In this report, we provide the following evidence that the protein kinase C PKCh activity is func-tionally involved in the AhR-independent signal transduction mechanism that participates
Trang 1tetrachlorodibenzo-p-dioxin-induced signal transduction
pathway leading to apoptosis in L-MAT, a human
lymphoblastic T-cell line
Sohel Ahmed, Masahiko Shibazaki, Takashi Takeuchi and Hideaki Kikuchi
Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
The immune system is recognized as a consistent and
sensitive target for the toxic widespread environmental
pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
and its congeners [1] Triggering of apoptosis in both
thymocytes [2] and T cells [3,4] has clearly emerged as
a hallmark of TCDD immunotoxicity, as shown by
in vivo studies in animal models Although an in vitro
study has also revealed that TCDD directly causes apoptotic death in immature thymocytes [5], no such direct effect of TCCD has been observed in vitro in T cells from animal models [6] However, we have clearly shown that TCCD can directly induce apoptosis in some cultured human T-cell lines [7] In addition, we have evaluated the immunotoxicity of TCDD and
Keywords
dioxin; apoptosis; PKCh; lymphoblastic T
cell; rottlerin
Correspondence
H Kikuchi, Department of Biochemistry and
Biotechnology, Faculty of Agriculture and
Life Science, Hirosaki University, 3
Bunkyo-cho, Hirosaki 036-8561, Japan
Fax: +81 172 39 3586
Tel: +81 172 39 3586
E-mail: hkikuchi@cc.hirosaki-u.ac.jp
(Received 17 June 2004, revised 7 September
2004, accepted 8 December 2004)
doi:10.1111/j.1742-4658.2004.04519.x
The aromatic hydrocarbon receptor (AhR)-dependent pathway involved
in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity has been studied extensively, but the AhR-independent molecular mechan-ism has not In previous studies we found that the AhR is not expressed
in L-MAT, a human lymphoblastic T-cell line In this report, we provide the following evidence that the protein kinase C (PKC)h activity is func-tionally involved in the AhR-independent signal transduction mechanism that participates in the TCDD-induced L-MAT cell apoptosis First, only rottlerin, a novel PKC (nPKC)-selective inhibitor, blocked the apoptosis completely, in a dose-dependent manner Second, PKCh was the major nPKC isoform (compared to PKCd) expressed in the L-MAT cell line Third, a cell-permeable myristoylated PKCh pseudosubstrate peptide inhibitor also blocked the apoptosis completely, in a dose-dependent man-ner Fourth, both rottlerin and myristoylated PKCh pseudosubstrate pep-tide inhibitor completely inhibited PKCh kinase activity in vitro at doses that effectively blocked TCDD-induced L-MAT cell apoptosis TCDD treatment induced a time-dependent activation of nPKC kinase activity in L-MAT cells, and moreover, TCDD induced a translocation of PKCh from the cytosolic fraction to the particulate fraction in L-MAT cells Finally, transient over-expression of a dominant negative PKCh (a kinase-dead mutant, K⁄ R 409) in L-MAT cells conferred significant protection against TCDD-induced apoptosis
Abbreviations
AcDEVD-AMC, acetyl-Asp-Glu-Val-Asp ⁄ 7-amino-4-methylcoumarin; AhR, aromatic hydrocarbon receptor; DN PKC, dominant negative protein kinase C; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; PKC, protein kinase C; NaCl ⁄ P i , phosphate-buffered saline; nPKC, novel protein kinase C; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; myr-PKCh-PPI, myristoylated-PKCh-pseudosubstrate peptide inhibitor.
