4-hydroxynonenal signalling to apoptosis in isolated rat hepatocytes: the role of pkc-δ

.elsevier.com/locate/bba

Biochimica et Biophysica Ac

4-Hydroxynonenal signalling to apoptosis in isolated rat hepatocytes:

The role of PKC-y

L. Castello a, B. Marengo d, M. Nitti b, T. Froio a, C. Domenicotti b, F. Biasi c, G. Leonarduzzi a,

M.A. Pronzato b, U.M. Marinari b, G. Poli a, E. Chiarpotto a,*

a Department of Clinical and Biological Sciences, University of Torino, Regione Gonzole 10, 10043 Orbassano (TO), Italyb Department of Experimental Medicine, University of Genova, Via L.B. Alberti 2, 16132 Genova, Italy

c CNR, Department of Clinical and Biological Sciences, University of Torino, Regione Gonzole 10, 10043 Orbassano (TO), Italyd Gaslini Institute, Genova, Italy

Received 30 November 2004; received in revised form 30 September 2005; accepted 18 October 2005

Available online 4 November 2005

Abstract

4-Hydroxynonenal, a significant aldehyde end product of membrane lipid peroxidation with numerous biochemical activities, has consistently

been detected in various human diseases. Concentrations actually detectable in vivo (0.1–5 AM) have been shown to up-regulate different genes

and modulate various enzyme activities. In connection with the latter aspect, we show here that, in isolated rat hepatocytes, 1 AM 4-

hydroxynonenal selectively activates protein kinase C-y, involved in apoptosis of many cell types; it also induces very early activation of Jun N-

terminal kinase, in parallel increasing activator protein-1 DNA-binding activity in a time-dependent manner and triggering apoptosis after only

120 min treatment. These phenomena are likely protein kinase C-y-dependent, being significantly reduced or annulled by cell co-treatment with

rottlerin, a selective inhibitor of protein kinase C-y. We suggest that 4-hydroxynonenal may induce apoptosis through activation of protein kinase

C-y and of Jun N-terminal kinase, and consequent up-regulation of activator protein-1 DNA binding.

D 2005 Elsevier B.V. All rights reserved.

Keywords: 4-hydroxy-2,3-nonenal (HNE); Protein kinase C; Jun N-terminal kinase; Apoptosis

1. Introduction

The oxidative breakdown of biological membrane phospho-

lipids, i.e., membrane lipid peroxidation, leads to the produc-

tion of a very complex mixture of carbonyl compounds, in

particular malondialdehyde (MDA), n-alkanals, 2-alkenals, and

4-hydroxy-alkenals. Of the hydroxyalkenal class, 4-hydroxy-

2,3-nonenal (HNE) has been shown to be the most interesting

molecule from the biological standpoint [1–3]. This aldehyde

is strongly electrophilic, thus readily reacting with sulphydryl

and amino groups of various bio-molecules, the adducts

formed with both plasma and tissue proteins being of primary

importance [4,5].

Interest in the role of this compound has grown considerably

after its detection in various chronic human diseases, often with

inflammatory and/or fibrotic features [6–8], including athero-

1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbalip.2005.10.003

* Corresponding author. Tel.: +39 11 6705423; fax: +39 11 6705424.

E-mail address: [email protected] (E. Chiarpotto).

sclerosis [9,10]. These findings have greatly stimulated the

investigation of the role of HNE in gene expression related to

chronic inflammation. In this connection, it has been demon-

strated that HNE treatment, at doses similar to those detectable

in vivo, induces expression and synthesis of the fibrogenic

cytokine transforming growth factor h1 in cells of the

macrophage lineage [11], and also of procollagen type I and

tissue inhibitor of metalloproteinases-1 in cultured human

stellate cells [12]. Moreover, HNE has been shown to modify

the expression of other genes, such as aldose reductase [13], g-

globin [14], g-glutamylcysteine synthetase [15,16] and alde-

hyde reductase [17].

We recently showed that different concentrations of HNE

were able to selectively modulate the activity of certain protein

kinase C (PKC) isoenzymes in isolated rat hepatocytes [18]

and in NT2 neurons [19], and that they induce MCP-1 release

by J774 macrophages [20]: high concentrations (10 AM)

inactivated and low concentrations (0.1 AM) activated the

classical (hI and hII) isoforms. Moreover, in isolated

hepatocytes 1 AM HNE activated one novel PKC isoenzyme,

ta 1737 (2005) 83 – 93

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L. Castello et al. / Biochimica et Biophysica Acta 1737 (2005) 83–9384

the y isoform. PKC isoforms differ considerably from the

structural standpoint and may be divided into four subfamilies,

based on primary structure and activation requirements: (1) the

classical isoenzymes (a, hI, hII and g), which are calcium

dependent; (2) the novel isoenzymes (y, (, D and u), which are

calcium independent; (3) the atypical isoenzymes (~ , L and E),which are independent of calcium, diacylglycerol and phorbol

esters; and (4) a class whose only member is PKC A, which is

also calcium independent and is structurally similar to the

novel and atypical isoforms [21]. PKC isoenzymes also

display functional variability, partially due to differences in

tissue distribution, sub-cellular localization and substrate

selectivity [21,22].

Selective involvement of PKC isoenzymes in the regulation

of apoptosis has been reported in a variety of cells, including

hepatocytes [23,24]. It has also been suggested that, during this

process, activation of novel isoforms and in particular of PCK-

y may be involved in the up-regulation of activator protein-1

(AP-1) [25,26], a transcription factor known to be redox-

sensitive [27,28]. However, the involvement of PKCs in HNE

signalling to apoptosis has not yet been demonstrated. The

overall pathway triggered by the aldehyde also still awaits full

elucidation.

Here, using the rat hepatocyte model, we show that HNE

triggers an apoptotic pathway that involves an increase in the

activity of PCK-y and Jun N-terminal kinase (JNK), and finally

a net increase in AP-1 nuclear binding.

2. Materials and methods

2.1. Materials

All chemicals were of reagent grade and were obtained from the following

sources: collagenase Type I, ethyleneglycol bis (h-aminoethylether)-N,N,NV,NV-tetraacetic acid (EGTA), N-2-hydroxyethylpiperazine-NV-2-ethanesulfonic acid

(HEPES), 2-mercaptoethanol, dithiotreitol (DTT), phenylmethylsulphonyl

fluoride (PMSF), leupeptin, aprotinin, phosphatidylserine, dioleylglycerol,

histone H1 and Visking dialysis tubing from Sigma Aldrich Italia (Milan,

Italy); phosphate-buffered saline from OXOID S.p.A., Milan, Italy; buffered

neutral formalin from Bioptica, Milan, Italy; rottlerin from INALCO, Milan,

Italy; protein-G sepharose from Sigma Aldrich Italia (Milan, Italy) ; AP-1

oligonucleotide from Promega Italia (Milan, Italy); ‘‘In situ cell death detection

kit’’, Boehringer, from Roche Diagnostics (Monza, Italy). [g32P]-ATP (specific

activity 3,000 Ci/mmol), nitrocellulose membrane (Hybond C-pure) and

chemiluminescence ECL+plus Western Blot Detection System were supplied

by Amersham International (Milan, Italy); mouse monoclonal antibody reacting

with pJNK, rabbit polyclonal antibody reacting with PKC-y and a single mouse

monoclonal antibody reacting with all classic PKC isoforms (a, hI, hII) weresupplied by Santa Cruz Biotechnology Inc. (Heidelberg, Germany); biotiny-

lated anti-mouse antibody and PBS/BSA were supplied by Dako Spa, Milan,

Italy. All other chemicals were from BDH Italia (Milan, Italy) or Merck

(Darmstadt, Germany).

