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Background Fibrous dysplasia is characterized by intense marrow fibrosis and increased rates of bone turnover. The lesions of fibrous dysplasia resemble those described in the long bones of transgenic mice overexpressing the c-fos proto-oncogene. Activating mutations in the
Methods We used in situ hybridization to determine the level of expression of c-fos in bone-biopsy specimens from two normal subjects, eight patients with fibrous dysplasia, and six patients with other bone disorders characterized by high rates of bone turnover. The probe used corresponded to the fourth exon of the c-fos gene.
Results High levels of c-fos expression were detected in the bone lesions from all eight patients with fibrous dysplasia. No expression of c-fos was detected in bone specimens from the normal subjects or from specimens of normal bone obtained from patients with fibrous dysplasia. The cells that expressed c-fos in the dysplastic lesions were fibroblastic and populated the marrow space. A very low level of c-fos expression was detected in the biopsy specimens from the patients with other bone diseases. One patient with polyostotic fibrous dysplasia and one patient with the McCune–Albright syndrome were tested for the previously described Gs
Conclusions Increased expression of the c-fos proto-oncogene, presumably a consequence of increased adenylate cyclase activity, may be important in the pathogenesis of the bone lesions in patients with fibrous dysplasia.
subunit of the stimulatory guanine-nucleotide–binding protein (Gs) linked to adenylate cyclase have recently been described in bone cells from patients with the McCune–Albright syndrome and fibrous dysplasia. gene mutations and were found to express these mutations in bone.
Recently, mutations in the subunit of the gene for the stimulatory guanine-nucleotide–binding protein (Gs) linked to adenylate cyclase have been described in bone cells from patients with the McCune–Albright syndrome2,3,4 who have polyostotic fibrous dysplasia as a clinical manifestation of their disease (other manifestations are café au lait spots and various endocrinopathies). These mutations lead to the constitutive activation of adenylate cyclase.5 The increased signaling through the cyclic AMP (cAMP) pathway is believed to be responsible for the clinical characteristics of the McCune–Albright syndrome, although the molecular events resulting from the constitutive production of cAMP have not been characterized.
The product of the c-fos proto-oncogene is a nuclear protein that forms heterodimers with the proteins encoded by the jun family of proto-oncogenes to form the transcription factor AP-1, which binds to specific sequences in the promoter region of target genes to modulate their expression.6 Examination of mutations involving both gain of function and loss of function has demonstrated that bone is a physiologic target tissue of the action of c-fos.7,8,9 In particular, transgenic mice overexpressing c-fos have abnormal bone remodeling characterized by bone marrow fibrosis and enhanced formation of woven bone.9 These lesions closely resemble those that occur in patients with fibrous dysplasia. In this study, we used in situ hybridization to measure the expression of c-fos in bone-biopsy specimens from normal subjects and patients with fibrous dysplasia and found that the expression of c-fos messenger RNA (mRNA) was increased in bone lesions from the patients.
Methods
Study Subjects
We studied biopsy specimens of iliac-crest bone from two healthy 8-year-old boys, eight patients with fibrous dysplasia (age range, 6 to 17 years), and six patients with other bone diseases (age range, 2 to 64 years). The biopsies were performed in the normal subjects and control patients during minor orthopedic procedures to build a reference data base for pediatric bone histomorphometry (unpublished data) and in the patients to confirm the diagnosis. In all patients with fibrous dysplasia, the biopsy specimens were obtained from areas of abnormal bone; in two patients with polyostotic fibrous dysplasia, we also obtained bone from the contralateral normal iliac crest (normal bone means histologically normal in this context). The lesions caused by fibrous dysplasia were histologically similar in all patients, whether they had monostotic or polyostotic disease, and were characterized by marrow fibrosis, trabeculae made of woven bone, high rates of bone turnover, and poorly organized cortexes.
Among the eight patients with fibrous dysplasia, one (a 14-year-old boy) had bone lesions involving the entire skeleton, a condition considered to be a discrete clinical entity — panostotic fibrous dysplasia10; two (a 10-year-old girl and a 7-year-old boy) had the McCune–Albright syndrome; three (a 17-year-old girl, an 11-year-old boy, and a 13-year-old boy) had polyostotic fibrous dysplasia; and two (a 6-year-old boy and a 15-year-old boy) had monostotic fibrous dysplasia. The disease was diagnosed in all patients after the occurrence of limb deformities or atraumatic fractures through a lesion. Among the six patients with other bone diseases, one (a 2-year-old girl) had hypocalcemic vitamin D–resistant rickets, one (a 64-year-old man) had Paget's disease, one (a 2-year-old girl) had the Proteus syndrome (hemihypertrophy, subcutaneous tumors, and macrodactyly), and three (a 5-year-old girl, a 13-year-old girl, and a 6-year-old boy) had osteogenesis imperfecta type IV. These patients were selected on the basis of variable degrees of marrow fibrosis and increased rates of turnover observed on biopsy.
