Fibrodysplasia Ossificans Progressiva (FOP), a Disorder of Ectopic Osteogenesis, Misregulates Cell Surface Expression and Trafficking of BMPRIA

Authors

  • Lourdes Serrano de la Peña,

    1. Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
    Current affiliation:
    1. Department of Genetics, Nelson Biological Laboratories, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA
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  • Paul C Billings,

    1. Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
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    • These authors contributed equally to this work

  • Jennifer L Fiori,

    1. Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
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    • These authors contributed equally to this work

  • Jaimo Ahn,

    1. Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
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  • Frederick S Kaplan,

    1. Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
    2. Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
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    • These authors contributed equally to this work

  • Eileen M Shore PhD

    Corresponding author
    1. Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
    2. Department of Genetics; University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
    • University of Pennsylvania, Department of Orthopaedic Surgery, 424 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104-6081, USA
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    • These authors contributed equally to this work


  • These data were presented in part at the 24th, 25th, and 26th Annual Meetings of the American Society for Bone and Mineral Research held, respectively, in San Antonio, TX, on September 20-24, 2002, Minneapolis, MN, on September 19-23, 2003, and Seattle, WA, on October 1-5, 2004.

  • The authors have no conflict of interest.

Abstract

FOP is a disorder in which skeletal muscle is progressively replaced with bone. FOP lymphocytes, a model system for exploring the BMP pathway in these patients, exhibit a defect in BMPRIA internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease.

Introduction: Fibrodysplasia ossificans progressiva (FOP) is a severely disabling disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the genetic defect and pathophysiology of this condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs for a subset of secreted BMP antagonists are not synthesized at appropriate levels in cultured lymphocytes from FOP patients. These data suggest involvement of altered BMP signaling in the disease. In this study, we investigate whether the abnormality is associated with defective BMP receptor function in lymphocytes.

Materials and Methods: Cell surface proteins were quantified by fluorescence-activated cell sorting (FACS). Protein phosphorylation was assayed by immunoprecipitation and immunoblotting. Protein synthesis and degradation were examined by [35S]methionine labeling and pulse-chase assays. mRNA was detected by RT-PCR.

Results: FOP lymphocytes expressed 6-fold higher levels of BMP receptor type IA (BMPRIA) on the cell surface compared with control cells and displayed a marked reduction in ligand-stimulated internalization and degradation of BMPRIA. Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation, whereas BMPRIA showed ligand-insensitive constitutive phosphorylation in FOP cells. Our data additionally support that the p38 mitogen-activated protein kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell lines and that expression of inhibitor of DNA binding and differentiation 1 (ID-1), a transcriptional target of BMP signaling, is enhanced in FOP cells.

Conclusions: These data extend our previous observations of misregulated BMP4 signaling in FOP lymphocytes and show that cell surface overabundance and constitutive phosphorylation of BMPRIA are associated with a defect in receptor internalization. Altered BMP receptor trafficking may play a significant role in FOP pathogenesis.

INTRODUCTION

FIBRODYSPLASIA OSSIFICANS PROGRESSIVA (FOP) is a rare genetic disease of heterotopic osteogenesis.(1,2) Extraskeletal bone formation in FOP begins with the appearance of large painful swellings associated with lymphocytic infiltration into skeletal muscle, followed by death of muscle cells and the development of highly vascular fibro-proliferative tissue that matures into bone through an endochondral pathway.(3,4) Heterotopic ossification leads to ankylosis of all major joints of the axial and appendicular skeleton severely restricting joint movement.(5) In FOP, the bone-forming process is normal, but the inductive events leading to bone formation are temporally and spatially inappropriate. Physical or surgical trauma to FOP patients can induce formation of heterotopic bone and has restricted the ability to safely obtain FOP tissue samples.(5–9) Consequently, most studies have been limited to the analysis of peripheral blood cells that can be safely obtained from patients.

