TISSUE‐SPECIFIC STEM CELLS
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College of Pharmacy, Yonsei Uni‐ versity, Incheon, Korea; 2 Department of Medical Science, CHA University, Kyunggi‐do, Korea; 3 College of Pharmacy, Ajou Univer‐ sity, Kyunggi‐do, Korea; 4 Translational Research Center, Inha University School of Medicine, Incheon, Korea Address correspondence to: Jong‐ Hyuk Sung, Ph.D, College of Phar‐ macy, Yonsei University, #162‐1, Songdo‐dong, Yeonsu‐gu, Incheon, 406‐840, Korea, Tel: +82‐32‐749‐ 4506, e‐mail:
[email protected]; *equal contribution; Abbreviation: ASCs Adipose‐derived stem cells, PDGF Platelet‐derived growth fac‐ tor, mtROS Mitochondrial reactive oxygen species, mito‐CP Mito‐ carboxy proxyl, Mdivi‐1 Mitochon‐ drial division inhibitor‐1, p66Shc 66‐kDa Src collagen homologue, BCL2A1 BCL2‐related protein A1, SERPINE1 Serpine peptidase inhibi‐ tor, clade E, member 1, FGF Fibrob‐ last growth factor, HBEGF Heparin‐ binding EGF‐like growth factor, IL11 Interleukin 11, INHBA Inhibin, beta A, LIF Leukemia inhibitory factor, VEGFA Vascular endothelial growth factor A Received June 27, 2014; accepted for publication September 14, 2014 ©AlphaMed Press 1066‐5099/2014/$30.00/0 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typeset‐ ting, pagination and proofreading process which may lead to differ‐ ences between this version and the Version of Record. Please cite this article as doi: 10.1002/stem.1865
Functional Regulation of Adipose‐derived Stem Cells by PDGF‐D JI HYE KIM1*, SANG GYU PARK2,3*, WANG‐KYUN KIM1, SUN U. SONG4, JONG‐HYUK SUNG1 Key words. PDGF‐D • adipose‐derived stem cells • mitogenic effect • para‐ crine effect • mitochondrial ROS generation ABSTRACT Platelet‐derived growth factor‐D (PDGF‐D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF‐D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF‐D in adipose‐derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF‐D in the present study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF‐D‐induced sti‐ mulation of ASCs. It is of interest that PDGF‐B is not expressed, but PDGF‐D and PDGF receptor‐β are expressed in ASCs. PDGF‐D showed the strongest mitogenic effect on ASCs, and PDGF‐D regulates the proliferation and mi‐ gration of ASCs through the PI3K/Akt pathways. PDGF‐D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS genera‐ tion and fission were mediated by p66Shc phosphorylation, and BCL2A1 and SERPINE1 mediated the proliferation and migration of ASCs. In addi‐ tion, PDGF‐D up‐regulated the mRNA expression of diverse growth factors such as VEGFA, FGF1, FGF5, LIF, INHBA, IL11 and HBEGF. Therefore, the preconditioning of PDGF‐D enhanced the hair‐regenerative potential of ASCs. PDGF‐D‐induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen‐activated protein kinase pathway. In summary, PDGF‐D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF‐D also up‐regulates growth factor expression in ASCs. Therefore, PDGF‐D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation. STEM CELLS 2014; 00:000–000
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Functional regulation of ASCs by PDGF‐D
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INTRODUCTION Adipose‐derived stem cells (ASCs) are a mesenchymal stem cell (MSC) obtained from subcutaneous adipose tissue that exhibits wound healing and anti‐wrinkling effects on skin [1‐3]. Recently, the hair regenerative potential of ASCs has been reported for secretomes of ASCs, which have been found to induce telogen‐to‐ anagen shifts in mice, while preconditioning with hy‐ poxia or vitamin C was observed to enhance the hair regenerative potential of ASCs [4, 5]. Of the growth fac‐ tors secreted, platelet derived growth factor (PDGF) expression in ASCs regulates hair follicular stem cell activity and induces the anagen phase of the hair cycle in vivo [6]. In addition, local injection of recombinant PDGF‐A and ‐B also induce and maintain the anagen phase of murine hair