Abstract

Heme oxygenase-1 (HO-1; encoded by Hmox1), a downstream target of the Nrf2 transcription factor, has been postulated to be a negative regulator of osteoclasts (OCLs) differentiation. Here, we further explored such a hypothesis by examining HO-1 effects in different stages of osteoclastogenesis. We confirmed the inhibition of the expression of OCLs markers by Nrf2. In contrast, both the lack of the active Hmox1 gene or HO-1 silencing in OCLs precursor cells, bone marrow macrophages (BMMs), decreased their differentiation towards OCLs, as indicated by the analysis of OCLs markers such as TRAP. However, no effect of HO-1 deficiency was observed when HO-1 expression was silenced in BMMs or RAW264.7 macrophage cell line pre-stimulated with RANKL (considered as early-stage OCLs). Moreover, cobalt protoporphyrin IX (CoPPIX) or hemin, the known HO-1 inducers, inhibited OCLs markers both in RANKL-stimulated RAW264.7 cells and BMMs. Strikingly, a similar effect occurred in HO-1−/− cells, indicating HO-1-independent activity of CoPPIX and hemin. Interestingly, plasma of HO-1−/− mice contained higher TRAP levels, which suggests an increased number of bone-resorbing OCLs in the absence of HO-1 in vivo. In conclusion, our data indicate that HO-1 is involved in the response of bone marrow macrophages to RANKL and the induction of OCLs markers, but it is dispensable in early-stage OCLs. However, in vivo HO-1 appears to inhibit OCLs formation.

Highlights

  • Osteoclasts (OCLs) are multinucleated myeloid cells crucial for constant bone remodelling because of their bone-resorbing activity

  • Among the whole population of bone marrow cells (BMCs) in the fresh bone marrow of heme oxygenase-1 (HO-1)−/− mice the percentage of monocytes defined as CD45+Ly6G−Ly6C+CD11b+MHCIIlow/−F4/80low/− was higher in comparison to HO-1+/+ mice (7.43 ± 0.19 vs. 5.26 ± 0.06, respectively, Fig. 1A)

  • A lower percentage of HO-1−/− macrophages defined as CD45+Ly6G−F4/80+CD11b+ was detected in fresh bone marrow (0.64 ± 0.08 vs. 2.33 ± 0.18 of HO-1+/+, Fig. 1D)

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Summary

Introduction

Osteoclasts (OCLs) are multinucleated myeloid cells crucial for constant bone remodelling because of their bone-resorbing activity. Oxidative stress conditions force cells to impel protective mechanisms, which, are thought to be attenuated during osteoclastogenesis to secure ROS signalling[17]. Such pathways are expected to be osteoclastogenic regulators of potential therapeutic significance for skeletal diseases. We showed that while HO-1 deficiency in OCLs precursors diminishes differentiation in response to RANKL, it is dispensable in RANKL-pre-stimulated cells considered as early-stage OCLs. HO-1 seems to mediate the response of OCLs precursors to RANKL and induction of OCLs markers but is dispensable in early-stage OCLs. In vivo, the advantage of the inhibitory effect of HO-1 on osteoclastogenesis might be concluded. Inhibition of the expression of OCLs markers by Nrf[2] was verified and confirmed

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