JOURNAL OF CELLULAR PHYSIOLOGY 194:225–236 (2002)

HOX Gene Network Is Involved in the TranscriptionalRegulation of In Vivo Human Adipogenesis

MONICA CANTILE, ALFREDO PROCINO, MARIA D’ARMIENTO, LUCA CINDOLO,AND CLEMENTE CILLO*

Department of Clinical and Experimental Medicine, Federico II University Medical School,Naples, Italy

Adipogenesis is regulated by the sequential activation of a series of transcrip-tion factors: the C/EBP proteins of type b and d trigger the process while PPARg andC/EBPa induce the differentiation from pre-adipocyte to adipocyte, followed byadipo-specific gene expression. A number of observations suggest the involvementof genes controlling embryonal development in adipogenesis. In human thyroidfollicular carcinoma, it has been recently identified an oncogenetic fusion proteinresulting from the interaction between the isoform PPARg1 of PPARg and thehomeoprotein encoded by the PAX-8 gene. Recent observations have pointed outthat gene expression associated with adipocyte differentiation in vivo and in vitro,although partially overlapping, is actually different.HOX genesmake up a networkof transcription factors (homeoproteins) controlling embryonal development aswell as crucial functions of adult eukaryotic cells. The molecular organization ofthis network of 39 genes appears to be unique in the genome and probably actsregulating phenotypic cell identity. In the present study we have analyzed theexpression of the complete HOX gene network, in vivo, in different deposits ofhuman white adipose tissue and in embryonal brown adipose tissues. Most of thegenes in the HOX network are active in white as well as brown adipose tissue.Furthermore HOX genes display a deposit-specific expression in white adiposetissue. Moreover, expression of the paralogous group 4 genes (HOX A4, HOX B4,HOXC4, andHOXD4), togetherwith that of isolated genes in the network, appearsto discriminate between white and brown adipose tissue. This data allows us topostulate the involvement of the HOX network in transcriptional regulation ofhuman adipogenesis and to hypothesize on the molecular mechanisms that couldbe implicated. J. Cell. Physiol. 194: 225–236, 2002. � 2002 Wiley-Liss, Inc.

Adipogenesis is one of the most studied cell differ-entiation models both for the existence of in vitro cellsystems (Green and Kehinde, 1974) and in order toidentify the molecular bases of such metabolic pathol-ogies as obesity or type 2 diabetes, which are becomingincreasingly widespread in western society (Must et al.,1999).

Transcriptional control of adipogenesis requires theexistence of a transcriptional cascade at whose origin wefind the CCAAT/enhancer binding proteins (C/EBP)(Yeh et al., 1995). In particular, the C/EBPd and b prot-eins trigger the differentiation process (Cao et al.,1991) resulting in the activation of the PeroxisomeProliferating Activated Receptor g (PPARg) (Wu et al.,1999), a crucial regulator of adipogenesis. PPARg,following hetero-dimerization with the retinoic acidreceptor RXR (Tontonoz et al., 1994), activates the adi-pocytic differentiation program inducing expression ofthe C/EBPa protein (Kubota et al., 1999; Rosen et al.,1999). C/EBPa, by means of positive feedback on theheterodimer PPARg-RXR (Rosen et al., 2002), in turnmaintains the differentiated state active. Furthermore,an external regulating protein ADD1/SREP1 is able

to activate both PPARg (Fajas et al., 1999) and a seriesof genes of lipogenesis, interconnecting nutritionalchanges, adipogenesis, and the lipogenic genetic pro-gram (Kim and Spiegelman, 1996).

While the transcriptional pathways involved inthe terminal differentiation of adipogenesis are wellcharacterized, the molecular mechanisms inducingprimitive mesenchymal precursors towards adipo-cytic differentiation are less well known (Rosen andSpiegelman, 2000). Moreover, there are still somedifficulties in identifying the processes that enablediversification of the brown and white phenotypes in

� 2002 WILEY-LISS, INC.

Contract grant sponsor: MURST 2001 projects.

*Correspondence to: Clemente Cillo, Department of Clinical andExperimental Medicine, Federico II University Medical School,Via S. Pansini 5-80131, Naples, Italy. E-mail: clecillo@unina.it

Received 6 August 2002; Accepted 11 October 2002

DOI: 10.1002/jcp.10210

adipogenesis, beyond the description of PGC-1, a co-factor of PPARg, which seems to be involved only inthe differentiation towards the brown adipose tissue(Puigserver et al., 1998).

