ARTHRITIS & RHEUMATISMVol. 60, No. 6, June 2009, pp 1704–1709DOI 10.1002/art.24522© 2009, American College of Rheumatology

Expression and Modulation of Ghrelin O-Acyltransferase inCultured Chondrocytes

Rodolfo Gomez,1 Francisca Lago,1 Juan J. Gomez-Reino,1 Carlos Dieguez,2 andOreste Gualillo1

Objective. To use reverse transcription–polymerasechain reaction to detect ghrelin O-acyltransferase(GOAT) transcripts in both murine and human chon-drocytes, to evaluate the effect of pharmacologic in vitrotreatments with lipopolysaccharide (LPS), growth hor-mone, ghrelin, and dexamethasone on GOAT messengerRNA (mRNA) expression, and to study the GOATmRNA profile during chondrocyte differentiation.

Methods. Murine and human GOAT and ghrelinmRNA levels were determined by the SYBR Green–based quantitative real-time polymerase chain reactionmethod.

Results. GOAT mRNA was expressed in murinecartilage explants as well as in the cultured murinechondrogenic ATDC-5 cell line. GOAT was also ex-pressed in human immortalized chondrocyte cell linesand in human cultured primary chondrocytes. In addi-tion, GOAT mRNA expression in differentiatingATDC-5 cells was lower at the early stage of differenti-ation (days 3–7), whereas GOAT mRNA levels increasedprogressively at the late stages. Finally, among the

drugs and hormones tested, only LPS was able tostrongly decrease GOAT mRNA expression.

Conclusion. These data indicate that chondro-cytes are equipped with biochemical machinery for thesynthesis of acylated ghrelin and suggest a novel role ofthe ghrelin axis in prehypertrophic and hypertrophicchondrocyte differentiation during endochondral ossifica-tion.

Ghrelin is a peptide hormone that is secretedprevalently from the stomach in response to hunger andstarvation and serves as a peripheral orexigenic signalinforming the brain, leading to increased food consump-tion (1). Ghrelin has been identified in almost allvertebrates examined so far. In its biologically activeform, ghrelin has 28 amino acids and a specific acylmodification of the third serine. This chemical modifi-cation is necessary for ghrelin to bind to the ghrelinreceptor and to exert biologic activity. The carboxylicchain that normally esterifies the hydroxyl of serine isprimarily n-octanoic acid, but other residues (6 to 10carbon chain) may modify the structure of ghrelin (2).

Acylated ghrelin represents �20–30% of circu-lating ghrelin molecules, whereas the remaining mole-cules circulate as unacylated ghrelin (3). Recent findingshave demonstrated that ghrelin, apart from the multi-plicity of its physiologic functions affecting growth hor-mone release, food intake, energy and glucose ho-meostasis, and gastrointestinal, cardiovascular,pulmonary, and immune functions (4), also has relevantphysiologic actions on bone (5) and cartilage (6). Inter-est in ghrelin has been fostered by characterization ofthe long-sought enzyme responsible for its acylation.Recently, Yang et al (7) and Gutierrez et al (8) inde-pendently described an enzyme named ghrelinO-acyltransferase (GOAT) that catalyzed the n-octanoylmodification of ghrelin in cultured cells. GOAT-knockout mice lack octanoylated ghrelin, which is con-

Mr. Gomez is recipient of a Pre-Doctoral Fellowship from theInstituto de Salud Carlos III (FIS-PI05/0525). Drs. Lago and Gualillo’swork was supported by the Instituto de Salud Carlos III and the Xuntade Galicia (SERGAS) through a research-staff stabilization contractand individual grants (PI050419, PI060919, PI08/0044, andPGIDIT06PXIB918307PR to Dr. Lago and PI05/0525, PI08/0040, andPGIDIT07PXIB918090PR to Dr. Gualillo).

1Rodolfo Gomez, BS, Francisca Lago, PhD, Juan J. Gomez-Reino, MD, PhD, Oreste Gualillo, PharmD, PhD: Santiago UniversityClinical Hospital, Santiago de Compostela, Spain; 2Carlos Dieguez,MD, PhD: Santiago University School of Medicine, Santiago deCompostela, Spain.

