Role of ghrelin in modulation of s-nitrosylation-Dependent akt inactivation induced in salivary gland acinar cells by porphyromonas gingivalis

doi:10.4236/health.2010.212215

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Vol.2, No.12, 1448-1455 (2010) Health

doi:10.4236/health.2010.212215

Role of ghrelin in modulation of S-nitrosylation-

dependent Akt inactivation induced in salivary gland

acinar cells by Porphyromonas gingivalis

Bronislaw L. Slomiany*, Amalia Slomiany

Research Center, University of Medicine and Dentistry of New Jersey, Newark, USA; *Corresponding Author: slomiabr@umdnj.edu

Received 23 August 2010; revised 8 October 2010; accepted 26 October 2010

ABSTRACT

Ghrelin, a peptide hormone, newly identified

in oral mucosal tissue, has emerged re-

cently as a principal modulator of the in-

flammatory responses to bacterial infection

through the regulation of nitric oxide syn-

thase system. In this study, using rat sub-

lingual salivary gland acinar cells, we report

that lipopolysaccharide (LPS) of periodon-

topathic bacterium, P. gingivalis- induced

enhancement in the activity of inducible ni-

tric oxide synthase (iNOS) was associated

with the suppression in Akt kinase activity

and the impairment in constitutive (c) cNOS

phosphorylation. Further, we show that the

detrimental effect of the LPS on Akt activa-

tion, manifested in the kinase protein

S-nitrosylation and a decrease in its phos-

phorylation at Ser473, was susceptible to

suppression by iNOS inhibitor, 1400W.

Moreover, we demonstrate that a peptide

hormone, ghrelin, countered the LPS-

induced changes in Akt activity and NOS

system. This effect of ghrelin was reflected

in the decreased in Akt S-nitrosylation and

the increase in its phosphorylation at Ser473,

as well as cNOS activation through phos-

phorylation. Our findings suggest that P.

gingivalis-induced up-regulation in iNOS

leads to Akt kinase inactivation through

S-nitrosylation that impacts cNOS activation

through phosphorylation. We also show

that the countering effect of ghrelin on P.

gingivalis-induced disturbances in Akt ac-

tivation are manifested in a decrease in the

kinase S-nitrosylation and the increase in its

phosphorylation.

Keywords: P. Gingivalis; Salivary Gland; iNOS; Akt

S-Nitrosylation; cNOS Phosphorylation; Ghreli n

1. INTRODUCTION

Recent advances in understanding the nature of fac-

tors that influence the extent of mucosal inflammatory

responses along the alimentary tract, including that of

oral cavity, have brought to focus the importance of a

peptide hormone, ghrelin [1-4]. This 28-amino acid pep-

tide, initially isolated from the stomach [1,2], and more

recently identified in oral mucosa, saliva and the acinar

cells of salivary glands [3], has emerged as a principal

modulator of the local inflammatory responses to bac-

terial infection through the regulation of nitric oxide

synthase (NOS) system responsible for nitric oxide (NO)

productio n [5 - 8].

NO is a short-lived signaling molecule that plays an

important role in a variety of regulatory pathways that

are of significance to cellular survival, integrity main-

tenance, and the extent of mucosal inflammatory in-

volvement in response to bacterial challenge [9,10]. The

physiological and pathophysiological implications of

NO depend on its local concentration, the type of NOS

isozyme involved in NO generation, sub strate availabili-

ty, and the enzy me compartmentalization with respect to

protein target [9,10]. A low level of NO generated by

membrane-associated constitutive nitric oxide synthase

(cNOS) appears to access a pool of substrates that are of

importance to the maintenance of normal physiological

functions, including the regulation of apoptogenic signal

propagation [7,10-12]. In contrast, a high output of NO

generated by more distant cytosolic inducible nitric

oxide synthase (iNOS) in response to proinflammatory

cytokines and bacterial LPS, has been implicated in host

response to sepsis and endotoxemia [9,13]. However,

sustained iNOS activation associated with persistence of

inflammatory stimulus, is known to have cytotoxic con-

sequences reflected in transcriptional disturbances and

the induction of apoptosis [9,11,12,14].

