Limb and skin abnormalities in mice lacking IKKalpha.

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.

ZIP kinase, a novel serine/threonine kinase which mediates apoptosis.

We have identified a novel serine/threonine kinase, designated ZIP kinase, which mediates apoptosis. ZIP kinase contains a leucine zipper structure at its C terminus, in addition to a kinase domain at its N terminus. ZIP kinase physically binds to ATF4, a member of the activating transcription factor/cyclic AMP-responsive element-binding protein (ATF/CREB) family, through interaction between their leucine zippers. The leucine zipper domain is necessary for the homodimerization of ZIP kinase as well as for the activation of kinase. Immunostaining study showed that ZIP kinase localizes in the nuclei. Overexpression of intact ZIP kinase but not catalytically inactive kinase mutants led to the morphological changes of apoptosis in NIH 3T3 cells, suggesting that the cell death-inducing activity of ZIP kinase depends on its intrinsic kinase activity. Interestingly, the catalytic domain of ZIP kinase is closely related to that of death-associated protein kinase (DAP kinase), which is a mediator of apoptosis induced by gamma interferon. Therefore, both ZIP and DAP kinases represent a novel kinase family, which mediates apoptosis through their catalytic activities.

Topologically protected surface states in a centrosymmetric superconductor ß-PdBi2.

The topological aspects of electrons in solids can emerge in real materials, as represented by topological insulators. In theory, they show a variety of new magneto-electric phenomena, and especially the ones hosting superconductivity are strongly desired as candidates for topological superconductors. While efforts have been made to develop possible topological superconductors by introducing carriers into topological insulators, those exhibiting indisputable superconductivity free from inhomogeneity are very few. Here we report on the observation of topologically protected surface states in a centrosymmetric layered superconductor, ß-PdBi2, by utilizing spin- and angle-resolved photoemission spectroscopy. Besides the bulk bands, several surface bands are clearly observed with symmetrically allowed in-plane spin polarizations, some of which crossing the Fermi level. These surface states are precisely evaluated to be topological, based on the Z2 invariant analysis in analogy to three-dimensional strong topological insulators. ß-PdBi2 may offer a solid stage to investigate the topological aspect in the superconducting condensate.

Duet is a novel serine/threonine kinase with Dbl-Homology (DH) and Pleckstrin-Homology (PH) domains.

We show here the identification of Duet, a novel molecule bearing serine/threonine kinase, Dbl-Homology (DH), and Pleckstrin-Homology (PH) domains. The kinase domain of Duet shows a homology to that of DAP kinase that is involved in apoptosis, and Duet is autophosphorylated by an in-vitro kinase assay. The DH- and PH-domains are closely related to those of Trio and Kalirin. Trad mRNA is specifically expressed in skeletal muscle. Duet protein was localized to actin-associated cytoskeletal elements. These data suggest a role of Duet in the cytoskeleton-dependent cell function.

The gene structure and promoter analysis of mouse lymphocyte signal transduction molecule alpha 4 that is related to the yeast TAP42 involved in a rapamycin-sensitive pathway.

The mouse alpha 4 phosphoprotein encoding a component associated with the B cell antigen receptor (BCR)-mediated signal transduction is suggested to be involved in a unique rapamycin-sensitive pathway. We studied the structure and the molecular mechanism of the expression of alpha 4 gene by isolating two phage clones, named #10 and #23, covering entire exons of the mouse alpha 4 gene. The alpha 4 gene is located within about 25 kb and composed of six exons. To analyze the regulation of alpha 4 gene expression, we determined the nucleotide sequence toward 2 kb upstream of the translation start site of the alpha 4 gene. The 5'-flanking region does not contain a typical TATA box or the initiation consensus sequence, but it contains a CCAAT box, E-boxes, and several DNA binding motifs such as c-Myc, c-Myb, and c-Ets. Transcription of the alpha 4 gene starts at four different sites, determined by primer extension analysis, that were surrounded by Y-rich sequences. We further characterized the functional promoter of the alpha 4 gene at the region between -263 and the transcription start site of alpha 4 gene by luciferase assay system and suggested that the 5' upstream region of alpha 4 gene contains the silencer element of MT repetitive sequence.

TLR6: A novel member of an expanding toll-like receptor family.

Drosophila Toll protein is shown to activate the innate immune system in adult and regulate the dorsoventral patterning in the developing embryo. Recently, five human homologs of Drosophila Toll, designated as Toll-like receptors (TLRs), have been identified and shown to play a role in the innate immune response. We report here the molecular cloning and characterization of a new member of Toll-like receptor family, Toll-like receptor 6 (TLR6). Human and murine TLR6 are type-I transmembrane receptors that contain both an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic Toll/IL-1 receptor (IL-1R)-like region. The amino acid sequence of human TLR6 (hTLR6) is most similar to that of hTLR1 with 69% identity. RT-PCR analysis revealed that murine TLR6 is expressed predominantly in spleen, thymus, ovary and lung. Like other TLR family members, constitutively active TLR6 activates both NF-kappaB and c-Jun N-terminal kinase (JNK). The TLR6 gene, as well as the TLR1 gene, mapped to the proximal region of murine chromosome 5 within 1.7cM of each other. These results suggest that TLR6 is a novel member of an expanding TLR family.