2.2. Incubation of isolated rat hepatocytes in the presence of

steady-state HNE concentrations

Male rats of the Wistar strain (180–200 g b wt) were used. All animals

received human care according to the criteria of the Italian animal welfare laws,

guidelines and policies. Hepatocytes were isolated by the collagenase perfusion

method described by Poli et al. [29], then resuspended in a balanced salt

solution to 106 cells/ml. Aliquots of 20 ml of cell suspension were introduced

into a piece of Visking 20/32 dialysis tube with suitable pore diameter to avoid

dispersion of the cells, immersed in 500 ml of the balanced salt solution and

incubated inside a rotating bottle at 37 -C for different times, either as such or

in the presence of 1 or 10 AM HNE. The presence of large amounts of HNE in

the bottle compartment provides a constant supply despite active consumption

by the cells [18].

To evaluate the actual HNE concentration outside and inside the dialysis

tube, aliquots (0.5 ml) of the incubation medium in the two compartments were

taken at different times (15, 30, 45 and 60 min) for direct h.p.l.c. monitoring of

HNE content [30].

2.3. Total glutathione content evaluation

After cell incubation and treatment for 60 min, 0.5 ml aliquots of

hepatocyte suspension were taken and the total glutathione content was

determined by the micromethod of Owens and Belcher [31].

2.4. Protein kinase C activity determination

After incubation, cells were centrifuged (80�g for 5 min), resuspended in 2

ml of 10 mM HEPES buffer at pH 7.5, containing 0.25 M sucrose, 5 mM

EDTA, 10 mM 2-mercaptoethanol, 2 mM PMSF and 1 mM leupeptin, and

lysed by sonication. After centrifugation at 13,000�g for 10 min, unbroken

cells and nuclei were discarded and cell lysis was checked by optical

microscopy. The soluble fraction was separated from the particulate fraction

by centrifugation at 100,000�g for 30 min; this fraction was further treated

with the above lysing buffer, containing 0.2% Triton X-100, for 20 min on ice,

and the membrane fraction was collected after centrifugation at 100,000�g for

a further 30 min.

Different PKC isoforms were immunoprecipitated with specific antibodies

and protein G sepharose, starting from cytosol or solubilized membrane

samples of 50 Ag of total proteins. The beads were washed three times in a

PKC buffer (10 mM Tris–HCl, 150 mM NaCl, 10 mM MgCl2 and 0.5 mM

DTT). The kinase assay was performed by adding 15 Al of PKC buffer

containing 0.1 mM ATP, [g32P]ATP (2 ACi per sample), 1 Ag of phosphati-

dylserine, 0.4 Ag of diacylglycerol, 0.5 mM CaCl2 and 10 Ag of histone H1 as

substrate [32]. When n-PKC activity was measured, the PKC buffer was

modified, omitting Ca2+. The reaction was continued for 10 min at 30 -C, thenstopped by addition of 3.5�Laemmli sample buffer. The reaction mixtures

were loaded onto 12.5% SDS-polyacrylamide gel and, after electrophoresis,

were dried and exposed to an autoradiographic film for 24 h at �80 -C. The

relative intensity of phosphorylated substrates was measured by densitometric

scanning of the autoradiographs.

2.5. Nuclear extract preparation

At different treatment times (30–60 min) with 1 or 10 AM HNE, nuclear

extracts were obtained following the method described by Parker and Topol

[33], with slight modifications, and protein content was determined by the

Bradford method (Bio-Rad, Milan, Italy). Cells were washed twice with cold

PBS (without calcium and magnesium) and resuspended in 1 ml of buffer A (15

mM KCl, 10 mM HEPES pH 7.6, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT,

0.1% Nonidet P-40, 1 mM PMSF, 10 Al/ml aprotinin, 2 Ag/ml leupeptin),

incubated for 10 min on ice, briefly mixed and centrifuged at 150�g for 10 min

at 4 -C. The nuclear pellet was lysed by incubation for 20 min at 4 -C in 20 Al ofbuffer B (2 mM KCl, 25 mM HEPES pH 7.6, 0.1 mM EDTA, 1 mM DTT, 1

mM PMSF, 10 Al/ml aprotinin, 2 Ag/ml leupeptin); 20 Al of buffer C (20%

glycerol, 25 mM HEPES pH 7.6, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10

Al/ml aprotinin, 2 Ag/ml leupeptin) were then added; the samples were left on

ice for 5 min and then centrifuged at 15,000�g for 15 min. The supernatant was

stored at �80 -C.

2.6. Electrophoretic mobility shift assay (EMSA)

Binding reactions were run in a mixture (20 Al) containing 20,000 cpm

(0.2–0.5 ng) of end-labeled DNA, equal amounts of nuclear protein extracts

(20 Ag), 20 Ag BSA, 2 Ag poly(dI-dC), 2 Al buffer D (20 mM HEPES pH 7.9,

Fig. 1. Time-course evaluation of HNE concentration in the medium inside the

dialysis tube in the presence of isolated rat hepatocytes. The aldehyde (1-10

AM) was added to 500 ml of incubation medium in a bottle in which the

dialysis tubes were then immersed and incubated at 37 -C for different times

(15, 30, 45 and 60 min). HNE concentration was evaluated by direct h.p.l.c.

monitoring. Values are expressed as meansTS.D. of three separate experiments.

L. Castello et al. / Biochimica et Biophysica Acta 1737 (2005) 83–93 85

20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM DTT

and 0.1 mM PMSF) and 4 Al of buffer F (20% Ficoll-400, 100 mM HEPES pH

7.9, 300 mM KCl, 10 mM DTT and 0.1 mM PMSF). AP-1 oligonucleotide

was labeled using [g32P]ATP and T4 polynucleotide kinase. After 20 min at

room temperature, the reaction products were subjected to electrophoresis at

200 V in 0.5 X Tris–borate at pH 8 through a 4% non-denaturating

polyacrylamide gel. After 150 min, the gel was dried and radioactivity detected

by exposure to Kodak XAR-5 film. Relative intensity of the bands was

measured by densitometric scanning of the autoradiographs. Competition

experiments were performed by incubating the extracts with the labeled probe,

in the presence of 100-fold excess of unlabeled AP-1 or nuclear factor kappa B

(NF-nB) oligonucleotide. The mixture was further incubated with 32P-labeled

probe.

