Yuya Terashima

We have examined the expression of chemokines and their receptors in the atopic dermatitis-like (AD-like) lesions of NC/Nga mice. Such lesions develop when the mice are kept in conventional conditions, but not when they are kept isolated from specific pathogens. The thymus- and activation-regulated chemokine TARC is unexpectedly highly expressed in the basal epidermis of 14-week-old mice with lesions, whereas it is not expressed in the skin without lesions. Production of TARC by keratinocytes was confirmed by culturing murine keratinocytic cell line cells (PAM212) with TNF-alpha, IFN-gamma, or IL-1beta. Expression of another Th2 chemokine, macrophage-derived chemokine (MDC), was observed in the skin from mice kept in both conventional and pathogen-free conditions, but expression of MDC was increased severalfold in the skin with lesions. The cellular origin of MDC was identified to be dermal dendritic cells. Infiltration of the skin by IL-4-producing T cells and mast cells, and the increase of CCR4 mRNA in the skin, coincided with the development of AD lesions. These observations indicate that TARC and MDC actively participate in the pathogenesis of AD-like lesions in NC/Nga mice and that these Th2 chemokines could be novel targets for intervention therapy of AD in humans.

Cellular disintegrin and metalloproteinases (ADAMs) are a family of genes with a sequence similar to the snake venom metalloproteinases and disintegrins. ADAMTS-1 is a unique ADAM family protein with respect to the presence of thrombospondin type I motifs and the capacity to bind to the extracellular matrix. Because ADAMTS-1 has a potential zinc-binding motif in the metalloproteinase domain, we examined in this study whether ADAMTS-1 is an active metalloproteinase by means of the proteinase trapping mechanism of alpha2-macroglobulin. We found that the soluble type of ADAMTS-1 protein is able to form a covalent-binding complex with alpha2-macroglobulin. Furthermore, the point mutation within the zinc-binding motif of ADAMTS-1 protein eliminates its capacity to bind to alpha2-macroglobulin. These data demonstrate that the metalloproteinase domain of ADAMTS-1 is catalytically active. In addition, we showed that the removal of the pro-domain from the ADAMTS-1 precursor is impaired in the furin-deficient cell line, LoVo, and that the processing ability of the cells is restored by the co-expression of the furin cDNA. These data provide evidence that the ADAMTS-1 precursor is processed in vivo by furin endopeptidase in the secretory pathway. Consequently, ADAMTS-1 is an active metalloprotease that is associated with the extracellular matrix. This study strongly suggests that ADAMTS-1 may play a role in the inflammatory process through its protease activity.

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 beta-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.