The sampling of human subcutaneous adipose tissue, isolation of ASCs, and characterization of ASCs with ASC-specific surface markers using flow cytometry were performed as previously described (28,29). Liposuction aspirates were obtained from a healthy female donor following the attainment of informed consent and approval from Bundang CHA Hospital (Seongnam, Korea; BD2011-152D). Human ASCs were cultured in α-minimum essential medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco Life Technologies) and 1% penicillin-streptomycin (Gibco Life Technologies) at 37°C in a humidified atmosphere containing 5% CO₂.

For chemical inhibition studies, the ASCs were co-treated with 5 mM U0126 (ERK12 inhibitor; EMD Millipore, Billerica, MA, USA), 5 mM LY294002 (PI3KAkt inhibitor; EMD Millipore), 0.1–1 mM Ki16425 (Sigma-Aldrich, St. Louis, MO, USA) or oleoyl-L-α-lysophosphatidic acid sodium salt (Sigma-Aldrich). For the inhibition of ROS generation, the ASCs were treated with 100 µM NADPH oxidase (Nox) inhibitor diphenyleneiodonium chloride (DPI; Sigma-Aldrich).

ASCs were seeded at 7×10³ cells/well in 48-well plates. Following incubation with the LPA (1–50 µM) for 48 or 72 h, cell numbers were measured using the Cell Counting kit (CCK)-8 Assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). Briefly, the medium in each well was replaced with medium containing the water-soluble tetrazolium salt WST-8 (10% vv). Following incubation for 2 h at 37°C, absorbance was measured at 450 nm using a Sunrise™ Microplate Absorbance Reader (Tecan Group Ltd., Männedorf, Switzerland).

Cell migration was measured using a transwell chamber. The transwell inserts with an 8 µm 3422 pore polycarbonate membrane (Corning Life Sciences, Cambridge, MA, USA), were coated with 0.1% gelatin (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 2 h at 37°C. The ASCs were seeded at 1×10⁴ cells per 200 µl in the upper chamber and incubated for 2 h at 37°C in an atmosphere containing 5% CO₂. The ASCs were allowed to migrate towards 500 µl medium containing 10 µM LPA in the lower chamber. Migration induced by serum-free medium was used as a negative control. Following 16 h incubation at 37°C, the transwell inserts were fixed with ice-cold 100% methanol for 20 min and stained with 0.23% crystal violet (Sigma-Aldrich) in 2% ethanol for 30 min. Following further washing with distilled water, the non-migratory cells were removed by gently wiping the upper face of the transwell inserts with a cotton swab. Images of the stained cells were captured using a light microscope (CKX41; Olympus, Tokyo, Japan) and were analyzed using Image J 1.47 software (National Institutes of Health, Bethesda, MD, USA), in order to determine the number of cells that had migrated to the lower side of membrane.

Intracellular ROS production was measured by flow cytometry using the ROS-sensitive dye 2′,7′-dichlorofluorescin diacetate (DCF-DA; Molecular Probes Life Technologies, Carlsbad, CA, USA), as previously described (25,26,30). Briefly, the ASCs were seeded at 2.5×10⁵ cells/60 mm dish. The following day, the cells were incubated with 20 µM DCF-DA for 10 min at 37°C, and treated with LPA for 20 min at 37°C. Following incubation, the cells were trypsinized and resuspended in PBS. Green fluorescence intensity was measured using a BD FACSCalibur flow cytometer and analyzed using CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ, USA).

Total RNA was isolated using the RNeasy kit (Qiagen, Inc., Valencia, CA, USA), and was reverse transcribed using a cDNA Synthesis kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions.

The synthesized cDNA was used in a PCR Array analysis using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's instructions. A PAHS-014ZD Human Signal Transduction Pathway Finder RT2 Profiler PCR Array (Qiagen, Inc.), which was composed of 84 transcripts representing 10 distinct signaling pathways, was used to identify the genes associated with signaling pathways that may be affected by LPA treatment. Differential gene expression and statistical analysis of the data were performed using PCR Array Data Analysis software (Qiagen, Inc.).