2.7. Detection of apoptosis

After 120 min incubation with or without 1 AM HNE, hepatocytes were

harvested by centrifugation (80�g for 5 min) and resuspended in balanced salt

solution; aliquots corresponding to 50,000 cells were cytocentrifuged at 30�g

for 7 min in a Cytospin cytocentrifuge (Shandon Inc., Pittsburgh, U.S.A.) and

fixed with 0.4 ml 4% buffered formalin, pH 7.4, for 10 min. For detection and

quantification of apoptosis at the single-cell level, the TUNEL (TdT-mediated

dUTP nick end labeling; ‘‘In situ cell death detection kit’’, Boehringer, Roche

Diagnostics S.p.A., Monza, Italy) and the Hoechst staining techniques were

used. The first method identifies DNA strand breaks by labeling free terminal

3_-OH with nucleotides conjugated with fluorescein in an enzymatic reaction:

terminal deoxynucleotidyl transferase (TdT) catalyses the polymerization of the

new strand of DNA using the labeled nucleotides [34]. Samples were analysed

by laser scanning confocal microscopy (LSCM, Zeiss, Germany), using 488 nm

excitation and 505 nm emission wavelengths. Apoptosis was quantified by

counting the number of labeled cells on each slide. To detect nuclear

morphology by Hoechst staining, after collection by cytocentrifugation as

described above, the slides were fixed in 95% ethanol for 10 min, incubated at

37 -C for 10 min in Hoechst solution (3.2 AM in PBS 1�), and washed with

PBS and 95% ethanol. Nuclear morphology changes were analysed using a

fluorescent microscope with an ultraviolet filter, 630� magnification.

2.8. Immunocytochemical evaluation of JNK activation

Hepatocytes in suspension (50,000 cells) were cytocentrifuged (30�g) for 5

min in a Cytospin cytocentrifuge (Shandon Inc., Pittsburgh, U.S.A.). The cells

were fixed in 95% ethanol for 5 min at room temperature and permeabilized

with sodium-cyano-boro-hydrate (100 mM in 140 mM NaCl and 10 mM PBS,

pH 7.4) for 10 min at 37 -C. After preincubation with 5% normal goat serum,

3% bovine serum albumin (BSA), and 0.3% Tween 20 in phosphate-buffered

saline (PBS; 0.01 M) for 30 min at room temperature to block non-specific

binding, cells were stained for indirect immunofluorescence using an anti-

pJNK mouse monoclonal primary antibody at 1:300 (v/v) dilution in PBS/BSA

(0.1% albumin, pH 7.6) in a humidified chamber for 1 h at room temperature.

Negative controls were incubated in the solution with normal goat serum. The

cells were then incubated with a biotinylated anti-mouse secondary antibody

(1:500 dilution v/v in PBS/BSA) for 30 min at room temperature and then with

fluorescein avidin D (10 Ag/ml in PBS/BSA) for 15 min at room temperature in

the dark. The bound immunocomplex was visualized by incubation with 0.1%

propyl gallate in PBS for 15 min. All incubations were preceded by two 5-min

washes with PBS/BSA. The slides were mounted with glycerol and observed

with a laser scanning confocal microscope (LSCM Zeiss, Germany) equipped

with an inverted microscope with a 40� objective. The instrument was set to

488 nm exciting light, with a filter barrier of 510 nm on the emission pathway.

Intracellular fluorescence was evaluated quantitatively by computerized image

analysis.

2.9. Statistical analyses

Student’s t test and one-way analysis of variance (ANOVA) associated with

Dunnett’s test were used to determine the statistical significance of the

differences between experimental groups.

3. Results

3.1. Maintenance of steady-state HNE concentration

During the experimental incubation, HNE continually

diffused into the dialysis tube and was taken up by the cells,

but the concentration in the 500 ml mother solution did not

show any significant variation (data not shown). Hence, when

1–10 AM HNE was added to the main compartment medium,

hepatocytes were effectively exposed to a steady-state concen-

tration of aldehyde for almost the entire duration of treatment

very close to the external concentration, i.e., about 0.8 AM and

about 9 AM respectively (Fig. 1).

3.2. Effect of HNE on total glutathione content

At least for the 60 min incubation time in our experimental

design, the total glutathione content of the cells was not

modified by low HNE concentrations (1–10 AM), while the

highest HNE concentration used (100 AM) markedly decreased

it (Fig. 2).

3.3. Effect of 1 lM HNE on the activity of classic and novel

PKC isoforms

To distinguish the different PKC isoforms, they were

immunoprecipitated with specific antibodies; when PKC-yactivity was measured the PKC buffer was modified, omitting

Ca2+.

Exposure of isolated rat hepatocytes to 1 AM HNE for 30

min exerted a differential effect on the activity of different PKC

isoforms: while HNE significantly inhibited the classic PKC

isoforms, it stimulated PKC-y activity by at least 40% in both

Fig. 2. Total glutathione content in HNE-treated hepatocytes. The aldehyde (1–

10–100 AM) was added to 500 ml of incubation medium in a bottle in which

the dialysis tubes were then immersed and incubated at 37 -C for 60 min. Total

glutathione content was evaluated by the micromethod of Owens and Belcher

[31]. Values are expressed as nmol/106 cells and are means S.D. of three

separate experiments in duplicate. *P <0.001 versus control.Fig. 4. Time-course of PKC-y isoform activity in the total cellular extracts o

control and HNE-treated hepatocytes. The aldehyde concentration used was 1

AM. PKC activity was assayed by the immunoprecipitation technique (see

Materials and methods): the autoradiographic bands from one representative

experiment (upper panel) show the phosphorylation of H1 histone. In the lowe

panel, isoenzyme activity is expressed as percentage variation versus the

control values, derived from densitometric analysis. Values are meansTS.D. ofour separate experiments. *P <0.05 versus control; **P <0.01 versus control


cytosol and membrane fractions (Fig. 3). Moreover, PKC-yactivity in total cell lysates was slightly but significantly

increased after only 15 min treatment with the aldehyde,

reaching a maximum after 30 min. This increase was seen to be

transient and was followed by a reduction at longer incubation

times (Fig. 4).

