Differential expression of cyclin G2, cyclin dependent kinase inhibitor 2C and peripheral myelin protein 22 genes during adipogenesis..

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1 The Ohio Stte University From the SeletedWorks of Jiin Zhng Summer My 5, 214 Differentil expression of ylin G2, ylin dependent kinse inhiitor 2C nd peripherl myelin protein 22 genes during dipogenesis..pdf Jiin Zhng, The Ohio Stte University Yeunsu Suh, The Ohio Stte University Young Min Choi, The Ohio Stte University Jinsoo Ahn, The Ohio Stte University Mihel E Dvis, The Ohio Stte University, et l. Aville t:

2 Animl (214), 8:5, pp 8 89 The Animl Consortium 214 doi:1.117/s niml Differentil expression of ylin G2, ylin-dependent kinse inhiitor 2C nd peripherl myelin protein 22 genes during dipogenesis J. Zhng 1, Y. Suh 1, Y. M. Choi 1, J. Ahn 1,2, M. E. Dvis 1 nd K. Lee 1,2 1 Deprtment of Animl Sienes, The Ohio Stte University, Columus, OH 4321, USA; 2 Interdisiplinry Ph.D. Progrm in Nutrition, The Ohio Stte University, Columus, OH 4321, USA (Reeived 23 Otoer 213; Aepted 13 Ferury 214) Inrese of ft ells (FCs) in dipose tissue is ttriuted to prolifertion of predipoytes or immture dipoytes in the erly stge, s well s dipogeni differentition in the lter stge of dipose development. Although oth events re involved in the FC inrese, they re ontrry to eh other, euse the former requires ell yle tivity, wheres the ltter requires ell yle withdrwl. Therefore, pproprite regultion of ell yle inhiition is ritil to dipogenesis. In order to explore the importnt ell yle inhiitors nd study their expression in dipogenesis, we dopted strtegy omining the Gene Expression Omnius (GEO) dtse ville on the NCBI wesite nd the results of quntittive rel-time PCR (qpcr) dt in porine dipose tissue. Three ell yle inhiitors ylin G2 (), ylin-dependent kinse inhiitor 2C (CDKN2C) nd peripherl myelin protein (PMP22) were seleted for study euse they re reltively highly expressed in dipose tissue ompred with musle, hert, lung, liver nd kidney in humns nd mie sed on two GEO DtSets (GDS596 nd GDS3142). In the ltter nlysis, they were found to e more highly expressed in differentiting/ed predipoytes thn in undifferentited predipoytes in humn nd mie s shown respetively y GDS2366 nd GDS2743. In ddition, GDS2659 lso suggested inresing expression of the three ell yle inhiitors during differentition of 3T3-L1 ells. Further study with qpcr in Lndre pigs did not onfirm the high expression of these genes in dipose tissue ompred with other tissues in mrket-ge pigs, ut onfirmed higher expression of these genes in FCs thn in the stroml vsulr frtion, s well s inresing expression of these genes during in vitro dipogeni differentition nd in vivo development of dipose tissue. Moreover, the reltively high expression of in dipose tissue of mrket-ge pigs nd inresing expression during development of dipose tissue ws lso onfirmed t the protein level y western lot nlysis. Bsed on the nlysis of the GEO DtSets nd results of qpcr nd Western lotting we onlude tht ll three ell yle inhiitors my inhiit dipoyte prolifertion, ut promote dipoyte differentition nd hold differentited stte y induing nd mintining ell yle inhiition. Therefore, their expression in dipose tissue is positively orrelted with ge nd mture FC numer. By regulting the expression of these genes, we my e le to ontrol FC numer, nd, thus, redue exessive ft tissue in nimls nd humns. Keywords: ylin G2 (), 22-kD peripherl myelin protein (PMP22), ylin-dependent inhiitor 2C (CDKN2C), dipoyte, GEO dtsets Implitions The ell yle is neessry for predipoyte prolifertion, wheres dipoyte differentition requires its inhiition. In this study, three ell yle inhiitors were found to e low in predipoytes nd inresingly expressed during dipogeni differentition nd dipose tissue development, serving s potentil inditors for rtes of prolifertion nd dipoyte differentition. Moreover, modultion of their expression or tivities of their enoding proteins during dipose growth E-mil: Lee.2626@osu.edu nd development my provide prospetive method for ontrolling ft deposition in nimls nd humns y reduing the ft ell numer nd size. Finlly, this study provides n effiient reserh strtegy omining n online dtse with experimentl dt. Introdution As the most flexile tissue in size nd weight, dipose tissue n rnge from 2% to 3% of BW in the fittest thletes to 6% to 7% of BW in extremely oese individuls 8

