Extracellular processing of proopiomelanocortin generates short beta endorphin that regulates rat keratinocytes via the delta opioid receptor

Abstract

Proopiomelanocortin (POMC), a precursor with multiple bioactive peptides, is expressed by keratinocytes and regulates various pathophysiological responses, including pruritus associated with atopic dermatitis (AD). In the skin, POMC is extracellularly processed into peptides like α-melanocyte-stimulating hormone (α-MSH); however, the processing and functional role of β-endorphin (β-END) remain unclear. Here, we investigated the molecular form and biological activity of β-END generated in the context of AD. We analyzed skin extracts from AD lesions using immunoprecipitation and MALDI-TOF MS, identifying β-END(1–9), a truncated peptide, as a major derivative. To explore opioid receptor expression in fetal rat skin keratinocytes (FRSK) using RT-PCR. Delta opioid receptor (DOR) was detected in FRSK cells. Functional assays showed β-END(1–9) reduced cAMP levels via DOR signaling, acting as a full agonist with about half the potency of methionine-enkephalin. To further explore its biological effects, DNA microarray analysis was conducted on β-END(1–9)-treated FRSK cells. Differential gene expression analysis revealed modulation of genes involved in collagen maturation and hyaluronic acid synthesis, suggesting roles in skin barrier function. Collectively, these findings suggest that POMC is processed into β-END(1–9) in atopic-inflammatory skin, and this peptide acts via DOR expressed in keratinocytes to promote the expression of skin-protective factors.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-31279-5.

Introduction

The biologically active peptide β-endorphin (β-END), such as other peptide hormones, is derived from its precursor proopiomelanocortin (POMC) via proteolytic cleavage by specific prohormone convertases (PCs), primarily PC1/3 and PC2^1,2. POMC is a well-characterized precursor that undergoes tissue-specific processing to yield multiple biologically active peptides, with its biosynthetic pathways and regulatory mechanisms having been extensively studied. In the anterior pituitary, expression of PC1/3 promotes the cleavage of POMC into adrenocorticotropic hormone (ACTH) and β-lipotropin. In contrast, in the intermediate lobe of the pituitary and the hypothalamus—where both PC1/3 and PC2 are expressed—POMC is further processed into α-melanocyte-stimulating hormone (α-MSH) and β-END, among other peptides.

While α-MSH in the skin contributes to pigmentation and originates from locally produced POMC, its activation mechanism differs from that in the pituitary³. In keratinocytes and fibroblasts, the expression levels of PC1/3 and PC2 are significantly lower compared with that in the pituitary tissues, resulting in limited intracellular processing. Consequently, POMC is released as an intact precursor in the extracellular environment. A previous study indicated that the POMC to α-MSH ratio in the skin is approximately 40:1⁴. Once secreted, extracellular POMC undergoes proteolytic cleavage by skin-associated proteases to generate bioactive peptides, such as α-MSH(1–8), a truncated peptide corresponding to amino acids 1–8 of α-MSH, which stimulates melanogenesis via the melanocortin 1 receptor³. Notably, the generation of α-MSH(1–8) is absent in mast cell-deficient mice, suggesting a role for mast cell-derived tryptase in this extracellular processing event. In addition to α-MSH, POMC encodes other functional peptides, notably β-END—a well-conserved opioid peptide across species. However, the precise mechanism of β-END activation in the skin remains insufficiently understood.

Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disorder characterized by intense pruritus and epidermal barrier dysfunction, which facilitates the penetration of allergens and irritants, leading to inflammation. Stress is a well-documented trigger for AD exacerbations and is associated with dysregulation of neuroimmune pathways, including the release of stress-related peptides such as those derived from POMC^5,6. These peptides modulate multiple pathological aspects of AD, including pruritus and pigmentation.

For example, α-MSH contributes to pruritus in AD via thromboxane A₂⁷ and promotes hyperpigmentation in lesional skin⁸. Similarly, β-END and other opioid peptides have been implicated in the modulation of itch in AD⁹. The roles of their respective receptors are distinct: the μ-opioid receptor (MOR) is associated with itch induction¹⁰, while the κ-opioid receptor (KOR) mediates itch suppression¹¹.