3.4. AP-1 nuclear binding in hepatocytes treated with 1 lMHNE

It is well known that activator protein-1 (AP-1) is a redox-

sensitive transcription factor [27,28] closely related to the

activity of protein kinase C y [35]; we studied the effect of

HNE concentrations within the pathophysiological range (1

and 10 AM) on AP-1 nuclear binding. In the presence of 10 AMHNE, DNA binding activity of this transcription factor did not

Fig. 3. PKC isoform activities in the cytosol and membrane fractions of control and HNE-treated hepatocytes. The aldehyde concentration used was 1 AM. PKC

activity was assayed by the immunoprecipitation technique (see Materials and methods): the autoradiographic bands from one representative experiment (uppe

panel) show the phosphorylation of H1 histone, substrate for all the PKC isoenzymes considered. In the lower panel, isoenzyme activities are expressed as percentage

variations versus the control values, derived from densitometric analysis. Values are meansTS.D. of four separate experiments. *P <0.05; **P <0.001 versus control

cPKCs: classic PKCs.

f

r

f

.

appear affected, being slightly decreased at 30 min, but

returning to control values after 45 and 60 min treatment. On

the contrary, 1 AM HNE markedly increased AP-1 nuclear

binding activity after only 30 min treatment, with a stimulatory

effect that appeared to be time-dependent at least in these

experimental conditions (Fig. 5).

Co-treatment of isolated rat hepatocytes with rottlerin, a

rather specific inhibitor of novel PKC isoforms [36], strongly

counteracted the HNE-induced increase in AP-1 nuclear

binding (Fig. 6). The concentration of rottlerin used was

chosen on the basis of its IC50 [36]; this concentration was

r

.

Fig. 5. Time-course of AP-1 DNA-binding in control and HNE-treated hepatocytes. The aldehyde concentrations used were 1 and 10 AM. AP-1 nuclear binding

activity was analysed by EMSA in nuclear cell extracts. The specificity of the shifted band was assessed by competition experiments. The autoradiographic bands

from one representative experiment are shown in the left panel (lanes 1–4–7: control; lanes 2–5–8: 1 AMHNE; lanes 3–6–9: 10 AMHNE). The histograms on the

right represent AP-1 binding activity, expressed in arbitrary units derived from the densitometric analysis. Values are meansTS.D. of three separate experiments.

*P <0.05 versus control.


shown to be non toxic (data not reported) and is in the range of

concentrations commonly used to obtain selective inhibition of

novel PKC isoforms [37–39].

3.5. Pro-apoptotic effect of 1 lM HNE on rat hepatocyte

suspensions

Apoptosis was examined by both confocal and fluorescence

microscopy in hepatocytes treated with 1 AM HNE, employing

the TUNEL test and Hoechst staining, respectively. As shown

in Fig. 7, HNE produced a large number of TUNEL positive

Fig. 6. Protective effect of rottlerin on AP-1 DNA-binding in control and HNE-treate

AM respectively. The autoradiographic bands from one representative experiment are

16 AM Rottlerin; lane 4: 16 AM Rottlerin). The histograms on the right represent AP

analysis. Values are meansTS.D. of three separate experiments. *P <0.05 versus co

cells (93% in treated cells versus 1% in control cells) already

after 120 min of incubation. Interestingly, co-treatment with

rottlerin (16 AM) markedly reduced the pro-apoptotic effect of

HNE since the percentage of TUNEL positive cells was

reduced by 60%. Notably, cell treatment with rottlerin alone

did not lead to any significant effect versus control hepatocytes.

The protective effect of rottlerin was confirmed by the more

direct approach of Hoechst staining, as reported in Fig. 8. Cell

treatment with 1 AM HNE was shown to induce the appearance

of several apoptotic bodies after 120 min of cell incubation,

while co-treatment with rottlerin (16 AM) once again protected

d hepatocytes. The aldehyde and rottlerin concentrations used were 1 AM and 16

shown on the left (lane 1: control; lane 2: 1 AM HNE; lane 3: 1 AM HNE plus

-1 binding activity, expressed in arbitrary units derived from the densitometric

ntrol; **P <0.05 versus HNE.

Fig. 7. DNA fragmentation in control and HNE-treated hepatocytes: protective

effect of rottlerin. The aldehyde and rottlerin concentrations used were those

reported in Fig. 4. Hepatocyte suspension was incubated for 120 min inside

dialysis tubes immersed in 500 ml of incubation medium with or without HNE

and/or rottlerin. At the end of incubation, cells were processed and analysed for

DNA fragmentation using the ‘‘In situ cell death detection kit’’, as described in

Materials and methods. Samples were analysed by laser scanning confocal

microscopy (LSCM, Zeiss, Germany), using 488 nm excitation and 505 nm

emission wavelengths. The images shown were recorded with a 40� objective,

and are 163 Am�163 Am�7.6 Am sections; they are from one of three

separate experiments. Scale bar: 20 Am. A: control; B: 1 AM HNE; C: 16 AMRottlerin; D: 1 AM HNE plus 16 AM Rottlerin.


against the proapoptotic effect of the aldehyde. Under Hoechst

staining, hepatocytes incubated with rottlerin alone did not

show any difference versus untreated cells.

Fig. 8. Apoptosis in control and HNE-treated hepatocytes: protective effect of rottler

Hepatocyte suspension was incubated for 120 min inside dialysis tubes immersed in

of incubation, cells were processed and analysed for apoptosis using the Hoechst

morphology changes were analysed using a fluorescent microscope with an ultraviol

A: control; B: 1 AM HNE; C: 16 AM Rottlerin; D: 1 AM HNE plus 16 AM Rottler

3.6. JNK activation in hepatocytes treated with 1–10 lM HNE

We used laser confocal microscopy to investigate HNE-

induced effects on JNKs, and at the same time the possible

nuclear translocation of these kinases, using an anti-pJNK

antibody coupled with a biotinylated secondary antibody and

fluorescent avidin. In the presence of 10 AM HNE, JNK

activity did not appear affected; on the contrary, there was a

marked increase in fluorescence after 10–15 min stimulation

with 1 AM HNE, with maximum increase at 15 min. Longer

exposure times were characterized by a progressive loss of this

effect (Fig. 9). Of note in this case too the stimulation of JNK

activation induced by HNE was largely prevented in HNE-

treated hepatocytes co-incubated with rottlerin (Fig. 10).

4. Discussion

There is increasing evidence that HNE, generated during the

lipid peroxidation process, is a key mediator of oxidative

stress-induced pathophysiological effects. In particular, at

doses compatible with those detected in vivo (1–10 AM),

HNE exhibits a wide array of biological activities, including

signal transduction, gene expression and modulation of cell

proliferation (for updated reviews, see [3,40]).

To contrast the rapid metabolism of the aldehyde shown by

the majority of cell types tested, including liver cells [30], we

applied to the rat hepatocytes a system designed to keep the

concentration of HNE in the cell medium steady for the whole

incubation period. In fact, by externally adding 1–10 AMHNE, we were certain to expose the cells to steady concentra-

tions very close to the external concentration, i.e., 0.8 and 9 AMrespectively (Fig. 1). Since in previous studies, we have

demonstrated that very low steady amounts of HNE differen-

in. The aldehyde and rottlerin concentrations used were those reported in Fig. 4.

500 ml of incubation medium with or without HNE and/or rottlerin. At the end

staining technique. One representative experiment of three is shown. Nuclear

et filter, 63�objective. Scale bar: 20 Am. The arrows indicate apoptotic bodies.

in.