The signaling mechanism that underlies the regulation

B. L. Slomiany et al. / Health 2 (2010) 1448-1455

1449

of NO by ghrelin involves the receptor (GHSR1a)- me-

diated activation of heterotrimeric G protein-dependent

pathway that results in signal propagation through a

multiple network of protein kinases, including that of

Akt kinase implicated in controllin g the activity of NOS

isozyme system [6,7,15]. The serine/threonine kinase

Akt, also known as protein kinase B (PKB) or Akt/PKB,

is a central player in the regulation apoptosis, cell cycle

and metabolic pathways, host inflammatory responses,

and signal transduction pathways activated by growth

factors and insulin [16-18]. Activation of Akt in response

to insulin or ghrelin occurs downstream of phosphoino-

sitide 3-kinase (PI3K) and involves the g eneration of the

lipid second messenger phosphatidylinositol-3,4,5-

triphosphate, which accumulates in the plasma mem-

brane and serves as a recognition site for the N-terminal

PH (pleckstrin homology) domain of Akt [17,18]. The

induced conformational changes in Akt result in the ex-

posure to phosphorylation within the activ ation (A) loop

at Thr308 and of Ser473 located within the HM (hydro-

phobic motif) region of the C-terminal regulatory do-

main of Akt [17,18]. Apparently, phosphorylation of the

Thr308 in the activation loop and the Ser473 in the hydro-

phobic motif results in stabilization of the kinase domain

in the active state, and hence is required for full activa-

tion of Akt [17,19] .

Moreover, recent studies indicate that the activity of

Akt may be also regulated through S-nitrosylation at the

kinase cysteine residues [16,20,21], and protein

S-nitrosylation, with the involvement of NO generated

by both constitutive and inducib le forms of NOS system,

is rapidly emerging as a signaling event of significance

to a variety of biological processes [7-10]. Indeed,

S-nitrosylation with the involvement of cNOS has been

linked to the ap optogenic signal inhibition and the events

of cytosolic phospholipase A2 activation, whereas the

NO generated by iNOS has been implicated in

S-nitrosylation of proteins involved in insulin signal

transduction, and the reduced Akt activity in muscle

cells of diabetic mouse [7,8,16,20-22]. Furthermore,

protein modification through targeted S-nitrosylation is

gaining recognition as an important post-translational

event that, like protein modification through post-

translational phosphorylation, mediates a variety of sig-

nal transduction events propagated by NO [7-10,20-22].

As the oral mucosal inflammatory responses to peri-

odontopathic bacterium, P. gingivalis, are characterized

by the disturbances in NO production, and since Akt

kinase plays a major role in the regulation of NOS iso-

zyme system [8,15,22,23], in this study we investigated

the effect of P. gingivalis, key virulence factor, LPS, on

the processes associated with the activation of kinase

Akt in sublingual salivary gland acinar cells. Our data

revealed that the LPS-induced up-regulation in iNOS

leads to Akt inactivation through S-nitrosylation that

exerts the detrimental effect on the processes of cNOS

activation through phosphorylation. Moreover, we also

revealed that the countering effect of ghrelin was mani-

fested in a marked increase in Akt activity that correlated

with a decrease in the kinase protein S-nitrosy-

lation and the increase in its phosphorylation.

2. MATERIALS AND METHODS

2.1. Salivary Gland Cell Incubation

Freshly dissected rat sublingual salivary glands were

trimmed of fat and connective tissue, and minced by

passage through a 50 mesh metal grid [23]. The minces

tissue was suspended in five volumes of ice-cold Dul-

becco's modified (Gibco) Eagle's minimal essential me-

dium (DMEM), supplemented with fungizone (50

µg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml),

and 10% fetal calf serum, and gently dispersed by tritu-

ration with a syringe, and settled by centrifugation [23].

