Molecular mechanism of nitric oxide-induced osteoblast apoptosis.

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ELSEVIER

Journal of Orthopaedic Research 23 (2005) 462468

Journal

of

Orthopaedic

Research

www.elsevier.com/locate/orthres

Molecular mechanism

of

nitric oxide-induced osteoblast apoptosis

*

Ruei-Ming Chen

a$b2*,

Ta-Liang Chen

a,

Wen-Ta Chiu

',

Chia-Chen Chang

a Department of Anesthesiology, Wan-Fang Hospital, College of Medicine, Taipei Medical University.

No. 11 1 , Hsing-Lung Rd.. Sec. 3, Taipei 116. Taiwan

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, No. 250, Wu-H.sing St.. Taipei 110, Taiwan

Department of Surgery, Division of Neurosurgery, Wan-Fang Hospital, College of Medicine, Taipei Medical University, Taipei 11 6, Taiwan

Accepted 5 August 2004

Abstract

Nitric oxide (NO) can regulate osteoblast activities. Our previous study showed that NO induced osteoblast apoptosis [Chen

RM, Liu HC, Lin YL, Jean WC, Chen

JS, Wang JH. Nitric oxide induces osteoblast apoptosis through the de novo synthesis

of Bax protein. J Orthop Res 2002;20:295-3021. This study was further aimed to evaluate the mechanism of NO-induced osteoblast

apoptosis from the viewpoints of mitochondrial functions, intracellular oxidative stress, and the anti-apoptotic Bcl-2 protein using

neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP), an NO donor,

significantly increased amounts of lactate dehydrogenase in the culture medium, and decreased cell viability in concentration- and

time-dependent manners. Administration of SNP in osteoblasts time-dependently led to DNA fragmentation. The mitochondrial

membrane potential was significantly reduced following SNP administration. SNP decreased complex

I NADH dehydrogenase

activity in a time-dependent manner. Levels of cellular adenosine triphosphate (ATP) were suppressed by SNP. In parallel with

the mitochondrial dysfunction, SNP time-dependently increased levels

of intracellular reactive oxygen species. Immunoblotting

analysis revealed that SNP reduced Bcl-2 protein levels. Exposure to lipopolysaccharide (LPS) and IFN-y significant increased

endogenous nitrite production. In parallel with the increase in endogenous NO, administration of LPS and IFN-y suppressed cell

viability, mitochondrial membrane potential, and ATP synthesis. Results of this study show that NO released from SNP can induce

osteoblast insults and apoptosis, and the mechanism may involve the modulation of mitochondrial functions, intracellular reactive

oxygen species, and Bcl-2 protein.

0 2004 Orthopaedic Research Society. Published by Elsevier Ltd.

All rights reserved.

Keywords: Osteoblasts; Nitric oxide; Apoptosis; Mitochondria1 functions; Reactive oxygen species; Bcl-2 protein

Introduction

*

Part of this study was presented at the 47th Annual Meeting of the Orthopedic Research Society, February 2001, San Francisco, CA, USA.

Nitric oxide (NO), synthesized from L-argenine by

NO

synthases, contributes to the regulation of tissue/

* Corresponding author. Address: Graduate Institute of Medical

Sciences, College of Medicine, Taipei Medical University, No. 250, Wu-Hsing St., Taipei 110, Taiwan, ROC. Tel.: +886 2 29307930x21 59; fax: +886 2 86621150.

cell activities, including vasodilation, neurotransmis-

sion, immunoresponses,

and death

control

~4,271. NO

E-mail addresses: [email protected], [email protected]

osteoblasts, N O is constitutively produced

I1

l1.

can also modulate bone remodeling [ l l ] . In untreated

Following pretreatment with inflammatory cytokines

(R.-M. Chen).

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R.-M. Chen et al. I Journal of’ Orthopaedic Research 23 (2005) 462-468 463

or mechanical stress, high levels

of NO

in osteoblasts

are synthesized [8,26].

NO

has biphasic effects on

osteoblast activities [11,27]. Constitutive NO can be

an effective mediator to regulate osteoblast prolifera-

tion and differentiation [29]. However, overproduction

of NO leads to osteoblast injuries [12,24,26].

