Interleukin-1 (IL-1) is a central mediator of the immune system involved in acute and chronic inflammatory responses. Although the sequences of two types of IL-1 receptors are known, the exact molecular events resulting in signal transduction and coupling to downstream signaling elements remain unclear. The recently cloned IL-1 receptor accessory protein (IL-1RAcP) has been suggested as a co-receptor molecule for IL-1RI, supported by the observation that its expression correlates to IL-1 responsiveness. We transfected the EL-4 subline D6/76 with IL-1RAcP cDNA. This cell line is an IL-1 non-responder expressing IL-1RI but lacking constitutive IL-1RAcP expression. The expression of IL1RAcP in EL-4 D6/76 was sufficient to restore IL-1-induced activation of interleukin-1 receptor-associated kinase and of stress-activated protein kinases, translocation of the transcription factors NFB and IL-1 NF to the nucleus, and induction of IL-2 mRNA synthesis. These results proved that IL-1RAcP is an indispensible molecule in the IL-1 receptor signal transduction complex, necessary to link events on the plasma membrane level to downstream signaling pathways, allowing IL-1-dependent activation of transcription factors and gene expression. IL-11 is a pro-inflammatory cytokine centrally involved in regulating local and systemic responses of the immune system. It exerts its many biological effects on a wide variety of target cells through specific plasma membrane receptors (1). Two types of IL-1 receptors have been cloned so far (2, 3), of which only the type I IL-1 receptor (IL-1RI) initiates signal transduction (4), whereas the type II IL-1 receptor (IL-1RII) presumably functions solely as a ligand sink, as a decoy receptor (5). Although it is clear that the cytoplasmic tail of IL-1RI is necessary to initiate cytoplasmic signaling (6), the molecules involved and the mechanisms utilized remain obscure. The recent identification of a second subunit to the IL-1RI, the IL-1 receptor accessory protein (IL-1RAcP) (7) places the IL-1 receptor into the group of multimeric cytokine receptors. Homology searches and sequence comparisons show a protein kinase C docking site and a putative GTPase domain in IL-1RAcP, but it remains to be shown that these play any role in the biological function of this molecule. Recently we found that IL-1RAcP mRNA is constitutively expressed in a wide variety of cell types and that its expression correlates with IL-1 responsiveness. Cells lacking IL-1RAcP did not respond to IL-1 (8), suggesting that the signal transduction machinery initiating IL-1 signaling is dependent on the presence of IL-1RAcP.Here we show that the IL-1RAcP is indispensible in linking the first step in IL-1 signal generation, namely the binding of IL-1 to IL-1RI, to two important elements of downstream signaling, the IL-1RI-associated protein kinase IRAK and the stress-activated protein kinases (SAP kinases). This results in activation and translocation of transcription factors as well as in IL-2 gene transcription and IL-2 production. W...

Interleukin-1 (IL-1) is a central molecule in inflammation and immune responses whose pleiotropic activities are mediated by the type I IL-1 receptor (IL-1RI). The IL-1RI alone on the cell surface is silent after binding of the ligand. We show that the recently identified IL-1RI accessory protein (IL-1RAcP) converts the silent into a fully functional IL-1RI complex. Although transfection of IL-1RAcP into IL-1RAcP-deficient EL4D6/76 cells did not alter the binding kinetics or dissociation constants of the 125I-labeled IL-1alpha/IL-1RI complex, a very early event, internalization of the activated receptor complex, and a late event, IL-1-stimulated IL-2 production, were successfully restored. Therefore, recruitment of IL-1RAcP is a critical early step in the signaling cascade mediated by the IL-1RI activation complex.