DRAKs, novel serine/threonine kinases related to death-associated protein kinase that trigger apoptosis.

The present study describes the cloning of two novel serine/threonine kinases termed DRAK1 and DRAK2, whose catalytic domains are related to that of death-associated protein kinase, a serine/threonine kinase involved in apoptosis. Both DRAKs are composed of the N-terminal catalytic domain and the C-terminal domain that is responsible for regulation of kinase activity. DRAK1 and DRAK2 show 59.7% identity and display ubiquitous expression. An in vitro kinase assay revealed that both DRAKs are autophosphorylated and phosphorylate myosin light chain as an exogenous substrate, although the kinase activity of DRAK2 is significantly lower than that of DRAK1. Both DRAKs are exclusively localized to the nucleus. Furthermore, overexpression of both DRAKs induces the morphological changes of apoptosis in NIH 3T3 cells, suggesting the role of DRAKs in apoptotic signaling.

Ig receptor binding protein 1 (alpha4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A.

Rapamycin is an immunosuppressant that effectively controls various immune responses; however, its action in the signal transduction of lymphocytes has remained largely unknown. We show here that a phosphoprotein encoded by mouse alpha4 (malpha4) gene transmitting a signal through B-cell antigen receptor (BCR) is associated with the catalytic subunit of protein phosphatase 2A (PP2Ac). The middle region of alph4, consisting of 109 amino acids (94-202), associates directly with PP2Ac, irrespective of any other accessory molecule. Rapamycin treatment disrupts the association of PP2Ac/alpha4 in parallel with the inhibitory effect of lymphoid cell proliferation. The effect of rapamycin was inhibited with an excess amount of FK506 that potentially completes the binding to FKBP. Rapamycin treatment also suppresses the phosphatase activity of cells measured by in vitro phosphatase assay. Introduction of the malpha4 cDNA into Jurkat cells or the increased association of PP2Ac/alpha4 by the culture with low serum concentration confers cells with rapamycin resistance. Moreover, glutathione S-transferase (GST)-alpha4 augments the PP2A activity upon myelin basic protein (MBP) and histone in the in vitro assay. These results suggest that alpha4 acts as a positive regulator of PP2A and as a new target of rapamycin in the activation of lymphocytes.

Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.

The human homologue of Drosophila Toll (hToll), also called Toll-like receptor 4 (TLR4), is a recently cloned receptor of the IL-1/Toll receptor family. Interestingly, the TLR4 gene has been localized to the same region to which the Lps locus (endotoxin unresponsive gene locus) is mapped. To examine the role of TLR4 in LPS responsiveness, we have generated mice lacking TLR4. Macrophages and B cells from TLR4-deficient mice did not respond to LPS. All these manifestations were quite similar to those of LPS-hyporesponsive C3H/HeJ mice. Furthermore, C3H/HeJ mice have, in the cytoplasmic portion of TLR4, a single point mutation of the amino acid that is highly conserved among the IL-1/Toll receptor family. Overexpression of wild-type TLR4 but not the mutant TLR4 from C3H/HeJ mice activated NF-kappaB. Taken together, the present study demonstrates that TLR4 is the gene product that regulates LPS response.

Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components.

Toll-like receptor (TLR) 2 and TLR4 are implicated in the recognition of various bacterial cell wall components, such as lipopolysaccharide (LPS). To investigate in vivo roles of TLR2, we generated TLR2-deficient mice. In contrast to LPS unresponsiveness in TLR4-deficient mice, TLR2-deficient mice responded to LPS to the same extent as wild-type mice. TLR2-deficient macrophages were hyporesponsive to several Gram-positive bacterial cell walls as well as Staphylococcus aureus peptidoglycan. TLR4-deficient macrophages lacked the response to Gram-positive lipoteichoic acids. These results demonstrate that TLR2 and TLR4 recognize different bacterial cell wall components in vivo and TLR2 plays a major role in Gram-positive bacterial recognition.

A Toll-like receptor recognizes bacterial DNA.

DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.

A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 in macrophages.

C-type lectins serve multiple functions through recognizing carbohydrate chains. Here we report a novel C-type lectin, macrophage-inducible C-type lectin (Mincle), as a downstream target of NF-IL6 in macrophages. NF-IL6 belongs to the CCAAT/enhancer binding protein (C/EBP) of transcription factors and plays a crucial role in activated macrophages. However, what particular genes are regulated by NF-IL6 has been poorly defined in macrophages. Identification of downstream targets is required to elucidate the function of NF-IL6 in more detail. To identify downstream genes of NF-IL6, we screened a subtraction library constructed from wild-type and NF-IL6-deficient peritoneal macrophages and isolated Mincle that exhibits the highest homology to the members of group II C-type lectins. Mincle mRNA expression was strongly induced in response to several inflammatory stimuli, such as LPS, TNF-alpha, IL-6, and IFN-gamma in wild-type macrophages. In contrast, NF-IL6-deficient macrophages displayed a much lower level of Mincle mRNA induction following treatment with these inflammatory reagents. The mouse Mincle proximal promoter region contains an indispensable NF-IL6 binding element, demonstrating that Mincle is a direct target of NF-IL6. The Mincle gene locus was mapped at 0.6 centiMorgans proximal to CD4 on mouse chromosome 6.