adipogenesis: from stem cell to...

BI81CH29-Lane ARI 9 May 2012 9:5

Adipogenesis: From Stem Cellto AdipocyteQi Qun Tang1,2 and M. Daniel Lane1

1Department of Biological Chemistry, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205; email: [email protected] Laboratory of Molecular Medicine, Ministry of Education, Department ofBiochemistry and Molecular Biology, Fudan University Shanghai Medical College,Shanghai 200032, People’s Republic of China; email: [email protected]

Annu. Rev. Biochem. 2012. 81:715–36

First published online as a Review in Advance onMarch 29, 2012

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev-biochem-052110-115718

Copyright c© 2012 by Annual Reviews.All rights reserved

0066-4154/12/0707-0715$20.00

Keywords

lineage commitment, BMP, C/EBP, PPARγ, Wnt

Abstract

Excessive caloric intake without a rise in energy expenditure promotesadipocyte hyperplasia and adiposity. The rise in adipocyte number istriggered by signaling factors that induce conversion of mesenchymalstem cells (MSCs) to preadipocytes that differentiate into adipocytes.MSCs, which are recruited from the vascular stroma of adipose tissue,provide an unlimited supply of adipocyte precursors. Members of theBMP and Wnt families are key mediators of stem cell commitment toproduce preadipocytes. Following commitment, exposure of growth-arrested preadipocytes to differentiation inducers [insulin-like growthfactor 1 (IGF1), glucocorticoid, and cyclic AMP (cAMP)] triggersDNA replication and reentry into the cell cycle (mitotic clonal expan-sion). Mitotic clonal expansion involves a transcription factor cascade,followed by the expression of adipocyte genes. Critical to these eventsare phosphorylations of the transcription factor CCATT enhancer-binding protein β (C/EBPβ) by MAP kinase and GSK3β to pro-duce a conformational change that gives rise to DNA-binding activity.“Activated” C/EBPβ then triggers transcription of peroxisomeproliferator–activated receptor-γ (PPARγ) and C/EBPα, which in turncoordinately activate genes whose expression produces the adipocytephenotype.

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Mesenchymal stemcell (MSC) lineagecommitment: MSCsare pluripotent stemcells, which have thecapacity to undergocommitment anddifferentiation intoadipocytes,chondrocytes,myocytes, andosteocytes

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 716THE ADIPOCYTE . . . . . . . . . . . . . . . . . . 716

Origin of Adipocytes . . . . . . . . . . . . . . . 717Pluripotent Stem Cell Lines . . . . . . . . 717Preadipocyte Cell Lines . . . . . . . . . . . . 717Mesenchymal Stem Cells . . . . . . . . . . . 718

COMMITMENT: PLURIPOTENTSTEM CELL TOPREADIPOCYTE . . . . . . . . . . . . . . . . 718Bone Morphogenetic Protein

Signaling . . . . . . . . . . . . . . . . . . . . . . . 718Wnt Signaling . . . . . . . . . . . . . . . . . . . . . 721Hedgehog Signaling . . . . . . . . . . . . . . . 722Retinoblastoma Protein Signaling. . . 722

DIFFERENTIATION:PREADIPOCYTE TOADIPOCYTE . . . . . . . . . . . . . . . . . . . . . 722Induction of Differentiation . . . . . . . . 723Mitotic Clonal Expansion . . . . . . . . . . 723Transcription Factor Cascade . . . . . . 724Histones and Chromatin

Remodeling . . . . . . . . . . . . . . . . . . . . 729Role of microRNAs

in Adipogenesis . . . . . . . . . . . . . . . . . 729

INTRODUCTION

Animals possess highly integrated systems toregulate energy storage and expenditure. Thesesystems have evolved to promote energy storageduring periods of food surplus and mobiliza-tion of these stores when food is scarce (1–4).Such systems are regulated both at the cellu-lar level and in the whole organism by the co-ordinated actions of circulating hormones andefferent neural signals from the central ner-vous system both to higher brain centers andto peripheral tissues, including the liver, mus-cle, and adipose tissue. When food is abun-dant, carbohydrates are stored as small reservesof glycogen in the liver and as a much largerreserve as fat—primarily in adipocytes. Whenfood is limited, these stores are mobilized tomeet energy needs (1, 2). Despite these control

mechanisms to survive feast-or-famine situa-tions, lifestyles in developed societies have ledto excessive consumption of energy-rich foodsand sedentary behavior. As a consequence obe-sity and its accompanying pathological conse-quences, most notably insulin resistance, type 2diabetes, and heart disease, have become seri-ous medical problems (5–8).

Excessive caloric intake relative to expendi-ture produces a metabolic state that promoteshyperplasia and hypertrophy of adipocytes (5).Hyperplasia involves stem cell recruitment tothe adipocyte lineage and a consequent increasein adipocyte number. Here, we review boththe mechanisms by which pluripotent mes-enchymal stem cells (MSCs) undergo commit-ment to become preadipocytes and the pro-gram by which preadipocytes differentiate intoadipocytes. Together, these processes are re-sponsible for the increase in adiposity producedby excessive energy intake, which is accompa-nied by low energy expenditure. The reader isreferred to several recent reviews that deal withrelated aspects of this topic (2, 6–9).

THE ADIPOCYTE

The mature adipocyte contains a single largefat droplet surrounded by a thin rim of cyto-plasm that lies between the droplet and theplasma membrane. When triggered by lipo-lytic hormones, cytoplasmic hormone-sensitivelipase (10) and adipocyte-triglyceride lipase (11,12) translocate to the surface of the fat droplet,where lipolysis of triglyceride occurs (13). Alsobound to the surface of the fat droplet are ac-cessory proteins, such as perilipin (14), that fa-cilitate this process. Thus, fatty acids derivedfrom triglyceride are released into the bloodstream to supply peripheral tissues, especiallythose of the skeletal and heart muscle and theliver, with an energy-rich fuel. During periodsof excess caloric intake, lipogenic enzymes, lo-calized in the cytoplasm and endoplasmic retic-ulum (ER), synthesize triglyceride, which is in-corporated into the fat droplet. As discussedin the section Differentiation: Preadipocyte toAdipocyte, see below, the genes encoding these

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Mitotic clonalexpansion: theprocess by whichG1-growth-arrestedpreadipocytes, inducedto differentiate,reenter the cell cycleand undergoapproximately tworounds of division, aprerequisite fordifferentiation

and other adipocyte proteins are coordinatelyexpressed during preadipocyte differentiationto produce the adipocyte phenotype.

Two technical approaches have been ofparamount importance in advancing progressin studies on the metabolism and differenti-ation with adipocytes and their progenitors.The use of collagenase dissociation to isolatefunctional adipocytes from adipose tissue(15–17) made possible the biochemical anal-ysis of adipocyte function. Using isolatedadipocytes, it was shown that these cells areexquisitely responsive to hormones, such asinsulin, that promote glucose uptake andlipogenesis as well as to adrenergic agents,such as epinephrine and cyclic AMP (cAMP),that promote triglyceride mobilization (5, 15,17). Studies on gene expression/regulationand differentiation, however, had to await thedevelopment of clonal preadipocyte cell linesthat could be differentiated into adipocytes incell culture (18–20). Established cell lines, e.g.,the 3T3-L1 preadipocyte line, that can be cul-tured indefinitely have served as faithful modelsystems for characterizing the differentiationprogram.

Recent studies have revealed that adipocytesalso have endocrine functions and secrete hor-mones and cytokines that play key roles inglobal energy metabolism. Certain of thesehormones act locally as paracrine factors, andothers, such as leptin (21–25) and adiponectin(26–28), have long-range effects and act onthe feeding centers of the central nervous sys-tem/brain, notably the hypothalamus. Leptin,which is expressed and secreted by adipocytes inproportion to adipose tissue mass, is anorecticand acts to limit energy storage when adiposetissue reserves have been filled (29). Leptininteracts with specific receptors in the hypo-thalamus (30) to reduce food intake. In theobese state, however, resistance to leptinoccurs, limiting its effectiveness. Adipose tissueis also under the control of the central nervoussystem because it is innervated by neurons of thesympathetic nervous system that secrete adren-ergic hormones (epinephrine/norepinephrine)that promote fat mobilization (17).