Address correspondence and reprint requests to Oreste Gua-lillo, PharmD, PhD, Santiago University Clinical Hospital, ResearchLaboratory 9: Laboratory of Neuro Endocrine Interactions in Rheu-matology and Inflammatory Diseases, Building C, Level-2, CalleChoupana s/n, 15706 Santiago de Compostela, Spain. E-mail:oreste.gualillo@sergas.es.

Submitted for publication December 19, 2008; accepted inrevised form February 17, 2009.

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sistent with the notion that GOAT is the acyltransferaserequired for the n-octanoylation of ghrelin.

GOAT is a porcupine-like enzyme belonging tothe membrane-bound O-acyltransferase family. MurineGOAT has been described as being localized within theendoplasmic reticulum, and its distribution is likely to belimited to the gastrointestinal tract and testes. Interest-ingly, however, human GOAT is expressed in the stom-ach and pancreas (7,8). Regarding the enzymatic activityof GOAT, the murine form specifically covalently bindsoctanoyl residues to Ser3 of ghrelin, whereas humanGOAT can also acylate ghrelin with carboxylic acidsother than octanoate, ranging from acetate to tetrade-canoic acid (for review, see ref. 3).

It has been demonstrated that primary osteo-blasts, as well as osteoblastic cell lines of various species,express ghrelin receptors (9). Evidence has also beenprovided that ghrelin treatment directly stimulates fetalrat calvarial osteoblast cell proliferation and differenti-ation, alkaline phosphatase activity, and calcium accu-mulation in the matrix of primary cell culture (10). Inaddition, ghrelin increased bone mineral density in bothnormal and growth hormone–deficient rats. These ob-servations show that ghrelin directly stimulates boneformation. In cartilage, ghrelin may have a significantrole in regulating chondrocyte metabolism in the growthplate, because it is localized in the proliferative andmaturation zones, inhibits the cell metabolic rate, pro-motes cAMP accumulation, and inhibits basal and in-duced fatty acid uptake in chondrocyte cultures (6).Thus, ghrelin produced by chondrocytes could influence,via an autocrine/paracrine pathway, the synthesis offactors that selectively promote osteogenesis, or alterna-tively, modulate the biosynthesis of eicosanoids in thecartilage.

The present study had 2 specific goals. The firstgoal was to investigate whether GOAT is expressed inmurine and human chondrocytes. If that were the case,it would mean that chondrocytes will have all of thebiochemical pieces needed to synthesize active ghrelin,further supporting a possible autocrine/paracrine role ofacylated ghrelin derived from chondrocytes. The secondgoal was to assess whether levels of the GOAT transcriptare regulated by factors influencing chondrocyte cellfunction. To this end, we measured the levels of GOATmessenger RNA (mRNA) during chondrocyte differen-tiation, using ATDC-5, the embryonic mousecarcinoma–derived cell line that is frequently used forthe in vitro model of chondrocyte differentiation (11).Furthermore, the effect on GOAT mRNA expression of

substances such as lipopolysaccharide (LPS), ghrelin,growth hormone, and dexamethasone was explored.

MATERIALS AND METHODS

Reagents. A First Strand Kit, polymerase chain reac-tion (PCR) Master Mix, and primers for GOAT and GAPDHwere purchased from SuperArray BioSciences (Frederick,MD). A NucleoSpin kit was purchased from Machery-Nagel(Duren, Germany), human growth hormone was obtainedfrom Serono (Madrid, Spain), and dexamethasone and LPSwere purchased from Sigma-Aldrich (St. Louis, MO).

Cell culture. The immortalized human juvenile costalchondrocyte cell lines T/C-28a2 and C-28/I2 (a kind gift fromMary B. Goldring, Hospital for Special Surgery, New York,NY) and the primary chondrocytes were cultured in Dulbec-co’s modified Eagle’s medium (DMEM)–Ham’s F-12 mediumsupplemented with 10% fetal calf serum, L-glutamine, andantibiotics (50 units/ml penicillin and 50 �g/ml streptomycin).

The murine chondrogenic cell line ATDC-5 was pur-chased from RIKEN Cell Bank (Tsukuba, Japan). Cells werecultured in DMEM–Ham’s F-12 medium supplemented with5% fetal bovine serum, 10 �g/ml human transferrin, 3 �10�8M sodium selenite, and antibiotics (50 units/ml penicillinand 50 �g/ml streptomycin).

