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Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide Michiko Kudo, Kumiko Kobayashi-Nakamura, Natsuko Kitajima, Kentaro Tsuji-Naito* DHC Corporation, Fundamental Research Laboratory, Division 2, 2-42 Hamada, Mihama-ku, Chiba, 261-0025, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2019 Accepted 5 November 2019 Available online xxx
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. © 2019 Elsevier Inc. All rights reserved.
Keywords: Keratinocytes Solute carrier 15 (SLC15) Oligopeptide transporter Glycylsarcosine (GlySar) Skin
1. Introduction Peptide transporters 1 and 2 (PEPT1 and PEPT2) are protoncoupled oligopeptide transporter (POT) members of the solute carrier 15 family and serve in cellular uptake of di/tri-peptides and peptidomimetics [1]. PEPT1 is predominantly expressed in the intestinal epithelium and functions for absorption of dietary nutrients [2,3]. PEPT2 is widely expressed in other tissues and has been reported to function in the kidney in reabsorption of dietary peptides [4,5]. These two peptide transporters have some differences in terms of substrate affinity and capacity. PEPT1 is characterized as low-affinity and high-capacity and therefore can transport large amounts of oligopeptides. In contrast, PEPT2 is characterized as high-affinity and low-capacity [6]. The differences in substrate affinity are due to different specificities for a- and b-amino carbonyl structures of the substrates [7]. Several recent studies have identified physiological functions of PEPT2 beyond its role in nutritional
* Corresponding author. E-mail address: [email protected] (K. Tsuji-Naito).
processes: in the choroid plexus for clearance of neuropeptide fragments from cerebrospinal fluid and in immune cells for uptake of bacterially derived peptides to trigger innate immune responses [8e10]. Recent research has therefore focused on the distinctive, tissue-specific functions of PEPT1 and PEPT2. As the outermost layer of the skin, the epidermis protects the body from the environment. Keratinocytes, which constitute the majority of cells in the epidermis, maintain immune homeostasis of the skin, together with epidermal dendritic cells [11]. Thus, keratinocytes act as a physical and immunological barrier. Innate immunity serves as the first-line defense against pathogenic invasion and detects infection through pattern recognition receptors (PRRs) [12]. PRRs are classified as membrane-bound or cytoplasmic-type PRRs on the basis of their location, including Toll-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), respectively. NOD1 and NOD2, which are members of the NLR family, detect specific bacterial peptidoglycan motifs in the host cytosol, leading to production of proinflammatory cytokines [13]. NOD1 is activated by peptidoglycan fragments containing the mesodiaminopimelic acid derived primarily from gram-negative and specific gram-positive bacteria. In contrast, NOD2 responds to
https://doi.org/10.1016/j.bbrc.2019.11.044 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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Abbreviations PEPT MDP SLC GlySar POT PRRs NOD NLRs IL Ntg Gbc
Peptide transporter Muramyl dipeptide Solute carrier Glycylsarcosine Proton-coupled oligopeptide transporter Pattern recognition receptors Nucleotide-binding oligomerization domain Nucleotide-binding oligomerization domain (NOD)like receptors Interleukin Nateglinide Glibenclamide
invasive bacteria by sensing peptidoglycan-related molecules containing muramyl dipeptide (MDP) that are produced by both gramnegative and gram-positive bacteria. Activation of the NOD signaling pathway induces the transcription factors nuclear factor-kappa B and activator protein-1, leading to enhancement of the production of proinflammatory cytokines (e.g., interleukin-6; IL-6, IL-8, and tumor necrosis factor-a). The NOD2 gene is implicated in the pathogenesis of several chronic inflammatory diseases [13]. Loss-offunction mutations in NOD2 are closely related to Crohn’s disease, and Blau syndrome and early-onset sarcoidosis are caused by gainof-function mutations. As for the epidermis, the NOD2 risk alleles are highly suggestive of several inflammatory diseases, including atopic eczema and dermatitis. Moreover, several research groups have demonstrated increased expression of inflammasome and innate immune receptors, including NOD2, in keratinocytes of psoriatic lesional skin [14,15]. Although accumulating evidence indicates a role for staphylococcal peptidoglycans and MDP in NLR signaling in keratinocytes, it is unknown how the NOD ligands enter the host cytoplasmic space. We previously showed that primary keratinocytes express PEPT2 more than PEPT1 [16]. But the possible variance of these two POT expressions during epidermal differentiation and also their role in innate immunity of the epidermis are still undefined. Thus, in this research we investigated the expression of PEPT1 and PEPT2 during differentiation and their role in NOD ligand-inducible immune responses using normal human epidermal keratinocytes (NHEKs). Additionally, we addressed two of the following points: 1) these two PEPTs are alternately expressed during differentiation in keratinocytes; and 2) they work together in facilitating the uptake of the bacterially derived peptide MDP into the cytosol of keratinocytes. These indicate that PEPT1 and PEPT2 are variably modulated in the epidermis to play a role in host defense responses. Our finding may provide a new insight into the cutaneous immune homeostasis. 2. Materials and methods 2.1. Materials Glycylsarcosine (GlySar) was purchased from Sigma-Aldrich (St. Louis, MO, USA). MDP was purchased from InvivoGen (San Diego, CA, USA). Nateglinide (Ntg) and glibenclamide (Gbc) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other chemicals were of the highest commercially available grade. 2.2. Cell cultures and human skin NHEKs (newborn/male, Thermo Fisher Scientific, Waltham, MA,
NHEKs KRT FLG qPCR RT-PCR PBS BSA DAPI FAAs LC-MS/MS MRM ELISA siRNA
Normal human epidermal keratinocytes Keratin Filaggrin Quantitative polymerase chain reaction Reverse transcription-polymerase chain reaction Phosphate-buffered saline Bovine serum albumin 4ʹ,6-Diamidino-2-phenylindole Free amino acids Liquid chromatography/tandem mass spectrometry Multiple reaction monitoring Enzyme-linked immunosorbent assay Small interfering RNA
USA) were maintained in keratinocyte growth medium consisting of EpiLife™ Medium, with 60 mM CaCl2 and human keratinocyte growth supplement (both from Thermo Fisher Scientific). Differentiation of NHEKs was induced by culturing them in MCDB 153 medium (Sigma-Aldrich) supplemented with 5 mg/ml insulin, 0.5 mg/ml hydrocortisone, 10 mg/ml transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 40 mg/ml bovine pituitary extract, and 1.35 mM CaCl2. Keratinocyte differentiation was confirmed based on morphologic changes and mRNA levels of keratin 5 (KRT5), KRT10, and filaggrin (FLG) determined by quantitative polymerase chain reaction (qPCR), as described below. Cells were grown in a humidified incubator at 37 C in an atmosphere containing 5% CO2. The medium was changed every other day, and the cells were passaged when they reached 80%e90% confluency. NHEKs were not used beyond passage 6. The medium was changed every day when cells reached 50% confluency. A human abdominal skin sample (40-year-old female, Caucasian) was purchased from Biopredic International (Rennes, France). This human experiment was approved by and followed the guidelines for the ethical use of human-origin organs and tissues of KAC (Kyoto, Japan), an alliance partner of Biopredic International. 2.3. Reverse transcription-polymerase chain reaction (RT-PCR) and qPCR Total RNA was isolated from cultured cells using an RNeasy Mini Kit (Qiagen, Mississauga, Canada), according to the manufacturer’s instructions. First-strand cDNA was synthesized with total RNA using a PrimeScript® II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan), according to the manufacturer’s instructions. For RTPCR gene expression studies, cDNA was mixed with KOD Plus (Toyobo, Osaka, Japan) as a DNA polymerase and gene-specific primers. The primers used were as follows: human PEPT1, 5ʹGGTAAAGTGGCCAAGTGCAT-30 and 5ʹ-CAAACAAGGCCCAGAACATT30 for a 193-bp fragment; human PEPT2, 5ʹ-TGACAGTGGTGGGAAATGAA-3ʹ and 5ʹ-TCCCATCTTCACGAATGACA-3ʹ for a 204-bp fragment; and human glyceraldehyde-3-phophate dehydrogenase (GAPDH), 5ʹ-GAGTCAACGGATTTGGTCGT-3ʹ and 5ʹTTGATTTTGGAGGGATCTCG-3ʹ for a 238-bp fragment. The PCR conditions on a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA) were as follows: at 98 C for 2 min; 30 cycles at 98 C for 10 s, at 60 C for 15 s, and at 72 C for 15 s; and finally at 72 C for 1 min. PCR products were separated on a 2% agarose gel. For qPCR, target-gene mRNA expression levels were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems) with the following TaqMan® Gene Expression Assays: FLG (assay ID Hs00856927_g1); KRT5 (assay ID Hs00361185_m1);
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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KRT10 (assay ID Hs00166289_m1); PEPT1 (assay ID Hs00192639_m1); PEPT2 (assay ID Hs01113665_m1); CXCL8 (assay ID Hs00174103_m1); and 18S (assay ID Hs99999901_s1). All reactions were performed in triplicate. 18S was used as a housekeeping gene for quantity normalization. The relative amounts of mRNA were calculated using the comparative CT method (2DDCt). The results are presented as fold change in each mRNA during keratinocyte differentiation. For absolute quantification of PEPT1 and PEPT2, the standard curve method was applied. The standard curves of PEPT1, PEPT2, and 18S were designed based on known quantities of synthetic DNA containing the specific sequences of each TaqMan® assay location disclosed by Applied Biosystems. 2.4. Immunohistochemical analysis For immunofluorescence histochemistry of PEPT1, a sample of human abdominal skin tissue was fixed to 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 C overnight. After rinsing with PBS, the tissue was immersed in 20% sucrose in PBS at 4 C overnight and then embedded in Tissue Freezing Medium (Leica Biosystems, Nussolch, Germany). Frozen tissues were cut in 7 mm slices with a cryostat. Sections were rinsed with PBS and subsequently treated with 0.1% Triton-X 100 in PBS for 30 min at room temperature. Nonspecific reactions were blocked by incubation of sections in 3% horse serum in PBS for 30 min. A polyclonal rabbit or anti-PEPT1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:150 in 1% bovine serum albumin (BSA) in PBS was applied overnight at 4 C. After washing in PBS, sections were incubated with Alexa Fluor 488 donkey anti-mouse IgG (Thermo Fisher Scientific) at a dilution of 1:400 in 1% BSA in PBS for 1 h at room temperature. Following a rinse in PBS, sections were treated with 1 mg/ml of 4ʹ,6-diamidino-2-phenylindole (DAPI) (Dojindo Laboratories, Kumamoto, Japan) in PBS for 3 min. After washing in PBS, sections were mounted using Fluoromount/Plus™ (Diagnostic BioSystems, Pleasanton, CA, USA). Fluorescence signals were examined with a Leica confocal fluorescence microscope (Leica Microsystems Japan, Tokyo, Japan). 2.5. Uptake measurements For the uptake studies, NHEKs were seeded at a density of 1.0 105 cells/dish on 60-mm plastic culture dishes. After a 7-day period for keratinocyte differentiation, the medium was changed to the new one adjusted to pH 6.0. NHEKs were preliminarily treated with Ntg, Gbc, or glycine (Gly) for 1 h and incubated with GlySar for an additional 1 h. The culture medium was removed from the dishes and the cells were washed three times with PBS. NHEKs were lysed using a lysis buffer (150 mM NaCl; 20 mM Tris, pH 7.5; 10 mM EDTA; 1% Triton-X; 1.0% sodium deoxycholate). Each extract was subjected to derivatization of free amino acids (FAAs) and GlySar. FAAs and GlySar were derivatized using the EZ:faast™ amino acid analysis kit (Phenomenex, Torrance, CA, USA), following the manufacturer’s instructions. Briefly, methionine-d3 was added as an internal standard to each cell extract, and FAAs and GlySar were derivatized using Sorbent Tips and extracted by the provided reagents from the EZ:faast™ kit. The GlySar content of the samples was determined by liquid chromatography/tandem mass spectrometry (LC-MS/MS), as described below. 2.6. GlySar measurement using LC-MS/MS The derivatives of FAAs and GlySar were detected and quantified by LC-MS/MS using selected ion monitoring in the positive-ion electron capture mode. MS was performed on an API 2000 tandem mass spectrometer (AB SCIEX, Tokyo, Japan) equipped with a
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standard API electrospray ionization source and interfaced with an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA). Samples were injected onto an EZ:faast™ AAA-MS column (2 250 mm) at a flow rate of 0.25 ml/min. The separation was performed with a two-pump gradient. Solvent A was 10 mM ammonium formate in water, and solvent B was 10 mM ammonium formate in water in methanol. The gradient program was as follows: 0 min, B 68%; 13 min, B 83%. Analyses were monitored in positive-ion mode using the API ion source at 400 C and with multiple reaction monitoring (MRM). Nitrogen was used as a curtain (setting 40) and collision (setting 7) gas. The derivatives of GlySar and methionine-d3 were eluted at 5 and 9 min, respectively, under these conditions. The following MRM transitions were selected: (m/z, Q1/Q3) of GlySar (m/z, 275 / 132) and methionine-d3 (m/z, 281 /193) derivatives. 2.7. Enzyme-linked immunosorbent assay (ELISA) For quantification of IL-8 protein levels, NHEKs were seeded at a density of 1.0 105 cells in six-well plates. After allowing the keratinocytes differentiate for 7 days, NHEKs were stimulated with 10 mg/ml of MDP in the absence or presence of Gbc or Gly. After culturing, the cell culture media were collected. Cytokine IL-8 secreted in the culture medium from NHEKs was quantified using a solid phase sandwich ELISA kit (Proteintech Group, Chicago, IL) according to the manufacturer’s instruction. 2.8. RNA silencing For the small interfering RNA (siRNA) studies, siRNAs targeting PEPT1 were purchased from Horizon Discovery (Cambridge, UK). Nontargeting siRNA (Horizon Discovery) was used as a negative silencing control. NHEKs were seeded at a density of 1.0 105 cells in six-well plates. After a 7-day period for keratinocyte differentiation, NHEKs were transfected with 20 nM of PEPT1 or scramble siRNA plus HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s instructions for mRNA silencing in the cells. The efficiency of siRNA-mediated repression of target mRNA levels was assessed by qPCR. 2.9. Statistical analyses Data are expressed as mean ± SE from at least three independent experiments. Statistical analyses were performed by the TukeyeKramer test. 3. Results and discussion We previously showed that PEPT2 is predominantly expressed within undifferentiated keratinocytes [16]. Using primary keratinocytes, we conducted RT-PCR analyses to clarify the variance of PEPT2 transcripts during differentiation. PEPT2 transcripts were strongly observed at the initial stage but declined with the progression of differentiation stage (Fig. 1A). In contrast, PEPT1 transcripts were significantly observed at the later stages (days 5e9). We speculated that, instead of PEPT2, PEPT1 was expressed in advanced differentiated keratinocytes. As shown in Fig. 1B, qPCR analyses showed that PEPT2 was highly expressed in the earlier stages similarly to KRT5 as a marker for basal and undifferentiated keratinocytes. The expression of PEPT1 was found to be inversely expressed in the later stages identical to the late markers of differentiation, such as KRT10 and FLG. In addition, absolute qPCR for PEPT1 and PEPT2 revealed that PEPT1 expression at the onset of differentiation was only less than one-fifth that of PEPT2, but the highest value of PEPT1 was reached at day 7 of differentiation and
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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Fig. 1. Peptide transporters 1 and 2 (PEPT1 and PEPT2) mRNA expression profiles during keratinocyte differentiation. Cell differentiation was induced by culturing in a culture medium containing a high concentration of calcium (see Materials and methods). (A) Total RNA samples from keratinocytes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using each specific primer. (B) Quantitative polymerase chain reaction (qPCR) analyses for PEPT1, PEPT2, and the indicated differentiation markers at various times (days) of calcium-induced differentiation. The results are presented as fold change in each mRNA. (C) Absolute qPCR for PEPT1 and PEPT2 at onset and day 7 of differentiation. Values represent the mean ± SE of triplicate determinations (**p < 0.01 versus the indicated group; TukeyeKramer test). KRT5, keratin 5; KRT10, keratin 10; FLG, filaggrin; GAPDH, glyceraldehyde-3phosphate dehydrogenase.
was more than 17 times higher than that of PEPT2 (Fig. 1C). The maximal value of PEPT1 at day 7 was approximately four times higher than that of PEPT2 at onset, suggesting that the outer layers express these transporters more than the inner ones. Our previous work immunohistochemically examined PEPT2 expression in human adult skin and showed that PEPT2 is localized in the epidermis, particularly in the basal layer [16]. Immunolocalization analysis of human skin revealed positive staining for PEPT1 in the epidermis, with the upper layers showing the strongest staining (Fig. 2), which suggested that keratinocytes alter the expression pattern of these two POTs from PEPT2 to PEPT1 in a stepwise manner during differentiation. Our previous work has shown the ability of undifferentiated keratinocytes to intracellularly absorb several oligopeptides, such as GlySar and collagen-derived peptides [16]. To investigate PEPT transport capacity in differentiated keratinocytes, we measured the cellular uptake of a synthetic dipeptide, GlySar, which is known as a substrate of PEPTs. After exposure of cultured differentiated keratinocytes to GlySar, we measured the intracellular levels of GlySar with the LC-MS/MS system. As shown in Fig. 3, we observed that GlySar uptake into keratinocytes was significantly blocked by the PEPT inhibitors Ntg and Gbc, but not by the amino
Fig. 2. Tissue localization of peptide transporter 1 (PEPT1) immunostaining in the human skin. Frozen sections were stained for PEPT1 (green) and DNA (blue). Bars, 50 mm. IgG, immunoglobulin G; DAPI, 40 ,6-diamidino-2-phenylindole.
Fig. 3. Effect of peptide transporter 1 (PEPT1) on glycylsarcosine (GlySar) uptake in differentiated keratinocytes. After a 7-day period for keratinocyte differentiation, cells were preliminarily treated with nateglinide (Ntg), glibenclamide (Gbc), or glycine (Gly) at the indicated concentrations for 1 h and incubated with GlySar for 1 h, after which GlySar concentrations in cell extracts were measured. Values represent the mean ± SE of triplicate determinations (*p < 0.05, **p < 0.01 versus control; TukeyeKramer test).
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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acid Gly. This result indicated that oligopeptides can be transported by PEPTs in advanced differentiated keratinocytes. MDP is a degradation product of bacterial peptidoglycan that triggers innate inflammatory responses upon ligand binding to NOD2 present in the cytosol [13]. MDP increased the expression of
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proinflammatory cytokine IL-8 mRNA and protein in cultured differentiated keratinocytes (Fig. 4A and B). These results indicate that MDP accesses the cytosol to activate NOD2 for IL-8 production. Based on gene expression in keratinocytes, we hypothesized that PEPT1 and 2 play a causative role in NOD2-mediated immune
Fig. 4. Effect of peptide transporter 1 (PEPT1) on the muramyl dipeptide (MDP)-stimulated immune response in differentiated keratinocytes. (A, B) Normal human epidermal keratinocytes (NHEKs) were treated for the indicated number of hours with 10 mg/ml MDP. (A) The expression levels of CXCL8 were quantified by quantitative polymerase chain reaction (qPCR). (B) Interleukin-8 (IL-8) concentrations in the cell culture media were measured by enzyme-linked immunosorbent assay (ELISA). Values represent mean ± SE of triplicate determinations (*p < 0.05, **p < 0.01 vs. hour 0; TukeyeKramer test). (C, D) For mRNA and protein expression analyses of IL-8, NHEKs were stimulated with 10 mg/ml MDP in the absence or presence of glibenclamide (Gbc) or glycine (Gly) for 2 and 8 h, respectively. (E) NHEKs were transfected with the indicated small interfering RNA (siRNA) (20 nM) plus HiPerFect Transfection Reagent for 48 h, after which the cells were incubated for 24 h. After incubation, NHEKs were stimulated with 10 mg/ml MDP for 2 h. Values represent mean ± SE of triplicate determinations (*p < 0.05, **p < 0.01 versus the indicated group; TukeyeKramer test). N.S., not significant.
