Toll-like receptors (TLRs) are the key molecular sensors used by the mammalian innate immune system to detect various types of pathogens. Tlr13 is a novel and uncharacterized member of the mammalian TLR family. Here we report the cloning and characterization of tlr13. Tlr13 is predominantly expressed in the spleen, particularly in dendritic cells and macrophages. Tlr13 appears to activate a MyD88-and TAK1-dependent TLR signaling pathway, inducing the activation of NF-B. This receptor can also activate type 1 interferon through IRF7. Furthermore, Tlr13 seems to be another intracellular TLR. Remarkably, cells expressing tlr13 fail to respond to known TLR ligands but instead respond specifically to vesicular stomatitis virus. Cells with the knockdown of tlr13 are highly susceptible to vesicular stomatitis virus infection. Thus, these results provide an important insight into the potential role of the novel Toll-like receptor tlr13 in the recognition of viral infection.The best characterized molecular sensors used by the mammalian innate immune system to detect invading pathogens are the Toll-like receptors (TLRs) 3 (1-3). TLRs are type I transmembrane proteins that contain an amino-terminal leucine-rich repeat (LRR) domain and a carboxyl-terminal Toll-interleukin-1 receptor (TIR) domain (4). The leucinerich repeat domain is responsible for the recognition of pathogen-associated molecular patterns, whereas the TIR domain is required for initiating intracellular signaling (3, 4). Signal transduction by TLRs after ligand engagement is initiated with the recruitment of the cytosolic TIR-containing adaptor proteins such as MyD88 and TRIF (also known as TICAM1) (5-7). MyD88 is utilized by all TLRs except for TLR3 (5). For the MyD88-dependent pathway, MyD88 subsequently recruits the serine/threonine interleukin-1 receptorassociated kinase (IRAK) to the receptor complex through a homophilic interaction of the death domains (8). The recruited IRAK is then auto-phosphorylated and, after associating with the cytosolic adaptor protein TRAF6, dissociates from the receptor and is degraded (5). Finally, TRAF6 activates the IB kinase complex through the adaptor protein TAK1 (5-7). The MyD88-independent pathway is the TRIF pathway. TLR3 and TLR4 recognize double-stranded RNA (dsRNA) and LPS, respectively, to activate this pathway. This results in the activation of IRF3 and the subsequent induction of type I interferons and IFN-inducible genes (9 -11). IRF7 is a key transcription factor for the induction of type I interferons, and its activation occurs via both the MyD88-dependent pathway and the TRIF-dependent pathway (12, 13).The subcellular localization of different TLRs correlates with the nature of their ligands. In the TLR family, TLR1, -2, -4, -5, -6, and -11 are present on the cell surface membrane, whereas TLR3, -7, -8, and -9 are expressed in the intracellular endosomal compartments. Intracellular TLRs are sensors of nucleic acids that have been well studied in the recognition of viral infection. After viruses are internal...

The synergistic action of cytosolic Ca2+ and inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ stores has been suggested to be responsible for the complex intracellular Ca2 signals observed during hormonal stimulation of many cell types. However, the ability of cytosolic Ca2+ to potentiate Ca2+ release has recently been questioned because of the observed inhibitory effects of Ca2+ chelators used in previous studies. In the present study, EGTA and BAPTA [1,2-bis-(2-amino-phenoxy)ethane- NNN'N'-tetra-acetic acid] poorly inhibited InsP3-induced Ca2+ release from permeabilized A7r5 smooth-muscle cells. Additionally, stimulatory effects of cytosolic and luminal Ca2+ were observed either in the complete absence of Ca2+ chelator or at constant Ca(2+)-free chelator concentration. These data suggest that potentiation of InsP3-induced Ca2+ release by Ca2+ in A7r5 cells reflects an interaction between Ca2+ and InsP3 receptors, rather than a decrease in chelator-dependent inhibition. The EC50 for activation of InsP3-induced Ca2+ release by cytosolic Ca2+ was unaffected by ATP, or by changing InsP3 concentration, although InsP3-induced Ca2+ release became less sensitive to the inhibitory effects of cytosolic Ca2+ as the InsP3 concentration was elevated. Increasing H+ or Mg2+ concentration shifted the Ca(2+)-activation curve towards higher Ca2+ concentrations. These data suggest that, in addition to the InsP3-binding site, the affinity of the Ca(2+)-binding site(s) on InsP3 receptors can be modulated by intracellular cations.