Most occurrences of FOP are attributed to spontaneous mutations, although several FOP families show an autosomal dominant pattern of inheritance.(10,11) The rarity of multigenerational inheritance of FOP has precluded identification of the causative gene by a positional cloning approach. FOP patients do not exhibit any karyotypic anomalies, and the nature of FOP mutations remains unknown. Because of the constraints on the study of this disease, a candidate gene approach has been used to identify genes that could have altered expression and/or function in FOP.(11,12)

BMPs, members of the TGF-β family of proteins, are potent osteogenic morphogens capable of inducing ectopic bone formation in animal models.(13–17) Recombinant BMPs induce mesenchymal cells to differentiate to bone through cellular events that parallel the stages of bone formation in FOP lesions.(3,18,19) BMPs interact with specific receptors in heterodimeric membrane receptor complexes that phosphorylate downstream effector molecules. Several combinations of type I (BMPRIA, BMPRIB, or ActRI) and type II (BMPRII or ActRII) receptors have been reported to mediate the BMP4 signaling pathway.(20–22) After ligand binding, constitutively activated type II BMP receptors phosphorylate type I BMP receptors.(23,24) The activated type I BMP receptor subsequently phosphorylates intracellular proteins of at least two signaling cascades: the Smad pathway and the p38 mitogen-activated protein kinase (MAPK) pathway.(25) Both pathways regulate nuclear transcriptional activity through co-factor association in a cell and developmental-specific context.(26)

BMP signaling is modulated through negative feedback loops involving regulation of ligand expression as well as BMP antagonist expression.(22,27) In FOP lymphocytes, we found increased expression of BMP4 mRNA and protein as well as insufficient BMP4-induced BMP antagonist expression.(28–31) These data suggested that a disruption of BMP signaling occurs in these cells and prompted us to examine the expression and activity of BMP4-receptor complexes.

MATERIALS AND METHODS

Cell culture

Peripheral blood samples were obtained after informed consent from FOP patients and unaffected individuals in accordance with institutional guidelines and after Institutional Review Board approval. Lymphoblastoid cell lines (LCLs) were established from peripheral blood mononuclear cells by transformation with Epstein-Barr virus as described previously(28) and were grown in Roswell Park Memorial Institute 1640 medium (RPMI; Invitrogen) supplemented with 15% FCS. Four FOP and four control cell lines were examined in all experiments except as noted. To assess cell proliferation rates, cells were harvested and counted at defined time periods. Other experiments, except as indicated, were performed with cells at the exponential phase of the growth curve. Before cytokine treatment, cells were washed in PBS and grown overnight in serum-free RPMI medium. Treatment with BMP4 (100 ng/ml; R&D Systems), Noggin (400 ng/ml; a gift of Regeneron Pharmaceuticals), or lipopolysaccharides (LPS; 1 μg/ml; Sigma) was in serum-free medium. For p38 MAPK inhibition, cells were pretreated with 1 μg SB203580 (Calbiochem) for 1 h and then with BMP4 as above.

RNA isolation and RT-PCR

Total RNA was isolated from LCLs with Trizol Reagent (Invitrogen) following the recommended protocol. Reverse transcription used 5 μg of total RNA, 50 ng oligo-dT primers (Promega), and Superscript reverse transcriptase II (Invitrogen), following the recommended protocol. PCR reactions used specific primer pairs, 2 μl of cDNA product, and Advantage 2 polymerase mix (Clontech). Optimal cycling number for linear amplification was determined for each target by sampling the PCR reactions every 5 cycles through a range of at least 15-30 cycles. For ActRII: forward, 5′-GCTGACTTTGGCTTGGCTGTTC-3′ and reverse, 5′-TCCTGGGCTTAGATGCTTGACTC-3′ at 58°C annealing for 28 cycles; for BMPRIA: forward, 5′-AAGGTGACAGTACACAGGAAAC-3′ and reverse, 5′-CACTGTTGTCCGTAAGGTCT-3′ at 58°C annealing for 30 cycles; for GAPDH: forward, 5′-CAGCCTCAAGATCATCAGCAAT-3′ and reverse, 5′-ACCCTGTTGCTGTAGCCAAAT-3′ at 55°C annealing for 18 cycles; for BMP4: forward, 5′-CCAGTCATTCCAGCCCACATC-3′ and reverse, 5′-AGGGGCTTCCACCGTATAAACA-3′ at 65°C annealing for 30 cycles. PCR products were electrophoresed through agarose gels. BMP4 and GAPDH mRNA (Fig. 1) were quantitated by phosphorimager after staining with Vistra Green. BMP4 mRNA levels were normalized to GAPDH mRNA.

Figure FIG. 1..