In this study, we aimed to investigate whether β-END-like peptides, similar to α-MSH, are generated in atopic-inflammatory skin. We further assessed whether the resulting peptide fragments bind to specific receptors and exert biological effects, focusing on the functional role of β-END in skin homeostasis and inflammation.

Results

Structural analysis of β-END-like immuno-active components produced in atopic inflammatory skin

β-END-like peptides were extracted from the skin of AD-induced mice using immunoprecipitation and analyzed using MALDI-TOF MS. An anti-β-END(1–9) antibody, which selectively recognizes the N-terminal active site of β-END, was employed for immunoprecipitation, given that this region harbors its bioactivity. This resulted in detection of a peptide fragment with a molecular weight of 1019.687 Da (Fig. 1A,B). In the skin, extracellular processing of POMC is regulated by tryptase, an enzyme with trypsin-like specificity, suggesting its involvement in generation of active peptides. The processing of POMC by tryptase is predicted to yield β-END(1–9), based on the immunoprecipitation antibody’s specificity. This predicted sequence aligns with the observed molecular weight from mass spectrometry (Fig. 1C). The identified cleavage sites at the C-terminal ends of lysine and arginine residues are consistent with the cleavage specificity of tryptase, which is also known to generate α-MSH(1–8) through similar mechanism. To confirm the identity of the β-END-like immunoreactive component in the AD skin extract, RP-HPLC was performed. The retention time of the immunoreactive component matched that of the synthetic β-END(1–9) standard (Fig. 1D–F), supporting the conclusion that the β-END-like peptide isolated from inflamed skin is indeed β-END(1–9).

Fig. 1.

Detection of β-END(1–9) in atopic dermatitis lesions. Detection of peptide fractions in affinity extracts from AD lesions (A) and non-inflammatory skin (B) using MALDI-TOF MS. The amino acid sequence of β-END is shown (C), with the basic amino acids recognized by tryptase highlighted in red. β-END(1–9) is the sequence cleaved at the C-terminal of the basic amino acids. The retention time of β-END(1–9) was examined by separating AD skin extracts using RP-HPLC. Absorbance at 215 nm during the separation of tissue extracts (D), β-END(1–9)-like immunoreactive components detected by ELISA (E), and absorbance at 215 nm during the separation of synthetic β-END(1–9) (F). AD, atopic dermatitis; β-END, β-endorphin; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; HPLC, high-performance liquid chromatography; RP, reverse phase; ELISA, enzyme-linked immunosorbent assay.

Exploration of opioid receptors expressed in epidermal keratinocytes

Opioid receptors are traditionally classified into three subtypes: mu (MOR), delta (DOR), and kappa (KOR). To investigate their expression in skin keratinocytes, RT-PCR was performed using specific primers for each receptor subtype (Fig. 2A). Among them, only the DOR was detected in keratinocytes. The identity of the RT-PCR product was verified through DNA sequencing, which confirmed complete homology with the rat DOR sequence. No detectable expression of MOR or KOR was observed under these experimental conditions. To validate DOR protein expression in keratinocytes, western blot analysis was performed (Fig. 2B). A specific band was observed at approximately 50 kDa, consistent with the expected molecular weight of DOR and matching the positive control derived from brain tissue extract. In addition, RT-PCR analysis of the human keratinocyte cell line HaCaT revealed detectable expression of MOR, DOR, and KOR (Supplementary Fig. 1). Furthermore, the expression of opioid receptors in the skin of NC/Nga mice exhibiting atopic dermatitis-like symptoms was examined by RT-PCR (Supplementary Fig. 2). Both MOR and DOR were expressed in mouse skin; however, no significant differences in expression levels were observed between inflamed and non-inflamed regions.

Fig. 2.

Expression of opioid receptors in FRSK cells. RT-PCR analysis of opioid receptor expression in FRSK cells (A). Lane 1, DNA markers; lane 2, MOR; lane 3, DOR; lane 4, KOR; lane 5, β-actin. Western blot analysis confirming the expression of DOR and β-actin in FRSK cells (B). Mouse brain tissue extracts were used as a positive control and β-actin was used as an internal standard. FRSK, fetal rat skin keratinocyte; RT-PCR, reverse transcription polymerase chain reaction; MOR, mu-opioid receptor; DOR, delta-opioid receptor; KOR, kappa-opioid receptor.