Following rinsing, the cells were resuspended in the me-

dium to a concentration of 2 x 107 cell/ml, transferred in

1 ml aliquots to DMEM in culture dishes and incubated

under 95% O2 – 5% CO2 atmosphere at 370C for 16h in

the presence of 0 – 200 ng/ml of P. gingivalis LPS [23].

In the experiments evaluating the effect of ghrelin (rat,

Sigma), cNOS inhibitor, L-NAME, iNOS inhibitor,

1400W, Src inhibitor, Akt inhibitor, SH-5 (Calbioche m),

and ascorbate (Sigma), the cells were first preincubated

for 30 min with the indicated dose of the agent or ve-

hicle before the addition of the LPS. The v iability of cell

preparations before and during the experimentation, as-

sessed by Trypan blue dye exclusion assay [23], was

greater than 98%.

2.2. P. gingivalis Lipopolysaccharide

P. gingivalis used for LPS preparation was cultured

from clinical isolates obtained from ATCC No. 33277

[24]. The bacterium was homogenized with liquid phe-

nol-chloroform-petroleum ether, centrifuged, and the

LPS contained in the supernatant was precipitated with

water, washed with 80% phenol solution and dried with

ether. The dry residue was dissolved in a small volume

of water at 45ºC, centrifuged at 100,000xg for 4 h, and

the resulting LPS sediment subjected to lyophilization.

The preparation was essentially free of nucleic acid and

its protein content was less than 0.2%.

2.3. NO Production, and cNOS and iNOS

Activity Assay

NO production in the acinar cells was determined by

B. L. Slomiany et al. / Health 2 (2010) 1448-1455

1450

measuring the stable NO metabolite, nitrite, accumula-

tion in the culture medium using Griess reaction [25]. A

100 µl of spent culture medium was incubated for 10

min with 0.1 ml of Griess reagent and the absorbance

was measured at 570 nm. The concentration of nitrite

was calculated with sodium nitrite as standard. The ac-

tivity of cNOS and iNOS enzymes was measured by

monitoring the conversion of L-[3H]arginine to

L-[3H]citrulline using NOS-detect kit (Stratagene). The

acinar cells from the control and experimental treatments

were homogenized in a sample buffer containing either

10 mM EDTA (for Ca2+ -independent iNOS) or 6 mM

CaCl2 (for Ca2+ -dependent cNOS), and centrifuged

[23]. The aliquots of the resulting supernatant were

incubated for 30 min at 25ºC in the presence of

50µCi/ml of L-[3H]arginine, 10 mM NAPDH, 5 µM

tetrahydrobiopterin, and 50 mM Tis-HCl buffer, pH 7.4,

in a final volume of 250µl. Following addition of stop

buffer and Dowex-50 W (Na+)resin, the mixtures were

transferred to spin cups, centrifuged and the formed

L-[3H]citrulline contained in the flow through was

quantified by scintillation counting.

2.4. Akt Activity Assay

The kinase activity of Ak t in su blingual salivary g land

acinar cells was measured with the Akt Activity Kit

(Calbiochem), by quantifying phosphorylation of a bio-

tinylated peptide substrate (GRPRTSSFAEG). The cells

were lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 150

mM NaCl, 10% glycerol, 1% Triton X-100, 1% deoxy-

cholate, 2 mM EDTA, 1 mM sodium orthovanadate, 1

mM PAF, and 1 mM NaF), containing protease inhibitor

cocktail (Sigma), at 4ºC for 30 min, centrifuged at

14,000 x g for 15 min, and immunoprecipitated with

anti-Akt antibody for 1 h at 4ºC. Protein A/G agarose

beads were then added for an additional 1 h, and the

immune complex was recovered by centrifugation and

thoroughly washed with lysis buffer. The agarose beads

were then suspended for 30 min at room temperature in

the assay buffer, centrifuged, and the supernatants used

for the Akt activity assay by following the manufactur-

er’s instruction.

2.5. Akt Phosphorylation Assay

Measurement of the phosphorylation status of Akt in

sublingual gland acinar cells was performed using Akt

(pThr308) and Akt (pSer473) ELISA kits (Calbiochem).