Chondrogenic ATDC-5 cells were differentiated intomature and hypertrophic chondrocytes, as previously described(12,13). Briefly, cells were plated at an initial density of 2 �104/well in 24-well plates. Cells were cultured in the above-mentioned medium supplemented with 10 �g/ml of humanrecombinant insulin (Novo Nordisk, Bagsvaerd, Denmark).After 15 days, the culture medium was switched to �-minimalessential medium (Invitrogen, San Diego, CA). Culture wascontinued for an additional 21 days, with medium replacementevery day. As expected, ATDC-5 cell cultures treated withinsulin underwent progressive differentiation from day 0 to day21, as compared with untreated cultures. This differentiationwas qualitatively characterized by increased formation of car-tilage nodules and enhanced staining with Alcian blue, whichare indicative of cartilage proteoglycan accumulation.

In other experiments (data not shown), differentiationwas further analyzed by sequential increases in the levels oftype II collagen, aggrecan, and type X collagen mRNA fromday 0 to day 21. Type II collagen, a marker of early and maturechondrocytes, was expressed in undifferentiated ATDC-5 cells;expression began to increase on day 3, peaked on days 7–10,and gradually declined after day 15. The expression profile ofaggrecan mimicked that of type II collagen, with a slight delayof a couple of days. The decline in expression of bothchondrocyte markers coincided with onset of the late stage ofchondrocyte differentiation. Expression of the hypertrophicchondrocyte marker type X collagen began on days 12–13. Theexpression patterns of the markers of early and mature chon-drocytes described here were consistent with previous find-ings using ATDC-5 cells and in vivo chondrocyte differen-tiation.

Cartilage harvest and human chondrocyte isolation.Normal human articular cartilage samples were obtained fromthe knee joints of patients undergoing knee amputations forperipheral vascular disease (with permission from the local

GOAT TRANSCRIPTS IN MURINE AND HUMAN CHONDROCYTES 1705

ethics committee). None of the patients had a clinical historyof arthritis or any other pathology affecting cartilage, and thespecimens appeared normal by morphologic examination (i.e.,no change in color and no fibrillation). Chondrocyte isolationwas performed as described previously (14).

Control tissue. For mRNA expression studies in hu-man tissue, gastric mucosa (fundus) was used as a positivecontrol. Gastric tissue samples were obtained from the Depart-ment of Morphological Sciences of Santiago University Clini-cal Hospital (a kind gift from Professor Tomas Garcia Cabal-lero). Mouse gastric mucosa from albino Swiss mice wasobtained from the Central Animal Breeding Department,University of Santiago.

RNA isolation and real-time reverse transcription–polymerase chain reaction (RT-PCR). Murine and humanGOAT and ghrelin mRNA levels were determined using theSYBR Green–based quantitative PCR method. RNA wasextracted using a NucleoSpin kit, according to the instructionsand protocol provided by the manufacturer. For relativequantification, we performed an RT reaction with a FirstStrand Kit from SuperArray. Next, real-time PCR was per-formed with SuperArray Master Mix and specific primers (formouse GOAT, 178 bp, PPM40440A, reference position 1127–1145, GenBank accession no. XM_001472220.1; for mouseghrelin, 111 bp, PPM31564A, reference position 505–526,GenBank accession no. NM_021488.4; for human GOAT, 168bp, PPH63501A, reference position 1050–1068, GenBank ac-cession no. XM_001125855.1; for mouse GAPDH, 140 bp,

PPM02946E, reference position 309–328, GenBank accessionno. NM_008084.2; for human GAPDH, 175 bp, PPH00150E,reference position 1287–1310, GenBank accession no.NM_002046.3). Results of comparative real-time PCR wereanalyzed with MxPro version 4 software (Stratagene, La Jolla,CA).

Statistical analysis. Results are reported as themean � SEM log2 fold changes from at least 3 independentexperiments (each with at least 3 independent observations).The algorithm used to determine the dRn (a parametercorresponding to the baseline subtracted fluorescence readingnormalized to the reference dye, for both control and treatedsamples) is as follows: normalized unknown/normalized con-trol � 2��Ct unknown/2��Ct control � 2���Ct. Statisticalanalysis was performed by analysis of variance, followed bypost hoc comparison testing (using the unpaired t-test and theStudent-Newman-Keuls test) with the InStat computer pro-gram. P values less than 0.05 were considered significant.