Apoptosis, an energy-dependent type of pro-

grammed cell death, has a critical role in evolutionarily

conserving physiologic cell death [14,15]. Hock et al. re-

ported that apoptosis determines osteoblast popula-

tions in the postanal and adult skeleton [18]. A

variety of intrinsic and extrinsic factors are involved

in the regulation of cell apoptosis [10,15,28].

NO

can

be an effector for death regulation [4,9]. In inflamma-

tion-induced osteoporosis, elevated levels of

NO

have

been shown to induce osteoblast apoptosis, and to

decrease bone mineral density [l]. Several lines of

evidence were provided by our previous study to

demonstrate that the NO-induced osteoblast insult

occurs via an apoptotic mechanism [6].

Mitochondria are energy-producing organelles.

Maintenance of the mitochondrial membrane potential

and metabolizing enzyme activities is critical to

adenosine triphosphate (ATP) synthesis [32,38]. Depo-

larization of the mitochondrial membrane potential in-

creases the release of apoptotic factors from the

mitochondria to the cytoplasm and leads to cell apopto-

sis [3,19,34]. Intracellular reactive oxygen species

(ROS),

one of several apoptotic factors, can augment oxidative

stress and damage cells [9,17,22]. Bcl-2 is an anti-apop-

totic protein [20,35]. A decrease in the ratio of Bcl-2

over Bax, an apoptotic protein, increases the risk that

cells will undergo apoptosis [16]. Our previous study

showed that NO increases Bax protein production and

induces osteoblast apoptosis [6]. In this study, we fur-

ther hypothesized that NO-induced osteoblast apoptosis

may occur through modulation of the mitochondrial

functions, intracellular oxidative stress, and Bcl-2

protein levels.

Materials and methods

Cell isolation and drug treatment

Rat osteoblasts were prepared from 3-day-old Wistar rat calvaria according to the method of Partridge et al. [31]. Osteoblasts were seeded in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Grand Island,

NY,

USA) supplemented with 10% heat-inactivated fetal bo- vine serum, L-glutamine, penicillin (1 00 IUIml), and streptomycin (lOOpg/ml) in 75-cm2 flasks at 37°C in a humidified atmosphere of 5% C02. Osteoblasts were grown to confluence prior to drug treatment. Only first passage of rat osteoblasts was used in the present study. Each osteoblast isolation represents a determination. Each experiment was repeated at least three times.

Sodium nitroprusside (SNP) purchased from Sigma Corporation (St. Louis, MO, USA) was freshly dissolved in phosphate-based saline

(PBS) buffer (0.14M NaC1,2.6mM KCI, 8mM Na2HP04, and 1.5mM KH2P04) and protected from light. Concentration-and time-dependent effects of SNP on osteoblasts were determined.

Quantijcation of lactate dehydrogenase

For evaluating the toxicity of SNP to osteoblasts, amounts of lactate dehydrogenase released in culture medium were determined. Osteoblasts (1 x 10’) were seeded in 24-well tissue culture plates (Corn- ing-Costar, Cambridge, MA, USA). After administration of SNP, the culture medium was collected and centrifuged. Levels of lactate dehydrogenase in supernatants were analyzed using a model 7450 automatic autoanalyzer system of Hitachi (Tokyo, Japan).

Assay of cell viability

A trypan blue exclusion method was carried out to determine the cytotoxicity of SNP to osteoblasts. Briefly, osteoblasts (2 x lo’) were cultured in 24-well tissue culture plates. After SNP administration, osteoblasts were trypsinized by 0.1% trypsine-EDTA (Gibco-BRL). Following centrifugation and washing, osteoblasts were suspended in

Ix PBS buffer and stained with an equal volume of trypan blue dye (Sigma). Fractions of dead cells with a blue signal were determined using a reverse-phase microscope.