Origin of Adipocytes

Adipocytes are derived from pluripotentMSCs that have the capacity to develop intoseveral cell types, i.e., adipocytes, myocytes,chondrocytes, and osteocytes (7, 31, 32). Thesestem cells reside in the vascular stroma ofadipose tissue as well as in the bone marrow,and when appropriately stimulated undergoa multistep process of commitment in whichthe progenitor cells become restricted to theadipocyte lineage. Recruitment to this lineagegives rise to preadipocytes, which, wheninduced, undergo multiple rounds of mitosis(mitotic clonal expansion) and then differenti-ate into adipocytes. Model cell culture systems(discussed below) have been indispensablein identifying/characterizing the steps in thecommitment and differentiation programs.

Pluripotent Stem Cell Lines

Several cell lines faithfully mimic the functionalcharacteristics of the mesenchymal pluripotentcell type in vivo and can be induced to undergocommitment to the adipose, muscle, cartilage,and bone lineages. The best characterized ofthese pluripotent lines is the CH310T1/2 lineestablished in 1973 in the Heidelberger labo-ratory at the University of Wisconsin (33). Inaddition, embryonic stem cell knockdowns andmouse knockouts have proven useful (32, 34).The adipocyte commitment process can be ini-tiated by factors such as BMP4 or BMP2 (bonemorphogenetic proteins) (35) or factors in thedownstream signaling pathway (36).

Preadipocyte Cell Lines

Preadipocyte lines that represent the nextstep (differentiation) in adipocyte developmentwere first established in Green’s laboratory (18,19, 37). Notable among these is the 3T3-L1preadipocyte line, which has become the“gold standard” for investigating preadipocytedifferentiation. This line (along with the 3T3-F442A line) faithfully recapitulates the stepsin adipocyte differentiation. Induction of

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Adipogenesis: theprocesses by whichmesenchymal stemcells commit to theadipose lineage anddifferentiate intoadipocytes

differentiation is triggered with a “cocktail” ofagents to initiate the cascade of synchronoussteps in the differentiation process.

Mesenchymal Stem Cells

Pluripotent MSCs derived from the embryonicmesoderm (7, 31) have the capacity to differ-entiate into adipocytes and have been used toverify many findings made with the established3T3-L1 preadipocyte line (38). It was defini-tively shown that a subpopulation of MSCs ispluripotent and capable of committing to theadipocyte lineage (36, 38).

COMMITMENT: PLURIPOTENTSTEM CELL TO PREADIPOCYTE

The vascular stroma of adipose tissue possessesa resident population of pluripotent MSCs thathas the potential to undergo commitment anddifferentiation into adipocytes, chondrocytes,myocytes, and osteocytes (7, 31, 35, 39). Useof these cells for genetic studies is limited,however, because of their short lifetime inculture. Pluripotent C3H10T1/2 cells inculture have served as a faithful MSC modelfor long-term genetic studies of the adipocytedevelopmental program. When appropri-ately induced, C3H10T1/2 stem cells can beprompted to commit and differentiate intocells of the adipocyte, myocyte, chondrocyteor osteocyte lineages.

Recruitment to the adipocyte lineage in vivois prompted by excessive energy intake and ele-vated glucose uptake (5) over an extended timeperiod. This metabolic state appears to gener-ate a signal (or signals), yet to be identified, thatinduces MSCs to enter the commitment path-way leading to hyperplasia and the preadipocytephenotype. Several factors have been identifiedthat commit or inhibit the conversion ofpluripotent stem cells to the adipocyte lineage.These include BMP family members BMP4 andBMP2 (6, 35), Wnt (40, 41), and Hh (hedgehog)(42–44). BMP4 and BMP2 have an activatingrole, whereas Hh signaling has an inhibitoryrole, and Wnt appears to have both an activat-

ing role in commitment (41) and an inhibitoryrole in adipocyte differentiation, see below (45).

Mounting evidence indicates that lineagedetermination is regulated by a network ofextracellular signaling factors that ultimatelyimpinge on the promoters of lineage-specifictranscription factors. It is the balance ofthese signaling molecules that determines thedevelopmental pathway, often simultaneouslypromoting one pathway while inhibitinganother. For example, Wnt10b promotesosteogenesis and possibly myogenesis, andinhibits adipogenesis (46); BMP4 promotesadipogenesis while inhibiting myogenesis (45).Conversely, peroxisome proliferator–activatedreceptor-γ (PPARγ) inhibits chondrogen-esis and stimulates adipogenesis, whereasMsx2 stimulates osteogenesis while inhibit-ing adipogenesis (47, 48) by inhibiting thetranscriptional activity of PPARγ.

Bone MorphogeneticProtein Signaling

BMP4 and BMP2 have been implicated inthe commitment of pluripotent stem cells tothe adipocyte lineage (32, 35, 36, 41, 49–52).Thus, exposure of dividing C3H10T1/2 stemcells to either BMP4 or BMP2 gives rise topreadipocyte-like cells which, when treated atgrowth arrest with differentiation inducers, en-ter the adipose development pathway, expressadipocyte markers, and acquire the adipocytephenotype (6, 32, 35).

The role for BMP4 in the commitmentprocess has been validated using anotherapproach, clonal selection after blocking DNAmethylation (6). Thus, exposure of proliferat-ing C3H10T1/2 stem cells to 5-azacytidine,an inhibitor of DNA methylation, generatedclonal subpopulations of cells, e.g., the A33line, that converted into adipocytes whenexposed to differentiation inducers in theabsence of exogenous BMP4 (6). Remarkably,A33 cells express and secrete BMP4 in the sameproliferation time window at which exogenousBMP4 must be added to induce adipocytecommitment of “naive” C3H10T1/2 cells.

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Moreover, exposure of A33 cells to the naturallyoccurring BMP4-binding antagonist, noggin,during this critical time window blockedconversion/differentiation into adipocytes(Figure 1). The role of BMP4 in adipocytelineage commitment is further supported bythe expression in proliferating C3H10T1/2stem cells and A33 preadipocytes of genes andproteins that are known to be involved in theBMP signaling pathway (6, 35, 36). Theseinclude BMP4; the BMP receptors, BMPr2and BMPr1a; and Smad-1, -5, -8. BMPs areknown to signal through two receptor types,BMPr1 and BMPr2, which form cell-surfacecomplexes with serine/threonine kinase activity(53, 54). Binding of BMP to the BMPr1:BMPr2complex induces phosphorylation and, thus,activation of the BMPr1 kinase. The BMPreceptor phosphorylates Smad-1,-5,-8, whichforms a complex with Smad4 that translocatesinto the nucleus and regulates gene expres-sion (32). Furthermore, overexpression ofa constitutively active (CA) BMP receptor,CA-BMPr1A or CA-BMPr1B, induces com-mitment in the absence of BMP2 or BMP4,whereas overexpression of a dominant-negativereceptor, dominant-negative-BMPr1A, sup-presses commitment induced by BMP. Also,knockdown of the expression of Smad4 (acoregulator in the BMP/Smad signaling path-way) with RNAi disrupts commitment by theBMPs (see Figure 2).