Recent comparisons of the expression patterns of alarge number of genes in pre-adipocytes and adipocytesin vitro and in vivo, using the microarrays technique,have pointed out that the gene programs associated toadipocyte differentiation turn out to be more complexthan had so far been established, and that many as yetunidentified regulator genes are active during in vivoadipogenesis, contrary to in vitro observations (Soukaset al., 2001).

Numerous observations have linked genes regulatingembryonal development to adipogenesis and lipidicmetabolism. The morphogenetic protein encoded bytheHedgehoggene is localized onthe lipid raftsof thecellmembrane (Simons and Toomre, 2001) and undergoespost-transductional modification linking: (i) cholesterolto its carbosillic end, which is essential for the protein’sreceptor activity; (ii) an acylated fatty acid to theprotein’s amminic end, in order to determine the extentof its signal (Ingham, 2001). Alterations in the Hedgehogprotein’s signaling caused by an inborn error of choles-terol metabolism generate the Smith-Lemli-Optiz syn-drome, archetypal of major alterations in embryonaldevelopment (Kelley and Hennekam, 2000). In humanthyroid follicular carcinoma, an oncogenic proteinresulting from the fusion of the product of the genecontaining homeobox PAX-8 and the PPARg1 isoform ofthe adipogenic transcription factor PPARg has recentlybeen isolated (Kroll et al., 2000). The pref-1 proteinacts on the pre-adipocytic membrane as a powerfulinhibitor of adipogenesis (Smas and Sul, 1993) and isencoded by the dlk (Delta like) gene, a homologue of thehomeotic Delta gene, involved in controlling neuronalsignaling during embryonal development in Drosophila(Kramer, 2001).

Homeobox genes are transcription factors that actduring normal development (Gehring and Hiromi, 1986)and contain the homeobox, a 183bp DNA sequencecoding for a 61amino acid domain defined as home-odomain (HD). Different HD types or classes may beidentified through sequence similarities within thehomeodomains (Duboule and Morata, 1994), eachcharacterizing a homeobox gene family. Among these,the HD of the homeotic gene of Drosophila Anten-napedia (Antp) defines a consensus sequence referredto as class I HD or Hox genes (Akam, 1987). In mice(Hox genes) and humans (HOX genes) there are at least39 genes (Fig. 1 right-side) organized in four genomicclusters of about 100 kb in length, defined Hox loci, eachlocalized on a different chromosome (HOX A at 7p15.3,HOX B at 17p21.3, HOX C at 12q13.3, and HOX D at2q31) (Apiou et al., 1996) and comprising from 9 to11 genes. On the basis of sequence similarity andposition on the locus, corresponding genes in the fourclusters can be aligned with each other and with genes ofthe HOM-C complex of Drosophila in 13 paralogousgroups (Scott, 1992). During mammalian development,Hox gene expression controls the identity of variousregions along the body axis, from the branchial areathrough to the tail (Graham et al., 1989). This isachieved according to the rules of temporal and spatial

co-linearity, with 30 Hox genes expressed early in deve-lopment and controlling anterior regions, followed byprogressively more 50 genes expressed later and con-trolling more posterior regions (Dekker et al., 1992).In particular, 30 Hox genes in groups 1–4 (cervical) pri-marily control the development of the branchial areaand the rhomboencephalon, the embryonic region cor-responding to the hindbrain (Lumsden and Krumlauf,1996). Central Hox genes in groups 5–8 control thethoracic portion of the body, whereas 50 Hox genes ingroups 9–13 control the lumbo-sacral region. The HOXgene network, the most repeat-poor regions of thehuman genome (Lander et al., 2001), is also expressedin normal adult human organs (Cillo, 1994–95).Hoxandhomeobox genes appear to regulate normal develop-ment, normal and abnormal cell proliferation as well ascrucial cellular processes, as proven by the recentdescription of congenital (Mortlock and Innis, 1997),somatic (Nakamura et al., 1996), metabolic (Ferberet al., 2000), and neoplastic alterations (Cillo et al., 2001)involving these genes.

