signal through proteolytic activation of downstream caspase zymogens (13). Studies done with FADD-deficient embryonic fibroblasts suggest that this is the major but not the only pro-apoptotic pathway engaged by TNFR-1, because instead of being completely resistant to TNF-induced apoptosis, 30% of FADD-null cells are still sensitive (14,15). Taken together, these studies suggest that there exists a subsidiary FADD-independent TNFR-1-initiated death pathway. Earlier biochemical studies indicated that the TNFR-1-associated adapter molecule RAIDD might fulfill this function by recruiting caspase-2 to the receptor signaling complex (16). However, caspase-2 null cells do not show any loss of sensitivity to TNF-induced cytotoxicity (17); therefore, the physiological significance of this interaction remains unclear. Initial biochemical studies also suggested a role for the TRADD-bindin...

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-γ. The induction of cytokine production by NK cells was preceded by activation of NF-κB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. In addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. mRNAs for both subunits are cell cycle regulated and inducible by DNA damage in all organisms examined, including E. coli, S. cerevisiae and H. sapiens. This DNA damage regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes. S. cerevisiae also encodes a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. Genetic analysis of the DNA damage response in S. cerevisiae has shown that RNR expression is under both positive and negative control. Among mutants constitutive for RNR expression are the general transcriptional repression genes, SSN6 and TUP1. Mutations in POL1 and POL3 also activate RNR expression, indicating that the DNA damage sensory network may respond directly to blocks in DNA synthesis. A protein kinase, Dun1, has been identified that controls inducibility of RNR1, RNR2 and RNR3 in response to DNA damage and replication blocks. This result suggests that the RNR genes in S. cerevisiae form a regulon that is coordinately regulated by protein phosphorylation in response to DNA damage.

In response to DNA damage and replication blocks, yeast cells arrest at distinct points in the cell cycle and induce the transcription of genes whose products facilitate DNA repair. Examination of the inducibility of RNR3 in response to UV damage has revealed that the various checkpoint genes can be arranged in a pathway consistent with their requirement to arrest cells at different stages of the cell cycle. While RADg, RAD24, and MEC3 are required to activate the DNA damage checkpoint when cells are in G1 or G2, POL2 is required to sense UV damage and replication blocks when cells are in S phase. The phosphorylation of the essential central transducer, Rad53p, is dependent on POL2 and RAD9 in response to UV damage, indicating that RAD53 functions downstream of both these genes. Mutants defective for both pathways are severely deficient in Rad53p phosphorylation and RNR3 induction and are significantly more sensitive to DNA damage and replication blocks than single mutants alone. These results show that POL2 and RAD9 function in parallel branches for sensing and transducing the UV DNA damage signal. Each of these pathways subsequently activates the central transducers Meclp/Esrlp/Sad3p and Rad53p/Mec2p/Sadlp, which are required for both cell-cycle arrest and transcriptional responses.

MDS is characterized by ineffective hema- IntroductionThe myelodysplastic syndromes (MDSs) are clonal stem cell disorders characterized by cytologic dysplasia and ineffective hematopoiesis. [1][2][3] Although approximately one third of patients may progress to acute leukemia, refractory cytopenias are the principal cause of morbidity and mortality in patients with MDS. 4 In fact, approximately two-thirds of patients present with lower risk disease characterized by hypercellular marrows with increased rates of apoptosis in the progenitor and differentiated cell compartments in the marrow. [5][6][7][8] Ineffective hematopoiesis arising from abortive maturation leads to peripheral cytopenias. Higher grade or more advanced disease categories are associated with a significant risk of leukemia transformation, with a corresponding lower apoptotic index and higher percentage of marrow blasts.Cytokines play important roles in the regulation of normal hematopoiesis, and a balance between the actions of hematopoietic growth factors and myelosuppressive factors is required for optimal production of different hematopoietic cell lineages. Excess production of inhibitory cytokines amplifies ineffective hematopoiesis inherent to the MDS clone. Transforming growth factor-␤ (TGF-␤) is a myelosuppressive cytokine that has been implicated in the hematopoietic suppression in MDS. The plasma levels of TGF-␤ have been reported to be elevated in some [9][10][11][12][13] but not all studies [14][15][16][17] and are supported by greater TGF-␤ immunohistochemical staining in selected studies. In addition to direct myelosuppressive effects, TGF-␤ has also been implicated in the autocrine production of other myelosuppressive cytokines (TNF, IL-6, and IFN␥) in MDS. 18 Conflicting data may arise from technical limitations of bone marrow immunohistochemical analyses of a secreted protein as well as the biologic heterogeneity of the disease itself. In addition, plasma levels of TGF-␤ may not be an accurate reflection of the biologic effects of this cytokine in the MDS bone marrow microenvironment. Thus we investigated the role of TGF-␤ in MDS by direct examination of receptor signal activation to conclusively determine its role in the pathogenesis of ineffective hematopoiesis in MDS.Our previous studies have shown that signaling pathways activated by myelosuppressive cytokines can serve as therapeutic targets in low-risk MDS. We showed that interferons (IFN␣, IFN␤, and IFN␥), TGF-␤, and tumor necrosis factor ␣ (TNF␣) can all activate the p38 mitogen-activated protein kinase (MAPK) in primary human hematopoietic progenitors and that activation of p38 is required for myelosuppressive actions of these cytokines on hematopoiesis. 19,20 We subsequently confirmed overactivation of p38 MAPK in the bone marrow progenitors of low-risk MDS patients. Our data showed that inhibition of this cytokinestimulated p38 MAPK pathway partially rescues hematopoiesis in MDS progenitors. This led to a clinical trial of a p38 inhibitor, SCIO-469, in low-risk MDS; the ...

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