3 Expression of ell yle inhiitors during dipogenesis (Husmn et l., 21). Growth of dipose tissue is hieved through inreses of ell size (hypertrophy) nd/or ell numer (hyperplsi). Although it seems tht hyperplsi only ours t erly developmentl stges in non-oese nimls, it ws lso found to e triggered in the dult oese nimls when the ell size nd lipid ontent rehed ertin level (Fust et l., 1978; Guo et l., 211). The different ptterns of dipose development re losely relted to timely ell yle inhiition, euse prolifertion of predipoytes nd immture dipoytes, whih is dependent on ell yle tivity, preedes differentition of predipoytes into mture ft ells (FCs), whih is hrterized y ell yle withdrwl (Ntmi nd Kim, 2), wheres promotion of ell prolifertion n inhiit dipogenesis (Hou et l., 213). Therefore, we postulted hypothesis tht there my e importnt ell yle inhiitors tht inhiit ell prolifertion in the erly stge of development of dipose tissue, ut llow FC differentition in the lter stge through ell yle inhiition. Therefore, they should e highly expressed in mture FCs nd well-differentited dipose tissue nd inresingly expressed during dipose development. If suh ell yle inhiitors n e found, they my eome potentil mrkers for seletion of redued ft tissue in the niml industry. Menwhile, sine inrese of FC numer is one of the ontriutors to severe oesity (Hirsh nd Knittle, 197; Fust et l., 1978), inresed expression of these ell yle inhiitors in the erly stge of dipose development nd deresed expression in the lter stge my provide venues for uring severe oesity. Although some ell yle inhiitors suh s retinolstom protein (Chen et l., 1996; Rihon et l., 1997) hve een well studied for their funtion oupling ell yle inhiition nd dipoyte differentition, the funtions of most other ell yle inhiitors in dipogenesis re still unler. In ddition, to the est of our knowledge, there hve een no omprehensive studies of ell yle inhiitors in dipose development. Fortuntely, the vilility of hundreds of mirorry dt in the Gene Expression Omnius (GEO) dtse on the NCBI wesite mkes it fesile to initite omprehensive study of ell yle inhiitors y ompring gene expression in vrious physiologil, developmentl or nutritionl onditions. In this study, three importnt ell yle inhiitors ylin G2 (), ylin-dependent kinse inhiitor 2C (CDKN2C) nd peripherl myelin protein 22 (PMP22) were found to e expressed to greter extent in dipose tissue y nlyzing mirorry dt from two GEO DtSets, GDS596 nd GDS3142, whih ompre gene expression in vrious tissues of humns nd mie. As n unonventionl ylin,, hs een demonstrted to not only inhiit ell prolifertion y loking trnsition from G1 to S phse in the ell yle, ut lso promotes trnsriptionl tivity of PPARγ y forming trnsriptionl omplex (Aguilr et l., 21). CDKN2C, whih is lso lled p18 INK4C, is lso well-known s key inhiitor of the ell yle nd n lok ell yle G1 progression y preventing tivtion of ylin-dependent kinse 4 or 6 (CDK4/6) (Hiri et l., 1995). CDKN2C hs een identified s protein tht is indiretly regulted y PPARγ in the differentition-dependent sde (Morrison nd Frmer, 1999). Therefore, nd CDKN2C re oth importnt regultors oupling ell yle rrest nd dipogeni differentition. PMP22 is involved in G growth rrest s homologue of the murine growth rrest-speifi 3 (gs3) (Mnfioletti et l., 199; Welher et l., 1991) nd hs een demonstrted to negtively regulte DNA synthesis nd retrd the trnsition from G/G1 to the S + G2/M phses in Shwnn ells (Zoidl et l., 1995). In ddition, gs3/pmp22 is upregulted in humn neontl firolsts rrested y serum strvtion or onfluene, ut is downregulted when these ells re-enter the ell yle (Krlsson et l., 1999), inditing tht it is importnt for mintining growth rrest stte. Although there re some reports regrding the three genes in the in vitro predipoyte ell ulture, there re no reports out the in vivo expression ptterns of these three genes during dipose development. In this study, the expression of the three genes in dipose tissue ws investigted y omining GEO DtSets for the humn nd mouse with qpcr nd Western lotting results for the pig. The sptil expression ws studied for distriution in different tissues nd omprison etween stroml vsulr (SV) ells nd FCs. Menwhile, the temporl expression ws exmined during in vitro ulture of porine primry predipoytes nd in vivo development of porine suutneous dipose tissue. Mteril nd methods Dt soures nd proessing The mirorry expression profiles for six tissues (hert, liver, lung, musle, kidney nd dipose) were derived from two GEO DtSets (GDSs) ville on