Enhanced Type I Collagen Synthesis in Fibroblasts by Dermal Stem/Progenitor Cell-Derived Exosomes

Abstract

The self-duplication and differentiation of dermal stem cells are essential for the maintenance of dermal homeostasis. Fibroblasts are derived from dermal stem cells and produce components of connective tissue, such as collagen, which maintains the structure of the dermis. Cell–cell communication is required for the maintenance of tissue homeostasis, and the role of exosomes in this process has recently been attracting increasing attention. Dermal stem cells and fibroblasts have been suggested to communicate with each other in the dermis; however, the underlying mechanisms remain unclear. In the present study, we investigated communication between dermal stem/progenitor cells (DSPCs) and fibroblasts via exosomes. We collected exosomes from DSPCs and added them to a culture of fibroblasts. With the exosomes, COL1A1 mRNA expression was up-regulated and dependent on the Akt phosphorylation. Exosomes collected from fibroblasts did not show the significant up-regulation of COL1A1 mRNA expression. We then performed a proteomic analysis and detected 74 proteins specific to DSPC-derived exosomes, including ANP32B related to Akt phosphorylation. We added exosomes in which ANP32B was knocked down to a fibroblast culture and observed neither Akt phosphorylation nor enhanced type I collagen synthesis. Additionally, an immunohistochemical analysis of skin tissues revealed that ANP32B expression levels in CD271-positive dermal stem cells were lower in old subjects than in young subjects. These results suggest that DSPCs promote type I collagen synthesis in fibroblasts by secreting exosomes containing ANP32B, which may contribute to the maintenance of skin homeostasis; however, this function of DSPCs may decrease with aging.

INTRODUCTION

The dermis is a tissue underlying the epidermis and is rich in collagen and elastin fibers, which maintain the structure and elasticity of skin. The fiber structure in the dermis is composed of extracellular matrix (ECM) molecules, including collagen, elastin and proteoglycans, which are produced by fibroblasts. The production of ECM was previously shown to be decreased in fibroblasts exposed to UV ray and oxidative stress as well as with aging, which may compromise the ability of these cells to maintain the dermal structure.¹⁾ Stem cells in the dermis play a role in preventing the destruction of the tissue structure. Stem cells, which exhibit the abilities of self-renewal and differentiation into various cells, supply new cells and, thus, contribute to the maintenance of tissue homeostasis and the repair of damaged tissues. We previously reported that CD271-positive cells in the dermis were stem cells with the ability to differentiate into multiple types of cells.²⁾ We also found that CD271-positive cells produced new fibroblasts and played an important role in maintaining skin homeostasis.³⁾

Cells in living organisms communicate with surrounding cells (cell–cell communication) to select the most appropriate behavior. Cell–cell communication is mediated by multiple mechanisms. Communication by the secretion of soluble factors and their recognition on cell surface receptors as well as direct cell–cell interactions between adjacent cells have been extensively examined.⁴⁾ Exosomes have recently been attracting increasing attention for their role in cell–cell communication. Exosomes are small vesicles with a diameter of approximately 50–150 nm that are secreted by cells and possess a lipid double membrane.⁵⁾ They were initially considered to remove unnecessary proteins from cells, but were subsequently found to include not only proteins, but also nucleic acids.^4,6) Furthermore, microRNA (miRNA) in exosomes was shown to be incorporated into and function in cells,⁷⁾ and the functions of exosomes have since been intensively investigated. Exosomes derived from mesenchymal stem cells have potential in therapeutic applications for tissue repair because they exhibit great stability and homing effects and are not immunologically rejected.^8–10) A previous study reported that exosomes from adipose-derived stem cells promoted wound healing by regulating the migration and proliferation of dermal fibroblasts and collagen synthesis.¹¹⁾ Emerging evidence has shown that exosomes are involved in skin homeostasis. Exosomes derived from epidermal keratinocytes were shown to play a role in skin pigmentation by regulating the synthesis of melanin in melanoctyes.¹²⁾ Furthermore, exosomes from dermal fibroblasts promoted the proliferation of dermal papilla cells.¹³⁾ The function of exosomes secreted by dermal stem/progenitor cells (DSPCs) currently remains unknown. Therefore, we herein investigated the effects of DSPC-derived exosomes on fibroblasts to elucidate their function in the maintenance of skin homeostasis.

