Vol. 9, 595-610, August 1998 Cell Growth & Differentiation 5�

Regulation of Cyclin-dependent Kinase 4 during AdipogenesisInvolves Switching of Cyclin D Subunits and ConcurrentBinding of p18””� and p27�’�

Dawn E. Phelps and Yue Xiong2Uneberger Comprehensive Cancer Center [D. E. P., V. X.], Departmentof Biochemistry and Biophysics [V. X.], Program in Molecular Biologyand Biotechnology (V. X.J, University of North Carolina at Chapel Hill,Chapel Hill, North Carolina 27599

AbstractTerminal differentiation of many cell lineages involvesan exit from the mitotic cycle and entry into, andmaintenance of, a permanent state of G1 arrest. Wefound that during terminal differentiation of mouse3T3-L1 preadipocytes, the level of cyclin-dependentkinase 4 (CDK4) remained constant, but the subunitcomposition of the CDK4 complex underwent adynamic rearrangement. As 313-LI cells differentiated,the levels of cyclin Dl and cyclin D1-CDK4 complexesdeclined to negligible levels. Meanwhile, cycllns D2 andD3 levels and their associations with CDK4 increasedtransiently and persistently, respectively, with cyclin D3becoming the predominant cyclin partner of CDK4 inmature adipocytes. At least five CDK inhibitors areexpressed during the differentiation program of 3T3-L1cells. Both p15�� and p16”� continuously declinedto undetectable levels immediately after differentiationinduction. p21 was transiently expressed during theexit of 3T3-L1 cells from mitotic clonal expansion andthen decreased to undetectable levels in matureadipocytes. The level of p27’�#{176}1and p27-CDK4complexes remain high during differentiation and inmature adipocytes. Distinctly, there is a remarkableinduction of pIWNK4C mRNA and protein that was notseen in the closely related nondifferentiating 3T3-C2cell line, suggesting that p18 induction in 313-LI cellsis related to cell differentiation, not cell cycle arrest.The pRb kinase activity of cyclin D3 and CDK4 was notdetected in quiescent 3T3-L1 cells and was theninduced as the cells entered the mitotic clonalexpansion phase. Unexpectedly, cyclin D3 and CDK4

Received 4/22/98; revised 6/9/98; accepted 6/15/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This study was supported by Public Health Service grant CA-68377 (toV. X.) from the NIH. D. P. is a recipient of the National Research ServiceAward from the NIH/National Institute of General Medical Science Grant1F32GM18437-01 . V. X. is a recipient of American Cancer Society JuniorFaculty Award and a Pew SChOlar in Biomedical Science.2 To whom requests for reprints should be addressed, at University ofNorth Carolina at Chapel Hill, Campus Box 7295, Uneberger Building,Mason Farm Road and Manning Drive, Chapel Hill, NC 27599. Phone:(919) 962-2143; Fax: (919) 966-8799; E-mail: yxiong@email.unc.edu.

pRb kinase activity remained high after 3T3-L1 cellscompleted their mitotic division and was still readilydetectable in mature adipocytes. Our study reveals anactive regulation, rather than passive inhibition, ofCDK4 activity during adipocyte differentiation. Twocentral features of this complex regulation areswitching of activating cyclin D subunits andconcurrent binding by the p18 and p27 CDK inhibitors.

IntroductionConcomitant with terminal differentiation, cells exit from thedivision cycle at the G1 phase and maintain a nonproliferativestate commonly known as G0. The precise regulation of entryinto, and maintenance of, permanent cell cycle arrest in G0 is

critical for cell differentiation, because its deregulation canpotentially lead to abnormal morphogenesis and a widerange of proliferative diseases, particularly cancer. The mo-lecular mechanisms coupling terminal cell differentiation andcell cycle arrest are, however, still poorly understood. Theeukaryotic cell cycle is primarily regulated by a family ofstructurally related serine/threonine protein kinases, whichconsists of a regulatory subunit, a cyclmn, and a catalyticsubunit, a CDK3 (reviewed in Ref. 1). In mammalian cells,CDK4 or CDK6, in combination with three D-type cyclins (Di,D2, and D3), and CDK2 in association with cyclin E, play keyroles in regulating G1 progression (reviewed in Refs. 1 and 2).Both CDK2 and cyclin E are expressed in all cell types. Theexpression of cyclin E, but not CDK2, is periodic during thecell cycle, with maximal levels at the G1-S boundary, and isnecessary for S phase entry (2-7). On the other hand, CDK4,CDK6, and D cyclins are differentially expressed. The ex-pression and pRb kinase activity of the cydin D gene isinduced during the delayed early response to mitogenicstimulation, prior to the accumulation and kinase activity ofcyclin E (8-1 0). These features suggest that CDK4/6-cyclin Dand CDK2-cyclin E enzymes have distinct functions in rag-ulating G1 progression, with CDK4/6-cyclin D coupling ex-tracellular growth signals with the cell cycle whereas CDK2-cyclin E controls the initiation of DNA replication.

The activity of CDK4 is primarily regulated posttranslation-ally at the level of protein-protein interactions. Binding to a Dcyclin is necessary for the activation of CDK4 (1 1 , 1 2) andmay be facilitated by the association of the catalytic subunitwith CDC37 and Hsp9O (13, 14). The principle negative rag-ulation of CDK4 is by physical association with inhibitoryproteins, i.e., CDK inhibitors (reviewed in Ref. 15). Presently,

3 The abbreviations used are: CDK, cyclmn-dependent kinase; IP,immunoprecipitation; UTA, untranslated region; TGF, transforming growthfactor.

596 CDK4 Regulation during Adipogenesms

seven CDK inhibitors have been identified in mammaliancells, and they fall into two distinct multigene families, rep-resented by the prototypes p21 and p16”’”<�, that differ inboth structure and mechanism of action. Members of thep21 family (p21 , p27’#{176}’�,and p57’#{176}’�) inhibit CDK activity by

forming a ternary p21 -cyclin D-CDK4 complex, whereasmembers of the INK4 family (p1 5INK4b, p1 61NK45, p1 8�%��(4c,and p1 9!NK4d) specifically inactivate CDK4 (and CDK6) activ-ity by forming a binary INK4-CDK4 complex (15). Within theCDK family, CDK4/6 are unique in that they are regulated by

both the CIP/KIP and lNK4 families of inhibitors. This regu-lation provides CDK4/6 with the ability to serve as integratorsfor the convergence of many cell growth control signals fromdifferent stimulatory and inhibitory regulation pathways.These findings suggest the possibilities that different cellgrowth control signals, such as terminal cell differentiation,

may exert their effects on the cell cycle by regulatingCDK4/6, pointing to the critical importance of investigatingCDK4/6-associated regulatory proteins.

Terminal adipogenesis is the process of converting prolif-erating adipoblasts into permanently cell cycle-arrested, fat-laden adipocytes, which play a central role in lipid homeo-stasis and the maintenance of energy balance. Abnormalregulation of adipocyte differentiation is linked to obesity, acommon disorder characterized by an increase in both thenumber and size of adipocytes (reviewed in Ref. 16). Inrecent years, significant progress has been made in identi-fying adipocyte-specific genes, adipogenic transcription fac-tors, and specific ligands. These studies have been greatlyfacilitated by the availability of several established mousepreadipocyte cell lines that represent faithful models of prea-

dipocyte differentiation in vivo. In vitro differentiated adipo-

cytes faithfully mimic the metabolism of adipocytes isolated

from adipose tissue, and s.c. injection of these culturedpreadipocytes led to the development of normal fat pads at

the site of injection (reviewed in Refs. 16 and 1 7). In contrast,the molecular regulation of the cell cycle during adipogenesisremains poorly understood. Committed preadipocytes, suchas 3T3-L1 cells, maintain a proliferative state when culturedin growth medium. 3T3-Li preadipocyte cells temporarily

arrested in a quiescent state upon confluence can be in-duced by the appropriate combination of mitogenic andadipogenic signals to first progress through a required mi-totic clonal expansion phase and then enter into a permanentstate of cell cycle arrest as terminally differentiated adipo-cytes (reviewed in Refs. 1 7 and 1 8). Therefore, differentiationof these established preadipocytes not only provides a usefulsystem for elucidating the mechanisms that couple terminalcell differentiation and permanent cell cycle arrest, it alsoprovides a unique model to study the molecular details oftemporary versus permanent cell cycle arrest. To understandthe mechanism that couples terminal cell differentiation andcell cycle arrest, we carried out a detailed analysis of CDK4protein complexes at different stages during adipogenesis of313-Li preadipocytes. Our study revealed a dynamic sub-unit rearrangement of CDK4 complexes during 313-Li celldifferentiation that involves switching of activating cyclin Dsubunits and cooperative binding by CDK inhibitors fromboth families.

ResultsCDK4 Associates with Multiple D-Cyclins and CDK Inhib-itors during Adipocyte Differentiation of 3T3-L1 Cells.313-Li cells were maintained as proliferating adipoblasts inbovine calf serum and then induced to synchronously pro-

gress through the differentiation program at high frequency(>90%) over 7 days (Fig. 1A). Adipocyte differentiation is

initiated by growing the cells to confluence, at which timethey enter a temporary state of quiescence in G1 . Uponexposure to an adipogenic and mitogenic mixture, quiescent313-Li preadipocyte cells reinitiate cell cycle progressionand undergo at least one round of mitotic division (clonalexpansion phase). At the end of clonal expansion, preadipo-cytes enter a permanent state of growth arrest. 313-Li adi-pocyte differentiation in culture was monitored by examiningthe accumulation of cytoplasmic lipid droplets, by bright field

microscopy and staining with Oil-Red-O (Fig. 1B), and theinduction of a program of adipocyte-specific genes, such asthe C/EBPa transcription factor by Western blot analysis(Fig. 1C). Consistent with published observations, the ap-pearance of cytoplasmic lipid droplets was first evident bypostinduction day 3 (Fig. iB, Induced; Ref. 19), and theexpression profile of C/EBPa was typical for 313-Li adipo-genesis, being first evident on day 2 of differentiation (Fig.1 C, Induced; Ref. 20). Uninduced 313-Li cells, which weremaintained as a confluent monolayer in growth medium for 8days, did not accumulate cytoplasmic triglyceride droplets(Fig. 1B, Uninduced) or express C/EBPa (Fig. 1C, Unin-

duced) and thus served as undifferentiated 313-Li controlcells. Cell cycle progression of both induced and uninduced313-Li cells was monitored by [�H]thymidine incorporation(Fig. 1D). In density-arrested quiescent cells (day 0), DNAsynthesis was low and then reached a peak 24 h after dif-ferentiation induction. By 48 h after adipogenic induction,DNA synthesis was low again, consistent with a single roundof cell division during the mitotic clonal expansion. The in-corporation of [�H]thymidine during the first 2 days of differ-entiation induction was consistent with published data (21,22). [�H]Thymidine incorporation remained at a low back-ground level over the next 5 days of the experiment, indicat-ing an entry of 313-Li cells into, and maintenance of, apermanent state of cell cycle arrest.

