SUPPRESSION OF PROTEIN KINASE C
␣
TRIGGERS APOPTOSIS
THROUGH DOWN-REGULATION OF BCL-XL IN A RAT HEPATIC
EPITHELIAL CELL LINE
Ya-Ching Hsieh, Hsiao-Ching Jao, Rei-Cheng Yang, Hseng-Kuang Hsu, and
Chin Hsu
Department of Physiology, Kaohsiung Medical University, Kaohsiung, Taiwan
Received 22 Oct 2002; first review completed 5 Nov 2002; accepted in final form 30 Dec 2002ABSTRACT—Inactivation of protein kinase C (PKC)␣plays an important role in modulating hepatic failure and/or apo-ptosis during sepsis. To determine whether and how PKC␣inactivation mediates the apoptosis, PKC␣was suppressed by antisense treatment or transiently transfection in Clone-9 rat hepatic epithelial cell line. Apoptosis was evaluated by cell survival rate, poly-adenyl ribonuclease polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling stain. The expressions of PKC␣and Bcl-xL were quantified by Western blot analysis after antisense treatment. In the transfection studies, cells were co-transfected with green fluores-cent protein cDNA as a transfection marker. The expressions of PKC␣and Bcl-xL were detected by immunohistochemical staining with second antibody conjugated with Texas red. Apoptosis was evaluated by tetramethyl-rhodamine labeling of DNA strand breaks and immunostaining of 85-kDa fragment of PARP. The results showed that cytosolic and membrane-associated PKC␣were decreased by 54.5% and 41.4%, respectively, after PKC␣antisense treatment. The apoptotic incidence and percentage of PARP cleavage were significantly increased, whereas protein expression of Bcl-xL was decreased after PKC␣-antisense treatment. In the transfection studies, the results showed that most of the cells express-ing green fluorescent protein revealed less PKC␣and Bcl-xL protein contents and more in situ PARP cleavage and DNA strand breaks. These findings indicated that decrease of PKC␣declines the Bcl-xL content and leads to the vulnerability of apoptosis in hepatic epithelial cells. Taken together, our data provide evidence that suppression of PKC␣plays a critical role in triggering caspase-dependent apoptosis, which may act through modulating the Bcl-xL expression.
KEYWORDS—PKC␣, apoptosis, Bcl-xL, PARP
INTRODUCTION
Our previous work has shown that inactivation of protein
kinase C (PKC) may play a critical role in modulating hepatic
failure during sepsis (1). However, the participation of PKC in
signal transduction pathways that regulate liver function is
likely to be a complex process because PKC is a multigene
family consisting of 11 closely related isoforms. Recently, we
screened the changes of all isoforms in the liver during sepsis
and found that alteration of PKC
␣
is the most prominent one
during sepsis (2). PKC
␣
is essentially involved in maintaining
cells in an antiapoptotic state such as glioma cell (3) and
myoblast (4). Our recent result also showed that inactivation of
PKC
␣
was accompanied by reduction in Bcl-xL levels during
sepsis that coincided with the appearance of apoptotic cell
death as detected by outlet exposure of phosphatidylserine (5).
However, despite these evidences, the causal relationship
between PKC
␣
and the downstream effectors that are involved
in the hepatic apoptosis have not been identified. Accordingly,
we design an in vitro system of PKC
␣
suppression that was
simulated by transient antisense treatment or transfected with
an expression vector containing the cDNA for rat PKC
␣
in the
antisense orientation to identify the in situ alterations after
PKC
␣
suppression for a long duration. Then,
caspase-acti-vated PARP cleavage, apoptotic incidence, and expression of
Bcl-xL, which is down-regulated by PKC
␣
inactivation (3),
were evaluated after PKC
␣
suppression. In the present study,
we demonstrated that inhibition of PKC
␣
leads to a decrease in
Bcl-xL protein content and induction of apoptosis.
