Lkb1/Stk11 regulation of mTOR signaling controls thetransition of chondrocyte fates and suppresses skeletaltumor formationLick Pui Laia,b, Brendan N. Lilleyc, Joshua R. Sanesc, and Andrew P. McMahona,b,c,d,1

aDepartment of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad–California Institute for Regenerative Medicine Center for RegenerativeMedicine and Stem Cell Research, University of Southern California Keck School of Medicine, Los Angeles, CA 90089; and Departments of bStem Cell andRegenerative Biology, cMolecular and Cellular Biology, and dHarvard Stem Cell Institute, Harvard University, Cambridge, MA 02138

Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved October 22, 2013 (received for review May 25, 2013)

Liver kinase b1 (Lkb1) protein kinase activity regulates cell growthand cell polarity. Here, we show Lkb1 is essential for maintaininga balance between mitotic and postmitotic cell fates in develop-ment of the mammalian skeleton. In this process, Lkb1 activitycontrols the progression of mitotic chondrocytes to a mature, post-mitotic hypertrophic fate. Loss of this Lkb1-dependent switch leadsto a dramatic expansion of immature chondrocytes and formationof enchondroma-like tumors. Pathway analysis points to a mamma-lian target of rapamycin complex 1-dependent mechanism that canbe partially suppressed by rapamycin treatment. These findingshighlight a critical requirement for integration of mammalian tar-get of rapamycin activity into developmental decision-making dur-ing mammalian skeletogenesis.

chondrocyte differentiation | endochondral ossification | cell death |hypoxia

Growth of the endochondral skeleton is dependent on a car-tilaginous growth plate. In the growth plate, mitotic chon-

drocytes transition to a postmitotic, terminal hypertrophicchondrocyte fate (Fig. S1A). Reciprocal signaling betweenprehypertrophic chondrocyte-derived Indian hedgehog (Ihh)and epiphyseal secreted parathyroid hormone-related peptide(Pthrp; also known as Pthlh) controls the spatial positioning ofthe hypertrophic transition and the normal growth propertiesof the skeleton (1–3). The present study demonstrates an un-expected role for liver kinase b1 (Lkb1; also known as Stk11) ingrowth plate regulation.Lkb1 is a multifunctional serine/threonine kinase inhibitor of

mTOR signaling whose activity regulates cell cycle progression,cellular energy homeostasis, and cell polarity (4, 5). Mouseembryos lacking Lkb1 die at midgestation with vascular and neuraltube defects (6), and germ-line inactivating mutations of Lkb1 inthe human population underlie Peutz–Jeghers syndrome, charac-terized by development of benign polyps in the gastrointestinaltract, and an increased risk of various types of epithelial cancers (7,8). Conditional ablation of Lkb1 in pancreatic, vascular, neuraland cardiac tissue links Lkb1 to tissue specific actions in a varietyof organ systems (9). Here, we provide evidence that Lkb1 regu-lation of mammalian target of rapamycin complex 1 (mTORC1)action is a critical step in the transition of mitotic chondrocytes topostmitotic hypertrophic fates suppressing cartilaginous tumor-likegrowths in the postnatal mammalian skeleton.

ResultsRemoval of Lkb1 in Chondrocytes Results in Expansion of ColumnarMitotic Chondrocytes, Delayed Hypertrophic Development, andFormation of Enchondroma-Like Tumors. We established a poten-tial link between Lkb1 activity and mammalian skeletogenesisunexpectedly through conditional removal of Lkb1 activity ina large region of the caudal mouse embryo. Given the pleiotropicactivity of the original Cre-driver line, we intercrossed mice car-rying a Cre-dependent conditional Lkb1 allele (Lkb1c/c) (10) with

a Col2a1-Cre transgenic strain (11); here, skeletal Cre-activity isinitiated in immature, mitotic, and early postmitotic chondrocytes(Fig. S1B). Through these crosses, mice were generated that lackedLkb1 activity specifically within chondrocytes of the endo-chondral skeleton (Col2a1-Cre;Lkb1c/c; hereafter referred toas Lkb1 mutants). In contrast to littermates that retained anactive Lkb1 allele (Col2a1-Cre;Lkb1c/+; hereafter referred toas control littermates), Lkb1 mutants displayed a prominentpostnatal phenotype.Lkb1 mutants were born at the expected Mendelian ratio, and