the NCBI wesite: GDS596 for dult humn (more thn 2 yers old) nd GDS3142 for dult mouse (1 to weeks old). The mirorry in GDS596 ontins spots other thn the housekeeping spots, wheres the mirorry in GDS3142 ontins spots other thn the housekeeping spots. There re two smples for eh tissue in GDS596 nd three to four smples for eh tissue in GDS3142. First, ll of the genes in the two mirorry dtsets were rnked in desending order sed on the rtio etween men expression vlue of eh gene in dipose tissue nd men expression vlue of tht gene in the other five tissues s previously desried (Song et l., 213). The 18-ell yle regultory genes pulished y Cell Cyle RT 2 Profiler TM PCR rry y the Qigen ompny ( om/rt_pr_produt/html/pahs-2z.html) nd 21 other importnt ell yle regultors mentioned in pulished pper (Shfer, 1998) were then seleted nd nlyzed in the two dtsets. Finlly, three inhiitors, CDKN2C nd PMP22, were seleted s they re diretly involved in the ell yle nd showed high rnks in oth dtsets. The expression levels of the three inhiitors were then further ompred mong different differentition stges of predipoytes y nlyzing GDS2743 nd GDS

4 Zhng, Suh, Choi, Ahn, Dvis nd Lee GDS2743 ompres the gene expression etween primry white predipoytes from epididyml white dipose tissue of mie ultured for 4 nd 7 dys, whih orresponds to the undifferentited stge nd differentiting stge (n = 8 for undifferentited predipoytes nd n = 6 for differentiting predipoytes) of dipoytes. GDS2366 ompres the gene expression etween undifferentited nd differentited predipoytes from suutneous dipose tissues in humns (n = 3 for eh of the two groups). In ddition, GDS2659, whih reords gene expression t severl time points (preonfluene, onfluene nd 1, 3, 7 nd 28 dys fter ddition of differentition oktil) during differentition of 3T3-L1 predipoytes, ws lso used to nlyze gene expression of the three inhiitors during differentition of dipoytes in vitro. Experimentl nimls All niml re nd use proedures were pproved y the Institutionl Animl Cre nd Use Committee (IACUC) t The Ohio Stte University. Pigs were rered t the Ohio Agriulturl Reserh nd Development Center (OARDC) Western Agriulturl Reserh Sttion in South Chrleston, OH. Neontl pigs were nursed y sows. Mrket-ge pigs were fed with three different diets during the three phses of their growth. When the pigs weighed 32 to 64 kg, the diet ontined 18% CP nd.95% lysine with metolizle energy (ME) of 3234 kl/kg. When the pigs weighed 64 to 91 kg, the diet ontined 16.3% CP nd.78% lysine with 3243 kl ME/kg. When the pigs weighed more thn 91 kg, the diet ontined 14.3% CP nd.68% lysine with 3247 kl ME/kg. Rering of the pigs used to detet the tissue distriution of gene expression ws the sme s tht for the pigs used to detet gene expression during dipose tissue development (Crwford et l., 21). To detet the tissue distriution of the three inhiitors, CDKN2C nd PMP22 in pigs, dipose tissue, musle, hert, lung, liver, kidney, spleen nd intestine were olleted from four Lndre pigs t dys of ge. In ddition, for Western lot nlysis of tissue distriution of, the sme tissues exept intestine were lso olleted from one Duro pig t2dysofge.todetetexpressionofthethreegenes during development of dipose tissue in vivo, suutneous dipose tissues were olleted from the middle of the k of 15-dy fetl pigs, 6-dy post-ntl pigs nd -dy mrketge Lndre pigs with three individuls in eh group. All of the olleted smples were kept t 8 C fter eing snp frozen in dry ie for totl RNA isoltion nd qpcr (Deiuliis et l., 28; Ahn et l., 213). Seprtion of SV nd FC frtion To ompre gene expression etween the SV nd FC frtions, the two frtions were seprted from three 5 g suutneous dipose tissues tht were olleted from eh of the three -dy mrket-ge Lndre pigs mentioned ove. The suutneous dipose tissues were first mined using rzor ldes efore inution with 3.2 mg/ml of ollgense II (Sigm-Aldrih, St. Louis, MO, USA) t 37 C. After inution for 1 h in shking wter th, the suspension ws pssed through 1 µm nylon ell striner to remove the lrge piees. Finlly, the floting FC frtion ws seprted from the SV frtion in the pellet fter entrifugtion of the filtrte t 5 g for 5 min (Deiuliis et l., 26). In vitro ulture of SV ells After the SV frtion, whih onsists mostly of predipoytes, ws isolted, the ells were diluted in DMEM ulture medium ontining 1% fetl ovine serum (Invitrogen In., Grnd Islnd, NY, USA) nd mixture of peniillin nd streptomyin (Invitrogen). The diluted ells were then mintined nd