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Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH) 2 D 3 and upregulating responsiveness to 1,25-(

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To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2.
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  BMP-2 Induces Chondrocyte Maturation  /Schwartz et al. Vol. 9, No. 3 273 Received August 17, 1998; Revised October 6, 1998; Accepted October 6, 1998.Author to whom all correspondence and reprint requests should be addressed:Dr. Barbara D. Boyan, Department of Orthopaedics, The University of Texas,Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX   78284-7774. E-mail: boyanb@uthscsa.edu Endocrine, vol. 9, no. 3, 273–280, December 1998  0969–711X/98/8:273–280/$10.00© 1998 by Humana Press Inc.All rights of any nature whatsoever reserved. 273 Treatment of Resting Zone Chondrocyteswith Bone Morphogenetic Protein-2Induces Maturation into a PhenotypeCharacteristic of Growth Zone Chondrocytesby Downregulating Responsiveness to 24,25(OH) 2 D 3 and Upregulating Responsiveness to 1,25-(OH) 2 D 3 Zvi Schwartz, 1,2,3  Victor L. Sylvia, 3  Yuhong Liu, 3  David D. Dean, 3  and Barbara D. Boyan 2,3,4 1  Department of Periodontics, Hebrew University Hadassah Faculty of Dental Medicine, P.O. Box 1172, Jerusalem, Israel 92-010;and Departments of  2  Periodontics,  3 Orthopaedics, and  4  Biochemistry,University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX  To determine if bone morphogenetic protein-2 (BMP-2)can induce the endochondral maturation of restingzone (RC) chondrocytes, confluent fourth-passagecultures of these cells were pretreated for 24, 36, 48,72, or 120 h with recombinant human BMP-2. At theend of pretreatment, the media were replaced withnew media containing 10 –10 –10 –8   M   1,25-(OH) 2 D 3  or10 -9 –10 -7  M   24,25-(OH 2 )D 3,  and the cells incubatedfor an additional 24 h. This second treatment waschosen, because prior studies had shown that the moremature growth zone (GC) chondrocytes and RC cellsrespond to 1,25-(OH) 2 D 3  and 24,25-(OH) 2 D 3  in dis-tinctly different ways with respect to the parametersexamined. The effect of BMP-2 pretreatment on cellmaturation was assessed by measuring alkaline phos-phatase specific activity (ALPase). In addition, changes inmatrix protein production were assessed by measuringcollagen synthesis, as well as [ 35 S]-sulfate incorporationinto proteoglycans. When RC cells were pretreated for72 or 120 h with BMP-2, treatment with 1,25-(OH) 2 D 3 caused a dose-dependent increase in ALPase specificactivity and collagen synthesis, with no effect onproteoglycan sulfation. RC cells pretreated with 1,25-(OH) 2 D 3  responded like RC cells that had not receivedany pretreatment. RC cells normally respond to 24,25-(OH) 2 D 3 ; however, RC cultures pretreated for 72 or120 h with BMP-2 lost their responsiveness to 24,25-(OH) 2 D 3 . These results indicate that BMP-2 directlyregulates the differentiation and maturation of RCchondrocytes into GC chondrocytes. These observationssupport the hypothesis that BMP-2 plays a significantrole in regulating chondrocyte maturation duringendochondral ossification.Key Words: Chondrocytes; differentiation; matura-tion; growth plate cartilage; BMP-2; 1,25-(OH) 2 D 3 ;24,25-(OH) 2 D 3 Introduction The process of endochondral bone formation consists of a developmental cascade of chondrocyte maturation statesthat culminates in extracellular matrix mineralizationbefore osteogenesis can occur. The process is required for normal growth and development of long bones and for certain kinds of bone repair. During the chondrogenic phaseof the process, chondrocytes are responsible for the syn-thesis, maintenance, and maturation of a calcifiable extra-cellular matrix that is composed mainly