BMP promoter switch during commitment.The mouse BMP4 gene is known to have twopromoters that are differentially regulated dur-ing development (55, 56). Analysis of RNAearly in the commitment program revealed thatpromoter 1 is used to drive expression of BMP4in A33 cells but not in C3H10T1/2 cells (6).On day seven when BMP4 synthesis decreases,use of promoter 1 and expression of BMP4ceases. In contrast, mRNA from uncommit-ted C3H10T1/2 cells showed that promoter2 was used exclusively to drive expression ofthe BMP4 gene (6). These findings suggestthat commitment to the adipocyte lineage in-duced by 5-azacytidine altered the methylation

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Figure 1Effect of the BMP4 antagonist, noggin, on the acquisition of adipocytecharacteristics by A33 cells [which are cloned CH310T1/2 cell derivatives withpreadipocyte characteristics; see the text (33)]. One day after plating, A33 cellswere exposed or not to recombinant mouse noggin. Treatment with nogginwas continued until growth arrest at confluence when cells were treated withdifferentiation inducers. Photomicrographs were taken after four days, andRNA was harvested at six days after induction. Panel (a) shows control A33cells, and panel (b) shows A33 cells treated with noggin. (c) Effect of noggin onthe expression of the adipocyte marker PPARγ2 mRNA. Figure is fromReference 6.

status of the BMP4 gene, rendering promoter1 more accessible for transcriptional activation,and indicate that a region of the BMP4 locus,rich in CpG islands, has fewer methylated cy-tosines in genomic DNA from A33 cells than


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Figure 2Proposed schema of events for the commitment of mesenchymal stem cells (MSCs) to the adipocyte lineage. BMP refers to bonemorphogenetic protein. Frizzled refers to the Wnt cell-surface receptor. The Wnt and BMP pathways are shown as both linear andparallel events since both pathways may be involved. Wnt appears to function both in an activating and an inhibitory capacity in MSCcommitment (10, 42, 50) and differentiation (41). Dashed lines indicate some uncertainty.

from 10T1/2cells. The BMP4 gene locus innaive C3H10T1/2 stem cells possesses a highlymethylated region of CpG islands, whereasthis region in committed 10T1/2cells possessesfewer methylated cytosines (6). This differencecorrelates with the switch in BMP promoter us-age (promoter 2 to promoter 1) during com-mitment that is induced by the methylation in-hibitor, suggesting that a change in methylationstatus renders promoter 1 more accessible fortranscriptional activation.

Downstream targets of BMP signal-ing. Proteomic analysis revealed that threecytoskeleton-associated proteins [i.e., lysyloxidase (Lox), translationally controlled tumorprotein 1 (Tpt1), and αB crystallin] are down-

stream target genes in the BMP signaling path-way and play important roles during adipocytelineage commitment (52). Eight proteins werefound to be upregulated by BMP2, and 27proteins were upregulated by BMP4. Fiveunique proteins were upregulated ≥10-foldby both BMPs, including three cytoskeleton-associated proteins (i.e., Lox, Tpt1, andαB-crystallin). Commitment was completelyblocked by knockdown of Lox, whereas it waspartially inhibited by knockdown of Tpt1 andαB-crystallin expression. Dramatic changes incell shape normally occur during commitment.Knockdown of these cytoskeleton-associatedproteins prevented these cell shape changes andrestored F-actin organization into stress fibersand inhibited commitment to the adipocyte

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lineage. These differentially expressed pro-teins may determine the ability of MSCs tocommit to the adipocyte lineage via cell shaperegulation.

Wnt Signaling

The Wnts comprise a family of secreted signal-ing glycoproteins whose effects are mediatedthrough the frizzled receptor and low-densitylipoprotein-related protein 5/6 coreceptor (57).The Wnt proteins can act via the more promi-nent “canonical” Wnt signaling pathway (58–60) or a “noncanonical” Wnt signaling path-way (61). A linkage between Wnt signaling andadipogenesis was first recognized through thefinding that expression of Wnt10b decreaseddramatically during adipocyte differentiation(40). Consistent with this finding, forced ex-pression of Wnt10b prevented adipocyte dif-ferentiation by blocking the expression of thekey adipogenic transcription factors, PPARγ

and C/EBPα. Surprisingly, the Wnts act attwo points in the adipose development program(Figure 2), both early in the program as an ac-tivator of lineage commitment (41) and late inthe program as an inhibitor of adipocyte differ-entiation (40, 45), perhaps through the actionsof different Wnt proteins.

Wnt activates lineage commitment ofMSCs. The canonical Wnt pathway functionsearly in the lineage commitment process of thecanonical pathway. In this pathway cytosolicβ-catenin is embedded in a “destructioncomplex” containing adenomatous polyposiscoli, axin, and glycogen synthase kinase-3β

(GSK-3β) (60). In the absence of Wnt stim-ulation, GSK-3β phosphorylates β-catenin,priming it for ubiquitination and proteasomaldegradation (60). Conversely, activation ofWnt signaling through binding of Wnts totheir cell-surface receptor, frizzled, and theassociated low-density lipoprotein receptor-related coactivator 5/6 promotes dissociationof the destruction complex, thereby allowingβ-catenin to accumulate and translocate to thenucleus (59, 62). Thus, during Wnt signaling,β-catenin accumulates in the nucleus, where

it activates the transcription factors lymphoidenhancer factor and/or T-cell factor (Figure2), triggering the transcription of downstreamgenes (including c-myc and Cyclin D1) (39).

Microarray studies combined with quantita-tive RT-PCR analyses identified several genesof the Wnt signaling pathway that are differen-tially expressed by A33 cells (preadipocytes de-rived from C3H10T1/2 stem cells, see above)(41). Of particular interest are the newly de-scribed R-spondins-2 and -3, which activatethe canonical Wnt signaling pathway, and aredramatically upregulated in proliferating A33preadipocytes compared with their progenitorC3H10T1/2 stem cells. Likewise, Lef1 and Tcf,downstream transcription factors of the Wntsignaling pathway, are expressed differently inA3 preadipocytes relative to their expression inC3H10T1/2 stem cells (41). These events oc-cur concurrently with the accumulation of β-catenin in the nuclei of proliferating A33 cells.Taken together, these findings implicate Wntsignaling as an early event in stem cell commit-ment to the adipose lineage.

Wnt inhibits terminal differentiation. Latein the adipogenic program, the canonical Wntsignaling pathway regulates the balance amongmyogenic, osteoblastogenic, and adipogeneticfates (40, 46, 57) and, by doing so, decreases adi-pogenesis. Myoblasts in cell culture retain theirplasticity in developmental potential becausethey can be induced to undergo myogenic,adipogenic, or osteoblastogenic differentiation(57). β-catenin-dependent signaling has beenreported to promote both myogenesis (40) andosteogenesis (46) while inhibiting the differen-tiation of preadipocytes into adipocytes (41, 63).Consistent with the inhibitory effect of Wnt onadipogenesis, myoblasts isolated from Wnt10b-null mice exhibit increased adipogenic potential(57). In addition, activation of the Wnt signal-ing pathway enhances myogenesis and inhibitsadipogenesis in cultured MSCs (40, 64).

The Wnt signaling pathway is more ac-tive in proliferating A33 cells (a clonal linewith preadipocyte characteristics) than inits C3H10T1/2 progenitors, perhaps because


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Wnt signaling serves to increase the number ofpreadipocytes during commitment. It has beenspeculated that Wnt signaling serves to increasethe number of preadipocytes during commit-ment and mitotic clonal expansion (41) butthat this signal must ultimately be terminatedas preadipocytes/A33 cells enter growth arrest,before the newly recruited cells can undergoterminal differentiation into adipocytes (see theMitotic Clonal Expansion section below).

Hedgehog Signaling

Three vertebrate Hh ligands have been iden-tified, Sonic (Shh), Indian (Ihh), and Desert(Dhh), which initiate a signaling cascade me-diated by Patched (Ptch-1 and Ptch-2) recep-tors (65, 66). In the presence of an Hh ligand,the membrane-spanning protein Smoothened(Smo), a homolog of G protein–coupled recep-tors, is activated, and the signal is transmittedvia phosphorylation and nuclear localization ofGliA (65, 66).

