Nuclear Accumulation of Prohibitin 1 in Osteoarthritic Chondrocytes Down‐Regulates PITX1 Expression

Abstract

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.

E2F1 and TFDP1 Regulate PITX1 Expression in Normal and Osteoarthritic Articular Chondrocytes

“…RNA extraction, cDNA synthesis, and real-time RT-PCR analysis were performed, as previously described [6]. Primer sequences are listed in Table 1.…”

“…Chromatin immunoprecipitation (ChIP) using primary chondrocytes or C28/I2 cells and anti-E2F1 antibodies was performed, as previously described [6]. …”

“…We recently demonstrated that the downregulation of PITX1 is due to the nuclear accumulation of Prohibitin 1 [6]. To further understand how PITX1 regulation could be associated with the progression of osteoarthritis, we investigated potential positive regulators.…”

Prohibitin Ligands in Cell Death and Survival: Mode of Action and Therapeutic Potential

“…Further evidence that PHB1 is a putative therapeutic target for treating IBD comes from the observations that adenovirus-directed administration by enema and nanoparticle-based colonic delivery of PHB1 effectively enhanced the levels of PHB1 at the surface of colonic epithelial cells and alleviated colitis induced by dextran sodium sulfate (Theiss et al, 2011). Picard et al (2013) recently demonstrated that PHB1 accumulates in the nucleus of osteoarthritic chondrocytes, where it represses the expression of the transcription factor PITX1, suggesting an involvement of PHB1 in the etiology of osteoarthritis.…”

Ret finger protein-like 3 promotes tumor cell growth by activating telomerase reverse transcriptase expression in human lung cancer cells

“…The streptavidin-agarose pulldown assay is a useful and feasible approach for analyzing the binding of an array of proteins on DNA sequence [ 15 – 17 ]. The method combined with high-throughput proteomics can generate an effective screening system to indentify the novel DNA sequence binding proteins [ 18 – 21 ]. Using this technology, we previously discovered novel expression regulating mechanisms of carcinogenic genes COX-2 [ 22 – 24 ].…”