RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis

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 crosslinking, 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.

RANKL Up-regulates Brain-type Creatine Kinase via Poly(ADP-ribose) Polymerase-1 during Osteoclastogenesis

“…In HeLa cells, PARP-1 has been shown to inhibit estrogen receptor ␣-activated gene transcription (39,40). In BMMs, PARP-1 was found to inhibit the expression of TRACP and Tcirg1 genes (32)(33)(34). Consistent with these observations, we demonstrated that PARP-1 bound directly to the ckb promoter region.…”

“…The binding sequence TTCCCA, which was responsible for the negative regulation of ckb by PARP-1, is similar to the PARP-1 consensus element identified by Beranger et al (32)(33)(34) on TRACP and Tcirg1 genes. PARP-1 binding sequences have been reported to regulate other genes, such as CATTCCT in the cardiac troponin T gene promoter (42) and TTGAXXACAA in the human T cell leukemia virus type I gene promoter (50).…”

“…This region contains TTCCCA, the known PARP-1 consensus sequence within the TRAP and Tcirg1 promoter regions (32)(33)(34)). An EMSA assay using a probe (Ϫ271/ Ϫ246 bp) containing the putative PARP-1 binding element demonstrated a specific binding (Fig.…”

Inflammatory osteolysis: a conspiracy against bone

“…While it has yet to be studied in the context of RA, PARP1 proteolysis occurs during osteoclastogenesis (98,99,112,113). A proteolytically stable PARP1 mutant antagonizes osteoclastogenesis, whereas PARP1 deficiency enhances this process (107, 114), indicating that PARP1 negatively regulates osteoclast differentiation.…”

Positive Transcriptional Regulation of the Human μ Opioid Receptor Gene by Poly(ADP-ribose) Polymerase-1 and Increase of Its DNA Binding Affinity Based on Polymorphism of G−172 → T

“…those for the murine inducible nitric-oxide synthase gene (5Ј-AAT-TATAATTT-3Ј), the rat Reg gene (5Ј-TGCCCCTCCCAT-3Ј), the mouse Tcirg1 gene (5Ј-TTCCCACAGC-3Ј), the human SNCA gene [5Ј-(TC) 10 (T) 2 (TC) 10 (TA) 8 (CA) 11 -3Ј], and the human SMARCB1 gene (5Ј-CTTTTGTTTGAGCTGCGGCG-CGCGCGTC-3Ј) (29,(31)(32)(33)(34). We speculate from these reports that PARP-1 tends to bind to the CCCC sequence like GC-box, but it is difficult to forecast the appropriate PARP-1-binding region from DNA sequences, as with other transcription factors.…”