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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responses by MDP. Thus, to clarify whether POTs mediate entry of MDP into the cytosol of keratinocytes, we investigated the influence of the PEPT inhibitor Gbc on IL-8 production by MDP. As shown in Fig. 4C and D, Gbc significantly suppressed MDP-induced IL-8 production at both the mRNA and the protein levels in cultured differentiated keratinocytes. To confirm the role of POTs in MDPNOD2 signaling, we next used siRNA against PEPT1. The siRNA knockdown of PEPT1 significantly reduced PEPT1 expression, but has no off-target effect on PEPT2 (Fig. 4E). We confirmed the involvement of PEPT1 in MDP-mediated IL-8 induction in keratinocytes followed by cultured differentiation. In anaplastic keratinocytes, treatment with siRNA against PEPT2 reduced MDPinducible IL-8 transcripts as well (data not shown). Taken together, these results suggest that PEPT1 and PEPT2 may have an important role in MDP-NOD2 signaling in the epidermis. In this study, we have shown that keratinocytes undergo a switch from PEPT2 to PEPT1 expression in the differentiation process. The entry of MDP into the keratinocytes is shown to be accomplished through both of these transporters, suggesting that PEPT1 and PEPT2 concertedly contribute to skin immune homeostasis via NOD-like receptor signaling pathways. The question remains why their expression pattern changes during keratinocyte differentiation. We speculate that the higher transporting capacity of PEPT1 compared with PEPT2 may provide a dedicated platform for protection against invasion by various pathogens. Indeed, differentiated keratinocytes showed a higher response against MDP than did proliferating keratinocytes, although there was no difference between differentiated and proliferating keratinocytes in NOD2 expression (data not shown). Taking into consideration the fact that the differences in sensitivity to MDP were well correlated with the expression pattern of PEPT1 in the process of differentiation, the entry of MDP into host cytosol via PEPTs is likely to affect the intensity of the immune response as one of the rate-limiting steps. In addition, because unlike the dermis, there are no blood vessels in the epidermis, these transporters in the epidermis are likely to work in a concerted manner for nutrient absorption of peptide-bound amino acids as well. PEPT1 and PEPT2 are differentially expressed in the proximal tubules of the kidney, which work to efficiently reabsorb oligopeptides from the tubular fluid. The upper layers of the epidermis are more distant from the blood vessels than are the basal layers and therefore require the high capacity of PEPT1 for nutritional absorption. In fact, these two transporters are abundantly expressed in keratinocytes but are hardly expressed in fibroblasts from the human dermis [16]. Impairments of NLR signaling are recognized risk factors for several chronic inflammatory diseases, including atopic dermatitis [13e15]. NOD1 and NOD2 polymorphisms have been related to elevated IgE levels and atopic dermatitis [17,18]. In addition, skin lesions of atopic dermatitis have been reported to exhibit decreased innate immune responses, including expression of b-defensin and IL-8 [19e21]. Aberrancy in epidermal differentiation is often linked to the disturbed barrier function observed in atopic dermatitis [22]. Thus, change in keratinocyte differentiation in atopic dermatitis may also affect susceptibility to bacteria-derived muramyl peptides through unbalanced expression of PEPT1 and PEPT2, leading to change in the epidermal immune system. Our results show that the expression pattern of PEPTs is tightly regulated during differentiation. These findings provide new insights into the understanding of skin innate immunity.
References [1] T. Terada, K. Inui, Recent advances in structural biology of peptide transporters, Curr. Top. Membr. 70 (2012) 257e274. [2] Y.J. Fei, Y. Kanai, S. Nussberger, V. Ganapathy, F.H. Leibach, M.F. Romero, S.K. Singh, W.F. Boron, M.A. Hediger, Expression cloning of a mammalian proton-coupled oligopeptide transporter, Nature 368 (1994) 563e566. [3] R. Liang, Y.J. Fei, P.D. Prasad, S. Ramamoorthy, H. Han, T.L. Yang-Feng, M.A. Hediger, V. Ganapathy, F.H. Leibach, Human intestinal Hþ/peptide cotransporter. Cloning, functional expression, and chromosomal localization, J. Biol. Chem. 270 (1995) 6456e6463. [4] H. Daniel, G. Kottra, The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology, Pflueg. Arch. Eur. J. Physiol. 447 (2004) 610e618. [5] D.E. Smith, C.E. Johanson, R.F. Keep, Peptide and peptide analog transport systems at the blood-CSF barrier, Adv. Drug Deliv. Rev. 56 (2004) 1765e1791. [6] S. Ramamoorthy, W. Liu, Y.Y. Ma, T.L. Yang-Feng, V. Ganapathy, F.H. Leibach, Proton/peptide cotransporter (PEPT 2) from human kidney: functional characterization and chromosomal localization, Biochim. Biophys. Acta 1240 (1995) 1e4. [7] T. Terada, K. Sawada, M. Irie, H. Saito, Y. Hashimoto, K. Inui, Structural requirements for determining the substrate affinity of peptide transporters PEPT1 and PEPT2, Pflugers Arch. 440 (2000) 679e684. [8] S.M. Ocheltree, H. Shen, Y. Hu, R.F. Keep, D.E. Smith, Role and relevance of peptide transporter 2 (PEPT2) in the kidney and choroid plexus: in vivo studies with glycylsarcosine in wild-type and PEPT2 knockout mice, J. Pharmacol. Exp. Ther. 315 (2005) 240e247. [9] U.V. Berger, M.A. Hediger, Distribution of peptide transporter PEPT2 mRNA in the rat nervous system, Anat. Embryol. 199 (1999) 439e449. ~ ez, D.E. Smith, SLC15A2 and SLC15A4 mediate [10] Y. Hu, F. Song, H. Jiang, G. Nun the transport of bacterially derived di/tripeptides to enhance the nucleotidebinding oligomerization domain-dependent immune response in mouse bone marrow-derived macrophages, J. Immunol. 201 (2018) 652e662. [11] A. Baroni, E. Buommino, V. De Gregorio, E. Ruocco, V. Ruocco, R. Wolf, Structure and function of the epidermis related to barrier properties, Clin. Dermatol. 30 (2012) 257e262. [12] T. Kawai, S. Akira, The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors, Nat. Immunol. 11 (2010) 373e384. [13] Y. Zhong, A. Kinio, M. Saleh, Functions of NOD-like receptors in human diseases, Front. Immunol. 4 (2013). Article 333. [14] H.D. de Koning, D. Rodijk-Olthuis, I.M. van Vlijmen-Willems, L.A. Joosten, M.G. Netea, J. Schalkwijk, P.L. Zeeuwen, A comprehensive analysis of pattern recognition receptors in normal and inflamed human epidermis: upregulation of dectin-1 in psoriasis, J. Investig. Dermatol. 130 (2010) 2611e2620. [15] M.H. Tervaniemi, S. Katayama, T. Skoog, H.A. Siitonen, J. Vuola, K. Nuutila, R. Sormunen, A. Johnsson, S. Linnarsson, S. Suomela, E. Kankuri, J. Kere, O. Elomaa, NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis, Sci. Rep. 6 (2016). Article 22745. [16] M. Kudo, T. Katayoshi, K. Kobayashi-Nakamura, M. Akagawa, K. Tsuji-Naito, H(þ)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport, Biochem. Biophys. Res. Commun. 