QuantiJkation of DNA fragmentation

DNA fragmentation in osteoblasts was quantified to evaluate if SNP damaged nuclear DNA. The BrdU-labeled histone-associated DNA fragments in the cytoplasm of cell lysates were detected according to the instructions of the cellular DNA fragmentation enzyme- linked immunosorbent assay (ELISA) kit (Boehrin er Mannheim, Indianapolis, IN, USA). Briefly, osteoblasts (2 x 10 ) were sub-cultured in 24-well tissue culture plates and labeled with BrdU overnight. Cells were harvested and suspended in the culture medium. One hun- dred microliters of cell suspension was added to each well of 96-well tissue culture plates. Osteoblasts were cocultured with SNP for another 8 h at 37°C in a humidified atmosphere of 5% C02. Amounts of BrdU- labeled DNA in the cytoplasm were quantified using an Anthos 2010 microplate photometer (Anthos Labtec Instruments, Lagerhausstrasse, WalsISalzburg, Austria) at a wavelength of 450nm.

B

QuantiJcation of the mitochondria1 membrane potential

The mitochondrial membrane potential was determined following the method of Chen [5]. Briefly, osteoblasts (5 x 10’) were seeded in 12-well tissue culture plates overnight, and then treated with SNP for different time intervals. After SNP administration, osteoblasts were harvested and incubated with 3,3‘-dihexyloxacarbocyanine (DiOC6(3)), a positively charged dye, at 37°C for 30min in a humidified atmosphere of 5% C02. After washing and centrifuging, cell pellets were suspended with Ix PBS buffer. Intracellular fluorescent intensities were analyzed using a flow cytometer (FACS Calibur, Becton Dickin- son, San Jose, CA, USA).

Assay of mitochondria1 NADH dehydrogenase activity

NADH dehydrogenase activity was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay following the method of Wu et al. [37]. Briefly, osteoblasts (5 x 10’) were seeded in 96-well tissue culture plates overnight. After drug treatment, cells were cultured with new medium containing OSmg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny~tetrazo~ium bromide for another 3 h. A blue formazan product in cells was dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 570nm.

Quantification of cellular adenosine triphosphate ( A T P ) levels

Levels of cellular ATP in osteoblasts were determined with a bioluminescence assay described previously [7]. This assay was based on luciferase’s requirement for ATP in producing emission light according to the protocol of Molecular Probes’ ATP determination kit (Molecu- lar Probes, Eugene, OR, USA). Luminent light (560nm) emitted by the

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464 R.-M. Chen et al. I Journal of Orthopaedic Research 23 (2005) 462468

luciferase-mediated reaction of ATP and luciferin was detected using a WALLAC VICTOR&,, 1420 multilabel counter (Welch Allyn, Turku, Finland).

Table 1

Concentration-dependent effects of sodium nitroprusside on osteoblast viability

Determination of intracellular ROS

Levels of intracellular ROS were uantified following a method described previously [23]. Briefly, 5 x 10 osteoblasts were cultured in 12- well tissue culture plates overnight, and then cotreated with SNP and 2’,7’-dichlorofluorescin diacetate, an ROS sensitive dye. After drug treatment, osteoblasts were harvested and suspended in Ix PBS buffer. Relative fluorescence intensities of osteoblasts were quantified using a flow cytometer (FACS Calibur).

’3

Gel electrophoresis and immunoblotting analysis

After SNP treatment, osteoblasts were washed with lx PBS buffer. Cell lysates were prepared in ice-cold radioimmunoprecipitation assay (RIPA) buffer (25mM Tris-HCl pH 7.2, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 0.15M NaC1, and 1mM EDTA). To avoid protein degradation, a mixture of proteinase inhibitors, including 1 mM phenyl methyl sulfonyl fluoride, 1 mM sodium orthovanadate, and 5pg/ml leupeptin, was added to the RIPA buffer. Protein concentrations were quantified by a bicinchonic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Cytosolic proteins (100 pg per well) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transfered to nitrocellulose membranes. Mem- branes were blocked with 5% non-fat milk at 37 “C for 1 h. Immunode- tection of Bcl-2 was carried out using a mouse monoclonal antibody against rat Bcl-2 (Transduction Laboratories, Lexington, KY, USA). Cellular p-actin protein was immunodetected using a mouse monoclonal antibody against mouse p-actin (Sigma) as an internal standard. Intensities of the immunoreactive bands were determined using an UVIDOCMW version 99.03 digital imaging system (Uvtec, Cam- bridge, England, UK).