appeared superficially normal at birth. However, marked growthretardation was evident by weaning, and, as a result of this growthdeficiency, and a lethargic phenotype, mutants were euthanizedby postnatal day (P) 40 to satisfy institutional guidelines on hu-mane animal care. Histological analysis of long bones afterweaning (at P30) revealed a profound disorganization of the Lkb1mutant skeleton (Fig. 1 and Fig. S2A). Alcian blue staining of longbones from normal individuals highlights nonhypertrophic chon-drocytes within the cartilaginous growth plate localized close to theepiphysis (Fig. S2A). Chondrocytes organize into stratified tiers ofmitotic Col2a1+/Sox9+ proliferative chondrocytes in the growthplate before transitioning to Col2a1−/Sox9−/Runx2+ postcolumnar,postmitotic, hypertrophic chondrocytes that undergo cell deathand replacement by bone-forming osteoblasts (Fig. 1A and Fig.S2A). Hypertrophic chondrocytes and osteoblasts of the outercortical and inner trabecular region of the main shaft of the long

Significance

The transition from a mitotic to a postmitotic, hypertrophicchondrocyte is a key regulatory event in the growing verte-brate skeleton. By using genetic approaches, cell culture, andcell transplantation models, we provide compelling evidencethat attenuating the energy-sensing mammalian target ofrapamycin complex 1 (mTORC1) pathway is critical for switch-ing chondrocyte states. A failure of mTORC1 suppression inLkb1 mutants leads to a dramatic disruption of the skeletalgrowth plate and the formation of cartilage tumors comprisingundifferentiated chondrocytes that display differential sensi-tivity to two key cartilage growth regulators, Indian hedgehogand Igf. The study highlights the interconnection betweenenergy sensing pathways, normal growth control, and tumor-igenesis in the skeletal program.

Author contributions: L.P.L. and A.P.M. designed research; L.P.L. performed research; L.P.L.and A.P.M. analyzed data; and L.P.L., B.N.L., J.R.S., and A.P.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE41898).1To whom correspondence should be addressed. E-mail: amcmahon@med.usc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1309001110/-/DCSupplemental.

19450–19455 | PNAS | November 26, 2013 | vol. 110 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1309001110

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 22

, 202

0

bone are mineralized and highlighted by Alizarin red (Fig. S2A). Incontrast to control littermates, Lkb1 mutants displayed prominentAlcian blue staining within normally bone-restricted regions of theendochondral skeleton (Fig. S2A). Detailed histological analysesof the Lkb1 mutant from P10 to P30 revealed a mass of pro-liferating immature Col2a1+/Sox9+ chondrocytes deep within theshaft of the long bone (Fig. 1B). At P10, the growth plate wasmarkedly disorganized: ectopic hypertrophic chondrocytes wereobserved at the core of the growth plate and next to the groove ofRanvier (Fig. S3). By P20, proliferating chondrocytes formedcolumns perpendicular to the normal longitudinal axis of growth.Tumor-like cell nodules were also found close to the primaryspongiosa (Fig. S3). Analysis at P30, showed that these are largelymade up of Sox9+/Osx+ chondrocytes that displayed low levels ofcollagen (X) indicative of immature chondrocytes (Fig. 1B andFig. S3).To investigate the genesis of this phenotype, we focused on the

period preceding the overt change in body size in Lkb1 mutants:

P3 and earlier (Fig. 1 and Fig. S2D). At P3, analysis of Alcianblue and Alizarin red staining revealed that skeletal growth wassimilar between Lkb1 mutants and control littermates, but theaxial (vertebrae) and appendicular (long bone) skeleton wasmarkedly deficient in mineralized matrix (Fig. 1 F–K). In linewith expectations from the genetic model, the osteoblast pro-gram was not primarily affected (Alizarin red and von Kossastains; Fig. S4A). In contrast, femur and vertebral sections (Fig. 1M, N, P, Q, S, and T) revealed a dramatic expansion of thegrowth plate region in Lkb1 mutants reflected by an extendeddomain of Alcian blue-stained immature cartilage (Fig. S4A).Although less marked, this phenotype was evident before birth,at embryonic day (E) 18.5 (Fig. 1 L, O, and R and Fig. S2B).Measurement of specific cartilage domains showed similar pro-portions of round resting zone and postcolumnar chondrocytesbetween control littermates and Lkb1 mutants (Fig. 1U), buta grossly extended domain of immature columnar chondrocyte inmutants (Fig. 1U). At E16.5, no clear phenotype was evident in thefemur, although a delay mineralization was evident in posteriorvertebrae (Fig. S2 B and C). In summary, in the absence of Lkb1,mutant chondrocytes retained an immature identity wherebynormal chondrocytes transition to a terminal hypertrophic fate. Anextended growth state is likely the underlying event in the estab-lishment of tumor masses in the later skeleton.