In view of the above considerations, we decided tostudy the expression of the whole HOX gene network(39 genes) in different deposits (extra-peritoneal, intra-peritoneal, and dermic) of normal human white adiposetissue and in fetal brown adipose tissue. The resultsshow that the HOX network is active for almost all thegenes of which it is made up, both in the white andthe brown adipose tissues. Moreover, the expression ofthe genes in the network appears to be deposit-specific,in white adipose tissue, and makes it possible to detectsignificant differences between white and brown adi-pose tissue.

MATERIALS AND METHODS

The genome sequences corresponding to the 39 genesin the HOX network were obtained from the GeneBank(www.ncbi.nlm.nih.gov) in order to identify the specificHOX gene primers. We analyzed the exon sequences soas to establish, for each gene, a pair of primers (senseand anti-sense) that can generate amplified fragmentsmeasuring between 150 and 700 bp in length and withannealing temperature between 55 and 628C. The senseand anti-sense primers were selected so as to include atleast one intron to prevent genomic DNA contaminationduring amplification (Table 1). Each pair of primers wastested using RT-PCR on total RNA from humanhaemopoietic cell lines available in the laboratory, andcompared with the expression of the same HOX gene,on poly (Aþ) RNA obtained from the same cell linesand determined by Northern Blotting using a radio-active probe specific for the corresponding HOX gene(data not shown).

Biopsies of adipose tissue

The biopsies of extraperitoneal, intra-peritoneal, anddermic normal, human, white adipose tissue wereobtained from the Surgery Departments of the ‘‘FedericoII’’ University of Naples. The biopsies of retro-renal,fetal brown adipose tissue were supplied by the Depart-ment of Pathology at the same university. Afterremoval, the bioptic material was immediately frozenin liquid nitrogen and then stored in a freezer at �808Cuntil RNA extraction.

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Fig. 1. Left. Diagram of HOX gene expression detected by RT-PCR in human samples of white adiposetissue, extra-peritoneal (E-WAT), intra-peritoneal (I-WAT), and dermic (D-WAT), and in human brownadipose tissue (BAT). Black and white small rectangle indicate active or silent HOX genes, respectively.Right. Schematic representation of the four HOX loci (see the text for details).

Extraction of RNA and expression analysisby means of duplex RT-PCR

Frozen tissues were pulverized with a blender. TotalRNA was extracted by the guanidinium thiocyanatetechnique. Four mg of total RNA were subjected to cDNAsynthesis for 1 h at 378C using the ‘‘Ready to go You-Primer First-Strand Beads’’ kit (Amersham PharmaciaBiotech, Piscataway, NJ, cod. 27-9264-01) in a reactionmixture containing 0.5 mg oligo-dT (Amersham Phar-macia Biotech cod. 27-7610-01).

PCR amplification of cDNA was performed in a reac-tion mixture containing 4 ml of cDNA sample anddifferent primer sets (20 p/mol each). The co-amplifica-tion of each HOX gene and human b-actin gene, as aninternal control, was achieved using 2 primer sets in asingle reaction mixture. We selected two pairs of b-actinprimers to obtain amplified fragments with differentmolecular weight (149 and 433 bp; see Table 1), to beused alternatively in the co-amplification reactionaccording to the size of the HOX signal.

Duplex-PCR products were separated by ethidium1.2% agarose gel electrophoresis.

RESULTS

We extracted total RNA from 38 samples of adiposetissue taken from different deposits on the human body.These were divided into: 11 samples of extra-peritonealwhite adipose tissue (E-WAT); 8 samples of intra-peritoneal white adipose tissue (I-WAT); and 11 samplesof dermic white adipose tissue (D-WAT). We alsoanalyzed 8 samples of human brown adipose tissue(BAT) taken from the retro-renal area of fetusesbetween the 15th and 30th week of development.

Although the yield in extracted RNA varied betweensamples taken from the same deposits, we observedhighly marked differences in the quantity of RNAextracted from samples of adipose tissue taken fromdifferent deposits. While the yield of RNA in intra-peritoneal and extra-peritoneal adipose tissue is in theorder of 10�5–10�6 g of RNA/g of tissue, in dermicadipose tissue 8 out of 11 samples did not yield

TABLE 1. Sequence of PCR primers

Gene Sense primer (50–30) Antisense primer (50–30)GeneBank

accession no. TM8CFragmentlength (bp)

