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Alpha-mangostin promotes myoblast differentiation by modulating the gene-expression profile in C2C12 cells a

a

Taro Horiba , Masahiro Katsukawa , Keiko Abe a

bc

& Yuji Nakai

b

Research and Development Division, Kikkoman Corporation, Noda, Japan

b

Graduate School of Agricultural and Life Sciences, Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo c

Kanagawa Academy of Science and Technology, Life Science & Environment Research Center, Kawasaki-ku, Kawasaki, Japan Published online: 25 Jul 2014.

To cite this article: Taro Horiba, Masahiro Katsukawa, Keiko Abe & Yuji Nakai (2014) Alpha-mangostin promotes myoblast differentiation by modulating the gene-expression profile in C2C12 cells, Bioscience, Biotechnology, and Biochemistry, 78:11, 1923-1929, DOI: 10.1080/09168451.2014.940832 To link to this article: http://dx.doi.org/10.1080/09168451.2014.940832

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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 11, 1923–1929

Alpha-mangostin promotes myoblast differentiation by modulating the gene-expression profile in C2C12 cells Taro Horiba1,*, Masahiro Katsukawa1, Keiko Abe2,3 and Yuji Nakai2 1

Research and Development Division, Kikkoman Corporation, Noda, Japan; 2Graduate School of Agricultural and Life Sciences, Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo; 3Kanagawa Academy of Science and Technology, Life Science & Environment Research Center, Kawasaki-ku, Kawasaki, Japan

Received March 20, 2014; accepted May 27, 2014

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http://dx.doi.org/10.1080/09168451.2014.940832

Alpha-mangostin, a xanthone contained mostly in mangosteen pericarp, has been reported to exert various biological functions. However, little is known about involvement of this xanthone in the muscle differentiation process. Here, we report the effect of α-mangostin on murine skeletal muscle-derived C2C12 myoblasts. α-mangostin stimulated myoblast differentiation leading to myotube formation. DNA microarray analysis revealed that genes associated with myoblast differentiation and muscle cell component formation were up-regulated in α-mangostintreated cells. These results indicate that α-mangostin promotes myoblast differentiation through modulating the gene-expression profile in myoblasts. Key words:

alpha-mangostin; DNA microarray; myoblast; myotube; xanthone

Mammalian skeletal myogenesis is a complex process consisting of the proliferation of mononucleated myoblasts, cell cycle withdrawal, cell fusion to form myotubes, and subsequent maturation of myotubes into myofibers. This myogenic differentiation process requires various interactions between diverse cellular processes.1–3) The process for whether myoblasts continue to proliferate or shift into myogenic differentiation is regulated by cell cycle regulators. After withdrawal from the cell cycle, myoblasts fuse to form multinucleated myotubes. This process is associated with the expression of differentiation markers, including myosin heavy and light chains. Although myogenesis has been widely studied, a number of steps and interactions between various genes involved in this process are not fully understood. C2C12 cells are murine myoblasts derived from satellite cells. These cells serve as a well established and reproducible model system to investigate complex biochemical processes that underlie the formation of

myotubes,4) since they spontaneously differentiate and fuse to form myotubes under low-serum conditions. Several reports have described the gene-expression profile during the onset of myogenic differentiation.5–7) Moran et al. conducted analysis of more than 10,000 transcripts over four time points during C2C12 cell differentiation.5) Shen et al. have focused on the events taking place during C2C12 myoblast cell cycle withdrawal.6) Tomczak et al. reported the analysis of 24,290 murine probe sets in a 12-d time course of differentiating C2C12 myoblasts.7) Mangosteen (Garcinia mangostana L.) is a tropical evergreen tree native to Southeast Asia. Mangosteen pericarp and the whole fruits have been used for centuries in Southeast Asia to treat several diseases such as diarrhea, dysentery, infections of the skin, inflammation, and fever.8,9) This tree contains a large amount of tricyclic isoprenylated polyphenols, referred to as xanthones, and the fruits have putative medicinal effects. In general, xanthones are found in a small group of higher plants, with at least 68 distinct xanthones identified in the mangosteen plant alone.9) The major constituents of xanthones in the pericarp of the mangosteen fruit are α- and γ-mangostin10); with α-mangostin being the most widely studied of the xanthones. Various biological activities of α-mangostin, including anti-apoptotic, anti-carcinogenic, anti-inflammatory, and anti-oxidant activities, have been reported.11) However, no information is available for this natural compound on the differentiation of myoblasts. The aim of the present study was to examine the effects of α-mangostin on C2C12 myoblast differentiation. To study global changes in gene expression, we conducted DNA microarray analysis to compare geneexpression profiles of C2C12 cells treated with or without α-mangostin during myogenic differentiation. The results of the gene ontology (GO) terms enrichment analysis of the differentially expressed genes (DEGs), revealed that α-mangostin stimulated expression of genes related to myotube formation.