To assess whether the DOR expressed in keratinocytes is functionally active, intracellular cAMP levels were measured in response to receptor stimulation. The intracellular cAMP concentration under forskolin stimulation was defined as 100%, and that under non-stimulated conditions as 0%; the results are presented in Fig. 3. Stimulation with forskolin led to a robust increase in intracellular cAMP concentration. However, co-treatment with β-END, Met-ENK, or β-END(1–9) attenuated this forskolin-induced increase in cAMP concentration in a dose-dependent manner. Notably, β-END(1–9) exhibited a half maximal effective concentration (EC₅₀) of approximately 44 nM, while Met-ENK had a slightly higher potency, with an EC₅₀ of approximately 25 nM. These findings demonstrate that DOR is not only expressed in keratinocytes but is also functionally active, and that β-END(1–9) acts via DOR to suppress intracellular cAMP levels.

Fig. 3.

Structure–activity relationship of β-END analogue on FRSK cells. The inhibitory effects of β-END analogue on DOR activation in FRSK cells were evaluated by measuring intracellular cAMP levels. Values are presented as the mean ± SD from at least three independent experiments. The cAMP level in response to forskolin stimulation was defined as 100%, while the basal cAMP level under non-stimulated conditions was defined as 0%. β-END, beta-endorphin; DOR, delta-opioid receptor; FRSK, fetal rat skin keratinocyte; cAMP, cyclic adenosine monophosphate; SD, standard deviation.

Effects of β-END(1–9) via DOR on keratinocytes

To examine the downstream effects of β-END(1–9) and Met-ENK on keratinocytes, gene expression profiling was performed using DNA microarray analysis. Genes exhibiting more than a two-fold increase or a reduction to less than half of baseline expression were considered significant (Table S1). Among the genes upregulated in response to both β-END(1–9) and Met-ENK, Meprin1α was identified as a key factor associated with skin barrier protection and regeneration. Conversely, SMPD2, a gene implicated in ceramide metabolism and potential skin barrier disruption, was found to be consistently For validating the microarray results, quantitative RT-PCR was conducted to assess the expression levels of Meprin1α and SMPD2 following treatment with 100 nM of either β-END(1–9) or Met-ENK. The RT-PCR results confirmed the microarray findings: both peptides significantly upregulated Meprin1α expression (Fig. 4A) and suppressed SMPD2 expression (Fig. 4B). These findings suggest that β-END(1–9), through activation of DORs, modulates gene expression in keratinocytes in a manner that may contribute to skin homeostasis by promoting protective factors and downregulating pathways associated with barrier impairment.

Fig. 4.

Real-time PCR analysis of meprin1a and smpd2 mRNA expression in FRSK cells following stimulation with Met-ENK and β-END(1–9). mRNA expression levels of meprin1a (A) and smpd2 (B) were quantified by real-time RT-PCR. Expression levels were normalized to β-actin. Data are presented as mean ± SD from four biological replicates. *p < 0.05, Dunnett’s t-test compared with none-treated group. RT-PCR, reverse transcription polymerase chain reaction; Met-ENK, methionine-enkephalin; β-END, beta-endorphin; FRSK, fetal rat skin keratinocyte; SD, standard deviation.

Discussion

POMC is a multifunctional precursor peptide that gives rise to various biologically active peptides, with its physiological roles modulated by tissue-specific enzymatic processing mechanisms^1,2. In this study, we identified a novel skin-specific processing pathway for POMC, distinct from the canonical PC-mediated maturation, leading to the generation of the bioactive peptide β-END(1–9). This peptide was shown to play a pivotal role in maintaining skin homeostasis via activation of the DOR in keratinocytes.