Cell proliferation and BMP4 mRNA expression. FOP and control lymphoblastoid cell lines (LCLs) were plated at 0.5 × 106 cell/ml (day 0). At 24-h intervals, cells were counted, and 5 μg of RNA was used for cDNA synthesis. (A) Growth curves of representative control and FOP cell lines are shown. (B) RT-PCR analysis of BMP4 mRNA in four FOP and four control cell lines. BMP4 mRNA expression is shown relative to day 1.

ID-1 mRNA levels were quantitated by real-time RT-PCR (ABI Prism 7000 Cycler). Primers were designed (Primer Express; Applied Biosystems) for ID-1; forward, 5′-GGTGGAGATTCTCCAGCACG-3′ and reverse, 5′-TCCAACTGAAGGTCCCTGATG-3′; GAPDH: forward, 5′-AGATCATCAGCAATGCCTCCTG-3′ and reverse, 5′-ATGGCATGGACTGTGGTCATG-3′. PCR reactions were performed in triplicate with 50 nM of each primer and SYBR Green Master Mix (ABI) at 95°C for 10 minutes to denature, and then 40 cycles of 95°C for 15 s and 60°C for 1 minute.

Protein purification

LCLs were lysed for 40 minutes on ice in RIPA buffer (PBS, 1% NP-40, 0.5% Na-deoxycholate) containing Protease Inhibitor cocktail (Sigma), 10 μg/μl phenylmethylsulfonylfluoride (PMSF), 1 mM Na-Orthovanadate, and 0.1 mM NaF. Protein was quantified by BCA protein assay (Pierce) using BSA as a standard.

Metabolic labeling

Proteins were labeled in methionine-free RPMI (ICN Biomedicals) containing 50 μCi/ml Easytag [35S]Methionine (NEN/Perkin Elmer Life Sciences) and 1% FCS at 37°C for 0.5-24 h. Labeled proteins were chased in complete RPMI medium with 5% FCS.

Fluorescence-activated cell sorter analysis

LCLs were resuspended in PBS + 1% BSA (PBSB; 106 cells/0.1 ml) on ice. Cells were blocked with preimmune IgG (Sigma) for 10 minutes. Biotinylated BMP receptor antibodies (BMPRIA/Alk3, BMPRIB/Alk6, and BMPRII; R&D Systems/ActRII; U.S. Biological) were added directly to the blocking mix for 40 minutes, detected with streptavidin (SA)-fluorochrome conjugate in PBSB for 20 minutes, and fixed in 0.5 ml PBS/1% paraformaldehyde. CD3/CD19 (markers for mature human T [CD3+] and B [CD19+] lymphocytes) were detected with a Simultest kit (BD Biosciences) following the recommended protocol to verify uniform B-cell populations in FOP and control cell lines. Antibody-bound cells were analyzed with a Becton Dickinson FACSCalibur Flurocescence-Activated Cell Sorter (FACS), in the Abramson Cancer Center Flow Cytometry and Cell Sorting Shared Resource at the University of Pennsylvania.

Immunoprecipitation and Western blots

Aliquots of RIPA cell extracts (250-500 μg protein) were diluted in 1 ml RIPA buffer, precleared with preimmune IgG and Protein A/G agarose (Santa Cruz Biotechnology), and incubated with primary antibody (1 h, 4°C) and Protein A/G agarose (overnight, 4°C). The Protein A/G-immune complexes were recovered, and precipitated proteins were resuspended in SDS-gel loading buffer and size fractionated on Novex SDS-polyacrylamide gels (Invitrogen) under reducing conditions before transfer to nitrocellulose. Membranes were blocked in PBST (PBS, 0.1% Tween 20) containing 5% milk, and specific proteins were detected with primary antibody in PBST, 0.3% BSA (overnight, 4°C). For anti-phosphothreonine antibodies (Santa Cruz Biotechnology), membranes were blocked and incubated with primary antibody in PBST containing 1% milk/1% BSA, incubated with anti-IgG-horseradish peroxidase (HRP) conjugate (1 h, room temperature), and developed with ECL + chemiluminescence reagents (Amersham Biosciences).

For Smad1 phosphorylation analysis, nuclear and cytoplasmic cell fractions were extracted using the NE-PER system (Pierce) and quantitated by BCA assay (Pierce). Immunoblotting used primary antibodies to phosphorylated Smad1 (Upstate), histone H1 (Santa Cruz), and BiP/GRP78 (BD Biosciences) and secondary antibodies to IgG-HRP.

