The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 7 3321-3326
Copyright © 2003 by The Endocrine Society
A Mutation Affecting the Latency-Associated Peptide of TGFß1 in Camurati-Engelmann Disease Enhances Osteoclast Formation in Vitro
Neil W. A. McGowan,
Heather MacPherson,
Katrien Janssens,
Wim Van Hul,
Julie C. Frith,
William D. Fraser,
Stuart H. Ralston and
Miep H. Helfrich
Department of Medicine and Therapeutics (N.W.A.M., H.M., J.C.F., S.H.R., M.H.H.), University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom; Department of Medical Genetics (K.J., W.V.H.), University of Antwerp, 2610 Antwerp, Belgium; and Department of Clinical Chemistry (W.D.F.), University of Liverpool, L69 3GA Liverpool, United Kingdom
Address all correspondence and requests for reprints to: Dr. Miep Helfrich, Department of Medicine and Therapeutics, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. E-mail: m.helfrich{at}abdn.ac.uk.
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Abstract
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Camurati-Engelmann disease (CED) is a rare autosomal dominant disorder characterized by bone pain and osteosclerosis affecting the diaphysis of long bones. CED is caused by various missense mutations in the TGFB1 gene that encodes TGFß1, the most common of which is an arginine-cysteine amino acid change at codon 218 (R218C) in the latency-associated peptide domain of TGFß1. We studied osteoclast formation in vitro from peripheral blood mononuclear cells obtained from three related CED patients harboring the R218C mutation, in comparison with one family-based and several unrelated controls. Osteoclast formation was enhanced approximately 5-fold (P < 0.001) and bone resorption approximately 10-fold (P < 0.001) in CED patients, and the increase in osteoclast formation was inhibited by soluble TGFß type II receptor. Total serum TGFß1 levels were similar in affected and unaffected subjects, but concentrations of active TGFß1 in conditioned medium of osteoclast cultures was higher in the three CED patients than in the unaffected family member. We concluded that the R218C mutation increases TGFß1 bioactivity and enhances osteoclast formation in vitro. The activation of osteoclast activity noted here is consistent with clinical reports that have shown biochemical evidence of increased bone resorption as well as bone formation in CED.
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Introduction
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CAMURATI-ENGELMANN DISEASE (CED; OMIM no. 131300) is a rare, autosomal dominant bone dysplasia characterized by increased bone turnover and osteosclerosis affecting the diaphysis of long bones. The disease typically presents in early childhood with pain, muscular weakness, and waddling gait, and in some cases, other features such as exophthalmos, facial paralysis, hearing difficulties, and loss of vision. To date, seven different mutations have been reported in CED, and most of these cluster at the C terminus of the latency-associated peptide (LAP) of TGFß1 (1, 2). TGFß1 is synthesized as a precursor, consisting of a signal peptide, the LAP region, and mature TGFß1. The full-length precursor molecule dimerizes and undergoes proteolytic cleavage to yield the three subdomains. First, the signal peptide is cleaved off, and this is followed by cleavage of the LAP from the mature peptide, which continues to remain associated with LAP until subjected to specific conditions of activation. Because many of the mutations that cause CED cluster around the region of LAP, which participates in dimerization, it has been suggested that CED mutations activate TGFß1 function by reducing the ability of the LAP to remain associated with mature TGFß1.
Although TGFß1 has traditionally been considered to be a stimulator of bone formation (3, 4), recent work indicates that it is also a potent stimulator of osteoclast formation and bone resorption in vitro (5, 6, 7). In view of this, we studied the ability of peripheral blood mononuclear cells (PBMCs) from CED patients harboring the R218C mutation to form osteoclasts in vitro in response to receptor activator of nuclear factor 
B ligand (RANKL) and macrophage colony-stimulating factor.
