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Heme oxygenase 1 (HO-1) regulates osteoclastogenesis and bone resorption

Jochen Zwerina^*, Sotiria Tzima, Silvia Hayer^*, Kurt Redlich^*, Oskar Hoffmann, Beatrice Hanslik-Schnabel, Josef S. Smolen^*, George Kollias and Georg Schett^*,1

^* Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria;
Alexander Fleming Biomedical Sciences Research Center, Vari, Greece;
Institute of Pharmacology and Toxicology, University of Vienna, Vienna, Austria; and
Department of Orthopedics and Orthopedic Surgery, Medical University of Vienna, Vienna, Austria

¹Correspondence: Department of Internal Medicine III, Division of Rheumatology, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria. E-mail: georg.schett{at}meduniwien.ac.at

SPECIFIC AIMS

Heme oxygenase-1 (HO-1), the rate-limiting step in heme catabolism, is a negative regulator of inflammation and oxidative stress. Since inflammation is an important trigger of bone loss, we hypothesized that HO-1 might affect bone metabolism and investigated the role of HO-1 on osteoclast formation and bone resorption.

PRINCIPAL FINDINGS

1. Induction of HO-1 inhibits osteoclast formation and bone resorption in vitro
To assess the principal ability of HO-1 to interfere with breakdown of bone, we used in vitro cell culture systems for osteoclastogenesis and bone resorption. Mononuclear cells representing osteoclast precursors were stimulated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-B ligand (RANKL) to differentiate into mature osteoclasts or, upon cultivation on bone slices, to induce the formation of resorption pits. In these cultures, numerous bone resorbing multinucleated osteoclasts were formed that were negative for HO-1. In contrast, addition of hemin, a metabolite of heme, not only leads to a dose-dependent induction of HO-1 via stimulation of MAPK but also to blocked osteoclast formation as well as resorption pit formation in these cultures (Fig. 1 ). Inhibitory effects started at concentrations of 10 µM and complete inhibition of osteoclastogenesis were achieved with hemin concentrations > 25 µM. Inhibition of osteoclastogenesis by hemin was based on a direct effect on the osteoclast precursor cell and was only partially mediated by biliverdin production since biliverdin, in contrast to hemin, did not achieve complete inhibition of osteoclast formation. Hemin inhibited early differentiation of osteoclast precursors into osteoclasts, as seen by the absent up-regulation of differentiation markers such as TRAP, cathepsin K, and the calcitonin receptor. Hemin, however, did not act on mature osteoclasts, as it did not influence apoptosis rate or proliferative capacity of cells. In contrast, hemin-induced increased HO-1 expression led to an unresponsiveness of osteoclast precursors to M-CSF and RANKL through down-regulation of their receptors c-fms and RANK, respectively, as well as downstream signaling molecules such as TRAF-6 and c-fos.

View larger version (44K): [in this window] [in a new window]   Figure 1. Hemin inhibits osteoclastogenesis in vitro. A, B) Dose response analysis: murine spleen cells were cultured with 30 ng/mL M-CSF and 50 ng/mL RANKL and various concentrations of hemin for 5 days. Osteoclasts are stained for TRAP and appear as purple-stained multinucleated cells (original magnification x40). C) Time-response analysis: murine spleen cells were cultured as described above with addition of hemin from days 0–5 (square), days 0–3 (triangle), or days 3–5 (triangle). D) Osteoclast formation from purified CD11b-positive osteoclast precursors upon addition of 30 ng/mL M-CSF and 50 ng/mL RANKL and various concentrations of hemin. E) Bone resorption assay on bovine cortical slices showing toluidine blue-stained resorption pits with various concentrations of hemin. Original magnification x40. F) Quantification of the area covered by resorption pits. All data are given as mean ± SE. *Significant difference (P<0.05).

2. Induction of HO-1 blocks inflammation-triggered osteoclast formation and bone loss in vitro and in vivo
To extend these findings on inflammatory bone loss and to identify a potential relevance of HO-1 for bone breakdown in vivo, we investigated the potential of HO-1 to block osteoclast formation and bone resorption triggered by proinflammatory cytokines. Considering that TNF is a major inducer of osteoclast differentiation, we stimulated osteoclast precursors from human tumor necrosis factor transgenic (hTNFtg) mice with M-CSF and RANKL with or without hemin (Fig. 2 A). Despite enhanced osteoclast differentiation in cells from hTNFtg mice, hemin achieved a dose-dependent blockade of osteoclast formation. After subperiosteal injection of lipopolysaccharide into calvarial bones of wild-type mice, osteoclast formation, which emerged in conjunction with an inflammatory cellular infiltrate, was dramatically blocked upon systemic treatment with hemin (Fig. 2B ). These data suggest that induction of HO-1 by hemin was able to interfere with osteoclast formation in vivo. To further define the role of HO-1 in inflammatory bone resorption in vivo, we treated hTNFtg mice, which develop a severe erosive arthritis with COPP, a strong activator of HO-1. Systemic treatment significantly inhibited osteoclast formation and bone resorption in the affected joints, resulting in preserved joint structure (Fig. 2C ).

View larger version (35K): [in this window] [in a new window]   Figure 2. Hemin inhibits inflammation-driven osteoclastogenesis in vitro and in vivo. A) Dose response analysis: spleen cells from human tumor necrosis factor transgenic (hTNFtg) mice were cultured with 30 ng/mL M-CSF, 50 ng/mL RANKL, and hemin for 5 days. Osteoclasts were stained for TRAP and quantified. Original magnification x40. B) Wild-type mice were injected subperiostally in the midline calvaria with saline or 25 mg/kg LPS. 20 mg/kg hemin was given i.p. 1 day before LPS injection. After 5 days, calvarial bones were assessed for osteoclast formation by TRAP staining and quantified by measuring osteoclast numbers per millimeter bone surface. C) 7-wk-old hTNFtg mice were treated 3 times daily i.p. with PBS or 20 mg/kg CoPP. After 1 wk, mice were killed and hind paws were quantitatively assessed by TRAP staining for osteoclast formation and area of bone erosion. All data are given as mean ± SE. *Significant differences (P<0.05).