Our findings demonstrated that β-END(1–9) is produced in the skin under atopic-inflammatory conditions. In classical endocrine tissues like the anterior pituitary, POMC is cleaved by PC1/3 to produce ACTH and β-lipotropin, while in the posterior pituitary, POMC undergoes cleavage by both PC1/3 and PC2, leading to the production of α-MSH and β-END. The β-END produced in the pituitary is released extracellularly as acetylated (Ac)-β-END, where the tyrosine at position 1 is acetylated by N-terminal acetyltransferase. However, our results suggest an alternative enzymatic pathway being active in the skin. The β-END(1–9) detected in this study had a molecular weight consistent with that of non-acetylated β-END(1–9), as confirmed by MALDI-TOF mass spectrometry. This finding suggests that the β-END(1–9) generated through the extracellular activation mechanism in the skin is not a derivative of Ac-β-END, which is typically formed via classical intracellular maturation and N-terminal acetylation. Rather, the molecule is generated by extracellular cleavage of POMC by a trypsin-like protease, likely tryptase.

Previous studies have reported that β-END can also be produced in the skin. Our present results do not contradict the existence of this conventional β-END production pathway. Similar to β-END, α-MSH, another peptide produced from POMC through classical processing, has been reported to be produced in keratinocytes. Rousseau et al. reported that intracellular concentrations of POMC and α-MSH in keratinocytes are nearly equivalent, whereas in the culture medium, POMC is present at high levels and α-MSH is only minimally detectable⁴. These findings suggest that in the skin, β-END may be produced through a cooperative mechanism involving both the classical intracellular processing pathway and a novel extracellular activation process, in which POMC released as a precursor is converted into β-END(1–9) outside the cell. The difference between these processing mechanisms results in distinct molecular species—β-END and β-END(1–9)—that exhibit differential receptor selectivity for MOR and DOR, respectively. This receptor shift may be of physiological significance, allowing the same precursor, POMC, to regulate diverse biological processes through different signaling pathways. In our study, β-END(1–9) was found to activate the DOR, demonstrating distinct receptor selectivity compared to full-length β-END. Sequence homology analysis revealed that β-END(1–9) corresponds to the N-terminal fragment of β-END(1–31), sharing its first five amino acids with Met-ENK (Fig. 5). Although both β-END(1–31) and Met-ENK are categorized as endogenous opioid peptides, they differ in receptor affinity: β-END(1–31) is the primary ligand for MOR, while Met-ENK preferentially activates DOR. Receptor selectivity is thought to be influenced by the peptide’s C-terminal sequence. β-END(1–9), produced via the novel extracellular processing mechanism described in this study, is shorter than full-length β-END but possesses a four-residue C-terminal extension relative to Met-ENK. This structural feature may confer enhanced affinity or selectivity for DOR while reducing activity at MOR. Although previous reports have suggested that β-END(1–9) can bind MOR, its potency is markedly lower than that of the full-length peptide. Moreover, the expression of Met-ENK in the skin of patients with atopic dermatitis has not been reported. Together, these findings suggest that the unique enzymatic processing of POMC in atopic dermatitis skin generates a functionally distinct peptide, β-END(1–9), which preferentially activates DOR and may play a skin-specific regulatory role in inflammation and homeostasis.

Fig. 5.

Amino acid sequences of β-END analogues. The sequences of β-END and Met-ENK were referenced from NP_647542 and A61445, respectively. Met-ENK, methionine-enkephalin; β-END, beta-endorphin.

In the skin, a subset of POMC undergoes processing similar to that observed in the pituitary intermediate lobe, leading to the production of α-MSH and β-END. These peptides primarily act through the melanocortin 1 receptor (MC1R) and MOR, respectively. However, skin-specific processing by trypsin-like enzymes such as tryptase results in the generation of shorter peptide fragments, including α-MSH(1–8) and β-END(1–9). Notably, α-MSH(1–8) exhibits approximately 30-fold lower activity compared to full-length α-MSH³, and β-END(1–9) functions as a partial agonist at MOR¹² but serves as a full agonist at the DOR. These findings suggest that POMC processing in the skin diverges from classical pathways, shifting receptor engagement from MC1R and MOR to DOR. This highlights the functional versatility of a single precursor peptide, capable of generating multiple biologically active fragments with distinct receptor specificities and physiological roles. While previous studies have reported the expression of MOR and KOR in human-derived keratinocyte cell lines such as HaCaT, the presence of DOR in skin keratinocytes has not been thoroughly investigated. In the present study, we demonstrated that HaCaT cells express not only MOR and KOR but also DOR. Furthermore, we showed that FRSK cells express DOR; whereas MOR and KOR expression was undetectable in FRSK cells, which may reflect species-specific differences, developmental stage at the time of cell isolation, or variations in keratinocyte differentiation.