The cells were lysed on ice for 30 min in lysis buffer (10

mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1

mM EGTA, 1 mM NaF, 2 mM sodium orthovanadate,

1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deox-

ycholate, and 1 mM PMSF), containing protease inhibi-

tor cocktail, and centrifuged at 14,000 x g for 15 min.

The supernatants diluted (1: 10) in standard diluent buf-

fer were pipetted in 100 µl aliquots into wells containing

immobilized capture antibody, and after washing the

complex was reacted with antibody specific for Akt

(pThr308) or Akt (pSer473). Following washing, the re-

tained complex was labeled with horseradish peroxidase

and probed with TMB reagent for spectrophotometric

quantificat i on at 450 nm.

2.6. Akt Protein S-Nitrosylation Assay

Detection of Akt kinase S-nitrosylation in the acinar

cells sublingual salivary gland was carried out using a

biotin switch procedure for protein S-nitrosylation [26,

27]. The cells were treated with iNOS inhibitor, 1400W

(30 µM) or ghrelin (0.6 µg/ml), and incubated for 16h in

the presence of 100ng/ml of P. gingivalis LPS. Follow-

ing centrifugation at 500xg for 5 min, the recovered cells

were lysed in 0.2 ml of HEN lysis buffer (250 mM

HEPES, 1 mM EDTA, 0.1 mM neocuprin, pH 7.7), and

the unnitrosylated thiol groups were blocked with

S-methyl methanethiosulfonate reagent at 50ºC for 20

min [27]. The proteins were precipitated with acetone,

resuspended in 0.2ml of HEN buffer containing 1% SDS,

and subjected to targeted nitrothiol group redu ction with

sodium ascorbate (100 mM). The free thiols were then

labeled with biotin and the biotinylated proteins were

recovered on streptavidin beads. The formed streptavidin

bead-protein complex was washed with neutralization

buffer, and the bound proteins were dissociated from

streptavidin beads with 50 µl of elution buffer (20 mM

HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.7) contain-

ing 1% 2-mercaptoethanol [27]. The obtained proteins

were then analyzed by Western blotting.

2.7. Immunoblotting Analysis

The acinar cells from the control and experimental

treatments were collected by centrifugation and resus-

pended for 30 min in ice-cold lysis buffer (20 mM

Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Tri-

ton X-100, 2 mM EDTA, 1 mM sodium orthovanadate, 4

mM sodium pyrophosphate, 1 mM PMSF, and 1 mM

NaF), containing 1 µg/ml leupeptin and 1 µg/ml pepsta-

tin [28]. Following brief sonication, the lysates were

centrifuged at 12,000g for 10 min, and the supernatants

were subjected to protein determination using BCA pro-

tein assay kit (Pierce). The samples, including those

subjected to biotin switch procedure, were then resus-

pended in loading bu ffer, boiled for 5 min, and subjected

to SDS-PAGE using 40 µg protein/lane. The separated

proteins were transferred onto nitrocellulose membranes,

blocked for 1 h with 5% skim milk in Tris-buffered

B. L. Slomiany et al. / Health 2 (2010) 1448-1455

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Tween (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1%

Tween-20), and probed with the antibody against phos-

phorylated protein at 4ºC for 16 h. After 1h incubation

with the horseradish peroxidase-conjugated secondary

antibody, the phosphorylated proteins were revealed

using an enhanced chemiluminescence. Membranes

were stripped by incubation in 1M Tris-HCl (pH 6.8),

10% SDS, and 10 mM dithiotreitol for 30 min at 55ºC,

and reprobed with antibody against total cNOS or Akt.

Immunoblotting was performed using specific antibodies

directed against cNOS and phospho-cNOS (Ser1179),

and Akt, phospho-Akt (Ser473) (Calbiochem).

2.8. Data Anal ysis

All experiments were carried out using duplicate sam-

pling, and the results are expressed as means ± SD.