Statistical analysis

Statistical differences between the control and SNP-treated groups were considered significant when the

P

value of Duncan’s multiple- range test was less than 0.05. Statistical analysis between groups over time was carried out using two-way ANOVA.

Results

Table 1 presents the concentration-dependent effects

of SNP on osteoblast viability. Administration of

0.5 and 1mM SNP in osteoblasts did not affect lactate

dehydrogenase release. SNP at 1.5 and 2mM

significantly increased amounts of lactate dehydroge-

nase by 85% and 154%, respectively. Analysis by a try-

pan blue exclusion method revealed that SNP at 0.5

and 1mM was not cytotoxic to osteoblasts (Table 1).

However, after administration of 1.5 and 2mM SNP,

viability of osteoblasts was decreased by 38% and

6270, respectively.

Table 2 shows the time-dependent effects of SNP

on

osteoblast viability. In 4-h-treated osteoblasts, SNP

did not affect lactate dehydrogenase release. After SNP

administration for

8 and 16h, amounts of lactate dehy-

drogenase were significantly augmented by 65% and

151%, respectively. Exposure to SNP for 4 h was not

cytotoxic to osteoblasts (Table 2). Viability of osteo-

SNP, mM Lactate dehydrogenase Cell viability

IUIL) (cell number x lo3)

0 41 f 11 1 8 8 f 44

0.5 3 8 f 10 187 f 38

1 35 f 8 179 f 45

1.5 76 f 8’ 116f 18‘

2 1 0 4 f 13’ 7 2 f 21’

Rat osteoblasts were exposed to 0.5, 1, 1.5, and 2mM sodium nitroprusside (SNP) for 16h. Amounts of lactate dehydrogenase in the culture medium were determined by an autoanalyzer as described in “Materials and Methods”. Cell viability was assayed by the trypan blue exclusion method. Each value represents the mean f SEM for

n = 12 from four independent cell preparations.

Values significantly differ from the respective control,

P

< 0.05.

Table 2

Time-dependent effects of sodium nitroprusside on osteoblast viability Time, h Lactate dehydrogenase Cell viability

( U W (cell number x

10”)

0 3 7 f 10 4 30 f 6 8 61 f 9’ 16 93 f 11’ 179 f 31 167 f 58 107 & 30‘ 81 f 19’

Rat osteoblasts were exposed to 2mM sodium nitroprusside for 4, 8, and 16h. Amounts of lactate dehydrogenase in the culture medium were determined by an autoanalyzer as described in “Materials and Methods”. Cell viability was assayed by the trypan blue exclusion method. Each value represents the mean f SEM for n = 12 from four independent cell preparations.

* Values significantly differ from the respective control,

P

< 0.05.

blasts was significantly reduced by 400/0 and 55%, respec-

tively, following SNP administration for

8 and 16h.

Fig. 1 presents the effects of SNP on DNA damage.

Exposure of osteoblasts to 0.5 and 1mM SNP for 16h

did not cause DNA injury (top panel). SNP at 1.5 and

2mM significantly increased levels of DNA fragments

by 92% and 267%, respectively. In 4-h-treated osteo-

blasts, SNP did not damage nuclear DNA

(bottom

panel).

After SNP administration for 8 and 16h, levels

of DNA fragments were enhanced by 65% and 205%,

respectively.

The mitochondria1 membrane potential of osteo-

blasts was determined and shown in Fig. 2. In l-h-trea-

ted osteoblasts, SNP did not change the membrane

potential of mitochondria. After administration of

SNP for 2 and 4h, the mitochondrial membrane

potential was significantly decreased by 16% and 36%,

respectively.