MATERIALS AND METHODS

Cell Culture

SF8428 cells, provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan, were cultured in Minimum Essential Medium Alpha (MEM α; Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 15% fetal bovine serum (FBS; Biosera, Ringmer, U.K.) and Antibiotic-Antimycotic (Wako, Osaka, Japan) at 37 °C with 5% CO₂. Human dermal fibroblasts (HDFs; KURABO, Osaka, Japan), a cell line established by Inoue et al.¹⁴⁾ Asian derived immortalized fibroblast 1 (AIF1) and human adipose derived stem cells (ASCs; KURABO) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS and Antibiotic-Antimycotic at 37 °C with 5% CO₂.

Isolation and Characterization of Exosomes

Exosomes were isolated from SF8428 cells, HDFs, AIF1 and ASCs by differential centrifugation. Cells were seeded at a concentration of 5 × 10⁵ cells in 100 mm dishes and cultured for three days. Cells were washed with phosphate-buffered saline (PBS) and were cultured in fresh FBS-free medium for another two days. Cell culture media were collected from 80 to 90% confluent cells under sterile conditions. Centrifugation was performed at 2000 × g for 10 min to remove dead cells, and the supernatants were passed through a 0.2-µm filter (Millipore, Billerica, MA, U.S.A.). Filtrates were concentrated to a 1/100 volume with VivaspinTurbo (Sartorius, Göttingen, German) at 2300 × g. The concentrates were then ultracentrifuged at 100000 × g for 70 min using a TLA110 rotor (Beckmann, Brea, CA, U.S.A.). Centrifugation was performed at 4 °C. Pellets were resuspended in 100 µL of PBS. To quantify exosomes, protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific). The size distribution of exosome preparations was analyzed by dynamic light scattering using nanoSAQLA (Otsuka Denshi, Osaka, Japan). The expression of the exosome markers, CD63, CD81, and Alix, was analyzed by Western blotting.

Preparation of Exosome-Depleted FBS

Exosome-depleted FBS was obtained by ultracentrifugation as previously reported¹⁵⁾ with a slight modification. FBS was ultracentrifuged at 100000 × g for 18 h. The supernatants were passed through a 0.2-µm filter (Millipore). The filtrate was used as exosome-depleted FBS.

Exosome Uptake

Purified exosomes were labeled using a PKH67 fluorescent labeling kit (Sigma-Aldrich, St. Louis, MO, U.S.A.) according to the manufacturer’s protocol. Labeled exosomes were added to Wheat Germ Agglutinin, Texas Red™-X Conjugate (Thermo Fisher Scientific)-labeled HDFs and cells were cultured for 24 h. Cells were then washed with PBS and fixed with 4% paraformaldehyde (PFA). Images were taken using a fluorescence microscope (IX71; OLYMPUS, Tokyo, Japan).

Western Blotting

Cells or exosomes were lysed with 2% sodium dodecyl sulfate (SDS). Total protein was measured using a BCA protein assay kit (Thermo Fisher Scientific). Samples were run on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk (Wako) or 5% bovine serum albumin (BSA) (Wako) at room temperature for 1 h and then incubated at 4 °C overnight with an anti-CD81 mouse monoclonal antibody (1 : 1000; Santa Cruz, Dallas, TX, U.S.A.), anti-CD63 mouse monoclonal antibody (1 : 1000, Santa Cruz), anti-Alix mouse monoclonal antibody (1 : 1000, Santa Cruz), anti-ANP32B rabbit monoclonal antibody (1 : 1000; OriGene, Rockville, MD, U.S.A.), anti-Akt mouse monoclonal antibody (1 : 1000, Santa Cruz), anti-p-Akt monoclonal rabbit antibody (1 : 500; GeneTex, Irvine, CA, U.S.A.), anti-smad3 mouse monoclonal antibody (1 : 500, Santa Cruz), anti-p-smad3 mouse monoclonal antibody (1 : 500, Santa Cruz), or anti β-actin mouse monoclonal antibody (1 : 5000, Santa Cruz). Membranes were washed three or four times with Tris-buffered saline containing 0.1% Tween 20 and then incubated with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.) at room temperature for 1 h. After an exposure to enhanced chemiluminescence substrates (Cytiva, Tokyo, Japan), immunoreactivity was visualized with LuminoGraph II (ATTO, Tokyo, Japan).

