RANKL treatment releases the negative regulation of the poly(ADP-ribose) polymerase-1 on Tcirg1 gene expression during osteoclastogenesis.

Abstract

UNLABELLED:

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 approximately 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.

• Authors: Guillaume E Beranger, David Momier, Nathalie Rochet, Danielle Quincey, Jean-Marie Guigonis, Michel Samson, Georges F Carle, Jean-Claude Scimeca
• Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research (J Bone Miner Res) Vol. 21 Issue 11 Pg. 1757-69 (Nov 2006) ISSN: 0884-0431 [Print] United States
• PMID: 17002555 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Atp6ap1 protein, mouse
• RANK Ligand
• RNA, Messenger
• Poly(ADP-ribose) Polymerases
• Vacuolar Proton-Translocating ATPases

Topics

• Amino Acid Sequence
• Animals
• Base Sequence
• Cell Line
• Cell Nucleus (metabolism)
• Gene Expression Regulation
• Mice
• Molecular Sequence Data
• Osteoclasts (metabolism)
• Poly(ADP-ribose) Polymerases (metabolism)
• Promoter Regions, Genetic
• RANK Ligand (physiology)
• RNA, Messenger (metabolism)
• Transcription, Genetic
• Vacuolar Proton-Translocating ATPases (biosynthesis, genetics)