Trang 2T-cell line, L-MAT, as the model, that the
AhR-medi-ated pathway is in no way involved in TCDD-induced
apoptosis, [7,8] Furthermore, neither TCDD-mediated
apoptosis in mouse thymoma cells (EL-4) [11] nor
poly-chlorinated biphenyl (Aroclor 1254)-mediated apoptosis
in mouse spleen cells [12], can be explained by the single
AhR pathway The molecular mechanism involved in
the AhR-independent pathway(s) leading to
TCDD-induced immunotoxicity is not clearly understood, and
indeed the lack of a suitable system in which this
immu-notoxicity can be readily detected and demonstrated in
a regulated manner has hindered the research efforts
The rapidity by which L-MAT cell apoptosis is
induced by TCDD, and the failure of actinomycin D
(an inhibitor of gene transcription) and cycloheximide
(an inhibitor of de novo protein synthesis) to block this
apoptosis, led us to focus on the possibility of a rapid
post-translational signal transduction mechanism [7,8]
Many reports in the last decade have suggested that
protein phosphorylation by protein kinase C (PKC)
plays a key role in the regulation of the signal
trans-duction mechanism involved in TCDD-induced cellular
responses [13,14] To date, a total of 11 isoforms of
PKC have been reported [15] Although a few studies
have shown isoform-specific PKC activation as a
cellu-lar response to TCDD [16,17], a functional role for
isoform-specific PKC activity in the mediation of
TCDD immunotoxicity has not yet been shown at the
molecular level
Recently, in an in vivo study, TCDD was implicated
in the enhancement of an activation-induced cell death
mechanism (AICD) involved in T-cell apoptosis [18]
Furthermore, activation of PKCh and its kinase
acti-vity have been implicated in the AICD mechanism in
human T-leukemic Jurkat cells [19,20] PKCh, a novel
PKC (nPKC) isoform, is characterized both by its
unique tissue distribution (in skeletal muscle, lymphoid
organs, and hematopoietic cell lines, particularly
T cells [21]) and by its isoenzyme-specific activation
requirements and substrate preferences in vitro [21,22]
The unique expression profile and functional
proper-ties of PKCh led us to believe that it may play a
spe-cialized role in many signal transduction events in
T cells
by rottlerin, an nPKC inhibitor 12-O-Tetradecanoyl phorbol-13-acetate (TPA) was used
in combination with TCDD to clarify the involvement
of PKC in the signal transduction mechanism participa-ting in TCDD-induced cellular responses [14] We also looked for evidence of the involvement of PKC in the signal transduction mechanism of TCDD-induced L-MAT cell apoptosis We used several PKC-selective inhibitors to determine whether PKC is functionally involved in the L-MAT cell apoptosis induced by TCDD and attempted to identify the specific PKC iso-form(s) involved in the process Pre-treatment with a nonspecific PKC inhibitor, staurosporine [23], caused only a partial inhibition of the apoptosis (Fig 1A), and
no inhibitory effect was observed in the case of the clas-sical PKC-selective inhibitor, Go¨6976 [24] (Fig 1B) Only pretreatment with rottlerin, an nPKC-selective inhibitor [25], resulted in the complete inhibition of TCDD-induced L-MAT cell apoptosis (Fig 1C) at doses suggestive of inhibition of PKCd and PKCh These results indicate that nPKC (PKCd and⁄ or PKCh)
is involved in the TCDD-induced apoptosis of L-MAT cells
The incubation of L-MAT cells with 20 nm TCDD resulted in morphological changes characteristic of apoptosis upon staining with the DNA-specific fluoro-chrome bis-benzinide (Fig 2, TCDD) However, the pretreatment of L-MAT cells with rottlerin (20 lm) inhibited the alteration of morphology in nuclei treated with TCDD (Fig 2, TCDD + Rottlerin)
PKCh is a major isoform of nPKC in the L-MAT cell line
The expression of PKCh was detected in L-MAT cells
at both mRNA (Fig 3A) and protein (Fig 3B) levels HepG2, a human hepatoma cell line, was used as a negative control for PKCh In HepG2 cells, neither PKCh mRNA nor PKCh protein were detected, as shown in Fig 3 The Jurkat cell line was used as a positive control for PKCh Next, the expression level
of PKCh was compared to that of PKCd by using iso-form-specific antibodies, with purified PKCh and
Trang 3PKCd as standards In L-MAT cells, PKCh was the
major isoform, the expression level of PKCd being less
than one-tenth that of PKCh (Fig 3C)
TCDD-induced L-MAT cell apoptosis is completely blocked by myristoylated-PKCh-pseudosubstrate peptide inhibitor (myr-PKCh PPI)
Having made the above experimental observations, we sought to confirm the functional involvement of nPKCs, specifically PKCh, in the L-MAT T-cell apoptosis induced by TCDD Synthetic peptides corresponding to the pseudosubstrate domains of PKC have been used as specific inhibitors of PKC in in vitro assays N-myristoy-lation of such synthetic PKC-specific pseudosubstrate peptides permits their use as selective, cell-permeable inhibitors of PKC in intact cells [26] Therefore, we examined the effect of a PKCh pseudosubstrate peptide-inhibitor myristoylated at its N-terminal site The L-MAT cell apoptosis induced by TCDD was com-pletely inhibited in the presence of 20 lm myr-PKCh PPI, as shown in Fig 4, confirming the involvement of nPKCs
PKCh kinase activity is completely inhibited by rottlerin and by myr-PKCh-PPI in vitro To test indirectly whether PKCh might be a target for both rottlerin and myr-PKCh-PPI in the inhibition of TCDD-induced L-MAT cell apoptosis, we performed
an in vitro kinase assay for PKCh This confirmed that both rottlerin and myr-PKCh PPI strongly inhibited PKCh kinase activity (Fig 5) at doses that completely blocked TCDD-induced L-MAT cell apoptosis
TCDD induces nPKC kinase activity in L-MAT cells
To establish whether TCDD increases nPKC kinase activity, we examined nPKC kinase activity by using PKCh pseudo-substrate, which is preferentially phos-phorylated by PKCh and PKCd, in whole L-MAT cells exposed to TCDD for different time-periods We found that the kinase activity of nPKC increased in a time-dependent manner, as shown in Fig 6 In a previ-ous report on the apoptotic cell-death mechanism in immature CD4+CD8+ mouse thymocytes, a specific activation of nPKCs was shown to be responsible for the induction of apoptosis by glucocorticoids and diterpine ester ingenol 3, 20-dibenzoate (IDB) [27] Therefore, both PKCh and PKCd may be activated in L-MAT cells following treatment with TCDD
PKCh is translocated in TCDD-treated L-MAT cells
We performed an nPKC translocation assay by fractiona-ting L-MAT cells into cytosol and particulate fractions and then examining the translocation of each of the nPKC isoforms in L-MAT cells treated with TCDD (by
Fig 1 Effects of protein kinase C (PKC) inhibitors on the
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced apoptosis of
L-MAT cells L-MAT cells (10 5 cellsÆ100 lL)1per well in a
96-micro-well plate in serum-free RPMI 1640) were preincubated with
PKC-selective pharmacological inhibitors (as indicated) for 30 min at
37 C in 95% air and 5% CO 2 , followed by treatment with TCDD
for 3 h Then, a caspase-3 activation assay was performed as
described in the Experimental procedures (A) Staurosporine, (B)
Go¨6976, and (C) rottlerin Data are shown as average values ± SD
(n ¼ 3) *P < 0.05; **P > 0.01 vs TCDD alone (Student’s t-test).
Trang 4comparison with untreated L-MAT cells) Our data
clearly revealed that PKCh, not PKCd, was the nPKC
isoform activated in L-MAT cells treated with TCDD,
as shown in Fig 7 Although the level of PKCd was low
because of the low expression of this isoform (Fig 7),
the loading amounts (40 lg of protein) were sufficient to
allow the detection of change of PKCd in the particulate
fraction Finally, we sought to verify the functional
involvement of PKCh kinase activity in TCDD-induced
L-MAT cell apoptosis at the molecular level
Transfected L-MAT cells express H-2KKon their
surface
To test whether PKCh kinase activity is required in the
pathway of TCDD-induced L-MAT cell apoptosis, we
examined the effect of a dominant negative PKCh (DN
PKCh; a kinase-dead mutant, K⁄ R 409) [22] To
separ-ate (on the miniMACS column) transfected cells from
the mixture of electroporated cells containing pMACS
plasmid DNA, expression of H-2KKon the cell surface
is essential Therefore, prior to their magnetic separation
we checked the L-MAT cells transfected with empty
pMACSKK.II or DN PKCh-FLAG⁄ pMACSKK.II
DNA to determine whether they expressed the H-2KK
molecule on their surface Direct immunofluorescence
microscopy of these transfected cells did indeed reveal
the expression of H-2KKon their surface (Fig 8A)
Over-expression of DN PKCh in L-MAT cells
confers protection against TCDD-induced apoptosis
Over-expression of DN-PKCh would interfere with
endogenously expressed PKCh and specifically
sup-press its kinase activity To confirm that PKCh kinase activity really does participate in the signal transduc-tion mechanism involved in TCDD-induced L-MAT cell apoptosis, transfected L-MAT cells expressing H-2KKwere separated from the nontransfected cells in the miniMACS column and then tested for caspase-3 activation by treatment with TCDD In this experi-ment, TCDD-induced caspase-3 activation (that is, apoptosis) was significantly reduced in L-MAT cells transfected with DN PKCh-FLAG⁄ pMACSKK.II DNA (as compared to that in cells transfected with empty pMACSKK.II) (Fig 8B) As the next step, the expression of DN PKCh-3·FLAG of the same con-struct in L-MAT cells was confirmed by immunopre-cipitation and Western blotting (Fig 8C)
Discussion
Participation of PKCh in TCDD-induced apoptosis The L-MAT cell apoptosis induced by TCDD was completely blocked by rottlerin, now well established
as an nPKC inhibitor [25] A number of studies have demonstrated that rottlerin acts solely as a specific inhibitor of many PKCh functions in T cells [28] Rottlerin has also been shown to block the activation-induced cell death process in T cells, indicating a func-tional role for PKCh in the cell-death mechanism [19,20] In our study, PKCh expression was detected at both the mRNA and protein levels in L-MAT T cells This observation strengthens the possibility of a func-tional involvement of PKCh in TCDD-induced L-MAT cell apoptosis However, rottlerin was origin-ally reported as a novel ATP-competitive protein
Fig 2 Morphological alterations in chroma-tin L-MAT cells were treated with solvent only (Cont), with 20 n M 2,3,7,8-tetrachlo-rodibenzo-p-dioxin (TCDD) or 20 l M rottlerin,
or with the combination of TCDD + rottlerin Cells were collected after 4 h and fixed in paraformaldehyde, then stained with the DNA-specific fluorochrome, bis-benzimide (Hoechst 33258).
Trang 5kinase inhibitor with a very high selectivity for PKCd
[25], and it was later used widely as a selective
inhib-itor for nPKCd, both in vitro and in studies of intact
cells [29] Human primary T cells have been reported
to express PKCd [30], and we found here that L-MAT cells did indeed express PKCd, as we were able to detect PKCd mRNA and protein by RT⁄ PCR and Western blotting methods, respectively (Fig 3A,B) However, the involvement of PKCd seemed to be less important, as judged from our analysis of the dose– response effect of rottlerin on L-MAT cell apoptosis
If PKCd really is involved, a much lower
concentra-B
A
C
Fig 3 Detection of the expression of endogenous protein kinase
Ch (PKCh) The expression of endogenous PKCh and PKCd was detected in the L-MAT cell line at the mRNA and protein levels (A) RT-PCR analysis of L-MAT cell mRNA for PKCh HepG2 cell mRNA was used as a negative control Glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) detection was performed as a control Total RNA was extracted from 2 · 10 7 L-MAT or HepG2 cells Then,
10 lg of the total RNA was reverse transcribed to cDNA and lyzed for PKCh mRNA along with GAPDH (B) Western blotting ana-lysis of endogenous PKCh and PKCd expression Whole cell lysates were prepared from 2 · 10 7 HepG2, L-MAT and Jurkat cells, and
100 lg of total protein was probed for PKCh expression by using the Western blotting method HepG2 and Jurkat cell lysates were used as negative and positive controls, respectively (C) Evaluation
of the protein levels of PKCh and PKCd in L-MAT cells by using a quantitative Western blotting method The assay system was opti-mized to resolve 20 and 40 lg of total L-MAT WCL (whole cell lysate) protein for the evaluation of comparable expressions of PKCh and PKCd, respectively Suitable dilutions of the purified enzymes were used as standards for PKCh and PKCd The upper panel shows the amount of PKCh (in 20 lg of total protein) expressed in L-MAT cells, which seemed to be around 5 ngÆlg)1of total L-MAT cell protein The lower panel shows the amount of pro-tein PKCd (in 40 lg of total propro-tein) expressed in L-MAT cells, which seemed to be 500 pgÆlg)1of total L-MAT cell protein.
Fig 4 The effect of myristoylated-PKCh pseudosubstrate peptide inhibitor (myr-PKCh-PPI) on the apoptosis of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD)-induced L-MAT cells L-MAT cells (10 5 cellsÆ
100 lL)1per well in serum-free RPMI 1640 in a 96-microwell plate) were pretreated with myr-PKCh-PPI (as indicated) for 30 min at
37 C in 95% air and 5% CO 2 , followed by treatment with TCDD for 3 h Then, a caspase-3 activation assay was performed for the evaluation of apoptosis Data are presented as average values ± SD (n ¼ 3) (*P < 0.05; **P > 0.01 vs TCDD alone; Student’s t-test.)
