Biochem Biophys Res Commun. 2016 Jul 8;475(4):335-41.

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

Kudo M1, Katayoshi T1, Kobayashi-Nakamura K1, Akagawa M2, Tsuji-Naito K3.
  • 1DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan.
  • 2Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531, Japan.
  • 3DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan. Electronic address: knaito@dhc.co.jp.

Abstract

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.

PMID: 27216463; DOI: 10.1016/j.bbrc.2016.05.093

 

Supplement 

Peptide transporter 1 (PEPT1) and peptide transporter 2 (PEPT2) belong to the proton-coupled oligopeptide transporter (POT) family involved in the uptake of small peptides (dipeptides and tripeptides) and peptide-like drugs across cytoplasmic membranes1. PEPT1 is primarily expressed in the intestinal epithelia and kidneys, whereas PEPT2 is primarily expressed in the kidneys and is widely distributed in other tissues, except the small intestine. Analyses of their functions have mainly examined the small intestine and kidneys and shown PEPT1 and PEPT2 to be involved in dietary peptide absorption and peptide filtration, respectively.

In recent years, there has been growing interest in the tissue-specific functions of these transporters other than in the small intestine and kidneys. In particular, because PEPT2 has been identified to be expressed in various tissues, several studies have reported its functions: PEPT2 acts in the efflux of small peptides and peptide-like drugs from cerebrospinal fluid into the choroid plexus2 and removes neuropeptide fragments broken down by peptidases following their neuronal release3. However, these expression profiles and functional activities in the skin remain unclear.

The expression profiles of PEPT1 and PEPT2 in skin cells were examined using three primary cells derived from the human skin: keratinocytes, fibroblasts, and melanocytes. The human intestinal cell line Caco-2 and human hepatoma cell line HepG2 were used as cells positive for the expression of PEPT1 and PEPT2, respectively. Although keratinocytes showed very low PEPT1 expression compared with Caco-2 cells (Fig. 1A), the level of PEPT2 expression in keratinocytes was nearly equal to that in HepG2 cells (Fig. 1B). These results suggested that keratinocytes express both PEPT1 and PEPT2 and that PEPT2 is expressed more in keratinocytes than in PEPT1. In contrast, dermal fibroblasts and melanocytes showed no or markedly lower PEPT1 and PEPT2 mRNA expression than did keratinocytes. To confirm PEPT2 expression in keratinocytes, we investigated PEPT2 gene expression profiles in three keratinocytes: malignant keratinocytes (A431), immortalized keratinocytes (PSVK1), and primary keratinocytes. PCR detection confirmed that PEPT2 is expressed in all keratinocytes (Fig. 1C). The specificity of PCR detection was confirmed by sequencing analysis of the detected mRNA-derived PCR products, and the DNA sequences of the PCR products were 100% identical to reference sequences in GenBank. Overall, the findings showed that PEPT2 was clearly expressed in epidermal keratinocytes, which were more common than other skin cells, such as dermal fibroblasts.

 

 

Figure 1. PEPT1 and PEPT2 mRNA expression profiles in three primary cells derived from the human skin: keratinocytes, fibroblasts, and melanocytes. (A, B) Quantitative real-time PCR analysis of PEPT1 and PEPT2 in keratinocytes, fibroblasts, and melanocytes. The results are presented as fold change of each mRNA in the other cells relative to those in Caco-2 or HepG2 cells used as cells positive for the expression of PEPT1 or PEPT2, respectively. Values represent the mean ± SE of triplicate determinations (N.D., not detectable). (C) Total RNA samples of malignant keratinocytes (A431), immortalized keratinocytes (PSVK1), and primary keratinocytes were analyzed with reverse transcription-PCR using specific primers.

 

 

Figure 2. Immunofluorescence localization of PEPT2 in human skin tissue. (A) Corresponding Nomarski images are also shown. White dotted lines represent basal membrane. (B) Frozen sections were stained for PEPT2. The area near the basal layer in the epidermis is stained for PEPT2 (red arrows), whereas the stain is absent in the stratum corneum and dermis (white arrows). Sc: stratum corneum. E: epidermis. D: dermis. Bars, 20 μm.

 

Using an anti-PEPT2 antibody, immunohistochemical analysis of the human skin revealed PEPT2 localization in the epidermis (Fig. 2). PEPT2 was shown to have the strongest expression in the area closest to the basal membrane. In contrast, PEPT2 staining was negative in the dermis or stratum corneum (white arrows in Fig. 2).

PEPT1 and PEPT2 function in dipeptide and tripeptide absorption. A preliminary uptake test using an oligopeptide confirmed that keratinocytes could absorb small peptides in the presence of an inward H+ gradient. Therefore, we investigated the peptide absorptive behaviors of keratinocytes at the tissue level. A skin-permeability test for peptides was performed using a Franz cell with a skin substitute (Fig. 3A), which is reconstructed as human epidermis alone. As shown in Fig. 3B, the transdermal amount of glycylsarcosine (GlySar) increased over 6 h. The skin-permeability test was also performed using alanyl-4-hydroxyproline (AlaHyp), which has been identified as a food-derived collagen peptide4. POTs in the small intestine are believed to be involved in the intestinal absorption of such collagen peptides after the ingestion of dietary gelatin hydrolysate5. Linear penetration of AlaHyp was detected as well as that of GlySar (Fig. 3C), suggesting that oligopeptides could be transdermally absorbed.

In conclusion, in the present study, we demonstrated that PEPT2 is highly expressed in human keratinocytes and that oligopeptides can penetrate the epidermal layer. These observations support the possibility that epidermal PEPT2 is involved in the transcellular transport of oligopeptides and peptide-like drugs and contributes to the development of the transdermal drug delivery system.

 

 

Figure 3. Permeation of oligopeptides through a skin substitute. (A), The skin permeation of oligopeptides was performed using a Franz cell with a skin substitute. (B, C) The time course of GlySar or AlaHyp permeation was measured at 37°C for 6 h. Values represent the mean ± SE of triplicate determinations.

 

 

References

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