HOX A1 ATG AAC TCC TTC CTG GAA TA CGT ACT CTC CAA CTT TCC AC004079 56 625/655HOX A2 GAC GCT TTC ACA CTC GAC TGG TGT AAG CAG TTC TCA G AC004079 55 151HOX A3 ATG CCA ATC AGC AGC CGT A TGT ACT TCA TGC GGC GAT AC004079 55 658HOX A4 TGC ATG CGA GCC ACG TCC T TTG ACC TGG CGC TCA GAC AA NM002141 61 402HOX A5 ATG GCA TGG ATC TCA GCG T GTA ACG GTT GAA GTG GAA CT AC004080 55 499HOX A6 CTG ATA AGG ACC TCA GTG TCA GGT AGC GGT TGA AGT G AC004080 55 299HOX A7 AAT GCC GAG CCG ACT TCT T AGA TCT TAA TCT GGC GCT CG AC004080 56 460HOX A9 ACG GCA GGT ACA TGC GCT GAA CCA GAT CTT GAC CTG C AC004080 57 499HOX A10 CTA CTG CCT CTA CGA CTC AAG TTG GCT GTG AGC TCC AC004080 56 330HOX A11 ACG TGC TGG CCA AGA GCT TGA CTT GAC GAT CAG TGA GG AC004080 57 551HOX A13 CGG ACA AGT ACA TGG ATA C TAT AGG AGC TGG CAT CCG A AC004080 55 337

HOX B1 CCT TCT TAG AGT ACC CAC TCT G GCA TCT CCA GCT GCC TCC TT X16666 61 826HOX B2 TCC TCC TTT CGA GCA AAC CTT CC AGT GGA ATT CCT TCT CCA GTT CC X16665 61 353HOX B3 TAC CAG TGC CAC TAG CAA CA GAA CCA GAT CTT GAT CTG C X16667 55 420HOX B4 CGA GGA ATA TTC ACA GAG CGA T CCA GAT CTT GAT CTG GCG CT AF287967 55 350HOX B5 GCT CTT ACG GCT ACA ATT ACA ATG GCT GTA GCC AGG CTC ATA CT AF287967 58 659HOX B6 AAG AGC AGA AGT GCT CCA CT TGA TCT GCC TCT CCG TCA g184302 56 225HOX B7 AGC CGA GTT CCT TCA ACA TG CGC GTC AGG TAG CGA TTG TA XM008559 58 250HOX B8 TTC TAC GGC TAC GAC CCG CT CGT GCG ATA CCT CGA TTC GC AY014293 55 291HOX B9 CGA TCA TAA GTC ACG AGA GTG TCC TTC TCT AGC TCC AGC GT AY014296 55 568HOX B13 CTG GAA CAG CCA GAT GTG TT TTG GCG AGA ACC TTC TTC TC NM006361 60 300

HOX C4 CTG AAC ACA GTC CGG AAT A TTG ATC TGC CTC TCA GAG AG NM014620 56 515HOX C5 TGG ATG ACC AAA CTG CAC ATG AGC CAA GTT GTT GGC GAT CTC TAT GCG X61755 62 149HOX C6 ACC TTA GGA CAT AAC ACA CAG A ACT TCA TCC GGC GGT TCT GGA A NM004503 58 317HOX C8 CCA CGT CCA AGA CTT CTT CCA CGG C CAC TTC ATC CTT CGA TTC TGA AAC C NM022658 55 449HOX C9 TCA GTC GTC CGT GGT ATA TCA C AGT TCC AGC GTC TGG TAC TTG AY014301 55 280HOX C10 TGT TGG CAG GCC GCT GTC CT CTC CAA TTC CAG CGT CTG GTG T AF255675 62 580HOX C11 CTT CGA CAA CGC CTA CTG CG GTC CGT CAG GTT CAG CAT CC AJ000041 61 359HOX C12 TGC GCT CGG CTT CAA GTA CG TGG CGT GTG ATG AAC TCG TTG AC AF328963 62 337HOX C13 TGT CGC ACA ACG TGA ACC TG CTT CAG CTG CAC CTT AGT GTA G NM017410 60 417

