J Oral Tissue Engin 2009;7(2):99-106

ORIGINAL ARTICLE Effect of Adiponectin on Expression Profiles in

Kaoru TANABE, Aiko KAMADA, Seiji GODA and Takashi IKEO

Department of Biochemistry, Osaka Dental University

SYNOPSIS Adiponectin is abundantly present as a plasma , and exhibits various bio- logical functions, such as regulating energy homeostasis and increasing insulin sensitivity in the liver and . Circulating adiponectin includes both a full-length type (AN) and a globular type (gAN). Recent studies have demonstrated both a positive and negative action of adiponectin on bone formation, and it was thought that these different actions might be attributed to different forms of adi- ponectin. In this study we investigated the profiles of osteoblasts stimulated by AN or gAN. Osteoblastic differentiation of a murine pro-osteoblastic cell line, MC3T3-E1 cells, was induced by ascorbic acid and -glycerophosphate with or without re- combinant murine gAN or AN. Gene expression profiles were obtained by DNA microarray analysis. Stimulation of AN or gAN showed different gene expression profiles in os- teoblasts. The different profiles of up-regulated gene expression in osteoblasts indicated that different forms of adiponectin act on osteoblastic differentiation through different pathways.

Key words: adiponectin, globular adiponectin, , microarray

INTRODUCTION teoblastic differentiation, and indicated Body fat and bone mass are known to that adiponectin rather than leptin may be directly related, and many studies be involved in osteoblastic differentia- 3 have shown a positive association of tion . bone mineral density with fat mass1. Adiponectin/Acrp30(adipocyte com- These associations are considered to plement- related protein of 30 kDa) be mediated by not only biomechanical contains three easily recognizable do- 4 but also biochemical factors, since adi- mains ; an N-terminal signal sequence, pocytes have been found to generate a collagen-like domain, and a C-terminal and secrete many adipocytokines in- globular domain (Fig. 1a). It exhibits cluding adiponectin, plasminogen acti- various biological functions, such as vator inhibitor-1, - regulating energy homeostasis, in- (TNF-), resistin, leptin and so on 2. creasing insulin sensitivity in the liver and skeletal muscle, and protecting Recently, we demonstrated the gene 5 expression of adipocytokine receptors in vascular walls from atherosclerosis . murine pro-osteoblastic cells during os- Circulating adiponectin includes both a

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full-length type (AN) and a globular type motifs, inference of signal pathways and (gAN), which is a proteolytic cleavage genetic networks in the context of GO. product of full-length adiponectin at its In this study, to investigate the ef- C-terminal globular domain, as shown in fects of adiponectin differential forms on Fig. 1a. Some studies have shown that osteoblastic differentiation, we used AN synergizes with insulin to inhibit murine pro-osteoblastic cells which are hepatic glucose production, however, differentiated into mature osteoblasts by gAN simulates fatty acid oxidation in the addition of ascorbic acid and skeletal muscle 6, 7. Recent studies have glycerophosphate. The gene expres- demonstrated both a positive and nega- sion profiles in osteoblasts stimulated by tive action of adiponectin on bone for- AN or gAN were analyzed using a mi- mation 8-13, and it was thought that these croarray, and suggested some possible different actions might be attributed to roles or pathways. different forms of adiponectin. Microarray technology is an ex- MATERIALS AND METHODS tremely powerful approach to accurately Cell culture measure changes in global mRNA ex- A murine pro-osteoblastic cell line, pression levels14 and can measure gene MC3T3-E1, was purchased from the expressions of tens of thousands of American Type Culture Collection discrete sequences in a single array. (CRL-2594; Manassas, VA, USA). Cells This technique has been used to dis- were maintained in -minimum essential cover novel , determine gene medium (-MEM) containing 10% fetal functions, drug evaluation, pathway bovine serum (FBS; Lot No 915150, dissections, and the classification of Biological Industries, Ashrat, Israel), clinical samples. The importance of mi- penicillin (100 U/mL) and streptomycin croarray data analysis lies in presenting sulfate (100 g/mL) (GibcoBRL, Grand functional annotations and classifica- Island, NY, USA) at 37°C in humidified tions. Cluster 3.0 15 and Java Treeview 16 5% CO2 conditions. Cells were plated in are widely used open-source programs 24-well (2 x 104 cells/well) tissue culture to group together genes with a similar plates and grown to subconfluency in pattern of expression, and to provide a -MEM supplemented with 10% FBS. computational and graphical environ- Confluent monolayers were precultured ment for analyzing data from DNA mi- 17 in control medium (-MEM containing croarray experiments . The Gene On- 0.1% FBS) for 24 h, and then cultured in tology project (http://www.geneontology. differential medium (-MEM containing org/) is a major bioinformatics initiative 0.1% FBS, 50 g/mL ascorbic acid (VC) with the aim of standardizing the repre- and 10 mMglycerophosphate (GP))18, sentation of gene and gene product at- 19 tributes across species and databases. with or without recombinant murine The project provides a controlled vo- gAN (gAcrp30; Pepro Tech EC, London, cabulary of terms (GO terms) for de- UK) or AN (Acrp30; BioVendor Lab. Med. scribing gene product characteristics. Inc., Brno, Czech Republic) for 3 days. (GO) organizes informa- tion for molecular function, biological Western blotto analysis processes, and cellular components for The molecular weights of commercially a number of different organisms. available adiponectin samples were Therefore the identifications of confirmed by Western blot analysis. The co-expression gene groups can lead to were applied to SDS-PAGE on the identification of common regulatory 4－20% gel and transferred to a polyvi-