Analysis of variance (AN OVA) followed by nonp arame-

tric Kruskal-Wallis test was used to determine signific-

ance and the significance level was set at P < 0.05.

3. Results

To investigate the role of Akt activation in the distur-

bances in NO production associated with oral mucosal

inflammatory responses to periodontopathic bacterium, P.

gingivalis, we used rat sublingual gland acinar cells ex-

posed to the bacterium key virulence factor, LPS. We

demonstrated that P. gingivalis LPS caused a

dose-dependent decrease in the acinar cell Akt activity,

accompanied by the increase in NO production, so that

at 100µg/ml of the LPS the NO production showed a

15.1-fold increase, while the activity of Akt decreased by

a 38% (Figure 1). Furthermore, we revealed that that the

inhibitory effect of the LPS on Akt activity was reflected

in a 41% decrease in th e k inase phospho rylation at Ser 473,

whereas the kinase phosphorylation at Thr308 was not

affected (Figure 2).

Moreover, we found that preincubation with ghrelin

lead to a concentration-dependent suppression of the

LPS-induced effect on Akt activity and the extent of its

protein phosphorylation at Ser473, which at 0.6 µg/ml

resulted in a 2.2-fold increase the activity of Akt, and a

2.5-fold increase in its phosphorylation at Ser473 over

that of the LPS (Figure 3). The Akt protein phosphory-

lation at Thr308, however, was not affected by ghrelin.

We also determined that the disturbances in NO produc-

tion elicited in the acinar cells by P. gingivalis LPS at

100µg/ml were manifested a 26.4-fold up-regulation in

iNOS activity and a 5.2-fold decrease in the activity of

cNOS (Figure 4). However, while the activity of cNOS

in the presence of 0.6 µg/ml ghrelin displayed an 80.4%

increase over that of the LPS, the activity of iNOS in the

presence of ghrelin showed a 94.3% decrease.

Figure 1. Effect of P. gingivalis LPS on Akt kinase ac-

tivity and nitrite production in rat sublingual salivary

gland acinar cells. The cells were treated with the indi-

cated concentrations of the LPS and incubated for 16 h.

Values represent the means ±SD of five experiments. *P

< 0.05 compared with that of control (LPS – 0).

Figure 2. Effect of P. gingivalis LPS on Akt kinase threo-

nine (Thr308) and serine (Ser473) phosphorylation in rat

sublingual salivary gland acinar cells. The cells were

treated with the indicated concentrations of the LPS and

incubated for 16 h. *P < 0.05 compared with that of con-

trol (LPS – 0).

Figure 3. Effect of ghrelin on P. gingivalis LPS-induced

changes in the acinar cell Akt activity and the phosphory-

lation at Thr308 and Ser473. The cells, preincubated with

the indicated concentrations of ghrelin, were treated with

the LPS at 100ng/ml and incubated for 16 h. Values

represent the means ± SD of five experiments. *P < 0.05

compared with that of control. **P < 0.05 compared with

that of LPS alone.

B. L. Slomiany et al. / Health 2 (2010) 1448-1455

1452

Figure 4. Effect of ghrelin on P. gingivalis LPS-induced

changes in the expression of cNOS and iNOS activities in

the salivary gland acinar cells. The cells, preincubated

with the indicated concentrations of ghrelin, were treated

with the LPS at 100 ng/ml and incubated for 16 h. Values

represent the means ± SD of five experiments. *P < 0.05

compared with that of control. **P < 0.05 compared with

that of LPS alone.

To gain further leads into the involvement of Akt in

the regulation of NOS system, we examined the influ-

ence of ghrelin on cNOS phosphorylation. For this, the

acinar cells prior to incubation with the LPS were pre-

treated with ghrelin or ghrelin plus Akt inhibitor, SH-5,

and the lysates were probed with antibodies directed

against cNOS and phosphorylated cNOS (Ser1179). As

shown in Figure 5, the LPS suppression in cNOS activ-

ity was associated with the inhibition in the enzyme

phosphorylation, while the countering effect of ghrelin

was reflected in a marked increase in the enzyme protein

phosphorylation at Ser1179. Moreover, the suppression

of ghrelin effect on cNOS phosphorylation was attained

with Akt inhibitor, SH-5, thus supporting the role of Akt

in ghrelin-induced cNOS activation through phosphory-

lation.

