ACR Meeting Abstracts

ACR Meeting Abstracts

Abstract Number: 35

Hydrogen Sulfide Inhibits Human Osteoclast Differentiation in Vitro By Triggering Sustained Antioxidant Response and Inhibiting the RANKL/OPG Ratio

Francesco Grassi1, Laura Gambari2, Andrea Facchini3 and Gina Lisignoli4, 1LAB. RAMSES, ISTITUTO ORTOPEDICO RIZZOLI, BOLOGNA, Italy, 2Lab. RAMSES, ISTITUTO ORTOPEDICO RIZZOLI, BOLOGNA, Italy, 3Lab of Immunorheumatology and Tissue Regeneration, ISTITUTO ORTOPEDICO RIZZOLI, BOLOGNA, Italy, 4LAB. OF IMMUNORHEUMATOLOGY AND TISSUE REGENERATION, ISTITUTO ORTOPEDICO RIZZOLI, BOLOGNA, Italy

Meeting: 2014 ACR/ARHP Annual Meeting

Keywords: , , ,

Session Information

Title: Biology and Pathology of Bone and Joint: Osteoclasts, Osteoblasts and Bone Remodeling

Session Type: Abstract Submissions (ACR)

Background/Purpose

Hydrogen sulfide (H2S) has been recently appreciated as a novel gasotransmitter with an important role in the regulation of tissues and organs. H2S is produced endogenously in mammalian cells from L-cysteine mainly by the enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) Several correlations were established between low levels of plasmatic H2S, reduced activity of H2S-generating enzymes and diseases, including erosive bone diseases, leading to the hypotheses that disregulation of H2S levels may be critical in the onset of specific diseases. However, the role of H2S in the regulation of bone homeostasis  has been scarcely investigated. Circulating osteoclasts (OCs) precursors are a population of clinical relevance in erosive bone diseases, such as rheumatoid arthritis or psoriatic arthritis, and a potential pharmacological target.

Our objective was to investigate the role of H2S in the regulation of OCs differentiation and function in vitro, by both direct effect on OCs precursors and indirect regulation of mesenchymal stem cells (MSC).

Methods

human monocytes were isolated and differentiated into osteoclasts  in the presence of M-CSF (10ng/ml) and RANKL (75ng/ml) and variable concentrations of NaHS, an H2S donor. TRAP assay and pit assay were performed to evaluate either h-OCs differentiation or “bone” resorption. h-OCs precursors were tested for cellular apoptosis (Annexin V/ PI staining),  acute toxicity (LDH assays), ROS production (DCF staining), mRNA and/or protein expression of NRF2, and NQO1 and PRDX1 (RT-PCR, IHC analyses). RANKL and OPG expression were evaluated by Real-Time PCR in human MSC stimulated with NaHS. GraphPad Prism5 software was used for statistical analysis.

Results

exogenous H2S (100-300 μM)  significantly inhibited the differentiation and function of h-OCs without affecting cell viability. H2S inhibited RANKL-induced ROS production in OC precursors; moreover, H2S induced significant nuclear translocation of NRF2, the master regulator of antioxidant response, and significantly increased mRNA and protein expression of PRDX-1 and NQO1,  two key NRF-2-dependent antioxidant genes. Furthermore, siRNA-based inhibition of NRF2 in OC precursors completely prevented the NaHS-dependent inhibition of OCs differentiation, showing that NRF2 is critical for H2S regulation of OCs differentiation. Finally, NaHS stimulation (100mM) significantly inhibited the RANKL/OPG mRNA ratio in human MSC, a key marker of the OCs-supporting ability of these cells.

Conclusion

Our findings show that H2S inhibits OC differentiation by both direct and MSC-mediated mechanisms, suggesting a possible therapeutical role of H2S donors in erosive bone diseases.

Disclosure:

F. Grassi,
None;

L. Gambari,
None;

A. Facchini,
None;

G. Lisignoli,
None.

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