Lkb1 Is Essential for Switching Between Chondrocyte States. To in-vestigate these regulatory events further, we examined key markersof chondrocyte identity. Col2a1 [collagen (II)]-producing non-hypertrophic chondrocytes were expanded in the E18.5 Lkb1mutant femur (Fig. 2 A and D), whereas the number of Col10a1[collagen (X)]-expressing hypertrophic chondrocytes was markedlyreduced, and Col10a1 protein was not detected (Fig. 2 B, E, I, andL). In addition, late-stage, Mmp13+ hypertrophic chondrocyteswere entirely absent from long bones of mutants at E18.5 (Fig. 2 Cand F; note Mmp13+ osteoblasts were not affected by Lkb1 re-moval). Production of transcriptional regulators linked to chon-drocyte developmental programs displayed a similar temporal andspatial displacement. Mef2c and Runx2, key determinants of hy-pertrophic differentiation, are activated together with Osx and Ihhin prehypertrophic chondrocytes. In Lkb1 mutants, expression ofall of these genes was first observed within chondrocytes at anextended position relative to the periarticular surface indicative ofa marked delay in chondrocyte differentiation (Fig. 2 G, H, J, andK and Fig. S4B).To examine cell proliferation, we visualized cyclin D1, a key

regulator of the G1-to-S phase transition, and the incorporationof exogenously supplied 5-ethynyl-2′-deoxyuridine (EdU) orBrdU, to identify chondrocytes undergoing DNA replication.Both approaches highlight an expanded domain of proliferatingundifferentiated chondrocytes (Fig. 2 M–R). However, the frac-tion of cells undergoing DNA replication within this domain wasnot altered, suggesting that the excessive number of flattenedchondrocytes likely reflects delayed hypertrophic differentiationrather than an increased rate of division (Fig. S4D). Collagen (X)protein was detected by P3; consequently, Lkb1 is not essentialfor the hypertrophic transition, but rather Lkb1 activity controlsthe normal developmental timing of this key cellular transitionwithin the growth plate (Fig. S4C).

The mTOR Pathway Mediates the Effects of Lkb1 in Chondrocytes.The mTOR pathway balances cell growth and proliferation withthe energy level of the cell (12), and is negatively regulated whenconditions are unfavorable (13, 14). To address mTOR signalingin chondrocytes, and to distinguish between mTOR action withinmTORC1 and mTORC2 complexes, we examined phosphory-lation of two key mTORC1 substrates, ribosomal protein S6(rpS6) and eukaryotic initiation factor 4e-binding protein

Fig. 1. Expansion of columnar mitotic chondrocytes results in formation ofenchondroma-like structure. (A and B) P30 femur sections stained with H&E.Immunohistochemistry was performed on adjacent sections with specificantibodies recognizing collagen (II) (A2 and B2) and Sox9 (A2′ and B2′).Areas boxed in red in A and B are magnified in A1, A2, B1, and B2, re-spectively. Areas boxed in green in A2 and B2 are magnified in A2′ and B2′,respectively. (C–K) P3 skeletal preparations stained with Alcian blue andAlizarin red, with higher-magnification views of the femur (F–H) and ver-tebra (I–K). Colored bars indicate the length of mineralized regions withinthe skeletal element. (L–T) Histological sections through the femur andlumbar vertebrae at E18.5 and P3, stained with H&E. (U) Bar graphs dis-playing the length of the zones of round, columnar, and postcolumnarchondrocytes. Error bars indicate the SD of the means (n > 3; *P < 0.01between columnar regions of Lkb1c/c and Col2a1-Cre;Lkb1c/c on E18.5; **P <0.001 between columnar regions of Lkb1c/c and Col2a1-Cre;Lkb1c/c at P3).(Scale bars: A and B, 1 mm; C–E, 0.5 cm; I–K, 1 mm; L–U, 200 μm.)