grown to onfluene (dy ) t 37 C in 5% CO 2. After indution of differentition y dexmethsone (8 nm) for 3 dys post onfluene, the differentition mixture ontining.5 mm of isoutylmethylxnthine, 5 µg/ml of trnsferrin nd 5 µg/ml of insulin ws dded for 6-dy ulture to promote differentition into dipoytes. The ells were olleted on dy, 3, 6 nd 9 fter indution of differentition for extrtion of totl RNA smples, whih were stored t 8 C for susequent RT-PCR. DNA synthesis nd qpcr The totl RNA smples from different tissues nd ell ultures were isolted using Trizol regent (Invitrogen) ording to the mnufturer s instrutions nd the qulity ws ssessed y eletrophoresis on 1% gels. Then the DNA ws synthesized using 1 μg of totl RNA, oligo dt nd moloney murine leukemi virus (M-MLV) reverse trnsriptse (Invitrogen) ording to the mnufturer s instrutions. The synthesized DNA ws then used for qpcr to mesure expression of (forwrd 5 -CAGCTGAAAGCTTGCAACTGC-3 nd reverse 5 -TGAAAATAGGCCAGATCTGATCTGA-3 ), CDKN2C (forwrd 5 -GGGACCTAGAGCAACTTACTAGTTTGT-3 nd reverse 5 -GTGTCCAGGAAACCTGCTCTG-3 ) nd PMP22 (forwrd 5 -CTCCACGATCGTCAGCCAAT-3 nd reverse 5 -GTGAAGAGCTGGCAGAAGAACAG-3 ) with primers shown in the prentheses. qpcr ws performed using AmpliTq Gold polymerse (Applied Biosystems, Grnd Islnd, NY, USA), SYBR green I s detetion dye nd ylophilin (y) s n internl ontrol with yling prmeters s follows: 95 C for 1 min, followed y 4 yles of 94 C for 3 s, 6 C for 1 min, nd 82 C for 3 s. The reltive gene expression ws then lulted s the rtio of trget gene to y expression. In ddition, in order to verify seprtion of the SV nd FC frtions, differentition of predipoytes in vitro nd development of dipose tissue in vivo, the expression of one predipoyte mrker, delt-like 1 homolog (DLK1), nd one dipoyte mrker, peroxisome prolifertor-tivted reeptor γ (PPARγ), ws lso mesured y qpcr. The sequenes of primers for y, DLK1 nd PPARγ in qpcr were desried in previous reports (Li et l., 27). Protein isoltion nd western lot nlysis In order to detet protein expression mong different tissues nd different time points during dipose development, 82

5 Expression of ell yle inhiitors during dipogenesis protein ws isolted from different tissues of one -dy Lndre pig nd one 2-dy Duro pig, s well s dipose tissues of two individuls t eh developmentl stge. Due to lk of ville peptide sequene nd ntiody, only expression of ws deteted. Western lot nlysis ws rried out following the proedure desried in our previous report (Li et l., 2). Protein ws extrted from ~1 mg of tissue fter homogeniztion in 1 ml of lysis uffer followed y entrifugtion t r.p.m. for 5 min t 4 C. Coomssie stin ws used to determine n equl mount of protein loded for eh smple efore Western lotting. For Western lotting, the proteins in eh smple were trnsferred to polyvinylidene fluoride memrne (GE Helthre, Pistwy, NJ, USA) fter seprtion in SDS-PAGE y the mini-protein system (Bio-Rd, Herules, CA, USA). The memrne ws then loked for 3 min in 4% non-ft dry milk in 1 tris-uffered sline Tween-2 (TBST;.1% Tween 2) nd inuted overnight t 4 C with primry ntiody rised ginst the region of humn protein (Bioss In., Wourn, MA, USA) in 4% non-ft dry milk. After eing wshed with 1 TBST for 4 min eh time for seven times, the memrne ws inuted with horserdish peroxidse-onjugted seondry ntiody (Cell Signlling Tehnology In., Dnvers, MA, USA) in 4% nonft dry milk for 1 h t room temperture, nd then wshed gin with 1 TBST for 4 min eh time for seven times. Finlly, nds were deteted with Hyperfilm (GE Helthre) in drk room fter the Amershm ECL Plus Western Blotting Detetion Regents were pplied on the memrne. To ensure tht the nds deteted were the trget nds (38.9 kd), the protein isolted from musle of postntl 2-dy mouse ws used s positive ontrol. Sttistil nlysis Sttistil nlysis for the tissue distriution of gene expression ws performed using mixed model (MIXED) proedure ville in the SAS softwre (version 9.3, SAS Institute In., Cry, NC, USA). The DIFF option ws used to detet signifint differenes etween pirs of lest squres mens. Comprison of gene expression etween two groups ws onduted in SAS using Student s t-test. Multiple omprisons mong the different time points during in vitro differentition nd in vivo development were rried out using one-wy ANOVA followed y Fisher s post ho test. P-vlues lower thn 5 were treted s signifint. All of the results re presented s lest squres mens plus or minus stndrd errors of the lest squres mens (s.e.m.). Results Dt