CDK4 and its closely related relative, CDK6, in combina-tion with the cyclin D cyclins, play a key role in coupling

extracellular growth signals with the cell cycle and have beenpostulated to control the G1-S transition. The mechanism bywhich the activity of CDK4 is regulated during a proliferativestate and both temporary and permanent cell cycle arrestwas examined using the well-characterized 313-Li cell line.313-Li cells express considerably higher levels of CDK4than CDK6 (data not shown), suggesting that CDK4 plays amore prominent role in regulating G1 progression in thesecells and prompting us to focus our effort on examining theregulation of CDK4. We first conducted [�5S]methionine met-abolic labeling coupled with immunoprecipitation e5S-IP)experiments to have a global view of CDK4 complexes atdifferent stages of 3T3-L1 cell differentiation. These studieswere performed using an anti-CDK4 polyclonal antibodygenerated against a synthetic peptide corresponding to the

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Cell Growth & Differentiation 597

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Fig. 1 . Adipocyte differentiation of3T3-Ll cells and cell cycle progressionduring adipogenesis. A, schematic of3T3-L1 adipogenic differentiation pro-gram. 3T3-Ll cells can be maintainedas proliferating adipoblasts. Whengrown to confluence (day 0), 3T3-L1cells are temporarily arrested in G1 andbegin to express early markers of adi-pocyte differentiation. Upon exposureto a mitogenic and adipogenic cocktail,3T3-L1 preadipocytes reenter the cellcycle and undergo mitotic clonal ex-pansion. By day 2, >80% of the cellshave permanently arrested in G1 (22),and they begin to express adipose-specific genes. In B, 3T3-L1 cells weregrown to confluence and induced to dif-ferentiate into mature adipocytes(Induced) or were maintained as a con-fluent monolayer in growth medium(Uninduced). The accumulation of cyto-plasmic lipid droplets was examined bystaining with Oil-Red-O on postinduc-tion day 8. In C, the expression of theC/EBPcr transcription factor, a well-characterized marker for the late stagesof 3T3-L1 adipocyte differentiation, wasexamined by immunoblot analysis. In D,cell cycle progression of both inducedand uninduced 3T3-L1 cells was moni-tored over the 7-day differentiation pro-gram by [�H]thymidine incorporation.

COOH-terminal sequence of mouse CDK4 (see “Materials

and Methods”), because several previously reported anti-

CDK4 antibodies against human CDK4 either failed to coim-munoprecipitate CDK-associated cellular proteins or cross-reacted with mouse CDK4 poorly. This newly generatedantibody recognizes specifically and efficiently mouse CDK4

protein and coimmunoprecipitates most, if not all, presently

known CDK4-associated cellular proteins (see below).

[35S]Methionine-labeled cell lysates, derived from 313-Li

cells cultured in growth medium as proliferating adipoblasts

or 313-Li cells induced to undergo adipogenic differentia-tion for various lengths of time, were immunoprecipitated

with the anti-CDK4 antibody and resolved by SDS-PAGE

(Fig. 2/1). To control for any subunit rearrangements of CDK4

Alnd uced

p DO Dl D2 D4 ____

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598 CDK4 Regulation during Adipogenesis

p16-

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1 2 345 67 8 9101112

Fig. 2. Analysis of CDK4 complexesduring adipocyte differentiation of3T3-L1 cells. r5S]Methionine-labeledlysates were prepared from proliferat-ing (P) 3T3-L1 adipoblasts, temporar-ily density-arrested (DO) 3T3-Ll prea-dipocytes, at different time points (indays) after the induction of the adipo-genic program (A) or at different timepoints (in days) from 3T3-Ll cellsmaintained as a confluent monolayer(B). Cell lysates were immunoprecipi-tated with a mouse anti-CDK4 anti-body, with (+) or without (--) preincu-bation with its corresponding antigenpeptide. The immunoprecipitates wereresolved to SDS-PAGE. The mobilityof protein molecular weight standardsand relevant proteins discussed in thetext are indicated. The band in Lane 4of both A and B that migrates abovep1 6 was not blocked by the compet-ing antigen peptide and most likelyrepresents a nonspecific backgroundband.

complexes that are not associated with adipogenic induc-

tion, parallel experiments were performed with uninduced

313-Li cells that were maintained in a confluent state (see

“Materials and Methods”; Fig. 2B). The identity of each rel-evant protein was confirmed by their gel mobility, by Western

blot analysis (see below), and/or by V8 proteolytic mapping

(data not shown). In proliferating 313-Li cells (Fig. 2A, Lanes1 and 2), CDK4 is complexed with three CDK inhibitors, p15,

p16, and p27, and at least two cyclin D proteins, cyclin Dl

and cyclin D2, as revealed by the antigen peptide competi-

tion experiments carried out in parallel using the same Ia-beled lysate (Fig. 2, Lane P). The identity of the two cyclin D

bands as cyclin Dl and 02 was based on two lines ofevidence: (a) the steady-state level of cyclin Di in CDK4immunoprecipitates largely parallels the association of newly

synthesized cyclin Dl with CDK4 (compare Fig. 2A with Fig.6A); and (b) in an independent series of metabolic labelingexperiments in which CDK4, cyclin Dl , and cyclin D3[35S]methionine-labeled immunoprecipitates were resolved

on the same SDS-PAGE gel, it was observed that the two

A B

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Day P

Induced

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Cell Growth & Differentiation 599

________ Unlnduced I I Induced Unlnduced I12468 P012470 1247

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CDK4 IP

I Induced Unlnduced IDay P 0 1 2 4 7 0 1 2 4 7

p21 IP

Induced Unlnduced IP 0 1 24 7 01 247

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Fig. 3. Expression of the p21 and p27 CDK inhibitors and their association with CDK4 during adipose conversion of 3T3-L1 cells. Lysates prepared from3T3-Ll at different time points during the differentiation induction protocol or from uninduced 3T3-L1 cells were immunoprecipitated with antibodies specificto CDK4 (A and C), p27 (B), or p21 (D). The immunoprecipitates were subjected to SOS-PAGE and immunoblotted with antibodies to CDK4, p21 , and p27as indicated.

cyclin D bands in the CDK4 IP comigrated with the cyclin Dl

and cyclin 02 proteins in the cyclin Dl IP, having establishedthat the polyclonal anti-Di antibody specifically recognizes

cyclin Dl and cyclin D2 but not cyclin D3 (data not shown,

Fig. 6E). It was also found that the electrophoretic mobility of

mouse cyclin D3 was not separable from that of CDK4 and

thus we turned to reciprocal IP-Western blot analysis to

detect the expression and association of these two proteins(see Fig. 6, C and D). As 3T3-Ll cells were induced toundergo adipogenic differentiation, the rate of CDK4 synthe-

sis, as determined by metabolic labeling, remained essen-tially unchanged. The association of CDK4 with its regulatory

proteins, however, underwent dynamic changes during the

course of 313-Li cell differentiation. The association ofCDK4 with newly synthesized cyclin Dl , p15, and p1 6 con-

tinuously declined to nearly undetectable levels as the cells

entered the terminally differentiated state (day 4), whereas itsassociation with newly synthesized p27 remained constant.The association of CDK4 with newly synthesized p21 tran-siently increases at a time when these cells are entering a

permanent state of cell cycle arrest (day 2 through day 4,

Lanes 7 and 9), but was undetectable in proliferating (Lane 1),

density-arrested (Lane 3), mitotically expanding (Lane 5), and

permanently arrested (Lane 1 1) 313-Li cells. The associationof CDK4 with p1 8 was not detected in proliferating cells,became visible after 24 h of differentiation induction, contin-uously increased throughout the 7-day differentiation pro-

gram, and was then maintained at high levels in terminally

arrested cells (Fig. 2A). The observed subunit rearrange-ments of CDK4 complexes, as determined by its association

with newly synthesized D cyclins and CDK inhibitor proteins,

were specific for the induction of 313-Li differentiation andwere not observed in uninduced 313-Li cells (Fig. 2, com-pare A, Induced with B, Uninduced). These results revealedthat during terminal adipocyte differentiation, there is a com-plex and dynamic regulation imposed on CDK4 that involveschanges of several CDK4-associated regulatory proteins.

The INK4 inhibitors have been shown to interact with cyclin

D-CDK4 complexes in insect cells (23) or by overexpressionto high levels in cultured mammalian cells (24). In a series of

reciprocal 35S-labeled IP experiments using cyclin Di , cyclin

D3, p1 6, and p1 8, we did not detect any cyclin D in INK4

immunoprecipitates or INK4 in cyclin D precipitates in313-Li cells (data not shown). The anti-cyclin D and anti-

INK4 antibodies are both capable of coimmunoprecipitating

their respective associated CDK4 and CDK6 proteins, argu-

ing against the possibility that failure to detect INK4-cyclin Dassociation is due to antibody disruption. We believe that iflNK4 does form a complex with D cyclins through CDK4/6 in

vivo, it must be very transient and greatly favor a CDK4/6-

INK4 complex, as D-cyclins, both free form and CDK4/6-

bound, are intrinsically unstable and undergo rapid turnover.

