Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the ß-Subunit of Acid Ceramidase.

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the ß-subunit of aCDase, which consists of α- and ß-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the ß-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the ß-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading ß-subunit that evokes the ceramide deficiency in AD skin.

Expression of CC-chemokine receptor 7 (CCR7) and CXC-chemokine receptor 4 (CXCR4) in head and neck squamous cell carcinoma.

OBJECTIVES: We tried to clarify the correlation of the expression of CCR7 and CXCR4 with lymph node and distant metastasis. MATERIALS AND METHODS: We examined expression of CCR7 and CXCR4 in 9 HNSCC cell lines and 25 HNSCC tissues by semi-quantitative RT-PCR and immunohistochemistry study. We examined the expression levels of CCR7 and CXCR4 in undifferentiated and differentiated human normal keratinocyte. RESULTS: All cell lines expressed CCR7 mRNA, and three expressed CXCR4 mRNA. CCR7 and CXCR4 mRNAs were significantly higher in HNSCC tissues than in non-neoplastic tissues (p<0.05, respectively) and correlated with lymph node metastasis (p<0.05, respectively). The level of CXCR4 mRNA also correlated with distant metastasis (p<0.05). Immunohistochemistry demonstrated localization of CCR7 and CXCR4 to carcinoma cells and lymphocytes and immunohistochemical staining scores of CCR7 and CXCR4 also showed similar correlation to lymph node and distant metastasis with CCR7 and CXCR4 mRNA levels. The level of CCR7 mRNA was significantly higher in poorly and moderately differentiated than in well-differentiated HNSCC (p<0.05). The level of CCR7 mRNA in undifferentiated keratinocyte was significantly higher than that in differentiated keratinocyte. CONCLUSION: The expression of CCR7 in HNSCC increases by dedifferentiation and plays an important role in lymph node metastasis of HNSCC and CXCR4 plays an important role in lymph node metastasis as well as distant metastasis.