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nylidine difluoride membrane (Bio-Rad primers (Applied Biosystems, Foster Laboratories, Hercules, CA, USA) for City, CA, USA). Western blot analysis. Blots were Two micrograms of the cDNA prod- probed with rabbit anti-mouse gAcrp30 ucts were labeled with Cys3 using an IgG (Alpha Diagnostic Int., San Antonio, Agilent Genomic DNA Enzymatic La- TX, USA), which detects the globular beling , and the labeled targets were domain of adiponectin, followed by hybridized to the Agilent Whole Genome HRP-goat anti-rabbit secondary anti- Array (4x44K) according to manufac- body, and processed for chemilumi- turer’s instructions (Agilent Technolo- nescence with an ECL-plus kit (GE gies, Santa Clara, CA, USA). Gene ex- Healthcare, Buckinghamshire, UK). The pression data were applied to hierar- images were recorded using an imaging chical clustering with correlation cen- analyzer (VersaDoc; Bio-Rad). tered similarity metrics. Complete link- age hierarchical clustering was per- Microarray analysis formed using Cluster3.0 15, and the re- Total RNA was isolated using the sults were visualized as a heatmap us- MagExtractor RNA purification kit (TO- ing Java Treeview 16, 17. YOBO, Osaka, Japan) from cultured cells 3 days after the addition of a dif- RESULTS ferential medium. The quantity and pu- Gene expression profiles in os- rity of extracted total RNA were deter- teoblasts mined spectrophotometrically at 260 It was confirmed by Western blotting and 280 nm. Total RNA was re- that gAN and AN used in this study were verse-transcribed into cDNA using the globular- type and full-length adi- High Capacity RNA-to-cDNA Master Mix, ponectin, respectively (Fig. 1b). including random primers and oligo(dT) Genes were considered to be up- or

a b

Fig. 1 Characterization of adiponectin (AN) and globular adiponectin (gAN). (a) Full-length adiponectin (AN) is an ~30-kDa polypeptide containing an N-terminal signal sequence, a variable domain, a collagen-like domain, and a C-terminal globular domain. Globular adiponectin (gAN) is a naturally occurring globular protein, obtained by proteolytic processing at AA 104 of AN. (b) Western blotting with globular do- main-specific anti-serum detected recombinant peptides for murine gAN and AN as ~18kDa- and ~35kDa-bands, respectively.

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down-regulated if the fold change in the heatmap, shown in Fig. 2a. Up- and log ratio was more than 1.5 in any down- regulated expressions are rep- comparisons among the three experi- resented as red and green, respectively, mental groups; the log ratio of differen- and black represents no change of tial medium to control medium expression. (DM/Control), differential medium with There were red and green seg- AN to differential medium (DM+AN/DM), ments in the gAN/AN heatmap, indicat- differential medium with gAN to differen- ing that gAN and AN affected different tial medium (DM+gAN/DM), and differ- osteoblast genes. Then, the character- ential medium with gAN to differential istic subsets for DM, AN or gAN addition medium with AN (gAN/ AN). All signifi- were selected as Cluster 1 - 6, and cant expression genes were hierar- zoomed (Fig. 2b). chically clustered with a similar ex- pression pattern and visualized as a

Fig. 2 Effects of adiponectin on the gene expression in osteoblasts. (a) All significant expression genes are expressed as heatmaps. (b) Characteristic gene expressions for DM, gAN or AN addition were clustered into Cluster 1 - 6. DM, addition of differential medium; AN+DM, addition of differential medium with full-length adiponectin; gAN+DM, addition of differential medium with globular adiponectin.