CDK4 Levels Remain Constant during Adipogenesis of3T3-L1 Cells. Analysis of CDK4 immunocomplexes after

metabolic labeling detects only the association of CDK4 with

newly synthesized proteins and does not determine the

steady-state level of each individual CDK4-interacting pro-

tein. A series of reciprocal, coupled immunoprecipitation-

immunoblotting (IP-Western) experiments were therefore

carried out to examine the steady-state levels of CDK4 and

CDK4 IPBA

I Induc.d Unlnducsd IDay P 0 1 2 4 7 0 1 2 47

p15- �� S �

C CDK4 P

p15 IP

I Induc#{149}d Unlnduced IDay P 0 1 2 4 7 0 1 2 4 7

p16�

Fig. 4. Expression of the p15

Induc#{149}d Unlnduced I and p16 INK4 CDK inhibitors andtheir association with CDK4 dur-

P 0 1 2 4 7 0 1 2 4 7 ing adipocyte differentiation of

3T3-L1 cells. Lysates prepared- �- - - - i-I- � � � � from 3T3-L1 at different time

points during the differentiationinduction protocol or from unin-duced 3T3-L1 cells were immu-

D noprecipitated with CDK4 (A andp16 IP C), p15 (B), or p16 (D). The im-

Inducsd Unlnducsd I munoprecipitates were sub-jected to SOS-PAGE and immu-

P 0 1 2 4 7 0 1 2 4 7 noblotted with antibodies toCDK4, CDK6, p15, and p16 as

� � - p16 indicated.�es

CDK4 Regulation during Adipogenesis

its regulatory proteins as well as their association during

313-Li differentiation. P-Western analysis demonstrated

that the steady-state level of CDK4 remained constant during

313-Li differentiation and that the level of CDK4 in prolifer-

ating, differentiation-induced, and uninduced 313-Li cell

was the same (Fig. 3, A and C). These results suggested that

during adipocyte differentiation of 313-Li cells, CDK4 is

primarily regulated through its interactions with other regu-

latory proteins rather than by changing the rate of CDK4

protein synthesis or the level of CDK4 protein within the cells.

That the steady-state level of CDK4 protein remains high in

terminally differentiated adipocytes raises a challenging

question as to what function CDK4 may perform in these

nondividing cells.

Distinct Expression Profiles of the p21 and p27 CDKInhibitors during 3T3-L1 Adipogenesis. The p21 family ofCDK inhibitors is presently composed of three genes, p21,

p27’�’1, and p57�<1P2, the protein products of which form

ternary complexes with and inhibit the activity of many cy-clin-CDK enzymes (1 5). At least two members of this family,

p21 and p27, were detected in [35Sjmethionine-labeled

CDK4 immunoprecipitates (Fig. 2). The steady-state level of

the p27 protein fluctuated slightly during both 313-Li cell

differentiation as well as in the control uninduced cells, withthe highest levels of p27 present in temporary density-

arrested cells on day 0 (Fig. 3B, Day 0). This is in agreement

with previously published observations that reported a mod-

est increase in the steady-state level of p27 protein in qui-

escent cells that were temporarily arrested under several

growth inhibitory conditions, including cell-cell contact inhi-bition (25-27). Reciprocal IP-Western experiments were car-

ned out to determine the association of p27 with CDK4

during adipogenesis of 313-Li cells (Fig. 3). Consistent with

the 35S-labeled IP studies, these experiments demonstrated

that in spite of an increase of p27 protein in density-arrested

cells, the association between CDK4 and p27 was similar

between, and remained constant in, proliferating (Lane P),

density-arrested (Day 0 and Uninduced lanes), mitotically

expanding (Day 1, Induced), and differentiating 313-Li cells

(Day 2 through Day 7, Induced). These results suggest that

p27 may play an important role in regulating CDK4 at all

stages of 313-Li cell differentiation and that another CDKinhibitor(s) may cooperate with p27 to regulate CDK4, be-

cause the level of CDK4 and CDK4-p27 association remains

constant in spite of a change of CDK4 kinase activity (seebelow).

In distinct contrast to the p27 protein, p21 displayed a

unique pattern of protein expression as well as association

with CDK4 during 313-Li adipocyte differentiation (Fig. 3, C

and D). p21 was transiently expressed only in induced

313-Li cells exiting from mitotic clonal expansion and en-

tering a permanent state of cell cycle arrest (day 2 through

day 4) and was then decreased to undetectable levels by day

7 (Fig. 3D). Reciprocal IP-Western blot analysis of CDK4 and

p21 immunoprecipitates demonstrated that the association

between p21 and CDK4 closely paralleled p21 expression(Fig. 3, C and D). The onset of p21 expression on day 2 of

differentiation induction, coupled with its transient expres-

sion and association with CDK4, suggests that the p21 CDK

inhibitor may play a role in the initiation of permanent cell

cycle arrest during 313-Li adipocyte differentiation. These

results also suggest that the p21 and p27 CDK inhibitors,

which share a very similar spectrum of CDK target specific-

ity, are regulated differently during adipogenesis.

Down-Regulation of the p15 and p16 CDK Inhibitorsduring Adipogenesis. Members of the p16 family of CDK

inhibitors (p1 5INK4b p1 6INK4a p1 8INK4c and p1 9!NK4d) bind to

the catalytic subunits CDK4 and CDK6 to form a stable

binary complex in a competing or potentially mutually exclu-

sive manner with the activating D-type cyclins, thereby in-

hibiting the kinase (28). At least three members of this family,p15, p16, and p18 were seen in [35S]methionine-labeled

CDK4 immunoprecipitates (Fig. 2). Both 35S-labeled IP and

Western blot analysis of CDK4 and p19 immunoprecipitates

from different time points of 313-Li adipocyte differentiation

failed to detect any p1 9 protein using an anti-pi 9 antibody

that can readily detect mouse p1 9 protein in other tissues

(29), indicating that p1 g�’�’4d is not likely to have a significantfunction during 313-Li cell differentiation. p1 5 and p1 6 were

readily detectable in proliferating and temporary, density-

arrested (day 0) 313-Li cells (Fig. 2 and 4). Following the

induction of 313-Li differentiation, the synthesis and steady-

state levels of both p1 5 and p16 declined to negligible levels

by day 2, whereas in uninduced 313-Li cells, their levels

remained constant (Fig. 4, B and D, compare Induced to

Uninduced). Reciprocally, the overall levels of p1 5 and p16

Bp18 IP

I lnducsd

Day P 0 1 2 3 4 7

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Day P 0 2 4 7 P 0 2 4 7

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Cell Growth & Differentiation 601

A

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lnducsd

Day P 0 1 2 3 4 7

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Fig. 5. Expression of the p18 INK4 CDK inhibitorand its association with CDK4 during 3T3-L1 adi-pogenesis. In A and B, lysates prepared from3T3-L1 at different time points during the differen-tiation induction protocol or from uninduced 3T3-L1cells were immunoprecipitated with CDK4 (A) orp1 8 (B). The immunoprecipitates were subjected toSOS-PAGE and immunoblotted with antibodies toCDK4, CDK6, and p18 as indicated. In C, totalcellular RNA was isolated from 3T3-L1 and 3T3-C2cells from the indicated days of the differentiationinduction protocol. Ten �g of total RNA from eachtime point were subjected to Northern blot analysiswith a 32P-labeled probe corresponding to the p18coding region (top). The 2.4- and 1.2-kb p18 tran-scripts are indicated. Equal RNA loading was yen-fled by ethidium bromide staining (bottom).

within the CDK4 complexes also declined to undetectablelevels in 313-Li cells after 2 days of differentiation induction,

whereas their levels remained essentially unchanged in con-

trol uninduced cells (Fig. 4, A and C, compare Induced with

Uninduced). These results suggest that p1 5 and p16 mayplay a role, in conjunction with p27, in the temporary growth

arrest of contact-inhibited 313-Li cells but not in initiating ormaintaining permanent cell cycle arrest during 313-Li differ-

entiation.Induction of the p18 CDK Inhibitor during Adipogenesis.

Distinct from the other CDK inhibitors, there is a dramatic

and sustained increase of p1 8 protein during adipogenesis of313-Li cells (Fig. 5). There was nearly undetectable p18 in

proliferating 313-Li cells, and as a result, there was no

detectable pi8-CDK4 complex in these cells. As asynchro-

nously dividing 313-Li cells reach confluence and tempo-

rarily arrest in a quiescence state (proliferating versus day 0),

p18 protein levels become detectable (Fig. 5B, top, Day 0).

After the induction of differentiation, the levels of p1 8 proteinremained unchanged for 2 days during the mitotic clonal

expansion period, dramatically increased 12-fold between

day 2 and day 3, and then remained high through day 7 (Fig.5B, Induced). As a control, the levels of p18 in uninduced313-Li cells remained unchanged throughout the timecourse such that the levels of p1 8 on day 7 were similar to

that in day 0 quiescent cells (Fig. 5B, Uninduced). Although

CDK4 was detectable in p1 8 immunoprecipitates betweenday 0 and day 2, there was an increased association of p18with CDK4 (7-fold increase) and CDK6 on day 3 of induction

that correlated with the sharp rise in p1 8 levels at this stage(Fig. 5B, bottom, compare Induced and Uninduced). Recip-

rocally, the level of p1 8 in CDK4 immunoprecipitates was

CDK4 Regulation during Adipogenesis

adipocytes but not myotubes.

negligible prior to day 2, dramatically increased on day 3, andthen remained relatively constant through day 7 as 313-Licells differentiated (Fig. 5A, compare Induced and Unin-

duced). The induction of p1 8 protein expression occurredabout 24 h later than the induction of p21 expression, whichbegan on day 2 (compare!nduced Day 2 and Day 3 of Fig. 3Dand Fig. 5B). Furthermore, pi 8 levels remained high in ter-minally differentiated cells, whereas p21 levels decreased tonegligible levels. These results suggest the possibility thatp18 may collaborate with p21 to inhibit CDK4 and initiate313-Li cell cycle arrest after the mitotic clonal expansionphase and may also collaborate with p27 to maintain thearrest in terminally differentiated 313-Li cells.