RT-qPCR was performed using a StepOne Plus Real-Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA) using the following primers: Adrenomedullin (ADM) forward, CTT ATT CGG CCC CAG GACAT, and reverse, ACT GGT AGA TCT GGT GTGCC; Serpine1 forward, CCG CCT CTT CCA CAA ATCAG, and reverse, AAT GTT GGT GAG GGC AGAGA; LPA receptor 1 (LPAR1) forward, TCA TCT GGA CTA TGG CCATC, and reverse, CAA GTT GAA AAT GGC CCAGA; LPAR2 forward, CAA TGC TGC TGT GTA CTCTT, and reverse, TGG GCA GAG GAT GTA TAGTG; LPAR3 forward, CTA CAA GGA CGA GGA CATGT, and reverse, ATC CTC TAT GTA CTG GCTGC; LPAR4 forward, TCT GCA AGA TCT CTG GAACT, and reverse, ACA CAA TGG CAG AAT TCCTC; LPAR5 forward, TTC TCC CGT GTC CTG ACTA, and reverse, ACA TGT ACA CGC TCA CCAC; LPAR6 forward, TTG TAT GGG TGC ATG TTCAG, and reverse, CAA GTC TGA CAT TGC CAAGT; and GAPDH forward, CGA GAT CCC TCC AAA ATCAA, and reverse, TGT GGT CAT GAG TCC TTCCA. GAPDH served as a loading control. Thermal cycling over 40 cycles consisted of an initial denaturation at 95°C for 10 min, then 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec, and was terminated by a final extension at 60°C for 1 min.

Total RNA was extracted using TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and 0.5 µg total RNA was reverse transcribed using a Mir-X miRNA First-Strand Synthesis kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer's instructions. The expression levels of miR-210 were measured by RT-qPCR using the miR-210 primer (CTG TGC GTG TGA CAG CGG CTGA; Genolution Pharmaceuticals, Inc., Seoul, Korea) as previously described (24). U6 small nuclear RNA served as a loading control (Clontech Laboratories, Inc.).

The Stealth RNAi™ siRNA specific to human Nox4 (sense, UUA UCC AAC AAU CUC CUG GUU CUCC; and antisense, GGA GAA CCA GGA GAU UGU UGG AUAA), the Silencer Select siRNA for human Serpine1 (ID# s10013), and the non-targeting negative control (scramble) siRNA (Silencer Select Negative Control#1 siRNA) were purchased from Invitrogen Life Technologies. The miR-210 inhibitor (hsa-miR210) and the miRNA inhibitor negative control were purchased from Genolution Pharmaceuticals, Inc. ASCs were seeded in 60 mm dishes at a density of 2×10⁶ with complete medium. The following day, confluent ASCs were maintained in serum-free medium without antibiotics for 2 h. Subsequently, the ASCs were transfected with 20 nM of each siRNA or miR-210 inhibitor using Lipofectamine^® 2000 transfection reagent (Invitrogen Life Technologies), according to the manufacturer's instructions in 24-well plates or 60 mm dishes.

Proteins of ASCs were isolated using SDS sample buffer. The soluble protein concentration was determined using the Quick start Bradford 1X dye reagent (Bio-Rad Laboratories, Inc.). The western blot analysis was performed as previously described (25) using antibodies targeting p-ERK12 (1:2,000; cat. no. 9106; in 5% milk), ERK12 (1:4,000; cat. no. 9102; in 5% milk), p-Akt (1:1,000; cat. no. 9271; in 5% milk), and Akt (1:4,000; cat. no. 9272; in 5% milk). All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish-peroxidase (HRP)-conjugated secondary mouse antibody (1:10,000) was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA) and HRP-conjugated secondary rabbit antibody was purchased from Cell Signaling Technology, Inc. (1:10,000; cat. no. 7074).

