c-Abl, Free Radicals, and Osteoporosis

RESULTS. The c-Abl kinase is activated by treatment with oxidants in addition to genotoxic stress, and is likely to participate in the response to oxidative stress. We tested whether induction of Prx I was affected by the absence of c-Abl by using primary calvarial osteoblasts isolated from day 20 c-abl-/- and control embryos. The osteoblasts were treated with different concentrations of sodium arsenate for 12 hours and expression of Prx I mRNA was assessed by Northern blot. We observed Prx I mRNA up-regulation at lower concentration of arsenate, and to higher maximal levels, in the c-abl-/- cells. Western blot analysis showed similar increases in Prx I protein in the mutant lines. Thus, the c-abl-/- cells are hypersensitive to arsenate, suggesting that c-Abl normally limits or negatively regulates Prx I induction. To test whether the absence of c-Abl affects the rate of induction of cell death caused by oxidative stress, we treated c-abl-/- and control osteoblasts for 24 hours with different concentrations of arsenate and the percentage of dead cells was scored by trypan blue exclusion. Untreated cell populations, as well as cells treated with only 0.05 mM arsenate, contained less than 10% dead cells. Mutant cells treated with 0.1 mM arsenate were hypersensitive: whereas wild-type cultures showed less than 30% dead cells, c-abl-/- cultures showed more than 60% dead cells. At all concentrations, c-abl-/- osteoblasts consistently showed more cell death than control cells. Treatment with H2O2, and CDDP, a DNA damage drug that also generates free radicals, produced similar results (data not shown). These results suggest that c-abl-/- osteoblasts are more sensitive than the wild-type cells to several oxidative stresses.


RESULTS.
The c-Abl kinase is activated by treatment with oxidants in addition to genotoxic stress, and is likely to participate in the response to oxidative stress. We tested whether induction of Prx I was affected by the absence of c-Abl by using primary calvarial osteoblasts isolated from day 20 c-abl-/-and control embryos. The osteoblasts were treated with different concentrations of sodium arsenate for 12 hours and expression of Prx I mRNA was assessed by Northern blot. We observed Prx I mRNA up-regulation at lower concentration of arsenate, and to higher maximal levels, in the c-abl-/-cells. Western blot analysis showed similar increases in Prx I protein in the mutant lines. Thus, the c-abl-/-cells are hypersensitive to arsenate, suggesting that c-Abl normally limits or negatively regulates Prx I induction.
To test whether the absence of c-Abl affects the rate of induction of cell death caused by oxidative stress, we treated c-abl-/-and control osteoblasts for 24 hours with different concentrations of arsenate and the percentage of dead cells was scored by trypan blue exclusion. Untreated cell populations, as well as cells treated with only 0.05 mM arsenate, contained less than 10% dead cells. Mutant cells treated with 0.1 mM arsenate were hypersensitive: whereas wild-type cultures showed less than 30% dead cells, c-abl-/-cultures showed more than 60% dead cells. At all concentrations, c-abl-/-osteoblasts consistently showed more cell death than control cells. Treatment with H 2 O 2 , and CDDP, a DNA damage drug that also generates free radicals, produced similar results (data not shown). These results suggest that c-abl-/-osteoblasts are more sensitive than the wild-type cells to several oxidative stresses.
Nitric oxide is generated in osteoblasts in response to pro-inflammatory cytokines, mechanical strain, and sex hormone. High concentrations of NO inhibits osteoblast growth and differentiation, and plays an important role in inflammation-induced osteoporosis. To test whether c-abl-/-osteoblasts show hypersensitivity to NO, three NO donor compounds were used to treat osteoblasts for 24 hours and the extent of cell death was determined. The mutant cells were significantly more sensitive to sodium nitroprusside, and also to SNAP and DETA NONOate (data not shown). These results indicate that c-abl-/-osteoblasts are hypersensitive to a variety of nitrosative stress.
In contrast to the killing by oxidative/nitrosative stress, serum starvation, which causes apoptosis in osteoblasts, induced slightly less cell death in the mutant osteoblasts than in controls, suggesting that the protective role of c-Abl is specific to oxidative/nitrosative stress. Furthermore, mouse embryonic fibroblasts (MEFs) isolated from c-abl-/-mice did not show hypersensitivity to arsenate, cisplatin, H 2 O 2 , nitroprusside, SNAP, or DETA NONOate (data not shown), indicating that the abnormal response is specific to osteoblasts. These results suggest that c-Abl may play very different roles depending upon the cell type.

DISCUSSION.
We propose that the hypersensitivity of c-abl-/-osteoblasts to oxidative and nitrosative stress contributes to the osteoporosis observed in mice lacking c-Abl. The hypersensitivity to free radicals and subsequent accumulation of oxidation and/or nitrosylation damage may contribute to the defects in maturation of c-abl-/-osteoblasts. Further, the number of osteoblasts would be reduced in the mutant mice due to their vulnerability to free radicals (oxidants and NO) generated in vivo. Our results suggest that c-Abl plays a special role in modulating the sensing of stress by osteoblasts. Phenotype of the c-abl knockout mice further indicates a correlation between osteoporosis and hypersensitivity of osteoblasts to free radicals.