RESULTS

Relative BMP4 mRNA expression levels and cell growth

In addition to being potent osteo-inductive agents, BMPs have also been associated with growth arrest and apoptosis in several human cell types, including B cells,(32) leading us to study the growth rate of lymphocytes cultured from FOP patients. Cells from affected and unaffected individuals were plated at an initial concentration of 0.5 × 106 cell/ml, and cell proliferation was assessed over a period of 4 days. Under these conditions, the maximum density was observed at day 4, with a cell concentration of ∼2 × 106 cell/ml. No significant difference in the growth rate between four FOP and four control cell lines was observed (Fig. 1A).

BMP4 mRNA expression was assayed by RT-PCR to determine the steady-state levels of BMP4 at 24-h intervals of the cell growth curve. In control cell lines, BMP4 mRNA levels were constant through the entire growth curve. However, in FOP cell lines, BMP4 message levels increased with increasing cell density. This increase in BMP4 expression was consistent for the four FOP cell lines analyzed (Fig. 1B).

BMP4 receptor expression

FACS analysis was used for identification and relative quantitation of BMP receptors on the cell surface. BMPRII protein was not detectable by FACS on the surface of control and FOP LCLs, although was detected on U-2 OS cells, confirming that the antibody can detect BMPRII when present. ActRII was detected at similar levels on control and FOP LCLs (Fig. 2A). Similar low levels of BMPRIB were detected in both control and FOP cells. BMPRIA was also detected at the cell surface in both FOP and control cells; however, the amount of BMPRIA was 6-fold higher on FOP cells by FACS analysis compared with cells from unaffected individuals (Figs. 2B and 2C).

Figure FIG. 2..

Cell surface expression of BMP receptors. BMP receptors were detected in control and FOP LCLs with biotin-labeled BMP receptor antibodies and streptavidin-FITC; receptors were quantitated by FACS analysis. Representative FACS histograms are shown for (A) ActRII and (B) BMPRIA. Cell number (y-axis) is plotted against BMPRIA signal level (x-axis). Isotype background (IgG biotin + avidin-FITC) is indicated (I). (C) The average linear value of relative quantitation of BMPRIA protein of four FOP and four control LCLs is shown. MFI, mean fluorescent intensity.

Steady-state mRNA levels for BMPRIA and ActRII receptors were evaluated by RT-PCR. Similar ActRII mRNA levels in FOP and control LCLs are consistent with the similar levels of ActRII protein detected. In comparison, no differences in BMPRIA mRNA levels were observed between affected and unaffected individuals (Fig. 3), despite clear differences in BMPRIA protein levels at the cell surface (Fig. 2).

Figure FIG. 3..

BMPRIA and ActRII mRNA expression. BMP receptor mRNAs were detected by RT-PCR analysis using RNA from four control and four FOP cell lines. PCR products were electrophoresed through agarose gels and stained with ethidium bromide. GAPDH mRNA served as a control.

BMP4 receptor complexes

BMPRIA complexes with BMPRII or ActRII(20); however, the composition of the BMP receptor heterodimers in lymphocytes has not been reported. Our identification of BMPRIA and ActRII on the surface of these cells (Fig. 2) suggested that BMPRIA/ActRII is a functional BMP4 receptor complex in lymphocytes.

To investigate this question, BMPRIA was immunoprecipitated from total cell protein extracts with a specific antibody and immunoblotted using ActRII and BMPRII antibodies to study the type II receptor components of the BMPRIA receptor complexes. ActRII co-immunoprecipitated with BMPRIA in both FOP and control lymphocytes (Fig. 4), with ActRII detected at higher amounts in FOP lymphocytes compared with control cells, presumably as a consequence of the higher levels of BMPRIA surface protein on FOP cells. The same amount of protein and an excess amount of specific antibody were used in each experiment. No co-immunoprecipitation of BMPRII with BMPRIA was observed (data not shown). These results identify BMPRIA/ActRII as a receptor complex for BMP4 in these cells.

Figure FIG. 4..

Identification of BMPRIA-ActRII receptor complexes in LCLs. BMPRIA protein was immunoprecipitated from total cell extracts of four control and four FOP cell lines and immunoblotted to detect co-immunoprecipitated ActRII (85 kDa).