Real-Time PCR

Total RNA was isolated from cultured HDFs using RNA ISO^® (TaKaRa Bio, Shiga, Japan), and cDNA was synthesized by reverse transcription. Real-time semi-quantitative RT-PCR was performed with SYBR Select Master Mix (Applied Biosystems, Tokyo, Japan) using a StepOnePlus Real-Time RT-PCR System (Applied Biosystems) in accordance with the manufacturer’s protocol. The sequences of primers used were as follows: 18s ribosomal RNA (rRNA) forward, 5′-CCGAGCCGCCTGGATAC-3′ and reverse, 5′-CAGTTCCGAAAACCAACAAAATAGA-3′; COL1A1 forward, 5′-GCTACCCAACTTGCCTTCATG-3′ and reverse, 5′-TTCTTGCAGTGGTAGGTGATGTTC-3′; ANP32B forward, 5′-CTTACCTACTTGGATGGCTATGAC-3′ and reverse, 5′-CTCATCTTCTCCTTCTTCGTCC-3′; ANP32A forward, 5′-CCTCTGATGTGAAAGAACTTGTC-3′ and reverse, 5′-TAAGTTTGCGATTGAGGTGAG-3′. Amplification was normalized to the housekeeping gene 18s rRNA, and differences between samples were quantified based on the ΔΔCt method. All PCR products were checked by a melting curve analysis to exclude the possibility of multiple products or an incorrect product size.

Exosome Enzyme-Linked Immunosorbent Assay (ELISA)

Isolated exosomes were quantified using a PS Capture™ Exosome ELISA Kit (Wako) according to the manufacture’s protocol. Anti-CD81 antibody (1 : 100; Novus Biologicals, Littleton, CO, U.S.A.) was used as primary antibody instead of Anti-CD63 antibody included in the kit.

Immunocytochemistry

HDFs were fixed with 4% PFA three days after treated with SF-Exo. HDFs were incubated with anti-procollagen I antibody (1 : 200, Santa Cruz) followed by incubation with 4′-6-diamidino-2-phenylindole (DAPI) and secondary antibody Alexa Fluor™ 488 donkey anti-goat immunoglobulin G (IgG) (H + L) (1 : 500, Thermo Fisher Scientific). Immunocytochemical images of two randomly chosen areas were observed under a fluorescence microscope (IX71) for each observation. The fluorescent intensity of the procollagen I signal was analyzed with ImageJ.¹⁶⁾

Proteomic Analysis by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Exosomes derived from SF8428 cells and HDFs were run on a 12% polyacrylamide gel electrophoresis (PAGE) gel and stained using a silver stain kit for MS (Integral, Tokyo, Japan) according to the manufacturer’s protocol. Four bands with different intensities between exosomes from SF8428 cells and HDFs were excised from each gel lane, and proteins in excised bands from each lane were extracted and desilverized using Silver stain KANTO gel washing solution for MS (Kanto Kagaku, Tokyo, Japan). Protein samples were reduced with 10 mM dithiothreitol at 60 °C for 30 min and incubated with 20 mM iodoacetamide to alkylate exposed side chains at room temperature for 30 min in the dark. Samples were digested with 10 ng/µL trypsin at 37 °C for 16 h. Peptides were purified using MonoSpinC18 (GL Science, Tokyo, Japan). Samples were loaded onto an AcclamPepMap100 trapping column (Thermo Fisher Scientific) connected to an Easy Spray LC Column (Thermo Fisher Scientific) in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology (Applied Flow Technology, Colorado Springs, CO, U.S.A.). Linear gradient conditions were as follows: 0–35% buffer B for 50 min, 35–95% buffer B for 5 min, and then 95% buffer B for 5 min. The LC-MS/MS analysis was performed for 1 h on a Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific) coupled with EASY-nLC 1000 (Thermo Fisher Scientific). MS data were analyzed using MASCOT (Matrix Science, London, GB). The parameters used were as follows: enzyme, trypsin; maximum missed cleavages,  2; precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.6 Da; dynamic modification, oxidation (M, +15.995 Da) and acetyl (N terminus, +42.011 Da); static modification, carbamidomethyl (C, +57.021 Da).