follicles [7]. PDGF is one of the growth factors that regulate cell growth and division [8, 9]. PDGF is mitogenic during early developmental stages, driving the proliferation and migration of undifferentiated mesenchyme and some progenitor populations [10, 11]. During later ma‐ turation stages, PDGF signaling has been implicated in tissue remodeling and cellular differentiation, and in patterning and morphogenesis. In particular, it plays a significant role in blood vessel formation. The PDGF signaling network consists of four ligands: PDGF‐A, ‐B, ‐ C, and ‐D. All PDGFs function as disulfide‐linked homo‐ dimers, and PDGF‐A and ‐B can form functional hetero‐ dimers. The PDGF receptor (PDGFR) is classified as a receptor tyrosine kinase, and two receptor types (i.e., PDGFR‐α and PDGFR‐β have been identified to date [12, 13]. Upon activation, PDGFRs dimerize, and are auto‐phosphorylated on their cytosolic domains, which subsequently activate signal transduction through the PI3K pathway or through the generation of reactive oxygen species (ROS) [14‐16]. PDGF‐C and D have re‐ cently been identified, and PDGF‐D mediates its func‐ tion through PDGFR‐β [17, 18]. PDGF‐D forms only ho‐ modimers and does not dimerize with the other three family members. PDGF‐D reportedly induces cellular transformation and promotes tumor growth [19]. Because PDGF has a potent mitogenic effect on me‐ senchymal cells, there are many reports on the effects of PDGF isoforms and their receptors on MSCs. For ex‐ ample, MSCs are source of PDGF and act as a niche for hematopoetic stem cells in bone marrow [20]. PDGF‐B was reported to regulate the proliferation and invasion of MSCs through ERK and Akt signaling pathways [21]. PDGFR‐α has been shown to be involved in the forma‐ tion of smooth muscle actin filaments in MSCs [22]. Inhibition of PDGFR by chemical inhibitors regulated Oct4 and Nanog expression, and modulated the cell shape and the potency of MSCs [23]. Vascular endo‐ thelial growth factor (VEGF) can signal through the PDGFR in MSCs [24]. In ASCs, PDGF induced the prolife‐ ration and migration of ASCs through c‐Jun N‐terminal kinase, and PDGF‐B increased the proliferation and mi‐ www.StemCells.com
gration of ASCs via ROS generation and miR‐210 up‐ regulation [25, 26]. PDGF was shown to mediate this paracrine effect, and PDGF secreted from ASCs helped to maintain the hair cycle and induce anagen [6]. Tis‐ sue‐resident ASCs secreted PDGF‐D and induced the epithelial‐mesenchymal transition in breast cancer [27]. Although PDGF and its receptor isoforms play autocrine and paracrine roles in ASCs, the mechanism of action for ASC regulation has not been fully identified. Of the PDGF isoforms, the mitogenic effect of PDGF‐ B has been well reported in ASCs. Although PDGF‐B is exogenous since ASCs do not express PDGF‐B, we found that PDGF‐B exhibits the strongest effect on ASC proli‐ feration and migration of all tested growth factors such as PDGF‐A, PDGF‐B, VEGF, epithelial growth factor, insu‐ lin‐like growth factor, and basic fibroblast growth factor [26, 28]. In addition, pharmacological inhibition of PDGFR‐β significantly reduced the proliferation and migration of ASCs [26]. PDGF‐A, ‐C, and ‐D are ex‐ pressed in ASCs; however, PDGF‐B is not [27]. There‐ fore, we should pay attention to the autocrine and pa‐ racrine role of PDGF‐D in ASCs. PDGF‐D functions as a potent transforming and angiogenic growth factor via the PDGFR‐β in cancer cells [19]. In addition, PDGF‐D has been shown to increase cell migration and invasion more effectively than PDGF‐B [29]. However, the func‐ tional role of PDGF‐D in ASCs has not been fully identi‐ fied, especially its effect on growth factor secretion. Therefore, we primarily investigated the stimulatory effect of PDGF‐D on the proliferation, migration and growth factor secretion of ASCs. Furthermore, we iden‐ tified the signaling pathways and the molecular me‐ chanisms involved in PDGF‐induced stimulation of ASCs.