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Functional classification of specific and it was notable that AN was closely osteoblast genes in response to linked to the downstream signaling adiponectin pathway as Jun. Cluster 3, in which Functional cluster annotations for the genes were up-regulated only by gAN, selected clusters were generated based included genes related to the G-protein on the significant GO terms (Table 1). coupled receptor signaling pathway, cell The significant genes up-regulated morphogenesis and neurological sys- by DM were included in Clusters 1, 2 tem processes as cadherins, BMPs and and 5 related to osteoblast differentia- NGF etc.. This cluster indicated that tion and cell growth. AN up-regulated gAN may be involved in neuronal dif- the genes in Clusters 4, 5, 6 related to ferentiation. several signaling pathways as Wnt etc., Table 1 Functional enrichment, including clusters

DM DM (number)* DM Function** Gene +AN +gAN

Ada, Csf1, Dab2, Elf3, Fgf2, Fgf9, Tbp, Il6, Pax6, Igf1, Smoc2, Socs3, cell development Stat5b, App, Ins, Map4k4, Mapk8, Gata6, Jak3, Mef2C, Cebpb, Sost et al.

bone differentiation Bhlhe40, Cebpb, Csf1, Fes, Fgf9, Igf1, Il6, Irs1, Jak3, Mef2C, Mef2D, Cluster 1 Nf1, Rgs2, SostT, Wwtr1 ↑ (1197) intercellular signaling, immune response Itga2, Elf3, Dab2, Igf1, Ins, IL6, Fgf2, Ccl24, Il18, Cd83, Mcam et al.

Atp2B1, Calm1, Gria2, Grin3a, Hdac3, Hdac8, Hdac11, Hdac9, Itprc2, calcium signaling pathway Mef2C, Mef2D, Myh3, Nfatc1, Pp3cb, Ppp3cc, Ppp3r1, Prkar1b, Tp63, Trpc7

cell development Runx1, Basp1, Cebpa, Itga9, Lipa, Map4k4, Egfr, Il12rb1, Il4r, Ltb, Stat1 et al. Cluster 2 ↑ intercellular signaling, immune response Adipor2, Aoc3, Axl, Cd248, Hla-a, Il4r, Itga9, Lama2, Ltb, Egfr, ILirl1, (380) Map3k5, Map4k4 et al.

transcription factor、gene expression Runx1, Wnt5a, Fzd2, Mras, Irf7, Ptpn1, Atf1, Srebf2 et al.

signaling pathway (G-protein coupled Adora3, Gpr174, Galr3, Vlrc1, Oprm1, Olfr611, Olfr1459, Olfr1176, receptor protein) Olfr330, Gpr37, Vlra2, Olfr33, Olfr74 et al.

neurological system process Sema3c, Sema3e, Sema4c, Sema6d, Nav1, Nav3, Ngf, Wnt8, Ncam, Cspg5, Rims2, Rims3, Klk2, Bmp4, Bmp6, Bmp7 Cluster 3 ↑ (920) cell morphogenesis, cell motility Cdh2, Cdh8, Cdh10, Cdh4, Vamp2, Angpt1, Cd40lt, Il10, Il1b, Muc1, Cd4, Cd3e, Tek, Ambn, Figf, Cxcl13, Cx3xr1, Ccr2, Arrb2 et al.

Csf2rb, Chrna1, Grik2, Cxcr1, Adrb1, Insr, Cubn, Ccr2, Nr3c2, Fgf22, cell surface receptor Il11, Sfrp4, Fgfr4, Accn1, Il1b, Map2, Ank2, Socs4, Socs6, Map3k2, Il7r, Cd4, Cd3e, Fcgr2b et al.

intercellular signaling, immune response Ccl19, Ccl21, Cd8A, Cdh9, Lta, Cxcl9, Ifna7, Itgb1, Mmp7, Adamts9, Notch4, Lama1, L4 et al. Cluster 4 ↑ ↑ cell motility, cell adhesion Cdh2, Cdh3, Cdh9, Cdh17, Alcam, Mllt4, MMP7, ITGB1, Mfap5, (1014) Amarcer1, ADAM19, Map2 et al.

carbohydrate metabolism Pygl, Inadl ,Gcg, Gmr5, Gip, Gria4, Grp4, Muc4, Pdx1, Apoa5, Adra1a, Ihh et al.

intercellular signaling, immune response Ddx58, Cdh13, Csf2rb, Itgal, B2m, Cd52, Col16a et al.

cell growth, cell cycle Sox9, Enc1, Crem, E2f3, Usp2 et al.