The observation that pi 8 protein and its association with

CDK4 are significantly induced led us to determine whetherthis induction was regulated at the transcriptional level dur-

ing 313-Li differentiation. Total RNA was isolated from313-Li cells at several stages of the differentiation program,and the steady-state level of p1 8 mRNA was determined byNorthern blot analysis (Fig. 5C). Two distinct p1 8 transcripts,2.4 and 1 .2 kb, were differentially expressed during the dif-ferentiation program. The p18 gene has been reported pre-viously to express two distinct transcripts (23, 30). We havedetermined recently that both transcripts contain the samecoding exons, exons 2 and 3, and encode the same pi 8protein. The 2.4-kb transcript uniquely contains an additionalnoncoding 1 .2-kb exon, exon 1 , corresponding exclusively tothe 5’ UTR. The presence of the long 5’ UTR in exon iattenuated the translation of the 2.4-kb pi 8 transcript,

whereas its absence from the 1 .2-kb pi 8 transcript resultedin significantly more efficient translation of the p1 8 protein(30). Judging from its similar size and expression patternduring differentiation (see below), we believe that the two p18transcripts expressed in 313-Li cells correspond to thesame two p1 8 transcripts expressed during myogenic differ-entiation of C2C12 cells. Similar to proliferating C2Ci2 myo-blasts, proliferating 313-Li adipoblasts express only the 2.4transcript (Fig. 5, Lane P). In 313-Li cells that have beencontact inhibited for 48 h, the 1 .2-kb transcript is inducedwhile the expression of the 2.4-kb transcript persists (day 0).The induction of short pi 8 transcript correlated with, andmay be responsible for, the induction of pi 8 protein in tem-porary arrested 313-Li cells (Fig. 5B). Because 313-Li cellsare hormonally induced to undergo differentiation, there is afurther increase in the steady-state level of both transcripts,which correlates with a second induction of p1 8 protein.These observations as well as the long half-life of INK4

proteins led us to conclude that the induction of p1 8 proteinduring 313-Li differentiation is primarily regulated at the levelof transcription. The induction and sustained expression ofboth p1 8 transcripts in permanently arrested 313-Li adipo-cytes is distinct from pi 8 mRNA expression during C2C1 2myoblast differentiation, in which induction of the short tran-script occurs concomitantly with a decrease of the largetranscript to nearly undetectable levels in mature myotubes.This observation indicates that the expression of the p18

gene is regulated differently during myogenesis and adipo-genesis and that both pi 8 promoters remain active in mature

To determine whether activation ofpl8 gene expression isconsequential to cell cycle withdrawal or specifically asso-ciated with 313-Li cell differentiation, we analyzed p18 geneexpression in 313-C2 cells that were similarly treated inparallel with 313-Li cells. 3T3-C2 cells were clonally derivedfrom murine 313 cells similar to 313-Li cells, but they do notundergo adipocyte conversion after the established adipo-genic induction protocol (3i). 3T3-C2 cells do enter a quies-cent state upon cell-cell contact and can then be induced toundergo mitotic clonal expansion upon hormonal stimulationbefore withdrawing from the cell cycle (Ref. 32 and ourconfirmatory observation). As in 313-Li adipoblasts, prolif-erating 3T3-C2 cells express only the 2.4-kb transcript, albeitat much lower levels (Fig. 5C). However, as 3T3-C2 cellswere induced to undergo mitotic clonal expansion, neitherp18 transcript is induced, indicating that the induction of

both pi 8 transcripts is specifically related to 313-Li celldifferentiation but not mitotic clonal expansion or permanentcell cycle withdrawal.

CDK4 Switches Cyclin D Subunits during 313-LI CellDifferentiation. A unique characteristic of the CDK4 (andCDK6) catalytic subunit is its preferential, if not exclusive,association with the D-cyclins (Di , D2, and D3) as opposedto the combinatorial interactions of some CDK proteins withmultiple cyclins (2). At least two members of the cyclin Dfamily, cyclin Di and D2, were detected in [esS]methionine�labeled CDK4 immunoprecipitates (Fig. 2). The electro-phoretic mobility of mouse cyclin D3 was not separable fromthat of CDK4, and thus its expression level can only be

determined by immunoblotting. Reciprocal IP-Westem blotanalysis of cyclin D and CDK4 immunoprecipitates revealedfour distinct forms of D-type cyclins (Fig. 6). Thus, we firstattempted to establish the identity of each form by usingmember-specific antibodies. The polyclonal anti-cyclin Diantibody 342 (12), which was raised against the NH2-terminaltwo-thirds of human cyclin Di , and a cyclin Di antibodygenerated against a COOH-terminal peptide of human cyclinDi (33, 34), detected three bands in 313-Li cells that havebeen differentiated for 2 days (Fig. 6, B, and E, Lanes 1, 2, 11,

and 12). These three bands correspond to two forms ofcyclin Di and cyclin D2 (not cyclin Di , D2, and D3), deter-mined as follows (Fig. 6E): (a) when the same anti-cyclin Diimmunoprecipitates were blotted with a monoclonal anti-body specific to cyclin Di , only two bands were detected,most likely corresponding to two different forms of cyclin Di(Fig. 6E, Lanes 1 1 and 12; Ref. 8); (b) a monoclonal antibodyspecific to cyclin D2 (DCS-3) precipitated only one band thatwas detected by the polyclonal anti-cyclin Di antibody 342and comigrated with the lower band seen in anti-cyclin Diimmunoprecipitates (Fig. 6E, Lanes 4-10), indicating that thefastest migrating band corresponds to cyclin D2; and (c) tofurther confirm that this lower band corresponds to cyclinD2, but not cyclin D3, the 2-day differentiated 313-Li celllysate was immunoprecipitated with a D3-specific antibody(Fig. 6E, Lanes 3 and 13), which was raised against a syn-

thetic peptide, and blotted with a polyclonal (Lanes 1-3) ormonoclonal (Lanes 1 1-13) anti-cyclin Di antibody. Althoughcyclin D3 is abundantly expressed in these cells (Fig. 6D), itwas not detected by the anti-Di antibodies (Fig. 6E, Lanes 3

I Induced ____________ ___________ _____________Day 0 1 2 3 4 7

BI � a�’ � � s� �fUL.., �D2�

C DCyclin D3 IP

Unlnduced

01247

E

1 I Induced UnlnducedP0124701247

Di � . Di-.Di I Di-�D2 ,� � , 1�

Blot: a-D1

gi��

a-D1 cx -D1(MAb)

Cell Growth & Differentiation 603

A BCDK4 IP

Unlnduced �012347

CDK4 IP

I InducedDayP 0 1 2 4 7

D3-#{248} dt.I.4�#{149}sm �

Cyclin Dl IP

Induced Unlnduced

P012468012468

IP: a-D1 a-D3

1 2 3�

a-D2 a-D1 a-D1 a-D3

4 5 6 7 8 9 10 11 12 13

Fig. 6. Expression of D-cyclins and their association with CDK4 during adipocyte differentiation of 3T3-L1 cells. Lysates prepared from 3T3-L1 at differenttime points during the differentiation induction protocol or from uninduced 3T3-L1 cells were immunoprecipitated with CDK4 (A and C), cyclin Dl (B), orcyclin 03 (D). The immunoprecipitates were subjected to SOS-PAGE and immunoblotted with antibodies to CDK4, cyclin Dl , and cyclin 03 as indicated.As shown in E, the anti-cyclin Dl antibody can immunoprecipitate and immunoblot both the cyclin Dl as well as cyclin 02 proteins but does not recognizethe mouse cyclin 03 protein. E, characterization of cyclin 0 antibodies. In Lanes 1-3, cyclin 0 proteins were immunoprecipitated from day 2-induced 3T3-L1cell lysates with either anti-cyclin Dl clone 342 (Lane 1), anti-cyclin Dl -CT (Lane 2), or anti-cyclin 03 (Lane 3) and then Westem blotted with anti-cyclin Dlclone 342. In Lanes 4-10, cyclin 0 proteins were immunoprecipitated from proliferating (Lane 5), day 0 (Lane 6), or induced day 1 (Lane 7), day 2 (Lanes4 and 8), day 4 (Lane 9), and day 7 (Lane 10) 3T3-Ll cell lysates using anti-cyclin Dl clone 342 (Lanes 5-10) or an anti-cyclin 02 monoclonal antibody cloneDCS-3 (Lane 4) and then Westem blotted with anti-cyclin Dl clone 342. In Lanes 1 1-13, cyclin 0 proteins were immunoprecipitated from day 2-induced3T3-L1 cell lysates with either anti-cyclin Dl clone 342 (Lane 1 1), anti-cyclin 01-CT (Lane 12), or anti-cyclin 03 (Lane 13) and then Westem blotted withan anti-cyclin Dl -specific monoclonal antibody (Santa Cruz 72-1 3G).

and 13). From this data, we concluded that the polyclonal

anti-cyclin Di antibody 342 does not cross-react with cyclin

D3, and that the upper two bands recognized by this anti-body correspond to different forms of cyclin Di , whereas the

lower band corresponds to cyclin D2. Because the poly-

clonal anti-cyclin Di antibody is more efficient in precipitat-

ing and blotting both cyclin Di and D2 than their respectivemonoclonal antibodies, it was used in subsequent analysis.

In proliferating 313-Li cells, both cyclin Di and D3 were

detected, whereas cyclin D2 was barely detectable (Fig. 6).

Upon the induction of adipocyte differentiation, cyclin Di

protein levels were induced and sustained at high levels as

the cells progressed through and completed the mitotic

clonal expansion phase (day 1 to day 2). As these cells

entered permanent cell cycle arrest (day 2 to day 4), the level

of cyclin Di declined and became nearly undetectable by

day 6 when all cells in the population have terminally differ-

entiated (Fig. 6B). In contrast, both cyclin D2 and cyclin D3

underwent a continuous increase throughout the course ofdifferentiation and remained high in differentiated cells (Fig.

6, B and D). A closer examination, however, revealed a

significant difference in the kinetics of cyclin D2 and D3

induction. The level of cyclin D3 protein was low in prolifer-

ating 313-Li cells, increased i 0-fold in density-arrested

preadipocytes, and further increased 5-fold during differen-

tiation induction to reach maximal levels on day 4 (50-fold

induction between proliferating and day 4). By day 7, the

level of cyclin D3 declined 6-fold to reach levels equivalent to

that present on day 0. On the other hand, cyclin D2 was

barely detectable in both proliferating and density-arrested

313-Li cells and became elevated and continuously accu-

mulated when 313-Li cells were induced for differentiation.

In parallel cultured 313-Li cells that were not induced for

differentiation and thus remained in a temporary arrested

A

B

C

D

604 CDK4 Regulation during Adipogenesis

state (Fig. 6, B and D, Uninduced), neither a decrease in the

level of cyclin Di nor the induction of cyclin D2 and D3 was

observed, providing further correlative evidence for the dif-

ferent regulation of the three D cyclins during adipocyte

differentiation.

As 313-Li cells are induced to differentiate, the associa-

tion of cyclin Di with CDK4 increased during the mitotic

clonal expansion phase and then decreased to negligible

levels between days 2 and 3, a time at which these cells are

completing mitotic division and permanently withdrawing

from the cell cycle (Fig. 6A). Despite being able to clearly

detect the expression and association of cyclin Di with

CDK4 on day 1 of differentiation by IP-Western blot, the

association of newly synthesized cyclin Di with CDK4 is

barely detectable at this time (compare day 1 of Fig. 2A,

upper cyclin D band and Fig. 6, A and B). The absence of a

cyclin Di band in [35S]methionine-labeled CDK4 immuno-precipitates on day i of differentiation most likely reflects a

difference between the level of protein synthesis (Fig. 2)

versus the steady-state level (Fig. 6). Therefore, the absence

of a cyclin Di band in day 1 [35S]methionine-labeled CDK4

immunoprecipitates suggests the possibility that the rate of

cyclin Di synthesis is significantly lower at this time. Cyclin

D2 transiently associates with CDK4, being first detected inthe CDK4 complex on day 2 and then declining to negligible

levels after day 4 (Fig. 6A). However, newly synthesized

cyclin D2 is present in CDK4 complexes in proliferating313-Li cells and throughout the differentiation program (Fig.