Lai et al. PNAS | November 26, 2013 | vol. 110 | no. 48 | 19451

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 22

, 202

0

(4e-bp1) and phosphorylation of serine 473 of Akt, a hallmarkof mTORC2 complex activity.In control littermates, phosphorylation of rpS6 and 4e-bp1 was

evident in the proliferating columnar chondrocytes within thelong bones, but their phosphorylation state was markedly re-duced on commitment to the hypertrophic chondrocyte program(Fig. 3 A–C; note that rpS6 displays a later burst of phosphory-lation within mature hypertrophic chondrocytes marked by anasterisk in Fig. 3C). In Lkb1 mutants, mTOR expression was notaltered; however, mTORC1 activity, highlighted by rpS6 and4e-bp1 phosphorylation, extended into regions where hypertro-phic development should normally have initiated (Fig. 3 E–G). Incontrast, phosphorylation of Akt (Ser473) was unaltered in Lkb1mutants, lending support for an mTORC1-specific role in theskeletal phenotype (Fig. 3 D and H).To explore mTORC1 action, the mTORC1 inhibitor rapamycin

was introduced into dams harboring Lkb1 mutant embryos be-tween 16.5 and 18.5 d of development. Interestingly, rapamycin

treatment decreased phosphorylation of mTORC1 substrates (Fig.S5), normalized proliferation and differentiation of chondrocytesin Lkb1 mutants, and restored a cyclinD1−/Osx−/Col10a1+ hyper-trophic chondrocyte zone by E18.5 (Fig. 3 I–T). Collectively,these data indicate that an Lkb1-dependent attenuation ofmTORC1 action is critical for the normal progression ofchondrocytes to a terminal hypertrophic fate.

Loss of Lkb1 Results in Chondrocyte Apoptosis at the Core of theGrowth Plate. By P3, the cell density at the core of the Lkb1mutant growth plate was noticeably lower. To determine whethercells are dying in this region, we performed TUNEL assay andexamined activation of caspase-3 to visualize apoptotic cells.TUNEL and cleaved caspase-3–positive cells localize within thecore of the extended growth plate (Fig. S6). EF5 staining (achemical indicator of hypoxia) and Vegfa expression (a hypoxia-induced target gene) indicate that the region surrounding thearea of cell death was markedly hypoxic, suggesting that low

Fig. 2. Lkb1 is essential for switching between chondrocyte states. (A–F) In situ hybridization of S35-labeled riboprobes specific for collagen (II), collagen (X),and Mmp13 on E18.5 femur sections. (G–L) E18.5 femur sections immunostained with antibodies specific to osterix, Mef2c, and collagen (X). Nuclei are vi-sualized with DAPI. White arrows indicate the length from the periarticular end of the bone to the start of the chondrocyte regions demarcated by eachprotein. The yellow arrows indicate osterix plus chondrocyte domain. (M–R) E18.5 femur sections following in vivo EdU labeling (red double-headed arrow)and immunostaining with cyclin D1, Sox9, and osterix antibodies (green double-headed arrow). (Scale bars: 200 μm.)

19452 | www.pnas.org/cgi/doi/10.1073/pnas.1309001110 Lai et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 22

, 202

0

oxygen levels likely underlie the observed apoptosis (Fig. S6).TUNEL-positive cells were detected in a similar region of the Lkb1mutant growth plate at P10, but, by P22, when the growth plate ishighly disorganized, scattered apoptotic cells predominantly local-ized at the edge of the cartilaginous zone abutting bone-formingareas (Fig. S6).

Loss of Lkb1 Results in Enchondroma-Like Tumors in the PostnatalSkeleton. To investigate the tumorigenic properties of theenchondroma-like mass that forms postnatally in Lkb1 mutantlong bones, we assayed chondrocyte growth in anchorage-independent conditions in vitro, and growth following trans-plantation into immune deficient [NOD scid gamma (NSG)]mice. Whereas control chondrocytes occasionally generated smallcolonies in nonadherent agar cultures, Lkb1mutant chondrocytesconsistently formed prominent colonies under identical con-ditions (Fig. 4 A–C). When Lkb1 mutant chondrocytes weretransplanted to the flank of NSG mice, safranin O-stained carti-lage matrix-secreting cells were recovered at the site of injection 3mo later (six of eight experiments; Fig. 4G, J, andM). In contrast,no cartilage nodules were observed in the only tissue recoveredfrom one of four control chondrocyte transplants (Fig. 4 F, I, andL). Consistent with an mTORC1 action, rapamycin inhibitedgrowth of Lkb1 mutant chondrocytes in nonadherent agar cultureand following in vivo transplantation (Fig. 4 D, E, H, K, and N).To gain additional insights into the mechanisms of enchon-