nlysis sed on the GEO DtSets A totl of genes in the GDS596 (humn) nd genes in the GDS3142 (mouse) dtsets were rnked in desending order sed on the rtio etween verge expression in dipose tissue nd verge expression in the other five tissues. By ompring high-rnking ell yle regultors ross the humn nd mouse, three ell yle inhiitors were seleted tht hd reltively high expression in the dipose tissue of oth the humn nd mouse (Tle 1). The rnks of, CDKN2C nd PMP22, respetively, were 2, 654 nd 223 in GDS596 nd 1878, 55 nd 27 in GDS3142. Although expression of the three genes ws not neessrily highest in dipose tissue, the three genes generlly hd high expression in lung or dipose tissue nd the lowest expression in liver. In the ell ulture of predipoytes from mouse epididyml dipose tissue in GDS2743, ll three ell yle inhiitors showed signifintly greter expression in differentiting predipoytes (7 dys in ell ulture) thn in undifferentited predipoytes (4 dys in ell ulture) (Figure 1). In ddition, the differentited predipoytes in humn suutneous dipose tissues in GDS2366 lso exhiited greter expression of the three ell yle inhiitors ompred with undifferentited predipoytes (Figure 1). The high expression of these genes in differentited predipoytes ws further onfirmed y the 28-dy timeline reords of gene expression during differentition of 3T3-L1 predipoytes in GDS2659, euse the three inhiitors were generlly expressed to greter degree t the end of differentition thn t the initition of differentition (Figure 1). However, there ws lso trnsient flutution in expression for PMP22 nd from preonfluene to 3 dys fter differentition Tle 1 The ell yle regultor expressed higher in dipose in oth humn nd mouse Speies Gene (rnk) Adipose Musle Hert Lung Liver Kidney A/O (2) 238 ± ± 54 ± ± ± ± Humn CDKN2C(654) 358 ± 4 58 ± ± ± 3 42 ± ± PMP22 (223) 26 ± ± ± ± ± 13 e 369 ± 65 d 1.43 (1878) 735 ± ± ± ± ± ± Mouse CDKN2C (55) 2677 ± ± ± ± ± ± PMP22(27) 1833 ± ± ± ± 68 3 ± 18 e 248 ± 14 d 1.65 A/O = rtio etween verge expression in dipose tissue nd tht in the other tissues; PMP22 = peripherl myelin protein 22; CDKN2C = ylin-dependent kinse inhiitor 2C; = ylin G2. -e Different supersript indite signifint differene (P < 5). 83

6 Zhng, Suh, Choi, Ahn, Dvis nd Lee () () 1 EU ED 8 *** ** * 8 SU SD 6 * *** * CDKN2C PMP22 CDKN2C PMP22 () CDKN2C PMP PC CF D1 D3 D7 D28 Figure 1 Gene expression nlysis of PMP22, CDKN2C nd sed on mirorry dtsets otined from NCBI wesite. () Expression omprison of the three genes etween undifferentited (epididyml undifferentited; EU, ultured for 4 dys, n = 8) nd differentiting (epididyml differentiting; ED, ultrued for 7 dys, n = 6) predipoytes from mouse epididyml dipose tissue sed on GDS2743. () Expression omprison of the three genes etween undifferentited (suutneous undifferentited; SU, n = 3) nd differentited (suutneous differentited; SD, n = 3) predipoytes isolted from suutneous dipose tissue of humn sed on GDS2366. () Expression of the three genes t vrious time points during differentition of 3T3-L1 predipoytes in GDS2659. In the histogrms, eh r reprents men ± s.e.m. nd sttistil signifine of Student s t-test is indited y *P < 5, **P < 1 nd ***P < 1. efore nother strong inrese, whih my hve een used y the ddition of differentition oktil. Expression of the three seleted genes in vrious tissues of pigs mrna expression of the three ell yle inhiitors in the Lndre pigs showed slightly different pttern from tht in humn (GDS596) nd mouse (GDS3142). None of the three genes showed the highest expression in dipose tissue. For PMP22, the highest expression ourred in the lung, ut without signifint differenes from musle nd dipose tissue. For nd CDKN2C, the highest expression ws oserved in musle with the expression in dipose tissue lose to the men vlue of eight tissues. This result ws espeilly ovious for CDKN2C, where expression in musle ws high with the expression in other tissues signifintly low. Therefore, expression of CDKN2C seemed to e muslespeifi. Although the three genes showed different distriutions of expression mong the eight tissues, they exhiited onsisteny in tht lowest expression ourred in the liver (Figure 2). However, the protein expression of in different tissues showed some disrepny from the mrna expression. Aording to Western lot nlysis, lthough the protein ws lso the most undnt in musle, it ws only deteted in ft, musle nd hert in oth Lndre nd Duro (Figure 2). Expression of the seleted genes in SV nd FC frtions