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Mechanisms of fibrous dysplasia

irnmature woven bone which is not replaced by maturelamellar bone (Marie et al . , 1997; Riminucci et al . ,1999). Th e lack of connectivity and the imm ature natureof the bone formed resul ts in decreased mechanicalproper ties of the bone, leading to deform at ions andfractures.

Osteoblast abnormalitles in flbrous dysplasia

The hi s to logica l analys i s of dysplas t i c l es ionssuggest the existente of abnormalities in the formationof os teoprogen i tor cel ls (Marie e t al ., 1 997; Marie,

1999a; Riminucci et al., 1999). Indeed, the number ofimmature osteob lasts is low. M oreover, cells locatedalong the bone surface are not oriented and elongated,and are distinct from normal cu boidal mature osteoblasts(Fig. lA,B). The marrow cavity is filled with elongated,immature, alkaline phosphatase-positive cells, and thiscellular invasion does not a llow the normal developm entof hematopoietic and adipogenic populations (Fig. 1C).These histological characteristics indicate that the pre-o s t e o b l a s t i c p o p u la t i on i s i n c r e as e d w h e r e a s t h epopula t ion of mature ce l l s i s decreased. De fect iveos teoblas t d i f f erent i a t ion i s a l so evidenced by theabnormal collagen organization in the immature bone.The co l l agen f i be r s a r e no t pa r a l l e l l y o r i en t ed , a

characteristic of immature woven bone. The collagenf iber s somet imes appear perpendicular to the bonesurface, showing a com b-like structure (Fig. 1A ). Theseabnormalities found in both monostotic and polyostoticles ions ref lect a disorganizat ion of col lagen f iberssynthes ized by dysplas t ic cel ls (Marie et al . , 1997;Riminucci et al . , 1999). In addition to the abnormal

formation and orientation of collagen, non-collagenousprote in s ynthe s i s i s a f f ec ted . Os teonect in l eve1 i sincreased, whereas osteopontin and bo ne sialoprotein aredecreased in dysplastic bone, reflecting the immaturecomposition of the matrix (Marie et al., 1997; Riminucciet al., 199 9; Sakam oto et al., 199 9). The reduced contentin som e of these bone proteins in the matrix may reducethe adherence of osteoblasts, leading to cell retractionand disorientation (Riminucci et al., 1999). As a result ofthe disorganized immature bone matr ix, os teocytesarising from dysplastic osteoblasts are more numerousthan normal, and are located within large lacunae. They

also do not form a wel l organized network throughintercellular extensions present in the canaliculi withinthe bone matrix (Fig. 1B). In addition to these alterationsof the osteoblast lineage, there are zones of active boneresorption in dysplastic bone that are caracterized by thepresence of multiple osteoclasts actively resorbing themineralized matrix and forming local lytic areas (Fig.1B). Some of the histological characteristics found infibrous dysplasia (marrow invasion, hyper-resorption,immature bone, lytic areas) are also found in severehyperparathyroidism and other cases of high turnover ofbone (Rasmussen and Bordier, 1974).

Cellular basls of fibrous dysplasia

The avai l abi l i ty and development of bone ce l lcultures obtained from dysplastic lesions allowed us toidentify the cellular abnormalities in osteoblasts in bothmonostotic and polyostotic fibrous dysplasia. We foundthat dysplastic cells show an increased proliferation rateas socia ted wi th e l evated in t r ace l lu lar cycl i c AMP

Flg. 1 stological abnormalities induced by Gsa mutations in polyostotic fibrous dysplasia in the McCune-Albright syndrome (A, C)

and in monostotic fibrous dysplasia (8). Bone lesions are characterized by immature fibrotic bone (F), irregular osteoid (o) anddisorganized collagen which presents a comb-like structure (A: arrows). Mature osteoblasts are rare and numerous osteocytes (B:Ocy) are found in large lacunae within the immature bone. Bone resorption by osteoclasts (B: Ocl) is locally increased. The marrow(M) space is invaded by immature osteoprogenitor cells wihich present alkaline phosphatase activity (C: arrows). A, B, x 125; C, x 250