Small Interfering RNA (siRNA) Transfection

siRNAs against ANP32B (siRNA1, 103237913; siRNA2, 103237916; siRNA3, 103237919) and a non-silencing negative control siRNA were purchased from Integrated DNA Technologies (Tokyo, Japan). Solutions of 10 nM siRNAs in OPTIMEM (Thermo Fisher Scientific) were used for transfection into SF8428 cells with Lipofectamine RNAiMAX (Thermo Fisher Scientific) at 37 °C for 4 h in a humidified atmosphere with 5% CO₂. Cells were cultured in complete medium for 2 d and total RNA was collected.

Human Skin Biopsies

As previously reported,¹⁷⁾ human skin samples were collected from surplus surgical tissues at Fujita Health University Hospital after ensuring that patients fully understood the study objective and other related information. Written informed consent was obtained from each subject. The present study was conducted with ethical approval by the Research and Ethics Committee of Fujita Health University (Approval No. 15-235). Skin samples of unexposed areas were obtained from the abdomen, thigh, back, and hip. Subjects younger than 40 years of age and those 60 years or older were classified into the young and old groups, respectively: young group, 5 males and 3 females, average 30.5 years; old group, 6 males and 2 females, average 71.4 years.

Immunohistochemistry

Skin samples were fixed in 4% PFA, embedded in paraffin, and then sectioned at a thickness of 4 µm. To activate antigens, a Proteinase K treatment (S3004; Dako North America, Santa Clara, CA, U.S.A.) was performed at a 1 : 50 dilution. Sections were incubated with anti-CD271 antibodies (1 : 150, OriGene) followed by an incubation with DAPI and the secondary antibody Alexa Fluor™ 488 donkey anti-mouse IgG (H + L) (1 : 500, Thermo Fisher Scientific). Sections were also incubated with an Alexa Fluor™ 594-labeled anti-ANP32B antibody (1 : 200, Santa Cruz). Immunohistological images of three randomly chosen areas for each observation were observed under a fluorescence microscope (IX71). The fluorescent intensity of the ANP32B signal in CD271-positive cells was analyzed with ImageJ.¹⁶⁾ In addition, the number of CD271-positive cells per unit area were counted.

Statistical Analysis

Data were presented as the mean ± standard error (S.E.). Statistical analyses were performed by the Student’s t-test where appropriate. p < 0.05 was considered to be significant. All experiments were performed at least 3 times.

RESULTS

Collection of Exosomes by Ultracentrifugation

To examine whether DSPC-derived exosomes are involved in maintaining skin homeostasis, we initially isolated exosomes from DSPCs and HDFs. We used SF8428 cells, a cell line established by Sudo et al.¹⁸⁾ that has multiple differentiation abilities, as a model of DSPCs. Exosomes may be isolated by various approaches, including ultracentrifugation, density-gradient centrifugation, and immunoprecipitation.¹⁹⁾ In the present study, exosomes were collected from the culture supernatants of SF8428 cells and HDFs by ultracentrifugation. To examine whether the pellet obtained contained exosomes, particle size distribution was measured using dynamic light scattering. Particles with around 50 nm and around 80 nm in diameter were obtained from culture media of HDFs and SF8428 cells, respectively (Fig. 1A). These were within the size range of exosomes, i.e., 50 to 150 nm in diameter.⁶⁾ We also performed a Western blot analysis of these particles and detected the exosome marker proteins, Alix, CD63, and CD81²⁰⁾ (Fig. 1B). Based on these results, since the pellet obtained exhibited the characteristics of exosomes,^6,20) we considered exosomes to be successfully isolated.