Trang 6tion of rottlerin should have blocked the apoptosis
completely, as demonstrated by others [29]
Moreover, we found that PKCh, but not PKCd, was
activated in TCDD-treated L-MAT cells We suggest
that TCDD treatment of L-MAT cells induces signal
transduction, leading to very rapid activation of PKCh,
as a significant translocation of PKCh to the
particu-late fraction from the cytosolic fraction (Fig 7) was
observed within 1 min of TCDD treatment in L-MAT
cells (with the peak being reached at 20 min and the
level sustained until 120 min, after which it decreased)
However, there was no significant change in the case of
PKCd We also compared the expression level of
PKCd, in the form of protein, to that of PKCh in
L-MAT cells (Fig 3C), and found the PKCd
expres-sion level ( 500 pgÆlg)1) to be at least 10 times lower
than that of PKCh (5 ngÆlg)1) Altogether, it was
suggested that the time-dependent increase of nPKC
kinase activity in TCDD-treated L-MAT cells (Fig 6)
was mainly a result of the activation of PKCh
Apoptosis is a multistage process The increase of
nPKC kinase activity (100 min, Fig 6) preceded the
apoptotic responses, as described below Early change
was observed in the induction of JNK activity within
30 min upon TCDD treatment [7] The caspase-3
acti-vation by proteolytic cleavage [the maximal decrease
of procaspase-3 at 240 min of treatment with TCDD
was detected by Western blotting, as was the caspase-3 activity (peak activity at 240 min of TCDD-treatment] was detected by using the kinetic assay (S Ahmed, PhD Thesis, 2004, Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan) The appearance of apop-totic morphology (peak at 180–240 min of treatment
of TCDD) was observed by fluorescence microscopy (shown in Fig 2, TCDD only at 240 min) Although
we do not have any evidence of a direct interaction, it
is possible that these events produce a cascade reaction
to the TCDD-mediated apoptosis of L-MAT cells Considering the results of all the experiments des-cribed above, we can conclude that a major part of the signal transduction to this apoptosis was mediated by PKCh, although we cannot completely rule out the participation of PKCd in the apoptosis Therefore, even if PKCd is involved in TCDD-induced L-MAT T-cell apoptosis, it would seem to be far less important than PKCh
Fig 5 Effects of rottlerin and myristoylated-PKCh pseudosubstrate
peptide inhibitor (myr-PKCh-PPI) on PKCh kinase activity in vitro.
The kinase assay was performed by using 10 ng of a purified
human recombinant PKCh enzyme in a reaction mixture that
con-tained 50 l M ATP, 40 l M of a biotinylated PKCh pseudosubstrate
peptide, and 4 lg of phosphatidylserine with or without rottlerin
(20 l M ) or myr-PKCh-PPI (20 l M ) Data are presented as average
values ± SD (n ¼ 3) (**P > 0.01 vs PKCh alone; Student’s t-test.)
Fig 6 Time-dependent effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on L-MAT cell novel protein kinase C (nPKC) activity Whole cell lysate was prepared from 1 · 10 7
L-MAT cells [treated (j) or not treated (s) with TCDD (20 n M ) on ice] by sonication for 5 s with a Branson Sonifier Cell Disruptor (Branson Ultrasonic Corpora-tion, Danbury, CT, USA) equipped with a microtip (output set at 4)
in 100 lL of buffer containing 50 m M Tris ⁄ HCl, pH 7.5, 150 m M NaCl, 0.1 m M Na3VO4, 0.1 m M Na4P2O712H2O, 1 m M NaF, 0.1 m M phenylmethanesulfonyl fluoride freshly supplemented with
1 · Complete EDTA-free Protease Inhibitor Cocktail (Roche Diag-nostics GmbH, Mannheim, Germany) Following centrifugation (16 000 g, 20 min, 4 C), the supernatant was transferred to fresh microcentrifuge tubes Then, 8 lg of total cell protein was examined in an in vitro nPKC kinase assay, using as the substrate biotin-PKCh pseudosubstrate peptide, which is a rather selective substrate for PKCh and PKCd.
