Epidermal keratinocytes regulate hyaluronan metabolism via extracellularly secreted hyaluronidase 1 and hyaluronan synthase 3.

Hyaluronan (HA) is a high-molecular-weight (HMW) glycosaminoglycan, which is a fundamental component of the extracellular matrix that is involved in a variety of biological processes. We previously showed that the HYBID/KIAA1199/CEMIP axis plays a key role in the depolymerization of HMW-HA in normal human dermal fibroblasts (NHDFs). However, its roles in normal human epidermal keratinocytes (NHEKs) remained unclear. HYBID mRNA expression in NHEKs was lower than that in NHDFs, and NHEKs showed no depolymerization of extracellular HMW-HA in culture, indicating that HYBID does not contribute to extracellular HA degradation. In this study, we found that the cell-free conditioned medium of NHEKs degraded HMW-HA under weakly acidic conditions (pH 4.8). This degrading activity was abolished by hyaluronidase 1 (HYAL1) knockdown but not by HYAL2 knockdown. Newly synthesized HYAL1 was mainly secreted extracellularly, and the secretion of HYAL1 was increased during differentiation, suggesting that epidermal interspace HA is physiologically degraded by HYAL1 according to pH decrease during stratum corneum formation. In HA synthesis, hyaluronan synthase 3 (HAS3) knockdown reduced HA production by NHEKs, and interferon-γ-dependent HA synthesis was correlated with increased HAS3 expression. Furthermore, HA production was increased by TMEM2 knockdown through enhanced HAS3 expression. These results indicate that NHEKs regulate HA metabolism via HYAL1 and HAS3, and TMEM2 is a regulator of HAS3-dependent HA production.

Pro-inflammatory cytokines suppress HYBID (hyaluronan (HA) -binding protein involved in HA depolymerization/KIAA1199/CEMIP) -mediated HA metabolism in human skin fibroblasts.

In the skin, the metabolism of hyaluronan (HA) is highly regulated. Aging leads to chronic low-grade inflammation, which is characterized by elevated levels of pro-inflammatory cytokines; however, the relationship between inflammation and HA metabolism is not clear. Herein, we investigated the effects of a mixture of pro-inflammatory cytokines containing TNF-α, IL-1ß, and IL-6 on HA metabolism in human skin fibroblasts. Treatment with the cytokine mixture for 24 h suppressed HA depolymerization via downregulation of HYBID (HA-binding protein involved in HA depolymerization/KIAA1199/CEMIP) and promoted HA synthesis via upregulation of HAS2 in human skin fibroblasts. Moreover, HAS2-dependent HA synthesis was driven mainly by IL-1ß with partial contribution from TNF-α. Transmembrane protein 2 (TMEM2/CEMIP2), which was previously reported as a candidate hyaluronidase, was upregulated by the cytokine mixture, suggesting that TMEM2 might not function as a hyaluronidase in human skin fibroblasts. Furthermore, the effects of the cytokine mixture on HA metabolism were observed in fibroblasts after 8 days of treatment with cytokines during three passages. Thus, we have shown that HYBID-mediated HA metabolism is negatively regulated by the pro-inflammatory cytokine mixture, providing novel insights into the relationship between inflammation and HA metabolism in the skin.

Role of fatty acid salts as anti Acanthamoeba agents for disinfecting contact lens.

　Acanthamoeba is found in seawater, fresh water, and soil and is an opportunistic pathogen that causes a potentially blinding corneal infection known as Acanthamoeba keratitis. The anti-amoeba activity of 9 fatty acid salts (potassium butyrate (C4K), caproate (C6K), caprylate (C8K), caprate (C10K), laurate (C12K), myristate (C14K), oleate (C18:1K), linoleate (C18:2K), and linolenate (C18:3K)) was tested on Acanthamoeba castellanii ATCC 30010 (trophozoites and cysts). Fatty acid salts (350 mM and pH 10.5) were prepared by mixing fatty acids with the appropriate amount of KOH. C8K, C10K, and C12K showed growth reduction of 4 log-units (99.99% suppression) in A. castellanii upon 180 min incubation at 175 mM, whereas the pH-adjusted control solution showed no effect. After the amoeba suspension was mixed with C10K or C12K, cell membrane destruction was observed. The minimum inhibitory concentration of C10K and C12K was also determined to be 2.7 mM. Confirmation tests were conducted using contact lenses to evaluate the effectiveness of C10K and C12K as multi-purpose solutions. Experiments using increasing concentrations showed reduced numbers of living cells in C10K (5.5 mM, 10.9 mM) and in C12K (5.5 mM, 10.9 mM). These results demonstrate the inhibitory activity of C10K and C12K against A. castellanii and indicate their potential as anti-amoeba agents.

Antibacterial Effect of Fatty Acid Salts on Oral Bacteria.

Fatty acid salts are a type of surfactant known to have potent antibacterial activity. We therefore examined the antibacterial activities of fatty acid salts against Streptococcus mutans. Potassium caprylate (C10K), potassium laurate (C12K), potassium myristate (C14K), potassium oleate （C18:1K), potassium linoleate (C18:2K), and potassium linolenate (C18:3K), used at a concentration of 175 mM, resulted in a 7 log-unit reduction of S. mutans after a 10-min incubation. The minimum inhibitory concentration (MIC) of C18:2K and C18:3K was 5.5 mM. C12K also demonstrated high antibacterial activity (MIC of 21.9 mM). These results indicate that C12K, C18:2K, and C18:3K have high antibacterial activity against S. mutans, and possess great potential as antibacterial agents.

Nickel-catalyzed intermolecular [3 + 2 + 2] cocyclization of ethyl cyclopropylideneacetate and alkynes. Synthesis of seven-membered carbocycles.

The [3 + 2 + 2] cocyclization of ethyl cyclopropylideneacetate (1a) and various alkynes proceeded smoothly in the presence of Ni(cod)2-PPh3. The cycloheptadiene derivatives were synthesized in highly selective manners. The unique reactivity of 1a was essential for the progress of the reaction. The observed regioselectivity of the product formation and the mechanism of the reaction are discussed.

Nickel-catalyzed intermolecular [3 + 2 + 2] cocyclization of ethyl cyclopropylideneacetate and alkynes.

The [3 + 2 + 2] cocyclization of ethyl cyclopropylideneacetate (1a) and terminal alkynes (2) proceeded smoothly in the presence of 10 mol % "Ni(PPh3)2", which was prepared in situ from Ni(cod)2 and PPh3. The high reactivity of 1a, which was induced by the introduction of an electron-withdrawing group, is very important for the progress of this reaction. The cycloheptadiene derivatives were synthesized in highly selective manner in good yields.