Fig. 1. Characterization of Exosomes Collected from HDF and SF8428 Cells Supernatants

(A) Distribution of exosome sizes measured by dynamic light scattering. HDF-Exo, dotted line; SF-Exo, line. (B) The expression of Alix, CD63, and CD81 in exosomes collected from HDFs and SF8428 cells (HDF-Exos and SF-Exos, respectively) was examined by Western blotting.

Enhanced Type I Collagen Synthesis in Fibroblasts by DSPC-Derived Exosomes

To clarify whether HDFs take up exosomes from SF8428 cells and HDFs (SF-Exos and HDF-Exos, respectively), exosomes isolated from each cell line were labeled with the fluorescent dye PKH67 and added to HDFs culture media. After 24 h, PKH67-labeled vesicles were detected in HDFs (Fig. 2A), indicating that exosomes were incorporated by fibroblasts. To elucidate the effects of the incorporated exosomes, we examined collagen mRNA expression in HDFs. When HDFs were cultured with SF-Exos, COL1A1 mRNA expression was up-regulated in a dose-dependent manner, with 50 µg/mL SF-Exos inducing the greatest increase (Fig. 2B). SF-Exos were hereafter added at a concentration of 50 µg/mL. HDF-Exos did not up-regulate COL1A1 mRNA expression, even at 50 µg/mL (Supplementary Fig. S1). To examine the effect of exosomes on collagen protein synthesis, immunocytochemistry of procollagen I was performed. We found that the addition of SF-Exos increased the protein expression level of procollagen I, although the addition of HDF-Exos did not (Supplementary Fig. S2). The difference observed in the effect of enhancing type I collagen synthesis between SF-Exos and HDF-Exos might be caused due to the difference in the numbers of exosomes even when the protein concentrations of the SF-Exos and HDF-Exos preparations were the same. Therefore, we examined the relative particle numbers of SF-Exos and HDF-Exos at various protein concentrations. It was found that the relative particle number of HDF-Exos was higher than that of SF-Exos when the protein concentrations were the same (Supplementary Fig. S3). From these findings, SF-Exos may specifically have the type I collagen synthesis promoting function. Prior to the preparation of SF-Exos by ultracentrifugation, SF8428 cells were cultured in FBS-free medium after cultured for proliferation in FBS-containing medium. To further exclude the possibility that residual FBS-derived exosomes contained in SF-Exos preparation may affect the experiment, exosomes were removed from FBS by ultracentrifugation.¹⁵⁾ Then, exosomes were newly prepared from SF8428 cells cultured in medium containing the exosome-depleted FBS. These FBS-Exo-free exosomes from SF8428 cells were added to HDFs and the expression of COL1A1 mRNA was analyzed. We found that similar to SF-Exos, SF-Exos (FBS-Exo-free) up-regulated COL1A1 mRNA expression (Supplementary Fig. S4), demonstrating that the effect of SF-Exos was not due to FBS-derived exosomes. Furthermore, exosomes derived from AIF1 cells¹⁴⁾ and ASCs²¹⁾ (AIF1-Exos and ASC-Exos, respectively), which are mesenchymal cells also reported to have pluripotency, were tested for their effect on enhancing type I collagen synthesis. Similar to SF-Exos, both AIF1-Exos and ASC-Exos up-regulated COL1A1 mRNA expression (Supplementary Fig. S5).