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Subjects and Methods
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Subjects
Blood samples were obtained from four individuals (three affected, one unaffected) from a CED kindred harboring an R218C mutation in the TGFß1 LAP and from 32 healthy controls. Two of the CED patients had presented in childhood with typical features of the disease including bone pain and muscular weakness. Patient 1 (a woman aged 29 yr at the time of evaluation) had received alternate-day prednisolone therapy 5–10 mg, between the ages of 8 and18 yr, whereas patient 2 (a man aged 38 yr at the time of evaluation) had received similar doses of prednisolone between the ages of 15 and 18 yr. Their father (patient 3, aged 78 yr at the time of evaluation) had radiographic signs of CED but had been relatively asymptomatic and had not required any specific therapy for the disease. Radiographs confirmed the presence of typical diaphyseal osteosclerosis of the long bones, but spine radiographs in patients 1 and 2 showed evidence of osteopenia (Fig. 1
). In keeping with the radiographic features, bone density measurements at the spine and hip (Norland XR-36) in two of the affected subjects showed greatly increased values at the femoral neck [bone mineral density (BMD) Z-score: patient 1, +3.9; patient 2, +2.5] and greatly reduced BMD values at the lumbar spine (BMD Z-score: patient 1, -3.05; patient 2, -2.32).
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FIG. 1. Example of spine and pelvic radiograph from CED patient. The radiographs shown are from patient 1 and illustrate the typical osteosclerotic lesions of CED in both femorae (A, arrows) with osteopenia in the spine (B).
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Osteoclast cultures
Blood samples were obtained with informed consent, and the study was approved by the Grampian Research Ethics Committee. PBMCs were isolated from heparinized venous blood using standard techniques and suspended in 
MEM supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Paisley, UK). The PBMCs were cultured on dentine slices at a density of 2.5–5 x 105 cells per well and incubated for 21 d in the presence of RANKL 10–100 ng/ml (Insight Biotechnology, Middlesex, UK); MCSF 20 ng/ml (R&D Systems, Abingdon, UK), 1,25(OH)2-vitamin D3 10-8 M; dexamethasone 10-8 M; and prostaglandin E2 10-8 M (all from Alexis Biochemicals, Nottingham, UK) as previously described (8). Experiments were also carried out in the presence of recombinant human (rh) TGFß1 (R&D Systems), a monoclonal neutralizing antibody to TGFß1 (R&D systems, cat. no. MAB240), and recombinant human soluble type II TGFß receptor (rhTGFß-sRII, cat. no. 241-R2). In these experiments, rhTGFß1 was used at concentrations of 0.1–10 ng/ml; TGFß1 antibody at 10 µg/ml, and rhTGFß-sRII at 1 µg/ml. In all experiments, half of the culture medium was removed and replaced with new medium containing fresh cytokines and other reagents (such as blocking antibodies or rhTGFß-sRII) every 3 d. Conditioned medium was pooled and stored frozen at -80 C for biochemical analysis. At the end of the culture period, actively resorbing osteoclasts were visualized by staining for the presence of F-actin rings with TRITC-labeled phalloidin (Sigma, Poole, Dorset, UK), followed by identification of all osteoclasts by immunostaining for the vitronectin receptor (VNR) with monoclonal antibody 23C6 (a kind gift from Dr. Michael Horton, University College, London, UK). Bone resorption was quantified by reflected light microscopy after removal of cells by immersing the dentine slices in a 20% solution of sodium hypochlorite.
Measurements of active and total TGFß1
Measurement of active TGFß1 in conditioned medium from osteoclast cultures was carried out using the Duoset human TGFß1 ELISA (R&D Systems; cat. no. DY240). Total TGFß1 was measured in 1:5 dilutions of conditioned medium using the same assay, following acid activation of the TGFß1 using 1 N HCl, followed by neutralization with 1.2 N NaOH and 0.5 M HEPES according to the manufacturer’s instructions. Measurement of total TGFß1 levels in patient sera were measured using the Quantikine hTGFß1 ELISA (R&D Systems; cat. no. DB100).