MATERIALS AND METHODS
Inhibition of PKC
␣ protein synthesis by
antisense treatment
Clone 9 rat hepatic epithelial cells were pre-treated with 1M phorbol 12, 13-dibutyrate (PDBu) for 12 h until 70–80% confluent. This procedure removes greater than 90% of PKC protein from the cells (6). Then medium was replaced with F12 K to remove PDBu and incubated with 1M PKC␣ antisense for 6 h (7). Then, the cells were sampled for analysis of expressions of PKC␣ and Bcl-xL as well as apoptosis 18 h later.
Extraction and purification of PKC
␣
PKC␣ was extracted and partial purified by the method of Wise et al. (8) with modification. Cells were homogenized in 300L of buffer (25 mM Tris-Base; 2 mM EDTA; 10 mM EGTA, pH 7.46; 10 mM DTT; 150 mM sucrose; 1% protein enzyme inhibitor) and then centrifuged at 30,000 g for 15 min. The supernatant containing enzyme was used as cytosolic fraction. The resulting pellet was reho-mogenized in buffer A containing 0.25% Triton X-100. The mixture was incubated for 15 min on ice followed by centrifugation at 30,000 g for 15 min and the resulting supernatant was used as enzyme of membrane-associated fraction.
Western analysis of PKC
␣ isoform
Equal amount of sample (5g) was subjected to SDS-PAGE using a 7.5% running gel (9). Proteins were transferred onto polyvinylidene difluoride transfer membrane by electroblotting for 55 min (120 v). PKC␣ antibody (1:1000, Trans-ductional Laboratory. Cat No. P16520) was incubated with the membrane for 1 h at room temperature. The membranes were washed in TBS-t and incubated with goat anti-mouse horseradish peroxidase antiserum (1:5000, Transduction Laboratory, cat no. M15345) for 1 h. Then, the membranes were detected by the ECL system.
Determination of cell survival rate
Cell survival was determined by trypan blue exclusion after treating cells with PKC␣ antisense oligonucleotides, and enumeration of viable cells using a hemo-cytometer.
Address reprint requests to Chin Hsu, Department of Physiology, Kaohsiung Medical University, Kaohsiung city, Taiwan.
This work was supported by NSC-90-2320-B-037-025 and NSC-91-2745-B-037-002 (Taiwan).
DOI: 10.1097/01.shk.0000065705.84144.ed
Terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate-digoxigenin nick end labeling
(TUNEL) staining
Cells were cultured on coverslips in 6-well plates and fixed in 4% paraformal-dehyde for 15 min. DNA strand breaks were identified by TUNEL assay using the KLENOW FragELTM DNA fragmentation detection kit (Calbiochem, cat no.
QIA21) following the manufacturer’s protocol. The cells were counter stained with methyl green. Negative controls were processed identically except that TdT was not added. The incidence of apoptosis was derived from the quotient of apoptotic nucleus number divided by the sum of total cell numbers in each coverslip. All measurements were performed by one person.
Detection of poly-(ADP-ribose) polymerase
(PARP) cleavage
Cells were suspended in 300L of lysis buffer (20 mM HEPES, pH 7.9; 0.2% 0.1% Triton X-100; 10% glycerol; 0.1 mM EDTA; 200 mM NaCl; 1 mM DTT; 0.1 M PMSF; 0.5g/mL leupeptin, 0.5 g aprotinin; Refs. 10, 11). After sonication, the lysate was cleared by centrifugation for 10 min at 12,000 g. Ten micrograms of protein was resolved by 7.5% SDS-PAGE after incubation for 15 min at 65°C. Then, membrane was incubated with anti-PARP (C-2-10; 1:1000, Zymed Labora-tory, Inc., San Francisco, CA, cat no. 33-3100) in 5% nonfat dry milk/TBS-t for 1 h and then anti-mouse IgG (1:5000, Transduction Laboratory, cat. no. 15345) for 1 h. The molecular weight of intact PARP and cleaved PARP were 116 KDa and 85 KDa, respectively.