Table 3 shows the effects of SNP on NADH dehy-

drogenase activity and ATP synthesis. In 4-h-treated

osteoblasts, SNP did not affect NADH dehydrogenase

activity. Administration of SNP for 8 and 16 h signifi-

cantly reduced activities of NADH dehydrogenase by

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R.-M. Chen et al. I Journal of Orthopaedic Research 23 (2005) 462-468 465

lS6

c

-

*

C E

=

1.2 P

t P

nc

gg

2

0.8

2

6

0.4

0.0

0 0.5

1

1.5

2

SNP,

mM

3 lime, h

Fig. 1. Effects of SNP on DNA fragmentation. Osteoblasts prepared from neonatal rat calvaria were exposed to 0.5, 1, 1.5, and 2mM SNP for 16 h ( f o p panel) or 2mM SNP for 4, 8, and 16h (bottom panel). DNA fragments in osteoblasts were quantified using a BrdU-labeled histone-associated DNA fragmentation ELISA kit as described in “Material and Methods”. Each value represents the mean ? SEM for

n = 9 from three independent cell preparations.

*

Values significantly differ from the respective control, P < 0.05.

Table 3

Effects of sodium nitroprusside on NADH dehydrogenase activity and cellular adenosine triphosphate levels

Time, h NADH dehydrogenase ATP

(OD value at 550nm) (PmoU

0 1.03 f 0.21 4 0.98

f

0.17 8 0.74 f 0.18’ 16 0.62 ? 0.14’ 38 f 8 31 f 9 24 f 4’ 17f.5’ Rat osteoblasts were exposed to 2mM sodium nitroprusside for 0,4,8, and 16h. NADH dehydrogenase activity was assayed by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method as described in “Materials and Methods”. Cellular adenosine triphosphate (ATP) levels were determined by a bioluminescence assay. Each value represents the mean f SEM for n = 12 from four independent cell preparations.

* Values significantly differ from the respective control, P < 0.05.

18%

and 58%, respectively. In 4-h-treated osteoblasts,

SNP did not change cellular ATP levels (Table 3). After

*

0

1

2

4 lime, h

Fig. 2. Effects of SNP on the mitochondrial membrane potential. Osteoblasts prepared from neonatal rat calvaria were exposed to 2 mM SNP for 1, 2, and 4h. The mitochondrial membrane potential of osteoblasts was analyzed using the fluorescent dye, DiOC6(3), and quantified by a flow cytometer. Each value represents the mean f SEM for n = 9 from three independent cell preparations.

*

Values significantly differ from the respective control, P < 0.05.

0

1

2

4 Time, h

Fig. 3. Effects of SNP on intracellular reactive oxygen species (ROS). Osteoblasts prepared from neonatal rat calvaria were exposed to 2 mM

SNP for 1, 2, and 4h. Levels of intracellular ROS were caught using the ROS-sensitive dye, 2’,7’-dichlorofluorescin diacetate, and quantified by a flow cytometer. Each value represents the mean ? SEM for

n = 9 from three independent cell preparations.

*

Values significantly differ from the respective control, P < 0.05.

administration of

S N P

for

8 and 16h, cellular ATP

levels were significantly decreased by 37% and 55%,

respectively

.

Intracellular levels of

ROS

were determined and are

shown in Fig.

3. In 1-h-treated osteoblasts,

SNP

caused

a significant increase in

ROS

levels by 76%. Levels of

intracellular ROS in osteoblasts were respectively

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466 R.-M. Chen et al. I Journal of Orthopaedic Research 23 (2005) 462468

1

2 Bcl-2

*

4 2 6 kD

f3-Actin

-26

kD

(A)

C

SNP

C

SNP

Fig. 4. Immunoblotting analysis of Bcl-2 protein. Osteoblasts prepared from neonatal rat calvaria were exposed to 2mM SNP. Cytosolic protein was prepared, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Immunodetection of Bcl-2 protein was carried out using a monoclonal antibody against rat Bcl-2 protein (A, rop panel). P-Actin was immunodetected as an internal control (bottom panel). Intensities of protein bands were quantified by a digtal imaging system (B). Each value represents the mean f SEM for n = 6 from three independent cell preparations.