Statistical analyses
The data were analyzed with Minitab version 12 using t test; general linear model ANOVA and one-way ANOVA as appropriate. Between-group comparisons in ANOVA were made using Dunnett’s posttest.
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Results
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Osteoclasts did not form in the absence of RANKL in either CED patients or controls, but RANKL-stimulated osteoclast formation and bone resorption was greatly increased in all three CED patients when compared with three age- and sex-matched controls, one of whom was an unaffected family member (Fig. 2, A and B). Furthermore, pooled data from the CED patients studied showed an 18-fold increase in bone resorption at a RANKL concentration of 50 ng/ml, compared with 32 healthy volunteers (P < 0.001, Table 1). Representative osteoclast cultures from a CED patient are shown in Fig. 3A (VNR-positive cells), 3C (F-actin rings), and 3E (bone resorption) and for a normal control in Fig. 3B (VNR-positive cells), 3D (F-actin rings), and 3F (bone resorption).
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FIG. 2. Osteoclast formation and bone resorption is enhanced in CED. PBMCs were isolated from three CED patients with the R218C mutation and three controls (one unaffected family member and two healthy age- and sex-matched volunteers) and cultured in the presence of RANKL and other reagents (see Subjects and Methods). A, Osteoclast formation, as assessed by F-actin rings was enhanced in CED at all RANKL concentrations (P < 0.001) with significant differences between CED and controls at 50 and 100 ng/ml RANKL. A significant increase in the number of VNR-positive cells was also observed (P < 0.001, data not shown). B, The increase in osteoclast formation was accompanied by a highly significant increase in the area resorbed at all RANKL concentrations with significant differences between CED and controls at 50 and 100 ng/ml RANKL.
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FIG. 3. Representative photomicrographs from osteoclast cultures in CED patients and controls. Representative photomicrographs illustrating the increase in osteoclast formation in a CED patient (A, C, and E), compared with a control (B, D, and F). A and B, VNR-positive cells (bar, 50 µm). C and D, F-actin ring positive cells (bar, 50 µm). E and F, Areas of bone resorption (dark gray) on dentine slices visualized by reflected light microscopy.
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Addition of rhTGFß1 to control cultures increased osteoclast formation and bone resorption in a concentration-dependent manner such that values for osteoclast formation in control cultures approached those observed in CED cultures at a concentration of 10 ng/ml TGFß1 (Fig. 4A). Addition of monoclonal antibody to TGFß1 at a final concentration of 10 µg/ml had no significant inhibitory effect on osteoclast formation and bone resorption in cultures from CED patients (data not shown) but significantly inhibited osteoclast formation in RANKL-stimulated cultures of healthy controls stimulated with 10 ng/ml TGFß1 (Fig. 4B). Addition of rhTGFß-sRII (1 µg/ml) to CED cultures significantly inhibited osteoclast formation in CED patients, and there was a trend for inhibition of osteoclast formation when the same concentration rhTGFß-sRII was added to cultures of unaffected controls (Fig. 5).
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FIG. 4. Effects of TGFß1 and TGFß1 neutralizing antibody on osteoclast formation from control PBMCs. A, PBMCs were isolated from two healthy volunteers (one unaffected CED family member and one age- and sex-matched control) and cultured for 21 d in the presence of rhTGFß1 at the concentrations noted along with RANKL at 100 ng/ml and other reagents (see Subjects and Methods). Osteoclast formation (as assessed by F-actin ring and VNR-positive cells) was significantly enhanced, compared with control without TGFß1 (one-way ANOVA with Dunnett’s post hoc test) in the presence of 10 ng/ml rhTGFß1. At this concentration osteoclast formation was similar to that observed in CED patients at the same concentration of RANKL. B, PBMCs were isolated from a healthy volunteer and cultured in quadruplicate for 21 d in the presence of RANKL at 100 ng/ml and other reagents (control) along with 10 ng/ml rhTGFß1 (TGFß1) and 10 ng/ml rhTGFß1 plus neutralizing antibody to TGFß1 at 10 µg/ml (TGFß1 + TGFß1 mAB). The monoclonal antibody to TGFß1 significantly inhibited rhTGFß1 stimulated osteoclast formation to below the levels observed in control cultures.