475 (2016) 335e341. [17] S. Weidinger, N. Klopp, L. Rummler, S. Wagenpfeil, N. Novak, H.J. Baurecht, W. Groer, U. Darsow, J. Heinrich, A. Gauger, T. Schafer, T. Jakob, H. Behrendt, H.E. Wichmann, J. Ring, T. Illig, Association of NOD1 polymorphisms with atopic eczema and related phenotypes, J. Allergy Clin. Immunol. 116 (2005) 177e184. [18] M. Kabesch, W. Peters, D. Carr, W. Leupold, S.K. Weiland, E. von Mutius, Association between polymorphisms in caspase recruitment domain containing protein 15 and allergy in two German populations, J. Allergy Clin. Immunol. 111 (2003) 813e817. [19] I. Nomura, E. Goleva, M.D. Howell, Q.A. Hamid, P.Y. Ong, C.F. Hall, M.A. Darst, B. Gao, M. Boguniewicz, J.B. Travers, D.Y. Leung, Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes, J. Immunol. 171 (2003) 3262e3269. [20] Y. Skabytska, S. Kaesler, T. Volz, T. Biedermann, The role of innate immune signaling in the pathogenesis of atopic dermatitis and consequences for treatments, Semin. Immunopathol. 38 (2016) 29e43. [21] A. De Benedetto, R. Agnihothri, L.Y. McGirt, L.G. Bankova, L.A. Beck, Atopic dermatitis: a disease caused by innate immune defects? J. Investig. Dermatol. 129 (2009) 14e30. €lster-Holst, A. Baranowsky, M. Schunck, S. Winoto-Morbach, [22] J.M. Jensen, R. Fo C. Neumann, S. Schütze, E. Proksch, Impaired sphingomyelinase activity and epidermal differentiation in atopic dermatitis, J. Investig. Dermatol. 122 (2004) 1423e1431.
Declaration of competing interest No potential conflicts of interest were disclosed.
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide Michiko Kudo, Kumiko Kobayashi-Nakamura, Natsuko Kitajima, Kentaro Tsuji-Naito* DHC Corporation, Fundamental Research Laboratory, Division 2, 2-42 Hamada, Mihama-ku, Chiba, 261-0025, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2019 Accepted 5 November 2019 Available online xxx
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. © 2019 Elsevier Inc. All rights reserved.
Keywords: Keratinocytes Solute carrier 15 (SLC15) Oligopeptide transporter Glycylsarcosine (GlySar) Skin
1. Introduction Peptide transporters 1 and 2 (PEPT1 and PEPT2) are protoncoupled oligopeptide transporter (POT) members of the solute carrier 15 family and serve in cellular uptake of di/tri-peptides and peptidomimetics [1]. PEPT1 is predominantly expressed in the intestinal epithelium and functions for absorption of dietary nutrients [2,3]. PEPT2 is widely expressed in other tissues and has been reported to function in the kidney in reabsorption of dietary peptides [4,5]. These two peptide transporters have some differences in terms of substrate affinity and capacity. PEPT1 is characterized as low-affinity and high-capacity and therefore can transport large amounts of oligopeptides. In contrast, PEPT2 is characterized as high-affinity and low-capacity [6]. The differences in substrate affinity are due to different specificities for a- and b-amino carbonyl structures of the substrates [7]. Several recent studies have identified physiological functions of PEPT2 beyond its role in nutritional
* Corresponding author. E-mail address: [email protected] (K. Tsuji-Naito).
processes: in the choroid plexus for clearance of neuropeptide fragments from cerebrospinal fluid and in immune cells for uptake of bacterially derived peptides to trigger innate immune responses [8e10]. Recent research has therefore focused on the distinctive, tissue-specific functions of PEPT1 and PEPT2. As the outermost layer of the skin, the epidermis protects the body from the environment. Keratinocytes, which constitute the majority of cells in the epidermis, maintain immune homeostasis of the skin, together with epidermal dendritic cells [11]. Thus, keratinocytes act as a physical and immunological barrier. Innate immunity serves as the first-line defense against pathogenic invasion and detects infection through pattern recognition receptors (PRRs) [12]. PRRs are classified as membrane-bound or cytoplasmic-type PRRs on the basis of their location, including Toll-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), respectively. NOD1 and NOD2, which are members of the NLR family, detect specific bacterial peptidoglycan motifs in the host cytosol, leading to production of proinflammatory cytokines [13]. NOD1 is activated by peptidoglycan fragments containing the mesodiaminopimelic acid derived primarily from gram-negative and specific gram-positive bacteria. In contrast, NOD2 responds to
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Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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Abbreviations PEPT MDP SLC GlySar POT PRRs NOD NLRs IL Ntg Gbc
Peptide transporter Muramyl dipeptide Solute carrier Glycylsarcosine Proton-coupled oligopeptide transporter Pattern recognition receptors Nucleotide-binding oligomerization domain Nucleotide-binding oligomerization domain (NOD)like receptors Interleukin Nateglinide Glibenclamide
invasive bacteria by sensing peptidoglycan-related molecules containing muramyl dipeptide (MDP) that are produced by both gramnegative and gram-positive bacteria. Activation of the NOD signaling pathway induces the transcription factors nuclear factor-kappa B and activator protein-1, leading to enhancement of the production of proinflammatory cytokines (e.g., interleukin-6; IL-6, IL-8, and tumor necrosis factor-a). The NOD2 gene is implicated in the pathogenesis of several chronic inflammatory diseases [13]. Loss-offunction mutations in NOD2 are closely related to Crohn’s disease, and Blau syndrome and early-onset sarcoidosis are caused by gainof-function mutations. As for the epidermis, the NOD2 risk alleles are highly suggestive of several inflammatory diseases, including atopic eczema and dermatitis. Moreover, several research groups have demonstrated increased expression of inflammasome and innate immune receptors, including NOD2, in keratinocytes of psoriatic lesional skin [14,15]. Although accumulating evidence indicates a role for staphylococcal peptidoglycans and MDP in NLR signaling in keratinocytes, it is unknown how the NOD ligands enter the host cytoplasmic space. We previously showed that primary keratinocytes express PEPT2 more than PEPT1 [16]. But the possible variance of these two POT expressions during epidermal differentiation and also their role in innate immunity of the epidermis are still undefined. Thus, in this research we investigated the expression of PEPT1 and PEPT2 during differentiation and their role in NOD ligand-inducible immune responses using normal human epidermal keratinocytes (NHEKs). Additionally, we addressed two of the following points: 1) these two PEPTs are alternately expressed during differentiation in keratinocytes; and 2) they work together in facilitating the uptake of the bacterially derived peptide MDP into the cytosol of keratinocytes. These indicate that PEPT1 and PEPT2 are variably modulated in the epidermis to play a role in host defense responses. Our finding may provide a new insight into the cutaneous immune homeostasis. 2. Materials and methods 2.1. Materials Glycylsarcosine (GlySar) was purchased from Sigma-Aldrich (St. Louis, MO, USA). MDP was purchased from InvivoGen (San Diego, CA, USA). Nateglinide (Ntg) and glibenclamide (Gbc) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other chemicals were of the highest commercially available grade. 2.2. Cell cultures and human skin NHEKs (newborn/male, Thermo Fisher Scientific, Waltham, MA,