2A). These data, coupled with the low levels of cyclin D2 in

proliferating 313-Li cells and during the early stages of dif-

ferentiation, suggest that cyclin D2-CDK4 complexes are

forming prior to day 2 but that they are highly unstable and

rapidly disassembled (compare Fig. 2A and Fig. 6, A and B).

The association of cyclin D3 with CDK4 is low in proliferating

313-Li cells, dramatically increases 12-fold between day 0

and day 4 of the differentiation program, and then declines

6-fold by day 7 to levels equivalent to that seen at day 1 (Fig.

6C). The described changes in the associations between the

cyclin D proteins and CDK4 were specific for 313-Li adipo-

cyte differentiation and were not evident in uninduced

313-Li cells (Fig. 6, C and D, compare Induced with Unin-

duced). The decreased association of cyclin Dl with CDK4

after 2 days of differentiation coincided with a sharp increase

of both the level and association of cyclin D3 with CDK4,

suggesting an intriguing possibility that during 313-Li cell

differentiation cyclin D3 may replace cyclin Di as the primary

partner of CDK4.

It is interesting to note that the decline in the level of

CDK4-associated cyclin Di preceded the decline in the over-

all steady-state levels of cyclin Di . Although there is stillabundant cyclin Di after 4 days of differentiation, the level of

CDK4-associated cyclin Dl has significantly decreased by

day 3 of differentiation (Fig. 6, A and B). Furthermore, the

association between CDK4 and cyclin D2 decreases be-

tween day 4 and day 7 of differentiation, although the overall

levels of cyclin D2 remain constant and significant cyclin D2

levels are present on day 1 , yet it is not found associated with

CDK4 until day 2 (Fig. 6, A and B). These dynamic patterns

of cyclin D expression and association with CDK4 suggests

IP: a-CDK4 a-p18peptlde: - + - +

CDK4

IP: a-CDK4 ci-p18peptlde: - + -

p18

lP:a-CDK4 a-p27peptlde: - + - ‘tr

p27 � � �. �, �it

IP: a-CDK4 a-cyc D3peptide: - 1: - +

Cyc D3 � �j .Fig. 7. Expression and association of CDK4, cyclin 03, p1 8, and p27 inadult mouse adipose tissue. Total cell lysate was prepared from adultmouse adipose tissue and immunoprecipitated with antibodies to CDK4(A-D), p18 (A and B), p27 (C), or cyclin 03 (0) in the presence (+) orabsence (-) of the corresponding antigen peptide. The immunoprecipi-tates were resolved by SOS-PAGE and immunoblotted with antibodies toCDK4, p18, p27, and cyclin 03 as indicated.

that the assembly and disassembly of cyclin D-CDK4 com-

plexes is an actively regulated process rather than a passiveprocess dependent solely on the overall levels of each pro-tein within the cell and that the three D cyclins, in spite of

their high degree of sequence similarity, may have distinct

biological roles during 313-Li adipocyte differentiation.CDK4 Complexes in Adult Mouse Adipose Tissue. To

confirm the results obtained from cultured 313-Li cells in-

duced to differentiate in vitro, we analyzed the in vivo ox-pression and association of CDK4, cyclin D3, p1 8, and p27

in adult mouse adipose tissue. Similar to terminally differen-

tiated 313-Li cells, CDK4, cyclin D3, p1 8, and p27 were

expressed in adult mouse adipose cells (Fig. 7), whereas

there was no detectable levels of p21 (data not shown).

Reciprocal IP-Western analysis demonstrate that there ex-

ists abundant CDK4-pi8 (Fig. 7, A and B), CDK4-p27 (Fig.7C), and CDK4-cyclin D3 (Fig. 7D) complexes in adipose

tissue similar to mature 313-Li adipocytes. Although wehave not determined quantitatively the molar ratio between

A B

Osy 0 1 2 3 4 7

I.Dsy 00.51 2 4 6 7

D NoNo

pspAb SubDsy 0 1 2 3 4 7 2 3 3

CDC2 Hi-�” #{149}mi �

CHour 0 4 8 1012162024

CDK4 Rb -�. . �

cyc D3 Rb -�

EODK4 and c�In 03 KIn... Acevity

I

Cell Growth & Differentiation t

Fig. 8. CDC2, CDK2, CDK4, and cyclin03 kinase activity during adipocyte differ-entiation. Lysates prepared from proliferat-ing 3T3-L1 adipoblasts (P), temporarilydensity-arrested 3T3-L1 preadipocytes (0),or at different time points [in hours (A andC) or days (B and D)] after the induction ofdifferentiation were immunoprecipitatedwith antibodies to CDC2 (A and B), CDK2(A and B), CDK4 (C and D), or cyclin 03 (Cand D). The immunoprecipitates were as-sayed for histone Hi kinase activity (A andB) or pRb kinase activity(C and D) and thensubjected to SOS-PAGE. To show speci-ficity of the IP-kinase assay, the antiserawas preincubated with its correspondingantigen peptide (pep), no substrate wasadded to the kinase reaction (no sub), or noantibody was added to the lysate (no Ab);E, CDK4 and cyclin D3 pRb kinase activityfrom D was quantitated by densitometerscanning. The kinase activity on day 0,temporarily density-arrested 3T3-Li prea-dipocytes, was set at one.

psp

Hour P P 0 4 8 121824

CDK2 Hi -� 0

CDK4 and each of its regulatory protein, use of excess

amount of each antibody in immunoprecipitations allowed us

to estimate that approximately one-third of pi8 and p27 is

complexed with CDK4, suggesting that both p1 8 and p27,

two major CDK4-associated CDK inhibitors in mature 313-Li

adipocytes and adipose tissues, are in excess of CDK4.CDK and Cyclin Kinase Activity during 3T3-L1 Differ-

entiation. The high level of cyclin D3 expression and abun-

dant CDK4-cyclin D3 complexes in both mature 313-Li adi-

pocytes and adipose tissue prompted us to determinewhether the CDK4-cyclin D3 complex contains kinase activ-

ity toward its known substrate, the retinoblastoma protein

(pRb), in these permanently arrested cells. The pRb activities

of CDK4 and cyclin D3 and the histone Hi activities of CDC2and CDK2 at different time points during the differentiation

program of these cells were examined by in vitro kinase

assay of immunoprecipitated individual protein complexes(Fig. 8). In proliferating (data not shown) and density-arrested

(Fig. 8, Day 0) 313-Li cells, there was no detectable CDK4 or

cyclin D3 pRb kinase activity, despite clear detection of

cyclin D-CDK4 complexes (Fig. 6). The mechanisms under-

lying CDK4 inhibition in these cells is not clear at present.

Density-arrested 313-Li cells also exhibit no detectableCDC2 or CDK2 histone Hi kinase activity. Because 313-Licells progressed through the mitotic clonal expansion phase

(Fig. 8, A and C), the pRb kinase activity of CDK4 and cyclin

D3 was detectable by 8 h, followed by CDK2 and CDC2

histone Hi kinase activity by i 2 and 24 h, respectively. These

kinetics are consistent with the established kinase activity

profile of these enzymes observed in other cell types after

mitogenic stimulation (6, 9, 10, 35, 36) as well as the cell

cycle profile of 313-Li cells determined by [3H]thymidine

incorporation (Refs. 21 and 32; Fig. iD). Consistent with a

very low level of CDK6 expression and lack of detectable

cyclin D-CDK6 complexes in 313-Li cells, no appreciable

amount of Rb kinase activity was detectable in CDK6 immu-

noprecipitates (data not shown). Concomitant with the corn-

pletion of mitotic clonal expansion (between days 2 and 3),

there is a dramatic down-regulation of CDK2 and CDC2

histone Hi kinase activity to negligible levels which then

remained undetectable in terminally differentiated 313-Li

adipocytes (Fig. 8B, Day 4 to Day 7). In addition, the pRb

kinase activity of CDK4 immunoprecipitates, which was in-

duced 1 2-fold between days 0 and 2, was down-regulated

5-fold between days 2 and 3 of the differentiation induction

program (Fig. 8, D and E). The inhibition of CDK4 kinase

activity after the mitotic clonal expansion phase correlateswith its increased association with p21 and p18 and is con-

sistent with the proposed role of these two CDK inhibitors in

initiating and maintaining permanent cell cycle arrest during

313-Li adipocyte differentiation. In the meantime, the pRb

kinase activity of cyclin D3 also underwent a decline (2-fold)

after reaching its peak 2 days after differentiation induction

(Fig. 8, D and E). Unexpectedly, however, there was sus-

tamed pRb kinase activity in both the anti-CDK4 and anti-

cyclin D3 immunocomplexes derived from 313-Li cells that

TermInal DIfferentIatIon of Adlpocyt..

CDK4 Regulation during Adipogenesis

Fig. 9. Subunit rearrangementof CDK4 complexes during3T3-Ll adipogenesis. p27 and itsassociation with CDK4 was notincluded because its expressionand association with CDK4 re-main constant during 3T3-Ll ad-ipogenesis.

have completed their mitotic expansion (day 3) and in termi-nally differentiated adipocytes (days 4 and 7) compared withdensity-arrested preadipocytes (Fig. 8, D and E, Day 0).

These results suggest that cyclin D3 and CDK4 have a func-tion as an active enzyme not related to cell cycle progression

in permanently growth-arrested adipocytes.

Discussion

Terminal differentiation of many cell types involves changesof cell morphology and gene expression to become func-tional specialized cells. Accompanying these changes is a

withdrawal from mitotic division and entry into a state ofpermanent cell cycle arrest. In contrast to the extensivestudies on the identification and characterization of tran-scription factors that are involved in lineage-specific cell

determination and terminal differentiation, the molecularmechanisms that couple terminal differentiation and cell cy-

cle withdrawal are still poorly understood. Using the well-characterized mouse 313-Li preadipocyte cell line as amodel system, we have conducted a comprehensive analy-sis on the regulation of CDK4, a major G1 cell cycle regulator,

the function of which has been suggested to couple extra-cellular signals to the cell cycle progression. The presentstudy revealed a dynamic regulation of CDK4 during terminaladipocyte differentiation that involves active rearrangementof multiple regulatory proteins. We suggest that during ter-minal adipocyte differentiation and in terminally differentiatedadipocytes, CDK4 functions as an active enzyme and thatthe complex regulation imposed on CDK4 is to provide atight regulation, rather than passive inhibition, of CDK4 ac-

tivity. Two central features of this complex regulation of

CDK4 are switching of activating cyclin D subunits and co-operation between the two families of CDK inhibitors (Fig. 9).