The studies were approved by the appropriate institutional review committees, and informed consent was obtained from all subjects or their parents.
In Situ Hybridization
The biopsy specimens were prepared with the use of standard techniques for bone histomorphometry.11 Undecalcified 6-µm sections were immersed four times in ethylene glycol monoethyl ether acetate for 10 minutes. After rehydration, the sections were denatured by incubation in 0.2 N hydrochloric acid for 20 minutes followed by incubation in 2x sodium citrate buffer (SSC) (1x SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) at 70°C. The tissues were then fixed for five minutes in 4 percent paraformaldehyde at room temperature and blocked with dithiothreitol, iodoacetamide, and N-ethylmaleimide.12 Hybridization was carried out at 42° C for 30 minutes in a hybridization mix.12 The human c-fos riboprobe was generated by subcloning the NcoI-to-ScaI fragment of the human c-fos gene, which spans the fourth exon, into the pGEM-3Z vector (Promega, Madison, Wis.). The 71-base-pair (bp) antisense c-fos probe labeled with uridine 5'--[35S]thiotriphosphate was then synthesized by in vitro transcription of the BsgI-digested plasmid with T7 polymerase according to the manufacturer's instructions. The 73-bp control probe used to detect nonspecific hybridization was prepared by transcribing the SmaI-linearized pBluescript SK(–) phagemid (Stratagene, La Jolla, Calif.) with T7 polymerase. The c-fos probe contained 12 labeled uridine residues, whereas the control probe contained 15 labeled uridine residues. After hybridization, the sections were washed five times at 45° C for 12 minutes each time in 2x SSC and twice for 15 minutes in 0.2x SSC dehydrated in ethanol, and exposed to Kodak NTB-2 photographic emulsion (Eastman Kodak, Rochester, N.Y.).
Immunofluorescence Assay
Cryosections from the bone lesion from the 10-year-old girl with fibrous dysplasia were mounted on slides coated with poly-l-lysine and stained with monoclonal anti–c-fos antibody Ab-1 (Oncogene Science, Manhasset, N.Y.), as described previously.13 The dilutions used were 1:5 for the primary antibody and 1:100 for the secondary antibody.
Restriction-Site Polymorphism
Total RNA was isolated14 from cells from lesions caused by fibrous dysplasia in the 7-year-old boy with the McCune–Albright syndrome and the 14-year-old boy with panostotic fibrous dysplasia.10 Cells were also obtained from normal bone (an iliac-crest–biopsy specimen from an eight-year-old boy) and grown according to published procedures.15 The RNA was reverse-transcribed and amplified with the polymerase chain reaction (PCR) as described elsewhere.16 The 5' end-labeled upstream primer and the downstream primer were from exon 7 and exon 10, respectively.16
Results
Extensive c-fos hybridization signals were detected in samples of the bone lesions from all eight patients with fibrous dysplasia, and the level of c-fos expression was similar in all sections of abnormal bone from these patients. The cells in the lesions that expressed c-fos were the fibroblastic cells that populate the bone marrow space (Figure 1A and Figure 1C). All eight samples of bone lesions studied had substantially more signals than the samples of histologically normal bone obtained from two of the patients (Figure 1E) or the bone samples from the two normal subjects. Few grains were seen when the sections were probed with an unrelated control probe under identical conditions (Figure 1B, Figure 1D, and Figure 1F).
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Only the cells in the bone lesions from patients with fibrous dysplasia showed increased expression of c-fos mRNA (Figure 2A and Figure 2B). The level of c-fos mRNA was not increased in the bone specimen from a patient with hypocalcemic vitamin D–resistant rickets (Figure 2C and Figure 2D). The specimen from a patient with Paget's disease showed no c-fos hybridization signal when the exposure time was short (Figure 2E and Figure 2F). Longer exposure to the photographic emulsion allowed detection of c-fos mRNA in this biopsy specimen, as recently reported17 (data not shown). A very low level of c-fos expression was detected in the biopsy specimen from a patient with the Proteus syndrome (Figure 2G and Figure 2H), and in regions of fibrous tissue in specimens from three patients with osteogenesis imperfecta type IV (data not shown).