3. Evidence for a role of HO-1 in bone resorption of human disease
Stimulated by the therapeutic potential of HO-1, we searched for evidence that HO-1 plays a role in breakdown of bone in human disease. When human joints from patients with rheumatoid arthritis receiving joint replacement surgery were assessed for the expression of HO-1, we found that the majority of osteoclasts attached to bone erosions are negative for HO-1, whereas other cells within the inflammatory infiltrate frequently express this enzyme. This is well in line with our previous in vitro data showing that osteoclasts once formed are negative for HO-1 and that induction of HO-1 inhibits differentiation of osteoclast precursors. When investigating a population of 148 RA patients, we found evidence that bilirubin, a metabolite resulting from heme degradation by HO-1, is associated with a bone erosive state of RA. Bilirubin levels were significantly lower in RA patients with bone erosions than in RA patients with no structural damage, suggesting that the activity of HO-1 may regulate bone erosion in RA.

CONCLUSIONS AND SIGNIFICANCE

In this study, we investigated the influence of HO-1 on osteoclastogenesis in vitro and in vivo. Our results suggest that 1) induction of HO-1 inhibits osteoclastogenesis in vitro and in vivo; 2) this effect is mediated through down-regulation of surface receptor levels of c-fms and RANK and reduced intracellular expression of TRAF-6 and c-fos leading to impaired osteoclast differentiation; and 3) activity of HO-1 appears to regulate inflammatory bone destruction through its effects on osteoclastogenesis.

HO-1 is the rate-limiting first step of heme degradation leading to generation of biliverdin, which is then further processed to bilirubin. For this reason, HO-1 is considered an important regulatory element of antioxidative processes. Since HO-1 is induced by oxidants and mediators of inflammation, the role of HO-1 has recently gained scientific interest in in vivo models of inflammatory diseases. Thus, induction of HO-1 was reported to have beneficial effects on experimental models of glomerulonephritis. Moreover, HO-1 modifies the severity of LPS-induced inflammatory response. The anti-inflammatory role of HO-1 has been addressed in models that deal with human diseases, such as lupus erythematosus and rheumatoid arthritis, which are typically associated with bone loss. It is unclear, however, whether HO-1 actually affects bone. Excessive iron deposition seems to be a trigger for bone loss, since patients with hemochromatosis have a low bone mass. Our experiments suggest a direct link between HO-1 and bone resorption.

Hemin dose-dependently up-regulated HO-1 in osteoclast precursors and inhibited osteoclast differentiation and thus bone resorption. Hemin affected osteoclastogenesis directly, through up-regulation of HO-1 via MAPK-mediated mechanisms in osteoclast precursors, and leads to a blockade of early osteoclast differentiation. Blocked osteoclastogenesis was due to decreased expression of the M-CSF receptor c-fms, the RANKL receptor RANK, as well as reduced expression of TRAF-6 and c-fos. These signals are essentially required for osteoclast differentiation. By interfering with c-fms and RANK, hemin-induced up-regulation of HO-1 thus appears to render osteoclast precursors inert for further differentiation to the osteoclast lineage.

Hemin blocked TNF-mediated osteoclastogenesis in vitro and in vivo. TNF is a major cofactor for osteoclast differentiation and favors bone loss in diseases such as rheumatoid arthritis, which combines inflammation and bone loss. In human RA, osteoclasts are found at sites of tissue invasion and the current hypothesis suggests that osteoclasts emerge from synovial inflammatory tissue, which provides signals allowing their differentiation from monocytic precursors. Bone loss in experimental models of arthritis resembling human RA depends on osteoclast formation, and inhibition of osteoclastogenesis is a powerful tool to prevent arthritic bone destruction. We observed that TNF-mediated osteoclastogenesis is dose-dependently blocked by hemin. LPS-triggered bone loss, which depends on the formation of proinflammatory cytokines, was highly sensitive to hemin treatment in vivo. These data suggest a central role of HO-1 to interfere with inflammatory bone loss. Indeed, treatment of arthritic TNF-overexpressing mice with COPP, another potent inducer of HO-1, not only impaired osteoclastogenesis but also resulted in less structural damage in diseased joints.

In joint samples from patients with RA, we found numerous osteoclasts attached to bone. These cells were usually HO-1 negative, indicating that the emergence of osteoclasts depends on the lack of expression of HO-1. When assessing a population of RA patients, we found that the serum levels of bilirubin, a metabolite of heme degradation by HO-1, are associated with the bone erosive state of these patients. It thus appears that high activity of HO-1, as manifested by a high serum level of bilirubin, protects from bone erosion in arthritis. These data underline the important regulatory role of HO-1 gained from the Framingham study, which has shown a that high bilirubin level, possibly due to high HO-1 activity, protects from cardiovascular disease. It remains to be clarified, however, whether therapeutic stimulation of HO-1 is a feasible tool to inhibit osteoclastogenesis and bone resorption in human inflammatory disease.

We have shown that induction of HO-1 negatively regulates osteoclastogenesis in vitro and in vivo. There is evidence for a regulation of inflammatory bone loss by HO-1 in human disease. The data presented in conjunction with previous evidence of the anti-inflammatory potential of HO-1 induction may open new therapeutic avenues for RA.

View larger version (42K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4278fje;

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