RESULTS

Expression of mouse GOAT. As shown in Figure1A, the ATDC-5 cell line expressed mouse GOATmRNA. In the ATDC-5 cell line, constitutive expressionof mouse GOAT mRNA was 6-fold lower than that inmouse stomach and similar to the level of expression inmouse cartilage explants.

Figure 1. Expression of mouse ghrelin O-acyltransferase (GOAT) in chondrocytes. A, Relative quantification of mouse GOAT mRNA expressionin mouse cartilage and the ATDC-5 cell line, as evaluated by quantitative reverse transcription–polymerase chain reaction (RT-PCR). B, Relativequantification of human GOAT mRNA expression in human primary chondrocytes and the T/C-28a2 and C-28/I2 cell lines, as evaluated byquantitative RT-PCR. All experiments were performed at least 3 times. Results are the mean and SEM log2 fold changes versus control (stomach)(log2 dRn). ��� � P � 0.001.

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Expression of human GOAT. As shown in Figure1B, both of the immortalized human juvenile costalchondrocyte cell lines, T/C-28a2 and C-28/I2, expressedhuman GOAT mRNA. Relative quantification studiesof human GOAT mRNA expression in human primaryculture, as well as in human cell lines, revealed that thelevels were lower than those in human stomach.

Expression of mouse GOAT during chondrocytedifferentiation. To gain further insights in the physio-logic role of GOAT, we investigated the pattern ofGOAT mRNA expression during differentiation of theATDC-5 cell line. As shown in Figure 2A, GOATmRNA expression was low during the first days of thedifferentiation process and increased further during thedifferentiation process, reaching a stable level at 15–21days. Intriguingly, GOAT mRNA expression was similarto that of ghrelin mRNA expression, particularly duringthe early phase (days 0–3) of chondrocyte differentiation(Figure 2B).

Modulation of GOAT expression. Finally, westudied the influence of several hormonal and drugtreatments on the expression of GOAT mRNA. Ourresults showed that LPS treatment (500 ng/ml) de-creased GOAT expression in both murine and humancell lines (Figures 3A and B). Furthermore, in theATDC-5 cell line, but not in T/C-28a2, 10 �M dexameth-asone tended to up-regulate the expression of mouseGOAT mRNA. Neither growth hormone (150 nM) nor

ghrelin (100 nM) induced a significant change in GOATmRNA expression in both human and murine chondro-cytes (Figures 3A and B).

DISCUSSION

Skeletal development is an integral and multistepprocess that comprises pattern formation, chondrocytedifferentiation, longitudinal bone growth, and remodel-ing (15). Most of the skeleton is formed by endochondralossification, a complex process during which undifferen-tiated prechondrogenic mesenchymal cells undergo well-ordered and controlled phases of proliferation, hyper-trophic differentiation, apoptosis, blood vessel invasion,and finally replacement of cartilage by bone (15). Theseprocesses are reflected in the temporal and spatialorganization of the epiphyseal growth plate. In principle,the following 3 zones of chondrocyte differentiation canbe distinguished during embryonic bone formation: thezone of low-proliferating distal chondrocytes (also des-ignated as the resting zone or zone of round periarticu-lar chondrocytes); the zone of flat, high-proliferating,column-forming chondrocytes; and the hypertrophiczone. Each zone is characterized by the expression ofspecific sets of genes, which can be used as molecularmarkers to distinguish specific chondrocyte differentia-tion stages. However, the list of molecular markersfor chondrocytes is very sparse, and the mechanisms

Figure 2. Expression of mouse GOAT and ghrelin during chondrocyte differentiation. Relative expression of mouse GOAT mRNA (A) and ghrelinmRNA (B) during differentiation of the ATDC-5 cell line was evaluated by quantitative RT-PCR. All experiments were performed at least 3 times.Results are the mean � SEM log2 fold changes. ��� � P � 0.001 versus undifferentiated cells (undifferentiated cells have a level of expressionidentical to that at day 15 and day 21). See Figure 1 for definitions.

GOAT TRANSCRIPTS IN MURINE AND HUMAN CHONDROCYTES 1707

that contribute to the distal cell fate are just beingelucidated (16).