Trang 7Possible events downstream of PKCh
in TCDD-induced apoptosis
In a previous report, we showed that c-Jun N-terminal
kinase 1 (JNK1) is rapidly activated in L-MAT cells,
and that a dominant negative mutant of JNK prevented
TCDD-induced cell death [7] Ghaffari-Tabrizi et al
and others have demonstrated that the transfection of
constitutively active PKCh A408E activates both JNK1
and its upstream activating kinase, SEK1⁄ MKK4, in
a T-cell-specific manner [31], although the immediate
target for PKCh-mediated phosphorylation in the
SEK1⁄ JNK pathway is unknown [28] Therefore, in
TCDD-induced L-MAT cell apoptosis it is possible that
PKCh activation somehow conveys its signal to JNK1,
leading finally to caspase 3 activation However, we still
do not know the details of the signal pathway upstream
of PKCh, or the identity of the first molecule that
inter-acts with TCDD and conveys the signal on the
down-stream side
Significance of TCDD-induced L-MAT cell
apoptosis
Although it is generally considered that the
AhR-dependent pathway mediates the major part of TCDD
immunotoxicity [32], not all examples of this immuno-toxicity can be explained by using the single AhR model, as AhR-independent mechanisms exist by which TCDD can exert immunotoxic effects [9,10] In previous studies, we attested that AhR cannot be involved in TCDD-induced apoptosis in the human T-cell line, L-MAT, because AhR does not exist in L-MAT [7,8] In view of our previous findings and the very high susceptibility to TCDD among T-cell lines (A Hossain, unpublished data), we would like to emphasize the value of L-MAT as a model for study-ing the AhR-independent molecular mechanism involved in the immunotoxicity of TCDD
In summary, we suggest that PKCh kinase activity is functionally involved in the TCDD-induced signal transduction mechanism leading to L-MAT cell apopto-sis This study clearly demonstrates the importance of the PKC pathway in TCDD-induced immunotoxicity
Experimental procedures
Cell culture L-MAT T cells were cultured, as described previously [7], with some modifications Briefly, they were grown in
25 mm Hepes-supplemented RPMI 1640 (ICN Biomedicals Inc., Irvine, CA, USA), pH 7.4, containing 5% fetal bovine serum, 100 IUÆmL)1 penicillin, and 0.1% (v⁄ v) streptomy-cin at 37C in 95% air and 5% CO2 Jurkat T cells were maintained under the same conditions in RPMI 1640 of similar composition, pH 7.4, containing 10% (v⁄ v) fetal bovine serum HepG2 cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium) (Gibco, Invitrogen Corporation, Grand Island, NY, USA), pH 7.4, supplemen-ted with 10% (v⁄ v) fetal bovine serum, 100 IUÆmL)1 peni-cillin, and 0.1% (v⁄ v) streptomycin at 37 C in 95% air and 5% CO2
Apoptosis assay by determination of acetyl-Asp-Glu-Val-Asp⁄ 7-amino-4-methylcoumarin (AcDEVD-AMC) cleavage
Throughout the study we used the detection of caspase-3 activation to evaluate apoptosis in L-MAT cells, as des-cribed previously [8] This entailed some modifications of the method of Nicholson et al [33] We observed that the apoptosis of L-MAT cells by TCDD could be induced in the presence of 5% (v⁄ v) fetal bovine serum; however, serum starvation resulted in sensitization of the cells to TCDD Therefore, serum starvation conditions were used
in these experiments Cells growing exponentially in RPMI
1640 containing 5% (v⁄ v) fetal bovine serum were collec-ted, and fresh medium without serum was added Under these conditions, cells were grown for another 4–6 h at
Fig 7 The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on
L-MAT cells Novel protein kinase C (nPKC) activation by
transloca-tion L-MAT cells (1 · 10 7
), treated or not treated with TCDD (as indicated in the text), were fractionated Then, 40 lg of the
particu-late (membrane + cytoskeleton) fraction protein was examined by
Western blotting to determine whether TCDD treatment caused
translocation of PKCh and PKCd from the cytosolic to the
particu-late fraction in L-MAT cells The upper panel represents PKCh, the
lower panel PKCd.