in pigs In qpcr with pig suutneous dipose tissue, ll three genes showed greter expression in the FC frtion. Expression of nd CDKN2C ws more thn 18 nd 2 times greter, respetively, in the FC frtion thn in the SV frtion (P < 1), wheres PMP22 ws expressed less thn two times s muh, nd, therefore, ws expressed nonsignifintly more in the FC frtion thn in the SV frtion (Figure 3). Menwhile, expression of the predipoyte mrker DLK1 ws nerly 5-fold greter in the SV frtion thn in the FC frtion (P < 5), wheres expression of the dipoyte mrker PPARγ ws more thn 15 times greter in the FC frtion thn in the SV frtion (P < 1) (Figure 3), inditing tht isoltion of the two frtions ws suessful. Expression of the seleted genes during dipogeni differentition During the 9-dy in vitro differentition of predipoytes in the SV frtion isolted from porine suutneous dipose tissue, the expression of showed ontinuous nd signifint inrese from 3 to 9 dy fter indution of differentition with expression douling every three dys (P < 5). For CDKN2C, there were two signifint inreses in expression (P < 5). The first inrese ourred from dy to dy 3, while the seond one ourred from dy 6 to dy 9. 84

7 Expression of ell yle inhiitors during dipogenesis () CDKN2C PMP d d d F M H Lu Li K S I F M H Lu Li K S I () Lndre Coomssie stining M(+) F M H Lu Li K S I 5 kd 37 kd 1..5 d d d Duro M(+) F M H Lu Li K S 5 kd 37 kd F M H Lu Li K S I Coomssie stining Figure 2 Expressions of, CDKN2C nd PMP22 in ft (F), musle (M), hert (H), lung (Lu), liver (Li), kidney (K), spleen (S) nd intestine (I) of pigs. () mrna expressions of the three genes were mesured y quntittive rel-time PCR with porine ylophilin (y) s ontrol for normliztion. Brs represent mens ± s.e.m. A mixed model ws used with DIFF option to ompre expressions in different tissues (n = 4 for eh tissue). Tissues with different letters ove the rs re signifintly different (P < 5). () protein levels in different tissues of one -dy Lndre pig nd one 2-dy Duro pig. Protein from musle of 2-dy postntl mouse (M + ) ws used s positive ontrol. DLK1 PPAR CDKN2C PMP *** ** 15 2 ** * 2.5 SVC FC Figure 3 Reltive gene expressions of DLK1, PPARγ,, CDKN2C nd PMP22 in the stroml vsulr (SV) nd ft ell (FC) frtions (n = 3 for eh group) from pig dipose tissue. The rs represent mens ± s.e.m. Sttistil signifine is indited y *P < 5 nd ***P < 1. The expression vlues re normlized y porine ylophilin (y) gene. For PMP22, lthough the inresed expression lso seemed ontinuous, it ws only signifint from dy 6 to dy 9 (P < 5) with n inrement of 83% (Figure 4). Menwhile, DLK1 expression deresed 94.5% from dy to dy 3 (P < 5) nd remined t very low level from 3 dys fter indution of differentition onwrd. In ontrst, expression of PPARγ ws low t dy nd 3, ut inresed more thn four times nd two times, respetively, y dy 6 nd 9 (P < 5) (Figure 4), inditing suessful differentition of predipoytes into dipoytes. Expression of the seleted genes during dipose development As the three ell yle inhiitors ll inresed during dipogeni differentition, ll of them lso inresed signifintly during dipose development in vivo from 6 dys to dys 85

8 Zhng, Suh, Choi, Ahn, Dvis nd Lee DLK1 PPAR CDKN2C PMP Dy Dy 3 Dy 6 2 Dy Figure 4 Expressions of DLK1, PPARγ,, CDKN2C nd PMP22 during pig primry dipoytes differentition in vitro. Gene expressions were deteted t initition of differentition (dy ) nd fter differentition (dy 3, 6 nd 9) nd normlized y porine y gene. ANOVA followed y Fisher s post ho test ws performed to exmine the differenes of gene expressions mong the four time points (n = 3 for eh time point) whih re indited y different letters ( ) if signifint (P < 5). () DLK1 PPAR CDKN2C PMP Fetus 6 d old Adult () 5 kd M(+) F1 F2 N1 N2 A1 A kd 9 6 Coomssie stining 3 Figure 5 Gene expression in suutneous dipose tissues of 15-dy fetl (Fetus), 6-dy neontl (6-dy-old) nd -dy pigs (dult). () mrna expression of DLK1, PPARγ,, CDKN2C nd PMP22 during pig dipose development in vivo. Gene expressions were normlized y porine y gene. ANOVA followed y Fisher s post ho test ws performed to exmine the differenes in gene expressions mong the three groups (n = 3 for eh group), whih re indited y different letters ( ) if signifint (P < 5). () Protein expression of in suutneous dipose tissues of 15-dy fetl (F1, F2), 6-dy neontl (N1, N2) nd -dy pigs (A1, A2). Coomssie stining results re provided to show equl mounts of proteins loded in different wells in the sme gel. Protein from musle of 2-dy postntl mouse (M + ) ws used s positive ontrol. fter irth (P < 5). The mrna expression of, CDKN2C nd PMP22 inresed more thn 33, 4 nd 9 times, respetively, during this period, oinident with seven-fold inrese of PPARγ, whih is ontrry to the 1-fold derese of DLK1 from the fetl stge to the neontl stge (P < 5) (Figure 5). However, ll three genes mintined low level of 86