HOX D1 TTC AGC ACG TTC GAG TGG AT TGC GTG TCA TTC AGG TGC AA AF202118 57 210HOX D3 CAT CAG CAA GCA GAT CTT C AGC GGT TGA AGT GGA ATT C NM002146 54 187HOX D4 TGG ATG AAG AAG GTG CAC GT AGA TGA GGA CGA TGA CCT G X17360 56 271HOX D8 GGA TAC GAT AAC TTA CAG AGA C TAG AGT TTG GAA GCG ACT GT X15507 56 219HOX D9 GAG TTC TCG TGC AAC TCG T CAG CTC AAG CGT CTG GTA T g32390 56 285HOX D10 TAG ACT GAG TCA GAC CTA CG TCC AAT CCT GGC CTC TGA T X59373 55 610HOX D11 CTT CGA CCA GTT CTA CGA G CAG ACG GTC TCT GTT CAG T AF154915 57 451HOX D12 AGC AGG CTA AGT TCT ATG CG CAA TCT GCT GCT TCG TGT AG AF154915 57 383HOX D13 AGA AGT ACA TGG ACG TGT CA GTC ACT TGT CTC TCA GAT AG AB032481 55 460

b-actin 149 TCT ACA ATG AGC TGC TGG T TGG ATA GCA ACG TAC ATG G M10277 55 149b-actin 433 CAC CAT GGA TGA TGA TAT CG TGG ATA GCA ACG TAC ATG G M10277 55 433

PPARg2 AGC ATT CGA CTC AAG CTG GT AAG TAG GCC TCG TAG ATT CT NM015869 58 260

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appreciable quantities of RNA and only 3 samples yield-ed between 10�6 and 10�7 g of RNA/g. Whereas, the RNAyield obtained from samples of brown adipose tissue ismuch higher than the RNA extracted from any otherdeposit of white adipose tissue and is in the order of10�3–10�4 g of RNA/g. Therefore, the D-WAT and theBAT supply a minimum and a maximum yield, respec-tively, compared to what can be obtained from any otherdeposit of adipose tissue we considered.

Analysis of the HOX gene expression points out thatthe complete network is, overall, active in adipose tissue(Fig. 1 left-side). Considering the HOX gene expressionin relation to the four chromosome locations, we canobserve that the locus A genes on chromosome 7 areactive in the posterior region including the thoracic andlombo-sacral area of the locus (groups 5–13). Whereasthe locus D genes on chromosome 2 are active in theanterior region of the locus including the cervical andthoracic area (groups 1–9). As for the loci B and C HOXgenes, on chromosomes 17 and 12, respectively, weobserved a more heterogeneous expression of the geneslocated in these loci, although in locus B a thoracic area(groups 5–8) with a more uniform expression can beidentified.

A comparison of the expression of the individual genesin the HOX network from the different types of adi-pose tissue, E-WAT, I-WAT, D-WAT, and BAT (Fig. 1left-side), shows that 11 out of 39 HOX genes areconstitutively active in all types of adipose tissueanalyzed. They include four genes on locus A: HOX A6,A7, A9, and A13; two genes on locus B: HOX B6 andB7; one gene on locus C: HOX C10; and four genes onlocus D: HOX D1, D3, D4, and D8. In contrast, twogenes in the network, HOX B4 and HOX C5 are consti-tutively silent.

As regards the specificity of the expression of indi-vidual HOX A genes in the various deposits of adiposetissue analyzed (Fig. 2), we observe that the gene HOXA1 appears silent in extra-peritoneal, intra-peritoneal,and dermic white adipose tissue, while it is active in5 out of 6 samples of brown fat. However, the gene HOXA3 can be active in E-WAT and I-WAT and tends to besilent in D-WAT and BAT. The gene HOX A4 is alwaysactive in deposits of white adipose tissue while it is silentin BAT. HOX A5 is active in D-WAT and BAT andoccasionally silent in E-WAT and I-WAT. Finally, inlocus A, the genes HOX A10 and A11 can be silent indermic and intra-peritoneal white adipose tissue.

For locus B (Fig. 3), the geneHOXB1 is expressed onlyin dermic fat. HOX B2 tends to be silent in I-WAT. HOXB3 is sporadically active in individual samples of extraand intra-peritoneal white adipose tissue. HOX B5,always active in I-WAT and E-WAT, is silent in isolatedsamples of D-WAT and BAT. HOX B8 appears silent inE-WAT and I-WAT but active in the analyzed samples ofD-WAT and BAT. HOX B9 appears sporadically activein all deposits and finally HOX B13 is silent in extra-peritoneal and dermic white adipose tissue but consti-tutively expressed in I-WAT and may be activatedin BAT.

