Hair plays an important role in biological functions, from providing protection to maintaining body temperature (Kim and Dao, 2020). Hair loss is a common disorder associated with hair thinning and loss of hair from the head, caused by a variety of factors, including stress, inflammation, hormonal imbalance, nutritional imbalance, diseases, and medication (Springer
Hair is generated in hair follicles over the three stages of the hair growth cycle to determine the growth, maintenance, and elimination of hair: (i) anagen phase (the active growth state of hair follicles); (ii) catagen phase (the regression state of hair follicles); (iii) telogen phase (the rest state of the hair follicles). Hair loss is caused by disorders in the hair growth cycle, such as the shortening of the anagen phase, the rapid entry of the catagen phase, and the prolongation of the telogen phase (Pantelireis and Higgins, 2018). Among the cells of the hair follicle, dermal papilla cells (DPCs), a mesenchymal-derived fibroblast located at the base of the hair follicle, play an important role in hair growth and in the regeneration of hair follicles (Greco
Myristoleic acid (MA), also known as 9-tetradecenoate or myristoleate, is an omega-5 monounsaturated fatty acid obtained from the seeds of plant from the Myristicaceae family. It is biosynthesized in organisms from myristic acid by the enzyme stearoyl-CoA desaturase (SCD)-1, also known as delta-9 desaturation. MA is present in all eukaryotic organisms and is found in human adipose tissue (Jiang
MA was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) (Fig. 1A). DMSO, bovine serum albumin (BSA), and MXD were purchased from Sigma-Aldrich. Fetal bovine serum (FBS), penicillin-streptomycin solution and trypsin-EDTA solution were obtained from Gibco (Grand Island, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (Logan, UT, USA). Dulbecco’s phosphate-buffered saline (DPBS) was obtained from WelGENE (Daegu, Korea). EZ-CYTOX, a water-soluble tetrazolium (WST)-based cell viability assay kit, was purchased from Daile Lab Service (Seoul, Korea). PRO-PREP protein extraction solution was obtained from iNtRON Biotechnology (Seoul, Korea). Polyvinylidene fluoride (PVDF) membranes were purchased from Bio-Rad (Hercules, CA, USA). Westar Nova 2.0 ECL solution was obtained from Cyanagen (Bologna, Italy). X-ray film was purchased from Agfa-Gevaert (Mortsel, Belgium). 4% paraformaldehyde (PFA) solution was purchased from Biosesang (Seongnam, Korea). The antibodies used for western blotting and immunofluorescent staining included anti-cyclin A, -Cdc2, -cyclin B1, -β-actin, -β-catenin, -α-tubulin, horseradish peroxidase (HRP)-labeled anti-rabbit IgG, and HRP-labeled anti-mouse IgG, purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho (Ser2448)-mTOR, -Atg7, -LC3I/II, -GSK3β, -phospho (Ser9)-GSK3β (glycogen synthase kinase 3β), -phospho (Ser552)-β-catenin, -phospho (Ser675)-β-catenin, -ERK, and -phospho (Thr202/Tyr204)-ERK were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3II was purchased from Abcam (Cambridge, MA, USA). Anti-mouse AlexaFluor® 488, -rabbit AlexaFluor® 594, and -rabbit AlexaFluor® 488 were purchased from Invitrogen (Carlsbad, CA, USA). 4’,6-diamidino-2-phenylindole (DAPI)-contained VECTASHIELD mounting solution was obtained from Vector Laboratories (Burlingame, CA, USA). U0126 was obtained from Calbiochem (Cambridge, MA, USA). XAV939 was purchased from Tocris Bioscience (Bristol, UK).
Rat vibrissa immortalized DPCs were kindly provided by the Skin Research Institute of Amore Pacific Corporation R&D Center (Yongin, Korea). DPCs were cultured in DMEM, supplemented with 10% FBS and 1% penicillin-streptomycin solution in a humidified atmosphere of 5 % CO2 at 37°C.