NHEKs KRT FLG qPCR RT-PCR PBS BSA DAPI FAAs LC-MS/MS MRM ELISA siRNA
Normal human epidermal keratinocytes Keratin Filaggrin Quantitative polymerase chain reaction Reverse transcription-polymerase chain reaction Phosphate-buffered saline Bovine serum albumin 4ʹ,6-Diamidino-2-phenylindole Free amino acids Liquid chromatography/tandem mass spectrometry Multiple reaction monitoring Enzyme-linked immunosorbent assay Small interfering RNA
USA) were maintained in keratinocyte growth medium consisting of EpiLife™ Medium, with 60 mM CaCl2 and human keratinocyte growth supplement (both from Thermo Fisher Scientific). Differentiation of NHEKs was induced by culturing them in MCDB 153 medium (Sigma-Aldrich) supplemented with 5 mg/ml insulin, 0.5 mg/ml hydrocortisone, 10 mg/ml transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 40 mg/ml bovine pituitary extract, and 1.35 mM CaCl2. Keratinocyte differentiation was confirmed based on morphologic changes and mRNA levels of keratin 5 (KRT5), KRT10, and filaggrin (FLG) determined by quantitative polymerase chain reaction (qPCR), as described below. Cells were grown in a humidified incubator at 37 C in an atmosphere containing 5% CO2. The medium was changed every other day, and the cells were passaged when they reached 80%e90% confluency. NHEKs were not used beyond passage 6. The medium was changed every day when cells reached 50% confluency. A human abdominal skin sample (40-year-old female, Caucasian) was purchased from Biopredic International (Rennes, France). This human experiment was approved by and followed the guidelines for the ethical use of human-origin organs and tissues of KAC (Kyoto, Japan), an alliance partner of Biopredic International. 2.3. Reverse transcription-polymerase chain reaction (RT-PCR) and qPCR Total RNA was isolated from cultured cells using an RNeasy Mini Kit (Qiagen, Mississauga, Canada), according to the manufacturer’s instructions. First-strand cDNA was synthesized with total RNA using a PrimeScript® II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan), according to the manufacturer’s instructions. For RTPCR gene expression studies, cDNA was mixed with KOD Plus (Toyobo, Osaka, Japan) as a DNA polymerase and gene-specific primers. The primers used were as follows: human PEPT1, 5ʹGGTAAAGTGGCCAAGTGCAT-30 and 5ʹ-CAAACAAGGCCCAGAACATT30 for a 193-bp fragment; human PEPT2, 5ʹ-TGACAGTGGTGGGAAATGAA-3ʹ and 5ʹ-TCCCATCTTCACGAATGACA-3ʹ for a 204-bp fragment; and human glyceraldehyde-3-phophate dehydrogenase (GAPDH), 5ʹ-GAGTCAACGGATTTGGTCGT-3ʹ and 5ʹTTGATTTTGGAGGGATCTCG-3ʹ for a 238-bp fragment. The PCR conditions on a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA) were as follows: at 98 C for 2 min; 30 cycles at 98 C for 10 s, at 60 C for 15 s, and at 72 C for 15 s; and finally at 72 C for 1 min. PCR products were separated on a 2% agarose gel. For qPCR, target-gene mRNA expression levels were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems) with the following TaqMan® Gene Expression Assays: FLG (assay ID Hs00856927_g1); KRT5 (assay ID Hs00361185_m1);
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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KRT10 (assay ID Hs00166289_m1); PEPT1 (assay ID Hs00192639_m1); PEPT2 (assay ID Hs01113665_m1); CXCL8 (assay ID Hs00174103_m1); and 18S (assay ID Hs99999901_s1). All reactions were performed in triplicate. 18S was used as a housekeeping gene for quantity normalization. The relative amounts of mRNA were calculated using the comparative CT method (2DDCt). The results are presented as fold change in each mRNA during keratinocyte differentiation. For absolute quantification of PEPT1 and PEPT2, the standard curve method was applied. The standard curves of PEPT1, PEPT2, and 18S were designed based on known quantities of synthetic DNA containing the specific sequences of each TaqMan® assay location disclosed by Applied Biosystems. 2.4. Immunohistochemical analysis For immunofluorescence histochemistry of PEPT1, a sample of human abdominal skin tissue was fixed to 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 C overnight. After rinsing with PBS, the tissue was immersed in 20% sucrose in PBS at 4 C overnight and then embedded in Tissue Freezing Medium (Leica Biosystems, Nussolch, Germany). Frozen tissues were cut in 7 mm slices with a cryostat. Sections were rinsed with PBS and subsequently treated with 0.1% Triton-X 100 in PBS for 30 min at room temperature. Nonspecific reactions were blocked by incubation of sections in 3% horse serum in PBS for 30 min. A polyclonal rabbit or anti-PEPT1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:150 in 1% bovine serum albumin (BSA) in PBS was applied overnight at 4 C. After washing in PBS, sections were incubated with Alexa Fluor 488 donkey anti-mouse IgG (Thermo Fisher Scientific) at a dilution of 1:400 in 1% BSA in PBS for 1 h at room temperature. Following a rinse in PBS, sections were treated with 1 mg/ml of 4ʹ,6-diamidino-2-phenylindole (DAPI) (Dojindo Laboratories, Kumamoto, Japan) in PBS for 3 min. After washing in PBS, sections were mounted using Fluoromount/Plus™ (Diagnostic BioSystems, Pleasanton, CA, USA). Fluorescence signals were examined with a Leica confocal fluorescence microscope (Leica Microsystems Japan, Tokyo, Japan). 2.5. Uptake measurements For the uptake studies, NHEKs were seeded at a density of 1.0 105 cells/dish on 60-mm plastic culture dishes. After a 7-day period for keratinocyte differentiation, the medium was changed to the new one adjusted to pH 6.0. NHEKs were preliminarily treated with Ntg, Gbc, or glycine (Gly) for 1 h and incubated with GlySar for an additional 1 h. The culture medium was removed from the dishes and the cells were washed three times with PBS. NHEKs were lysed using a lysis buffer (150 mM NaCl; 20 mM Tris, pH 7.5; 10 mM EDTA; 1% Triton-X; 1.0% sodium deoxycholate). Each extract was subjected to derivatization of free amino acids (FAAs) and GlySar. FAAs and GlySar were derivatized using the EZ:faast™ amino acid analysis kit (Phenomenex, Torrance, CA, USA), following the manufacturer’s instructions. Briefly, methionine-d3 was added as an internal standard to each cell extract, and FAAs and GlySar were derivatized using Sorbent Tips and extracted by the provided reagents from the EZ:faast™ kit. The GlySar content of the samples was determined by liquid chromatography/tandem mass spectrometry (LC-MS/MS), as described below. 2.6. GlySar measurement using LC-MS/MS The derivatives of FAAs and GlySar were detected and quantified by LC-MS/MS using selected ion monitoring in the positive-ion electron capture mode. MS was performed on an API 2000 tandem mass spectrometer (AB SCIEX, Tokyo, Japan) equipped with a