Fig. 9. Time-course of JNK activation in control and HNE-treated hepatocytes. The aldehyde concentrations used were 1 and 10 AM. JNK activation was assayed by

an immunocytochemical technique (see Materials and Methods) and analysed by laser scanning confocal microscopy (LSCM, Zeiss, Germany), using 490 nm

excitation and 525 nm emission wavelengths. The images shown in the upper panel were recorded with a 40� objective, and are 163 Am�163 Am�7.6 Amsections; they are from one of two separate experiments. Scale bar: 20 Am. A: control; B: 1 AM HNE; C: 10 AM HNE. The lower panel shows quantification of

fluorescence intensity. Values are meansTS.D. of the fluorescence values of 100 cells. *P <0.001 versus control.


tially modulate PKC isoforms in the rat hepatocyte model [18],

we focused our attention on this low range of concentrations.

Here, we show that 1 AM HNE rapidly triggers a signalling

pathway leading to programmed cell death: this pathophysio-

logical concentration of the aldehyde induces apoptosis of

isolated rat hepatocytes after 120 min and the process appears

related to the increased PKC-y activity (Figs. 3, 4), since it is

prevented by rottlerin co-treatment (Figs. 7, 8).

It has been reported variously that higher concentrations of

HNE and long-term treatment cause apoptosis in different cell

types [41–44]; however, an additional and important aim is to

define the molecular pathways regulating its proapoptotic

effect.

In this context, PKC is a family of related, but structurally

and functionally different, polypeptides, playing key regulatory

roles in cell function, such as gene expression, and cell

proliferation and differentiation [45]. In particular, PKC�y hasbeen implicated in the apoptotic process in many cell types,

such as epidermal cells, neutrophils, myeloid cells and

fibroblasts [26,46–48].

An early event in HNE-signalling to apoptosis is increased

PKC-y activity (Figs. 3, 4) since selective inhibition of this

isoform by rottlerin efficiently counteracts the apoptotic

process (Figs. 7, 8). As regards the possible mechanism/s of

PKC-y modulation by HNE, involvement of reactive oxygen

species (ROS) as mediators of the enhanced enzymatic activity

Fig. 10. Time-course of JNK activation in control and HNE-treated hepatocytes: effect of rottlerin. The aldehyde and rottlerin concentrations used were those

reported in Fig. 4. JNK activation was assayed as reported in Fig. 9. Scale bar: 40 Am. A: control; B: 1 AM HNE; C: 16 AM Rottlerin; D: 1 AM HNE plus 16 AMRottlerin. The lower panel shows quantification of fluorescence intensity. Values are meansTS.D. of the fluorescence values of 100 cells. *P <0.001 versus control;‘P <0.01 versus HNE.


is ruled out by the low diclorofluorescein accumulation in both

control and HNE-treated hepatocytes (data not shown).

Moreover, HNE showed no effect on hepatocyte total

glutathione content, except at the 100 AM concentration (Fig.

2), further confirming that no oxidative stress is caused by very

low HNE concentrations. This is unlike what occurs with high

micromolar HNE concentrations which have been reported to

induce ROS production [49,50] or to induce GSH depletion,

shown in turn to induce a marked increase in PKC-y activity

through ROS generation [51].

It has been extensively demonstrated that virtually all of the

biochemical effects of HNE can be explained by its high

reactivity towards thiol and amino groups. Primary reactants

for HNE are the amino acids cysteine, histidine and lysine

[1,3,5]. It is thus conceivable that very low doses of HNE can

activate PKC-y through a direct interaction of the aldehyde

with these thiol-rich regions of the kinase regulatory domain,

by decreasing auto-inhibition and permitting cofactor-indepen-

dent catalytic activity [52]. This hypothesis is supported by our

previous work demonstrating that HNE interacts directly with

single PKC isoenzymes inducing changes in their functional

activity [20]. Moreover, since both the regulatory and catalytic

domains of PKC contain cysteine-rich regions [52,53], the

accumulated evidence suggests a model in which selective

oxidative modification of the regulatory domain leads to

activation, whereas higher concentrations of oxidants react


with the catalytically important cysteine residues and inactivate

the enzyme. In this context, previous studies from our group

have confirmed the biphasic behaviour of PKC in response to

different concentrations of pro-oxidant compounds [54,55].

We also show that 1 AM HNE leads to rapid induction of

AP-1 nuclear binding, which is already evident after 30–60

min (Fig. 5) and appears to be related to PKC�y activity, sincerottlerin co-treatment annuls it completely (Fig. 6). It is

noteworthy that the effect of HNE on AP-1 activation in rat

liver cells is concentration-dependent, 10 AM HNE being

ineffective in this respect (Fig. 5). This behaviour is peculiar to

HNE which has been demonstrated to modulate AP-1 activity

stimulating it at low concentrations and decreasing it at higher

ones, in different cell types [56,57].

Again, as a consequence of the stimulated PKC-y activity, 1AM HNE subsequently induces JNK activation; this effect

likewise is counteracted by co-treatment with rottlerin, which

per se does not influence this kinase (Fig. 10). Moreover, at 60

min, when PKC-y activity decreases versus control levels, JNK

is down regulated. These results are in partial agreement with

previous studies showing JNK nuclear translocation and

activation to be independent of its phosphorylation in HNE-

treated human hepatic stellate cells [58]. However, other

studies have shown that HNE also induces JNK activation in

neuronal cells through its phosphorylation [59,60], and in

HL60 cells, where activation is related to induction of

apoptosis [41]. It is however noteworthy that the HNE

concentration used in these latter studies was one order of

magnitude higher than that used here. Moreover, once again,

the effect of HNE on JNK activity is concentration-related, low

concentrations (1 AM) activating JNK whereas higher ones (10

AM) are without effect (Fig. 9). This could be related to a

differential effect of HNE on c-jun and c-fos, the two major

constituents of the transcriptional factor. In fact, Kakishita and

Hattori have shown that in vascular smooth muscle cells HNE

increases c-fos gene expression from 1 to 2.5 AM then

decreasing it at higher concentrations, while c-jun expression

increases as a function of HNE concentration [56].

In conclusion, these data indicate that, at a concentration

compatible with those detected in several human diseases,

HNE induces apoptosis in liver parenchymal cells. HNE-

triggered programmed cell death has been reported to be

involved in several oxidative stress-related human diseases

[43,61,62]. However, analysis of the molecular events under-

lying this effect has so far been mainly limited to specific

individual steps, without achieving an adequate overview.

The rat hepatocyte model allowed us to show that HNE-

induced apoptotic cell death proceeds through activation of

PKC-y, JNK and AP-1 nuclear binding.

Moreover, in agreement with results obtained for other cell

types [63,64], we show that PKC-y plays a key role in the

machinery of hepatocyte apoptotic death: selective inhibition of

this novel PKC isoform prevents both early events, such as

JNK activation, and relatively late events, such as increased

AP-1 DNA binding activity and apoptotic morphological

features. Recent studies suggest that PKC-y may play a role

in two or more steps in the apoptotic pathway both upstream

and downstream of caspase activation [65,66]. These biological

functions of PKC-y may be distinct, full-length PKC-y playing

a role in the initiation of apoptosis, and cleavage and activation

of PKC-y by caspase 3 resulting in the amplification of

apoptosis [67].

