Oxidative stress stimulates apoptosis and activates NF-kappaB in osteoblastic cells via a PKCbeta/p66shc signaling cascade: counter regulation by estrogens or androgens

Abstract

Aging or acute loss of estrogens or androgens increases the levels of reactive oxygen species, activates nuclear factor-κB (NF-κB), and promotes the phosphorylation of p66(shc), a redox enzyme that amplifies mitochondrial reactive oxygen species generation and stimulates apoptosis. We report that in mesenchymal progenitor and osteoblastic cell models, H(2)O(2) activated a protein kinase C (PKC)β/p66(shc)/NF-κB signaling cascade and that p66(shc) was an essential mediator of the stimulating effects of H(2)O(2) on the apoptosis of osteoblastic cells as well as their ability to activate NF-κB. 17β-Estradiol (E(2)) or the nonaromatizable androgen dihydrotestosterone abrogated the effects of H(2)O(2) on p66(shc) and NF-κB activation by attenuating the phosphorylation of the redox-sensitive cytoplasmic kinase PKCβ. Additionally, both E(2) and dihydrotestosterone prevented H(2)O(2)-induced apoptosis by a mechanism that involved attenuation of p66(shc) resulting from decreased phosphorylation of PKCβ. Consistent with a kinase-mediated mechanism of sex steroid action, the effects of E(2) were reproduced by a polymeric form of estradiol that is not capable of stimulating the nuclear-initiated actions of ERα. These results demonstrate that p66(shc) is an essential mediator of the effects of oxidative stress on osteoblastic cell apoptosis, NF-κB activation, and cytokine production. The ability of either estrogen or androgen to attenuate the effects of oxidative stress on osteoblastic cell apoptosis, NF-κB activation, and cytokine production results from their common property to suppress PKCβ-induced p66(shc) phosphorylation via a mechanism that does not require stimulation of the nuclear-initiated actions of sex steroids.

Figure 1

H₂O₂-induced osteoblast apoptosis requires p66^shc. A, Shc protein levels by Western blot in C2C12 cells transduced with empty viral particles (EV) shRNA or one of four clones expressing shRNA directed against p66^shc mRNA. B, Shc protein levels of the three shc isoforms by Western blot in scramble or p66^shc clone 3 silenced cells. C, Caspase 3 activity in indicated cells, cultured in the presence of vehicle (veh), 50 μm H₂O₂, or 50 μm etoposide for 6 h. Bars indicate means ± sd of triplicate determinations. *, P < 0.05 vs. respective veh; #, P < 0.05 vs. veh in EV-silenced cells.

Figure 2

H₂O₂-induced NF-κB activation is stimulated by p66^shc. A, IκB phosphorylation determined by Western blot in empty viral particles (EV) or p66^shc -silenced UAMS-32 cells incubated with 100 μm H₂O₂ or 10 ng/ml TNFα for the indicated time. B, Luciferase activity in EV- or p66^shc-silenced cells transfected with a NF-κB-luc reporter construct after treatment with vehicle (veh), H₂O₂, or TNFα, as above, for 24 h. C, mRNA levels of TNFα and IL-6 by qRT-PCR in EV- or p66^shc-silenced UAMS-32 cells treated with veh or 100 μm H₂O₂ for 1 h. Bars indicate means ± sd of triplicate determinations. *, P < 0.05 vs. veh; †, P < 0.05 vs. equivalent treatment in EV-silenced cells.

Figure 3

PKCβ activity is required for H₂O₂-induced apoptosis and NF-κB activation. A, IκB and p66^shc phosphorylation determined by Western blot in UAMS-32 cells pretreated without or with 0.5 mm LY333531 (LY) for 1 h, followed by 100 μm H₂O₂ or 100 μm PMA for 6 h. B, Caspase 3 activity in cells described in panel A, cultured in the presence of vehicle (veh) or 100 μm H₂O₂ for 6 h. C, Luciferase activity in cells transfected with the NF-κB-luc reporter construct after pretreatment with 10 μm hispidin or LY333531 followed by H₂O₂ or PMA, as above, for 24 h. D, Caspase 3 and luciferase activity in empty vector (EV) or p66^shc-silenced UAMS-32 cells, as in panels B and C, treated with veh or 10 μm PMA. Bars indicate means ± sd of triplicate determinations. *, P < 0.05 vs. veh; †, P < 0.05 vs. equivalent treatment in veh-treated or EV-silenced cells.

Figure 4

Sex steroids antagonize H₂O₂-induced activation of NF-κB. A, IκB phosphorylation determined by Western blot in OB-6 cells pretreated with vehicle (veh), E₂ (10⁻⁸ m) or DHT (10⁻⁸ m) for 1 h and treated without or with H₂O₂ (100 μm) for the indicated time. B, ERα protein levels determined by Western blot in the indicated cell lines. C, Luciferase activity in cells transfected with the NF-κB luc reporter construct after pretreatment with veh, E₂, DHT (as above), 10⁻⁸ m dendrimer control (DC) or estradiol dendrimer conjugate (EDC) for 1 h followed by 50 μm H₂O₂ for 24 h. D, mRNA levels of TNFα and IL-6 by qRT-PCR in UAMS-32 cells pretreated with veh or 10⁻⁸ m E₂ for 1 h followed by 100 μm H₂O₂ for 5 h. Bars indicate means ± sd of triplicate determinations. *, P < 0.05 vs. respective veh; †, P < 0.05 vs. equivalent treatment in veh-treated cells.

Figure 5

PKC-induced p66^shc activation is prevented by sex steroids. A, p66^shc phosphorylation determined by Western blot in UAMS-32 cells pretreated with vehicle (veh), 10⁻⁸ m of each dendrimer control (DC), E₂, DHT, or estradiol dendrimer conjugate (EDC) for 1 h and treated with veh or 100 μm PMA for 1 h. B, Caspase 3 activity in UAMS-32 cells pretreated as in panel A and treated with PMA for 6 h. C, Luciferase activity in cells transfected with the NF-κB-luc reporter construct after pretreatment as in panel A followed by PMA for 24 h. D, PKCβ phosphorylation determined by Western blot in UAMS-32 cells pretreated as in panel A and treated with or without H₂O₂ for 1 h. Bars indicate means ± sd of triplicate determinations. *, P < 0.05 vs. respective veh; †, P < 0.05 vs. equivalent treatment in veh-treated cells.