A crucial role for thiol antioxidants in estrogen-deficiency bone loss
Jenny M. Lean, Julie T. Davies, Karen Fuller, Christopher J. Jagger, Barrie Kirstein, Geoffrey A. Partington, Zoë L. Urry, Timothy J. Chambers
Jenny M. Lean, Julie T. Davies, Karen Fuller, Christopher J. Jagger, Barrie Kirstein, Geoffrey A. Partington, Zoë L. Urry, Timothy J. Chambers
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Article Bone biology

A crucial role for thiol antioxidants in estrogen-deficiency bone loss

Abstract

The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone. Thus, estrogen might prevent bone loss by enhancing oxidant defenses in bone. We found that glutathione and thioredoxin, the major thiol antioxidants, and glutathione and thioredoxin reductases, the enzymes responsible for maintaining them in a reduced state, fell substantially in rodent bone marrow after ovariectomy and were rapidly normalized by exogenous 17-β estradiol. Moreover, administration of N-acetyl cysteine (NAC) or ascorbate, antioxidants that increase tissue glutathione levels, abolished ovariectomy-induced bone loss, while L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, caused substantial bone loss. The 17-β estradiol increased glutathione and glutathione and thioredoxin reductases in osteoclast-like cells in vitro. Furthermore, in vitro NAC prevented osteoclast formation and NF-κB activation. BSO and hydrogen peroxide did the opposite. Expression of TNF-α, a target for NF-κB and a cytokine strongly implicated in estrogen-deficiency bone loss, was suppressed in osteoclasts by 17-β estradiol and NAC. These observations strongly suggest that estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts. This directly sensitizes osteoclasts to osteoclastogenic signals and entrains ROS-enhanced expression of cytokines that promote osteoclastic bone resorption.

Authors

Jenny M. Lean, Julie T. Davies, Karen Fuller, Christopher J. Jagger, Barrie Kirstein, Geoffrey A. Partington, Zoë L. Urry, Timothy J. Chambers

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17-β estradiol modulates osteoclastic thiol antioxidant system, and oste...
17-β estradiol modulates osteoclastic thiol antioxidant system, and osteoclasts are influenced by modulation of thiol antioxidant system. (a and b) 17-β estradiol stimulated glutathione reductase (GR) and thioredoxin reductase (TrxR) in osteoclasts. This stimulation was reversed by ICI 182,780 (ICI) (three cultures per variable). Statistically, significant stimulation of GR and TrxR by 17-β estradiol (E2) and inhibition of stimulation by ICI 182,780 was observed in each of two further experiments. (c) BSO stimulated TRAP-positive multinucleate cell formation (TRAP-pos MNC), while this was suppressed by NAC. Like BSO, the ROS hydrogen peroxide also stimulated TRAP-positive multinucleate cell formation (10 cultures per variable). (d) EMSA was used to show effect of BSO and NAC on NF-κB activity in in vitro–formed osteoclasts. Activity (arrowheads) was increased by BSO and suppressed by NAC. We confirmed that the bound material contained NF-κB p50 by supershifting both bands with p50 Ab (arrow). The binding was further confirmed to be specific by competing binding of NF-κB with 100-fold excess of unlabeled self probe (S), but not by a mutant species (H). M, M-CSF; RL, RANKL. (e and f) 17-β estradiol and NAC suppress expression of RNA for TNF-α in in vitro-derived osteoclasts. *P < 0.05 versus other groups.