(cAMP) levels (Marie et al., 1997). Osteoblastic cellsisolated from the more severe bone lesions, composed ofimmature bone matrix, show a less differentiatedphenotype and increased proliferative rate compared tocells from the less affected lesions composed of a

mixture of mature and immature bone matrix. Thisindicates that the increased cell proliferation isassociated with the less differentiated osteoblastphenotype in the dysplastic lesions. In addition to the

increased cell proliferation, dysplastic cells showdecreased differentiation, as evidenced by the alterationof osteocalcin mRNA expression and protein synthesis(Marie et al., 1997; Sakamoto et al., 1999; Hopyan et al.,1999). However, dysplastic cells respond normally to1,25-dihydroxyvitamin D which enhances osteocalcin

expression in osteoblasts (Marie et al., 1997). Theexpression of Runx21Cbfa1, a master transcription factor

known to stimulate osteocalcin expression (Karsenty,2000), was found to be increased in dysplastic bone cells

(Sakamoto et al., 1999). One possible explanation for the

increased Runx2lCbfal expression with regard to thereduced osteocalcin expression is that dysplastic cellsexpress high levels of Msx2, a transcription factor that is

expressed early during osteoblast differentiation and thatinhibits osteocalcin expression (Newberry et al., 1997).The concomittant decrease in cell differentiation andincreased proliferation of pre-osteoblastic cells indysplastic lesions lead to a rapid deposition of animmature, poorly organised woven bone characteristic of

the bone lesion at the histological leve1 (Marie et al.,1997; Riminucci et al., 1999).

Molecular basis of fibrous dy splasia

The molecular basis of fibrous dysplasia has been

clarified when mutations affecting the stimulatory a sub-unit of G protein (Gs) have been found in dysplasticbone lesions. In the cell membrane, adenylate cyclase iscoupled to hormonal receptors by a G protein whichbinds ATP (Fig. 2). This protein is composed of a , 13 andy subunits. When inactivated, the a-subunit binds GTP.When a ligand binds to the receptor, the a-subunit isreleased from the remaining By protein complex. Thisprocess is reversed through GTP hydrolysis by the

intrinsic GTPase activity of the Gsa protein (Spiegel etal., 1992). The a chain of the G protein binds andhydrolyses GTP, and thereby activates adenylate cyclasethat produces cAMP (Fig. 2). Mutations in the Gsa havebeen initially identified in multiple endocrinopathies

(Landis et al., 1989; Lyons et al., 1990). In thesepathologies, a constitutive activation of Gsa leads toaccumulation of cAMP in endocrine tissues, leading toincreased cell proliferation and hormonal secretion(Klibanski, 1990; Landis et al., 1990; Spada et al.,1990). This was found in autonomous thyroid and

pituitary tumors, as a result of somatic mutations of Gsa(arginin20lcystein or glutamin227leucin). Theseactivating mutations inhibit the GTPase activity of Gsa,resulting in increased adenylate activity and increased

cAMP levels (Spiegel et al., 1993; Spiegel, 2000) (Fig.2). In the McCune-Albright syndrome, activatingmutations were found in exon 8 of the Gsa gene inpatients with endocrinopathies (Weinstein et al., 1991;Ringel et al., 1996). In this syndrome, somatic mutationsof Gs a were found at variable levels in severalendocrine or non endocrine tissues, leading to a mosaicdistribution of the somatic mutation early duringembryogenesis, and resulting in multiple and variableendocrinopathies later on (Cuttler et al., 1989; Shenker

et al., 1993, 1994; Malchoff et al., 1994; Levine, 1999).The mutations consist of the substitution of cystein(R201C) or histidin (R201H) by arginin at position 201in Gsa. Another mutation such as R201G (Shenker et al,1995; Candeliere et al., 1997; Riminucci et al., 1997) ormutations which do not affect Gsa have also been found

in patients with fibrous dysplasia (Gessl et al., 1994).However, fibrous dysplasia is not associated withaberrent chromosomic recombinations (Da1 Cin et al.,

2000).Mutations of Gsa have been found by RT-PCR

analysis in dysplastic bone lesions (Shenker et al., 1995;Candeliere et al., 1997; Riminucci et al., 1997). Theculture of osteoblastic cells isolated from dysplasticbone lesions allowed us to show the expression ofR201C and R201H mutations in dysplastic bone cells

Adivated Receptor

GDP GTPf iGDP GGTP

m& J+ r ""i""'Adenylate Cyclase

Protein Kinase A

Fig. 2. Schematic representation of the effects of Gsa mutation

on adenylate cyclase activity. Adenylate cyclase i s coupled to

hormonal receptors by the Gsa protein. When the receptor is

activated, the a chain of the protein binds and hydrolyses GTP

to GDP, thereby activating adenylate cyclase which leads to

cAMP production. Gsa-activating mutations (Gsa*) inhibit the

GTPase activity. result ing i n increased adenylate cyclase

activity and increased cAMP accumulation. This leads to

activation of proteine kinase A and phosphorylation of a number

of target rnolecules.