Western blotting analysis of Bcl-xL
Four volumes of extraction buffer (20 mM Tris-HCl, pH 7.4; 2 mM EDTA; 2 mM EGTA; 0.1 mM PMSF; 10g/mL aprotonin; 10 g/mL leupeptin; 10 mM NaF; 6 mM mercaptoethanol; 1% Triton X-100; 0.1% SDS, 10 mM NaCl) was added to the sample and homogenized by sonication (12). The suspension was subsequently centrifuged for 80 min at 16000 g and the clear lysate was ready for protein concentration analysis. Five micrograms of protein was separated by 12.5% SDS-PAGE. Blots were then incubated with Bcl-xL antibody (1:1000, monoclonal; Transduction Laboratory, cat no. B61220, Lexington, KY) in TBS-t containing 5% nonfat milk and then incubated with goat antimouse IgG (1:5000, HRP conjugated, Transduction Laboratory, cat no. M15345). Immunoreactive proteins were visual-ized by enhanced chemiluminescence.
Construction of PKC
␣ antisense expression vector
and transfection
The total RNA from rat liver was isolated using TRIzol reagent (Life Technolo-gies, Grand Island, NY). First-strand cDNA was synthesized using reverse tran-scription polymerase chain reaction (RT-PCR) kit (Promega, Madison WI). PCR was performed using a Perkin-Elmer thermocycler (Model 2400) for 35 cycles. The forward primer 5⬘-CCC AAG CTT CAG CTG GTC ATC GCT AAC AT-3⬘ and reverse primer 5⬘-GGA ATT CGA TCA CTT ATG GAC TAA TAT CC-3⬘ for rat PKC␣ were used in PCR with modifications by adding a HindIII site and a EcoRI site at 5⬘ end of the forward and reverse primers, respectively. An amplification program of denaturation (95°C, 1 min), annealing (55°C, 1 min), and extension (72°C, 2 min) was used. A 264-bp PKC␣ cDNA fragment obtained from PCR amplification was ligated into the TA cloning vector (Invitrogen, San Diego, CA). DNA sequencing was performed to confirm the sequence of the insert. A 254-bp fragment was released from TA cloning vector by digesting with HindIII and EcoRI. After digestion, the fragment was purified from an agarose gel and then inserted into pcDNA3.1(−)/neo expression vector (Promega) in an antisense orientation named as pAS-PKC␣ (13).
Clone-9 cells were washed with wash medium (Gibco) and plated onto poly-L-lysine-coated culture well in attachment medium. After 3 h, the medium was changed to DMEM containing 5% fetal calf serum, 1 × 10−8M dexamethasone, 10
ng/mL epidermal growth factor, 5g/mL insulin, 2.5 g/mL fungizone, 50 g/mL gentamycin, 67g/mL penicillin, and 100 g/mL streptomycin. Cells were cultured in their usual, serum-containing medium until they reached 80% confluency. The cells were then washed twice with Opti-MEM I reduced-serum medium, and trans-fected with 2g of plasmid DNA/2 mL culture well using 10 L of Lipofectamin. In each experiment, 1g of a green fluorescent protein (GFP) vector (Promega) was co-transfected as a transfection marker. After transfection, the cells were incubated in reduced-serum medium for 8 h then replaced with normal growth medium and immunofluorescence was detected 72 h later.
Immunofluorescence
Cells were fixed with 4% (w/v) paraformadehyde for 15 min and incubated for 1 h at room temperature with primary antibody of PKC␣ (1:100, Transduction Laboratory), Bcl-xL Ab (1:50, Transduction Laboratory), or antibody recognizing PARP 85-kDa fragment (1:100 dilution, Promega, cat no. G7341) in phosphate-buffered saline (PBS) containing 5% bovine serum albumin. After washing with
PBS, cells were incubated for 1 h with secondary antibody-conjugated Texas red (1: 50, ICN Pharmaceuticals, Inc., cat no. 55544). Finally, cells were mounted with PBS containing 80% glycerol and observed for green or red fluorescence using a fluorescent microscope. Ten fields were examined in each sample. Green fluores-cence of GFP was used as a transfection marker and red fluoresfluores-cence after immu-nostaining of PARP or TUNEL identified an apoptotic-positive cell. However, cells exhibiting GFP fluorescence but not red fluorescence after PKC␣ or Bcl-xL immu-nostaining were identified as by suppressed by PKC␣ or down-regulated by of Bcl-xL, respectively.
