de la Peña Lourdes Serrano, Billings Paul C, Fiori Jennifer L, Ahn Jaimo, Kaplan Frederick S, Shore Eileen M
Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6081, USA.
J Bone Miner Res. 2005 Jul;20(7):1168-76. doi: 10.1359/JBMR.050305. Epub 2005 Mar 7.
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.
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.
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.
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.
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.
进行性骨化性纤维发育不良（FOP）是一种骨骼肌逐渐被骨替代的疾病。FOP淋巴细胞是探索这些患者中骨形态发生蛋白（BMP）信号通路的模型系统，其在BMPRIA内化方面存在缺陷，且下游信号激活增加，这表明BMP受体运输改变是该疾病异位骨形成的基础。
进行性骨化性纤维发育不良（FOP）是一种严重致残的疾病，其特征为结缔组织进行性异位骨化。尽管这种疾病的基因缺陷和病理生理学仍然不明，但BMP4 mRNA和蛋白过度表达，并且在FOP患者的培养淋巴细胞中，一部分分泌型BMP拮抗剂的mRNA未在适当水平合成。这些数据表明BMP信号改变与该疾病有关。在本研究中，我们调查这种异常是否与淋巴细胞中BMP受体功能缺陷有关。
通过荧光激活细胞分选（FACS）对细胞表面蛋白进行定量。通过免疫沉淀和免疫印迹分析蛋白磷酸化。通过[35S]甲硫氨酸标记和脉冲追踪分析检测蛋白合成和降解。通过逆转录聚合酶链反应（RT-PCR）检测mRNA。
与对照细胞相比，FOP淋巴细胞在细胞表面表达的I型BMP受体（BMPRIA）水平高6倍，并且在配体刺激下BMPRIA的内化和降解显著减少。此外，在对照细胞中，BMP4处理增加BMPRIA磷酸化，而在FOP细胞中BMPRIA显示出对配体不敏感的组成型磷酸化。我们的数据还支持p38丝裂原活化蛋白激酶（MAPK）信号通路是这些细胞系中的主要BMP信号通路，并且BMP信号的转录靶点DNA结合与分化抑制因子1（ID-1）在FOP细胞中的表达增强。
这些数据扩展了我们之前对FOP淋巴细胞中BMP4信号失调的观察结果，并表明BMPRIA在细胞表面的过量表达和组成型磷酸化与受体内化缺陷有关。BMP受体运输改变可能在FOP发病机制中起重要作用。