Regulation of CDK4 during Terminal Cell Differentia-

tion. Four lines of evidence suggest concertedly that during

adipogenesis of 313-Li cells and in terminally differentiated313-Li cells, which are permanently cell cycle arrested,CDK4 is tightly regulated, rather than simply inhibited (Fig. 9):(a) the synthesis and steady-state protein level of CDK4remained constant throughout the process of 313-Li adipo-

genesis, such that the levels of CDK4 in terminally differen-

tiated adipocytes (day 7) was similar to that in proliferating

adipoblasts (Figs. 2 and 3); (b) two members of the D-typecyclins, cyclins D2 and D3, are induced during 313-Li dif-ferentiation (Fig. 6). This induction conversely correlates witha decline in the levels of cyclin Di and its association withCDK4 at a time when 313-Li cells are completing theirmitotic clonal expansion and beginning to enter a state ofpermanent cell cycle arrest (Fig. 6). One explanation forswitching of the cyclin regulatory subunit would be thatcyclins D2 and D3 target CDK4 to different sets of substratesin differentiated cells from that targeted by cyclin Di in

proliferating precursor cells; (c) there is an active regulationof several members of both CDK inhibitor families exerting aninfluence on CDK4. p21 transiently associates with CDK4 as313-Li cells exit from mitotic clonal expansion and begin toinitiate a state of permanent cell cycle arrest, whereas p27 is

complexed with a significant fraction of CDK4 throughout thedifferentiation process (Fig. 3). Of three INK4 inhibitors ex-pressed in 313-Li cells, the decrease of both pi 5 and p16proteins levels after the reinitiation of cell cycle progressionin density-arrested preadipocytes is accompanied by a dra-

matic increase of p1 8 at a time when 313-Li cells are corn-pleting mitotic expansion. In terminally differentiated 313-Licells, a significant portion of CDK4 is complexed with p18(Figs. 5 and 7); and (d) an appreciative amount of pRb kinaseactivity of CDK4 was present in differentiated 313-Li cells,6-fold higher than that detected in temporary density-ar-rested 313-Li cells (Fig. 8). Reciprocally, the pRb kinaseactivity of cyclin D3 is also higher (iO-fold) in terminallydifferentiated cells compared with density-arrested 313-Lipreadipocytes (Fig. 8). These observations suggest that dur-ing adipogenesis, CDK4 functions as an active enzyme, theactivity of which is tightly regulated by both activating Dcyclins and CDK inhibitors. We have previously observed asimilar regulation of CDK4 during terminal muscle differenti-ation (29), including high levels of CDK4 and induction ofcyclin D3, suggesting a broad role of CDK4 in regulating aswell as maintaining terminal cell differentiation.

Regulation of p18 Induction. The induction of p1 8 during313-Li cell differentiation is likely to be a regulatory eventrelated to adipogenesis: (a) the level of p1 8 protein induction

Cell Growth & Differentiation 607

is remarkable, from an undetectable level in proliferating cellsto a considerable amount in density-arrested cells and anadditional 12-fold increase as 313-Li cells exit from mitoticexpansion (Fig. 5B). Equally significant is the maintenance ofhigh p1 8 levels in terminally differentiated 313-Li adipocytes(Fig. 5, Day 7) and in adult mouse adipose tissue (Fig. 7).Accompanying this induction is a significant increase of theassociation of pi 8 with CDK4 and CDK6 (Fig. 5, A and B); (b)

there is a similar increase in the level of p1 8 mRNA during313-Li cell differentiation. This induction was not seen in theclosely related 3T3-C2 cell line, which similarly undergoescontact inhibition, mitotic clonal expansion, and cell cyclewithdrawal but not adipocyte conversion, after adipogenicinduction. Notably, this is in contrast with the pRb-relatedp1 30:pi 07 switch in E2F complexes that occurs with essen-tially the same pattern in both hormonally stimulated 313-Liand 3T3-C2 cells (32). Hence, the mechanism(s) regulatingpi 8 induction correlate with adipocyte differentiation,whereas the pi 30:pi 07 switch in E2F complexes are relatedto cell cycle progression; and (c) p18 mRNA normally accu-mulates during S phase of the cell cycle in proliferating cells(23), thus the accumulation of p1 8 mRNA as 313-Li cells exitfrom the cell cycle argue against the possibility that theinduction of p1 8 is the consequence of an enrichment of a G1cell population.

Like most other CDK inhibitor genes, the mechanism thatregulates p1 8 induction is presently unknown. In two sepa-rate cell lineages, muscle (29, 30) and adipocyte (this study),we observed a sharp induction of pi 8 at a time when differ-entiating cells are entering a state of permanent cell cyclearrest and sustained expression in terminally differentiatedcells. pi 8 mRNA and protein also accumulated to a high levelin many other celltypes (29, 37, 38), suggesting that pi 8 maybe induced broadly during the G1 exit associated with theterminal differentiation of other cell lineages. We showedrecently that the p18 gene expresses two similarly sizedmRNA transcripts during C2C1 2 myogenesis, as in 313-Licells (30). In proliferating C2C12 myoblasts, only the larger2.4-kb p1 8 transcript is expressed from an upstream pro-moter. As C2Ci 2 cells are induced to differentiate into per-

manently arrested myotubes, the abundance of the 2.4-kbtranscript decreases, and a smaller 1 .2-kb pi 8 transcriptexpressed from a downstream promoter is induced to be-come the predominant transcript expressed in terminallydifferentiated myotubes. These two transcripts contain the

same coding exons, but the large transcript uniquely con-tains an additional noncoding i .2-kb exon, exon 1 , corre-sponding exclusively to the 5’ UTR. The presence of the long5’ UTR attenuated the translation of large 2.4-kb pi8 tran-script, whereas its absence from the shorter p1 8 transcriptresulted in significantly more efficient translation of the p18protein. These results demonstrate that during terminal mus-cle cell differentiation, the induction of p18 protein is regu-lated by promoter switching coupled with translational con-trol. The marked induction of the 1 .2-kb p1 8 transcript during313-Li cell differentiation correlates with a sharp increase ofp1 8-CDK4 complexes at a time when these cells are exitingfrom mitotic clonal expansion phase (Fig. 5B, Day 2 to Day 3),

suggesting that a similar translational control is also used

during adipogenesis. There is, however, one distinct differ-ence in p18 mRNA expression between these two cell types.Concurrent with the increase of the short 1 .2-kb p1 8 tran-script, the large 2.4-kb p18 transcript continuously declinedto nearly undetectable low levels in permanently arrestedmature myotube (30), whereas both transcripts increase withsimilar kinetics during 313-Li adipogenesis and remain highin differentiated adipocytes (Fig. 5C). These observationssuggest that the two pi 8 promoters may be regulated an-tagonistically during myogenesis but analogously during313-Li adipogenesis.

Cooperation between CDK Inhibitors during TerminalCell Differentiation. Two families of CDK inhibitors, com-prising seven genes, have been identified in mammaliancells. Although the biochemical mechanism by which thesetwo families inhibit CDK activity is different, the ectopic ex-pression of a single CDK inhibitor from either family is gen-erally sufficient to arrest the cell cycle. These observationshave led to the thinking that individual CDK inhibitors mayplay a critical role in causing and/or maintaining cell cyclearrest during cell differentiation and morphogenesis. Of fourCDK inhibitor genes, p16, 21, p27, and p57, that have beendisrupted, only mice lacking p57K�P2 display serious devel-opmental defects (39, 40). In spite of its high expression levelin both muscle and adipocyte cells, both tissues develop

normally in mice lacking p27. We suggest two explanationsfor the lack of significant developmental defects associatedwith the inactivation of a single CDK inhibitor gene: (a) ac-cumulation of CDK inhibitor genes associated with cell cyclewithdrawal during terminal cell differentiation is to maintain,rather than cause, the permanent cell cycle arrest. Cell cyclearrest brought about during terminal cell differentiation isdominant over increased cell proliferation caused by the lossof the growth suppressive activity of the CDK inhibitor. Onlywhen cells are challenged by mitogenic stimuli can the effectof loss of a CDK inhibitor function become evident; and (b)

more than one CDK inhibitor may be involved in maintainingcell cycle arrest in an individual cell type, and loss of functionof a single CDK inhibitor gene may be compensated by otherCDK inhibitor genes. Our studies on terminal adipogenesis(this study) and myogenesis (29) provide observations thatare consistent with the notion of functional cooperation be-tween CDK inhibitors.

There are at least the three CDK inhibitors, p18, p2i andp27, expressed during the differentiation of 313-Li adipo-blasts. Although p21 is transiently induced, both p1 8 andp27 remain high in terminally differentiated mature 313-Liadipocytes and complex with most, if not all, CDK4 andCDK6. A similar pattern of CDK inhibitor accumulation wasobserved during C2C1 2 myogenesis and in adult muscletissues (29). In terminally differentiated adipocytes (Fig. 7)and myotubes (29), both p1 8 and p27 appear to be in excessof CDK4 and CDK6, suggesting the possibility that pi 8 andp27 may collaboratively inhibit CDK4 in these cells. Coop-eration between two CDK inhibitors has been observed pre-viously in TGF-f3-caused G1 arrest (4i). Induction of p15 byTGF-p releases p27 from CDK4/6 complexes, which thentransfers to and inhibits the activity of CDK2. Although313-Li (and C2C1 2) cell differentiation represents a substan-

CDK4 Regulation during Adipogenesis

tially different G1 arrest from that caused by TGF-(3, onebeing permanent and one temporary, a striking similar co-operation involving two key components was seen in bothevents: a precipitous induction of an lNK4 inhibitor (p15 byTGF-�3 and pi 8 by terminal cell differentiation) and sustainedhigh levels of p27. We propose that such cooperation involv-ing both CDK inhibitor families may act as a compensatorymechanism to secure a stable cell cycle arrest and provides

a plausible molecular explanation for the lack of gross de-velopmental defects in mice lacking a single CDK inhibitor. Inthis regard, the unique feature of CDK4/6 being regulated byboth inhibitor families, as opposed to other CDKs that onlyinteract with the p21 family, would make them particularlybefitting for such compensation.