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standard API electrospray ionization source and interfaced with an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA). Samples were injected onto an EZ:faast™ AAA-MS column (2 250 mm) at a flow rate of 0.25 ml/min. The separation was performed with a two-pump gradient. Solvent A was 10 mM ammonium formate in water, and solvent B was 10 mM ammonium formate in water in methanol. The gradient program was as follows: 0 min, B 68%; 13 min, B 83%. Analyses were monitored in positive-ion mode using the API ion source at 400 C and with multiple reaction monitoring (MRM). Nitrogen was used as a curtain (setting 40) and collision (setting 7) gas. The derivatives of GlySar and methionine-d3 were eluted at 5 and 9 min, respectively, under these conditions. The following MRM transitions were selected: (m/z, Q1/Q3) of GlySar (m/z, 275 / 132) and methionine-d3 (m/z, 281 /193) derivatives. 2.7. Enzyme-linked immunosorbent assay (ELISA) For quantification of IL-8 protein levels, NHEKs were seeded at a density of 1.0 105 cells in six-well plates. After allowing the keratinocytes differentiate for 7 days, NHEKs were stimulated with 10 mg/ml of MDP in the absence or presence of Gbc or Gly. After culturing, the cell culture media were collected. Cytokine IL-8 secreted in the culture medium from NHEKs was quantified using a solid phase sandwich ELISA kit (Proteintech Group, Chicago, IL) according to the manufacturer’s instruction. 2.8. RNA silencing For the small interfering RNA (siRNA) studies, siRNAs targeting PEPT1 were purchased from Horizon Discovery (Cambridge, UK). Nontargeting siRNA (Horizon Discovery) was used as a negative silencing control. NHEKs were seeded at a density of 1.0 105 cells in six-well plates. After a 7-day period for keratinocyte differentiation, NHEKs were transfected with 20 nM of PEPT1 or scramble siRNA plus HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s instructions for mRNA silencing in the cells. The efficiency of siRNA-mediated repression of target mRNA levels was assessed by qPCR. 2.9. Statistical analyses Data are expressed as mean ± SE from at least three independent experiments. Statistical analyses were performed by the TukeyeKramer test. 3. Results and discussion We previously showed that PEPT2 is predominantly expressed within undifferentiated keratinocytes [16]. Using primary keratinocytes, we conducted RT-PCR analyses to clarify the variance of PEPT2 transcripts during differentiation. PEPT2 transcripts were strongly observed at the initial stage but declined with the progression of differentiation stage (Fig. 1A). In contrast, PEPT1 transcripts were significantly observed at the later stages (days 5e9). We speculated that, instead of PEPT2, PEPT1 was expressed in advanced differentiated keratinocytes. As shown in Fig. 1B, qPCR analyses showed that PEPT2 was highly expressed in the earlier stages similarly to KRT5 as a marker for basal and undifferentiated keratinocytes. The expression of PEPT1 was found to be inversely expressed in the later stages identical to the late markers of differentiation, such as KRT10 and FLG. In addition, absolute qPCR for PEPT1 and PEPT2 revealed that PEPT1 expression at the onset of differentiation was only less than one-fifth that of PEPT2, but the highest value of PEPT1 was reached at day 7 of differentiation and
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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Fig. 1. Peptide transporters 1 and 2 (PEPT1 and PEPT2) mRNA expression profiles during keratinocyte differentiation. Cell differentiation was induced by culturing in a culture medium containing a high concentration of calcium (see Materials and methods). (A) Total RNA samples from keratinocytes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using each specific primer. (B) Quantitative polymerase chain reaction (qPCR) analyses for PEPT1, PEPT2, and the indicated differentiation markers at various times (days) of calcium-induced differentiation. The results are presented as fold change in each mRNA. (C) Absolute qPCR for PEPT1 and PEPT2 at onset and day 7 of differentiation. Values represent the mean ± SE of triplicate determinations (**p < 0.01 versus the indicated group; TukeyeKramer test). KRT5, keratin 5; KRT10, keratin 10; FLG, filaggrin; GAPDH, glyceraldehyde-3phosphate dehydrogenase.
was more than 17 times higher than that of PEPT2 (Fig. 1C). The maximal value of PEPT1 at day 7 was approximately four times higher than that of PEPT2 at onset, suggesting that the outer layers express these transporters more than the inner ones. Our previous work immunohistochemically examined PEPT2 expression in human adult skin and showed that PEPT2 is localized in the epidermis, particularly in the basal layer [16]. Immunolocalization analysis of human skin revealed positive staining for PEPT1 in the epidermis, with the upper layers showing the strongest staining (Fig. 2), which suggested that keratinocytes alter the expression pattern of these two POTs from PEPT2 to PEPT1 in a stepwise manner during differentiation. Our previous work has shown the ability of undifferentiated keratinocytes to intracellularly absorb several oligopeptides, such as GlySar and collagen-derived peptides [16]. To investigate PEPT transport capacity in differentiated keratinocytes, we measured the cellular uptake of a synthetic dipeptide, GlySar, which is known as a substrate of PEPTs. After exposure of cultured differentiated keratinocytes to GlySar, we measured the intracellular levels of GlySar with the LC-MS/MS system. As shown in Fig. 3, we observed that GlySar uptake into keratinocytes was significantly blocked by the PEPT inhibitors Ntg and Gbc, but not by the amino
Fig. 2. Tissue localization of peptide transporter 1 (PEPT1) immunostaining in the human skin. Frozen sections were stained for PEPT1 (green) and DNA (blue). Bars, 50 mm. IgG, immunoglobulin G; DAPI, 40 ,6-diamidino-2-phenylindole.