All data are presented as the mean ± standard deviation. Statistical analysis of the data was performed using Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA). A Student's t-test was used to analyze the data. P<0.05 was considered to indicate a statistically significant difference.

The present study initially investigated whether LPA treatment increased the proliferation and migration of ASCs. As expected, 1–50 mM LPA significantly increased the proliferation (Fig. 1A) and migration (Fig. 1B) of ASCs in a dose-dependent manner. Since PI3K/Akt and mitogen-activated protein kinase (MAPK) signaling pathways have previously been reported to be associated with the mitogenic effects of LPA, the phosphorylation levels of Akt and ERK12 were measured following LPA treatment, and phosphorylation of both proteins increased in a time-dependent manner (Fig. 1C). In the inhibition study, pharmacological inhibition of PI3K/Akt by LY294002 significantly reduced the LPA-induced proliferation of ASCs (Fig. 1D). In addition, pharmacological inhibition of PI3K/Akt by LY294002, and of ERK by U0126 significantly reduced the LPA-induced migration of ASCs (Fig. 1E). These results suggest that LPA acts as a strong mitogenic factor in ASCs.

The expression levels of LPAR were measured in the ASCs (Table I), indicating that LPAR1 is highly expressed in ASCs, as compared with the other isoforms. To measure the involvement of LPAR in the mitogenic function of LPA, pharmacological inhibition of LPAR by Ki16425 was performed. Co-treatment with Ki16425 (0.1 and 1 mM) significantly attenuated LPA-induced proliferation (Fig. 2A) and migration (Fig. 2B) of ASCs.

The present study also investigated whether the LPA-induced proliferation and migration of ASCs was mediated by ROS generation. As expected, LPA treatment significantly increased the fluorescence intensity of DCF-DA, as determined by flow cytometric analysis (Fig. 3A); and pharmacological inhibition of Nox following treatment with DPI significantly attenuated the fluorescence intensity of DCF-DA (Fig. 3B). In addition, DPI treatment significantly reduced LPA-induced proliferation (Fig. 3C) and migration (Fig. 3D) of ASCs. As Nox4 is highly expressed in ASCs and primarily mediates ROS generation (26), downregulation of Nox4 expression in ASCs by siRNA transfection and Nox4 silencing significantly decreased LPA-induced cell proliferation (Fig. 3E) and migration (Fig. 3F). These results suggest that Nox4 may be associated with LPA-mediated ROS generation and downstream mitogenic effects in ASCs.

Our previous study demonstrated that ROS generation induced by hypoxia, or chemicals, induced miR-210 expression and increased the proliferation and migration of ASCs (24); therefore, the present study investigated the involvement of miR-210 in the LPA-induced proliferation and migration of ASCs. As expected, LPA treatment increased miR-210 expression (Fig. 4A); however, the extent of upregulation was not as great as that induced by hypoxia and chemical ROS donors. In the inhibition study, miR-210 inhibitors significantly attenuated LPA-induced proliferation (Fig. 4B) and migration (Fig. 4C) of ASCs. These results suggest that miR-210 is involved in the LPA-induced proliferation and migration of ASCs.

The human Signal Transduction Pathway Finder RT2 Profiler PCR Array was used to identify the LPA target genes that mediated the proliferation and migration of ASCs. A total of 4 h following LPA treatment, the expression levels of ADM increased 5.12-fold, as demonstrated by the PCR array (Fig. 5A). RT-qPCR analysis confirmed that the expression levels of ADM were upregulated ~6-fold (Fig. 5B). A total of 24 h following LPA treatment, the expression levels of Serpine1 increased 8.28-fold, as determined by the PCR array (Fig. 5C), and ~18-fold, as determined by the RT-qPCR (Fig. 5D). Therefore, the present study investigated whether ADM and/or Serpine1 mediated LPA-induced proliferation and migration of ASCs using siRNAs specific for ADM and Serpine1. Although silencing of ADM did not affect proliferation or migration (data not shown), transfection with Serpine1 siRNA significantly attenuated the LPA-induced migration of ASCs (Fig. 5E).