LL-37 boosts fat tissue stem cell functions through early growth response 1 and MAPK signaling

Subcutaneous adipose tissue was obtained during elective surgeries with written informed consent, as approved by the Samsung Medical Center Institutional Review Board. All donors were < 40 years old and did not have diabetes or acute inflammation. The mean body mass index of the donors was 25.2 ± 3.64. Human ASCs were isolated according to a previous protocol [23] and cultured in low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5 % CO₂. ASCs were characterized by the presence of the cell surface markers CD73, CD90, and CD105 and the absence of CD11b, CD34, CD45, and HLA-DR [24].

Cells were treated with human LL-37 (Phoenix Pharmaceuticals, USA) for 48 hr under serum deprivation conditions. Cell viability was determined by Trypan blue staining. Cell proliferation was measured with the cell counting kit (CCK)-8 according to the manufacturer’s protocol (Dojindo, Japan). ASCs (5 × 10³ cells/well) were treated with 2.5–20 μg/mL LL-37 for 24 and 48 hr prior to adding CCK-8 solution. Absorbance at 450 nm was determined with a multi-plate reader (Molecular Devices, CA, USA).

A cell migration assay was performed using Transwell plates (8 μm pore size; Costar, Corning, NY, USA) according to a previous study [25]. Briefly, ASCs were suspended in serum-free medium and 100 μL of the cell suspension (7 × 10⁵ cells/mL) was added to each upper well. LL-37 at the indicated concentrations (5, 10, and 20 μg/mL) was placed in the lower wells of a 24-well tissue culture plate. After incubation for 6 hr at 37 °C, cells that had migrated were stained with 0.15 % crystal violet and counted in five random microscopy fields using the Scanscope scanning system (Aperio Scanscope, CA, USA).

Cells were transfected with EGR1-targeting small interfering RNA (siRNA) (Santa Cruz Biotechnology, USA) or negative control siRNA (Bioneer, Daejeon, Korea) using Lipofectamine RNAi (Invitrogen, CA, USA). Briefly, when cells reached 60–70 % confluency, siRNA (final concentration, 100 nM) was combined with Lipofectamine RNAi and allowed to complex for 20 min. The transfection mixture was then applied to ASCs and incubated for 6 hr at 37 °C. Subsequently, cells were maintained in complete medium for 36 hr before being subjected to the cell migration assay and enzyme-linked immunosorbent assays (ELISAs).

Total protein from cell lysates was separated by SDS-PAGE and transferred to a nitrocellulose membrane, which was incubated with the corresponding primary antibodies (anti-EGR1 (Santa Cruz Biotechnology) and anti-GAPDH (Cell Signaling, USA)) overnight at 4 °C. Washed membranes were incubated for 1 hr with a horseradish peroxidase-conjugated anti-mouse secondary antibody. Bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). The band intensities were quantified using TotalLab software (UK).

FPRL1 expression in ASCs from four donors was evaluated by surface staining. Cells were washed with fluorescence-activated cell sorting (FACS) buffer, stained with a mouse anti-FPRL1 antibody (R&D Systems, USA), and then stained with anti-mouse IgG-FITC (Sigma, USA). Labeled cells were measured with a FACSCalibur instrument (Becton Dickinson Biosciences, CA, USA) and analyzed using the Win MDI program (Win MDI version 2.8).

Immunofluorescence analysis was performed as previously described [23]. Briefly, ASCs (5 × 10³ cells/well) were seeded on four-well Lab-Tek II chamber slides (Nalge Nunc International, IL, USA), treated with LL-37 for 48 hr, fixed, and permeabilized for 20 min with 0.1 % Triton X-100 prepared in phosphate-buffered saline. After washing, cells were blocked and incubated with an anti-proliferating cell nuclear antigen (PCNA) antibody (Abcam, Cambridgeshire, UK) for 1 hr. Next, cells were incubated with goat anti-mouse IgG-Alexa Fluor 488 (Invitrogen) for 30 min at 37 °C. A nucleic acid dye (DAPI; 0.5 μg/mL) was added to stain the nuclei. PCNA immunofluorescence was detected with an LSM700 confocal microscope system (Carl Zeiss, NY, USA; 400× objective).

