RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis
Open Access
- 1 November 2006
- journal article
- research article
- Published by Oxford University Press (OUP) in Journal of Bone and Mineral Research
- Vol. 21 (11), 1757-1769
- https://doi.org/10.1359/jbmr.060809
Abstract
The Tcirg1 gene encodes the osteoclast‐specific a3 isoform of the V‐ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP‐1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. Introduction: The TCIRG1 gene encodes the a3 isoform of the V‐ATPase a subunit, and mutations at this locus account for ∼60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4‐kb region upstream of the transcription starting point. Materials and Methods: The promoter activity of serial‐deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross‐linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. Results: The −3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to −1589 retained this response to RANKL. A deletion up to −1402 induced a 3‐fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the −1589/−1402 region, a 10‐nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL‐treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP‐ribose) polymerase‐1 (PARP‐1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA‐binding domain of PARP‐1 on RANKL treatment. Finally, both PARP‐1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP‐1 activity induced an increase of a3 mRNA expression. Conclusions: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP‐1 protein to its promoter region in mouse pre‐osteoclast. On RANKL treatment, PARP‐1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function.Keywords
This publication has 56 references indexed in Scilit:
- Inhibition of Hormone and Cytokine-stimulated Osteoclastogenesis and Bone Resorption by Interleukin-4 and Interleukin-13 Is Associated with Increased Osteoprotegerin and Decreased RANKL and RANK in a STAT6-dependent PathwayPublished by Elsevier BV ,2006
- BSP and RANKL Induce Osteoclastogenesis and Bone Resorption SynergisticallyJournal of Bone and Mineral Research, 2005
- Specific Binding of Poly(ADP-ribose) Polymerase-1 to Cruciform HairpinsJournal of Molecular Biology, 2005
- Osteoclast signalling pathwaysBiochemical and Biophysical Research Communications, 2005
- Regulation of α-Synuclein Expression by Poly (ADP Ribose) Polymerase-1 (PARP-1) Binding to the NACP-Rep1 Polymorphic Site Upstream of the SNCA GeneAmerican Journal of Human Genetics, 2005
- YY1 is involved in RANKL-induced transcription of the tartrate-resistant acid phosphatase gene in osteoclast differentiationGene, 2004
- Analysis of Nucleotide Sequence-Dependent DNA Binding of Poly(ADP-ribose) Polymerase in a Purified SystemBiochemistry, 2003
- Osteoclast differentiation and activationNature, 2003
- a4, a Unique Kidney-specific Isoform of Mouse Vacuolar H+-ATPase Subunit aPublished by Elsevier BV ,2001
- Proteinase expression during differentiation of human osteoclasts in vitroJournal of Cellular Biochemistry, 2000