We previously reported a loss-of-PITX1 expression in patients suffering of knee/hip osteoarthritis (OA). Search for the mechanism underlying this event led us to discover that PITX1 repression was triggered by the aberrant nuclear accumulation of Prohibitin (PHB1), an E2F1 co-repressor, in OA articular chondrocytes. In the current study, we assessed in details the involvement of E2F transcription factors in regulating PITX1 expression. We also analyzed other genes that are similarly regulated by E2F in regard to osteoarthritis. The transcriptional regulation of the PITX1 promoter by E2F1 was analyzed with the luciferase reporter assay, and chromatin immunoprecipitation assays, which confirmed direct E2F1-PITX1 interactions. The probable binding sites for E2F1 in the PITX1 promoter were identified by DNA pulldown experiments. In silico and in vitro analyses show that the PITX1 proximal promoter region contains 2 specific sequences that are bound by E2F1. Overexpression of E2F1 enhances PITX1 promoter activity and mRNA transcription. In primary control and osteoarthritis chondrocytes, real time RT-PCR was used to measure the mRNA expression levels of candidate genes under E2F1 transcriptional control. Transcription Factor Dp-1 (TFDP1) knockdown experiments confirmed that the E2F1-TFDP1 complex regulates PITX1. Knockdown of TFDP1, an E2F1 dimerization partner, inhibits the activating effect of E2F1 and reduces both PITX1 promoter activity and mRNA transcription. Real time RT-PCR results reveal reduced expression of TFDP1 and a similar downregulation of their targets PITX1, BRCA1, CDKN1A, and RAD51 in mid-stage OA chondrocytes. Collectively, our data define a previously uncharacterized role for E2F1 and TFDP1 in the transcriptional regulation of PITX1 in articular chondrocytes. Additional E2F1 targets may be affected in OA pathogenesis.

Objective To decipher the molecular mechanisms down-regulating PITX1 expression in primary osteoarthritis (OA). Methods 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 transcriptionpolymerase 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. Results 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. Conclusion Collectively, these data define an unrecognized role for PHB1 in repressing PITX1 expression in OA chondrocytes.

Introduction: AIS is characterized by severe spinal deformity with an unknown cause. Studies in Pitx1+/- mice reveal a similar phenotype, which prompted us to study the role of Pitx1 and its regulation in AIS. Hypoxia down-regulates Pitx1 expression in certain cell types, which lead us to investigate its role in the regulation of Pitx1 in AIS.

The adolescent idiopathic scoliosis (AIS) is the most common deformity arising during childhood. Although its aetiology remains unknown, we have demonstrated a differential dysfunction of melatonin signalling through Gi proteins in osteoblasts isolated from AIS patients, leading to their stratification into three functional subgroups. Herein we examine the molecular profiles of these AIS groups using the functional classification developed by Moreau et al.

Presently, the genetic cause of adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, remains unclear. Among many hypotheses, the neuroendocrine hypothesis involving a melatonin deficiency as the source for AIS generated the greatest interest and controversy since no decrease in circulating melatonin level has been observed in a majority of studies. Previously, we have reconciled the role of melatonin in AIS by demonstrating a melatonin signaling dysfunction occurring in osteoblasts derived from AIS patients, which contrasted with similar cells isolated from healthy subjects. We found that this difference is caused in AIS cells by increased phosphorylation of serine residues affecting the activity of G inhibitory proteins normally associated with melatonin cell surface receptors. Here we propose a preliminary molecular classification of patients with AIS based on the cellular response to the melatonin (cAMP) and distinct protein-protein interactions. These interactions include those between protein kinase C delta (PKCdelta) and MT2 melatonin receptors or PKCdelta and the receptor for activated protein C kinase 1. This finding could help in future molecular classification of patients with AIS.

Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis that affects a significant number of young teenagers, mainly females (0.2-6 % of the population). Historically, several hypothesis were postulated to explain the aetiology of AIS, including genetic factors, biochemical factors, mechanics, neurological, muscular factors and hormonal factors. The neuroendocrine hypothesis involving a melatonin deficiency as the source for AIS has generated great interest. This hypothesis stems from the fact that experimental pinealectomy in chicken, and more recently in rats maintained in a bipedal mode, produces a scoliosis. The biological relevance of melatonin in idiopathic scoliosis is controversial since no significant decrease in circulating melatonin level has been observed in a majority of studies. Analysis of melatonin signal transduction in musculoskeletal tissues of AIS patients demonstrated for the first time a defect occurring in a cell autonomous manner in different cell types isolated from AIS patients suffering of the most severe form of that disease. These results have led to a classification of AIS patients in three different functional groups depending on their response to melatonin, suggesting that the cause of AIS involves several genes. Molecular analysis showed that melatonin signaling dysfunction is triggered by an increased phosphorylation of Gi proteins inactivating their function. This discovery has led to development of a first scoliosis screening assay. This test, using blood sample, is currently in clinical validation process in Canada and could be used for screening children at high risk of developing AIS.

La scoliose est la déformation la plus fréquente en orthopédie pédiatrique. Elle survient principalement à l'adolescence affectant les filles en plus grand nombre et avec le plus de sévérité. Nos travaux ont mis en évidence un défaut dans la transmission du signal de l'hormone mélatonine chez des patients gravement atteints de scoliose idiopathique de l'adolescent (SIA). Cette anomalie est causée par la phosphorylation de résidus sérine au niveau des protéines G inhibitrices (Gi) couplées aux récepteurs à mélatonine. Nous avons observé chez les patients SIA trois types de réponses anormales en présence de mélatonine ce qui suggère que ce défaut est attribuable à des gènes distincts en cause dans cette voie de signalisation. Cette découverte nous a permis de mettre au point un premier test de dépistage précoce à partir d'une simple prise de sang. Actuellement en cours de validation clinique au Canada, ce test devrait permettre l'identification des enfants porteurs du risque de développer une SIA