Locus C (Fig. 4) has the gene HOX C4 which appearsconstitutively silent in white adipose tissue while it isactive in the majority of biopsies of brown adipose tissueanalyzed; the geneHOXC6, is always active in the other

deposits but is occasionally silent in I-WAT. Finally, thegene HOXC11, while heterogeneously expressed, tendsto be silent in intra-peritoneal white adipose tissue andin BAT.

In locus D (Fig. 5), besides a certain heterogeneousexpression of HOX D10 and D11 in the various types ofadipose tissue, the gene HOX D12 tends to be silent inbrown adipose tissue and occasionally silent in D-WATand I-WAT.

A comparison of the HOX gene expression in the13 paralogous groups into which the network can bedivided makes it possible to identify the paralogousgroups 4 and 1 as those of greater interest in the wholenetwork. As reported in Figure 1 (left side), the genes inparalogous group 4, HOX A4, HOX B4, HOX C4, andHOX D4 manifest a characteristic expression related tothe different deposits and types of adipose tissue. HOXA4 is always active, but not in brown fat; HOX B4appears constitutively silent in every type of adiposetissue; HOX C4 is always silent but it is active in BAT;HOX D4 appears constitutively expressed in everytype of adipose tissue analyzed. In paralogous group1, HOX A1 is active only in brown fat, HOX B1 can beexpressed only in dermic white adipose tissue whileHOX D1 is constitutively active in all deposits and typesof adipose tissue.

Finally, for all the samples of adipose tissue studied,we determined the expression of the main transcriptionfactor of adipogenesis, PPARg. The results reportedin Figure 6 point out that the gene PPARg is constitu-tively expressed in the different types of adipose tissueanalyzed.

DISCUSSION

The biological characteristics and gene expression ofwhite adipose tissue varies according to the locationof the deposit (Adams et al., 1997; Lefebvre et al., 1998).In particular, dermic WAT appears to be mostly made upof differentiated adipocytes, with a minimal presence ofpre-adipocytes showing a low transcriptional activity.This corresponds to: (a) a minor involvement in thesynthesis and release of signal molecules such as leptin(Arner, 2001); (b) the negligible clinical consequencesthat the accumulation of subcutaneous fat provokes inman, with regard to the increased risk of insulin-resistance, dyslipidemia, and cardiovascular diseaseas a result of an increase in visceral adiposity (Reaven,1988); (c) the small quantities of RNA that can beobtained from dermic deposits. In contrast, brown adi-pose tissue is metabolically more active both because ofits crucial role in embryonal development (Rosen andSpiegelman, 2000), and because it makes possibleadaptive thermogenesis (Spiegelman et al., 2000), aphysiological process which requires an intense tran-scriptional activity, as proven by the high RNA yieldsthat can be obtained from BAT.

In addition to the role of HOX genes in controllingembryonal development, a less well documented butequally important function of the network is to controlcell growth and proliferation (Scott, 1997) and deter-mine the cell’s phenotypical identity (Cillo et al., 1999).Under this hypothesis, the HOX network acts as asystem for decoding external signals so that the geno-type can react to the microenvironment. It is in this

IN VIVO HUMAN ADIPOGENESIS 229

Fig. 2. RT-PCR expression of locus A HOX genes in human samples of white adipose tissue, extra-peritoneal (E-WAT), intra-peritoneal (I-WAT), and dermic (D-WAT), and in human brown adipose tissue(BAT). Each lane contains amplified cDNA fragments from single biopsies of separate patients, male andfemale randomly selected. Control co-amplifications with 149 or 433bp b actin primers are reported.Duplex PCR products were separated by ethidium 1.2% agarose gel electrophoresis.

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framework that the HOX network is able to commu-nicate with the main signal transduction pathways(Miller et al., 2001) and manifest certain peculiaritiestowards any other area of the genome. Indeed, thealmost completed sequence of the human genome hasidentified the HOX loci as the regions of the whole DNAwith the smallest number of repetitions in the entire

genome (Lander et al., 2001). Moreover, the HOX geneexpression reflects differentiation stages that are char-acteristic of specific cell populations, as demonstrated inthe haemopoietic system (Magli et al., 1991). The datawe present with regard to expression of the HOXnetwork in adipose tissue seems to confirm the samefunction in adipogenesis. The expression of paralogous

Fig. 3. RT-PCR expression of locus B HOX genes in human samples of white adipose tissue, extra-peritoneal (E-WAT), intra-peritoneal (I-WAT), and dermic (D-WAT), and in human brown adipose tissue(BAT). Each lane contains amplified cDNA fragments from single biopsies of separate patients, male andfemale randomly selected. Control co-amplifications with 149 or 433bp b actin primers are reported.Duplex PCR products were separated by ethidium 1.2% agarose gel electrophoresis.