Using FRSK cells that express DOR, we evaluated their responsiveness to endogenous ligands such as Met-ENK and β-END(1–9). Our findings confirmed that DOR is functionally expressed in skin keratinocytes and is activated by these ligands. DNA microarray analysis further revealed that stimulation with Met-ENK or β-END(1–9) led to upregulation of Meprin1α and downregulation of SMPD2.

Meprin1α plays a key role in collagen maturation by digesting procollagen to generate mature collagen. Type IV collagen and type VII collagen, which are found in the epidermal and basal layers where keratinocytes reside, are crucial for skin structure¹³. Type VII collagen, produced by keratinocytes and fibroblasts, serves as anchoring fibers that link the basement membrane to the dermis. A decrease in type VII collagen has been associated with both physiological aging and photoaging, contributing to skin aging. Additionally, Meprin1α regulates collagenase MMP-1 activity, reducing collagen degradation. Thus, the upregulation of Meprin1α in response to atopic inflammation likely contributes to the maintenance of collagen levels in the skin, influencing skin homeostasis^13,14. Moreover, β-END(1–9) suppressed the expression of SMPD2, an enzyme involved in sphingomyelin degradation and ceramide production. Sphingolipids, including sphingomyelin, are vital for maintaining the skin barrier and preventing transepidermal water loss. Ceramide, a key component of extracellular lipids in the stratum corneum, regulates keratinocyte proliferation, differentiation, and apoptosis¹⁵. Abnormalities in sphingolipid metabolism, such as those observed in psoriatic lesions, have been reported, suggesting a potential role for SMPD2 in the pathophysiology of skin disorders¹⁶. The suppression of SMPD2 by β-END(1–9) may help to modulate sphingolipid metabolism, contributing to skin barrier function and cellular processes involved in keratinization.

The involvement of opioid receptors in pruritus associated with atopic dermatitis (AD) has been reported previously. Tominaga et al. demonstrated the expression of MOR and KOR in human epidermal keratinocytes and showed that truncated β-endorphin levels specifically increase in AD lesions⁹. In addition to ligand alterations, changes in receptor expression induced by inflammation and immune responses also warrant consideration. Epidermal expression levels of opioid receptors have been shown to fluctuate in patients with AD and to recover following effective treatment⁹. Functionally, MOR and KOR exert opposing effects on pruritus: activation of MOR enhances itch sensation, whereas KOR activation suppresses it. In patients with AD, reduced KOR expression leads to a relative predominance of MOR signaling, thereby contributing to exacerbated pruritus. Similar opioid-mediated mechanisms have also been implicated in the pruritus associated with psoriasis and uremia^17–19. Our present findings suggest that β-END(1–9)-mediated activation of DOR represents an additional regulatory axis within the cutaneous opioid system. This pathway may contribute to epidermal homeostasis by modulating the production and degradation of keratinocyte-derived protective factors, thereby providing a novel mechanism for maintaining skin integrity under inflammatory conditions.

This study has some limitations. The biological activity of the β-END(1–9) fragment was examined using rat-derived keratinocytes. Previous studies have shown that MOR and KOR are expressed in mouse and human epidermis, where they play distinct roles in the regulation of pruritus. As our experiments were performed in a different species and in a cultured cell system, receptor expression patterns may not fully reflect those in human or mouse skin. Therefore, potential interactions of β-END(1–9) with MOR- or KOR-mediated signaling pathways remain to be clarified. Furthermore, the in vivo analyses in this study were conducted using a mouse model of atopic dermatitis-like inflammation, which may not completely recapitulate the complex pathophysiological mechanisms of human atopic dermatitis. Future investigations using human skin samples or primary keratinocytes will be necessary to validate the physiological relevance of the β-END(1–9)—DOR axis identified here.