of proteoglycanand collagen (1,2) .Bone morphogenetic proteins (BMPs) are the onlyknown factors capable of inducing this process in an ectopicsite (3–5) . They are believed to initiate the expression of theendochondral cartilage cell phenotype by inducing the com-mitment of pluripotent mesenchymal and chondro-progenitor cells. Several studies have shown that purifiedand recombinant BMPs, including recombinant humanBMP-2, can cause these cells to express a cartilage cellphenotype in vivo (6–11) . In vitro, BMP-2 has been shownto promote expression of a chondrogenic phenotype by amultipotent mesenchymal cell line (12) .  274 Endocrine BMP-2 Induces Chondrocyte Maturation  /Schwartz et al. BMPs can also enhance endochondral development bydirect action on chondrocytes in the growth plate (13–15) .BMP-2 has been shown to inhibit collagenase-3 mRNAproduction during human fetal cartilage growth anddevelopment (16) . We have shown that BMP-2 affectsproliferation and differentiation of rat costochondralgrowth plate chondrocytes (17) . The effects are dose-dependent, but they also depend on the maturation state of the responding cell population. Resting zone cells exhibitgreater sensitivity to BMP-2 than do cells derived from theprehypertrophic and upper hypertrophic zones (growthzone) of the growth plate. BMP-2 upregulates BMP-2production in both populations of cells. BMP-2 also causesan increase in BMP-4 mRNA levels in resting zone cells,whereas it causes a decrease in growth zone cells.This differential expression of BMPs by growth platecells in postfetal development is also seen in fetal tissues (18) . In the developing limb bud, there is a differentialdistribution of BMP-2 and BMP-13 transcripts (5,19) .Similarly, Chang et al. (20)  have shown that two other members of the transforming growth factor- β  (TGF- β )superfamily are also differentially expressed in limb buddevelopment. These observations support the hypothesisthat BMPs are produced locally and act on the cells in aparacrine/autocrine manner, suggesting that they functionto promote the transition of growth plate chondrocytes fromone maturation state to another.The effects of BMP-2 on mesenchymal cell responsealso depend on the duration of exposure. Puleo (21)  hasshown that the C3H1OT1/2 cell line exhibits an initialproliferative response to BMP-2 followed by increasedalkaline phosphatase activity, osteocalcin production, andmineralized matrix dependent on the length of exposure toBMP-2. In contrast, BMP-2 inhibited proliferation of bonemarrow stromal cells, but elicited a similar effect onmarkers of osteoblastic differentiation that became greater as exposure to the factor was prolonged. Similarly, BMP-2modulates differentiation of fetal rat calvarial cells in a time-and dose-dependent manner (22) , suggesting that BMP-2enhances expression of other BMP genes during the processin much the same way as noted in the growth plate. Theseresults also suggest that BMP-2 may cause cells to becomesensitive to other regulatory agents. BMP-2 has been shownto cause increased synthesis of insulin-like growth factors1 and 2 by skeletal cells in a dose- and time-dependentmanner (23)  and to enhance the response of osteoblast-like2T9 cells to 1,25-(OH) 2 D 3   (24) . Whether this is true for chondrocytes as well is not known.Recent attention has focused on the transition of cellsfrom the proliferative zone to the zone of prehypertrophy,since it is at this juncture that cells in the growth platebecome demonstrably endochondral rather than hyaline intheir phenotypic expression.  