Cells were plated in six-well plates (300,000 cells/well) and treated with 20 μg/mL LL-37 in serum-free medium. After 48 hr, the culture medium was collected and ELISAs were performed for thymosin beta-4 (TB4; Immune Diagnostics, Canada), vascular endothelial growth factor (VEGF; Invitrogen), monocyte chemoattractant protein-1 (MCP-1; eBioscience, USA), stromal cell-derived factor-1 (SDF-1; RayBiotech, USA), and EGR1 (EIAab, China), in accordance with the manufacturers’ recommendations. For detection of EGR1, the cells were lysed by the addition of 200 μL of cell lysis buffer after LL-37 treatment for 6 hr.

Total RNA was extracted using TRIzol reagent (Invitrogen). After removing possible DNA contamination by DNAse treatment of the extracted RNA, cDNA was synthesized using 2 μg of total RNA and SuperScript II reverse transcriptase according to the manufacturer’s instructions. The primers used are provided in Additional file 1: Table S1. For real-time PCR, quantitative PCR was performed using the 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the Power SYBR Green qPCR Master Mix Kit (Life Technologies, CA, USA). The cycling profile for real-time PCR (50 cycles) was as follows: 95 °C for 10 min, 95 °C for 15 sec, and 60 °C for 60 sec. The comparative threshold cycle (Ct) method (i.e., 2^–ΔΔCt) was used to calculate fold amplification.

The microarray data are accessible in the Gene Expression Omnibus (GEO) database under accession number GSE76392↗. For the microarray, human ASCs were treated with 20 μg/mL LL-37 for 1 hr. Each total RNA sample (200 ng) was labeled and amplified using the Low Input Quick Amp Labeling Kit (Agilent Technologies, CA, USA). Cy3-labeled aRNAs were resuspended in 100 μL of hybridization solution (Agilent Technologies). Labeled aRNAs were placed on the Agilent SurePrint G3 Human GE 4x44K array (Agilent Technologies) and covered with a Gasket 8-plex slide (Agilent Technologies). Slides were hybridized for 17 hr at 65 °C. The hybridized slides were sequentially washed in 2× SSC containing 0.1 % SDS for 2 min, 1× SSC for 3 min, and 0.2× SSC for 2 min at room temperature. Finally, the slides were centrifuged at 3000 rpm for 20 sec to dry. Arrays were analyzed using an Agilent scanner with associated software. Gene expression levels were calculated with Feature Extraction v10.7.3.1 (Agilent Technologies). The relative signal intensities of each gene were generated using the Robust Multi-Array Average algorithm. Data were processed based on the quantile normalization method using GeneSpring GX 13.0 (Agilent Technologies). This normalization method aims to make the distribution of intensities for each array in a set of arrays the same. The normalized and log-transformed intensity values were analyzed using GeneSpring GX 13.0. Fold-change filters included the requirement that the genes be present in at least 200 % of controls for upregulated genes and less than 50 % of controls for downregulated genes. Hierarchical cluster analysis was conducted using the Cluster 3.0 program, the Euclidean distance, and average linkage algorithm.

Five-week-old male C57BL/6 mice were purchased from SLC Inc. (Haruno, Japan) and allowed to adapt to their new environment for 2 weeks. The fur on the backs of 7-week-old mice (n = 14) was shaved with hair clippers and removed with hair removal cream. Conditioned medium (CM) was topically applied daily for up to 18 days. Pigment darkening and the hair growth rate were monitored every 3 days for 2 weeks. Hair growth was evaluated by three independent dermatological scientists. The hair growth score was determined using the method described by Vegesna et al. [26, 27]. The animal experiments were approved by the Institutional Animal Care and Use Committee of Samsung Medical Center (approval number: SMC-IACUC-2013-0103-003) and all experiments followed regulatory standards.

hEGR1 was subcloned into the Hind III/Xho I sites of pcDNA3.1(+). After digestion of the pcDNA3.1(+)-hEGR1 plasmid with the restriction enzymes, the resulting hEGR1 gene along with the BamHI and SfiI sites was inserted into the multiple cloning site of the expression plasmid pLenti6/V5-D-TOPO (Invitrogen), which contains a cytomegalovirus promoter upstream of the inserted gene. The resulting plasmid was named pLenti6/V5-hEGR1.

The lentiviral expression system based on four plasmids was obtained from Invitrogen. Briefly, 2.5 μg of pLP1, 2.5 μg of pLP2, 2.5 μg of pLP/VSVG, and 2.5 μg of the lentiviral vector pLenti6/V5-hCAMP were co-transfected with Fugene6 (Roche) into HEK293T cells cultured in 10 cm plates. The medium was changed every 24 hr. After 48 hr, supernatants were pooled, filtered through a 0.45-μm filter, and centrifuged at 6000 rpm at 4 °C for 16 hr. The pellet was resuspended in 1 mL of complete medium and stored at −80 °C. The resulting recombinant lentivirus was added to ASCs cultured in 10 cm plates. At 48 hr after seeding, the medium was replaced with complete medium containing 10 μg/mL blasticidin as a selective agent. At 18 days after transfection, selected cell colonies were split into 60 mm petri dishes. Isolated cell colonies were cultured and expanded.