In conclusion, our findings suggest that in atopic dermatitis lesions, β-END(1 − 9), generated from POMC, plays a key role in maintaining skin homeostasis by regulating collagen maturation and sphingomyelin metabolism through DOR expressed in keratinocytes. This novel activation mechanism highlights the complexity of peptide processing and its impact on skin function and pathology.

Materials and methods

Experimental animals and sample preparation

All animal experiments were approved by the Animal Care and Use Committee of Nihon Pharmaceutical University (Approval No. 201915) and were conducted in accordance with the university’s Guidelines for the Care and Use of Laboratory Animals. The study protocol complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All methods were carried out in accordance with relevant institutional regulations. Male NC/Nga mice (8 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and housed under standard laboratory conditions (temperature: 23 ± 1 °C, humidity: 55 ± 5%) with ad libitum access to food and tap water. A 12-h light–dark cycle was maintained, with lights on at 8:00 and off at 20:00. To induce atopic dermatitis-like skin inflammation, house dust mite antigens (Institute of Tokyo Environmental Allergy, Tokyo, Japan) were topically applied to the auricular and dorsal regions. Sensitization was performed four times at 3-day intervals. Three weeks after the final sensitization, ear thickness (Supplementary Fig. 3A), and scratching behavior (Supplementary Fig. 3B) were measured as indicators of inflammation to confirm the development of atopic-like symptoms. For sample preparation, the auricles were excised from mice euthanized by exsanguination under isoflurane anesthesia and immediately boiled in 5 mL of 0.1 M acetic acid for 10 min. The tissues were then cooled on ice, homogenized in 0.1 M acetic acid, and centrifuged at 10,000 rpm for 30 min. The resulting supernatant was desalted using a Sep-Pac C18 column (Waters, Milford, MA), followed by elution with 0.1% trifluoroacetic acid (TFA)/acetonitrile (1:2, v/v). The eluates were lyophilized and stored at − 80 °C until further use for immunoprecipitation or high-performance liquid chromatography (HPLC) analysis.

Cell culture

The FRSK and HaCaT cell lines were used in this study. FRSK cells were obtained from the Health Science Research Resources Bank (HSRRB, Japan), and HaCaT cells were kindly provided by the German cancer research center (DKFZ, Germany). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS) (Moretate Biotech, Bulimba, Australia). Cultures were maintained in a humidified incubator at 37 °C with 5% CO₂ and 95% air. When cells reached approximately 80% confluency, they were detached using 0.05% (w/v) trypsin in phosphate-buffered saline (PBS) and harvested for further analysis.

Affinity chromatography by immunoprecipitation

Skin extracts were subjected to immunoprecipitation by incubation with antisera against β-END for 24 h. The resulting immune complexes were precipitated using Protein A-Sepharose CL-4B (GE Healthcare, Chicago, IL, USA), followed by washing with 0.5 M NaCl in PBS. Immunoprecipitates were then eluted by boiling in 0.1% TFA.

Analytical reverse-phase HPLC (RP-HPLC)

Analytical RP-HPLC was performed using a SLC-6B system (Shimadzu, Kyoto, Japan) equipped with an SPD-7A detector. Desalted crude auricle extracts (via Sep-Pak C18 column) were loaded onto an ODS column (4.6 × 150 mm, Mightysil RP-18 GP 5 µm; Kanto Chemical Co., Inc., Tokyo, Japan). A linear gradient elution was carried out using acetonitrile in 0.1% TFA, increasing from 0 to 60% over 40 min at a flow rate of 1 mL/min. The fractionated samples were lyophilized and then reconstituted in the standard diluent for enzyme-linked immunosorbent assay (ELISA) targeting β-END(1–9), a truncated peptide corresponding to amino acids 1–9 of β-END.

ELISA for β-END(1–9)

β-END(1–9) levels were quantified using an ELISA kit for Methionine-Enkephalin (Met-ENK) (Cusabio, Houston, TX, USA). The Met-ENK ELISA kit used in this study exhibits 100% cross-reactivity with β-END(1–9) (Supplementary Fig. 4).