*

Values significantly differ from the respective control,

P

< 0.05.

augmented by 3- and 5-fold following SNP administra-

tion for 2 and 4 h.

Fig. 4 shows the effects of SNP

on

Bcl-2 protein.

In

untreated osteoblasts, Bcl-2 protein was detectable

(Fig. 4A, fop panel, lane 1). Administration of SNP in

osteoblasts decreased Bcl-2 protein production (lane

2). p-actin was immunodetected as an internal control

(bottom panel). Quantificantion of these immunodetec-

ted protein bands revealed that levels of Bcl-2 protein

were significantly decreased by 82% following SNP

administration (Fig. 4B).

Table 4

Effects of lipopolysaccharide and IFN-y on nitrite production, cell viability, mitochondrial membrane Dotential. and ATP synthesis

Analysis Control LPS

+

IFN-y

Nitrite, pM 4 f 1 28 f 8'

Cell viability, cell number x lo3 58 ? 15'

Mitochondria1 membrane 100 61 ? 18'

ATP, pmol 4 6 f 13 2 4 2 6 '

Rat osteoblasts were exposed to a mixture of 1 pglml lipopolysaccharide (LPS) and IOOIUlml IFN-y for 16h. Amounts of nitrite in the culture medium were determined by the Griess reaction. Cell viability was assayed by the trypan blue exclusion method. The mitochondrial membrane potential was quantified by a flow cytometer. Levels of adenosine triphosphate (ATP) were assayed by a bioluminescence assay. Each value represents the mean f SEM for n = 6 from three independent cell preparations.

* Values significantly differ from the respective control,

P

< 0.05.

137 f 39

potential

(YO

of control)

Table 4 presents the effects of endogenous NO pro-

duction on cell viability, the mitochondrial membrane

potential, and ATP synthesis. Administration of lipo-

polysaccharide (LPS) and IFN-y significantly increased

nitrite production by 6.5-fold. Viability of osteoblasts

was decreased by 58% following administration of

LPS

and IFN-y. After administration of LPS and IFN-y,

the mitochondrial membrane potential and cellular

ATP levels were reduced by 39% and 48%, respectively.

Discussion

SNP can be decomposed to NO under light exposure

or

in

the presence of a biological reducing system [2,21].

As presented in our previous study, administration of

SNP significantly enhances the amounts of nitrite, which

corresponds to an increase in NO [6]. In parallel with the

increase in NO, the amounts of lactate dehydrogenase

released from osteoblasts into the culture medium were

significantly augmented. Analysis by the trypan blue

exclusion method showed that the membrane permeabi-

lity

of osteoblasts was disturbed following SNP admin-

istration. The breakage of cell membranes increased

the release

of

lactate dehydrogenase. Thus, NO decom-

posed from SNP broke down plasma membranes,

increasing levels of lactate dehydrogenase in the cul-

ture medium, and leading to osteoblast insults or even

death.

Our previous study provided several lines of evidence

which identified that NO-induced osteoblast death

mainly occurs via an apoptotic pathway [6]. This study

used a cellular DNA fragmentation ELISA to further

demonstrate that the nuclear DNA of osteoblasts was

fragmented following SNP administration. Fragmented

breakage of chromosome DNA is a critical characteris-

tic which indicates that cells are undergoing apoptosis

[

15,341. Our data reveal that NO can damage osteoblast

DNA and induce cell apoptosis. Dypbukt et al. reported

that high concentrations of NO donors increased the re-

lease of lactate dehydrogenase and caused cell necrosis

[13]. This does not rule out the possibility that SNP

partially induces osteoblast necrosis.

SNP significantly reduced cellular ATP levels. Mito-

chondria are critical ATP-synthesizing organelles. This

study shows that SNP can decrease the mitochondrial

membrane potential of osteoblasts. Previous studies re-

ported that disruption

of

the mitochondrial membrane

potential results in mitochondrial depolarization and

blocks the respiratory chain reaction [30,38]. Thus, one

of the possible mechanisms involved in the NO-induced

depletion of ATP levels in osteoblasts is through the

suppression of the mitochondrial membrane potential.

Administration of SNP significantly decreased mitoch-

ondrial complex

I

NADH dehydrogenase activity.

NADH dehydrogenase contributes to the respiratory

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R.-M. Chen et al. I Journal of Orthopuedic Re.wurch 23 (2005) 462468 467

chain reaction and ATP synthesis [34]. A decrease in

NADH dehydrogenase activity is another possible

mechanism involved in the NO-induced ATP depletion

in osteoblasts. Reduction of ATP synthesis has been

shown to induce cell apoptosis [3]. Therefore, NO can

decrease cellular ATP levels through suppression of

the mitochondrial membrane potential and complex

I

enzyme activity in osteoblasts and thus induce cell

apoptosis.

This study shows that levels

of

intracellular

ROS

were significantly augmented following SNP administra-

tion.

ROS

is one of the mitochondrial apoptotic factors

[24]. Previous studies reported that depolarization of the

mitochondrial membrane potential enhances the release

of apoptotic factors, including ROS and cytochrome c,

from mitochondria to the cytoplasm and drives cells

undergoing apoptosis [21,32,37]. Li et al. showed that

rotenone, an inhibitor of NADH dehydrogenase, en-

hanced mitochondrial

ROS

and induced cell apoptosis

[22]. Therefore, SNP can increase intracellular

ROS

through suppression

of

the mitochondrial membrane

potential and NADH dehydrogenase activity. However,

the NO radical is one ROS. DCFH-DA was used in this

study to catch ROS. Previous studies demonstrated that

NO and peroxynitrite ( N 0 0 0 - ) can also directly react

with DCFH-DA [17,33]. Therefore, the elevation of

intracellular ROS in SNP-treated osteoblasts is partially

due to the enhancement of intracellular NO.

SNP decreased Bcl-2 protein production. Bcl-2, an

anti-apoptotic protein, can determine if cells undergo

apoptosis [35,36]. A decrease in Bcl-2 protein levels will

increase the risk that cells will undergo apoptosis.

Bax

is

a pro-apoptotic protein [25]. Immunocytochemical and

immunoblotting analyses were carried out in our previ-

ous study to validate that NO increased Bax protein lev-

els in osteoblasts [6]. Mitochondria1 apoptotic factors,

including ROS and cytochrome c, can regulate the cellu-

lar Bcl-2/Bax ratio [9,10]. Therefore, SNP can modulate

mitochondrial functions and increase the release of mit-

ochondrial apoptotic factors. The SNP-caused suppres-

sion of Bcl-2/Bax proteins can induce osteoblasts

apoptosis.

Osteoblasts were exposed to a mixture of LPS and

IFN-y to determine if endogenous NO has similar effects

as those of SNP. After administration of LPS and IFN-

y,

levels of nitrite in osteoblsts were significantly

augmented. In parallel with the increase in NO, admin-

istration of LPS and IFN-y disrupted the mitochondrial

membrane potential, reduced cellular ATP levels, and

ultimately induced cell death. This study has shown that

the exogenous and endogenous forms of NO have the

same effects on the induction of mitochondrial dysfunc-

tion and cell death.

In summary, NO decomposed from SNP can cause

DNA fragmentation, thus the NO-induced death mech-

anism mainly occurs via an apoptotic pathway. SNP can

modulate mitochondrial functions through inhibition of

the mitochondrial membrane potential, NADH dehy-

drogenase activity and cellular ATP levels. Levels of

intracellular

ROS

and Bcl-2 protein are also regulated

by SNP. The modulating effects

of

NO decomposed

from SNP are also observed with endogenous NO.

Therefore, this study presents further data to validate

our hypothesis that NO can modulate mitochondrial

functions, intracellular oxidative stress, and Bcl-2 pro-

tein production to induce osteoblast apoptosis. The

roles of caspases in NO-induced osteoblast apoptosis

will be a further study in our laboratory.

Acknowledgements

This study was supported by grants NSC91-2314-B-

038-031 and NSC92-23 14-B-038-010 from the National

Science Council, Taipei, Taiwan, ROC.

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