Materials and MethodsCell Culture and Differentiation InductIon. The mouse 3T3-L1 (CL-i 73;American Type Culture Collection, Rockville, MD) and 3T3-C2 (kindlyprovided by Dr. Howard Green, Harvard Medical Schcol, Boston, MA) celllines were maintained in growth medium [DMEM with 4500 mg/L glucose

(DMEM-H), 10% bovine calf serum (Hyclone)]. The induction of differen-tiation was performed as described previously (20). Cells were grown to

confluence and then refed with growth medium. Forty-eight h after con-fluence (referred to as day 0 of the differentiation program), the mediumwas replaced with differentiation medium [DMEM-H, 10% FBS (Hyclone),10 pg/mI insulin (Life Technologies, Inc.), 0.5 m� 3-isobutyl-i-methyl-xanthine (Aldrich), and 1 �u,i dexamethasone (Aldrich)]. Forty-eight h later

(day 2), the differentiation medium was withdrawn and replaced withDMEM-H containing 10% FBS and 10 �g/ml insulin. On days 4 and 6 ofthe differentiation program, the cells were fed with DMEM-H containingonly 10% FBS. The appearance of cytoplasmic triglyceride droplets was

monitored by bright-field microscopy and confirmed by staining withOiI-Red-O (19, 42). The expression of C/EBPa, a well characterized

marker of adipocyte differentiation (20), was examined by Western blotanalysis (Santa Cruz; sc-61). In parallel, 3T3-L1 cells were grown toconfluence and maintained in growth medium. These cells did not accu-mulate cytoplasmic lipid droplets and did not express C/EBPa and, thus,served as undifferentiated 3T3-Li control cells.

AntibodIes and ImmunochemIstry Procedures. Within the mamma-ian cyclin, CDK, and CDK inhibitor gene families, the encoded proteins

are closely related in sequence and have similar molecular weights. Mom-bers from each family often interact with several proteins from otherfamilies in a combinatorial manner (e.g., CDK4 can interact with all threeD-type cyclins and seven CDK inhibitors). Therefore, a major effort wasmade to generate and characterize gene-specific antibodies to accuratelydetermine the expression level of a specific protein and its interaction withother proteins. All antibodies, with the exception of anti-cyclin Di clone

342, which was raised against the NH2-terminal two-thirds of humancyclin Di (12), and anti-p16, which was raised against a GST-p16 fusionprotein (this study), were generated using a synthetic peptide derived fromeach protein. Cross-reactivity with other family members was determinedby immunoprecipitation of [�S]methionine-Iabeled in vitro translated pro-

teins (see Fig. 5 in Ref. 29) or by IP-Westem blot analysis (this study, Fig.6E). In particular, the p18 antibody used extensively in this study wasraised against a COOH-terminal human p18 peptide (LMQANGAGGAT-NLQ) that is very similar to the corresponding region of mouse p18 (1 1 of14 identical) but does not share any detectable similarity with the corre-spending COOH-terminal region of other INK4 proteins. This anti-p18antibody cross-reacts with mouse p1 8 efficiently but does not cross-reactwith any other INK4 protein (29). In addition, the anti-cyclin Di antibody(clone 342) cross-reacts efficiently with both mouse cyclin Di as well ascyclin 02 but not cyclin D3 (Fig. 6E). Importantly, all antibodies used in thisstudy are capable of coimmunoprecipitating known associated proteinsas determined by coupled immunoprecipitation and immunoblothng. An-tibodies used in this study that were generated using a human peptide as

an antigen cross-react efficiently with the mouse counterpart protein.Anti-peptide antibodies were generated by covalently coupling a synthetic

antigen peptide to activated keyhole limpet hemocyanin (Pierce, Rock-

ford, IL), which was then used to immunize rabbits. The sequence for eachsynthetic peptide was derived from the COOH-terrninal part of eachprotein and as follows: human CDC2 (cNQIKKM, the underiined regioncorrespondstothe COOH-terminal region of human CDC2, and a cysteineresidue was added to the NH2 terminus for covalent coupling of thepeptide to keyhole limpet hemocyanin; Ref. 35); human CDK2 (cQDVTK-PVPHLRL; Ref. 43); human CDK6 (cSQNTSELNTA; Ref. 29); humanpi8�’� (cLMQANGAGGATNLQ; Ref. 29); human cyclin Di (cTPTDVRD-y�t; Ref. 33); human cyclin D3 (CSQTSTPTDVTAIHL this study); mouseCDK4 (cNLMDILQGHMMIPM; Ref. 29); mouse pi5W�� (cHRDIARYL-HAATGD; Ref. 29); mouse p27 (cvEQTPKKPGLRRQT; this study; andmouse p2i (cSNPGDVRPVPHRSK, corresponding to the NH2 terminus ofp21 ; this study). An anti-human p21 antibody generated against a syn-thetic COOH terminus peptide (amino acids 146-164; Santa Cruz #sc-397) reacted with denatured mouse p21 protein with higher sensitivity

than the anti-mouse p2i antibody and was therefore used for immuno-blothng, whereas the anti-mouse p21 antibody was used for immunopre-cipitation. The mouse monoclonal cyclin Di antibody(Santa Cruz 72-13G)is specific for cyclin Di , does not cross-react with cyclin D2 or D3, and isreactive with cyclin Di of mouse origin. The anti-cyclin Di antibody

(anti-cyclin Di CT) generated against a COOH-terminal peptide of humancyclin Dl was kindly provided by Fred Hall (33, 34). The cyclin D2-speciflcmonoclonalantibody, DCS3, was kindly provided byJiri Bartek (Universityof Southern California, Los Angeles, CA; 44). Because the polyclonalanti-cyclin Dl antibody (clone 342) was more efficient in precipitating andimmunoblotting both cyclin Di and cyclin 02 than their respective mono-

clonal antibodies, it was used in subsequent analysis.Procedures for [�5S]methionine metabolic labeling, immunoprecipita-

tion, and immunoblothng have been described previously (45). Protein

Iysate was prepared from 3T3-Li cells or aduit mouse adipose tissue inNP-40 lysis buffer [50 mM Tris-HCI (pH 7.5), 150 mp,i NaCI, 0.5% NP-40,50 mM NaF, i mM DTT, I mp�i Na3VO4, 1 m�,i phenylmethylsulfonyl fluoride,25 pg/mI leupeptin, 25 �g/ml aprotinin, 1 mu benzamidine, and 10 pg/mItrypsin inhibitor]. The protein concentration of cell lysates derived from

different time points during adipogenic induction or from aduit mouseadipose tissue was determined by Bradford Assay (Bio-Rad, Hercules,CA), and equal amounts of protein from each time point were used forimmunoprecipitation and immunoblotting.

Kinase Assays. Protein lysate was prepared in ice-cold NP-40 lysisbuffer from different time points of 3T3-L1 adipocyte differentiation andprecipitated with a specific antibody for 2 h at 4#{176}Cwith rotation. Five �gof affinity-purified anti-mouse CDK4 or anti-cyclin D3 antibodies wereused to immunoprecipitate 2 mg of 3T3-L1 cell lysate. The anti-CDC2 andanti-CDK2 antibody are capable of supporting kinase activity withoutrequiring affinity purification, and i �d of crude sera was used to immu-noprecipitate 0.5 mg of 3T3-L1 cell lysate. Protein A-agarose beads wereadded and incubated for 1 h at 4#{176}Cwith rotation to precipitate immuno-globulin. The beads were washed twice with NP-40 wash buffer [50 m�Tris-HCI (pH 7.4), 150 mM NaCI, 0.5% NP4O, 50 m,,i NaF, 1 mr�i Na3VO4,1 mM Dir, and i mM phenylmethylsulfonyl fluoride] and once with kinaseassay buffer [50 mM HEPES (pH 7.0), 10 m� MgCI2, 5 m� MnCI2, 1 m�iOTT, and 5 ,iM AW]. The washed beads were resuspended in 25 �l ofkinase assay buffer containing 5 �Ci [‘y-�P] AlP and 2 �g of glutathioneS-transferase-pRb substrate (a fusion protein of glutathione S-transferaseand the COOH-terminal 137 amino acids of pRb) for CDK4 and cyclin D3kinase assays or 4 �g of histone Hi substrate (Boehringer Mannheim) forCDC2 and CDK2 kinase assays. The reaction was incubated at 30#{176}Cfor30 mm and then terminated by adding 20 �I of 2X loading dye [100 m�i

Tris-HCI (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromphenol blue, and 0.2M DTTJ. Ten ILl (CDC2 and CDK2) or 20 �tl (CDK4 and cyclin D3) of thesample was resolved on a 12.5% SDS-polyacrylamide gel. To verify equalloading, the gel was stained with Coomassie blue to visualize immuno-globulin and substrate proteins before it was dried and exposed to X-rayfilm.

RNA Analysis. Total RNA was prepared by using TRI-REAGENT in

accordance with the manufacturer’s (Molecular Research Center, Inc.,Cincinnati, OH) instructions. Ten �g of each sample were separated on a1% agarose-formaldehyde-morpholinepropanesulfonic acid gel andtransferred to a nylon fliter. Hybridization were carried out at 68#{176}Cfor 2 hin QuikHyb (Stratagene, La Jolla, CA), followed by two washes in 2X

SSC-0.i% SDS and then two washes in 0.2x SSC-O.i% SDS for i5 mmeach at 50#{176}C.The p18 probe was a 0.6-kb cDNA fragment derived from

Cell Growth & Differentiation

the mouse p1 8 coding region. Ethidium bromide staining was used todemonstrate even RNA loading.

AcknowledgmentsWe thank Dr. Howard Green (Harvard Medical School) for providing the3T3-C2 cell line, Drs. Ormond MacDougald and M. Daniel Lane (JohnHopkins University) for advice on the cuituring and differentiation mnduc-tion of the 3T3-L1 cell line, Dr. Jiri Bartek for providing the cyclin 02monoclonal antibody DCS-3, Dr. Fred Hall for providing the anti-cyclin Di(COOH-terminal peptide) antibody, and David Franklin for critical readingof the manuscript.

References1 . Hunter, T., and Pines, J. Cyclmns and cancer II: cyclin D and CDKinhibitors come of age. Cell, 79: 573-582, i994.