Statistics
The statistical analysis of the data was performed by using the one-tailed Student
t test. A 95% confidence limit was accepted as statistically significant.
RESULTS
Suppression of PKC
␣ by transient antisense treatment
The efficiency of PKC
␣
antisense in suppressing the PKC
␣
protein expression was confirmed by Western blot analysis.
Both the protein expressions of the cytosolic and
membrane-associated fractions of PKC
␣
were significantly (P < 0.01)
decreased by 36% and 34%, respectively, after treatment of
PKC
␣
-antisense (Fig. 1).
Effect of PKC
␣ antisense treatment on the cell viability,
PARP cleavage, and apoptosis
PKC
␣
antisense treatment for 6 h resulted in a significant
decrease (P < 0.01) of cell count (Fig. 2a). The PARP cleavage
from a 116-kDa protein to 85-kDa fragment was detected by
Western blot analysis using antibody recognizing both the
cleavage and intact PARP. A 100.8% increase of PARP
cleav-age (P < 0.01) was observed after treatment of PKC
␣
-antisense
(Fig. 2b). Furthermore, TUNEL stain was performed to detect
nuclear DNA strand breaks. The result showed that TUNEL
positive cells were significantly (P < 0.01) increased after
PKC
␣
-antisense treatment (Fig. 2c and d).
Effect of PKC
␣ antisense treatment on protein expression
of Bcl-xL
To understand the response of Bcl-xL to PKC
␣
suppression,
protein expression of Bcl-xL was detected by Western blot
FIG. 1. Protein expression of PKC␣ in clone 9 cells treated with or
without PKC␣ antisense. (a) cytosolic fraction, (b) membrane-associated
fraction. Cells were incubated with 1 µM PKC␣-antisense for 6 h and samples were collected at 18 h after treatment and then were fractionated into cyto-solic and membrane-associated fractions 18 h later. The molecular weight of PKC␣ is 86 kDa. The data shown indicates mean ±SD of six samples in each group. **P < 0.01.
analysis. Result showed that the expression of anti-apoptotic
Bcl-xL protein was significantly (P < 0.01) decreased by 32%
after PKC
␣
-antisense treatment (Fig. 3).
Suppression of PKC
␣ by transfection with vector
containing PKC
␣ cDNA in antisense
orientation (pAS-PKC
␣)
To confirm the long-lasting suppression of PKC
␣
expression
by antisense transfection, the protein expression of PKC
␣
was
detected by immunostaining with antibody conjugated with
Texas red at 72 h after transfection. Cells showing green
fluo-rescence represented as a successful transfection marker. The
result showed that after pAS-PKC
␣
transfection, most of the
cells expressing GFP (Fig. 4, upper right panel) revealed
significantly (P < 0.01) lower level of PKC
␣
protein content
(Fig. 4, lower right panel) compared with that of the vector
control transfection (Fig. 4, lower left panel).
Effect of pAS-PKC
␣ transfection on apoptosis
Apoptotic cells were evaluated by tetramethyl-rhodamine
labeling of DNA break to identify if DNA strand breaks
occurred in situ after successfully transfected with pAS-PKC
␣
.
The result showed that the percentage of both GFP expression
and TUNEL staining double-positive cells after pAS-PKC
␣
transfection was significantly (P < 0.01) higher than that of
vector control transfection (Fig. 5).
FIG. 2. Effects of PKC␣ antisense treatment on (a) cell viability, (b)
PARP cleavage, (c) apoptosis, and (d) quantitative apoptotic incidence.
Cells were incubated with 1 µM PKC␣-antisense for 6 h and then replaced with fresh medium. Cell viability, PARP cleavage, and apoptotic incidence were evaluated at 18 h after replacement. Cell viability was determined by trypan blue exclusion test. PARP cleavage was detected by immunostain-ing with Ab against both 116-kDa PARP and 85-kDa fragment of PARP. Apoptotic cells showed in (c) was identified by TUNEL staining. Arrow indi-cates cell with apoptotic nucleus. The quantitative data of apoptotic incidence was derived from the quotient of apoptotic nucleus divided by the sum of total cell number. The data shown indicates mean ± SD of six samples in each group. **P < 0.01.