DPCs (1.5×103 cells/well) were seeded onto 96-well plates with DMEM containing 1% FBS. After 24 h, DPCs were treated with MA (1, 5, and 10 µM) or MXD (10 µM) for 48 h. To investigate whether ERK or Wnt/β-catenin signal pathway affects MA-induced DPC proliferation, DPCs were pre-treated with U0126 (ERK inhibitor, 10 µM) or XAV939 (Wnt/β-catenin inhibitor, 20 µM) for 30 min, and then treated with MA (5 µM) for 48 h. WST (10 µL/well) was added to medium and incubated for 2 h in a 5 % CO2 at 37°C. The absorbance was measured at 450 nm using a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA).
DPCs (1.5×105 cells/dish) were seeded in DMEM containing 1% FBS for 24 h and then treated with different concentrations (1 and 5 µM) of MA for 24 h. The cells were harvested, washed with PBS, fixed in 70% ethanol, and stored at ‒20°C for at least 30 min. After fixation, the cells were washed with PBS and stained with propidium iodide (PI, 50 µg/mL) in PBS containing 50 µg/ml RNase A for 30 min at 37°C. The cell cycle distribution was analyzed using a FACStar ﬂow cytometer (BD Biosciences, San Jose, CA, USA).
DPCs (1.5×105 cells/dish) were seeded in DMEM containing 1% FBS for 24 h, and then treated with different concentrations (1 and 5 µM) of MA for 24 h or 5 µM of MA for various times up to 6 h. To determine whether the ERK or Wnt/β-catenin signal pathways affect the action of MA on the regulation of protein levels, DPCs were pre-treated with U0126 (10 µM) or XAV939 (20 µM) for 30 min, followed by treatment with MA for 30 min or 24 h. To extract intracellular proteins, the cells were harvested and lysed using PRO-PREP protein extraction solution in ice for 30 min. Centrifugation was performed at 21,000×g for 15 min at 4°C, and the resulting supernatant was collected. The protein concentration was measured using the Bradford method based on BSA, a reference protein. An equal amount of proteins (10 µg) of total lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked in a TBS-Tween-20 (TBS-T: 50 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Tween-20) solution containing 5% non-fat dry milk at room temperature for 1 h and incubated with the specific primary antibodies overnight at 4°C. After washing the membranes five times with TBS-T, the membranes were incubated with HRP-labeled anti-mouse IgG or -rabbit IgG secondary antibodies at room temperature for 90 min. Westar Nova 2.0 ECL solution was used to expose the membrane signal to the X-ray film.
DPCs were seeded in DMEM containing 1% FBS on 6-well plates with cover-glasses and stabilized for 24 h. To evaluate the translocation of β-catenin to the nucleus, cells were treated with different concentrations of MA (1 and 5 µM) or with MA (5 µM) in the absence or presence of XAV939 (20 µM) for 24 h. In addition, to confirm the LC3 puncta, DPCs were treated with inhibitor (U0126; 10 µM or XAV939; 20 µM) for 30 min, and then treated with 5 µM of MA for 24 h. For immunofluorescent staining, the cells were washed with cold PBS and fixed with 4% PFA for 10 min. After washing the cells with cold PBS three times, the cells were reacted with 0.5% Triton X-100 for 7 min for permeability. The cells were then blocked with blocking solution (10% FBS and 1% BSA in PBS containing 0.1% Tween-20) at room temperature for 2 h, and then incubated with primary antibody (anti-β-catenin (1:50), anti-α-tubulin (1:50), or anti-LC3 (1 µg)) at 4°C overnight. The cells were washed three times with cold PBS and stained with the corresponding AlexaFluor® 488 or 594-conjugated secondary antibody for 1 h at room temperature. After washing, the cells were mounted in Vectastain (Vector Laboratories) containing DAPI. Images were acquired using a confocal microscope (FluoView® FV1200; Olympus, Tokyo, Japan).