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FIG. 5. The rhTGFß-sRII inhibits osteoclast formation in PBMC cultures prepared from CED patients. PBMCs were isolated from two healthy volunteers (Control) and three patients with the R218C mutation (CED) and cultured for 21 d in the presence of RANKL at 100 ng/ml and other reagents (see Subjects and Methods) in the presence or absence of rhTGFß-sRII at a concentration of 1 µg/ml. Osteoclast formation was significantly reduced in CED cultures, and there was a nonsignificant trend for reduction in control cultures. Values are expressed as mean ± SD percent of the values obtained in non-rhTGFß-sRII containing cultures.
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Measurements of active and total TGFß1 concentrations were made in conditioned medium from PBMC cultures prepared from three affected CED patients and one unaffected family member. The levels of active TGFß1 in tissue culture medium with 10% FCS were undetectable. Concentrations of active TGFß1 in culture supernatants from the three patients were 270.1 pg/ml, 77.2 pg/ml, and 355.1 pg/ml, whereas the level in culture supernatant from the one unaffected family member was 19.0 pg/ml. It was not possible to accurately measure total TGFß levels in tissue culture supernatants because of the high levels found in medium with 10% FCS alone. Concentrations of total TGFß1 in the patient sera were 26.8 ng/ml, 34.1 ng/ml, and 45.6 ng/ml, respectively (mean ± SD = 35.8 ± 9.95 ng/ml). These values did not differ significantly from those observed in 20 normal controls, including the unaffected family member (mean ± SD = 45.5 ± 11.3 ng/ml).
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Discussion
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The present study has shown that RANKL-induced osteoclast formation is greatly enhanced in CED patients carrying the R218C mutation, compared with family-based and unrelated controls. The PBMCs from CED patients formed increased numbers of osteoclasts, compared with PBMCs from normal controls over a range of RANKL concentrations, effectively shifting the RANKL concentration response curve to the left, with a difference between CED patients and controls that was equivalent to the presence of 10 ng/ml TGFß1 in the cultures. In agreement with the findings of previous workers (6, 7), osteoclasts did not form in the absence of RANKL, neither in CED patients nor controls, indicating that TGFß1 cannot substitute for RANKL in stimulating osteoclast differentiation and function.
The observations reported here complement and add to the results of previous studies that indicate that the TGFß1 mutations in CED activate TGFß1 signaling. Saito et al. (9) reported that growth of MG-63 cells was enhanced by coculture with CED fibroblasts, consistent with enhanced release of bioactive TGFß1 from the CED cells. The R218C expression constructs produced less of the small latent form of TGFß1 than wild-type constructs, indicating that the mutation results in production of a LAP molecule that does not associate well with the rest of the complex (9). We have also found that human embryo kidney 293 cells transfected with R218C expression constructs activate Smad signaling in vitro, compared with wild-type and they secrete increased amounts of active but not total TGFß1 into the culture medium (10).
In this study, levels of active TGFß1 in conditioned medium were higher in CED osteoclast cultures than in those from the unaffected family member, whereas no difference was found in total TGFß1 levels in serum between patients and controls. Addition of rhTGFß-sRII to PBMC cultures of CED patients significantly inhibited osteoclast formation, supporting the notion that the increased osteoclast generation was mediated by increased TGFß1 signaling as a result of higher levels of active TGFß1. To our surprise, osteoclast formation in CED cultures was not inhibited by a monoclonal antibody to TGFß1. The reasons for this are unclear because the antibody inhibited osteoclast formation induced by 10 ng/ml TGFß1 in normal cultures and was previously found to reverse inhibition of cell growth mediated by transfection of the R218C mutant into fibroblasts (9). Possible explanations for the differences between the TGFß1 antibody and soluble receptor might include different kinetics of antibody degradation vs. degradation of soluble receptor in the osteoclast cultures or differences in the ability of the antibody and soluble receptor to inhibit the mutant TGFß1 in the relatively acidic environment of osteoclast cultures.