Hh signaling has an inhibitory effect on adi-pogenesis in murine cells, e.g., C3H10T1/2,KS483, calvaria MSC lines, and mouse adipose-derived stromal cells (67) as visualized by de-creased cytoplasmic fat accumulation and theexpression of adipocyte marker genes (42–44).In genetically obese (ob/ob) mice, a negativeeffect on adipogenesis is accompanied by a re-duction in the expression of Smo, Gli1, Gli2,and Gli3. Moreover, reduced total white fatmass and epididymal adipocyte cell size wereobserved in naturally occurring spontaneousmesenchymal dysplasia (mes) adult mice (Ptch1mes/mes), which carry a deletion of Ptch1, anegative regulator of Hh signaling, at the C-terminal cytoplasmic region (68). Although itis generally agreed that expression of Hh hasan inhibitory effect on preadipocyte differenti-ation, the mechanisms linking Hh signaling andadipogenesis remain poorly defined.

Retinoblastoma Protein Signaling

The retinoblastoma protein (Rb) inhibits thecell cycle by binding to and repressing the

transcriptional activity of E2F (69). Upon hy-perphosphorylation of Rb (⇒ pRb) by cyclin-dependent kinases, E2F is released and pro-motes transcriptional activation of genes thatencode cell-cycle regulators required for S-phase entry and progression of the cell cycle(70). These events are critical for mitotic clonalexpansion (see the Mitotic Clonal Expansionsection below), an obligate step in the adipocytedifferentiation program (38).

Additionally, pRb has been shown to reg-ulate several transcription factors that are keydifferentiation inducers (38, 71). Dependingon the differentiation factor and its cellularcontext, pRb can either suppress or promotetranscriptional activity of such factors. Thus,pRb binds to Runx2 and potentiates its abilityto promote osteogenic differentiation (72).In contrast, Rb acts with E2F to suppressperoxisome proliferator-activated receptor c(PPARγ2-c) subunit, the master activator ofadipogenesis (73, 74). Because osteoblasts andadipocytes can both arise from MSCs, theseobservations suggest that pRb may play a rolein the choice between these two lineage fates.And these findings indicate that Rb status playsa key role in establishing fate choice betweenbone and brown adipose tissue in vivo (75).

DIFFERENTIATION:PREADIPOCYTE TO ADIPOCYTE

The synchronous events undergone bypreadipocytes during differentiation intoadipocytes are described below. Cell culturestudies with established preadipocyte lineshave been indispensable for the identificationand characterization of the key steps in thedifferentiation program. A large body ofevidence shows that these preadipocyte modelsfaithfully recapitulate differentiation of mouseembryonic fibroblasts (MEFs) in cell culture(34, 38, 76). Likewise, the authenticity ofmodel systems has been validated in vivoby demonstrating that these preadipocytelines give rise to normal adipose tissue whenimplanted subcutaneously into athymic micewithout exogenous inducers (40, 77, 78).

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G1 S G2 M

G1

M

Figure 3Synchrony of DNA replication during mitotic clonal expansion of the adipocyte differentiation program. After staining with propidiumiodide, DNA content was determined by FACS analysis and expressed ( y axis) in arbitrary units. Time (h) refers to the elapsed time (inhours) after induction of differentiation of 3T3-L1 preadipocytes with the inducer cocktail. G1 refers to the growth-arrested phase ofthe cell cycle. S, M, and G2 refer to the subsequent phases of the cell cycle. The plots were constructed using data from Reference 38.Abbreviations: 1n and 2n, one- to twofold; FLZ, fluorescence intensity.

Induction of Differentiation

Protocols have been developed to inducedifferentiation and track the synchronousprogression of cells through the program.Preadipocytes, e.g., 3T3-LI preadipocytes,or MEFs are grown to growth arrest, i.e., atthe G1 phase of the cell cycle. At this point,differentiation is initiated with a cocktail ofinducers, including a high level of insulin1 (orlow level of IGF1) (79) and dexamethasone, aswell as an agent to elevate cellular cAMP in fe-tal calf serum-containing medium (80). Theseinducers activate the IGF1-, glucocorticoid-,and cAMP-signaling pathways, respectively.Induction initiates a series of events thatregulate staging of the differentiation program.Following a delay of ∼16–20 h after induction,

1It has been established that the IGF1, rather than insulin,is the true inducer ligand. IGF1 is active as an inducer atmuch lower concentrations than insulin binding; IGF1 binds∼100-fold more tightly than insulin to the IGF receptor (79).

preadipocytes synchronously reenter the cellcycle (Figure 3) (39, 80) and undergo severalrounds of mitosis, referred to as mitotic clonalexpansion (see below). The cells then exit thecell cycle, lose their fibroblastic morphology,accumulate cytoplasmic triglyceride, and ac-quire the appearance and metabolic features ofadipocytes (18, 80). Triglyceride accumulationis closely correlated with an increased rate ofde novo lipogenesis and a coordinate rise inexpression of the enzymes of fatty acid andtriacylglycerol biosynthesis (79–81). Likewise,numerous regulatory proteins, characteristic ofadipocytes in situ, are coordinately expressed,including insulin receptors (82, 83), the insulin-responsive glucose transporter GLUT4 (84),leptin (85, 86), and others (79, 86, 87).

Mitotic Clonal Expansion

Following induction of G1-phase growth-arrested cells, preadipocytes reenter the cell


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P

P

P

P

N N

C

ATP

16–24 hP P

Active DNA binding

CC

N C

OHOH

N

Leucine zipper

G1 S

Dimerization

InactiveC/EBPβ

Figure 4Schema for the dual phosphorylation-induced conformational changes inC/EBPβ, which activates DNA binding. Dual phosphorylation (indicated by P)of C/EBPβ occurs on Thr188 by MAPK and on Ser184 or Thr179 by GSK3β;this induces a conformational change (or changes) that promotes dimerizationand gives rise to binding activity. G1 −> S refers to the mitotic clonalexpansion phase of the cell cycle 16–24 h after the induction of differentiationThe data is from References 93 and 114. Abbreviation: C/EBPβ,CCAAT/enhancer-binding protein.

cycle and undergo about two rounds of divi-sion, referred to as mitotic clonal expansion.Mitotic clonal expansion is a required step inthe adipocyte differentiation program. Thus,blocking DNA replication by various means,e.g., by inhibitors of DNA polymerase or byblocking progression of the cell cycle (38, 88–91), prevents differentiation. For example, theconstitutive overexpression of the cell-cycle in-hibitor p27 prevents cells from entering theS-phase of the cell cycle and thereby disruptsall subsequent steps of differentiation (88).

Growth arrest. Immediately (≤5 min) afterinduction of differentiation, cyclic AMPresponse element-binding protein (CREB)becomes phosphorylated and activates the ex-pression of C/EBPβ (34, 90). However, at thispoint, C/EBPβ lacks DNA-binding activity.

G1- to S-phase transition. At 14–16 h afterinduction, C/EBPβ acquires DNA-bindingactivity as the preadipocytes reenter the cellcycle (Figure 3). Between 16–20 h afterinduction, the cell-cycle markers of S-phase

entry are synchronously expressed (or inacti-vated) concomitant with the initiation of DNAreplication (38, 92). Acquisition of DNA-binding activity is delayed for until ∼16 hwhen C/EBPβ undergoes phosphorylation byGSK3β (see the CCAAT/enhancer-bindingprotein-β and -δ section below and Figure 4)(93) coincident with reentry into the cell cycleat the G1/S boundary (Figure 5). These eventsare closely correlated with the expression ofhistone H4 (94), the coordinated expression ofcell-cycle proteins (39), and DNA replication(92). Expression of histone H4 increasesdramatically 12–14 h after induction, just priorto entry into the S phase, concurrent with thegain of function by C/EBPβ. It is noteworthythat the histone H4 gene (hist4h4) promoterpossesses a functional C/EBP-binding site (94).Moreover, knockdown of C/EBPβ with siRNAprevents the induction of histone H4 expres-sion and arrests the cells in G1-growth arrest asindicated by bromodeoxyuridine incorporationand FACS analysis of DNA content (94).