MATERIALS AND METHODS
Materials Human PDGF isoforms (PDGF‐A, B and C) were obtained from Sigma‐Aldrich Co. (Saint Louis, MO, USA). Human PDGF‐D was obtained from R&D systems (Minneapolis, MN, USA). In the pharmacological inhibition study, vari‐ ous inhibition conditions such as LY294002 (PI3K/AKT inhibitor, Calbiochem, San Diego, CA, USA), U0126 (ERK inhibitor, Calbiochem), cp673451 (PDGFR‐β inhibitor, Selleckchem, Houston, TX, USA) and Mdivi‐1 (mito‐ chondria fission inhibitor, Enzo Life Sciences, Farming‐ dale, NY, USA) were used. Mito‐carboxy proxyl (Mito‐ CP, mtROS scavenger) was synthesized as previously described [30]. Antibodies recognizing Akt (1:4000), phospho‐Akt (1:2000), ERK (1:4000), phospho‐ERK (1:3000) were purchased from Cell Signaling Technology (Danvers, MA, USA). p66Shc‐ser34 (1:100 or 1:1000) was purchase from Calbiochem. Horseradish‐peroxidase (HRP)‐ conjugated secondary mouse antibody (1:10000) and HRP‐conjugated secondary rabbit antibody (1:1000)
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3 were purchased from Cell Signaling Technology (Danv‐ ers, MA, USA). Human signal transduction pathwayfinder RT2 profi‐ ler PCR Array (PAHS‐041ZD) and human growth factor RT2 profiler PCR array (PAHS‐041ZA) were obtained from SABiosciences (Qiagen, Hilden, Germany).
ASC Culture Human ASCs were isolated via liposuction of subcuta‐ neous fat, as described previously [1], after informed consent was obtained (Boondang CHA hospital, BD2011‐152D). ASCs were cultured in Minimum Essen‐ tial Medium Alpha Medium (α‐MEM, Hyclone, Thermo Scientific, Logan, UT, USA) with 10% fetal bovine serum (FBS; GIBCO, Invitrogen, Carlsbad, CA, USA), 1% penicil‐ lin and streptomycin (GIBCO). ASCs were maintained at 37 °C in a humidified 5% CO2 incubator, and ASCs at passage 7‐9 were used in this study. Characterization of ASCs was performed using flow cytometry. ASCs were positive for CD44, CD73, CD90, CD105, HLA‐I, and PODXL, but were negative for hematopoietic markers such as CD34 and CD45 [26, 30]. Multipotent differen‐ tiation potential was examined as described previously [31], and ASCs could be differentiated into adipocytes, osteocytes, and chondrocytes.
Cell proliferation assay
ASCs were seeded on 48‐well plates at density of 7×103 cells per well. After 24 h, the medium was replaced with 0.2% FBS in α‐MEM. The day following the medium change, cells were treated with PDGF isoforms (10 ng/ml) or inhibitors (10 μM LY294002, 10 μM U0126, 1 μM cp673451 and 1 μM Mdivi‐1) for 48 h. Then, the medium was removed and cell number was measured using CCK‐8 assay kit (Dojindo, Rockville, MD, USA). Cells were treated with 10% of CCK‐8 solution for 2 h at 37 °C in a humidified 5% CO2 incubator. The absorbance was measured at 450 nm by a microplate reader ma‐ chine (Tecan, Gordig, Austria). In addition, the kinetics of cell proliferation were measured using the IncucyteTM (Essen Bioscience, Mich‐ igan, USA) live cell imaging system. ASCs were seeded 5 on six‐well plates at a density of 1×10 cells per well with 0.2% FBS in α‐MEM. The following day, cells were treated with PDGF isoforms (10 ng/ml) and ASCs were monitored with Incucyte. Cell confluence was measured at 4‐h intervals for 72 h.