Statistical significance was estimated using the Student’s t-test. Mean differences were considered to be significant when P < 0.05.

To investigate the role of LL-37 in ASC proliferation, we examined the effect of LL-37 on the proliferative ability of ASCs using the CCK-8 assay. LL-37 treatment markedly stimulated ASC proliferation in a dose-dependent manner without causing cytotoxicity (Fig. 1b and Additional file 2: Figure S1a). Furthermore, we determined the number of proliferating cells by performing immunofluorescence staining of PCNA, a nuclear protein associated with cell proliferation. The proportion of ASCs with positively stained nuclei (green fluorescence) was markedly enhanced by LL-37 treatment (Fig. 1c).

To investigate the effects of LL-37 on ASC migration, a Transwell migration assay was performed. LL-37 treatment rapidly increased human ASC migration within 6 hr; this effect was dose-dependent, with maximal stimulation at 20 μg/mL among the concentrations tested (Fig. 1d). ASCs were pretreated with pertussis toxin (Ptx), a Gαi inhibitor, or a neutralizing anti-LL-37 antibody (αLL-37) before activation with LL-37. Ptx or αLL-37 treatment prior to LL-37 stimulation significantly inhibited ASC migration and proliferation (Fig. 1e and f). Taken together, these data suggest that LL-37 induces ASC proliferation and migration through the Gαi-coupled receptor FPRL1.

Next, to study the involvement of EGR1 in LL-37-enhanced ASC proliferation and migration, ASCs were transfected with EGR1-targeting siRNA. We confirmed EGR1 gene silencing at the protein level (Fig. 2c). LL-37-enhanced proliferation and migration of ASCs were markedly attenuated by EGR1-targeting siRNA transfection (Fig. 2d and e). These data suggest that EGR1 is important for LL-37-enhanced migration and proliferation of ASCs.

To further evaluate the paracrine effects of LL-37 in vivo, the hair growth of C57BL/6 mice with hair loss was tested in vivo. We compared hair growth after topically applying CM of ASCs pre-activated with or without LL-37 (20 μg/mL). After 2 weeks, the hair growth score was higher in the group treated with CM of ASCs than in the negative control group treated with medium alone. Moreover, the hair growth score was higher in the group treated with CM of ASCs pre-activated with LL-37 than in the group treated with CM of non-activated ASCs (Fig. 4i and j), with dark pigmentation and hair regeneration observed on the initially pink hairless skin. Treatment with minoxidil (MNX), which is widely used to treat hair loss, was also effective for hair regeneration. Specifically, 95–100 % of hair was regenerated to full length in the group treated with CM of ASCs pre-activated with LL-37 in comparison to the MNX-treated group, whereas the level of regeneration was lower in the group treated with CM of non-activated ASCs (30–35 %; Fig. 4i). Additionally, to elucidate the biological function of EGR1 in vitro and in vivo, EGR1 was lentivirally introduced into human ASCs. Cell lines stably expressing the control vector and EGR1 were designated pLenti6/V5-D-TOPO and pLenti6/V5-hEGR1, respectively. As expected, the secretion levels of VEGF, TB4, MCP-1, and SDF-1 were significantly higher in the medium of cells overexpressing EGR1 than in the medium of wild type (WT) cells or those expressing the control vector (Additional file 2: Figure S2a–2d). To confirm the paracrine effects of stable EGR1-overexpressing cells in vivo, we performed an additional investigation of hair regeneration. Mice were treated with CM of WT ASCs, ASCs expressing the control vector, or EGR1-overexpressing ASCs. Compared with CM of WT ASCs and ASCs expressing the control vector, treatment with CM of EGR1-overexpressing ASCs considerably increased hair regeneration (Additional file 2: Figure S2e and 2f).

Taken together, LL-37 might mediate paracrine actions by stimulating the secretion of growth factors such as VEGF, TB4, SDF-1, and MCP-1 by ASCs. In addition, CM from ASCs pre-activated with LL-37 and EGR1-overexpressing cells strongly promotes hair growth in vivo.