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gene subunit by either histidine or cysteine results in an abnormal Gs protein that stimulates adenylate cyclase in a constitutive fashion.5 These mutations have been described in bone cells obtained from the lesions caused by fibrous dysplasia and from other affected tissues in patients with the McCune–Albright syndrome.2,3,4 We assessed whether activating Gs mutations were present in one patient with the McCune–Albright syndrome and one patient with panostotic fibrous dysplasia.10 The missense mutation causing the substitution of histidine for arginine at position 201 introduces a novel cleavage site for the NlaIII restriction endonuclease.18 Reverse-transcribed RNA from cultured bone cells from lesions in these two patients and from a normal subject was amplified by PCR with labeled primers flanking the mutation site, digested with NlaIII, and analyzed by polyacrylamide-gel electrophoresis. Figure 3 shows the diagnostic NlaIII restriction fragment detected in one of the patients (lane 3), confirming the Arg-to-His mutation. The other patient did not have this mutation (Figure 3, lane 2), but was confirmed to express the Arg-to-Cys mutation on the basis of the hybridization of reverse-transcribed RNA with allele-specific oligonucleotide probes16 (data not shown). Samples from a normal subject tested negative for both mutations in all assays (Figure 3, lane 1; data not shown). The Gs gene was not studied in any tissue from any of the other patients.
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We found that the expression of c-fos mRNA and protein was increased in bone lesions from eight patients with fibrous dysplasia, at least two of whom had activating mutations of the Gs gene in bone cells. The increase in c-fos mRNA appears to be specific for this bone disease and not linked to a phenotype of a high rate of bone turnover or to fibrotic tissue in general. These results support a role for c-fos in fibrous dysplasia.
In patients with Paget's disease c-fos is expressed in osteoclasts,17 but only in cells attached to the bone surface and not in cells from the marrow cavity.17 In our study, no c-fos signal was detected in the biopsy specimen from a patient with Paget's disease. We were able to detect c-fos mRNA in bone sections from this patient when longer exposure times were used, thus confirming the published report.17 The expression of c-fos was not increased in the biopsy specimens from patients with hypocalcemic vitamin D–resistant rickets, the Proteus syndrome, or osteogenesis imperfecta type IV. Thus, our results suggest that overexpression of c-fos in fibrotic cells from bone lesions is specific for fibrous dysplasia.
Transgenic mice overexpressing c-fos have abnormal bone remodeling,9 and osteosarcomas develop in some.19 The tissue content of c-fos protein is also increased in human osteosarcomas.20 Osteosarcomas eventually develop in about 0.5 percent of patients with fibrous dysplasia and 4 percent of patients with the McCune–Albright syndrome.21 The increased expression of c-fos mRNA in lesions caused by fibrous dysplasia suggests that the overexpression of c-fos may represent the first step in the multistep carcinogenesis of bone sarcomas.22
A number of recent reports have linked mutations in both G protein–coupled receptors and G proteins to disease phenotypes,23 but the molecular targets of the activated signal-transduction pathways remain to be identified. The accumulation of intracellular cAMP activates protein kinase A to phosphorylate specific protein substrates and leads to the regulation of gene transcription by modulating the phosphorylation state of cAMP-response-element–binding protein members of the family of transcription factors.24 These nuclear proteins bind specific sequences, termed "cAMP response elements," in the promoter regions of target genes to modulate their transcription.25 The human c-fos promoter contains a functional cAMP response element.26 The increased production of cAMP in bone cells with the Gs mutations most likely leads to elevated c-fos expression through the c-fos cAMP response element. Indeed, expression of an exogenous activated Gs protein leads to increased transcription of c-fos.27
Supported by a grant from the Shriners of North America. Dr. Candeliere was the holder of a studentship from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche, and Dr. St.-Arnaud is a Chercheur-Boursier of the Fonds de la Recherche en Santé du Québec.
We are indebted to Dr. B. Morin for providing a bone-biopsy specimen from a patient with fibrous dysplasia, to Mr. Guy Charette for preparing the biopsy sections, to Mr. Mark Lepik and Ms. Jane Wishart for preparing the figures, and to Dr. Hideo Cho from the Tokyo Metropolitan Children's Hospital (Tokyo, Japan), who as a visiting scientist participated in the early phase of this study.
Source Information
From the Genetics Unit, Shriners Hospital (J.P.), and the Departments of Surgery (G.A.C., F.H.G., R.S.) and Human Genetics (F.H.G., R.S.), McGill University — both in Montreal.
Address reprint requests to Dr. St.-Arnaud at the Genetics Unit, Shriners Hospital, 1529 Cedar Ave., Montreal, QC H3G 1A6, Canada.
References
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mutation is present in fibrous dysplasia of bone in the McCune-Albright syndrome. J Clin Endocrinol Metab 1994;79:750-755. [Abstract]
chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 1989;340:692-696. [CrossRef][Medline]
subunit of the stimulatory G protein of adenylyl cyclase in McCune-Albright syndrome. Proc Natl Acad Sci U S A 1992;89:5152-5156. s: chronic transcriptional activation of the c-fos proto-oncogene in endocrine cells. J Biol Chem 1994;269:22663-22671.
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