Transcription Factor Cascade

The key features of the transcription factor cas-cade of the differentiation program are shownbelow (see also Figure 5):

Differentiation inducers ⇒⇑ [CREB

⇒ P − CREB] ⇒⇓ CHOP10

⇒ ⇑ [C/EBPβ ⇒ P2 − C/EBPβ]

⇒ ⇑ [C/EBPα/PPAR]γ

⇒ ⇑ SREBP1c ⇒⇑ adipocyte genes.

Cyclic AMP response element-bindingprotein. Because cAMP itself or forskolin orother agents, which elevate the cAMP l level,can substitute for methylisobutylxanthine inthe differentiation inducer cocktail, CREBwas considered a likely intermediate in thesignaling pathway. Many lines of evidencesupport this view; these include the following:

1. The cellular target of cAMP, protein ki-nase A, catalyzes the phosphorylation andactivation of CREB (95).

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Expression of adipocyte genes

SREBP1c

CDC25B Cyclin B Aurora B

+

Cyclin ECyclin A p27/Kip cdk2pRb

Time afterinduction

Stage/cell cycle

DIFF inducers(DEX, IGF1, cAMP)

Initiates DIFFprogram

Cyclin D1

G1

G1

G1

G2

GD

M

S

P-CREB

MAPKGSK-3β

C/EBPαPPARγ

PKA

E2F release initiates S phase

Autoactivation

ActiveP2-C/EBPβ

InactiveC/EBPβ

Mitotic clonal expansion

CREB0 h

<5 min

14 h

28 h

60 h

Figure 5The adipocyte differentiation program. The adipocyte differentiation activation cascade. Abbreviations:CDC25B, cell division cycle 25 homolog B; cdk2, cyclin-dependent kinase 2; C/EBP, CCAAT/enhancer-binding protein; CREB, cyclic AMP regulatory element-binding protein; DEX, dexamethasone; DIFF,differentiation; E2F, eukaryotic transcription factor 2; GSK-3β, glycogen synthase kinase-3β; IGF1,insulin-like growth factor 1; MAPK, mitogen-activated protein kinase; P, phosphoryl group; PPARγ,peroxisome proliferator–activated receptor-γ; pRb, hyperphosphorylated-retinoblastoma binding protein;SREBP1c, sterol regulatory element binding protein 1c; G1 and S, G1- and S-phases of the cell cycle,respectively. Dashed lines indicate some uncertainty.

2. Forced expression of CREB in 3T3-L1preadipocytes promotes differentiation(34).

3. The proximal promoter of the C/EBPβ

gene possesses dual cis-regulatory ele-ments that contain core CREB-bindingsites (34, 96). C/EBPβ promoter-reporter genes with 5′-truncations orsite-directed mutations in the TGAregulatory elements revealed that bothare required for maximal promoterfunction.

4. Electromobility shift assay and chromatinimmunoprecipitation analyses of wild-type MEFs and 3T3-L1 preadipocytesshow that CREB associates with theproximal promoter and that interaction

of phospho-CREB, the active form ofCREB, with the C/EBPβ gene promoteroccurs only after induction of differenti-ation of 3T3-L1 preadipocytes or MEFs(34, 90).

5. Constitutively active CREB activatesC/EBPβ promoter-reporter genes (34,90), induces expression of endogenousC/EBPβ, and promotes adipogenesis inthe absence of hormonal inducers.

6. Conversely, dominant-negative CREBblocks promoter-reporter activity, aswell as the expression of C/EBPβ andadipogenesis (90).

7. Subjecting wild-type MEFs to the stan-dard differentiation protocol inducesdifferentiation into adipocytes, whereas


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CREB−/− MEFs treated similarly ex-hibit reduced expression of C/EBPβ anddifferentiation (90).

Consistent with the role of CREB in the dif-ferentiation process, C/EBPβ expression andaccumulation of cytoplasmic triacylglycerol aremarkedly reduced in CREB−/− MEFs. To-gether these findings show that CREB func-tions early in the adipocyte differentiationprogram by transcriptionally activating theC/EBPβ gene.

The CCAAT/enhancer-binding family.Although C/EBPα was the first C/EBP familymember to be cloned and to have its functionshown in adipogenesis, three other isoformshave also been implicated, notably C/EBPα

(97–99) C/EBPβ (100–105); C/EBPδ (104,105); and CHOP10 (106). Three of theisoforms contain a DNA-binding domain(C/EBPα, C/EBPβ, and C/EBPδ) and anadjacent C-terminal leucine zipper dimeriza-tion domain that allows formation of homo-or heterodimers with other C/EBP familymembers; dimerization is required for DNAbinding (107). CHOP10 has a short nonfunc-tional DNA-binding domain and b-ZIP typeC-terminal leucine zipper dimerization domainand can heterodimerize with the other isoformsto produce dominant-negative C/EBP dimers(106). Both C/EBPα and C/EBPβ also occuras N-terminally truncated polypeptides whosefunctions remain obscure (49, 108, 109).

CCAAT/enhancer-binding protein-β and -δ.C/EBPβ and C/EBPδ are rapidly (in <4 h)expressed following induction of differenti-ation (38). The importance of C/EBPβ isindicated by its ability to promote adipogenesiswhen overexpressed in 3T3-L1 preadipocytesor in NIH-3T3 cells in the absence of hor-mone inducers—an activity not shared byC/EBPδ (110). Although rapidly expressedupon induction of differentiation, C/EBPβ isinactive and unable to bind DNA until laterin the differentiation program. Acquisition ofDNA-binding activity is achieved at ∼16 h

after induction, approximately concomitantwith entry into S phase and mitotic clonalexpansion (see below). Another indicator ofmultiple C/EBP consensus-binding sites is thecentromeric satellite DNA (38, 111). The roleof this binding to centromeres has not beenestablished.

Although C/EBPβ is required for adipocytedifferentiation in cell culture (38, 93, 112,113), a knockout of the C/EBPβ gene in micehas little effect on adipose tissue accumulation(113). Nevertheless, adipose tissue mass in thedouble knockout [C/EBPβ−/−/C/EBPδ−/−] ismarkedly reduced (113). This reduction of adi-pose tissue mass is because of the decreasedcell number, supporting the view that C/EBPβ

functions in mitotic clonal expansion. Thatthe double C/EBPβ−/−/C/EBPδ−/− knockoutproduces a significant effect, whereas singleknockouts of C/EBPβ−/− or C/EBPδ−/− donot, suggests redundancy of function by mem-bers of the C/EBP family (113).

Supporting the dependence of adipogene-sis upon C/EBPβ, disruption of the gene pre-vents mitotic clonal expansion, which is itselfrequired for differentiation. Thus, C/EBPβ−/−

MEFs fail to undergo mitotic clonal expansionand do not differentiate into adipocytes, whileC/EBPβ−/− MEFs undergo mitotic clonal ex-pansion and differentiate normally (89, 92).Further support for the role of C/EBPβ inclonal expansion is derived from the use of A-C/EBP, which contains a leucine zipper butlacks the functional DNA-binding and transac-tivation domains and thus acts as a dominant-negative of C/EBP proteins. Forced expressionof A-C/EBP disrupts both mitotic clonal expan-sion and differentiation in 3T3-L1 cells (90).Moreover, the turnover of p27—a requirementfor progression from G1-growth arrest to Sphase—is blocked by the forced expression ofA-C/EBP (88, 114). The mechanism for thiseffect is that formation of a tight C/EBPβ:A-C/EBP heterodimer prevents the translocationof C/EBPβ into the nucleus, a process thatis necessary for mitotic clonal expansion (90).Thus, the primary site of A-C/EBP—the firstC/EBP family member to be expressed in the

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transcription factor cascade—is most likely toblock C/EBPβ function. Moreover, when A-C/EBP, under the control of the aP2 adipocyte-specific promoter, is expressed in transgenicmice, the animals become fatless, i.e., devoidof white adipose tissue (115). This finding pro-vides further compelling proof that C/EBPβ isrequired for adipogenesis in vivo.

