Publications by authors named "Kumiko Kobayashi-Nakamura"

3 Publications

  • Page 1 of 1

Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide.

Biochem Biophys Res Commun 2020 01 19;522(1):151-156. Epub 2019 Nov 19.

DHC Corporation, Fundamental Research Laboratory, Division 2, 2-42 Hamada, Mihama-ku, Chiba, 261-0025, Japan. Electronic address:

Peptide transporters 1 and 2 (PEPT1 and PEPT2) are proton-coupled oligopeptide transporter members of the solute carrier 15 family and play a role in the cellular uptake of di/tri-peptides and peptidomimetics. Our previous work showed that PEPT2 is predominantly expressed within undifferentiated keratinocytes. Here we show that PEPT2 expression decreases as keratinocyte differentiation progresses and that PEPT1 alternately is expressed at later stages. Absolute quantification using quantitative polymerase chain reaction revealed that the expression level of PEPT1 is about 17 times greater than that of PEPT2. Immunohistochemical study of human skin provided evidence of PEPT1 in the epidermis. The uptake of glycylsarcosine into keratinocytes was significantly blocked by PEPT inhibitors, including nateglinide and glibenclamide. Moreover, we found that PEPT1 knockdown in differentiated keratinocytes significantly suppressed the influence of a bacterial-derived peptide, muramyl dipeptide (MDP), on the production of proinflammatory cytokine interleukin-8, implying that bacteria-derived oligopeptides can be transported by PEPT1 in advanced differentiated keratinocytes. Taken together, PEPT1 and PEPT2 may concertedly play an important role in MDP-NOD2 signaling in the epidermis, which provides new insight into the mechanisms of skin homeostasis against microbial pathogens.
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January 2020

Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport.

PLoS One 2017 9;12(2):e0171513. Epub 2017 Feb 9.

Fundamental Research Laboratory, Fundamental Research Department, DHC Corporation, Chiba, Japan.

The growing interest in skin lightening has recently renewed attention on the esthetic applications of Chinese herbal medicine. Although Scutellaria baicalensis Georgi is used for antipyretic and antiinflammatory purposes, its whitening effect remains unclear. This study reports three major findings: (1) S. baicalensis has a potent inhibitory effect on melanogenesis; (2) wogonin and its glycoside are the active components of S. baicalensis; and (3) O-methylated flavones from S. baicalensis, such as wogonin, inhibit intracellular melanosome transport. Using a melanin quantification assay, we showed that S. baicalensis potently inhibits melanogenesis in B16F10 cells. Componential analyses revealed that the main components of S. baicalensis are baicalin, wogonoside, baicalein, wogonin, and oroxylin A. Among these five flavones, wogonin and wogonoside consistently inhibited melanogenesis in both B16F10 melanoma cells and primary melanocytes. Wogonin exhibited the strongest inhibition of melanin production and markedly lightened the color of skin equivalents. We identified microphthalmia-associated transcription factor and tyrosinase-related proteins as potential targets of wogonin- and wogonoside-induced melanogenesis suppression. In culture, we found that the melanosomes in wogonin-treated B16F10 cells were localized to the perinuclear region. Immunoblotting analyses revealed that wogonin significantly reduced in melanophilin protein, which is required for actin-based melanosome transport. Other actin-based melanosome transport-related molecules, i.e., Rab27A and myosin Va, were not affected by wogonin. Cotreatment with MG132 blocked the wogonin-induced decrease in melanophilin, suggesting that wogonin promotes the proteolytic degradation of melanophilin via the calpain/proteasomal pathway. We determined that the structural specificities of the mono-O-methyl group in the flavone A-ring and the aglycone form were responsible for reducing melanosome transport. Furthermore, wogonin and two wogonin analogs, mono-O-methyl flavones, strongly suppressed melanosome transport. Our findings suggest the applicability of S. baicalensis in the esthetic field. Thus, we propose a novel pharmacologic approach for the treatment of hyperpigmentation.
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August 2017

H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

Biochem Biophys Res Commun 2016 07 20;475(4):335-41. Epub 2016 May 20.

DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan. Electronic address:

Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake.
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July 2016