IN VIVO HUMAN ADIPOGENESIS 231

group 4 HOX gene is evidently configured in a way toinduce two directions of differentiation in the same stemcell (a mesenchymatic adipocytic precursor). The HOXgenes B4 and D4, constitutively silent and active,respectively, in all types of adipose tissue act as lineagemarkers. The HOX A4 gene, active in white adiposetissue and silent in brown fat, and vice versa HOX C4,silent in white adipose tissue and active in brown fat, actas markers of the diversification between white andbrown adipose tissue.

The transcription factor PPAR-g, which is crucial inthe transcriptional control of adipogenesis, has beenidentified as an enhancer of the aP2 gene (Graves et al.,1992). The aP2 gene encodes the adipo-specific protein,which allows adipocytes to bind fatty acids (Hotamisligilet al., 1996). The promoter of the HOXA4 gene presentsa series of binding sites along the aP2 sequence(Doerksen et al., 1996) and belongs to the paralogousgroup 4 HOX gene in the network, whose expres-sion appears to differentiate between white and brown

Fig. 4. RT-PCR expression of locus C HOX genes in human samples of white adipose tissue, extra-peritoneal (E-WAT), intra-peritoneal (I-WAT), and dermic (D-WAT), and in human brown adipose tissue(BAT). Each lane contains amplified cDNA fragments from single biopsies of separate patients, male andfemale randomly selected. Control co-amplifications with 149 or 433bp b actin primers are reported.Duplex PCR products were separated by ethidium 1.2% agarose gel electrophoresis.

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adipose tissue. Moreover, the only data available in theliterature regarding the expression of some HOX genesin the NIH3T3L1 pre-adipocytic cell line points to arole played by HOX A4 gene in adipogenesis (Cowherdet al., 1997). It appears that HOX A4 is expressed inpre-adipocytic cells (as we have detected in vivo in whiteadipose tissue) but its expression tends to decreaseduring differentiation. Our data stresses the impor-tance of this gene along with its paralogs, in thedifferentiation stages of adipocytic stem cells where

phenotypical diversification between white and brownadipose tissue probably occurs.

The possibility of diversifying (white-brown) pheno-types of adipose tissue through paralogous group 4HOXgene expression, or of characterizing different whiteadipose tissue deposits (dermic, intra-peritoneal, andextra-peritoneal) concerns other genes in the HOXnetwork. In paralogous group 1, the HOX A1 geneseems to be a marker of brown adipose tissue, whereasthe HOX B1 gene characterizes the dermic deposits of

Fig. 5. RT-PCR expression of locus D HOX genes in human samples of white adipose tissue, extra-peritoneal (E-WAT), intra-peritoneal (I-WAT), and dermic (D-WAT), and in human brown adipose tissue(BAT). Each lane contains amplified cDNA fragments from single biopsies of separate patients, male andfemale randomly selected. Control co-amplifications with 149 or 433bp b actin primers are reported.Duplex PCR products were separated by ebridium 1.2% agarose gel electrophoresis.

IN VIVO HUMAN ADIPOGENESIS 233

white adipose tissue, and the HOX D1 gene appearsubiquitously expressed. The HOX B13 gene appearslinked to the intra-peritoneal site and can be active inBAT. The HOX C11 gene is silent in intra-peritonealdeposits and HOX D12 is only slightly active in brownadipose tissue. In addition to the behavior of individualgenes, it is clear that the combination of the entireHOX network expression characterizes every depositof white adipose tissue, and white versus brownadipose tissue.