A previous study on rat hepatocytes exposed to 0.1 AMHNE, (one order of magnitude lower than the present

concentration) showed this aldehyde to up-regulate the activity

of classic isoforms with a positive influence on cell functions

such as protein secretion and differentiation [18].

Until the very recent past, HNE was considered to be a toxic

compound to cells and tissues, primarily because in vitro

biochemical studies generally used HNE concentrations above

10 AM, to counteract the rapid metabolism of the aldehyde

shown by the majority of cell types tested. On the contrary, on

the basis of the extensive research of recent years [2,3,40] and

of our present findings, the role of HNE in the signalling

process appears to be an intriguing one because its effect is

concentration dependent. In particular, in the low concentration

range found in human physiopathology, it may act as a

molecular intermediate in the complex regulation of cell

function, differentiation, proliferation or apoptotic program,

as they occur in the various tissues.

Acknowledgements

This research was supported by grants from the Italian

Ministry for Education, University and Research, National

Projects, ‘‘Lipid oxidation and peroxidation products as

modulators of cellular functions’’, ‘‘Molecular signals of cell

growth regulation triggered by glutathione depletion’’ (Re-

search Projects of National Interest) and ‘‘Oxidative stress in

the pathogenesis of chronic complications of diabetes’’

(Investment Fund for Basic Research), from the National

Research Council (CNR), Rome, Finalised Project on Biotech-

nology, from the Universities of Turin and Genoa and from the

Piedmont Regional Government.

References

[1] H. Esterbauer, R.J. Schaur, H. Zollner, Chemistry and biochemistry of 4-

hydroxynonenal, malonaldehyde and related aldehydes, Free Radical Biol.

Med. 11 (1991) 81–128.

[2] M.U. Dianzani, 4-Hydroxynonenal and cell signalling, Free Radical Res.

28 (1998) 553–560.

[3] G. Poli, R.J. Schaur, 4-Hydroxynonenal in the pathomechanisms of

oxidative stress, IUBMB Life 50 (2000) 315–321.

[4] K. Uchida, E.R. Stadtman, Quantitation of 4-hydroxynonenal protein

adducts, Methods Enzymol. 233 (1994) 371–380.

[5] G. Waeg, G. Dimsity, H. Esterbauer, Monoclonal antibodies for detection

of 4-hydroxy-nonenal modified proteins, Free Radical Res. 25 (1996)

149–159.

[6] S. Seki, T. Kitada, T. Yamada, H. Sakaguchi, K. Nakatani, K. Wakasa, In

situ detection of lipid peroxidation and oxidative DNA damage in non-

alcoholic fatty liver diseases, J. Hepatol. 37 (2002) 56–62.

[7] S. Arlt, U. Beisiegel, A. Kontush, Lipid peroxidation in neurodegenera-

tion: new insights into Alzheimer’s disease, Curr. Opin. Lipidol. 13 (2002)

289–294.

[8] K. Zarkovic, 4-hydroxynonenal and neurodegenerative diseases, Mol.

Aspects Med. 24 (2003) 293–303.


[9] C. Napoli, F.P. D’Armiento, F.P. Mancini, A. Postiglione, J.L. Witzum, G.

Palumbo, W. Palinski, Fatty streak formation occurs in human fetal aortas

and is greatly enhanced by maternal hypercholesterolemia. Intimal

accumulation of low density lipoprotein and its oxidation precede

monocyte recruitment into early atherosclerotic lesions, J. Clin. Invest.

100 (1997) 2680–2690.

[10] K. Itakura, T. Oya-Ito, T. Osawa, S. Yamada, S. Toyokuni, N. Shibata, M.

Kobayashi, K. Uchida, Detection of lipofuscin-like fluorophore in

oxidized human low-density lipoprotein. 4-hydroxy-2-nonenal as a

potential source of fluorescent chromophore, FEBS Lett. 473 (2000)

249–253.

[11] G. Leonarduzzi, A. Scavazza, F. Biasi, E. Chiarpotto, S. Camandola, S.

Vogl, R. Dargel, G. Poli, The lipid peroxidation end-product 4-hydroxy-

2,3-nonenal up-regulates transforming growth factor-h1 expression in the

macrophage lineage: a link between oxidative injury and fibrosclerosis,

FASEB J. 11 (1997) 851–857.

[12] E. Zamara, E. Novo, F. Marra, A. Gentilini, R.G. Romanelli, A.

Caligiuri, G. Robino, E. Tamagno, M. Aragno, O. Danni, R. Autelli, S.

Colombatto, M.U. Dianzani, M. Pinzani, M. Parola, 4-Hydroxynonenal

as a selective pro-fibrogenic stimulus for activated human hepatic stellate

cells, J. Hepatol. 40 (2004) 60–68.

[13] J. Ruef, S.Q. Liu, C. Bode, M. Tocchi, S. Srivastava, M.S. Runge, A.

Bhatnagar, Involvement of aldose reductase in vascular smooth muscle

cell growth and lesion formation after arterial injury, Arterioscler. Thromb.

Vasc. Biol. 20 (2000) 1745–1752.

[14] V.M. Fazio, G. Barrera, S. Martinotti, M.G. Farace, B. Giglioni, L. Frati,

V. Manzari, M.U. Dianzani, 4-Hydroxynonenal, a product of cellular lipid

peroxidation, which modulates c-myc and globin gene expression in K562

erythroleukemic cells, Cancer Res. 52 (1992) 4866–4871.

[15] R.M. Liu, Z. Borok, H.J. Forman, 4-Hydroxy-2-nonenal increases

gamma-glutamylcysteine synthetase gene expression in alveolar

epithelial cells, Am. J. Respir. Cell Mol. Biol. Physiol. 24 (2001)

499–505.

[16] D.A. Dickinson, K.E. Iles, N. Watanabe, T. Iwamoto, H. Zhang, D.M.

Krzywanski, H.J. Forman, 4-Hydroxynonenal induces glutamate cysteine

ligase through JNK in HBE1 cells, Free Radical Biol. Med. 33 (2002)

974–987.

[17] Y.H. Koh, Y.S. Park, M. Takahashi, K. Suzuki, N. Taniguchi, Aldehyde

reductase gene expression by lipid peroxidation end products, MDA and

HNE, Free Radical Res. 33 (2000) 739–746.

[18] E. Chiarpotto, C. Domenicotti, D. Paola, A. Vitali, M. Nitti, M.A.

Pronzato, F. Biasi, D. Cottalasso, U.M. Marinari, A. Dragonetti, P.

Cesaro, C. Isidoro, G. Poli, Regulation of rat hepatocyte protein kinase C

( isoenzymes by the lipid peroxidation product 4-hydroxy-2,3-nonenal: a

signaling pathway to modulate vesicular transport of glycoproteins,

Hepatology 29 (1999) 1565–1572.

[19] D. Paola, C. Domenicotti, M. Nitti, A. Vitali, R. Borghi, D. Cottalasso, D.

Zaccheo, P. Odetti, P. Strocchi, U.M. Marinari, M. Tabaton, M.A.

Pronzato, Oxidative stress induces increase in intracellular amyloid h-protein production and selective activation of hI and hII PKCs in NT2

cells, Free Radical Biol. Med. 268 (2000) 642–646.

[20] M. Nitti, C. Domenicotti, C. d_Abramo, S. Assereto, D. Cottalasso, E.

Melloni, G. Poli, F. Biasi, U.M. Marinari, M.A. Pronzato, Activation of

PKC-h isoforms mediates HNE-induced MCP-1 release by macrophages,

Biochem. Biophys. Res. Commun. 294 (2002) 547–552.

[21] H. Mellor, P.J. Parker, The extended protein kinase C superfamily,

Biochem. J. 332 (1998) 281–292.

[22] E.C. Dempsey, A.C. Newton, D. Mochly-Rosen, A.P. Fields, M.E.

Reyland, P.A. Insel, R.O. Messing, Protein kinase C isoenzymes and the

regulation of diverse cell responses, Am. J. Physiol.: Lung Cell Mol.

Physiol. 279 (2000) L429–L438.

[23] B.A. Jones, Y.P. Rao, R.T. Stravitz, G.J. Gores, Bile salt-induced apoptosis

of hepatocytes involves activation of protein kinase C, Am. J. Physiol. 272

(1997) G1109–G1115.

[24] M. Villalba, A possible role for PKC delta in cerebellar granule cells

apoptosis, NeuroReport 13 (1998) 2381–2385.

[25] S. Hirai, Y. Izumi, K. Higa, K. Kaibuchi, K. Mizuno, S. Osada, K. Suzuki,

S. Ohno, Ras-dependent signal transduction is indispensable but not

sufficient for the activation of AP1/jun by PKCy, EMBO J. 13 (1994)

2331–2340.

[26] N. Chen, W. Ma, C. Huang, Z. Dong, Translocation of protein kinase (

and protein kinase y to membrane is required for ultraviolet B-induced

activation of mitogen-activated protein kinases and apoptosis, J. Biol.

Chem. 274 (1999) 15389–15394.

[27] M. Karin, Z.G. Liu, E. Zandi, AP-1 function regulation, Curr. Opin. Cell

Biol. 9 (1997) 240–246.

[28] K.E. Iles, D.A. Dickinson, N. Watanabe, T. Iwamoto, H.J. Forman, AP-1

activation through endogenous H2O2 generation by alveolar macrophages,

Free Radical Biol. Med. 32 (2002) 1304–1313.

[29] G. Poli, E. Gravela, E. Albano, M.U. Dianzani, Studies on fatty liver with

isolated hepatocytes: II. The action of carbon tetrachloride on lipid

peroxidation, protein, and triglyceride synthesis and secretion, Exp. Mol.

Pathol. 30 (1979) 116–127.

[30] H. Esterbauer, H. Zollner, J. Lang, Metabolism of the lipid peroxidation

product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic

fractions, Biochem. J. 228 (1985) 363–373.

[31] C.W.I. Owens, R.V. Belcher, A colorimetric micro-method for the

determination of glutathione, Biochem. J. 94 (1965) 705–711.

[32] F. Salamino, B. Sparatore, R. De Tullio, P. Mengotti, E. Melloni, S.

Pontremoli, Respiratory burst in activated neutrophils is directly correlated

to the intracellular level of protein kinase C, Eur. J. Biochem. 200 (1991)

573–577.

[33] C.S. Parker, A. Topol, A Drosophila RNA polymerase II transcription

factor contains promoter-region-specific DNA-binding activity, Cell 36

(1984) 357–369.

[34] R. Sgonc, G. Boeck, H. Dietrich, J. Gruber, H. Recheis, G. Wick,

Simultaneous determination of cell surface antigens and apoptosis, Trends

Genet. 10 (1994) 41–42.

[35] S.S. Chuang, J.K. Lee, P.A. Mathew, Protein kinase C is involved in 2B4

(CD244)-mediated cytotoxicity and AP-1 activation in natural killer cells,

Immunology 109 (2003) 432–439.

[36] M. Gschwendt, H.J. Muller, K. Kielbassa, R. Zang, W. Kittstein, G.

Rincke, F. Marks, Rottlerin, a novel protein kinase inhibitor, Biochem.

Biophys. Res. Commun. 199 (1994) 93–98.

[37] J. Zhang, N. Liu, J. Zhang, S. Liu, D. Zheng, PKCdelta protects human

breast tumor MCF-7 cells against tumor necrosis factor-related apopto-

sis-inducing ligand-mediated apoptosis, J. Cell. Biochem. 96 (3) (2005)

522–532.

[38] A. Basu, H. Tu, Activation of ERK during DNA damage-induced

apoptosis involves protein kinase Cdelta, Biochem Biophys Res Commun.

334 (2005) 1068–1073.

[39] E.A. Woolfolk, S. Educhi, H. Ohtsu, H. Nakashima, H. Ueno, W.T.

Gerthoffer, E.D. Motley, Angiotensin II-induced activation of P21-

activated kinase 1 requires Ca2+ and protein kinase C {delta} in vascular

smooth muscle cells, Am. J. Physiol.: Cell Physiol. 289 (5) (2005)

C1286–C1294.

[40] G. Poli, G. Leonarduzzi, F. Biasi, E. Chiarpotto, Oxidative stress and cell

signalling, Curr. Med. Chem. 11 (2004) 1163–1182.

[41] Y.C. Awasthi, R. Sharma, J.Z. Cheng, Y. Yang, A. Sharma, S.S. Singhal,

S. Awasthi, Role of 4-hydroxynonenal in stress-mediated apoptosis

signalling, Mol. Aspects Med. 24 (2003) 219–230.

[42] J.D. West, C. Ji, S.T. Duncan, V. Amarnath, C. Schneider, C.J. Rizzo, A.R.

Brash, L.J. Marnett, Induction of apoptosis in colorectal carcinoma cells

treated with 4-hydroxy-2-nonenal and structurally related aldehydic

products of lipid peroxidation, Chem. Res. Toxicol. 17 (2004) 453–462.

[43] I. Kruman, A.J. Bruce-Keller, D. Bredesen, G. Waeg, M.P. Mattson,

Evidence that 4-hydroxynonenal mediates oxidative stress-induced neu-

ronal apoptosis, J. Neurosci. 17 (1997) 5089–5100.

[44] U. Herbst, M. Toborek, S. Kaiser, M.P. Mattson, B. Henning, 4-

Hydroxynonenal induces dysfunction and apoptosis of cultured endothe-

lial cells, J. Cell. Physiol. 181 (1999) 295–303.