Cell migration assay For the migration assay, ASCs were seeded on the up‐ per site of trans‐well membrane plates (Corning Inc., Corning, NY, USA) at density of 2×104 cells per well. Af‐ ter 2 h, PDGFs (10 ng/ml) or inhibitors (10 μM LY294002, 10 μM U0126, 1 μM cp673451 and 1 μM Mdivi‐1) in α‐MEM medium containing 0.2% FBS were introduced on the lower site of trans‐well membrane plates for 16‐20 h. Then, migrated cells remaining in the trans‐well membrane were fixed with ice‐cold methanol www.StemCells.com
Functional regulation of ASCs by PDGF‐D for 20 min. Fixed cells were stained using 10% crystal‐ violet (Sigma‐Aldrich Co.) for 30 min at room tempera‐ ture and cells in the membrane were counted by light microscopy. The number of migrated cells in a 6.5‐mm (diameter) trans‐well with an 8.0‐μm pore polycarbo‐ nate membrane was also counted. In addition, the kinetics of cell migration were measured using scratch migration assay [32]. ASCs were seeded on 6‐well plates at a density of 2×105 cells per well. After 24 h, wounds were made using a 200‐μl pi‐ pette tip. Cell migration was determined by microscopy (Olympus, Shinjuku, Tokyo, Japan) 20 h after wounding. For evaluation of ASC migration, ten randomly selected points along each wound were marked, and the hori‐ zontal distance of migrating cells from the initial wound was measured.
Cellular and mitochondria‐specific ROS gen‐ eration assay ROS generation was measured using DCF‐DA (Molecular Probes, Eugene, OR, USA) as previously described [33, 34]. Similarly, mtROS generation was measured using Mito‐Sox (Molecular Probes). Cells were seeded on 60‐ mm dishes in 0.2% FBS in α‐MEM medium. Cells were co‐treated with PDGF‐D (10 ng/ml) and 5 μM Mito‐Sox for 2 h at 37 °C, incubated in complete darkness, and harvested with trypsin‐EDTA. Fluorescence was meas‐ ured using a flow cytometer (Becon Dickinson, Franklin Lakes, NJ, USA) and fluorescent images were taken by confocal microscope (Carl Zeiss, Oberkochen, Germa‐ ny).
Mitochondrial fission staining ASCs were seeded on circular cover glass and treated with PDGF‐D (10 ng/ml) with or without inhibitors (Mdivi‐1, 1 μM) for 72 h. For mitochondrial staining, cells were incubated with 500 nM of MitoTracker Red (Molecular Probes) in α‐MEM for 30 min. The morphol‐ ogy of mitochondria and fluorescence intensity of Mito‐ Tracker Red were examined by confocal microscopy (Carl Zeiss). For nuclear staining, cells were fixed with 4% para‐ formaldehyde for 15 min and permeabilized using 0.5% PBS‐T for 5 min. Fixed cells were treated with DAPI (Sigma‐Aldrich Co.) for 10 min at room temperature.