How does the finding of increased osteoclast activity fit with the clinical presentation of CED with diaphyseal osteosclerosis? Although CED is characterized by a net increase in bone mass in some regions of the skeleton, bone resorption is also increased, as reflected by biochemical markers of bone resorption that are increased 3- to 10-fold above the normal range in affected patients (11). This indicates that the activating mutations of TGFß1 that cause CED result in a state of increased bone turnover, rather than just an increase in bone formation. In this regard, it is of interest that two of the CED patients reported here showed evidence of marked osteoporosis in the lumbar spine while also exhibiting typical osteosclerotic lesions in the diaphysis of the long bones. This indicates that the skeletal response to the TGFß1 mutations that cause CED may differ at different skeletal sites. These differences probably are due to the fact that TGFß1 has complex effects on bone remodeling, with actions on cells of both the osteoblast and osteoclast lineage (12).
Both stimulatory (5, 6, 7, 13, 14) and inhibitory (13, 15, 16, 17, 18) effects of TGFß1 on osteoclast formation and bone resorption have been reported in different model systems. This discrepancy appears to be because the stimulatory effects of TGFß1 on osteoclast activity are counteracted in some experimental systems by increased production of osteoprotegerin and decreased expression of RANKL by osteoblasts and stromal cells (19, 20, 21). Presumably, the localization of osteosclerotic lesions to the diaphysis of the long bones with sparing of the axial skeleton or axial osteoporosis (as noted here) is due to local differences in RANKL/osteoprotegerin production and/or amounts of TGFß1 released from bone matrix during the process of bone resorption. Such a scenario has already been documented for transgenic mice overexpressing TGFß2, which exhibit increased bone loss in some areas of the skeleton and increased bone formation in others (22, 23). Further work using transgenic models that reproduce the CED mutations will probably be required to further address this issue. Whatever the explanation for the site specific differences in patterns of bone involvement, the data presented here, when combined with previous work, indicate that R218C is an activating mutation (9, 10), which up-regulates the formation and activity of osteoclasts generated from peripheral blood cells of CED patients in vitro. This rather unexpected finding emphasizes the importance of increased bone resorption as well as increased bone formation as a pathogenic feature of CED and raises the possibility that treatment with antiresorptive drugs such as bisphosphonates might be worth investigating in this condition.
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Acknowledgments
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We are grateful to Claire Clarkin and Dr. Rob van’t Hof for help with the analysis of bone resorption.
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Footnotes
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This work was supported by grants from the Arthritis Research Campaign of the United Kingdom (studentship RO558 to N.W.A.M., fellowship H535 to M.H.H., and ICAC grant RO544 to S.H.R.). K.J. holds a predoctoral research position with the Fonds voor Wetenschappelijk Onderzoek (FWO).
Present address for N.W.A.M.: Department of Craniofacial Development, King’s College, London, United Kingdom.
Present address for H.M.: Department of Comparative and Developmental Genetics, MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom.
Abbreviations: BMD, Bone mineral density; CED, Camurati-Engelmann disease; FCS, fetal calf serum; LAP, latency-associated peptide; PBMC, peripheral blood mononuclear cell; RANKL, receptor activator of nuclear factor B ligand; rh, recombinant human; rhTGFß-sRII, recombinant human soluble type II TGFß receptor; VNR, vitronectin receptor.
Received April 9, 2002.
Accepted March 21, 2003.
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Abstract
Introduction