Trang 837C in 95% air and 5% CO2 Then, after collection and
washing once with phosphate-buffered saline (NaCl⁄ Pi),
cells were incubated at a density of 105Æ100 lL)1per well in
96-microwell plates (Falcon 3072; Becton Dickinson, Franklin Lakes, NJ, USA) in serum-free RPMI 1640, either
in the presence of TCDD or in the presence of an equal
Fig 8 Effect of the over-expression of dominant negative protein kinase C h (DN PKCh) on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced L-MAT cell apoptosis (A) Detection of H-2KK expression in L-MAT cells by direct immunofluorescence microscopy L-MAT cells were transfected with empty pMACSKK.II or with DN PKCh-FLAG ⁄ pMACSKK.II DNA (10 lg) by using electroporation After 44 h of transfec-tion, L-MAT cells were directly immunostained with anti-mouse H-2KK immunoglobulin conjugated to fluorescein isothiocyanate (FITC), then examined under a fluorescence microscope The two pools of L-MAT cells were photographed using a digital camera at the same exposure (A1,3) Bright field, (A2,4) green fluorescence (A1,2) L-MAT cells transfected with empty pMACSKK.II (A3,4) L-MAT cells transfected with
DN PKCh-FLAG ⁄ pMACSKK.II DNA (B) Over-expression of DN-PKCh suppressed TCDD-induced L-MAT cell apoptosis L-MAT cells trans-fected with empty pMACSKK.II or with DN PKCh-FLAG ⁄ pMACSKK.II DNA were collected at 44 h, starved for 4 h in serum-free RPMI 1640, labeled with MACSelect KK MicroBeads, then separated magnetically by means of the miniMACS Separation System L-MAT cells retained
on the miniMACS column were immediately eluted with serum-free RPMI 1640 and counted for viability by using the Trypan blue exclusion assay These L-MAT cells were then distributed as 10 5 cellsÆ100 lL)1of serum-free RPMI 1640 per well in a 96-microwell plate, and subse-quently treated with TCDD (as indicated) for 3 h Finally, the effect of over-expression of DN-PKCh in L-MAT cells on TCDD-induced apopto-sis was evaluated by assessing caspase-3 activation Data are shown as average values ± SD (n ¼ 3) (**P > 0.01 vs TCDD alone; Student’s t-test.) The filled columns represent L-MAT cells transfected with empty pMACSKK.II; the open columns represent those trans-fected with DN PKCh-FLAG ⁄ pMACSKK.II DNA (C) Detection of the over-expression of DN PKCh in L-MAT cells by immunoprecipitation and Western blotting methods Whole cell lysates were prepared from L-MAT cells at 48 h of transfection, and 1.0 mg of total cell protein was then probed by immunoprecipitation and Western blotting to detect the 3·FLAG peptide fused at the C terminus of DN PKCh.
Trang 9volume of the solvent dimethylsulfoxide [the
concentra-tion of which never exceeded the 1% (v⁄ v) level)] or
NaCl⁄ Pi Three hours later, the cells were centrifuged at
1190 g for 10 min at room temperature Then, 75 lL of
the medium was removed and frozen at )80 C for
30 min, followed by thawing on ice for 30 min Next,
50 lL of 100 mm Hepes (pH 7.25), 20% (w⁄ v) sucrose,
5 mm dithiothreitol, 0.1% (v⁄ v) CHAPS, 10–6
% (v⁄ v) Nonidet P-40 (NP-40) containing 100 lm AcDEVD–AMC
(Calbiochem, San Diego, CA, USA) was added to each
well Substrate cleavage to release free AMC was
monit-ored against time at 37C, by using a Fluoroscan Ascent
(Labsystems, Helsinki, Finland) The amount of AMC
released was calculated from the emission at 460 nm
(excitation at 355 nm), using a standard curve for AMC
Fluorescence units were converted to pmoles of AMC
with the aid of a standard curve generated using free
AMC
RNA isolation and RT-PCR
Total RNA was extracted from exponentially growing
L-MAT and HepG2 cells (2· 107
) by using the acid guani-dium thiocyanate⁄ phenol ⁄ chloroform method, as described
by Chomczynski & Sacchi [34] The prepared RNA (50 lg)
was first treated with RNase-free DNaseI (Boehringer
Mannheim GmbH, Mannheim, Germany) to remove the
genomic DNA contaminant Then, 10 lg of total RNA
was reverse-transcribed to synthesize cDNA by means of
AMV (avian myeloblastosis virus)-reverse transcriptase
from Pharmacia using random hexamer, oligo
(dN)6-pri-ming in a final reaction volume of 50 lL supplemented