All experimental data are denoted as the mean ± standard deviation (SD) from three independent experiments. A
To determine whether MA affects the proliferation of hair follicle cells, DPCs were treated with MA at different concentrations (1, 5, and 10 μM) for 48 h, followed by a WST assay. As a result, MA treatment was found to enhance the proliferation of DPCs by 106.9% ± 1.0% (
Autophagy plays an important role in hair growth by maintaining the anagen phase during the hair cycle (Parodi
The Wnt/β-catenin pathway regulates cell proliferation, and is essential for the hair cycle, hair morphogenesis, and hair regeneration (Ito
To confirm the role of the Wnt/β-catenin pathway on the MA-induced proliferation of DPCs, XAV939, a tankyrase inhibitor that targets Wnt/β-catenin signaling, was used. MA (5 μM) increased the translocation of β-catenin to the nucleus, while XAV939 decreased the MA-induced nuclear β-catenin levels (Fig. 4A). As shown in Fig. 1 and 2, MA enhanced the levels of proteins that activate cell cycle progression and autophagy. Thus, we investigated whether the activation of Wnt/β-catenin signaling affects cell cycle progression and autophagy. MA increased the levels of Cdc2, a cell cycle-related protein, and Atg7, an autophagy-related protein, while XAV939 treatment attenuated the MA-induced increase in the levels of these proteins (Fig. 4B). In addition, when DPCs were pre-treated with XAV939 for 30 min, XAV939 attenuated the MA-induced LC3 puncta (Fig. 4C). Moreover, XAV939 significantly reduced MA-induced DPC proliferation, while only XAV939 treatment did not affect DPC proliferation (Fig. 4D). The results using XAV939, a Wnt/β-catenin signaling inhibitor, suggest that MA stimulated cell-cycle progression and autophagy via the activation of the Wnt/β-catenin pathway in DPCs, which was followed by an increased DPC proliferation.
Next, we evaluated whether MA activates ERK pathway. ERK is a signal transduction pathway that regulates the cell cycle, autophagy, and proliferation of many cells (Chambard
The present study showed that MA enhances anagen signaling by autophagy and G2/M phase cell cycle progression through the activation of the Wnt/β-catenin and ERK pathways in DPCs.
Although the regulatory mechanisms underlying hair growth are not yet fully understood, hair growth is known to be regulated by the interactions between DPCs and various types of cells, including keratinocytes, hair germ cells, and stem cells (Stenn and Paus, 2001). In particular, the interactions between DPCs and keratinocytes, which are known to be important for the regulation of both the hair cycle and hair growth (Sennett and Rendl, 2012). However, it has been reported that different sizes and types of hair are generated if the number of DPCs is different, even with the same number of keratinocyte stem pools (Chi
There are many studies related to hair growth and regeneration that control the microenvironment. However, little is known regarding the regulation of intracellular metabolic pathways (Chai
Autophagy is an essential cellular process tasked with maintaining homeostasis against various stresses by breaking down damaged or unfolded proteins and aged organelles. In the process of autophagy, early or initial autophagic vacuoles (AVi, autophagosome) and late or degradative autophagic vacuoles (AVd, autolysosome) are formed. The autophagosome is a double membrane structure that separates cytoplasmic material via autophagy initiation signals. It fuses with lysosomes to form autolysosomes, which then decomposes internal materials (Kraft and Martens, 2012). The importance of autophagy for the self-renewal and differentiation of epidermal and dermal stem cells in the skin has already been demonstrated (Belleudi
In conclusion, MA appears to promote DPC proliferation by inducing cell cycle progression and autophagy via the Wnt/β-catenin and ERK pathways. Our study provides scientiﬁc evidence for the applicability of MA as a treatment that could potentially alleviate hair loss.
This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region (P0002162).
The authors declare that there is no conflict of interest.