BMPRIA protein synthesis and turnover

Several mechanisms, such as anomalies in receptor protein synthesis or degradation, could give rise to increased BMPRIA protein on the surface of FOP cells despite similar steady-state levels of BMPRIA mRNA in affected and unaffected cells. To examine receptor synthesis, cellular proteins were metabolically labeled with [35S]methionine, and newly synthesized BMPRIA was detected by immunoprecipitation from total cell extracts at defined time-points. BMPRIA was synthesized at similar rates in control and FOP lymphocytes (Fig. 5A).

Figure FIG. 5..

BMPRIA synthesis and degradation. At the indicated time-points, BMPRIA protein was immunoprecipitated from total cell protein extracts and analyzed by SDS-PAGE. BMPRIA was quantified by phosphorimager scanning. (A) Rates of BMPRIA synthesis in representative FOP (squares) and control (circles) cells. Proteins were labeled with [35S]methionine for 0-24 h. (B) BMPRIA turnover in response to BMP4. Proteins in control and FOP LCLs were steady-state labeled with [35S]methionine for 15 h and chased in the presence (• and ▪) or absence (○ and □) of 100 ng/ml BMP for 0-6 h. Data are from four FOP and four control cell lines.

To study receptor degradation, cell proteins were steady-state labeled with [35S]methionine and incubated in chase medium (without label) in the absence or presence of ligand. At defined time-points during the chase period, BMPRIA was immunoprecipitated from total cell extracts and quantified. No difference in the rate of receptor turnover was observed in control or FOP LCLs in the absence of ligand (Fig. 5B). After treatment of control cells with BMP4, BMPRIA was rapidly degraded; downregulation of BMPRIA occurred within 1 h after BMP4 treatment. In contrast, BMP treatment of FOP cells did not stimulate BMPRIA degradation (Fig. 5B). No significant differences in BMPRIA degradation were observed in the absence or presence of ligand in FOP cells.

Ligand-mediated BMPRIA internalization

To determine whether the observed failure of receptor turnover was caused by a primary defect in receptor degradation or a failure of receptor delivery to the intracellular degradation machinery, we examined ligand-mediated receptor internalization in FOP and control lymphocytes. For these experiments, cycloheximide was used to block new receptor synthesis during BMP4 treatment. Quantification of surface receptor was performed before and after BMP4 treatment using FACS analysis with BMPRIA antibodies. In control cells, BMPRIA was significantly reduced after 1 h in the presence of the ligand (Figs. 6A and 6B). However, in FOP cells, BMP4 treatment did not affect the amount of the surface protein in that period of time (Fig. 6B). These data suggest a defect in ligand-mediated internalization of BMPRIA in FOP lymphocytes.

Figure FIG. 6..

Internalization of BMPRIA in response to BMP4. FACS analysis detects cell surface BMPRIA using biotinylated-BMPRIA antibody and streptavidin-FITC. (A) Representative histogram from a control cell line with (control + BMP) and without (control − BMP) BMP4 treatment (100 ng/ml for 1 h in the presence of cycloheximide). Isotype (I) background (IgG biotin + avidin-FITC) is indicated. The shift to the left of the histogram (arrow) after BMP4 treatment indicates a 1.6-fold decrease of BMPRIA on the cell surface. (B) Fraction of BMPRIA remaining at the cell surface. The data are mean values for four FOP and four control LCLs. Student's t-tests showed a significant difference for controls with vs. without BMP treatment (p < 0.01), but no significance for FOP samples.

BMPRIA phosphorylation in FOP cells

Activation of BMP receptor complexes normally occurs when ligand binding stimulates the type II receptor to phosphorylate the type I receptor. To examine phosphorylation of BMPRIA in LCLs grown in the absence or presence of ligand, BMPRIA was immunoprecipitated from total cell protein extracts and examined by immunoblotting with a phospho-threonine antibody. In control cells, BMPRIA phosphorylation was increased in response to BMP4 treatment. In FOP cells, BMPRIA was phosphorylated at a high level in the absence of the ligand and showed no increase in the level of phosphorylation in response to BMP4 treatment (Fig. 7A).

Figure FIG. 7..