Phosphorylation is an important posttrans-lational modification of C/EBPβ and leadsto the acquisition of DNA-binding functionas preadipocytes traverse to the G1-S check-point at the onset of mitotic clonal expan-sion (93). C/EBPβ is phosphorylated sequen-tially, first by MAP kinase and then muchlater by GSK-3β (93). Phosphorylation onThr188 by MAP kinase occurs ∼4 h afterinduction of differentiation and is requiredfor mitotic clonal expansion, C/EBPβ DNA-binding activity, and terminal differentiation;however, Thr188 phosphorylation is insuffi-cient on its own to produce DNA-binding ac-tivity by C/EBPβ (93). Phosphorylation onThr179 or Ser184 by GSK3β occurs between12 and 16 h after induction. Dual phosphoryla-tion of C/EBPβ at two of these sites (Thr188and Thr179 or Thr188 and Ser184) leads toacquisition of DNA-binding activity. Hence, itappears that a phosphorylation-induced con-formational change is involved. On the basis ofthese and other findings (116), it is thought that,following induction of differentiation, C/EBPβ

is first rapidly (in ∼2 h) phosphorylated byMAPK, and then phosphorylated at a secondsite ∼14 h later by GSK3β (Figure 4). A con-formational change is induced that renders theleucine zipper of monomeric C/EBPβ acces-sible for dimerization (117). It appears thatdimerization brings the basic regions of thetwo monomers of C/EBPβ together to create a“scissors-like” DNA-binding pocket just abovethe coiled-coil leucine zipper as suggested byVinson et al. (118).

CCAAT/enhancer-binding protein-α. C/EBPα

and PPARγ function together as pleiotropictranscriptional activators of the largegroup of genes that produce the adipocyte

phenotype (79, 86, 119). Within their proximalpromoters, both the C/EBPα and PPARγ

genes possess C/EBP regulatory elementsat which C/EBPβ binds to coordinatelyactivate transcription (111, 120–122). Onceexpressed, C/EBPα is thought to maintainexpression of both the C/EBPα and PPARγ

genes via transactivation mediated by theirrespective C/EBP regulatory elements (111,120–122).

The promoters of many adipocyte genescontain C/EBP and PPAR regulatory ele-ments and are trans-activated by C/EBPα andPPARγ (79). Forced expression of C/EBPα

or PPARγ in 3T3-L1 preadipocytes inducesadipogenesis in the absence of hormonalinduction (123–125). Furthermore, blockingexpression of C/EBPα with antisense RNAsuppressed adipogenesis (126), and knockingout the C/EBPα gene in mice led to decreasedlipid accumulation (127, 128). These and otherfindings indicate the requirement for C/EBPα

in the adipocyte differentiation program.Beginning 18–24 h after the induction ofdifferentiation, the C/EBPα and PPARγ genesare transcriptionally activated by C/EBPβ

through C/EBP regulatory elements in theirproximal promoters.

Once C/EBPα is expressed, its expression ismaintained through autoactivation (120). Thefollowing question is raised: Why is there suchredundancy in the expression of the C/EBPgenes during adipogenesis? One possible ex-planation is that C/EBPα is antimitotic, andthus, its premature expression would preventpreadipocytes from entering mitotic clonal ex-pansion, a required step for subsequent differ-entiation. Therefore, it appears that C/EBPα

must remain repressed until the opportune timewindow. Several mechanisms cause a delay inthe expression of C/EBPα. Binding of AP-2α

(129) and Sp1 (130) to the C/EBPα promoterrepress promoter activity, and delay acquisitionof DNA binding by C/EBPβ and C/EBPδ (seethe section titled CCAAT/enhancer-bindingprotein-β and -δ, above). Both AP-2α and Sp1are downregulated concurrent with the upreg-ulation of C/EBPβ.


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CHOP10. CHOP10 was cloned from a 3T3-L1 adipocyte cDNA library on the basis of itsability to interact with the C/EBPβ C-terminalleucine zipper domain (106). CHOP10 con-tains proline and glycine residues in the DNA-binding region, which abolishes its ability tobind DNA but not its ability to form het-erodimers. Thus, CHOP10 acts as a dominant-negative isoform (106) and when expressed ec-topically in 3T3-L1 cells blocks adipogenesis(131). CHOP10 is normally expressed by G1-growth-arrested preadipocytes, but is downreg-ulated as the expression of C/EBPβ declines.Because heterodimerization of CHOP10 withC/EBPβ prevents it from acquiring DNA-binding activity (132), CHOP10 provides a“fail-safe” mechanism to prevent the acquisi-tion of DNA-binding activity by C/EBPβ un-til preadipocytes have entered mitotic clonalexpansion.

Peroxisome proliferator–activated receptor-γ.PPARγ exists as three isoforms (PPARγ1,PPARγ2, and PPARγ3) that are transcribedfrom the same gene through alternative splicingand promoter usage (133, 134); PPARγ2 is theprimary adipocyte-specific isoform (9). All iso-forms possess transactivation, DNA-binding,and dimerization domains (9). To bind at perox-isome proliferator response elements in targetgenes, PPARs must first form heterodimerswith the retinoid X receptor (9). Althoughnaturally occurring ligands for PPARγ havenot yet been identified, several potent syntheticthiazolidinediones and prostanoids, e.g., 15-deoxy-�12,14 prostaglandin J2 (37, 38), bindwith high affinity. It is unlikely, however, thatthe 15-deoxy-�12,14 prostaglandin J2 concen-tration in vivo is of biological importance (9).

Although PPARγ2 appears to act as a “mas-ter” regulator of the adipogenesis program,like C/EBPα, it also participates in other di-verse systems, including hepatic lipogenesis (9).The ectopic expression of PPARγ2 in naivepreadipocytes and nonadipogenic fibroblastsactivates expression of adipocyte genes and dif-ferentiation (9, 10, 135). Moreover, dominant-

negative mutants (49, 136) and knockouts of themouse gene block these functions.

As indicated above, C/EBPβ (and C/EBPδ)are rapidly (in <4 h) expressed after inductionof differentiation. Following a delay of 16–20 h,C/EBPβ coordinately activates the expressionof PPARγ2 and C/EBPα through C/EBP reg-ulatory elements in the proximal promoters oftheir respective genes (38, 89, 137). Follow-ing their expression, PPARγ2 and C/EBPα

coordinately transactivate a large group ofgenes that produce the adipocyte phenotype.Once expressed, PPARγ and C/EBPα posi-tively cross activate each other through their re-spective C/EBP regulatory elements (136, 138,139). Presumably, this action perpetuates theadipocyte phenotype in the mature adipocyte.

Sterol regulatory element-binding protein.The sterol regulatory element-binding proteins(SREBPs) are basic helix-loop-helix-leucinezipper proteins. SREBP2 regulates transcrip-tion of the genes of cholesterol metabolism,while SREBP1c and its mouse homolog,ADD1, regulate lipogenesis (140, 141). In theadipocyte differentiation program, the expres-sion of SREBP1c/ADD1 mRNA is activated af-ter the expression of C/EBPα and PPARγ, i.e.,at ∼20 h after induction of differentiation (142).Current evidence implicates SREBP1c/ADD1in the following terminal events of adipocytedifferentiation. (a) SREBP1c/ADD1 is ex-pressed as a membrane-bound precursor boundto SCAP (SREBP-cleavage-activating protein)and tethered in the ER by Insig-2a. (b) Uponits release from the ER (stimulated by insulin),SCAP·SREBP1c/ADD1 moves to the Golgiapparatus, where proteolytic cleavage frees itsbasic helix-loop-helix component for translo-cation to the nucleus. (c) Once in the nucleus,transcription of genes encoding lipogenic en-zymes occurs—events that produce adipocytecharacteristics.