droma development, we compared the transcriptional profilebetween control and Lkb1 mutant chondrocytes. Although

broadly similar (Fig. S7B and Dataset S1), Gene Ontology (GO)analysis of differentially expressed gene highlighted significantdifferences among genes associated with skeletal system de-velopment (ID number GO:0001501; P = 1.06 × 10−11), regula-tion of cell proliferation (ID number GO:0042127; P = 1.71 ×10−5), and positive regulation of mesenchymal cell proliferation(ID number GO:0002053; P = 3.5 × 10−4). Igf1 and Igf2, whichencode broad regulators of cell growth and proliferation, andGli2, a transcriptional regulator of Hedgehog pathway targets,whose activity is linked to malignant transformation of chon-drosarcomas (15), displayed an elevated transcriptional profile inLkb1 mutant chondrocytes (Fig. S7A).Igf1r was present at the highest levels in the zone of pro-

liferating chondrocytes (Fig. S8). Phosphorylation of Tyr1161 onIgf1r, a site of autophosphorylation, indicated active Igf signalingin these cells (Fig. S8). This conclusion is supported further byanalysis of Igf signaling dependent phosphorylation of Thr308 onAkt (Fig. S8). The extension of this domain in the skeletal ele-ments of Lkb1 mutants is in agreement with a continued Igfsignaling input with the expanded domain of proliferating, im-mature chondrocytes, and may contribute the maintenance ofthe proliferative state (Fig. S8).To examine the potential role of Igf, we examined the effects

of picropodophyllotoxin and PQ401, specific Igf pathway inhib-itors, on anchorage-independent growth of Lkb1 mutant chon-drocytes. Consistent with continued Igf-dependent control, bothcompounds partially inhibited colony formation. In contrast,GDC-0449 and XAV939, inhibitors of Hh and Wnt signaling

Fig. 3. The mTORC1 pathway mediates the effects of Lkb1 in chondrocytes. (A–H) E18.5 femur sections immunostained with mTOR, phosphorylated 4e-bp1,phosphorylated rpS6, and phosphorylated Akt-specific antibodies. (I–T) E18.5 femur sections immunostained with osterix-, cyclin D1-, and collagen (X)-specificantibodies. Nuclei were visualized with DAPI. Red asterisk indicates the phosphorylated rpS6 protein within the hypertrophic chondrocytes. Blue arrowsindicate the distance from the periarticular surface to chondrocyte zones demarcated by each protein. Areas boxed in yellow are shown as high-magnificationimages (Right). (Scale bars: 200 μm.)

Lai et al. PNAS | November 26, 2013 | vol. 110 | no. 48 | 19453

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 22

, 202

0

[Wnt signaling is linked to chondrocyte maturation (16)], re-spectively, had no effect (Fig. 4 D and E) (17–20). Furthermore,although GDC-0449 abolished chondrocyte proliferation incontrol skeletal elements in vivo, GDC-0449 failed to block EdUincorporation into Lkb1 mutant chondrocytes (Fig. S9). Thus,aberrant chondrocyte proliferation in Lkb1mutants is dependenton mTOR and Igf signaling, but independent of Hh and Wntsignaling inputs. Further, loss of Lkb1 appears to abrogate therequirement for an Ihh input, suggesting that deregulation of Igf-mediated proliferative control is likely a key component of theLkb1 skeletal phenotype.

DiscussionThe coordination of chondrocyte proliferation and hypertrophicdifferentiation is crucial to the longitudinal growth, cellular or-ganization, and appropriate mineralization of the developingendochondral skeleton. The evidence presented here indicatesthat Lkb1-dependent inhibition of mTORC1 promotes the tran-sition of mitotic chondrocytes to a mature postmitotic fate. Theconsequence of the loss of Lkb1 action is an uncoupling of thenormal growth and differentiation program within the growthplate of the endochondral skeleton that leads to the establish-ment of enchondroma-like tumors throughout the long bones.