9 Expression of ell yle inhiitors during dipogenesis expression in 15-dy fetuses nd 6-dy old piglets, s in the se of PPARγ. This trend differed from tht of DLK1, whih ws highly expressed in 15-dy fetuses (Figure 5). Western lot nlysis of lso showed results onsistent with those of qpcr, sine the protein expression t different time points of development showed the sme trend s the mrna expression (Figure 5). In 15-dy fetuses nd 6-dy old piglets, there ws only smll mount of mrna without detetle protein, wheres in the -dy pigs, there ws undnt mrna nd protein expression. Disussion In this study, we pplied powerful strtegy tht omined mirorry dt deposited in NCBI s GEO puli dtse with the dt from our qpcr nd western lotting experiment, nd, therefore, onduted more omprehensive nlysis nd mde stronger onlusions with less time nd finnil ost due to experimenttion. There re nerly 1 GEO DtSets on FCs nd dipose tissues; therefore, we n often find useful dt supporting our reserh onlusions y exploring these dtsets. In ddition, these dtsets lso provide free tests of our hypothesis, nd, thus, guide our study in the right diretion without wsting time, money nd effort in the initil explortion of the hypothesis. For instne, seletion of the three importnt ell yle inhiitors in this study would hve een diffiult nd ostly without the five GEO DtSets, euse there re so mny ell yle inhiitors. The differentition of predipoytes to FCs hs een knowledged to e tightly oordinted with hnges in the ell yle, euse the predipoytes undergo ell prolifertion, whih is rrested in the mture FCs (Fjs et l., 1998). Therefore, it is resonle to hypothesize greter expression of some importnt ell yle inhiitors in mture FCs thn in the dipoyte preursors. This hypothesis ws proven y exmintion of the GDS2743 nd GDS2366 dtsets, euse the three ell yle inhiitors tht we studied exhiited muh greter expression in differentiting or differentited predipoytes thn in undifferentited predipoytes in oth humns nd mie. This result is lso supported y the higher expression of the three genes in the FC frtion ompred with the SV frtion, whih minly ontins predipoytes, suggesting tht the three ell yle inhiitors my e relted to ell yle inhiition during dipogeni differentition nd in the mintenne of the quiesent stte of mture FCs. In ddition, results of the 9-dy porine predipoyte primry ulture lso greed with results of the 3T3-L1 ell ulture in the GDS2659 dtset in terms of the inresing expression of the three genes during differentition. The highest level of DLK1 gene expression nd the lowest level of PPARγ expression t dy indited n undifferentited predipoyte stge, wheres the drmti derese in DLK1 expression fter indution of differentition, whih oinided with the grdul inrese in PPARγ expression, indited tht differentition ws promoted (Sms nd Sul, 1993; Deiuliis et l., 28; Li et l., 29). The grdul inrese in expression of the three ell yle inhiitors my therefore indite tht inhiition of the ell yle during the differentition proess is grdul rther thn immedite. This onlusion is lso supported y the disovery of smll prolifertive dipoytes (SPA), whih hve tiny or no lipoid droplets, expression of dipoyte mrkers nd limited prolifertive ility, nd, thus, my represent the middle stge of differentition (Hnmoto et l., 213). Moreover, it seems tht signifint inrese of PMP22/gs3 only ourred in the lter stge of differentition, suggesting tht it my e relted more to the growth rrest stte in the mture dipoytes thn in the differentition proess. Bsed on the ove nlysis, it seems tht the three ell yle inhiitors promote the differentition proess minly y ell yle inhiition, even though there re some reports of positive orreltions of (Morrison nd Frmer, 1999) nd CDKN2C (Hiri et l., 1995) with PPARγ, euse PPARγ lso indues ell yle withdrwl during dipoyte differentition (Altiok et l., 1997). Therefore, we speulte tht nd CDKN2C my form positive feedk loop with PPARγ oupling ell yle inhiition nd the dipogeni differentition proess. The PPARγ-dependent expression pttern of the three ell yle inhiitors lso seems to e ge-dependent during in vivo