Yohtaro Katagata

Helicobacter pylori VacA Enhances Prostaglandin E2 Production through Induction of Cyclooxygenase 2 Expression via a p38 Mitogen-Activated Protein Kinase/Activating Transcription Factor 2 Cascade in AZ-521 Cells

ABSTRACT Treatment of AZ-521 cells with Helicobacter pylori VacA increased cyclooxygenase 2 (COX-2) mRNA in a time- and dose-dependent manner. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, blocked elevation of COX-2 mRNA levels, whereas PD98059, which blocks the Erk1/2 cascade, partially suppressed the increase. Consistent with involvement of p38 MAPK, VacA-induced accumulation of COX-2 mRNA was reduced in AZ-521 cells overexpressing a dominant-negative p38 MAPK (DN-p38). Phosphatidylinositol-specific phospholipase C, which inhibits VacA-induced p38 MAPK activation, blocked VacA-induced COX-2 expression. In parallel with COX-2 expression, VacA increased prostaglandin E2 (PGE2) production, which was inhibited by SB203580 and NS-398, a COX-2 inhibitor. VacA-induced PGE2 production was markedly attenuated in AZ-521 cells stably expressing DN-p38. VacA increased transcription of a COX-2 promoter reporter gene and activated a COX-2 promoter containing mutated NF-κB or NF-interleukin-6 sites but not a mutated cis-acting replication element (CRE) site, suggesting direct involvement of the activating transcription factor 2 (ATF-2)/CREB-binding region in VacA-induced COX-2 promoter activation. The reduction of ATF-2 expression in AZ-521 cells transformed with ATF-2-small interfering RNA duplexes resulted in suppression of COX-2 expression. Thus, VacA enhances PGE2 production by AZ-521 cells through induction of COX-2 expression via the p38 MAPK/ATF-2 cascade, leading to activation of the CRE site in the COX-2 promoter.

Anti-aging effects of high molecular weight proteoglycan from salmon nasal cartilage in hairless mice

Proteoglycans comprise a family of complex macromolecules consisting of a core protein with covalently attached glycosaminoglycan (GAG) chains. The skin anti-aging effects of oral administration of proteoglycan fractions with different molecular weights from salmon nasal cartilage were investigated in a hairless mouse model of skin aging; aging was caused by repeated ultraviolet B (UVB) irradiation. Three proteoglycan fractions of different molecular weights were prepared from salmon nasal cartilage water extract by ion-exchange column chromatography and gel filtration column chromatography. Physiological and histological analysis of the skin indicated that oral administration of high molecular weight proteoglycan inhibited UVB-induced skin aging, defined as increased erythema, increased transepidermal water loss (TEWL), decreased hydration, and epidermal and dermal hypertrophies. The serum and dorsal skin inflammatory cytokine levels indicated that high molecular weight proteoglycan acts on gut immunity and improves skin by inhibiting surplus inflammatory cytokines produced by UVB irradiation. These results suggest that high molecular weight proteoglycan from salmon nasal cartilage is effective in preventing skin aging.

Hsp40 regulates the amount of keratin proteins via ubiquitin-proteasome pathway in cultured human cells

Keratins represent important structural components of intermediate filament proteins. Their expression profiles are remarkably tissue-specific. Recent data have shown that keratins associate with many proteins including heat shock proteins (HSP). We recently identified cell-specific keratin and HSP expression. We aimed to gain further insight into the regulation of keratins by specific inhibition through knockdown of Hsp40 in human keratinocyte cells. Keratin-HSP interaction in HaCaT cell lysate was evaluated by immunoprecipitation followed by Western blotting. Immunofluorescence, was used to examine the co-localization of keratins and Hsp40. Hsp40 depletion led to an increase in the levels of keratin proteins (K5, K14, K10) and a decrease in keratin ubiquitination without influencing keratin gene expression. Our results demonstrate direct or indirectly association of Hsp40 and imply that expressed keratin proteins were regulated by Hsp40 depending on the ubiquitin-proteasome pathway in HaCaT. Furthermore, the K10 differentiation marker was increased by knockdown of Hsp40. The results presented in this study indicate that Hsp40 is related to the differentiation exchange of keratin pairs.

Kuromoji (Lindera umbellata) essential oil inhibits LPS-induced inflammation in RAW 264.7 cells.

Kuromoji (Lindera umbellata) essential oil (KEO) has long been used in Japan as a traditional medicine. It contains linalool (C10H18O), a naturally occurring small terpenoid. For this study, we investigated the anti-inflammatory effect of KEO in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Mouse macrophage-like RAW 264.7 cells were stimulated with LPS. Then they were treated with 25 or 50 µg/mL of KEO for 24 h. KEO suppressed LPS-induced pro-inflammatory cytokine production such as that of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in a dose-dependent manner. In addition, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression and protein levels were suppressed by treatment with KEO cells. In addition, by treatment with 25 or 50 µg/mL of linalool showed the same anti-inflammatory effect. The results suggest that KEO and linalool can be regarded as a natural resource for use in anti-inflammatory therapeutic products.