Fig. 3. Effect of peptide transporter 1 (PEPT1) on glycylsarcosine (GlySar) uptake in differentiated keratinocytes. After a 7-day period for keratinocyte differentiation, cells were preliminarily treated with nateglinide (Ntg), glibenclamide (Gbc), or glycine (Gly) at the indicated concentrations for 1 h and incubated with GlySar for 1 h, after which GlySar concentrations in cell extracts were measured. Values represent the mean ± SE of triplicate determinations (*p < 0.05, **p < 0.01 versus control; TukeyeKramer test).
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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acid Gly. This result indicated that oligopeptides can be transported by PEPTs in advanced differentiated keratinocytes. MDP is a degradation product of bacterial peptidoglycan that triggers innate inflammatory responses upon ligand binding to NOD2 present in the cytosol [13]. MDP increased the expression of
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proinflammatory cytokine IL-8 mRNA and protein in cultured differentiated keratinocytes (Fig. 4A and B). These results indicate that MDP accesses the cytosol to activate NOD2 for IL-8 production. Based on gene expression in keratinocytes, we hypothesized that PEPT1 and 2 play a causative role in NOD2-mediated immune
Fig. 4. Effect of peptide transporter 1 (PEPT1) on the muramyl dipeptide (MDP)-stimulated immune response in differentiated keratinocytes. (A, B) Normal human epidermal keratinocytes (NHEKs) were treated for the indicated number of hours with 10 mg/ml MDP. (A) The expression levels of CXCL8 were quantified by quantitative polymerase chain reaction (qPCR). (B) Interleukin-8 (IL-8) concentrations in the cell culture media were measured by enzyme-linked immunosorbent assay (ELISA). Values represent mean ± SE of triplicate determinations (*p < 0.05, **p < 0.01 vs. hour 0; TukeyeKramer test). (C, D) For mRNA and protein expression analyses of IL-8, NHEKs were stimulated with 10 mg/ml MDP in the absence or presence of glibenclamide (Gbc) or glycine (Gly) for 2 and 8 h, respectively. (E) NHEKs were transfected with the indicated small interfering RNA (siRNA) (20 nM) plus HiPerFect Transfection Reagent for 48 h, after which the cells were incubated for 24 h. After incubation, NHEKs were stimulated with 10 mg/ml MDP for 2 h. Values represent mean ± SE of triplicate determinations (*p < 0.05, **p < 0.01 versus the indicated group; TukeyeKramer test). N.S., not significant.
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044
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responses by MDP. Thus, to clarify whether POTs mediate entry of MDP into the cytosol of keratinocytes, we investigated the influence of the PEPT inhibitor Gbc on IL-8 production by MDP. As shown in Fig. 4C and D, Gbc significantly suppressed MDP-induced IL-8 production at both the mRNA and the protein levels in cultured differentiated keratinocytes. To confirm the role of POTs in MDPNOD2 signaling, we next used siRNA against PEPT1. The siRNA knockdown of PEPT1 significantly reduced PEPT1 expression, but has no off-target effect on PEPT2 (Fig. 4E). We confirmed the involvement of PEPT1 in MDP-mediated IL-8 induction in keratinocytes followed by cultured differentiation. In anaplastic keratinocytes, treatment with siRNA against PEPT2 reduced MDPinducible IL-8 transcripts as well (data not shown). Taken together, these results suggest that PEPT1 and PEPT2 may have an important role in MDP-NOD2 signaling in the epidermis. In this study, we have shown that keratinocytes undergo a switch from PEPT2 to PEPT1 expression in the differentiation process. The entry of MDP into the keratinocytes is shown to be accomplished through both of these transporters, suggesting that PEPT1 and PEPT2 concertedly contribute to skin immune homeostasis via NOD-like receptor signaling pathways. The question remains why their expression pattern changes during keratinocyte differentiation. We speculate that the higher transporting capacity of PEPT1 compared with PEPT2 may provide a dedicated platform for protection against invasion by various pathogens. Indeed, differentiated keratinocytes showed a higher response against MDP than did proliferating keratinocytes, although there was no difference between differentiated and proliferating keratinocytes in NOD2 expression (data not shown). Taking into consideration the fact that the differences in sensitivity to MDP were well correlated with the expression pattern of PEPT1 in the process of differentiation, the entry of MDP into host cytosol via PEPTs is likely to affect the intensity of the immune response as one of the rate-limiting steps. In addition, because unlike the dermis, there are no blood vessels in the epidermis, these transporters in the epidermis are likely to work in a concerted manner for nutrient absorption of peptide-bound amino acids as well. PEPT1 and PEPT2 are differentially expressed in the proximal tubules of the kidney, which work to efficiently reabsorb oligopeptides from the tubular fluid. The upper layers of the epidermis are more distant from the blood vessels than are the basal layers and therefore require the high capacity of PEPT1 for nutritional absorption. In fact, these two transporters are abundantly expressed in keratinocytes but are hardly expressed in fibroblasts from the human dermis [16]. Impairments of NLR signaling are recognized risk factors for several chronic inflammatory diseases, including atopic dermatitis [13e15]. NOD1 and NOD2 polymorphisms have been related to elevated IgE levels and atopic dermatitis [17,18]. In addition, skin lesions of atopic dermatitis have been reported to exhibit decreased innate immune responses, including expression of b-defensin and IL-8 [19e21]. Aberrancy in epidermal differentiation is often linked to the disturbed barrier function observed in atopic dermatitis [22]. Thus, change in keratinocyte differentiation in atopic dermatitis may also affect susceptibility to bacteria-derived muramyl peptides through unbalanced expression of PEPT1 and PEPT2, leading to change in the epidermal immune system. Our results show that the expression pattern of PEPTs is tightly regulated during differentiation. These findings provide new insights into the understanding of skin innate immunity.
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Declaration of competing interest No potential conflicts of interest were disclosed.
Please cite this article as: M. Kudo et al., Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.044