Poly(adp-ribose) polymerase-1 regulates Tracp gene promoter activity during RANKL-induced osteoclastogenesis.
The Tracp gene encodes an acid phosphatase strongly upregulated during osteoclastogenesis on RANKL treatment. Using the mouse osteoclastic model RAW264.7, we studied Tracp gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis.


BACKGROUND
The Tracp gene encodes an acid phosphatase strongly expressed in differentiated osteoclasts. TRACP enzyme has a dual role and is involved in (1) the regulation of the biological activity of the bone matrix phosphoproteins osteopontin and bone sialoprotein and (2) the intracellular collagen degradation. Based on our previous work on Tcirg1 gene expression, and using data available in the literature, we focused on a 200-bp sequence located upstream the Tracp gene transcriptional start to identify binding activities.


METHODS
We first performed siRNA transfections and RAW264.7 cell treatment with an inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) activity. After EMSA and supershift experiments, we measured the promoter activity of wildtype and mutant constructs throughout the osteoclastic differentiation.


RESULTS
We first showed that depleting PARP-1 mRNA in the pre-osteoclastic cell line RAW264.7 results in an increase of both matrix metalloproteinase 9 and TRACP mRNA expression (3.5- and 2.5-fold, respectively). Moreover, in response to 3-aminobenzamide treatment, we measured a weak stimulation of MMP9 mRNA expression, whereas up to a 2-fold enhancement above the control condition of TRACP mRNA expression was observed. We next identified in the -839/-639 Tracp promoter region a PARP-1 binding site, and supershift experiments showed the interaction of a PARP-1 binding activity with the Tracp promoter sequence -830/-808. Finally, RAW264.7 cell transfection with a promoter construct mutated for this PARP-1 interacting sequence showed the functionality of this site within intact pre-osteoclastic cells.


CONCLUSIONS
In this study, we provide evidence that the transcriptional activity of the Tracp gene, in pre-osteoclastic cells, is negatively regulated by the binding of PARP-1 protein to a potential consensus sequence located in its promoter region. Taken together with our previous results related to the control of Tcirg1 gene expression, our data suggest that PARP-1 exerts a pivotal role in the basal repression of genes that are upregulated during RANKL-induced osteoclastogenesis.
Beranger
Guillaume E
GE
GéPITOs, Université Nice Sophia-Antipolis, CNRS, Nice, France.
Momier
David
D
Rochet
Nathalie
N
Carle
Georges F
GF
Scimeca
Jean-Claude
JC
D000135:Acid Phosphatase / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000235:genetics*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D010010:Osteoclasts / Q000166:cytology*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D000071681:Tartrate-Resistant Acid Phosphatase; D014158:Transcription, Genetic
D007527:Isoenzymes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D053245:RANK Ligand; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C505689:Tnfsf11 protein, mouse; D011065:Poly(ADP-ribose) Polymerases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase