Carbon monoxide and nitric oxide protect against tumor necrosis factor-alpha-induced apoptosis in osteoblasts: HO-1 is necessary to mediate the protection.
BACKGROUND
Carbon monoxide (CO) and nitric oxide (NO) each have unique roles for various inflammatory states, including inflammatory bone resorption. Although it is known that NO can induce the expression of the cytoprotective enzyme HO-1, there is no information as to whether the protective effect of CO requires NO production or whether CO must induce the expression of HO-1 to exert its functional effects.


METHODS
Murine osteoblast cells, MC3T3E1 osteoblasts, were cultured for CO and NO-associated HO-1 experiments and were transfected with pcDNA 3, pcDNA 3-HO-1, control siRNA or HO-1 siRNA using Nucleofector. For cell death measurement, MTT and annexin V assays were used. We performed Western blotting to check the expressions of HO-1 and iNOs and measured the HO-1 enzyme activity. We also measured the amounts of nitrite and nitrate using Griess reagents.


RESULTS
The increased expression of HO-1 is required for the protective effect of NO and a single treatment of CO can increase the expression of HO-1, and this is also important for the protective effect of CO in MC3T3E1 osteoblasts. CO as well as NO attenuates the TNF-alpha-induced apoptosis in osteoblasts. The anti-apoptotic effect of CO or NO is not mediated by cGMP, and CO has no effect on the release of NO. The inhibition of HO-1 with using the HO-1 inhibitor ZnPP or HO-1 siRNA resulted in a striking increase of apoptosis in the CO/TNF-alpha-treated cells. Furthermore, HO-1 overexpression showed resistance against the TNF-alpha-induced cytotoxicity in the MC3T3E1 osteoblasts.


CONCLUSIONS
There is a need for HO-1 expression to mediate the protection provided by exogenous CO or NO in osteoblasts.
Chae
H J
HJ
Department of Pharmacology and Institute of Cardiovascular Research, School of Medicine, Chonbuk National University, Jeonju, South Korea.
Chin
H Y
HY
Lee
G Y
GY
Park
H R
HR
Yang
S K
SK
Chung
H T
HT
Pae
H O
HO
Kim
H M
HM
Chae
S W
SW
Kim
H R
HR
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002248:Carbon Monoxide / Q000502:physiology*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology*; D010006:Osteoblasts / Q000166:cytology* / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D002248:Carbon Monoxide; D006419:Heme Oxygenase (Decyclizing)