Mass spectrometric analysis

Immunoprecipitated extracts were desalted using a ZipTipC18 peptide tip (Millipore, Billerica, MA, USA) and eluted with 0.1% TFA/acetonitrile (1:2, v/v). The eluent was mixed with an equal volume of saturated α-cyano-4-hydroxycinnamic acid (α-CHCA; Sigma-Aldrich, St. Louis, MO, USA) in the same solvent mixture. The sample was then applied to an MALDI target plate and air dried. MS experiments were carried out on a Bruker Autoflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).

Expression of opioid receptors mRNA

Total RNA was extracted from FRSK cells, HaCaT cells and NC/Nga mice skin using TriPure Isolation Reagent (Roche Life Science). Genomic DNA was removed with DNase I, and complementary DNA (cDNA) was synthesized using ReverTra Ace (Toyobo, Osaka, Japan) following the manufacturer’s instructions. PCR amplification was performed using Quick Taq^® HS DyeMix (Toyobo). Specific forward and reverse primers were designed to amplify the following genes: rat MOR (5′-TTAGCGACCAGTACACTGCC-3′ and 5′-ACCGGCATGATGAAAGCGAA-3′); rat DOR (5′-GCCTCAACCCGGTTCTCTAC-3′ and 5′-GTGGCCACTGCATCACTTCA-3′); rat KOR (5′-CACCAAAGTCAGGGAAGATGTG-3′ and 5′-ACTCTATTGGTGCTCTGGCG-3′); rat β-actin (5′-TGACAGGATGCAGAAGGAGA-3′ and 5′-CATCTGCTGGAAGGTGGACA-3′); human MOR (5′-CCCTTAGCTCCTGCAAGTTG-3′ and 5′-TCTGCCAGAGCAAGGTTGAA-3′); human DOR (5′-TGGCCGTTTGGCGAGCTGCTG-3′ and 5′-CTTGGCACTCTGGAAAGGCA-3′); human KOR (5′-CAAGATTGTCATTTCCATTG-3′ and 5′-CGATCTTTCTCTCGGGAGCC-3′); human β-actin (5′-GCGGGAAATCGTGCGTGACATT-3′ and 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′); mouse MOR (5′-CCTTCCATGGTCACAGCCAT-3′ and 5′-TGCAGAAGTGCCAGGAAACA-3′); mouse DOR (5′-CCCATCATGGTCATGGCAGT-3′ and 5′-CGTTTAAGGGGAAGGTCGGG-3′); mouse KOR (5′-CTGTGTGCCACCCTGTGAAA-3′ and 5′-AGAAGTCCCTAAAACACCGCT-3′) and mouse β-actin (5′-AACCCTAAGGCCAACCGTGAAAAG-3′ and 5′-CGACCAGAGGCATACAGGGACAGC-3′). The PCR conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation 94 °C for 1 min, annealing at 58–62 °C for 1 min, and extension at 72 °C for 1 min. PCR products were analyzed by electrophoresis on a 1.2% agarose gel containing ethidium bromide and visualized under UV illumination. The integrity of the RNA and specificity of the amplification were confirmed by amplifying β-actin and sequencing the PCR products. DNA bands were excised from the gel and purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Purified DNA was submitted to FASMAC (Kanagawa, Japan) for sequencing using the Sanger (dideoxy) method.

Western blotting analysis

Cell extracts were reduced with β-mercaptoethanol and heated at 95 °C for 10 min. Equal amounts of protein (10 μg/lane) were separated on a 12% sodium dodecyl sulfate–polyacrylamide gel²⁰. Following electrophoresis, proteins were transferred to a nitrocellulose membrane (ClearTrans; Fujifilm Wako Pure Chemical) using a semi-dry blotting system (NA-1512S; Nihon Eido, Tokyo, Japan)²¹. Membranes were blocked with 2% skim milk in Tris-based saline (pH 7.4) and incubated with rabbit anti-DOR antibody (1:1000; Bioss, Boston, MA, USA), or mouse anti-β-actin monoclonal antibody (1:10,000; Fujifilm Wako Pure Chemical). After washing, membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1:10,000; Seracare, Milford, MA, USA) or HRP-conjugated goat anti-mouse IgG antibody (1:2500; Biosource, Camarillo, CA, USA). Immunoreactive bands were visualized using the ImmunoStar Zeta chemiluminescent substrate (Fujifilm Wako Pure Chemical) and detected with a LuminoGraph imaging system (Atto, Tokyo, Japan).