Effect of BMP4 on BMPRIA phosphorylation. FOP or control LCLs were untreated (−) or treated (+) with (A) 100 ng/ml BMP4 or (B) 400 ng/ml Noggin for 30 minutes. BMPRIA was immunoprecipitated from total cell protein extracts and immunoblotted with anti-phospho-threonine antibody to detect phosphorylated BMPRIA (55 kDa). Representative results are shown.

To study whether the insensitivity of FOP lymphocytes to exogenous BMP4 is a consequence of overproduced endogenously expressed BMP4 that constitutively activates the system, we used Noggin protein, a potent BMP antagonist that binds BMP rendering it functionally inactive,(33) to remove any endogenous BMPRIA stimulus. After Noggin treatment, BMPRIA phosphorylation decreased in control cells, as expected, but remained constant in FOP cells (Fig. 7B), thus supporting the conclusion that BMPRIA hyperphosphorylation is independent of ligand stimulation in FOP cells. These data support that promiscuous phosphorylation of BMPRIA occurs in the presence or absence of BMP4 ligand in FOP lymphocytes.

BMP4 downstream signal transduction

BMP signal transduction can occur through the canonical Smad pathway or through p38 MAPK. To evaluate the relative functional activity of BMPRIA in FOP and control LCLs, BMP4 downstream signal transduction was analyzed by immunoblotting total cell protein extracts with antibodies for phosphorylated Smad1 (Fig. 8A). Control C2C12 cells showed enhanced Smad1 phosphorylation in response to BMP4 as expected. In control and FOP LCLs, however, we did not observe increased Smad1 phosphorylation after BMP4 stimulation in any cell line (Fig. 8A). In both control and FOP cells, the relative basal levels of Smad1 phosphorylation were variable.

Figure FIG. 8..

Effect of BMP4 on Smad1 phosphorylation. Cells were untreated (−) or treated (+) with 100 ng/ml BMP4 for 60 minutes. (A) Total proteins were isolated from C2C12 cells and from control and FOP cell lines and electrophoresed by SDS-PAGE and immunoblotted with anti-phospho-Smad1 antibody to detect phosphorylated Smad1 (60 kDa). (B) Nuclear and cytoplasmic protein fractions were separated and examined for the presence of phosphorylated Smad1 as in A. The blots were reprobed with antibodies for the nuclear-specific histone H1 protein (33 kDa) and the cytoplasmic-specific BiP protein (78 kDa) as sample loading controls and markers for cytosolic and nuclear fractions.

To determine whether BMP4 treatment induced translocation of phosphorylated Smad1 to the nucleus, we isolated cytoplasmic and nuclear protein fractions from FOP and control LCLs. Detection of the nucleus-specific protein histone H1 and the cytoplasmic-specific BiP protein confirmed separation of each subcellular fraction. No differences were observed in the levels of phosphorylated Smad1 in either protein fraction (Fig. 8B), suggesting that little or no signaling through the Smad1 pathway occurs in control or FOP lymphocytes.

Despite this apparent inactivity of Smad signaling in response to BMP4, we observed, by quantitative real-time RT-PCR, that BMP4 increases ID-1 mRNA levels in control and FOP LCLs (Fig. 9). BMP4-induced ID-1 mRNA levels in FOP cells were significantly greater than those in control cells. BMP4-induced ID-1 synthesis was effectively blocked by SB203580, a specific inhibitor of p38 MAPK. These data show that the p38 MAPK signaling pathway is a predominant BMP signaling pathway over Smad signaling in both control and FOP LCLs and suggest that activation of the p38 MAPK pathway is downstream of BMPRIA in FOP lymphocytes.

Figure FIG. 9..

BMP4-induced ID-1 expression through the p38 MAPK pathway. Cells were untreated (−) or treated (+) with 100 ng/ml BMP4 for 60 minutes or treated with the p38 MAPK inhibitor SB203580 and BMP4. Levels of ID-1 mRNA were determined by real-time PCR and normalized to GAPDH mRNA.

DISCUSSION

Our previous studies suggested the likelihood of a fundamental disruption of BMP signaling in FOP lymphocytes and pointed to a possible defect in BMP receptor physiology. This study shows that LCLs from FOP patients express increased levels of phosphorylated BMPRIA on their surface in the absence of ligand and exhibit a defect in ligand-stimulated internalization/degradation of this receptor. Our findings further show ligand-independent chronic phosphorylation of BMPRIA and suggest that altered BMP receptor regulation, trafficking, and/or signaling underlie the ectopic bone formation observed in this disease.