FIG. 3. Protein expression of Bcl-xL with of without PKC␣ antisense. Cells were treated with 1 µM PKC␣ antisense for 6 h and sampled at 18 h after fresh medium replacement for immunoblotting. The molecular weight of Bcl-xL is 26 kDa. The data shown indicate means ± SD of sixe samples in each group. **P < 0.01.
FIG. 4. Decrease of PKC␣ protein expression after transfection with
vector containing PKC␣ antisense (pAS-PKC␣). (a) Immunofluorescent
image of GFP expression (upper panel) and PKC␣ immunoreactive (lower panel) cells. (b) Quantitative ratio of PKC␣ negative-expression cells per GFP (+) cells. Cells transfected with vector control and pAS-PKC␣ was showed in left and right panel, respectively. Green fluorescence represented as a transfection marker. Red fluorescence indicates PKC␣ immunoreactive cells, which was stained with antibody conjugated with Texas red. Arrow indicates the relative PKC␣ reactivity in the cell, which is simultaneously GFP (+). The percentages of PKC␣ negative whereas GFP (+) cells were presented as means ±SD of 5 samples in each group. **P < 0.01.
PARP cleavage induced by transfection with pAS-PKC
␣
To further understanding the possible pathways of apoptosis
triggered by PKC
␣
suppression, the caspase-dependent
cleav-age of PARP was detected by immunostaining with antibody
recognizing only the 85-kDa fragment of PARP conjugated
with Texas red after pAS-PKC
␣
transfection. The result
showed that the percentage of cells expressing GFP associated
with PARP cleavage after pAS-PKC
␣
transfection was
signifi-cantly (P < 0.01) higher than that of vector control transfection
(Fig. 6).
Effect of pAS-PKC
␣ transfection on
Bcl-xL immunoreactivity
The expression of Bcl-xL, which affects cytochrome C
release and caspase activation, was detected after pAS-PKC
␣
transfection by immunostaining with antibody conjugated with
Texas red. Green fluorescence was also used as a transfection
marker. The result showed that, after pAS-PKC
␣
transfection,
the percentage of cells expressing GFP concomitant with a
negative expression of Bcl-xL protein was significantly (P <
0.01) higher than those of vector control transfection as
showed in Figure 7.
DISCUSSION
The present study provided evidence that suppression of
PKC
␣
by antisense increased the apoptotic incidence in hepatic
epithelial cell line. Furthermore, in situ down-regulation of
Bcl-xL, PARP cleavage, and DNA strand breaks occurred
when the cell was successively transfected with pAS-PKC
␣
. It
suggests that PKC
␣
plays a critical role in cell survival and the
expression of the cell survival molecule, Bcl-xL, is modulated
by the PKC
␣
signaling pathway.
The liver is essential in regulating host defenses to septic
challenge and the degree of hepatic failure during sepsis is
related to the extent of cell damage and death resulting from
either necrosis or apoptosis. Although hepatic apoptosis has
been reported to be a less apparent phenomenon during sepsis
in some previous reports (14, 15), induction of apoptosis
corre-lated with a reduced Bcl-2 content had been reported in
endo-toxin-treated animals (16). Our previous results showed that
hepatic apoptosis was apparently increased at early sepsis and
persisted to the late stage of sepsis whether it was evaluated by
flow cytometry, PARP cleavage, TUNEL stain or even by the
less sensitive DNA ladder gel electrophoresis. Because of the
rapid turnover of apoptotic cells by Kupffer cell, the prolonged
and constant hepatic apoptosis might contribute to hepatic
fail-ure during polymicrobial sepsis.