[45] Y. Nishizuka, Protein kinase C and lipid signaling for sustained cellular

responses, FASEB J. 9 (1995) 484–496.

[46] J. Pongracz, P. Webb, K. Wang, E. Deacon, O.J. Lunn, J.M. Lord,

Spontaneous neutrophil apoptosis involves caspase 3-mediated activation

of protein kinase C-delta, J. Biol. Chem. 274 (1999) 37329–37334.


[47] I. Gutcher, P.R. Webb, N.G. Anderson, The isoform-specific regulation of

apoptosis by protein kinase C, Cell. Mol. Life Sci. 60 (2003) 1061–1070.

[48] I. Dal Pra, J.F. Whitfield, A. Chiarini, U. Armato, Increased activity of the

protein kinase c-y holoenzyme in the cytoplasmic particulate fraction

precedes the activation of caspases in polyomavirus-transformed pyF111

rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors, Exp.

Cell Res. 255 (2000) 171–183.

[49] Q. Feng, T. Kumagai, Y. Torii, Y. Nakamura, T. Osawa, K. Uchida,

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative

stress, Free Radical Res. 35 (2001) 779–788.

[50] Y. Uchida, K. Ohba, T. Yoshioka, K. Irie, T. Muraki, Y. Maru, Cellular

carbonyl stress enhances the expression of plasminogen activator

inhibitor-1 in rat white adipocytes via reactive oxygen species-dependent

pathway, J. Biol. Chem. 279 (2004) 4075–4083.

[51] C. Domenicotti, B. Marengo, M. Nitti, D. Verzola, G. Garibotto, D.

Cottalasso, G. Poli, E. Melloni, M.A. Pronzato, U.M. Marinari, A novel

role of protein kinase C-y in cell signaling triggered by glutathione

depletion, Biochem. Biophys. Res. Commun. 66 (2003) 1521–1526.

[52] R. Gopalakrishna, S. Jaken, Protein kinase C signaling and oxidative

stress, Free Radical Biol. Med. 28 (2000) 1349–1361.

[53] G.C. Blobe, S. Striblimg, L.M. Obeid, Y.A. Hannun, Protein kinase C

isoenzymes: regulation and function, Cancer Surv. 27 (1996) 213–248.

[54] M.A. Pronzato, C. Domenicotti, E. Rosso, A. Bellocchio, M. Patrone,

U.M. Marinari, E. Melloni, G. Poli, Modulation of rat liver protein kinase

C during ‘‘in vivo’’ CCl4-induced oxidative stress, Biochem. Biophys.

Res. Commun. 194 (1993) 635–641.

[55] C. Domenicotti, D. Paola, A. Vitali, M. Nitti, D. Cottalasso, E. Melloni, G.

Poli, U.M. Marinari, M.A. Pronzato, Mechanisms of inactivation of

hepatocyte protein kinase C isoforms following acute ethanol treatment,

Free Radical Biol. Med. 25 (1998) 529–535.

[56] H. Kakishita, Y. Hattori, Vascular smooth muscle cell activation and

growth by 4-hydroxynonenal, Life Sci. 69 (2001) 689–697.

[57] K. Kaarniranta, T. Ryhanen, H.M. Karjalainen, M.J. Lammi, T.

Suuronen, A. Huhtala, M. Kontkanen, M. Terasvrita, H. Uusitalo, A.

Salminen, Geldanamycin increase 4-hydroxynonenal (HNE)-induced cell

death in human retinal pigment epithelial cells, Neurosci. Lett. 382

(2005) 185–190.

[58] M. Parola, G. Robino, F. Marra, M. Pinzani, G. Bellomo, G. Leonarduzzi,

P. Chiarugi, S. Camandola, G. Poli, G. Waeg, P. Gentilini, M.U. Dianzani,

HNE interacts directly with JNK isoforms in human hepatic stellate cells,

J. Clin. Invest. 102 (1998) 1942–1950.

[59] S. Camandola, G. Poli, M.P. Mattson, The lipid peroxidation product 4-

hydroxy-2,3-nonenal increases AP-1 binding activity through caspase

activation in neurons, J. Neurochem. 74 (2000) 159–168.

[60] B.J. Song, Y. Soh, M.-A. Bae, J.-E. Pie, J. Wan, K.-S. Jeong, Apoptosis of

PC12 cells by 4-hydroxy-2-nonenal is mediated through selective

activation of the c-Jun N-terminal protein kinase pathway, Chem. Biol.

Interact. 130 (2001) 943–954.

[61] H. Yoshino, N. Hattori, T. Urabe, K. Uchida, M. Tanaka, Y. Mizuno,

Postischemic accumulation of lipid peroxidation products in the rat brain:

immunohistochemical detection of 4-hydroxy-2-nonenal modified pro-

teins, Brain Res. 767 (1997) 81–86.

[62] M.L. Selley, (E)-4-hydroxy-2-nonenal may be involved in the pathogen-

esis of Parkinson’s disease, Free Radical Biol. Med. 25 (1998) 169–174.

[63] N. Bertho, V.M. Blancheteau, N. Setterblad, B. Laupeze, J.M. Lord, B.

Drenou, L. Amiot, D.J. Charron, R. Fauchet, N. Mooney, MHC class II-

mediated apoptosis of mature dendritic cells proceeds by activation of the

protein kinase C-delta isoenzyme, Int. Immunol. 14 (2002) 935–942.

[64] A. Cataldi, S. Miscia, L. Centurione, M. Rapino, D. Bosco, G. Grifone,

V.D. Valerio, F. Garaci, R. Rana, Role of nuclear PKC delta in mediating

caspase-3-upregulation in Jurkat T leukemic cells exposed to ionizing

radiation, J. Cell. Biochem. 86 (2002) 553–560.

[65] M. Reyland, S. Anderson, A. Matassa, K. Barzen, D. Quissell, Protein

kinase C is essential for etoposide-induced apoptosis in salivary acinar

cells, J. Biol. Chem. 274 (1999) 19115–19123.

[66] T. Fujii, M.L. Garcia-Bermejo, J.L. Berabop, J. Caamano, M. Ohba, T.

Kuroki, L. Li, S.H. Yuspa, MG. Kazaniet, Involvement of protein kinase

C-y (PKCy) in phorbol ester-induced apoptosis in LNCaP prostate cancer

cells. Lack of proteolytic cleavage of PKCy, J. Biol. Chem. 275 (2000)

7574–7582.

[67] E.C. Dempsey, A.C. Newton, D. Mochly-Rosen, A.P. Fields, M.E.

Reyland, P.A. Insel, R.O. Messing, Protein kinase C isozymes and the

regulation of diverse cell responses, Am. J. Physiol.: Lung Cell Mol.

Physiol. 279 (2000) L429–L438.

4-hydroxynonenal signalling to apoptosis in isolated rat hepatocytes: the role of pkc-δ

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