Hence, to reveal the nature of the detrimental effect of

P. gingivalis LPS on the acinar cell Akt activity, we fo-

cused on the influence of NO on the events of Akt acti-

vation. For this, we analyzed the effects of cNOS inhi-

bitor, L-NAME, and iNOS inhibitor, 1400W, as well as

nitrosothiols reducing agent, ascorbate. We found that

the detrimental effect of the LPS on the acinar cell Akt

activity was subject to suppression not only by the pre-

treatment with ghrelin, but also displayed susceptibility

to suppression by iNOS inhibitor, 1400W, and ascorbate,

but not to the cNOS inhibitor, L-NAME (Figure 6).

Furthermore, we observed that ascorbate as well as iN-

OS inhibitor, 1400W, produced amplification in the ef-

fect of ghrelin on Akt activity, thus suggesting that in

addition to the activation through Ser/Thr phosphoryla-

tion, the Akt kinase activity may be also dependent upon

its protein S-nitrosylation with the involvement of iNOS.

To assess further the course of events resulting in the

Figure 5. Effect of Akt kinase in-

hibitor, SH-5 (SH) on ghre-

lin-induced cNOS phosphorylation

in the acinar cells exposed to P.

gingivalis LPS. The cells were

treated with ghrelin (Gh) at 0.6

µg/ml or Akt inhibitor, SH-5 (30

µM)+Gh, and incubated for 16 h in

the presence of 100 ng/ml of the

LPS. Cell lysates were resolved on

SDS-PAGE, transferred to nitro-

cellulose and probed with phos-

phorylation specific cNOS pcNOS)

antibody, and after stripping re-

probed with anti-cNOS antibody.

The immunoblots shown are rep-

resentative of three experiments.

Figure 6. Effect of iNOS inhibitor, 1400W, cNOS inhibitor,

L-NAME, and ascorbate on the ghrelin (Gh)-induced

changes in Akt kinase activity in the acinar cell exposed to

P. gingivalis LPS. The cells, preincubated with 30 µM

1400W (14W), 200µM L-NAME (LN), or 300µM ascor-

bate (As), were treated with Gh at 0.6µg/ml) and incubated

for 16 h in the presence of 100ng/ml LPS. Values represent

the means ± SD of five experiments. *P < 0.05 compared

with that of control. **P < 0.05 compared with that of LPS

alone. ***P < 0.05 compared with that of GhLPS.

suppression of Akt activity by P. gingivalis LPS as well

as to provide additional leads as to the mechanism of

ghrelin countering effect, we examined the effect of

cNOS and iNOS inhibitors, as well as nitrosothiols

re-ducing agent, ascorbate, on Akt enzyme protein

phosphorylation at Thr308 Ser473. We found that while

Akt phosphorylation at Thr308 was not affected by the

LPS or ghrelin, the LPS-induced suppression in Akt

phosphorylation at Ser473 was subject to a partial reversal

in the presence of iNOS inhibitor, 1400W and ascorbate,

but not the cNOS inhibitor, L-NAME (Figure 7). Fur-

B. L. Slomiany et al. / Health 2 (2010) 1448-1455

1453

Figure 7. Effect of iNOS inhibitor, 1400W, cNOS in-

hibitor, L-NAME, and ascorbate on the ghrelin

(Gh)-induced changes in Akt kinase Thr308 and Ser473

phosphorylation in the acinar cells exposed to P. g i n -

givalis LPS. The cells, preincubated with 30 µM

1400W (14W), 200 µM L-NAME (LN), or 300 µM

ascorbate (As), were treated with Gh (0.6µg/ml) and

incubated for 16h in the presence of 100ng/ml LPS.