In situ  hybridization andimmunolocalization studies can distinguish between thesecell maturation states on the basis of tissue morphology.However, in cell culture, both the hyaline-like resting zonecells and the postproliferative growth zone cells undergoproliferation before they re-express their in vivo phenotype.To understand the transition from the resting zone to thezone of prehypertrophy, we have established a chondrocytemodel in which these two cell types are cultured separatelyafter discarding the intervening proliferative cell zone (25–28) . Characterization of these two distinct populationshas shown that they produce matrix vesicles with differentcompositions, including activity of alkaline phosphatase (29,30)  and matrix metalloproteinases (31) ; their basal Caion flux (32,33)  and membrane fluidity (34)  are different;phospholipid composition of their membranes is different (26) , as is their basal production of prostaglandin E 2   (35)  and1,25-(OH) 2 D 3  and 24,25-(OH) 2 D 3   (36) . Also, they exhibitmarked differences in their response to vitamin D metabolites.Whereas resting zone cells respond primarily to 24,25-(OH) 2 D 3 , growth zone cells respond primarily to 1,25-(OH) 2 D 3   (37,38) .Even though resting zone chondrocytes will formnodules in long-term culture and exhibit increased alkalinephosphatase as a function of time, these cells do not miner-alize their matrix, nor do they synthesize type X collagen,even after 28 d postconfluence, indicating that their hyalinechondrocyte phenotype is retained (39,40) . However, if these cells are treated for 36 h or more with 24,25-(OH) 2 D 3 ,they will exhibit a growth zone phenotype and becomeresponsive to 1,25-(OH) 2 D 3   (37) . rhTGF- β 1 also promotesthis shift in phenotypic expression, but the effect requires72 h of exposure (41) . In contrast, resting zone cells expo-sed to 1,25-(OH) 2 D 3  for up to 5 d do not become responsiveto 1,25-(OH) 2 D 3   (37,41) . These observations suggest thatacquisition of a 1,25-(OH) 2 D 3 -responsive phenotype is ahallmark of the shift from the resting zone maturation stateto the growth zone maturation state.Once the phenotypic shift occurs, at least with respectto TGF- β 1, it does not appear to be reversible, since thecells lose responsiveness to 24,25-(OH) 2 D 3  as they acquireresponsiveness to 1,25-(OH) 2 D 3 . Because of the sensitivityof resting zone cells to BMP-2 (17) , we hypothesized thatthis growth factor may also play an important role inpromoting the phenotypic transition of these cells. To testthis hypothesis, we assessed whether resting zone cellsacquired responsiveness to 1,25-(OH) 2 D 3  following expo-sure to BMP-2, if the effect was dose- or time-dependent,and if the cells retained responsiveness to 24,25-(OH) 2 D 3 after acquiring responsiveness to 1,25-(OH) 2 D 3 . Results  Alkaline Phosphatase BMP-2 induced resting zone cells to become responsiveto 1,25-(OH) 2 D 3 , but the effect was dependent on BMP-2concentration and time of exposure. In resting zone chon-drocyte cultures treated with BMP-2 alone for 24 h (Fig.1A), alkaline phosphatase specific activity was increased,  BMP-2 Induces Chondrocyte Maturation  /Schwartz et al. Vol. 9, No. 3 275 but only at the highest concentration of growth factor. Asnoted previously (37,41) , alkaline phosphatase was unre-sponsive to 1,25-(OH) 2 D 3  treatment alone. In addition, treat-ment of the cultures with BMP-2 for 24 h had no effect on thecells’ responsiveness to 10  –10  –10  –8    M   1,25-(OH) 2 D 3 .At 72 h (Fig. 1B), cultures treated with either 10 or 50ng/mL BMP-2 had elevated alkaline phosphatase specificactivity, and the effect was dose-dependent. The level of alkaline phosphatase specific activity in cultures treatedwith 50 ng/mL BMP-2 for 72 h was further augmented by10  –9  M   1,25-(OH) 2 D 3 , whereas cultures treated with 10 or 50 ng/mL BMP-2 followed by 10  –8    M   1,25-(OH) 2 D 3  alsoshowed increased enzyme specific activity.The effect of BMP-2 on alkaline phosphatase specificactivity was time-dependent (Fig. 2). Alkaline