It is well documented that PKC inhibitor is an effective
apoptotic-inducing agent. Previous report indicated that
calphostin, a specific PKC inhibitor, induced apoptosis
asso-ciated with down-regulation of Bcl-xL as well as activation of
caspase-3 (17). However, the role of each isoform of PKC in
FIG. 5. Increase of apoptotic incidence after transfecting with vectorcontaining PKC␣ cDNA in antisense orientation (pAS-PKC␣). (a)
Immu-nofluorescent image of GFP expression (+) (upper panel) and TUNEL stain (+) (lower panel) cells. (b) Quantitative ratio of TUNEL (+) cells per GFP (+) cells. Cells transfected with vector control and pAS-PKC␣ was showed in left and right panel, respectively. GFP cDNA was co-transfected as a transfec-tion marker. Red fluorescence indicates apoptotic cells, which were evalu-ated by tetramethyl-rhodamine labeling of DNA break. Arrow indicates the DNA strand breaks in the cells, which is simultaneously GFP (+). The percentages of double-positive cells were presented as mean ± SD of five samples in each group. **P < 0.01.
FIG. 6. Increase of the PRAP cleavage after transfecting with vector
containing PKC␣ cDNA in antisense orientation (pAS-PKC␣). (a)
Immu-nofluorescent image of GFP expression (+) (upper panel) and 85kD PARP immunoreactive (lower panel) cells. (b) Quantitative ratio of PARP (+) cells per GFP (+) cells. Cells transfected with vector control and pAS-PKC␣ was showed in left and right panel, respectively. GFP cDNA was co-transfected as a transfection marker. Red fluorescence indicates the cleaved PARP 85 kDa, which was detected by immunostaining with 85-kDa PARP antibody conjugated with Texas red. Arrow indicates the relative PARP cleavage in the cell, which is simultaneously GFP (+). The percentages of double-positive cells were presented as mean ± SD of five samples in each group. **P < 0.01.
apoptosis is not identified. Previous studies have demonstrated
that loss of PKC
␣
function correlated with the induction of
apoptosis in COS cells (18). Inhibition of PKC
␣
gene
expres-sion by ribozyme also resulted in a significant apoptosis in
glioma cells through suppressing Bcl-xL gene expression (3).
Although treatment with phorbol 12-myristate 13-acetate
(TPA), PKC activator, upregulated expression of Bcl-xL in
human erythropoietin-dependent cell line (19). Our previous
report showed that, during sepsis, inactivation of PKC
␣
was
accompanied by reduction in Bcl-xL levels that coincided with
the appearance of apoptotic cell death in liver (5). The present
result showed that the protein content of Bcl-xL was
signifi-cantly decreased by in situ PKC
␣
suppression. It strongly
supports a potential interaction between PKC
␣
and the Bcl-xL
protein. Recently, our preliminary result showed that the
expression of Bcl-xL mRNA under PKC
␣
suppression was
also decreased (unpublished data). It suggests that PKC
␣
may
modulate the transcriptional regulation of Bcl-xL. However,
the molecular mechanism involved in apoptosis triggered by
PKC
␣
suppression need further study. In addition, a previous
study showed that over-expression of PKC
␣
resulted in
increased mitochondrial PKC
␣
localization and Bcl-2
phos-phorylation, which is required for antiapoptotic function (20).
Therefore, the expression of the apoptosis-related proteins in
addition to Bcl-xL may be modulated by PKC
␣
suppression. In
this respect, comparison analysis between control and stably
PKC
␣
-antisense transfected cells by the PCR-based
differen-tial display technique identified some genes, which are under
investigation in our laboratory.
The immunohistochemical results of our previous study
showed that, during sepsis, PKC
␣
was significantly decreased
especially within the nucleus (2). It is possible that the
decli-nation of nuclear PKC
␣
might regulate the expression of
apoptosis-related protein, i.e., Bcl-xL, through modulating the
phosphorylation/dephosphorylation state of transcriptional
factors. Transcriptional factors, i.e., Ets, nuclear factor-
B,
STAT, and activator protein-1, play essential roles in
deter-mining the fate of a cell by affecting the expression of bcl-x
gene encoding the anti-apoptotic Bcl-xL protein (21). Several
hypotheses offer potential mechanisms how Bcl-xL exerts its
survival effects. Previous reports implicated that Bcl-xL
promotes cell survival by regulating the electrical and osmotic
homeostasis of mitochondria (22). Besides, Bcl-xL functions
as a facilitator of ADP supply to the mitochondrial matrix,
allowing for continued F1F0-ATPase activity (23). If the
mito-chondrial respiration terminates through loss of F1F0-ATPase
activity, mitochondria will release cytochrome C, which induce
apoptopsome formation and subsequent caspase activation
associated with endonucleosomal PARP cleavage and
apopto-sis. Previous study showed that Bcl-xL prevents the
staurospo-rin-induced apoptosis of dopaminergic neurons by inhibiting
the caspase activation (24), which is a subsequent process after
damage of the outer mitochondrial membrane and cytochrome
c release. In addition, the bulk of the bcl-xL product localizes
to the periphery of mitochondria (25). Bcl-xL may also
func-tion as, or associafunc-tion with, a mitochondria ion channel to
regulate apoptosis (22, 26). It can also close the
voltage-dependent anion channel preventing cytochrome c to pass
through the channel (27). Taken together, these observations
suggest that mitochondria may play a crucial role in Bcl-xL
regulated apoptosis caused by PKC
␣
suppression. Further
evaluation of the causal relationship between PKC
␣
and
mito-chondrial function will be performed in the near future.