development of dipose tissue, euse ll three genes nd PPARγ exhiited muh greter expression in mrket-ge pigs thn in the erly ge groups. For, this is further onfirmed t the protein level. In the 15-dy fetl pigs, the development of dipose tissue is minly hrterized y n inrese of FC numer y prolifertion of predipoytes nd immture FCs (Desnoyers et l., 198). Therefore, the expression of DLK1 ws high, wheres expression of the three ell yle inhiitors nd PPARγ ws low. In the 6-dy postntl pigs, DLK1 nd PPARγ s well s the three ell yle regultors, hd low expression levels, euse this developmentl stge is hrterized y n inrese of SPA ells, whih express dipoyte mrkers, ut still n proliferte (Anderson nd Kuffmn, 1973). In the 4-month old pigs, the drmti inrese in expression of the three ell yle inhiitors nd PPARγ my indite n inresing numer of mture FCs during this period, euse the inrese in FC size eomes dominnt in the development of dipose tissue, wheres the inrese in FC numer grdully slows during this period (Anderson nd Kuffmn, 1973). However, even in this stge, ell yle tivity my not ompletely stop in dipose tissue, euse there re still predipoytes or SPA ells with the potentil to differentite into mture FCs. This is perhps the reson why expression of the three genes in dipose tissue in the tissue distriution nlysis for 4-month old pigs is not s high s tht for dult humns (>2 yers old) nd mie (1 to weeks old) in the GDS596 nd GDS3142 dtsets. The FC numer seems to minimlly inrese fter 18 yers of ge in humns (Knittle et l., 1979) or fter 3 weeks of ge in mie (Johnson nd Hirsh, 1972), ut still inreses in pigs efore 7 months of ge (Anderson nd Kuffmn, 1973). In ddition to the lose reltionship of these three genes in dipoyte prolifertion nd differentition, the low expression 87

10 Zhng, Suh, Choi, Ahn, Dvis nd Lee of the three ell yle regultors in the liver of the humn, mouse nd pig my lso reflet high potentil of heptoytes to proliferte ompred with the differentited ells in other tissues, euse they n re-enter the ell yle soon fter injury to filitte regenertion (Alreht et l., 1998). Moreover, the high expression of PMP22 in the lung of the humn, mouse nd pig my lso e importnt for norml funtion in the quiesent stte of lung ells, euse expression of PMP22 ws reported to e down-regulted in lung tumors of mie (Re et l., 1992). Finlly, the high mrna expression nd low protein expression of in lung nd intestine my indite tht some post-trnsriptionl mehnisms re involved in the regultion of this gene nd tht my not e the mjor regultor of ell yle inhiition in these tissues. In onlusion,, CDKN2C nd PMP22 re three importnt genes in dipogenesis, euse they my promote dipoyte differentition through ell yle inhiition. Hyperplsi, whih requires ell yle tivity, preedes differentition from predipoytes into FCs. Therefore, overexpression of these ell yle inhiitors in the erly developmentl stge nd inhiition of their expression in the lter stge of dipogenesis my limit the inrese of FC numer in nimls nd humns used y FC prolifertion or differentition (Hirsh nd Knittle, 197; Fust et l., 1978), nd, therefore, provide potentil methods of reduing exessive ft tissue in dietry met nd uring oesity in humns. In ddition, sine the ell yle is tivted in differentited nd dedifferentited liposrom ompred with norml ft tissue (Singer et l., 27), indution of terminl differentition nd ell yle inhiition of liposrom my provide possile therpeuti method for the tretment of liposrom (Tontonoz et l., 1997). For exmple, CDK4 is the min trget for mplifition of hromosome in well-differentited or dedifferentited liposroms (Louis-Brennetot et l., 211), wheres CDKN2C is one of the min inhiitors of CDK4, nd, thus, my provide prospetive remedy for this disese. However, more in-depth studies still need to e done efore pplition of these ell yle inhiitors in these res. Aknowledgments This work ws supported y stte nd federl funds pproprited to the Ohio Agriulturl Reserh nd Development Center SEEDS grnt, The Ohio Stte University nd grnt from the Agend Progrm (No. PJ8587), Rurl Development Administrtion, Repuli of Kore. Referenes Aguilr V, Anniotte JS, Esote X, Vendrell J, Lngin D nd Fjs L 21. Cylin G2 regultes dipogenesis through PPARγ otivtion. Endorinology 151, Ahn J, Oh SA, Suh Y, Moeller SJ nd Lee K 213. Porine G /G 1 swith gene 2 (GS2) expression is regulted during dipogenesis nd short-term in-vivo nutritionl interventions. 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