Cluster 5 Gpr183, Sipr2, Nfkb1e, Ucn2, Gnai1, Cdh13, Pi3k, Pik3r5, Atp2a2, ↑↑ signaling pathway (G-protein, Rho protein, Sox8, Sox9, Sox5, Nfkb1e, Igfbp4, Crem, Furin, Nrp3, Gpr183，Sipr2, (158) MAPK, Ca2+, WNT et al.) Ucn2，Dusp16，Col4a2, Csf2rb, Ppp1r14c, Fkbp1a, Mcf2l, Arhgap17 et al. protein processing (synthesis, binding, Arf6, Furin, Cltb, Stx1b, G4galt5, Fut4, Nxf3, Hnrnph3, Bhlhe22, Dalrd3 transport) et al.

Cluster 6 Jun, Atf2, Fosl2, Alox12, Pglyrp2, Crem, Creb5, Trip4, Il2rg ↑ signaling pathway (Jun, MAPK, WNT.) Prok1, Dusp19, Map2k6, Jun (1103) Wnt9a, Wnt10b, Fzd4, Dkk4, Rspo3, Lrrfip2, Csnk1g1 ( ): number of genes in each cluster * Functions contain some GO terms. 103

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DISCUSSION adiponectin by adenoviral infection in We used the subclonal cell line C57BL/6J mice increased trabecular (CRL-2594) of MC3T3-E1, which was bone volume in the distal femur com- confirmed to form a mineralized matrix pared with controls. In contrast, Shinoda two weeks after the addition of VC and et al.12 found that transgenic mice GP in our previous study19. Microarray overexpressing globular mouse adi- analysis in this study demonstrated that ponectin exclusively from the liver were the addition of DM (VC and GP) induced not different from wild-type mice in bone the expression of genes known as a morphology. major source of osteoblastic differentia- On the other hand, gAN seemed to tion20. Our previous study using siRNA induce neurological characteristics in for adiponectin receptors, AdipoR1 and pro-osteoblastic cells, since microarray AdipoR2, demonstrated that AdipoR2 analysis showed significant gene ex- rather than AdipoR1 may be involved in pressions related to neurological system osteoblastic differentiation21. Also in this processes in Cluster 3. Recently, adi- study, the gene for AdipoR2 included ponectin has been shown to be a neu- Cluster 2, which is related to osteoblas- roendocrine hormone23, acting directly tic differentiation and cell growth. on the brain to regulate autonomic func- Adiponectin differential forms in- tion24 and sympathetic nerve activity25. duced different gene expression in os- Interestingly, the three-dimensional teoblasts during osteoblastic differentia- structure of the C-terminal globular do- tion. Full-length adiponectin, AN, main of adiponectin has homology to up-regulated the expression of genes that of TNF- A recent study demon- related to various signaling pathways, strated that globular adiponectin (gAN) although globular adiponectin, gAN, strongly inhibited up-regulated the expression of genes TNF-/RANKL-induced differentiation of related to cell morphogenesis and neu- by interfering with TNF re- rological system processes. ceptor-associated factor production and The Wnt signaling pathway is well calcium signaling26. These character- known to induce osteoblastic differen- istic effects of gAN may be attributed to tiation of mesenchymal stem cells and its structural resemblance to TNF-. 20 to lead to bone formation . Recent Microarray analysis in this study pro- findings also revealed that activation of posed a novel effect of the globular the MAPK p38 is required for osteoblast domain of adiponectin on osteoblasts. 22 differentiation , and reported that adi- Further detailed research is required to ponectin induces the activation of MAPK reveal the involvement of AN in neuro- 9 in cultured osteoblasts . Clusters 5 and logical system. 6, which were up-regulated by AN, in- Taken together, our present study clude many genes related to these sig- suggests that different forms of adi- naling pathways; therefore indicating ponectin act on osteoblastic differentia- that not the C1q-like globular domain tion through different pathways, and that but the collagen-like domain or signal microarray analysis is useful and helpful sequence in the adiponectin molecule to investigate molecular functions and may be involved in osteoblast differ- metabolisms. entiation. The two studies looked at the simi- ACKNOWLEDGMENTS lar effects of adiponectin overexpression This study was supported by a 10 on bone in vivo. Oshima et al. showed Grant-in-Aid for Scientific Research (C) that overexpression of full-length mouse

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(Received, November 5, 2009/ Accepted, December 24, 2009)

Corresponding author: Aiko KAMADA, Ph,D Department of Biochemistry, Osaka Dental University 8-1 Kuzuhahanazonocho, Hirakata-shi, Osaka, 573-1121 Japan Tel: +81-72-864-3055 Fax: +81-72-864-3155 E-mail: [email protected]

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