In conclusion, the present results indicated that suppression
of PKC
␣
decreased the cellular contents of Bcl-xL and leaded
to a caspase-dependent apoptosis in hepatic epithelial cells.
Therefore, PKC
␣
may be a therapeutic target for prevention of
apoptosis during sepsis.
REFERENCES
1. Hsu C, Jao HC, Yang SL, Hsu HK, Liu MS: Inactivation of protein kinase C in rat liver during late hypoglycemic phase of sepsis. Mol Cell Biochem 181:181– 189, 1998.
2. Hsu C, Hsieh YC, Hsu HK, Jao SC, Yang RC: Alteration of protein kinase C isoforms in the liver of septic rat. Shock 17:41–46, 2002.
3. Leirdal M, Sioud M: Ribozyme inhibition of the protein kinase C alpha triggers apoptosis in glioma cells. Br J Cancer 80:1558–1564, 1999.
4. Capiati DA, Vazquez G, Tellez Inon MT, Boland RL: Antisense oligonucleo-tides targeted against protein kinase c alpha inhibit proliferation of cultured avian myoblasts. Cell Prolif 33:307–315, 2000.
5. Jao HC, Yang RC, Hsu HK, Hsu C: The decrease of PKCalpha is associated with hepatic apoptosis at early and late phases of polymicrobial sepsis. Shock 15:130–134, 2001.
6. Dean NM, McKay R, Condon TP, Bennett CF: Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits FIG. 7. Decrease of Bcl-xL protein expression after transfecting with
vector containing PKC␣ cDNA in antisense orientation (pAS-PKC␣). (a)
Immunofluorescent image of GFP expression (+) (upper panel) and Bcl-xL immunoreactive (lower panel) cells. (b) Quantitative ratio of Bcl-xL negative-expression cells per GFP (+) cells. Cells transfected with vector control and pAS-PKC␣ was showed in left and right panel, respectively. GFP cDNA was co-transfected as a transfection marker. Red fluorescence indicates Bcl-xL immunoreactive cells, which was stained by antibody conjugated with Texas red. Arrow indicates the relative immunoreactivity of Bcl-xL in the cells, which is simultaneously GFP (+). The percentages of Bcl-xL negative whereas GFP (+) cells were presented as mean ± SD of five samples in each group. **P < 0.01.
induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters. J Biol Chem 269:16416–16424, 1994.
7. Baxter GT, Miller DL, Kuo RC, Wada HG, Owicki JC: PKC epsilon is involved in granulocyte-macrophage colony-stimulating factor signal transduction: evidence from microphysiometry and antisense oligonucleotide experiments.
Biochemistry 31:10950–10954, 1992.
8. Wise BC, Glass DB, Chou CH, Raynor RL, Katoh N, Schatzman RC, Turner RS, Kibler RF, Kuo JF: Phospholipid-sensitive Ca2+-dependent protein kinase
from heart. II. Substrate specificity and inhibition by various agents. J Biol
Chem 257:8489–8495, 1982.
9. Ways DK, Cook PP, Webster C, Parker PJ: Effect of phorbol esters on protein kinase C-zeta. J Biol Chem 267:4799–4805, 1992.