(Shenker et al., 1995). These mutations are expressed inboth monostotic and polyostotic fibrous dysplasia(Shenker et al., 1995; Alman et al., 1996) and representthe molecular basis of the cellular abnormalities in thetwo types of fibrous dysplasia. Interestingly, furtheranalyses indicated that not al1 bone cells within thedysplastic lesions express such mutations (Riminucci et

al., 1997). This mosaic distribution allows cell survivalbecause the mutations are lethal (Happle, 1986). It istherefore believed that Gsa mutations which appear

early during embryogenesis induce the generalizedabnormalities found in the McCune-Albright syndrome,

whereas the somatic mutations appearing later in lifeinduce more restricted pathologies such as thyroid andpituitary adenomas, and monostotic fibrous dysplastic

lesions. Thus, the endocrine and skeletal lesionsassociated with these mutations reflect a variablephenotypic expression of the mutations resulting from asomatic mosaicism of a same molecular alteration(Ringel et al., 1996; Spiegel, 1996; Cohen and Howell,1999). In dysplastic lesions, Gsa mutations areexpressed in osteoprogenitor cells present in the marrowstroma (Marie et al., 1997; Bianco et al., 2000). Thefrequence of expression of mutations is higher in

polyostotic than in monostotic fibrous dysplasia (Stantonet al., 1999). In terms of functional activity, dysplastic

cells from monostotic and polyostotic lesions are able tobe osteogenic in vitro in the presence of glucocorticoidswhich stimulate osteoblastic cell differentiation.

However, mutated dysplastic cells are not osteogenic invivo, probably because of the lethality induced by the

mutation. Only mixed mutated and non mutated cellsreconstituting the natural mosaic are able to formdysplastic lesions in vivo (Bianco et al., 1998). Thisindicates that Gsa mutations are responsible for theformation of dysplastic bone lesions, and that the

presence of mutated and nonmutated cells is required forthe development of the lesion (Bianco et al., 1998).

Molecular mecha nisms of fibrous dysplasia

The formation of dysplastic bone lesions resultingfrom Gsa mutations involves severa1 mechanisms. Intheory, activating mutations of Gsa should inducestimulation of adenylate cyclase and increased cAMPaccumulation (Fig. 2). Accordingly, we found that Gsa

mutations induce a constitutive accumulation of cAMPin osteoblastic cells in the McCune-Albright syndrome(Marie et al., 1997) and this was confirmed by othergroups (Candeliere et al., 1997; Bianco et al., 1998). Thedownstream events leading to the abnormal cellularphenotype in osteoblastic cells are linked to cAMPsignaling. The signaling pathways induced by cAMP are

known to tightly control gene expression (Borrelli et al.,

1992). cAMP accumulation activates protein kinase Awhich then phosphorylates transcription factors that aremembers of the cAMP-response-e lement-b ind ing-protein (CREB) family. This leads to binding of these

proteins to DNA domains called cAMP-responsive

element (or CRE) in the promoter of target genes(Montminy et al., 1990). This pathway controls the

expression of multiple genes in several endocrine andnonendocrine tissues. For example, in pituitary cells, theincreased activity of PKA induced by Gsa-activatingmutations stimulate the promotor activity of prolactinand growth hormone (Tian et al., 1994; Gaiddon et al.,1995), leading to endocrinopathies associated with themutation. In osteoblastic cells, a rise in cAMP induced

by Gsa mutations also induce multiple effects (Fig. 3).One of the more rapid effects is cell retraction which isobserved in dysplastic cells (Riminucci et al., 1997).

This may be explained by the rapid disassembling effectof cAMP on cytoskeletal proteins leading to cell

retraction (Lomri and Marie, 1990). The rise in cAMP inmutated dysplastic cells may also be responsible for thedecreased expression of bone matrix proteins, such asosteopontin (Noda and Rodan, 1989) whose promotercontains cAMP-responsive elements (Kopp et al., 1989;Kerr et al., 1993). The rise in cAMP is also likely to beinvolved in the increased bone resorption found in

dysplastic lytic lesions. Such local stimulation of boneresorption is found in severe cases ofhyperparathyroidism in which high levels of parathyroid

hormone induces elevated intracellular cAMP levels inosteoblasts (Lomri and Marie, 1990) and results in

increased osteoclastic bone resorption by increasingbone resorbing cytokines (see below).