Fig. 2. Enhanced Type I Collagen Synthesis in HDFs by SF-Exos

(A) HDF-Exos or SF-Exos labeled with PKH67 (green) were added to HDFs stained with wheat germ agglutinin (WGA) (red). Cells were cultured for 24 h. Exosomes were observed in HDF cells. Scale bar, 10 µm. (B) COL1A1 mRNA expression levels were examined after HDFs were cultured with SF-Exos for 24 h. Data shown are the mean ± S.E. (n = 3). * p < 0.05 (C) SMAD3 and phospho-SMAD3 (pSAMD3, serine 425) expression in HDFs cultured with SF-Exos for 18 h was examined by Western blotting. (D) COL1A1 mRNA expression was examined in HDFs cultured with SF-Exos and the Akt inhibitor 17-AAG (10 nM) or MK2206 (5 µM) for 24 h. (E) Akt and phospho-Akt (pAkt, serine 473) expression in HDFs cultured with SF-Exos and the Akt inhibitor 17-AAG (10 nM) or MK2206 (5 µM) for 18 h was examined by Western blotting. Data shown are the mean ± S.E. (n = 3). * p < 0.05, ** p < 0.01.

To elucidate the mechanisms by which SF-Exos regulate collagen expression, we investigated whether the downstream signaling pathways of TGF-β, a major regulator of collagen synthesis, were affected.²²⁾ Multiple pathways are downstream of TGF-β, namely, the Smad pathway and non-Smad pathways, including the Akt-mediated pathway.²³⁾ We initially examined the phosphorylation of Smad3 by Western blotting and found that SF-Exos did not exert any effects (Fig. 2C). We then investigated the phosphorylation of Akt and noted an increase when SF-Exos was added (Fig. 2D), suggesting that Akt was activated by SF-Exos. In addition, when the Akt phosphorylation inhibitor 17-AAG²⁴⁾ and MK2206^25,26) was added to the culture, Akt phosphorylation by SF-Exos was not observed (Fig. 2D) and up-regulation of COL1A1 mRNA expression by SF-Exos was suppressed (Fig. 2E). These results suggest that DSPC-derived exosomes promote type I collagen synthesis in fibroblasts in a manner that is dependent on Akt signaling.

ANP32B in DSPC-Derived Exosomes Enhanced Type I Collagen Synthesis

The contents of exosomes include proteins and mRNAs^4,7,8); however, the component of exosomes that activates Akt signaling to promote type I collagen synthesis has not yet been identified. The serine/threonine kinase Akt has been shown to play a role in numerous important cellular functions, including proliferation, migration, and metabolism.²⁷⁾ The activation of Akt signaling is triggered by the binding of ligands that are growth factors, including insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), cytokines, and hormones, to receptors on the cell membrane.^28,29) In the present study, we performed a proteomic analysis of exosomes to identify the molecules that function in the promotion of type I collagen synthesis. Since SF-Exos, but not HDF-Exos, enhanced COL1A1 mRNA expression (Fig. 2B, Supplementary Fig. S1), we hypothesized that proteins specific to SF-Exos are important for promoting type I collagen synthesis. Proteins extracted from the collected exosomes were separated by SDS-PAGE, and silver staining was performed. Four protein bands with different intensities between SF-Exos and HDF-Exos were excised from each gel lane, and the proteins in the excised bands were comprehensively detected by LC-MS/MS (Fig. 3A). A total of 373 proteins were identified (Supplementary Tables S1–S3), 74 of which were only present in SF-Exos (Fig. 3B and Supplementary Table S1). Among SF-Exo-specific proteins, we focused on ANP32B, which is involved in Akt signaling,³⁰⁾ because Akt signaling has been suggested to mediate the up-regulation of COL1A1 mRNA expression by SF-Exos. The expression of ANP32B in SF-Exos and HDF-Exos was examined by Western blotting, and ANP32B was only observed in SF-Exos, which was consistent with the results of the comprehensive analysis (Fig. 3C).

Fig. 3. Proteomic Analysis of Exosomes

(A, B) HDF-Exos and SF-Exos were run on a 12% SDS-PAGE gel, and silver staining was performed. Arrows indicate the bands of proteins with different expression levels between HDF-Exos and SF-Exos, and four bands were excised from the gel. Proteins extracted from the excised bands were subjected to a proteomic analysis (A). Venn diagram representing proteins detected in HDF-Exos and SF-Exos (B). HDF-Exos, yellow; SF-Exos, red. (C) ANP32B and CD81 protein expression in HDF-Exos and SF-Exos was examined by Western blotting.