Structural-activity relationship of β-END(1–9) using the cAMP assay system

FRSK cells were seeded into 6-well plates at a density of 1 × 10⁵ cells per well. After 24 h cultivation, 10 μM forskolin and 1–1000 nM of either Met-ENK (Peptide Institute, Osaka, Japan), β-END(1–9) (GenScript, Piscataway, NJ, USA), or β-END (Peptide Institute, Osaka, Japan), followed by incubation for 30 min. Intracellular cAMP levels were then measured using the cAMP-Glo Assay (Promega, Madison, WI, USA).

Microarray analysis

FRSK cells were seeded onto 10-cm culture dishes at a density of 1 × 10⁶ cells per dish and cultured under static conditions to allow for cell attachment. Following attachment, cells were treated with 100 nM of either Met-ENK or β-END(1–9) and incubated for 24 h. Total RNA was extracted using the TriPure Isolation Reagent (Roche Life Science). Purified RNA samples were sent to Filgen Inc. (Aichi, Japan) for microarray analysis using the GeneChip Clariom S Assay (Thermo Fisher Scientific, Waltham, MA). Gene expression changes greater than two-fold were considered significant.

Quantitative RT-PCR

FRSK cells were seeded into 6-well plates at a density of 1 × 10⁵ cells per well. Following attachment, cells were treated with 100 nM of either Met-ENK or β-END(1–9) and incubated for 24 h. Total RNA was extracted using the TriPure Isolation Reagent (Roche Life Science). First-strand cDNA synthesis was performed 1 μg of total RNA using ReverTra Ace (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Quantitative PCR was performed using the Thermal Cycler Dice Real-Time System III (Takara, Shiga, Japan) with THUNDERBIRD^® Next SYBR™ qPCR Mix (TOYOBO). Specific forward and reverse primers were used to amplify rat meprin1A (5′-CATCTGGGTGAGAAGGGACG-3′ and 5′-CCGCCATCTGAGTTACCAGG-3′), rat sphingomyelin phosphodiesterase 2 (smpd2) (5′-CATCTAAGCGGACTGGTGCT-3′ and 5′-AGGTCTTTGGGGTGCATGTT-3′), and rat β-actin (5′-AACCCTAAGGCCAACCGTGAAAAG-3′ and 5′-CGACCAGAGGCATACAGGGACAAC-3′). PCR conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The Ct values were normalized to β-actin, and the relative expression levels were calculated using the comparative Ct method.

Statistical analysis

Statistical analyses were performed using Dunnett’s t-test in SPSS Statistics (version 29.0.2.0; IBM, Chicago, Il, USA). A p-value of less than 0.05 was considered statistically significant.

Abbreviations

POMC

Pro-opiomelanocortin

α-MSH

α-Melanocyte-stimulating hormone

β-END

β-Endorphin

ACTH

Adrenocorticotropic hormone

PC

Prohormone convertase

AD

Atopic dermatitis

MOR

μ-Opioid receptor

DOR

δ-Opioid receptor

KOD

κ-Opioid receptor

EC₅₀

Half maximal effective concentration

MALDI-TOF

Matrix-assisted laser desorption/ionization time-of-flight

MS

Mass spectrometry

HPLC

High-performance liquid chromatography

RP

Reverse phase

ELISA

Enzyme-linked immunosorbent assay

FRSK

Fetal rat skin keratinocyte

Met-ENK

Methionine-enkephalin

Author contributions

Conceptualization: H.Y., T.Y. and Y.S.; funding acquisition: H.Y.; investigation: H.Y. and A.K.; methodology: H.Y., T.Y. and Y.S.; writing—original draft preparation: H.Y.; writing—review and editing: H.Y., A.K., T.Y. and Y.S.

Funding

This work was supported by funding from the KOSÉ Cosmetology Research Foundation to HY and by JSPS KAKENHI (Grant numbers: JP23K05029 and JP20K05829) to HY.

Data availability

All the data are available upon reasonable request. Microarray data from this study are available in the NCBI Gene Expression Omnibus (GEO) repository under the accession number GSE297678.

Competing interests

The authors declare no competing interests.