phosphatasespecific activity was increased over controls after 24, 48,72, and 120 h of exposure to BMP-2. Addition of BMP-2for 24 or 48 h did not induce responsiveness to 1,25-(OH) 2 D 3 . However, responsiveness to 1,25-(OH) 2 D 3  wasseen after 72 and 120 h of treatment with BMP-2. Maxi-mal stimulation of alkaline phosphatase in 1,25-(OH) 2 D 3 -challenged cultures was observed in cells pretreated for 72 h with BMP-2.Treatment with BMP-2 resulted in a loss of cellular response to 24,25-(OH) 2 D 3  (Fig. 3). In cultures pretreatedwith control media followed by 24,25-(OH) 2 D 3 , restingzone cells had increased alkaline phosphatase specificactivity at all times examined, but similar treatment with1,25-(OH) 2 D 3  had no effect. In cultures pretreated withBMP-2 for 24 or 48 h, 24,25-(OH) 2 D 3  caused no further increase in enzyme activity over that seen in cultures treatedwith 24,25-(OH) 2 D 3  alone, whereas 1,25-(OH) 2 D 3  had aneffect that was comparable to that seen with 24,25-(OH) 2 D 3 .In cultures treated for 72 or 120 h with BMP-2, however,addition of 1,25-(OH) 2 D 3  caused an increase in alkalinephosphatase, whereas enzyme activity in the cultures chal-lenged with 24,25-(OH) 2 D 3  was comparable to that of theuntreated controls. Collagen Production Collagen synthesis was inhibited by 10  –9  –10  –8    M   1,25-(OH) 2 D 3 in control cultures, and by 10  –8    M   1,25-(OH) 2 D 3 in BMP-2-pretreated cultures at 24 h (Fig. 4A). However, Fig. 1.  Effect of BMP-2 pretreatment for 24 h (A)  or 72 (B)  onalkaline phosphatase specific activity in cell layer lysates of rest-ing zone chondrocytes following challenge with 1,25-(OH) 2 D 3 .Cultures were treated for 24 or 72 h with 1, 10, or 50 ng/mL of BMP-2 and then treated with 10  –10  –10  –8 M 1,25-(OH) 2 D 3  for an additional 24 h. At harvest, alkaline phosphatasespecific activity in the cell layer was measured as described inthe Materials and Methods section. The data are from one of three replicate experiments yielding similar results. Each datapoint is the mean ±  SEM of six cultures. #  p  < 0.05, treatment withBMP-2 vs cultures not treated with BMP-2; *  p  < 0.05, treatmentwith 1,25-(OH) 2 D 3  vs cultures not treated with 1,25-(OH) 2 D 3 ;•  p  < 0.05, 50 ng/mL BMP-2 vs 10 ng/mL BMP-2. Fig. 2.  Effect of BMP-2 pretreatment time on alkaline phos-phatase specific activity of resting zone chondrocytes followingchallenge with 1,25-(OH) 2 D 3 . Cultures were pretreated with 50ng/mL BMP-2 for 24, 48, 72, or 120 h and then treated with 10 -8  M  1,25-(OH) 2 D 3  for an additional 24 h. At harvest, alkaline phos-phatase specific activity in the cell layer was measured asdescribed in the Materials and Methods section. The data arefrom one of three replicate experiments yielding similar results.Each data point is the mean ±  SEM for six cultures. #  p  < 0.05treatment with BMP-2 vs cultures not treated with BMP-2;*  p  < 0.05 vs 24 h; •  p  < 0.05 vs cells pretreated with BMP-2,but not subsequently treated with 1,25-(OH) 2 D 3 .  276 Endocrine BMP-2 Induces Chondrocyte Maturation  /Schwartz et al. this decrease was independent of BMP-2 concentration. Incultures treated with control media for 72 h, 10  –9  and 10  –8    M  1,25-(OH) 2 D 3  inhibited collagen production (Fig. 4B). Incontrast, when cultures were pretreated with 10 or 50 ng/mLBMP-2, there was a dose-dependent increase in collagen syn-thesis after challenge with 10  –8    M   1,25-(OH) 2 D 3 . The stimu-latory effect of BMP-2 on cell response to 1,25-(OH) 2 D 3  wasalso noted after 120 h of BMP-2 treatment (Fig. 5).  