Pthrp signaling is also a critical determinant of the transitionpoint between mitotic and postmitotic chondrocyte programswhereby Ihh governs Pthrp levels coupling chondrocyte pro-liferation (i.e., direct Ihh action) with chondrocyte differentiation(i.e., indirect action through Ihh control of Pthrp) (21). As withloss of Lkb1 activity, enhanced Pthrp signaling leads to a markedextension of the proliferative zone of immature chondrocytes atthe expense of hypertrophic chondrocyte development. We failedto observe any change in the Ihh pathway aside from the appo-sitional activation of Ihh reflecting the marked delay in formationof postmitotic prehypertrophic chondrocytes. Further, the failureof a Hh pathway antagonist to block chondrocyte proliferationspecifically in Lkb1 mutants suggests that loss of Lkb1 removesthe dependence on Ihh signaling for normal proliferative controlof chondrocytes. The absence of a direct readout of Pthrp sig-naling precludes an assessment of Pthrp signaling within the Lkb1mutant model; however, the endochondroma-like end state ob-served in Lkb1 mutant mice is distinct from the skeletal pheno-type observed on constitutive activation of Pthrp signaling inchondrocytes (22).Lkb1 is a multifunctional kinase: by activating different AMP

kinase family members, Lkb1 regulates cellular polarity andcoordinates cell growth and proliferation with the energy state ofthe cell (4, 5). Our data show elevated levels of mTORC1 activityin Lkb1 mutant chondrocytes that suggest a central role for Lkb1in suppression of mTORC1 action in the transition betweenmitotic and postmitotic hypertrophic cell states. Consistent withthis view, high mTORC1 activity, as measured by phosphoryla-tion of 4e-bp1 and rpS6, normally associates with proliferativecolumnar chondrocytes. Further, rapamycin-mediated inhibitionof mTORC1 normalizes the Lkb1 mutant phenotype in vivo, andinhibits expansion of Lkb1 mutant chondrocytes in nonadherentculture, and on transplant into mice. Interestingly, the Igf path-way is a critical regulator of mTOR action (23), and inhibition ofIgf pathway activity inhibits proliferation of Lkb1 mutant chon-drocytes suggesting that the observed phenotype is dependent onan upstream Igf input.The avascular growth plate has limited nutrient and oxygen

supply, which may affect the energy balance of chondrocytesconstraining the zone of active chondrocyte proliferation andpotentially contributing to apoptosis of mature hypertrophiccells. At P3, we observed a core of apoptotic cells within theextended growth plate surrounded by cells exhibiting molec-ular signatures of hypoxia. Likely, extreme hypoxia within thecore underlies the observed apoptotic phenotype (Fig. S6).Despite the change in environment, ultimately proliferatingchondrocytes transition to a hypertrophic cell fate. Thus, Lkb1is not essential for making the hypertrophic switch, but coor-dinates the timing and position of this critical cellular transi-tion within the normal growth plate.In conclusion, the coordination of chondrocyte proliferation

and hypertrophic differentiation is crucial to the longitudinalgrowth, cellular organization, and appropriate mineralization ofthe developing endochondral skeleton. Our work demonstratesthat Lkb1 is critical for the normal function and organization ofthe growth plate, suggesting a link between the integration ofbasic pathways of energy balance and growth control in a keydevelopmental decision-making process, and raising the possi-bility that Lkb1/mTORC1 deregulation may contribute to carti-laginous tumor formation in men.

Materials and MethodsAnimal Breeding and Procedures. To generate the Lkb1 conditional KO (Col2a1-Cre; Lkb1c/c), Col2a1-Cre mice weremated with Lkb1c/c mice to obtain Col2a1-Cre;Lkb1c/+ mice, which were then mated with Lkb1c/c mice. Rapamycin was injectedinto the peritoneum of pregnant mice to block mTORC1 activity from E16.5 toE18.5. GDC-0449 was delivered by gavage 4 d before the mouse was euthanizedon P30. All experiments and procedures were approved by the animal and

Fig. 4. Loss of Lkb1 results in enchondroma in postnatal skeleton. (A and B)Colony assay for anchorage-independent growth of chondrocytes. (Scalebar: 1 mm.) (C) High-magnification view of an Lkb1 mutant colony. (Scalebar: 10 μm.) Bar graphs comparing cell mass through a colorimetric cell de-tection assay (D) and colony formation (E) in Lkb1 mutant and controlchondrocyte cultures. Error bars indicate the SD of the means of three in-dependent experiments. (F–N) Histological analysis of chondrocyte trans-plants recovered from NSG mice. Sections were stained with H&E to viewgeneral histology and with safranin O to highlight cartilage matrix. (Scalebars: 1 mm.) (O) Graphical plot displaying the size of recovered tissue masses(*P < 0.01, mutant vs. control populations; **P < 0.01, indicated treatmentvs. mutant population).

19454 | www.pnas.org/cgi/doi/10.1073/pnas.1309001110 Lai et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 22

, 202

0

care and use committees of Harvard University and the University ofSouthern California.