A genome-wide phenotype screen was used to identify factors and pathways that induce proliferation of human umbilical vein endothelial cells (HUVEC). HUVEC proliferation is a recognized marker for factors that modulate vascularization. Screening ''hits'' included known proangiogenic factors, such as VEGF, FGF1, and FGF2 and additional factors for which a direct association with angiogenesis was not previously described. These include the kinase TBK1 as well as Toll-like receptor adaptor molecule and IFN regulatory factor 3. All three proteins belong to one signaling pathway that mediates induction of gene expression, including a mixture of secreted factors, which, in concert, mediate proliferative activity toward endothelial cells. TBK1 as the ''trigger'' of this pathway is induced under hypoxic conditions and expressed at significant levels in many solid tumors. This pattern of expression and the decreased expression of angiogenic factors in cultured cells upon RNA-interference-mediated ablation suggests that TBK1 is important for vascularization and subsequent tumor growth and a target for cancer therapy.cancer ͉ cDNA͞libraries ͉ expression cloning ͉ human umbilical vein endothelial cells ͉ screening

The cytokine interleukin-1 (IL-1) plays an important role in inflammation and regulation of immune responses, but the mechanisms of its signal transduction and cell activation processes are incompletely understood. Ceramide generated by sphingomyelinases

Interleukin-1 (IL-1) 1 possesses a wide spectrum of inflammatory, metabolic, physiological, hematopoetic, and immunological properties. IL-1 binds to two different receptors of the Ig superfamily: IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII). Both receptors bind the ligands with distinct affinities (1). Whereas IL-1RI is necessary for signal transduction (2), IL-1RII is not capable of transducing an activation signal but rather acts as a decoy receptor serving as a ligand sink and competing for IL-1 with the IL-1RI (3-5). An additional member of the IL-1 receptor family, IL-1 receptor accessory protein (IL-1RAcP), has also been identified. The 66-kDa IL-1RAcP shares limited homology with IL-1RI and IL-1RII but does not bind IL-1 (6). It has been shown that co-expression of the IL-1RAcP is essential for a fully functional IL-1RI complex (7-10).In the last years the elucidation of early IL-1 signaling events has progressed. After ligand-induced complex formation of IL-1RI and IL-1RAcP (7-11), the Ser/Thr kinase IRAK is recruited to the receptor complex, where it becomes highly phosphorylated (12, 13). Recruitment of IRAK requires the intracellular domains of both receptor chains (8,10,14). Dominant-negative forms of MyD88 block IL-1 signaling (15). After phosphorylation by itself (12) or by additional kinases (16), IRAK leaves the receptor complex and interacts with tumor necrosis factor receptor-associated factor 6 (17). Recently, the MAP kinase kinase kinase TAK1 has been identified to interact with tumor necrosis factor receptor-associated factor 6 in association with , thereby providing a link to the machinery that activates nuclear factor B (NF-B) via stimulation of IB kinases (IKK␣ and IKK␤) (23-28). One critical component in the signaling cascade is MyD88 (29, 30), because mice lacking this adaptor molecule do not show cytokine-induced activation of NF-B and c-Jun N-terminal kinase (JNK) (31).We have previously described an IL-1RI-positive subclone of EL4 cells, EL4D6/76, which binds IL-1 with high affinity but fails to activate NF-B or produce IL-2 following IL-1 stimulation (7,32). This defect is due to the lack of IL-1RAcP expression and can be overcome by transfection with IL-1RAcP, thus reconstituting IL-1-specific functional defects in EL4D6/76 cells (7,9). In the present study we investigated regions within the cytoplasmic domain of IL-1RAcP that are required for perpetuating IL-1 responses. We present data demonstrating that, in addition to box 3 (aa 538 -542) of the TIR domain, aa 527-534 within the cytoplasmic domain of IL-1RAcP are essential for IL-1 signaling. EXPERIMENTAL PROCEDURES Construction of Truncated and Mutated Forms of IL-1RAcP-Trun-cated forms of IL-1RAcP were constructed by PCR technique. The vector pEF-IL-1RAcP containing the murine IL-1RAcP coding sequence was used as a template (9). Truncated fragments were cloned into pFLAG-IL-1RAcP (kind gift of Michael Martin, Hannover, Germany), a *