The Hsp90 inhibitor 17-AAG represses calcium-induced cytokeratin 1 and 10 expression in HaCaT keratinocytes

Hsp90 is essential for maintaining the activity of numerous signaling factors, and plays a key role in cellular signal transduction networks. 17‐Allylamino‐17‐demethoxygeldanamycin (17‐AAG) is an ansamycin antibiotic that binds to Hsp90 and inhibits its function. HaCaT human keratinocytes were used to investigate the cellular and molecular functions of Hsp90 in keratinocyte differentiation. Inhibition of Hsp90 by 17‐AAG leads to downregulation of the differentiation markers cytokeratin 1 and cytokeratin 10 at the protein and mRNA levels.

Impaired activation of caspase cascade during cell death induced by newly synthesized singlet oxygen generator, 1-buthylnaphthalene-4-propionate endoperoxide.

Endoperoxides of naphthalene derivatives generate singlet oxygen under physiological conditions. Here we have synthesized a new endoperoxide of a naphthalene derivative, 1‐buthylnaphthalene‐4‐propionate endoperoxide (BNPE), and studied its cytotoxic properties on HepG2 and HaCaT cells. BNPE induced cell death at much lower concentration than 1‐methylnaphthalene‐4‐propionate endoperoxide (MNPE) and naphthalene dipropionate endoperoxide (NDPE). A positive correlation exists between the amount of endoperoxide incorporated into cells and its cytotoxic ability. The cytotoxic effect of BNPE was attenuated by α‐tocopherol but not by sodium azide. In contrast, the effects of MNPE and NDPE were attenuated by both α‐tocopherol and sodium azide. The caspase cascade in cells treated with endoperoxide was impaired. Caspase activity in a soluble protein fraction were inhibited similarly by the above three endoperoxides. These results suggest an abortive apoptotic pathway due to the suppression of caspase activation is a general feature of cell death induced by singlet oxygen.

Glycosylation of tyrosinase is a determinant of melanin production in cultured melanoma cells

The majority of malignant melanoma cell types are able to produce melanin and the degree of melanin synthesis in various types of cultured cell line differs. In this study, we evaluated three types of cultured cell line, MNT‑1, HM3KO and G‑361, with differing melanin production levels. The level was greatest in the MNT‑1 cells, lower in the HM3KO cells and lowest in the G‑361 cells. In addition, a positive correlation between melanin production and tyrosinase activity was observed. The molecular masses of tyrosinases from HM3KO and G‑361 cells were marginally lower than those from MNT‑1 cells. Glycosylation inhibitor treatment on MNT‑1 cells caused decreases in the molecular mass of tyrosinase, its activity and melanin production. An immunoprecipitation assay using anti‑tyrosinase indicated that the immature glycosylated tyrosinases were associated with a type of chaperone, Hsp70. The interaction between tyrosinase and Hsp70 was also detected in HM3KO and G‑361 cells. The results indicated that the immature glycosylation of tyrosinase has a critical effect on the melanin-producing ability of melanoma cells.

Antiproliferative activity of extracts prepared from three species of Reishi on cultured human normal and tumor cell lines

The present study investigated the growth of human fibrosarcoma (HT-1080) and fibroblast (SF-TY) cells in combination with water-soluble (WS) and high molecular component (HMC) fractions prepared from Reishi (R), Rokkaku-Reishi (2R) and Apple Rokkaku-Reishi (A2R). Each WS fraction exhibited dose-and time-dependent inhibition of the growth of the HT-1080 and SF-TY cells. The extracts exhibited marked antiproliferative activity against the HT-1080 cells. The HMC fractions inhibited cell growth dose-and time-dependently in the HT-1080 cells only, and not in the SF-TY cells, suggesting that HMC fractions selectively inhibit HT-1080 cells. Among the HMC fractions, A2R is a strong candidate for anti-tumor targeting since its fraction exhibited better inhibition than the R and 2R fractions. Furthermore, the volume of the A2R fraction was approximately five times greater than that of the others, and included four proteins (molecular mass 9, 13, 22 and 40 kDa) detected by SDS-PAGE. Three of these (13, 22 and 40 kDa) were confirmed to be glycosylated with the Periodic Acid-Schiff Stain kit. These results suggest that A2R may possess anti-tumor activity and, in particular, that the protein components of A2R may act to selectively inhibit the growth of HT-1080 cells.