Pagets disease of bone (PDB) is the second most common metabolic bone disorder, after osteoporosis. Optineurin (OPTN) gene, which is located within the PDB6 locus, and appears to be upregulated by TNFα and NF-kB, has been found to be associated with PDB in several European populations with PDB in genome-wide association studies. Several nucleotidic variations in OPTN were previously associated with PDB and may contribute to PDB pathogenesis. Recently, we have identified in the DNA of a patient with PDB, a SNP (RV-9906 G/A) in the proximal promoter of OPTN that could affect the transcriptional activity of this gene. In this work we have analysed the basal activity of the OPTN promoter and whether RV -9906 altered the activity of that promoter, and consequently OPTN transcription. DNA fragments of 1100 bp with and without the variant, were cloned in a reporter vector, and used in transient transfection assays with HEK293 cells. We also performed co-transfections using some of the transcription factors predicted by in silico analysis to bind the OPTN promoter in the presence or absence of RV-9906 Sp1, RAR and RXR. Our results showed that RV -9906 was responsible for a decrease in OPTN promoter associated to a new Sp1 binding site created in the presence of the A allele. We also showed that both RAR and RXR bind to the OPTN promoter construct used although this binding was not affected by the presence of the RV-9906 (either the G or A allele). Since OPTN was described as having a putative inhibitory role on NF-kB pathway, our results suggest that the A variant of this SNP, found in a PDB patient, by decreasing the activity of OPTN transcription, could activate the NF-kB signalling and contribute to PDB pathogenesis. This work also provides new insights into OPTN gene regulation.
17 May 2014 - 20 May 2014