Recent evidence (143, 144) indicatesthat insulin regulates the release ofSCAP·SREBP1c/ADD1 from the ER bydownregulating the level of Insig-2a throughincreased turnover of its mRNA. Reducing

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the level of Insig-2a facilitates export ofSCAP·SREBP1c/ADD1 from the ER to theGolgi for proteolytic cleavage and subsequenttranslocation of the basic helix-loop-helixcomponent to the nucleus. As a result, thetranscription of lipogenic genes is activated,including those that support fatty acid syn-thesis, desaturation, and uptake, as well astriacylglycerol synthesis. In a metabolic sense,this is consistent with the recent finding thatSREBP1c/ADD1 is phosphorylated by AMPkinase (145), which promotes energy mobi-lization. Insulin, on the other hand, promotesenergy storage by activating lipogenic enzymeexpression.

Histones and Chromatin Remodeling

Genome-wide mapping studies have revealedchanges in chromatin structure that oc-cur during the differentiation of 3T3-L1preadipocytes into adipocytes (145, 146).DNase I-hypersensitive site analysis revealedthat alteration of chromatin structure occursearly in the differentiation program, coincid-ing with the cooperative binding of early tran-scription factors, such as C/EBPβ and C/EBPδ,to regulatory element hot spots. Of particu-lar interest, C/EBPβ hot spots were observedprior to induction of differentiation and chro-matin remodeling. Furthermore, a subset of theearly remodeled C/EBPβ hot spots persistedthroughout differentiation and was later occu-pied by PPARγ, suggesting that early C/EBPfamily members may act as initiating factorsfor subsequent binding of PPARγ. These find-ings are, however, inconsistent with those ob-tained in cell culture studies (93). These studiesshowed that, in the G1-growth-arrested stateprior to induction, C/EBPβ is in its dephos-

phorylated inactive state and unable to bindDNA (i.e., to consensus C/EBP promoter-binding sites); synchronous entry into S phaseoccurs. Synchronous entry into S phase occursonly after completion of dual phosphorylationof C/EBPβ and acquisition of DNA-bindingactivity (Figure 4) (93). A possible explanationfor this discrepancy is that the preadipocytes inthis study were not in the G1-growth-arrestedstate (147). Dividing preadipocytes are knownto express high levels of active phosphory-lated C/EBPβ. Thus, C/EBPβ hot spots, at-tributed to “uninduced” preadipocytes, maywell have been due to cells that had traversedthe G1-S cell-cycle checkpoint. See the sec-tions above (i.e., Mitotic Clonal Expansion andCCAAT/enhancer-binding protein-β and –δ,above). C/EBPβ must be phosphorylated mul-tiple times to bind DNA. Thus, it will be neces-sary to definitively prove by cell-cycle analysisthat all preadipocytes prior to induction werein the G1-growth-arrested state as illustratedin Figure 3.

Role of microRNAs in Adipogenesis

A number of microRNAs have been identifiedthat appear to play a role in adipogenesis. Someof these microRNAs seem to accelerate adi-pogenesis (148, 149), while others negativelyregulate adipogenesis (150, 151). Mammalianhomologs of miR-8 promote adipogenesis byinhibiting Wnt signaling (148). Ectopic intro-duction of let-7 into 3T3-L1 cells inhibitedclonal expansion as well as terminal differentia-tion (151). However, the mechanisms by whichthese microRNAs act have not been definitivelylinked to specific aspects of the differentiationprogram. This should be a fertile area for futureresearch.

SUMMARY POINTS

1. Excessive caloric intake leads to adipocyte hyperplasia and adiposity.

2. Adipocyte hyperplasia is caused by recruitment of pluripotent mesenchymal stem cells(MSCs) from the vascular stroma of adipose tissue.


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3. The BMP and Wnt families are key mediators of MSC commitment to producepreadipocytes.

4. Exposure of growth-arrested preadipocytes to differentiation inducers (IGF1, glucocor-ticoid, and cAMP) triggers DNA replication, reentry of the cell cycle (a process knownas mitotic clonal expansion), and a transcription factor cascade, which leads to expressionof adipocyte genes.

5. The transcription factor cascade includes the following: Induction →⇑[CREB → P-CREB] →⇓CHOP10 → ⇑[C/EBPβ → P2-C/EBPβ] → ⇑[C/EBPα / PPAR]γ →⇑SREBP1c → ⇑adipocyte genes.

FUTURE ISSUES

1. A more detailed understanding is needed of the mechanisms by which lineage commit-ment and differentiation occur.

2. Are the Wnt and BMP pathways, involved in MSC commitment, linear/sequential orparallel?

3. Characterization is lacking of the conformational changes, induced by phosphorylationof C/EBPβ, that promote dimerization and acquisition of DNA-binding activity. Thethree-dimensional structures of phospho- and dephospho-C/EBPβ by X-ray crystallog-raphy/magnetic resonance imaging are needed.

4. The role of microRNAs in lineage commitment and differentiation needs to be deter-mined.

DISCLOSURE STATEMENT

The authors have nothing to disclose regarding potential bias and are not aware of any affiliations,memberships, funding, or financial holdings that might be perceived as affecting the objectivityof this review.

LITERATURE CITED

1. Cahill GF Jr. 2006. Fuel metabolism in starvation. Annu. Rev. Nutr. 26:1–222. Otto TC, Lane MD. 2005. Adipose development: from stem cell to adipocyte. Crit. Rev. Biochem. Mol.

Biol. 40:229–423. Wolfgang MJ, Lane MD. 2006. The role of hypothalamic malonyl-CoA in energy homeostasis. J. Biol.

Chem. 281:37265–694. Wahren J, Ekberg K. 2007. Splanchnic regulation of glucose production. Annu. Rev. Nutr. 27:329–455. Shepherd PR, Gnudi L, Tozzo E, Yang H, Leach F, Kahn BB. 1993. Adipose cell hyperplasia and

enhanced glucose disposal in transgenic mice overexpressing GLUT4 selectively in adipose tissue.J. Biol. Chem. 268:22243–46

6. Bowers RR, Kim JW, Otto TC, Lane MD. 2006. Stable stem cell commitment to the adipocyte lineageby inhibition of DNA methylation: role of the BMP-4 gene. Proc. Natl. Acad. Sci. USA 103:13022–27

7. Covas DT, Panepucci RA, Fontes AM, Silva WA, Orellana MD, et al. 2008. Multipotent mesenchymalstromal cells obtained from diverse human tissues share functional properties and gene-expression profilewith CD146+ perivascular cells and fibroblasts. Exp. Hematol. 36:642–54

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145. Aagaard MM, Siersbaek R, Mandrup S. 2011. Molecular basis for gene-specific transactivation by nuclearreceptors. Biochim. Biophys. Acta 1812:824–35

146. Steger DJ, Lazar MA. 2011. Adipogenic hotspots: where the action is. EMBO J. 30:1418–19147. Siersbaek R, Nielsen R, John S, Sung MH, Baek S, et al. 2011. Extensive chromatin remodelling and

establishment of transcription factor ‘hotspots’ during early adipogenesis. EMBO J. 30:1459–72148. Kennell JA, Gerin I, MacDougald OA, Cadigan KM. 2008. The microRNA miR-8 is a conserved negative

regulator of Wnt signaling. Proc. Natl. Acad. Sci. USA 105:15417–22149. Wang Q, Li YC, Wang J, Kong J, Qi Y, et al. 2008. miR-17-92 cluster accelerates adipocyte differentiation

by negatively regulating tumor-suppressor Rb2/p130. Proc. Natl. Acad. Sci. USA 105:2889–94150. Kim SY, Kim AY, Lee HW, Son YH, Lee GY, et al. 2010. miR-27a is a negative regulator of adipocyte

differentiation via suppressing PPARgamma expression. Biochem. Biophys. Res. Commun. 392:323–28151. Sun T, Fu M, Bookout AL, Kliewer SA, Mangelsdorf DJ. 2009. MicroRNA let-7 regulates 3T3-L1