Western blotting Cell extractions were isolated with SDS lysis buffer. Pro‐ teins were separated by 10% SDS‐PAGE gel and trans‐ ferred to PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non‐fat milk for 1 h at room temperature and incubated with prima‐ ry antibody overnight at 4 °C. The following day, the membrane was washed with TBS‐T (0.1% Tween 20 in Tris‐buffered saline) and incubated with HRP‐ conjugated secondary antibody for 1 hr. The membrane reacted to ECL solution (Millipore) and was exposed by X‐ray film (AGFA, Gevaert, Mortsel, Belgium). ©AlphaMed Press 2014
Functional regulation of ASCs by PDGF‐D
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Immunostaining of p66Shc‐ser34 For staining of p66Shc‐ser34, ASCs were seeded on cir‐ cular cover glass. The following day, cells were incu‐ bated with PDGF‐D (10 ng/ml) for 30 min. Then, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.5% PBS‐T for 5 min. After wash‐ ing with 0.1% PBS‐T, cells were blocked using blocking solution (10% FBS and 0.5% gelatin in PBS) for 30 min. Primary antibody (p66Shc‐ser34) was diluted to a con‐ centration of 1:100 and secondary antibody (FITC, Invi‐ trogen) to 1:200. For nuclear staining, cells were treated with DAPI. Fluorescence signals were detected by fluo‐ rescence confocal microscope.
siRNA transfection Twenty nanomolar mixtures of p66Shc siRNA, BCL2A1 siRNA and SERPINE1 siRNA (Dharmacon, Lafayette, LA, USA) were prepared with Lipofectamine 2000 (Invitro‐ gen) before cell trypsinization step. Then, cells were seeded on 6‐well plates or 60‐mm dishes, and siRNA mixtures were transfected into the cells for 48 hrs. Si‐ lencing was evaluated by quantitative PCR (QPCR), and significantly down‐regulated p66Shc mRNA level (data not shown).
RNA isolation and QPCR
Total RNA was extracted using RNA prep kit (RNeasyTM, Qiagen) and isolated mRNA was reverse‐transcribed with a cDNA synthesis kit (A3500, Promega, Madison, WI, USA). cDNA was synthesized from 500 ng of total RNA by 1000 U reverse transcriptase and 50 ng/μl oli‐ go(dT) primers. Thermal cycling took place over 35 cycles of 95 °C for 5 min, then 95 °C for 30 sec, 56 °C for 20 sec, 72 °C for 40 sec, and terminated at 72 °C for 5 min. QPCR reactions were performed on a Step One Plus Real‐Time PCR system (Applied Biosystems, Invi‐ trogen) using SYBR Green PCR Master Mix (Takara Bio, Inc., Otsu, Japan). The level of GAPDH was also quanti‐ fied for sample standardization. Analysis of fold change was calculated by ΔCt value.
PCR array For analysis with the human transduction pathwayfind‐ er RT2 profiler PCR array (signal transduction pathway: PAHS‐041ZD; growth factor pathway: PAHS‐041ZA), cells were seeded on 60‐mm dishes at density of 2.5×105 cells in 0.2% FBS in α‐MEM medium. Total RNA was harvested with an RNA prep kit (Qiagen) and cDNA was synthesized using reverse transcriptase. Gene ex‐ pression was detected by a PCR array kit according to the manufacturer’s instructions (Qiagen).
Animal experiment Mice were maintained and anesthetized according to a protocol approved by the United States Pharmacopoeia (USP) and the Institutional Animal Care and Use Com‐ mittee of CHA University (IACUC120002). The dorsal www.StemCells.com
area of 7‐week‐old C3H/HeN mice in the telogen stage of the hair cycle was shaved with a clipper and electric shaver, with special care taken to avoid damaging the bare skin. ASCs (1×104) or PDGF‐D‐treated cells (24 h pretreatment with 10 ng/ml PDGF‐D without FBS, 1×104) were injected into the dorsal skin of shaved mice [5]. Any darkening of the skin (indicative of hair cycle induction) was carefully monitored by photography. After 15 days, dorsal hair was shaved and its weight was measured.
Statistical analysis Generally, in vitro data were representative of indepen‐ dent experiments performed in triplicate. The statistical significance of the differences among groups was tested using one‐way analysis of variance (ANOVA) or the Stu‐ dent’s t‐test. Error bars are indicative of standard devia‐ tion. P‐values of