In a previous study, it was suggested that ghrelinmay have a significant role in regulating chondrocytemetabolism in the growth plate, because it was localizedin the proliferative and maturation zones, inhibited themetabolic rate of cultured chondrocytes, promotedcAMP accumulation, and inhibited basal and inducedfatty acid uptake in chondrocyte cultures (6). Thus,ghrelin produced by chondrocytes could influence, viaan autocrine/paracrine pathway, the synthesis of factorsthat selectively promote osteogenesis, or alternatively,could modulate the biosynthesis of eicosanoids in thecartilage (17). Therefore, it was reasonable to hypothe-size that ghrelin in cartilage could participate in chon-drocyte metabolism by promoting hypertrophy processesby increasing proteoglycan synthesis and programmedcell death, thus clearing the way for osteoblastic boneformation (18). Based on previous evidence and therecent identification of GOAT, the present study inves-tigated the expression of GOAT in different chondro-cytic cell lineages and analyzed its pharmacologic mod-ulation as well as its expression in an in vitro model ofchondrocyte differentiation.

Our data provide the first evidence that GOATis expressed in both murine and human chondrocytes as

well as in cultured cells of chondrocytic lineage. We alsoobserved that GOAT expression is low during the earlyphase of differentiation. As differentiation proceeds,GOAT becomes up-regulated. This result providesmechanistic support for the hypertrophic and antiprolif-erative roles of acylated ghrelin in cultured chondro-cytes, and suggests that GOAT expression may play acritical role as a regulator of the late phase of chondro-cyte differentiation in endochondral ossification. Inter-estingly, GOAT mRNA expression overlapped withghrelin mRNA expression, suggesting a coordinatemechanism between the expression of the acylatingenzyme and its physiologic substrate. Of note, GOATmRNA expression, along with ATDC-5 differentiation,have a temporal profile almost identical to those of typeX collagen (a marker for hypertrophic chondrocytes)and aggrecan (the major noncollagenous protein incartilage matrix) (19). Nonetheless, roles for GOATother than differentiation in chondrocytes might be atplay. Indeed, chondrocytes from human normal cartilagereflect what is happening at the hyaline cartilage levelbut not in growth plate cartilage.

Finally, we observed marked down-regulation ofGOAT transcripts following challenge with LPS. There-fore, it is possible that GOAT is involved in the proin-flammatory response to LPS challenge in chondrocytes.

Figure 3. Modulation of expression of mouse and human GOAT mRNA. A, Relative quantification of mouse GOAT mRNA expression in theATDC-5 cell line, as evaluated by quantitative RT-PCR. B, Relative quantification of human GOAT mRNA expression in the T/C-28a2 cell line,as evaluated by quantitative RT-PCR. All experiments were performed at least 3 times. Results are the mean � SEM log2 fold changes versus control(Cont; unstimulated cells). � � P � 0.05; ��� � P � 0.001, versus control. LPS � lipopolysaccharide; GH � growth hormone; GHREL � ghrelin;DX � dexamethasone (see Figure 1 for other definitions).

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Intriguingly, LPS is able to induce a sustained decline inthe level of circulating ghrelin, thereby supporting theconcept that ghrelin might be an important factor in thedevelopment of systemic symptoms of inflammationsuch as the suppression of appetite (20). In contrast, thefact that dexamethasone did not show a consistent effectin all of the cell lines tested and the complete lack ofeffect of ghrelin and growth hormone support the con-cept that the transcript is regulated in a specific manner.

In summary, the discovery of GOAT appears tobe a breakthrough in the understanding of ghrelinacylation that in turn will help with understanding thephysiologic role of the ghrelin system in different tissues,including chondrocytes, as well as the pathophysiologyof different clinical entities. In this study, we providedevidence that GOAT is expressed in chondrocytes, andthat its levels are markedly influenced by the degree ofchondrocyte differentiation and by LPS challenge. Thesefindings offer support for the hypothesis that acylatedghrelin derived from chondrocytes plays an importantrole in chondrocyte cell biology.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Gualillo had full access to all ofthe data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Study conception and design. Gualillo, Lago.Acquisition of data. Gomez.Analysis and interpretation of data. Gomez, Gomez-Reino, Dieguez,Gualillo.

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