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BI81-FrontMatter ARI 9 May 2012 7:26

Annual Review ofBiochemistry

Volume 81, 2012Contents

PrefacePreface and Dedication to Christian R.H. Raetz

JoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �xi

Prefatories

A Mitochondrial OdysseyWalter Neupert � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

The Fires of LifeGottfried Schatz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �34

Chromatin, Epigenetics, and Transcription Theme

Introduction to Theme “Chromatin, Epigenetics, and Transcription”Joan W. Conaway � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �61

The COMPASS Family of Histone H3K4 Methylases:Mechanisms of Regulation in Developmentand Disease PathogenesisAli Shilatifard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �65

Programming of DNA Methylation PatternsHoward Cedar and Yehudit Bergman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �97

RNA Polymerase II Elongation ControlQiang Zhou, Tiandao Li, and David H. Price � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 119

Genome Regulation by Long Noncoding RNAsJohn L. Rinn and Howard Y. Chang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 145

Protein Tagging Theme

The Ubiquitin System, an Immense RealmAlexander Varshavsky � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 167

Ubiquitin and Proteasomes in TranscriptionFuqiang Geng, Sabine Wenzel, and William P. Tansey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 177

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The Ubiquitin CodeDavid Komander and Michael Rape � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 203

Ubiquitin and Membrane Protein Turnover: From Cradle to GraveJason A. MacGurn, Pi-Chiang Hsu, and Scott D. Emr � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 231

The N-End Rule PathwayTakafumi Tasaki, Shashikanth M. Sriram, Kyong Soo Park, and Yong Tae Kwon � � � 261

Ubiquitin-Binding Proteins: Decoders of Ubiquitin-MediatedCellular FunctionsKoraljka Husnjak and Ivan Dikic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 291

Ubiquitin-Like ProteinsAnnemarthe G. van der Veen and Hidde L. Ploegh � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 323

Recent Advances in Biochemistry

Toward the Single-Hour High-Quality GenomePatrik L. Stahl and Joakim Lundeberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 359

Mass Spectrometry–Based Proteomics and Network BiologyAriel Bensimon, Albert J.R. Heck, and Ruedi Aebersold � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 379

Membrane Fission: The Biogenesis of Transport CarriersFelix Campelo and Vivek Malhotra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Emerging Paradigms for Complex Iron-Sulfur Cofactor Assemblyand InsertionJohn W. Peters and Joan B. Broderick � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 429

Structural Perspective of Peptidoglycan Biosynthesis and AssemblyAndrew L. Lovering, Susan S. Safadi, and Natalie C.J. Strynadka � � � � � � � � � � � � � � � � � � � � 451

Discovery, Biosynthesis, and Engineering of LantipeptidesPatrick J. Knerr and Wilfred A. van der Donk � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 479

Regulation of Glucose Transporter Translocationin Health and DiabetesJonathan S. Bogan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 507

Structure and Regulation of Soluble Guanylate CyclaseEmily R. Derbyshire and Michael A. Marletta � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 533

The MPS1 Family of Protein KinasesXuedong Liu and Mark Winey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 561

The Structural Basis for Control of Eukaryotic Protein KinasesJane A. Endicott, Martin E.M. Noble, and Louise N. Johnson � � � � � � � � � � � � � � � � � � � � � � � � � � 587

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Measurements and Implications of the Membrane Dipole PotentialLiguo Wang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 615

GTPase Networks in Membrane TrafficEmi Mizuno-Yamasaki, Felix Rivera-Molina, and Peter Novick � � � � � � � � � � � � � � � � � � � � � � � 637

Roles for Actin Assembly in EndocytosisOlivia L. Mooren, Brian J. Galletta, and John A. Cooper � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 661

Lipid Droplets and Cellular Lipid MetabolismTobias C. Walther and Robert V. Farese Jr. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 687

Adipogenesis: From Stem Cell to AdipocyteQi Qun Tang and M. Daniel Lane � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 715

Pluripotency and Nuclear ReprogrammingMarion Dejosez and Thomas P. Zwaka � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 737

Endoplasmic Reticulum Stress and Type 2 DiabetesSung Hoon Back and Randal J. Kaufman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 767

Structure Unifies the Viral UniverseNicola G.A. Abrescia, Dennis H. Bamford, Jonathan M. Grimes,

and David I. Stuart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 795

Indexes

Cumulative Index of Contributing Authors, Volumes 77–81 � � � � � � � � � � � � � � � � � � � � � � � � � � � 823

Cumulative Index of Chapter Titles, Volumes 77–81 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 827

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found athttp://biochem.annualreviews.org/errata.shtml

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AnnuAl Reviewsit’s about time. Your time. it’s time well spent.

AnnuAl Reviews | Connect with Our expertsTel: 800.523.8635 (us/can) | Tel: 650.493.4400 | Fax: 650.424.0910 | Email: [email protected]

New From Annual Reviews:

Annual Review of Statistics and Its ApplicationVolume 1 • Online January 2014 • http://statistics.annualreviews.org

Editor: Stephen E. Fienberg, Carnegie Mellon UniversityAssociate Editors: Nancy Reid, University of Toronto

Stephen M. Stigler, University of ChicagoThe Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that allow for their implementation. It will include developments in the field of statistics, including theoretical statistical underpinnings of new methodology, as well as developments in specific application domains such as biostatistics and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical sciences.

Complimentary online access to the first volume will be available until January 2015. table of contents:•What Is Statistics? Stephen E. Fienberg•A Systematic Statistical Approach to Evaluating Evidence

from Observational Studies, David Madigan, Paul E. Stang, Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage, Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema, Patrick B. Ryan

•The Role of Statistics in the Discovery of a Higgs Boson, David A. van Dyk

•Brain Imaging Analysis, F. DuBois Bowman•Statistics and Climate, Peter Guttorp•Climate Simulators and Climate Projections,

Jonathan Rougier, Michael Goldstein•Probabilistic Forecasting, Tilmann Gneiting,

Matthias Katzfuss•Bayesian Computational Tools, Christian P. Robert•Bayesian Computation Via Markov Chain Monte Carlo,

Radu V. Craiu, Jeffrey S. Rosenthal•Build, Compute, Critique, Repeat: Data Analysis with Latent

Variable Models, David M. Blei•Structured Regularizers for High-Dimensional Problems:

Statistical and Computational Issues, Martin J. Wainwright

•High-Dimensional Statistics with a View Toward Applications in Biology, Peter Bühlmann, Markus Kalisch, Lukas Meier

•Next-Generation Statistical Genetics: Modeling, Penalization, and Optimization in High-Dimensional Data, Kenneth Lange, Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobel

•Breaking Bad: Two Decades of Life-Course Data Analysis in Criminology, Developmental Psychology, and Beyond, Elena A. Erosheva, Ross L. Matsueda, Donatello Telesca

•Event History Analysis, Niels Keiding•StatisticalEvaluationofForensicDNAProfileEvidence,

Christopher D. Steele, David J. Balding•Using League Table Rankings in Public Policy Formation:

Statistical Issues, Harvey Goldstein•Statistical Ecology, Ruth King•Estimating the Number of Species in Microbial Diversity

Studies, John Bunge, Amy Willis, Fiona Walsh•Dynamic Treatment Regimes, Bibhas Chakraborty,

Susan A. Murphy•Statistics and Related Topics in Single-Molecule Biophysics,

Hong Qian, S.C. Kou•Statistics and Quantitative Risk Management for Banking

and Insurance, Paul Embrechts, Marius Hofert

Access this and all other Annual Reviews journals via your institution at www.annualreviews.org.

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