*Corresponding author. Email: [email protected] Abbreviations: DAVID, Database for Annotation, Visualization, and Integrated Discovery; DEG, differentially expressed genes; qFARMS, Factor Analysis for Robust Microarray Summarization; FBS, fetal bovine serum; FDR, false discovery rate; GO, gene ontology. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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Materials and methods

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Materials. Alpha-mangostin was purchased from ChromaDex (Irvine, CA, USA). All other chemicals used were obtained from Sigma (St. Louis, MO, USA). Cell culture and treatment. C2C12 cells (Dainippon Pharmaceutical, Osaka, Japan) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) (Invitrogen Japan, Tokyo, Japan), at 37 °C in a humidified incubator with 5% CO2. After reaching confluence, cells were differentiated into myotubes by changing the culture medium to differentiation medium (DMEM containing 2% FBS) in the presence or absence of α-mangostin at 5 μM, which was replenished every 2 d. After 4 d, differentiated myotubes were photographed at ×40 magnification using an SP350 digital camera (Olympus, Tokyo, Japan) attached to an IMT-2 microscope (Olympus). Total RNA was extracted for quantitative realtime reverse transcription polymerase chain reaction (RT-PCR) and DNA microarray analysis 4 d after induction of differentiation. These experiments were performed in triplicate. Quantitative real-time RT-PCR. Changes in expression of the Myl2, titin-cap, and follistatin genes were assessed using a SYBR® PrimeScript® RT-PCR Kit (Takara-Bio, Shiga, Japan), according to the manufacturer’s instructions. Briefly, total RNA was reverse transcribed into cDNA using a PrimeScript® RT reagent Kit (Takara-Bio). Subsequently, quantitative real-time RT-PCR was performed with SYBR® Premix Ex Taq™ (Takara-Bio), with PCR amplification carried out for 1 cycle at 95 °C for 10 s, then 45 cycles at 95 °C for 5 s and at 60 °C for 22 s. A dissociation curve was generated to ensure amplification of the true PCR product only. All real-time reactions were carried out on an MxPro 3005P QPCR System (Stratagene, CA, USA), and analysis was done using MxPro software (Stratagene). Relative expression levels were calculated according to standard curves obtained from real-time PCR using serial dilutions of templates, and normalized to the housekeeping gene cyclophilin. The primer sequences used were: Myl2 5′-CCC TAG GAC GAG TGA ACG TG-3′ (forward) and 5′-TCC CGG ACA TAG TCA GCC TT-3′ (reverse), titin-cap 5′AAG AGG GAT GCT CCT TGC AC-3′ (forward) and 5′-GGT ATT CCT GTA GCC CAC GG-3′ (reverse), follistatin 5′-GTG ACA ATG CCA CAT ACG CC-3′ (forward) and 5′-TCC GAG ATG GAG TTG CAA GA-3′ (reverse), cyclophilin 5′-TGG TGA CTT TAC ACG CCA TA-3′ (forward) and 5′-CAG TCT TGG CAG TGC AGA TA-3′ (reverse). DNA microarray analysis. DNA microarray analysis was performed using the Mouse Genome 430 2.0 Array (Affymetrix Santa Clara, CA, USA), according to the manufacturer’s protocol. Briefly, 100 ng of total RNA was reverse transcribed into cDNA using a poly (dT) oligonucleotide attached to a T7 promoter and converted to dsDNA. This was then used as a template

for the synthesis of biotinylated aRNA by T7 RNA polymerase. Labeled aRNA was fragmented and hybridized to the array at 45 °C for 16 h. After hybridization, the array was washed and stained with streptavidin–phycoerythrin. Fluorescent signals were scanned using the Affymetrix GeneChip System. Analysis of DNA microarray data. Affymetrix GeneChip Command Console software was used to reduce the array images to the intensity of each probe (CEL files). CEL files were quantified using the Factor Analysis for Robust Microarray Summarization (qFARMS) algorithm12) using the statistical language R13) and Bioconductor.14) DEGs were identified by applying the Rank Products method15) to the qFARMS quantified data. The annotation file for the Mouse Genome 430 2.0 Array was downloaded from the Affymetrix website. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to detect overrepresented GO terms in each group of DEGs.16,17) We compared the DEGs with the Mouse Genome 430 2.0 Array background. The functional annotation chart, a tool integrated in DAVID, was applied to examine significantly overrepresented GO terms. A modified fisher exact p-value (Expression Analysis Systematic Explorer [EASE] score18)) of