As regards the mechanisms through which HOXgenes are involved in adipogenesis, at present we canonly hypothesize. Substantial differences in gene ex-pression have been found between in vitro and in vivocells of adipose tissue, and it is well-known that pre-adipocyte cell lines in culture do not produce TNFa andleptin, in proportion to cell size and lipid content, as isnormally observed in vivo (Friedman and Halaas, 1998;Rosen and Spiegelman, 1999). Double knock out mice forC/EBP-b and d, nevertheless display white and brownadipose tissue and express normal levels of PPARg andC/EBP (Tanaka et al., 1997). This all suggests theexistence of alternative mechanisms that can inducePPARg and are independent of C/EBP. Another unclearpoint in the transcriptional control of adipogenesis isthe way in which ADD1/SREBP1 is able to induceadipocytic differentiation by acting directly on PPARgprobably through the production of an endogenousligand of PPARg (Kim et al., 1998). Each of these phasesof transcriptional control of adipogenesis might implythe involvement of the HOX gene network.

Another possible link between HOX genes andadipogenesis is related to the important role thatretinoic acid and its receptors play in both systems. Itis well known that retinoic acid can induce expression ofHOX network genes, if they are silent, according to therules of spatio-temporal co-linearity (Simeone et al.,1990). Moreover, the crucial point of transcriptionalcontrol in adipogenesis is the formation of the hetero-dimer by PPAR-g and the retinoic acid receptor RXR(Tontonoz et al., 1994). Our data seems to show that itwould be of primary interest to study whether the actionof HOX genes on adipogenesis takes place directlythrough interaction on the heterodimer PPARg-RXR.

An emerging concept in biology tends to aggregate, inthe genome, genes performing functions stemming fromcommon molecular mechanisms and convergent meta-bolic processes. Moreover, adipogenesis is part of a moregeneral process of regulation of the organism’s energy

balance which is linked both to fat and carbohydratemetabolism, as proven by adipocytes’ sensitivity to insu-lin (Spiegelman and Flier, 2001). Insulin-like growthfactor-binding proteins (IGFBPs) are a group of struc-turally similar proteins that specifically modulate theaction of insulin growth factors (IGFs), and displaybinding sites for integrins on their sequences (Joneset al., 1993). The genes encoding the IGFBPs are locatedon chromosomes 7,17,12, and 2 which are physicallycontiguous to the loci of the HOX network and thussuggest a possible evolutionary association betweenIGFBP and HOX families (Lee et al., 1997). It has alsobeen shown by means of phylogenetic analysis thatHOXgenes and integrins evolved in parallel and in closephysical proximity. There are four integrin clusterslocated on chromosomes 7, 17, 12, and 2, contiguous tothe loci on which the genes of the HOX network arelocated (Wang et al., 1995). Therefore the genes of theHOX network, of integrins and of IGFBP proteins arephysically associated in their chromosomal location andmay be associated in the metabolic processes related toenergy metabolism. Gerald Edelmann has long hypothe-sized the existence of a morpho-regulatory control looplinking homeoproteins, growth factors and adhesionmolecules in determining phenotypical cell identity(Edelman and Jones, 1993). In light of these considera-tions and as a consequence of the crucial role the HOXnetwork plays in the life of a cell, it might be involved notonly in the transcriptional control of adipogenesis, butalso and more generally in the metabolic processesregulating energy balance.

In conclusion, we have analyzed the expression ofthe whole HOX gene network in different deposits ofnormal adult human white adipose tissue (intra-peritoneal, extra-peritoneal, and dermic) and in embry-onal brown adipose tissue. The results indicate thatthe HOX network overall shows a highly marked ex-pression in adipose tissue and, moreover, this expres-sion seems to vary in the different bodily deposits ofwhite adipose tissue and all the more between white andbrown adipose tissue. We can thus conclude that theHOX network plays a non-secondary role in transcrip-tional regulation of human adipogenesis.

ACKNOWLEDGMENTS

We thank Lucia Lobraico for the helpful participationin this project. C.C. acknowledges the financial supportof MURST 2001 projects.

Fig. 6. RT-PCR expression of PPARg2 in human samples of white adipose tissue, extra-peritoneal(E-WAT), intra-peritoneal (I-WAT), and dermic (D-WAT), and in human brown adipose tissue (BAT).Each lane contains amplified cDNA fragments from single biopsies of separate patients, male and femalerandomly selected. Control co-amplification of PPARg2 with a 433bp b actin primer is reported. DuplexPCR products were separated by ethidium 1.2% agarose gel electrophoresis.

234 CANTILE ET AL.

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