We found that BMPRIA was expressed at 6-fold higher levels on the surface of FOP LCLs compared with cells from unaffected individuals. Despite this substantially higher BMPRIA protein level on the cell surface, the rates of protein synthesis and degradation of BMPRIA were similar in both control and FOP cells in the absence of ligand. However, treatment of control cells with BMP4 resulted in rapid internalization/degradation of this receptor, whereas cell surface receptor levels in FOP cells remained constant in the presence of ligand. These data suggest that the proximate cause of overabundant BMPRIA at the cell surface in FOP LCLs is a defect in ligand-mediated receptor internalization.

Reduced BMPRIA internalization in response to BMP4 in FOP lymphocytes is associated with increased phosphorylation of BMPRIA, suggesting an important relationship between receptor endocytic activity and receptor phosphorylation/dephosphorylation. For many single-pass transmembrane receptors, such as BMPRIA, dephosphorylation occurs within the cytoplasm, specifically within endosomal compartments or on the surface of the endoplasmic reticulum.(34,35) Whereas recent studies have provided important details on the relationship between receptor trafficking and signaling in the tyrosine kinase and TGF-β signal transduction pathways,(35–38) very little insight is yet available for similar relationships in the BMP signaling pathway. However, studies on BMP2-mediated signal transduction in C2C12 muscle satellite cells show that osteogenic differentiation is enhanced by inhibition of BMP2/BMP receptor endocytosis.(39) This is consistent with a potential correlation of a BMP receptor internalization defect with enhanced phosphorylation and osteogenic proclivity in FOP cells.

BMPRIA on the surface of FOP lymphocytes is increased relative to levels in control cells, but levels of ActRII, the cognate type II BMP receptor in our experimental LCL assay system, are not correspondingly increased, thereby altering the ratio between hetero/homodimeric type I/II receptor complexes. In contrast to TGF-β receptors that have been reported to be fully homodimeric (type I/type I) in the absence of the ligand, both homo- and heterodimeric (type I/type II) BMP receptor complexes form in the absence of ligand.(23,24) Both heterodimeric and homodimeric combinations of BMP receptors are able to bind ligand, most likely with different affinities,(40) although only the heterodimers are likely to be signaling-competent under normal circumstances.

These findings raise important questions as to how promiscuous signaling is normally prevented in preformed receptor hetero-complexes in the absence of ligand. The ability of such hetero-complexes to remain inactive may be facilitated by the binding of inhibitory proteins that are released or degraded after ligand binding(24) in normal cells. One reasonable postulate is that a mutation in a receptor-associated protein (such as FKBP12, BRAM1, BAMBI, cell surface heparan sulfate proteoglycans, or another as yet undiscovered adaptor protein) may plausibly be deficient in quantity or activity in FOP cells. A cause/effect relationship between promiscuous receptor phosphorylation and the failure of FOP cells to appropriately internalize and degrade BMPRIA is plausible, but not yet proven.

Flexibility in the pattern of receptor oligomerization and receptor-ligand interactions could be an important functional determinant of BMP action, with the anomalous amount and activity of BMPRIA in FOP cells unbalancing this network. Recent studies have shown that the specific pattern of receptor oligomerization preceding ligand binding determines the selection of downstream BMP signal transduction pathways.(41,42) The Smad signaling pathway is activated after BMP binding to preformed BMP type I/II heterodimeric receptor complexes, whereas the p38 MAPK signaling pathway is activated after BMP-induced hetero-oligomerization of homodimeric complexes activates.

Our data strongly suggest that FOP cells harbor a set of ligand-independent phosphorylated BMPRIA/ActRII heterodimers as well as an excessive pool of BMPRIA homodimers capable of being recruited into ligand-stimulated hetero-complexes that signal through p38 MAPK. We do not suggest that Smad signaling plays an unimportant role in FOP, but rather that it does not seem to play an important role in the LCL experimental system that we have used to study BMP signaling. Ligand-independent phosphorylation of BMPRIA in FOP cells suggests that the “primary” BMP receptor trafficking defect in FOP could affect both p38 MAPK and Smad signaling pathways. Although not active in our experimental LCL system, the Smad pathway could be important in other cell types, such as connective tissue cells, muscle cells, peripheral nerve cells, or other physiologic targets of BMP4 pathway overactivity in FOP.(17) However, such cell types are not currently available for analysis because of the restrictions imposed by the natural history of FOP and by the consequent dangers to the patients.