To investigate whether the up-regulation of COL1A1 mRNA expression by SF-Exos was dependent on ANP32B, we knocked down ANP32B using siRNA. ANP32B expression levels were examined by qPCR and Western blotting, and the results obtained confirmed the knockdown of ANP32B (Figs. 4A, B). In addition, we collected exosomes from SF8428 cells in which ANP32B was knocked down, and the results obtained again confirmed the knockdown of ANP32B (Fig. 4C). Furthermore, when SF-Exos in which ANP32B was knocked down were added to HDFs, neither the up-regulation of COL1A1 mRNA expression nor Akt phosphorylation was observed (Figs. 4D, E). These results suggest that DSPCs promote type I collagen synthesis in fibroblasts by secreting exosomes containing ANP32B, thereby contributing to the maintenance of skin homeostasis. ANP32A, a family of ANP32 protein, was also specifically detected in SF-Exos (Supplementary Table S1), but ANP32B knockdown did not affect ANP32A mRNA expression (Supplementary Fig. S6).

Fig. 4. ANP32B Mediated the Up-Regulation of COL1A1 mRNA Expression in HDFs by SF-Exos; siRNA against ANP32B or the Non-silencing siRNA Control Was Introduced into SF8428 Cells

(A) The expression of ANP32B mRNA in siRNA-introduced SF8428 cells was examined. Data shown are the mean ± S.E. (n = 3). ** p < 0.01 (B, C) The protein expression of ANP32B in siRNA-introduced SF8428 cells (B) and siRNA-introduced SF-Exos (C). (D) COL1A1 mRNA expression in HDFs after 24 h culture with siRNA-induced SF-Exos was examined. Data shown are the mean ± S.E. (n = 3). * p < 0.05, **P < 0.01. (E) The expression of Akt and phospho Akt (pAkt, serine 473) in HDFs cultured with SF-Exos with or without the knockdown of ANP32B for 18 h was examined by Western blotting.

Effects of Chronological Aging on the Function of DSPC-Derived Exosomes

To examine whether the function of DSPC-derived exosomes changes with aging, we analyzed the expression of ANP32B in an in vitro aging model. Since oxidative stress is known to increase in aged skin,³¹⁾ aged environment was reproduced by adding hydrogen peroxide to SF8428 cells. ANP32B expression levels decreased in a hydrogen peroxide dose-dependent manner (Fig. 5A). In addition, to assess the expression of ANP32B in human tissues, immunohistochemistry was performed against ANP32B and CD271, a dermal stem cell marker.²⁾ The results obtained showed that ANP32B expression levels in CD271-positive dermal stem cells were lower in old subjects than in young subjects (Figs. 5B, C). In addition, as previously reported,³⁾ the number of CD271-positive dermal stem cells was found to decrease with age (Fig. 5D). These results indicate that aging decreases the functions of DSPC-derived exosomes.

Fig. 5. Effects of Aging on the Function of DSPC-Derived Exosomes

(A) ANP32B mRNA expression levels in SF8428 cells were examined 24 h after the addition of hydrogen peroxide. Data shown are the mean ± S.E. (n = 3). * p < 0.05. (B) Immunohistochemistry was performed against CD271 (left, green) and ANP32B (middle, red) in unexposed parts of skin tissue sections from young (<40 years) and old (>60 years) human subjects. Arrowheads show CD271-positive cells. Scale bar, 10 µm. (C) Relative ANP32B fluorescence intensity in CD271-positive cells in the old group was calculated with that in the young group as 1. The young group (mean age = 30.5 ± 5.1 years), the old group (mean age = 71.4 ± 3.9 years). Data shown are the mean ± S.E. (n = 8). * p < 0.05. (D) The number of CD271-positive cells per unit area was counted. Data shown are the mean ± S.E. (n = 8). * p < 0.05.

DISCUSSION

In the present study, we used SF8428 cells, a fibroblast lineage cell line with multiple differentiation abilities, as a DSPCs model and showed that ANP32B in exosomes secreted from SF8428 cells activated Akt signaling and enhanced type I collagen synthesis in fibroblasts. We also found that ANP32B expression levels in dermal stem cells decreased in human dermal tissues with age.