Aims: Calcium-sensing receptor (CaR) acts as a G protein coupled receptor that mediates the increase of the intracellular Ca²⁺ concentration. The expression of CaR has been confirmed in various cell types, including cardiomyocytes, smooth muscle cells, neurons and vascular endothelial cells. However, whether CaR is expressed and functions in cardiac fibroblasts has remained unknown. The present study investigated whether CaR played a role in cardiac fibroblast proliferation and extracellular matrix (ECM) secretion, both in cultured rat neonatal cardiac fibroblasts and in a model of cardiac hypertrophy induced by isoproterenol (ISO). Methods and Results: Immunofluorescence, immunohistochemistry and Western blot analysis revealed the presence of CaR in cardiac fibroblasts. Calcium and calindol, a specific activator of CaR, elevated the intracellular calcium concentration in cardiac fibroblasts. Pretreatment of cardiac fibroblasts with calhex231, a specific inhibitor of CaR, U73122 and 2-APB attenuated the calindol- and extracellular calcium-induced increase in intracellular calcium ([Ca²⁺]_i). Cardiac fibroblast proliferation and migration were assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), cell count and the cell scratch assay. ECM production was detected by expression of matrix metalloproteinase-3 and -9 (MMP-3 and -9). Activation of CaR promoted cardiac fibroblast proliferation and migration and ECM secretion. More importantly, calhex231, suppressed cardiac fibroblast proliferation and migration and MMP-3 and -9 expression. To further investigate the effect of CaR on cardiac fibrosis, a model of ISO-induced cardiac hypertrophy was established. Pretreatment with calhex231 prevented cardiac fibrosis and decreased the expression of MMP-3 and -9 expression. Conclusions: Our results are the first report that CaR plays an important role in Ca²⁺ signaling involved in cardiac fibrosis through the phospholipase C- inositol 3,4,5 phosphate (PLC-IP₃) pathway.

Little has been known about Tlr13 (Toll-like receptor 13), a novel member of the Toll-like receptor family. To elucidate the molecular basis of murine Tlr13 gene expression, the activity of the Tlr13 gene promoter was characterized. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that Tlr13 gene transcription was regulated through three cis-acting elements that interacted with the Ets2, Sp1, and PU.1 transcription factors. Furthermore, our work suggests that these transcription factors may cooperate, culminating in maximal transcription of the Tlr13 gene. In contrast, NF-B appeared to act as an inhibitor of Tlr13 transcription. Overexpression of Ets2 caused a strong increase in the transcriptional activity of the Tlr13 promoter; however, overexpression of NF-B p65 dramatically inhibited it. Additionally, interferon-␤ is capable of acting Tlr13 transcription, but the activated signaling of lipopolysaccharide/TLR4 and peptidoglycan/TLR2 strongly inhibited the Tlr13 gene promoter. Thus, these findings reveal the mechanism of Tlr13 gene regulation, thereby providing insight into the function of Tlr13 in the immune response to pathogen.Upon infection, microorganisms are first recognized by cells of the host innate immune system, such as macrophages and dendritic cells, as well as mucosal epithelial cells (1-6). Recognition of pathogens is primarily mediated by a set of germ lineencoded molecules on innate immune cells that are referred to as pattern recognition receptors (7,8). These pattern recognition receptors are expressed as either membrane-bound or soluble proteins that recognize invariant molecular structures from the pathogen called pathogen-associated molecular patterns (7,8).Recent studies on the recognition of microbial pathogenassociated molecular patterns have highlighted the vital role of one group of pattern recognition receptors, the Toll-like receptors (TLRs) 2 (9, 10). It is already clear that TLRs play a crucial role in the recognition of "molecular signatures" produced by infecting microbes to engage differential signaling pathways (11,12). Signaling through TLRs activates various transcription factors, such as nuclear factor-B (NF-B), activating protein-1 (AP-1), and interferon regulatory factors to induce an immunological response (3, 11).Tlr13 is a novel and poorly characterized member of the Tolllike receptor family (3, 13). Although the elucidation of the function of Tlr13 depends mainly on the identification of its natural ligand, its transcriptional regulation also provide some clues. For example, which type of cells expresses Tlr13? Which transcription factors control Tlr13 expression? How do different pathogen-associated molecular patterns from different pathogens regulate Tlr13 expression? This information will perhaps help us understand not only how this novel TLR responds to different infections but also which pathogens might be recognized by Tlr13 to activate the innate immune response. Recently, Aderem et al. (14) reported that Tlr13 belongs to the Tlr1...

No abstract