Picard Cynthia, Pellicelli Martin, Taheri Maryam, Lavoie Jean-Francois, Doucet Roxanne, Wang DaShen, Bernard Lauriane, Bouhanik Saadallah, Lavigne Patrick, Moreau Alain
Sainte-Justine University Hospital Research Center and University of Montreal, Montreal, Quebec, Canada.
Arthritis Rheum. 2013 Apr;65(4):993-1003. doi: 10.1002/art.37837.
To decipher the molecular mechanisms down-regulating PITX1 expression in primary osteoarthritis (OA).
The functional activity of different PITX1 promoter regions was assessed by luciferase reporter assay. Tandem mass spectrometry coupled to protein sequencing was performed using nuclear extracts prepared from OA chondrocytes, in order to identify proteins bound to DNA regulatory elements. Expression analyses of selected candidate proteins were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry methods, using cartilage sections and articular chondrocytes from non-OA control subjects and patients with OA. Gain-of-function and loss-of-function experiments were performed in normal and OA chondrocytes, respectively, to study their effects on PITX1 regulation. The results were validated by real-time RT-PCR and immunohistochemistry in STR/Ort mice, a well-known animal model of OA.
PITX1 promoter analyses led to the identification of prohibitin 1 (PHB1) bound to a distal E2F1 transcription factor site. Aberrant accumulation of PHB1 was detected in the nuclei of OA articular chondrocytes, and overexpression of PHB1 in control cells was sufficient to inhibit endogenous PITX1 expression at the messenger RNA and protein levels. Conversely, knockdown of PHB1 in OA articular chondrocytes resulted in up-regulation of PITX1. Studies of early molecular changes in STR/Ort mice revealed a similar nuclear accumulation of PHB1, which correlated with Pitx1 repression.
Collectively, these data define an unrecognized role for PHB1 in repressing PITX1 expression in OA chondrocytes.
解读原发性骨关节炎（OA）中下调PITX1表达的分子机制。
通过荧光素酶报告基因检测评估不同PITX1启动子区域的功能活性。使用从OA软骨细胞制备的核提取物进行串联质谱联用蛋白质测序，以鉴定与DNA调控元件结合的蛋白质。使用来自非OA对照受试者和OA患者的软骨切片及关节软骨细胞，通过实时逆转录-聚合酶链反应（RT-PCR）和免疫组织化学方法对选定的候选蛋白进行表达分析。分别在正常和OA软骨细胞中进行功能获得和功能丧失实验，以研究它们对PITX1调控的影响。结果在STR/Ort小鼠（一种著名的OA动物模型）中通过实时RT-PCR和免疫组织化学进行验证。
PITX1启动子分析导致鉴定出与远端E2F1转录因子位点结合的抑制素1（PHB1）。在OA关节软骨细胞核中检测到PHB1异常积累，并且在对照细胞中过表达PHB1足以在信使RNA和蛋白质水平抑制内源性PITX1表达。相反，在OA关节软骨细胞中敲低PHB1导致PITX1上调。对STR/Ort小鼠早期分子变化的研究揭示了类似的PHB1核积累，这与Pitx1抑制相关。
总体而言，这些数据确定了PHB1在抑制OA软骨细胞中PITX1表达方面的一个未被认识的作用。