ANP32B is a nuclear phosphoprotein ANP32 family member that plays an important role during mouse development.³²⁾ We demonstrated that ANP32B was present in exosomes derived from SF8428 cells and suggested its involvement in the activation of Akt signaling, which is regulated by a number of molecules, including PH domain leucine-rich repeat protein phosphatase,³³⁾ pyruvate dehydrogenase kinase,³⁴⁾ PP2A,³⁵⁾ and PTEN.³⁶⁾ However, a previous study reported that ANP32B did not interact with PP2A or PTEN.³⁰⁾ Therefore, further studies are needed to elucidate the mechanisms by which ANP32B activates Akt signaling and promotes type I collagen synthesis.

ANP32A, a member of the ANP32 family, has been reported to be involved in the phosphorylation of Akt³⁷⁾ and was specifically identified in SF-Exos in this study (Fig. 3C, Supplementary Table S1). SF-Exos from SF8428 cells in which ANP32B was knocked down neither enhanced Akt phosphorylation (Fig. 4E) nor changed ANP32A mRNA expression (Supplementary Fig. S6). ANP32A may not be involved in the promotion of type I collagen synthesis by SF-Exos or may only affect a little.

In addition, it currently remains unclear how ANP32B is transported into exosomes. Although the nuclear proteins FOXH1³⁸⁾ and HIF1α³⁹⁾ have been detected in exosomes, the mechanisms underlying exosome transport have yet to be clarified. Therefore, further studies on the exosome transport of ANP32B will provide insights into the transport of nuclear proteins, and the selective or accelerated transport of exosome constituents, if achievable, may enhance the effects of exosomes.

Akt signaling affects skin aging not only by its involvement in the regulation of type I collagen synthesis, but also other cellular functions.⁴⁰⁾ UVB irradiation increases the secretion of LGI3, which promotes keratinocyte differentiation via Akt signaling and plays a role in the recovery of the skin barrier function.⁴¹⁾ Wound healing is accelerated by the enhanced migration of fibroblasts via Akt signaling activated by nerve growth factor.⁴²⁾ Therefore, Akt signaling is involved in various skin features and functions. The activation of Akt signaling by DSPC-derived exosomes may be important for ameliorating the effects of aging on skin.

Collectively, the present results demonstrated that DSPC-derived exosomes regulated type I collagen synthesis in dermal fibroblasts via the activation of Akt signaling by ANP32B incorporated in exosomes. Since the self-renewal and differentiation potentials of dermal stem cells are required for skin homeostasis, we herein suggest a third function for dermal stem cells, which is cell–cell communication via exosomes. Dermal stem cells may not only supply new fibroblasts, they may also enhance the function of fibroblasts by secreting exosomes for the maintenance of skin homeostasis.

Unlike SF-Exos, HDF-Exos did not show the effect of enhancing type I collagen synthesis (Supplementary Fig. S1). On the other hand, exosomes derived from pluripotent mesenchymal cells, AIF1 cells and ASCs, showed the effects similar to SF-Exos (Supplementary Fig. S5). That is, it can be said that the potential to enhance type I collagen synthesis in fibroblasts is specific to exosomes derived from pluripotent mesenchymal cells.

Since the number of CD271-positive dermal stem cells decreases with age³⁾ (Fig. 5D), the number of CD271-positive dermal stem cell-derived exosomes incorporated by fibroblasts may also be reduced in aged tissues. The present study showed age-related decreases in ANP32B expression in CD271 positive dermal stem cells, suggesting that the function of DSPC-derived exosomes also declines with aging. Therefore, the preservation of dermal stem cell numbers, which is generally considered to be important, as well as cell–cell communication via exosomes, a newly discovered function of stem cells in the dermis, is important for the maintenance of skin homeostasis.

Acknowledgments

We are grateful to Goshima N (Cellisis, Aichi, Japan) for her support preparing the manuscript. We would like to thank Center for Joint Research Facilities at Fujita Health University for proteomic analysis. We wish to thank Kawagishi-Hotta M for technical support and helpful advices.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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