Constitutive phosphorylation of BMPRIA in the absence of exogenous ligand in FOP cells suggests that BMPRIA receptors are constitutively activated in these cells, predicting that transcriptional targets of BMPRIA activation would be overexpressed in FOP LCLs and that little or no additional increase in transcription of these targets would occur on BMP treatment. Unexpectedly, our data show that ID-1 mRNA is not expressed at high basal levels (without ligand) in FOP cells; however, ID-1 transcription is stimulated by treatment with BMP to higher levels in FOP compared with control LCLs. We hypothesize that a subset of BMPRIA receptors is constitutively phosphorylated in FOP cells in the absence of ligand but that these receptors fail to signal because of the lack of an intact BMP-specific Smad signaling pathway in the LCLs. On ligand stimulation of the receptors, robust signaling occurs through the p38 MAPK pathway, which has been reported to be the pathway that mediates BMP signaling by receptor stimulated complexes and is the primary BMP signaling pathway in our LCL system. We propose that it is this ligand-sensitive pool of excessive BMPRIA that fails to properly internalize and degrade in the presence of BMP4 ligand and that is responsible for its excessive downstream signaling in FOP LCLs.

It is important to note that, although lymphocytes are present in the earliest stages of FOP lesion formation, their role as causative factors in the disease process remains uncertain. Nevertheless, the lymphoblastoid cell is a useful model system for studying the BMP pathway in these patients. Our data clearly support that the effects of the underlying genetic mutation in FOP is “unmasked” in FOP LCLs, as reflected by consistent differences in the BMP pathway in FOP and control LCLs. These cells therefore provide a useful system with which to gain insights into this disease.

We previously reported that FOP lymphocytes overexpress BMP4 mRNA and protein and do not appropriately upregulate multiple BMP4 antagonists. In this study, we show that FOP cells are unable to appropriately regulate ambient levels of BMP4 in their environment, suggesting a primary defect in receptor-mediated regulation of BMP4 signaling. Our studies show that BMPRIA levels are substantially higher on the surface of FOP LCLs, that these receptors exhibit ligand-independent phosphorylation, and that the presence of overabundant BMPRIA is caused by the inability of FOP cells to appropriately internalize and degrade BMPRIA. Furthermore, the absence of mutations in the protein coding regions of the BMPRIA gene (unpublished data), coupled with the lack of linkage of the BMPRIA gene to the FOP locus,(11,12) argues against a primary receptor mutation as the cause of FOP.

The data presented here suggest fertile new avenues of study into BMP receptor oligomerization, trafficking, and downstream signaling. Such studies in FOP have the potential to provide important insight into intracellular trafficking of developmental signals that induce normal and ectopic osteogenesis. The difficulty of finding substantial numbers of additional multigenerational FOP families for definitive positional cloning and the inability to safely obtain and establish FOP connective tissue cell lines for parallel analyses further points to the importance of studying the candidate BMP4 signaling pathway in the available LCL system to understand the primary molecular pathogenesis of this disabling and enigmatic disorder of ectopic osteogenesis.

Acknowledgements

The authors thank Dr Phoebe Leboy at the University of Pennsylvania School of Dental Medicine, Dr Mickey Marks at the University of Pennsylvania School of Medicine, and Dr Michael Zasloff at Georgetown University for helpful discussions. We are indebted to Dr Jonni Moore and the members of the Abramson Cancer Center Flow Cytometry and Cell Sorting Shared Resource at the University of Pennsylvania for assistance with FACS analysis. We also thank members of our laboratory, especially Marc Tarrus, Leota Terry, and Meiqi Xu, for invaluable contributions. This research was supported in part by the International FOP Association, the Center for Research in FOP and Related Disorders, the Ian Cali Endowment, the Betty Laue Resource Center, the Stephen Roach-Whitney Weldon Endowment, the Roemex and Grampian Fellowships, the Isaac and Rose Nassau Professorship of Orthopaedic Molecular Medicine, and National Institutes of Health Grant R01-AR41916.

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