Proteoglycan Sulfation Proteoglycan sulfation by resting zone cells was unaffec-ted by treatment with BMP-2, or by challenge with 1,25-(OH) 2 D 3 , whether or not the cells were treated with BMP-2for up to 120 h (Fig. 6). Discussion The results of this study demonstrate that BMP-2 caninduce resting zone cells to acquire a 1,25-(OH) 2 D 3 responsive phenotype characteristic of more mature growthzone chondrocytes. This effect is dose- and time-dependentand is specific to the parameter being examined. Respon-siveness of alkaline phosphatase to 1,25-(OH) 2 D 3  requiredat least 72 h of exposure to the growth factor. Althoughcells pretreated with BMP-2 for 24 h appeared to becomeresponsive to 1,25-(OH) 2 D 3 , the effect was comparable tothat seen in cultures pretreated with BMP-2 alone, indicatingthat 1,25-(OH) 2 D 3  did not elicit a further effect until 72 h.Responsiveness of collagen production to 1,25-(OH) 2 D 3 also required at least 72 h of exposure. This suggests that Fig. 3.  Effect of BMP-2 pretreatment time on 24,25-(OH) 2 D 3 -dependent alkaline phosphatase specific activity of resting zonechondrocytes. Cultures were pretreated with 50 ng/mL BMP-2for 24, 48, 72, or 120 h and then treated with 10  –8    M   1,25-(OH) 2 D 3 or 10  –7  M   24,25-(OH) 2 D 3  for an additional 24 h. At harvest, alka-line phosphatase specific activity in the cell layer was measuredas described in the Materials and Methods section. The data arefrom one of three replicate experiments yielding similar results.Each data point is the mean ±  SEM for six cultures. #  p  < 0.05,treatment vs control; *  p  < 0.05 vs 24 h. Fig. 4.  Effect of BMP-2 pretreatment for 24 (A)  or 72 (B)  h onpercent collagen production by resting zone chondrocytes fol-lowing challenge with 1,25-(OH) 2 D 3 . Cultures were pretreatedwith BMP-2 (1, 10, or 50 ng/mL) for 24 or 72 h and then treatedwith 10  –10  –10  –8  M   1,25-(OH) 2 D 3  for an additional 24 h. At har-vest, % collagen production was measured as described in theMaterials and Methods section. The data are from one of threereplicate experiments yielding similar results. Each data pointis the mean ±  SEM for six cultures. #  p  < 0.05, + BMP-2 vs –BMP-2; *  p  < 0.05, +1,25-(OH) 2 D 3  vs -1,25-(OH) 2 D 3 . BMP-2 initiates a cascade of responses leading to upregulationof receptors to this vitamin D metabolite.The effects of BMP-2 were similar to those noted previouslyin response to TGF- β 1, suggesting that similar mechanismsmay be involved. Whereas 24,25-(OH) 2 D 3  induced the pheno-typic shift by 36 h (37) , both BMP-2 and TGF- β 1 (41)  required72 h to elicit a comparable response. The requirement for longexposures to differentiating factors has been noted by others (42–44) , supporting the hypothesis that a cascade of events isinvolved. BMP-2 exerts its effects on cells via specific recep-tors activating a kinase cascade leading to new gene expres-sion, sharing certain common mediators with other membersof the TGF- β  superfamily (45,46) . We have shown that TGF- β 1 stimulates PKC α  activity in resting zone chondrocytes viaa mechanism that is independent of phospholipase C andtyrosine kinase, but requires new gene expression and pro-tein synthesis (47) . Activation of PKC can also lead to new  BMP-2 Induces Chondrocyte Maturation  /Schwartz et al. Vol. 9, No. 3 277 gene expression (48–50) . Thus, it is likely that TGF- β 1and BMP-2 initiate a comparable series of reactions, atleast with respect to the parameters measured here, potentia-lly including crosstalk (46,51) .At least some of the pathways modulated by TGF- β 1and BMP-2 may be in common with those activated by24,25-(OH) 2 D 3 . This vitamin D 3  metabolite has a rapid,direct effect on plasma membrane PKC α  in resting zonecells (52) . In addition, 24,25-(OH) 2 D 3  stimulates PKC α activity by activating gene expression and protein synthesis (53) . Given the importance of regulating this transitionduring endochondral bone formation in vivo, it is not sur-prising that some redundancy may exist.It is also possible that 