2. Sherr, C. Mammalian Gi cyclmns. Cell, 73: i059-1065, 1993.

3. Duronio, R. J., Brook, A., Dyson, N., and O’Farrell, P. H. E2F-inducedS phase requires cyclmn E. Genes Dcv., 10: 2505-25i3, i996.

4. Ohtsubo, M., and Roberts, J. M. Cyclmn-dependent regulation of Gi inmammalian fibroblasts. Science (Washington DC), 259: 1908-1912, 1993.

5. Ohtsubo, M., Theodoras, A. M., Schumacher, J., Roberts, J., andPagano, M. Human cyclin E, a nuclear protein essential for the Gi to Sphase transition. Mol. Cell Biol., 15: 2612-2624, 1995.

6. Pagano, M., Pepperkok, R., Lukas, J., Baldin, V., Ansorge, W., Bartek,J., and Draetta, G. Regulation of the cell cycle by the cdk2 protein kinasein cuitured human fibroblasts. J. Cell Biol., 121: 101-111, 1992.

7. Resnitzky, D., Gossen, M., Bujard, H., and Reed, S. Acceleration of theG1-S phase transition by expression of cyclins Di and E with an induciblesystem. Mol. Cell. Biol., 14: 1669-1679, 1994.

8. Matsushime, H., Roussel, M. F., Ashmum, R. A., and Sherr, C. J.Colony-stimulating factor 1 regulates a novel gene (CYL1) during the Giphase of the cell cycle. Cell, 65: 701-713, 1991.

9. Matsushime, H., Quelle, 0. E., Shurtleff, S. A., Shibuya, M., Sherr, C. J.,and Kato, J. V. D-type cyclmn-dependent kinase activity in mammaliancells. Mol. Cell Biol., 14: 2066-2076, 1994.

10. Meyerson, M., and Harlow, E. Identification of Gi kinase activity forcdk6, a novel cyclmn D partner. Mol. Cell. Biol., 14: 2077-2086, 1994.

11 . Matsushime, H., Ewen, M. E., Strom, D. K., Kato, J. V., Hanks, S. K,Roussel, M. F., and Sherr, C. J. Identification and properties of an atypicalcatalytic subunit (�34PSK�J3/cdk4) for mammalian D-type Gi cyclins. Cell,71: 323-334, 1992.

12. Xiong, V., Zhang, H., and Beach, D. D-type cyclins associate withmuitiple protein kinases and the DNA replication and repair factor PCNA.

Cell, 71: 505-514, 1992.

13. Stepanova, L, Leng, X., Parker, S. B., and Harper, J. W. Mammalianp5O�7 is a protein kinase-targeting subunit of hsp90 that binds and

stabilizes Cdk4. Genes Dcv., 10: 1491-1502, 1996.

14. Dai, K., Kobayashi, R., and Beach, D. Physical interaction of mam-

malian CDC37 with CDK4. J. Biol. Chem., 271: 22030-22034, 1996.

i5. Sherr, C. J., and Roberts, J. M. Inhibitors of mammalian Gi cyclmn-dependent kinases. Genes 0ev., 9: 1 149-i 163, 1995.

16. Spiegelman, B. M., and Flier, J. S. Adipogenesis and obesity: round-ing out the big picture. Cell, 87: 377-389, 1996.

17. Smas, C. M., and Sul, H. S. Control of adipocyte differentiation.Biochem. J., 309: 697-710, 1995.

18. MacDougald, 0. A., and Lane, M. D. Transcriptional regulation ofgene expression during adipocyte differentiation. Annu. Rev. Biochem.,64: 345-373, 1995.

19. Rubin, C., Hirsch, A., Fung, C., and Rosen, 0. Development of hor-mono receptors and hormonal responsiveness in vitro: insulin receptors

and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-Licells. J. Biol. Chem., 253: 7570-7578, i978.

20. Yeh, W., Ceo, Z., Classon, M., and McKnlght, S. L Cascade regula-tion of terminal adipocyte differentiation by three members of the C/EBP

family of leucine zipper proteins. Genes 0ev., 9: i68-i8i, 1995.

21 . Sadowski, H. B., Wheeler, T. T., and Young, 0. A. Gene expressionduring 3T3-Li adipocyte differentiation. J. Cell Biochem., 266: 4722-4731, 1992.

22. Freytag, S. 0. Enforced expression of the c-myc oncogene inhibits

cell differentiation by precluding entry into a distinct predifferentiationstate in G�/G1. Mol. Cell. Biol., 8: 16i4-i624, i988.

23. Hirai, H., Roussel, M. F., Kato, J., Ashmun, R. A., and Sherr, C. J.Novel 1NK4 proteins, p19 and p18, are specific inhibitors of cyclin 0-dependent kinases CDK4 and CDK6. Mol. Cell. BioI., 15: 2672-2681,

1995.

24. Reynisdottir, I., and Massagu#{233},J. The subcellular locations ofp1 5Ink4b and p27KiPl coordinate their inhibitory interactions with cdk4 andcdk2. Genes Dcv., 1 1: 492-503, 1997.

25. Polyak, K., Kato, J., Solomon, M. J., Sherr, C. J., Massague, J.,Roberts, J. M., and Koff, A. p27t�Pl, a cyclin-Cdk inhibitor, links trans-

forming growth factor-p and contact inhibition to cell cycle arrest. Genes0ev., 8: 9-22, 1994.

26. Agrawal, D., Hauser, P., McPherson, F., Dong, F., Garcia, A., andPledger, W. J. Repression of p27Kipi synthesis by platelet-derivedgrowth factor in BALB/c 3T3 cells. Mol. Cell. Biol., 16: 4327-4336, 1996.

27. Hengst, L, and Reed, S. I. Translational control of p27’�Pl accumu-lation during the cell cycle. Science (Washington DC), 271: 1861-1864,

1996.

28. Guan, K. L, Jenkins, C. W., Li, Y., O’Keefe, C. L, Noh, S., Wu, X.,Zariwala, M., Matera, A. G., and Xiong, Y. Isolation and characterization ofp1g1NI�d, a p16-related inhibitor specific to CDK6 and CDK4. Mol. Cell.Biol., 7: 57-70, 1996.

29. Franklin, 0. 5., and Xiong, Y. Induction of p18IN� and its predomi-nant association with CDK4 and CDK6 during myogenic differentiation.Mol. Biol. Cell, 7: 1587-1599, 1996.

30. Phelps, 0., Hsiao, K-M., Li, Y., Hu, N., Franklin, 0. 5., Westphal, E.,Lee, E. Y-H. P., and Xiong, Y. Coupled transcriptional and translationalcontrol of the CDK inhibitor p1 81N�’5 expression during myogenesis.Mol. Cell. Biol., 18: 2334-2343, 1998.

31 . Green, H., and Kehinde, 0. Spontaneous heritable changes leading toincreased adipose conversion in 3T3 cells. Cell, 7: 105-i 13, 1976.

32. Richon, V. M., Lyle, R. E., and McGehee, R. E. Regulation and ex-pression of retinoblastoma proteins p107 and p130 during 3T3-Li adi-

pocyte differentiation. J. Biol. Chem., 272: 101 17-10124, 1997.

33. HaIl, F. L, Williams, R. T., Wu, L, Wu, F., Carbonaro Hall, D. A.,Harper, J. W., and Warburton, D. Two potentially oncogenic cyclins, cyclinA and cyclmn Dl , share common properties of subunit configuration,tyrosine phosphorylation and physical association with the Rb protein.Oncogene, 8: 1377-1384, 1993.

34. Williams, A. T., Wu, L, Carbonaro Hall, 0. A., Tolo, V. T., and Hall, F. LIdentification of a novel cyclmn-like protein in human tumor cells. J. Biol.

Chem., 268: 8871-8880, 1993.

35. Draetta, G., and Beach, 0. Activation of cdc2 protein kinase duringmitosis in human cells: cell cycle-dependent phosphorylation and subunit

rearrangement. Cell, 54: 17-26, 1988.

36. Teal, L H., Lees, E., Faha, B., Harlow, E., and Riabowol, K The CDK2kinase Is required for the Gi -to-S transition in mammalian cells. Once-gene, 8: 1593-i602, 1993.

37. Guan, K-L, Jenkins, C. W., Li, Y., Nichols, M. A., Wu, X., O’Koefe,C. L, Matera, A. G., and Xiong, Y. Growth suppression by p18, ap16�4�Sl� and pi4�4�’�-reIated CDK6 inhibitor, correlates withwild-type pRb function. Genes Dcv., 8: 2939-2952, 1994.

38. Zindy, F., Soares, H., Herzog, K-H., Morgan, J., Sherr, C. J., andRoussel, M. F. Expression of INK4 inhibitor of cyclmn 0-dependent kinasesduring brain development. Cell Growth Differ., 8: 1 i39-1 150, 1997.

39. Zhang, P., Liegeois, N., Wong, C., Finegold, M., Hou, H., Thompson,J. C., Silverman, A., Harper, J. W., DePinho, R. A., and Elledge, S. J.Altered cell differentiation and proliferation in mice lacking p57’#{176}�mdi-cates a role in Beckwith-Wiedemann syndrome. Nature (Lond.), 387:151-158, 1997.

610 CDK4 Regulation during Adipogenesis

40. Yan, Y., Frisen, J., Lee, M-H., Massagu#{233},J., and Barbacid, M. Abla-

tion of the CDK inhibitor p57KiPl resuits in increased apoptosis and

delayed differentiation during mouse development. Genes Dcv., 11: 973-

983, i997.

41 . Reynisdothr, I., Polyak, K., lavarone, A., and Massagu#{233},J. Kip/Cip

and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in responseto TGF-j3. Genes Dcv., 9: 1831-1845, 1995.

42. Preece, A. Manual for Histologic Technicians. Boston, MA: Little,Brown, and Co., 1992.

43. Xiong, Y., Zhang, H., and Beach, 0. Subunit rearrangement of cyclin-dependent kinases is associated with cellular transformation. Genes Dcv.,7: 1572-1583, 1993.

44. Lukas, J., Bartkova, J., Welcker, M., Peterson, 0. W., Peters, G.,Strauss, M., and Bartok, J. Cyclmn 02 is a moderately oscillating nucleo-protein required for Gi phase progression in specific cell types. Onco-gene, 10: 2125-2134, 1995.

45. Jenkins, C. W., and Xiong, Y. Immunoprecipitation and immunoblot-ting in cell cycle studies. In: M. Pagano (ed.), Cell Cycle: Material andMethods, pp. 250-263. New York: Springer-Veriag, 1995.