*P < 0.05 compared with that of control. **P < 0.05

compared with that of LPS alone. ***P < 0.05 com-

pared with that of GhLPS.

ther, both ascorbate and 1400W elicited enhancement in

the effect of ghrelin on Akt protein phosphorylation at

Ser473. These findings indicate that the LPS-induced Akt

protein S-nitrosylation exerts the detrimental effect on

the kinase activation through pho sp horylation at Ser473.

To address further the relationship between Akt

S-nitrosylation and its activation through phosphoryla-

tion, the acinar cells prior to incubation with P. gin-

givalis LPS were pretr eated with iNOS inhibito r, 1400W

or ghrelin, and the lysates following the biotin switch

procedure were probed with antibodies directed against

phospho-Akt (Ser473) and total Akt (Figure 8). We ob-

served that the acinar cells exposed to the LPS alone

showed a marked increase in Akt protein S-nitro sylation,

while the effect of ghrelin was reflected in the loss in

Akt S-nitrosylation and the increase in its protein phos-

phorylation. Furthermore, the blockage of iNOS activity

with 1400W, lead to a substantial decrease in the

LPS-induced Akt S-nitrosylation. Thus, ghrelin counter-

ing effect of the detrimental consequences of P. gingiva-

lis LPS on the Akt activity is reflected in a decrease in

the kinase protein S-nitrosylation and the increase in its

phosphorylation.

4. Discussion

The oral mucosal responses to periodontopathic bac-

terium, P. gingivalis and its key virulence factor, cell

wall LPS, are manifested by a massive rise in epithelial

cell apoptosis, increase in proinflammatory cytokine

expression and the disturbances in NO signaling path

Figure 8. Effect of Ghrelin (Gh) on P. gin-

givalis LPS—induced Akt S-nitrosylation.

The acinar cells were treated with 30 µM

of iNOS inhibitor, 1400W (14W), or 0.6

µg/ml Gh, and incubated for 16h in the

presence of 100ng/ml LPS A portion of the

cell lysates was processed by biotin switch

procedure for protein S-nitrosylation and,

along with the reminder of the lysates,

subjected to SDS-PAGE, transferred to ni-

trocellulose and probed with phospho-Akt

(Ser473) antibody, and after stripping re-

probed with anti-Akt antibody. The immu-

noblots shown are representative of three

experiments.

ways [8,23,29]. As serine/threonine kinase Akt plays a

central role in the regulation of NOS isozyme system

responsible for NO generation [8,15,23], in this study we

investigated the processes associated with the activation

of Akt in rat sublingual salivary gland acinar cells and

the influence of P. gingivalis LPS.

Our findings revealed that the LPS-induced decrease

in cNOS activity and up-regulation in iNOS was asso-

ciated with the suppression in th e activity of Akt kinase.

Moreover, we found that of the two phosphorylation

sites required for the expression of full Akt activity [17,

19], the effect of the LPS was manifested by a decrease

in Akt protein phosphorylation at Ser473, whereas phos-

phorylation at the kinase Thr308 was not affected.

These finding are thus in keeping with the literature data

indicating that inactivation of Akt by bacterial toxins

correlates with the reduced phosphorylation at the kinase

Ser473 located within HM region of the C-terminal do-

main of the kinase [21,30]. Further, preincubation with a

peptide hormone, ghrelin (1,2), recently identified in

saliva and recognized for its modulatory effect on the

inflammatory responses to bacterial infection [3-5,7,8],

elicited countering effect on the LPS-induced changes in

Akt activity and the extent o f the kinase p hosphorylation

on Ser473. Moreover, ghrelin countered the LPS-

induced suppression in cNOS activity and elicited redu c-

tion in the activity of iNOS. We also observed that in

agreement with well documented role of Akt participa-

tion in posttranslational cNOS activation through phos-

phorylation at Ser1179 [6,7,15], the induced up-regula-

tion in cNOS activity by ghrelin was susceptible to sup-

B. L. Slomiany et al. / Health 2 (2010) 1448-1455

1454

pression by Akt inhibitor, SH-5. These findings together

with the recently demonstrated involvement of Akt in

LPS-activated mechanism of iNOS expression induction

[31], attest to central role Akt in the regulation of NOS

system and add further credence as to the importance of

ghrelin in controlling the extent of inflammatory in-

volvement in response to bacterial infection .