24,25-(OH) 2 D 3  produced by theresting zone chondrocytes plays a role. TGF- β 1 upreg-ulates 24-hydroxylase in resting zone cells, resulting inincreased production of 24,25-(OH) 2 D 3  within 1 h (54) .Increased local production of this vitamin D metabolitecould contribute to the phenotypic shift. It is unknown if BMP-2 elicits a similar change in local production of 24,25-(OH) 2 D 3 .Concomitant with the increase in responsiveness to 1,25-(OH) 2 D 3  there was a reduction in responsiveness to 24,25-(OH) 2 D 3 , further supporting the presence of a phenotypicshift in maturation state. In the absence of BMP-2, restingzone chondrocytes continue to express increased alkalinephosphatase activity as a function of time, and they retaintheir ability to respond to 24,25-(OH) 2 D 3 , but not 1,25-(OH) 2 D 3 . When treated with BMP-2 for 48 h, they continueto exhibit a 24,25-(OH) 2 D 3  responsive phenotype. How-ever, once the maturation shift occurs, these cells no longer respond to 24,25-(OH) 2 D 3 .Our data suggest that the transition event is definitive inthis regard. The transition involves multiple phenotypicmarkers. Not only is alkaline phosphatase affected, butcollagen production is affected as well. At 5 d, collagensynthesis was still inhibited in control cultures challengedwith 10 -9  –10 -8    M   1,25-(OH) 2 D 3 , a phenotypic response of the resting zone cell (28) . However, BMP-2 treatment for 72 h induced a dose-dependent increase in collagen inresponse to 1,25-(OH) 2 D 3 . These data are consistent withthe resting zone cultures acquiring a growth zone-like phe-notype, since 1,25-(OH) 2 D 3 stimulates collagen synthesisin growth zone chondrocyte cultures.Unlike the results obtained for collagen synthesis,proteoglycan production was not affected by BMP-2 pre-treatment, suggesting that this marker of chondrocytedifferentiation may either require additional culture time withBMP-2 or additional factors are involved in the acquisitionof this phenotypic marker. TGF- β 1 pretreatment also had noeffect on sulfate incorporation following challenge by 1,25-(OH) 2 D 3   (41) , indicating that it, too, does not modulate thisaspect of the cell. In contrast, following pretreatment with24,25-(OH) 2 D 3  for 36 h, resting zone cells respond to 1,25-(OH) 2 D 3  with a dose-dependent increase in [ 35 S]-sulfateincorporation typical of the growth zone cell phenotype (37) .Thus, although crosstalk among differentiating factors mayexist, they are not interchangeable. Moreover, the effects of BMP-2 appear to be more limited than those of 24,25-(OH) 2 D 3 , suggesting that it regulates a subset of cells or that Fig. 5.  Effect of BMP-2 pretreatment time on percent collagenproduction by resting zone chondrocytes following challengewith 1,25-(OH) 2 D 3 . Cultures were pretreated with 50 ng/mLBMP-2 for 24, 48, 72, or 120 h and then treated with 10  –8  M  1,25-(OH) 2 D 3  for an additional 24 h. At harvest, % collagenproduction was measured as described in the Materials andMethods section. The data are from one of three replicateexperiments yielding similar results. Each data point is themean ±  SEM for six cultures. #  p  < 0.05, treatment vs control;*  p  < 0.05; vs 24 h. Fig. 6. Effect of BMP-2 pretreatment time on [ 35 S]-sulfate incor-poration by resting zone chondrocytes following challenge with1,25-(OH) 2 D 3 . Cultures were pretreated with 50 ng/mL BMP-2for 24, 48, 72, or 120 h and then treated with 10  –8    M   1,25-(OH) 2 D 3 for an additional 24 h. At harvest, [ 35 S]-sulfate incorporationwas measured as described in the Materials and Methods sec-tion. The data are from one of three replicate experiments yield-ing similar results. Each data point is the mean ±  SEM for sixcultures. *  p  < 0.05 vs 24 h; •  p  < 0.05 vs 48 h.
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