10. Desnoyers S, Shah GM, Brochu G, Poirier GG: Erasable blot of poly(ADP-ribose) polymerase. Anal Biochem 218:470–473, 1994.
11. Keppler-Hafkemeyer A, Brinkmann U, Pastan I: Role of caspases in immuno-toxin-induced apoptosis of cancer cells. Biochemistry 37:16934–16942, 1998. 12. Arber N, Han EK, Sgambato A, Piazza GA, Delohery TM, Begemann M, Weghorst CM, Kim NH, Pamukcu R, Ahnen DJ, Reed JC, Weinstein IB, Holt PR: A K-ras oncogene increases resistance to sulindac-induced apoptosis in rat enterocytes. Gastroenterology 113:1892–1900, 1997.
13. Zhang X, Wen J, Aletta JM, Rubin RP: Inhibition of expression of PKC-alpha by antisense mRNA is associated with diminished cell growth and inhibition of amylase secretion by AR4-2J cells. Exp Cell Res 233:225–231, 1997. 14. Hiramatsu M, Hotchkiss RS, Karl IE, Buchman TG: Cecal ligation and
punc-ture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway. Shock 7:247–253, 1997.
15. Hotchkiss RS, Swanson PE, Cobb JP, Jacobson A, Buchman TG, Karl IE: Apoptosis in lymphoid and parenchymal cells during sepsis: findings in normal and T- and B-cell-deficient mice. Crit Care Med 25:1298–1307, 1997. 16. Haendeler J, Messmer UK, Brune B, Neugebauer E, Dimmeler S: Endotoxic
shock leads to apoptosis in vivo and reduces Bcl-2. Shock 6:405–409, 1996. 17. Ozaki I, Tani E, Ikemoto H, Kitagawa H, Fujikawa H: Activation of
stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 kinase in calphos-tin C-induced apoptosis requires caspase-3-like proteases but is dispensable for cell death. J Biol Chem 274:5310–5317, 1999.
18. Whelan RD, Parker PJ: Loss of protein kinase C function induces an apoptotic response. Oncogene 16:1939–1944, 1998.
19. Tsushima H, Urata Y, Miyazaki Y, Fuchigami K, Kuriyama K, Kondo T, Tomonaga M: Human erythropoietin receptor increases GATA-2 and Bcl-xL by a protein kinase C-dependent pathway in human erythropoietin-dependent cell line AS-E2. Cell Growth Differ 8:1317–1328, 1997.
20. Ruvolo PP, Deng X, Carr BK, May WS: A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis.
J Biol Chem 273:25436–25442, 1998.
21. Sevilla L, Zaldumbide A, Pognonec P, Boulukos KE: Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NFkap-paB, STAT and AP1 transcription factor families. Histol Histopathol 16:595– 601, 2001.
22. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB: Bcl-xL regulates the membrane potential and volume homeostasis of mito-chondria. Cell 91:627–637, 1997.
23. Vander Heiden MG, Chandel NS, Schumacker PT, Thompson CB: Bcl-xL prevents cell death following growth factor withdrawal by facilitating mito-chondrial ATP/ADP exchange. Mol Cell 3:159–167, 1999.
24. Kim JE, Oh JH, Choi WS, Chang II, Sohn S, Krajewski S, Reed JC, O’Malley KL, Oh YJ: Sequential cleavage of poly(ADP-ribose)polymerase and appear-ance of a small Bax-immunoreactive protein are blocked by Bcl-X(L) and caspase inhibitors during staurosporine-induced dopaminergic neuronal apoptosis. J Neurochem 72:2456–2463, 1999.
25. Gonzalez-Garcia M, Perez-Ballestero R, Ding L, Duan L, Boise LH, Thompson CB, Nunez G: bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development 120:3033– 3042, 1994.
26. Muchmore SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon HS, Nettesheim D, Chang BS, Thompson CB, Wong SL, Ng SL, Fesik SW: X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.
Nature 381:335–341, 1996.
27. Shimizu S, Shinohara Y, Tsujimoto Y: Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator. Oncogene 19:4309–4318, 2000.