The elevation in cAMP induced by the Gsa is a animportant mechanism leading to bone cell abnormalities

Gsa* + c fos

t - + c Jun-+ AP-1

Alterat ionof

BoneMatrix

Proteins

Lesions

Fig. 3. Molecular rnechanisrns involved in fibrous dysplasia. The

rnutations in Gsa (Gsa*) result in increased intracellular cAMP

levels that rnodulate the expression of several genes whose

promoter contains a cAMP-responsive elernent (CRE) . This

pathway leads to abnormal production of bone rnatrix proteins

and to increased c- fos expression, leading to abnormal

recruitrnent and function of osteoblastic cells, and to abnormalbone rnatrix production. The cAMP overproduction induced by

the mutation also leads to increased production of interleukin-6

and -1 1 (11-6, 11-1 1) wh ich in turn stim ula te osteoclastic bone

resorption. These rnolecular mechanisms induced by the Gsa

rnutations result in the forrnation of dysplastic bone lesions.




in fibrous dysplasia (Fig. 3). One mechanism involves c-fos, a protein that forms heterodimers with proteins ofthe jun family, forming a complex that bin ds to specificsequences in the promoter of several genes (Angel andKarin, 1991). The c-fos promoter contains a cAMP-reponsive element (Sassone-Corsi et al., 1988), and istherefore a potential target for Gsa mutations in fibrousdysplasia. Indeed, Gsa-activating mutations induce c-fosoverexpression in vitro (Sassone-Corsi et al., 1988). Inendocrine cells, the Gsa-induced stimulation of c-fostranscription involves multiple sequences such as CREand the serum responsive element SRE (Gaiddon et al.,1994). In bone, Gsa mutations increase c-fos expressionand c-jun, which acts by binding c-fos and forms theAP-1 he te rod imer com plex . In endocr in e ce l ls ,mutations of Gsa increase c-jun and Jun B expression(Gaiddon e t a l . , 1994) . His to log ica l ana lys isdemonstrated an increased expression of both c-fos andc-jun in dysplastic lesions (Sakamoto et al., 1999) whichmay in turn induce abnormal expression of genescontrolled by AP-1 (Angel and Karin, 1991; Sassone-Corsi, 1995). Members of the AP-1 family are impo rtantfactors regulating bone form ation. For examp le, deletionor overexpression of c-fos in transgenic mice inducessevere bone abnorm alities (Grigoriadis et al., 1 995). c-fos over-expression in mice results in increased boneformation, with characteristics similar to those found indysplastic bone lesions (Grigoriadis et al., 1995). Invitro, AP-1 controls genes that are involved in osteoblastdifferentiation, and c-fos expression modulates theexpression of osteoblast genes such as osteocalcin and

osteopontin whose promoter contains AP-1 sites (Lian etal., 1991). In situ hybridization and immunohisto-chemical analyses showed that c-fos mRNA and proteinlevels are high in bone lesions of dysplastic patients withGsa mutations (Candeliere et al., 1995). Thus, c-fosoverexpression in fibrous dysplasia may be involved inthe molecular m echanisms leading to the bone lesions. c-fos is often overexpressed in human osteosarcomas (Wuet al., 1990). Moreover, experimental studies showedthat c-fos can induce oncogenic transformation in m ouseosteoblasts (Grigoriadis et al., 1993, 1995). Thus, c-fosoverexpression may be part of the mechanisms leadingto tumor development in some cases of fibrous dysplasia(Yabut et al., 1988). Several mechanisms can be evoked

to explain how c-fos overexpression can induce thecellular abnormalities found in dysplastic bone lesions.The increased c-fos protein by cAMP may induce cellsto en te r in to the G1 phase o f the ce l lu la r cyc le(Herschman, 1991). This is consistent with the findingthat c-fos is expressed by immature osteoblasts andcontrols osteoblast proliferation and differentiation (Lianet al . , 1991; Machw ate e t al . , 199 5a,b). I t seem stherefore possible that the increased osteoblastic cellproliferation and the abnormal osteoblast differentationobserved in fibrous dysplasia results in part from theincreased expression of c-fos and other members of theAP-1 family.