Moreover, the accumulating evidence indicates that

physiological and pathophysiological implications of

NO generated by NOS isozyme system may also involve

protein modification through S-nitrosylation at cysteine

residues that results in functional alterations

[7,9,16,20,21]. The NO generated by cNOS has been

linked to the inhibition of apoptogenic signal propaga-

tion through S-nitrosylation of the key executioner cas-

pase, caspase-3, and the events of cytosolic phospholi-

pase A2 activation [7,22,32]. The NO produced by iNOS,

on the other hand, has been implicated in S-nitrosylation

of the insulin receptor and proteins involved in early

steps of the insulin signal transduction, and Akt

S-nitrosylation was reported to be responsible for the

reduced kinase activity in muscle cells of diabetic mice

[8,16,20,21].

Hence to gain more insights into the mechanism of P.

gingivalis LPS-induced disturbances in the acinar cell

Akt kinase activation, we examined the effect of cNOS

and iNOS inhibitors, as well as nitrosothiols reducing

agent, ascorbate, on Akt activity and its protein phos-

phorylation at the critical Thr308 and Ser473. Our findings

revealed that, while phosphorylation at the Akt Thr308

was not affected, the LPS-induced suppression in Akt

activity and the extent of its protein phosphorylation at

Ser473 showed susceptibility to iNOS inhibitor, 1400W,

and ascorbate, but not to cNOS inhibitor, L-NAME.

Moreover, both 1400W and ascorbate elicited amplifica-

tion in ghrelin effect on Akt phosphorylation at Ser473 as

well the activity. Considering well-known susceptibility

of S-nitrosylated proteins to reduction by ascorbic acid

[26,27], the presented data support the involvement of

iNOS in the acinar cell Akt protein S-nitrosylation and

indicate that S-nitrosylation interferes with the kinase

activation through phosphorylation at Ser473. It is also

apparent, that the countering effect of ghrelin on the

LPS-induced changes in Akt activity are reflected in the

loss in the kinase S-nitrosylation and the increase in its

phosphorylation at Ser473 located within the HM region

of the C-terminal domain of Akt [30]. Thus Akt protein

S-nitrosylation, like that of its phosphorylation at

Ser/Thr, appears to be of significance to a variety of

physiological and pathophysiological situations that re-

lay on signaling even ts which utilize this kinase.

Additional evidence as to the role of ghrelin in coun-

tering P. gingivalis LPS interference with Akt activation

through S-nitrosylation comes from the results of biotin

switch assay. We found that the acinar cells exposed to

incubation with the LPS showed a marked increase in

Akt protein S-nitrosylation. The countering effect of

ghrelin on the LPS-induced suppression in Akt activity

was reflected in the loss in S-nitrosylation and the in-

crease in the kinase phosphorylation at Ser473. Moreover,

we observed the dependence of Akt S-nitrosylation on

the LPS-induced up-regulation in iNOS activity, as the

suppression of iNOS with a specific inhibitor, 1400W,

lead to a substantial decrease in Akt S-nitrosy-

lation. These results are thus indicative of the involve-

ment of iNOS-generated NO in the suppression of Akt

activity through S-nitrosylation. Hence, the persistent

induction in iNOS may be of major consequence defin-

ing the extent of oral mucosal inflammato ry inv olvement

in response to P. gingivalis infection.

Together, the data provided in our study demonstrate

that P. gingivalis LPS-induced up-regulation in the aci-

nar cell iNOS expression leads to Akt inactivation

through S-nitrosylation that exert the detrimental effect

on cNOS activation through phosphorylation. We also

show that the countering effect of ghrelin on the

LPS-induced disturbances in Akt kinase activity are ma-

nifested in a decrease in the kinase protein S-nitrosy-

lation and the increase in its phosphorylation at Ser473.

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