Skeletal Staining, Histology, in Situ Hybridization, and Immunostaining. Skel-etons were stained with Alizarin red and Alcian blue as described previously(24). H&E staining and safranin O staining were performed according tostandard protocols. For in situ hybridization procedures, and hybridiza-tion with 35S-labeled probes was carried out as described previously (24).Immunostaining was performed according to standard protocols, andprimary antibodies used in this study are listed in Table S1. For visuali-zation, an HRP-conjugated secondary antibody, ABC Kit, and DAB sub-strate were used. For fluorescent visualization, secondary antibodiesconjugated with Alexa Fluor were used.

BrdU and EdU Analysis. BrdU or EdU (50 μg/g body weight) was injected 2 hbefore the animals were killed. BrdU analysis of cell proliferation was carriedout as described previously (24). EdU labeling was carried out according tothe protocol from the Click-it EdU Cell Proliferation Assays Kit (C10339;Life Technologies).

Chondrocyte Isolation. Epiphyseal ends of femurs and tibiae from P30 micewere removed and subjected to serial digestion at 37 °C with Liberase TMresearch grade (no. 0540111900; Roche). The digestion duration for fraction1 was 45 min, and those for fractions 2 to 5 were 30 min each. Fractions 4and 5 were collected, and were used for transcription profiling, anchorage-independent agar culture, and allotransplantation.

Transcription Profiling. Total RNA was isolated with RNeasy Mini Kit (no.74104; Qiagen) from freshly isolated chondrocytes. cDNAwas generated withthe Ambion WT Expression Kit (no. 4411973; Ambion), and was ultimatelyhybridized with the Affymetrix Mouse 1.0 Gene ST array.

Anchorage-Independent Agar Culture. Anchorage-independent agar culturewas carried out according to the protocol from the Cell TransformationDetection Kit (ECM570; Millipore), with minor modification. The seedingdensity was 2,000 cells per well of a 24-well plate. A cell detection kit

(ECM570; Millipore) was used to estimate cell mass through a spectropho-tometric assay (OD, 490 nm), and colony number by histochemical staining.Culture media with or without chemical inhibitors were changed twice perweek for 4 wk. Chemicals used in this study are listed in Table S2.

Allotransplantation. Freshly isolated chondrocytes (500,000 cells) were sus-pended in 200 μL of culture medium, and was mixed with equal volume ofMatrigel (no. 356234; BD Biosciences). The mixed suspension was injected s.c.into the right lower flank of the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)mouse (female, age 6–8 wk). Rapamycin treatment (delivered throughdrinking water; 5 mg/kg body weight/d) of NSG mice started 1 d after in-jection. Three months after transplantation, tumors were dissected, fixed,sectioned, and stained as described earlier.

Statistical Analyses. One-way ANOVA with post hoc Bonferroni test wasperformed unless otherwise specified. Microarray data were normalized andanalyzed with d-Chip, as well as the Database for Annotation, Visualizationand Integrated Discovery software tools (25, 26).

ACKNOWLEDGMENTS. We thank Dr. R. A. DePhino for sharing the Lkb1conditional KO mouse; Jennifer Couget for her help and technical supporton the microarray experiment; members of the Massachusetts General Hos-pital Endocrine Unit Histology Core for histology services; Drs. R. M. Whiteand N. Ono for advice and suggestions on the transplantation experimentand chondrocyte isolation, respectively; Dr. C. Koch for providing EF5 andthe EF5 antibody; members of the laboratory of Dr. E. Schipani for their helpand technical support on in situ hybridization; and members and advisors ofthe Tabin, Kronenberg, and A. P. McMahon P01 group for discussions andongoing support during the preparation of the manuscript. The type II col-lagen antibody developed by Dr. Linsenmayer was obtained from the De-velopmental Studies Hybridoma Bank developed under the auspices of theNational Institute of Child Health and Human Development and main-tained by the Department of Biology at the University of Iowa. Work inthe laboratory of A.P.M. was supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases GrantP01 DK056246. L.P.L. was supported by an Arthritis Foundation Postdoctoralfellowship.

1. Lanske B, et al. (1996) PTH/PTHrP receptor in early development and Indian hedge-hog-regulated bone growth. Science 273(5275):663–666.

2. St-Jacques B, Hammerschmidt M, McMahon AP (1999) Indian hedgehog signalingregulates proliferation and differentiation of chondrocytes and is essential for boneformation. Genes Dev 13(16):2072–2086.