As discussed above, localized lytic lesions are found

in dysplastic lesions. At least two cytokines may beresponsible for this local increase in bone resorption infibrous dysplasia. Osteoprogenitor cells bearing theR201H G s a mutation show a constitutive increase ininterleukin-6 (11-6) production (Yamamoto et al., 1996).In addition, less differentiated cells present in the bonelesions produce more IL-6 levels (Stanton et al., 1999).Inhibition of cAMP levels in mutant cells reduce 11-6production, suggesting that the Gsa mutation-inducedincreased cAMP levels results in 11-6 overexpression(Yamamoto et al., 1996). This is consistent with thef ind in g tha t t rans fec t ion of the mutated G s a inosteoblastic cells induces 11-6 production (Motomura etal., 1 998). The 11-6 promoter presents cA MP-responsiveelements, as well as AP-1 and NF-KB sites (Kishimoto,1989). The Gsa mutation increases binding of AP-1,nuclear factor NF-IL-6, N F-KB and CREB to the IL-6promoter, which can thus account for the increasedexpression of this cytokine in dysplastic osteoblasts(Motomura et al., 1998). The implication of 11-6 in theincreased bone resorption in dysplastic cystic lesions(Yamamoto et al., 19 96) is supported by the finding that,in other bone diseases characterized by increased boneresorption such as postmenopausal osteoporosis andPaget's disease, increased 11-6 stimulates osteoclastdifferentiation and bone resorption (Jilka et al., 1992;Roodman, 1992). Interestingly, glucocorticoids werefound to decrease 11-6 production by dysplastic cells andto reduce bone resorption, which may be of potentialtherapeutic interest in the treatment of fibrous dysplasia(Stan ton et al., 1999 ). 11-11 production was also found tobe increased in mutated dysplastic cells (Yamamoto etal., 1996), suggesting that this bone resorbing cytokinemay a lso be invo lved in the ly t ic le s ions in th issyndrome. Recent data indicate that local moleculesproduced by stromal cells and osteoblasts play a majorrole in osteoclast differentiation and bone resorption(Marie e t a l . , 2000; Hofbauer e t a l . , 2000). Suchmolecules are involved in the increased bone resorptioninduced by several local factors such as IL-6 and 11-11(Hofbauer et al., 2000). It is thus possible that thesemolecules may be the final step of IL-6- and 11-11-induced bone resorption induced by Gsa mutations infibrous dysplasia.

The cellular alterations observed in fibrous dysplasia

may inv olve other mechanisms. Two studies reported anincreased leve1 for estrogen expression in o steoblasts inboth mo nostotic fibrous dysplasia and McCune-Albrightsyndrome (Kaplan et al., 1988 ; Pensler et al., 1 990). Thiswas not linked to the end ocrine status of the subjects andcould thus reflect abnormal regulation of these receptors(Pensler et al., 1990). This abnormality may contributeto the bone pa tho logy in th is syndrome becauseestrogens stimulate the proliferation of osteoblastic cellsin part by increasing the expression of local growthfactors such as IGF-1 and TGF-B (Rickard et al., 1999)which are important factors controling osteogenesis(Marie, 1999b). It is not known, however, whether theexpression of these growth factors is increased locally in




fibrous dysplasia. Finally, a rise in the expression of theparathyroid hormone related peptide (PTHrP) has beenrecently reported in osteoblastic cells of patients with theMcCune-Albright syndrome. A treatment with 1,25-dihydroxy-vi tamin D3 can reduce the production ofPTHrP in vitro a s well a s bone markers in this disease. Itis thus possible that a local increase in the expression ofPTHrP, which acts on bone cells in a similar way toPTH, could contribute to the bone cell alterations in theMcCu ne Albright synd rome (Fraser et al., 2000).

Conclusion

The discovery that G s a mutations are expressed indysplastic bone cells provided a basis for the cellular andm o l e c u l a r m e c h a n i s m s i n v o l v e d i n t h e o s t e o b l a s ta l te ra t ions obse rved in f ib rous dysp las ia . I t i s nowapparent that the abnormal osteoblastic cell proliferationand differentiation obsewed in fibrous dysplasia resultf r o m i n c r e a s e d c A M P ac c u m u l a t i o n , l e a d i n g t oalteration in the expression of target genes including c-fos , c- jun, 11-6 , 11-11, which in turn modulate thetranscription and expression of downstream genes andresult in the alterations of osteoblast and osteoclastrecruitment and function in the dysplastic bone lesions.These pathways provide a cellular and molecular basis

for the bone alterations in fibrous dysplasia. Furtheranalysis of the molecular pathways activated by the G s amuta t ions in dysp las t ic bone ce l l s may lead to theidentification of target genes for cellular therapies andallow us in the future to develop a therapeutic approachof the skeletal alterations in fibrous dysplasia.

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Accepted May 9, 2001

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