Dysregulation of the BMP-p38 MAPK signaling pathway in cells from patients with fibrodysplasia ossificans progressiva (FOP).
FOP is a disabling disorder in which skeletal muscle is progressively replaced with bone. Lymphocytes, our model system for examining BMP signaling, cannot signal through the canonical Smad pathway unless exogenous Smad1 is supplied, providing a unique cell type in which the BMP-p38 MAPK pathway can be examined. FOP lymphocytes exhibit defects in the BMP-p38 MAPK pathway, suggesting that altered BMP signaling underlies ectopic bone formation in this disease.


BACKGROUND
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the primary genetic defect in this condition is unknown, BMP4 mRNA and protein and BMP receptor type IA (BMPRIA) protein are overexpressed in cultured lymphocytes from FOP patients, supporting that altered BMP signaling is involved in this disease. In this study, we examined downstream signaling targets to study the BMP-Smad and BMP-p38 mitogen-activated protein kinase (MAPK) pathways in FOP.


METHODS
Protein phosphorylation was assayed by immunoblots, and p38 MAPK activity was measured by kinase assays. To examine BMP target genes, the mRNA expression of ID1, ID3, and MSX2 was determined by quantitative real-time PCR. Statistical analysis was performed using Student's t-test or ANOVA.


RESULTS
FOP lymphocytes exhibited increased levels of p38 phosphorylation and p38 MAPK activity in response to BMP4 stimulation. Furthermore, in response to BMP4, FOP cells overexpressed the downstream signaling targets ID1 by 5-fold and ID3 by 3-fold compared with controls. ID1 and ID3 mRNA induction was specifically blocked with a p38 MAPK inhibitor, but not extracellular signal-related kinase (ERK) or c-Jun N-terminal kinase (JNK) inhibitors. MSX2, a known Smad pathway target gene, is not upregulated in control or FOP cells in response to BMP, suggesting that lymphocytes do not use this limb of the BMP pathway. However, introduction of Smad1 into lymphocytes made the cells competent to regulate MSX2 mRNA after BMP4 treatment.


CONCLUSIONS
Lymphocytes are a cell system that signals primarily through the BMP-p38 MAPK pathway rather than the BMP-Smad pathway in response to BMP4. The p38 MAPK pathway is dysregulated in FOP lymphocytes, which may play a role in the pathogenesis of FOP.
Fiori
Jennifer L
JL
Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6081, USA.
Billings
Paul C
PC
de la Peña
Lourdes Serrano
LS
Kaplan
Frederick S
FS
Shore
Eileen M
EM
D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000378:metabolism; D051796:Inhibitor of Differentiation Proteins / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D009221:Myositis Ossificans / Q000209:etiology / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction; D051898:Smad1 Protein / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D048051:p38 Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C497453:ID1 protein, human; D051798:Inhibitor of Differentiation Protein 1; D051796:Inhibitor of Differentiation Proteins; C084119:MSX2 protein; D009363:Neoplasm Proteins; D012333:RNA, Messenger; C497399:SMAD1 protein, human; D051898:Smad1 Protein; C075640:ID3 protein, human; D048051:p38 Mitogen-Activated Protein Kinases