3. Vortkamp A, et al. (1996) Regulation of rate of cartilage differentiation by Indianhedgehog and PTH-related protein. Science 273(5275):613–622.

4. Hezel AF, Bardeesy N (2008) LKB1; linking cell structure and tumor suppression. On-cogene 27(55):6908–6919.

5. JansenM, Ten Klooster JP, Offerhaus GJ, Clevers H (2009) LKB1 and AMPK family signaling:The intimate link between cell polarity and energy metabolism. Physiol Rev 89(3):777–798.

6. Ylikorkala A, et al. (2001) Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice. Science 293(5533):1323–1326.

7. Hemminki A, et al. (1998) A serine/threonine kinase gene defective in Peutz-Jegherssyndrome. Nature 391(6663):184–187.

8. van Lier MG, et al. (2010) High cancer risk in Peutz-Jeghers syndrome: A systematicreview and surveillance recommendations. Am J Gastroenterol 105(6):1258–1264.

9. Udd L, Mäkelä TP (2011) LKB1 signaling in advancing cell differentiation. Fam Cancer10(3):425–435.

10. Bardeesy N, et al. (2002) Loss of the Lkb1 tumour suppressor provokes intestinalpolyposis but resistance to transformation. Nature 419(6903):162–167.

11. Ovchinnikov DA, Deng JM, Ogunrinu G, Behringer RR (2000) Col2a1-directed ex-pression of Cre recombinase in differentiating chondrocytes in transgenic mice.Genesis 26(2):145–146.

12. Tee AR, Blenis J (2005) mTOR, translational control and human disease. Semin CellDev Biol 16(1):29–37.

13. Corradetti MN, Inoki K, Bardeesy N, DePinho RA, Guan KL (2004) Regulation of theTSC pathway by LKB1: Evidence of a molecular link between tuberous sclerosiscomplex and Peutz-Jeghers syndrome. Genes Dev 18(13):1533–1538.

14. Shaw RJ, et al. (2004) The LKB1 tumor suppressor negatively regulates mTOR sig-naling. Cancer Cell 6(1):91–99.

15. Ho L, et al. (2009) Gli2 and p53 cooperate to regulate IGFBP-3-mediated chondrocyteapoptosis in the progression from benign tomalignant cartilage tumors. Cancer Cell16(2):126–136.

16. Yang Y, Topol L, Lee H, Wu J (2003) Wnt5a and Wnt5b exhibit distinct activities incoordinating chondrocyte proliferation and differentiation. Development 130(5):1003–1015.

17. Gable KL, et al. (2006) Diarylureas are small-molecule inhibitors of insulin-likegrowth factor I receptor signaling and breast cancer cell growth. Mol Cancer Ther5(4):1079–1086.

18. Girnita A, et al. (2006) The insulin-like growth factor-I receptor inhibitor pic-ropodophyllin causes tumor regression and attenuates mechanisms involved ininvasion of uveal melanoma cells. Clin Cancer Res 12(4):1383–1391.

19. Huang SM, et al. (2009) Tankyrase inhibition stabilizes axin and antagonizes Wntsignalling. Nature 461(7264):614–620.

20. Rudin CM, et al. (2009) Treatment of medulloblastoma with hedgehog pathway in-hibitor GDC-0449. N Engl J Med 361(12):1173–1178.

21. Kronenberg HM (2006) PTHrP and skeletal development. Ann N Y Acad Sci 1068:1–13.

22. Weir EC, et al. (1996) Targeted overexpression of parathyroid hormone-relatedpeptide in chondrocytes causes chondrodysplasia and delayed endochondral boneformation. Proc Natl Acad Sci USA 93(19):10240–10245.

23. Levine AJ, Feng Z, Mak TW, You H, Jin S (2006) Coordination and communication betweenthe p53 and IGF-1-AKT-TOR signal transduction pathways. Genes Dev 20(3):267–275.

24. Long F, Zhang XM, Karp S, Yang Y, McMahon AP (2001) Genetic manipulation ofhedgehog signaling in the endochondral skeleton reveals a direct role in the regu-lation of chondrocyte proliferation. Development 128(24):5099–5108.

25. Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis oflarge gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57.

26. HuangW, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: Paths towardthe comprehensive functional analysis of large gene lists. Nucleic Acids Res 37(1):1–13.

Lai et al. PNAS | November 26, 2013 | vol. 110 | no. 48 | 19455

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 22

, 202

0