with RNase inhibitor (Boehringer Mannheim GmbH) For
human PKCh and PKCd, primer sequences were from a
published source [35] For human glyceraldehyde
3-phos-phate dehydrogenase (GAPDH), primers were designed as
follows: forward primer, 5¢-CATCACCATCTTCCAGG
AGC-3¢; reverse primer, 5¢-GGATGATGTTCTGGAGC-3¢
PCR reactions were prepared as a final volume of 20 lL
containing 1.0 lL of the reverse-transcribed sample, 2.0 lL
of 10· Taq buffer, MgCl2 (1.5 mm for PKCh and 2.0 mm
for GAPDH), 200 lm of each dNTP mixture in the
pres-ence of 0.5 lm of each primer, and 2.5 units of Taq DNA
polymerase (TaKaRa Bio Inc., Tokyo, Japan) The PCR
reaction was performed under the following conditions:
ini-tial denaturation for 5 min at 94C (1 cycle), followed by
35 cycles of amplification, each comprising denaturation
for 30 s at 94C, annealing for 1 min at 55 C for GAPDH,
and elongation for 1 min plus a 5 s extension at 72C,
then finally one more cycle of a 5 min elongation at 72C,
followed by cooling to 4C The PCR products (10 lL)
were then subjected to electrophoresis in a 1.5% (w⁄ v)
agarose gel supplemented with ethidium bromide
(0.5 lgÆmL)1) GAPDH expression was checked as the
internal control
Cell lysis, immunoprecipitation, and Western blotting analysis
Cells were lysed as previously described [36], with some modifications, for detection of the endogenous expression
of PKCh and PKCd by using direct Western blotting About 2· 107
cells were collected in a 15 mL conical tube (Nalge Nunc International, Rochester, NY, USA) at
154 g for 5 min and, after the removal of culture medium, were washed once with NaCl⁄ Piand collected again All the subsequent steps for protein preparation were conducted in
a cold room The cell pellet was resuspended by gentle pipetting in 1.0 mL of ice-cold cell lysis buffer (20 mm Tris⁄ HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 5 mm
Na4P2O712H2O, 1 mm Na3VO4, and 1% NP-40) freshly supplemented with 1· Complete EDTA-free Protease Inhib-itor Cocktail (Roche Diagnostics GmbH, Mannheim, Ger-many) Then, 10 lL of 10 mgÆmL)1phenylmethanesulfonyl fluoride was added, and the cells were further disrupted and homogenized by passing them 15 times through a 1.0 mL syringe fitted with a 21-gauge needle They were then main-tained on ice for 30 min, transferred to microcentrifuge tubes, and centrifuged (4C, 20 min, 16 000 g) The super-natant was collected by filtration through a 0.45 lm Acro-disk Syringe Filter (Pall Corporation, East Hills, NY, USA), and total protein was estimated by using the Brad-ford protein assay method Then, 100 lg of protein was mixed with an equal volume of 2· SDS ⁄ PAGE buffer [100 mm Tris⁄ HCl, pH 6.8, 4% (w ⁄ v) SDS, 1.728 mm b-mercaptoethanol, 20% (v⁄ v) glycerol, and 0.2% (w ⁄ v) Bromophenol blue], boiled for 3 min, resolved by electro-phoresis on a 7.5% (w⁄ v) SDS gel, and transferred onto a poly(vinylidene difluoride) membrane The membrane was blocked with 5% (w⁄ v) skimmed milk in 1 · TTBS [50 mm Tris⁄ HCl, pH 7.5, 0.15 m NaCl, 0.1% (v ⁄ v) Tween 20] for
30 min at room temperature on a shaker, then subjected to immunoblot analysis by incubation overnight with anti-human PKCh immunoglobulin (goat polyclonal, sc-1875; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at
4C The membrane was washed four times (15 min each wash) with 1· TTBS at room temperature, followed by another incubation with an anti-goat immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated antibody (donkey polyclonal; Santa Cruz Biotechnology Inc.) for
60 min at room temperature Finally, the signal was detec-ted by using an enhanced chemiluminescence kit (ECL Plus; Amersham Biosciences, London, UK)
For detection of the exogenous expression of PKCh)3·FLAG fusion protein, protein was prepared from transfected L-MAT cells as described above About 1.0 mg
of protein was immunoprecipitated overnight at 4C with
50 lL of anti-FLAG M2 affinity gel (Product No A2220; Sigma, St Louis, MO, USA) Before immunoprecipitation, anti-FLAG M2 affinity gel resin was prepared as a 2 : 1 ratio of suspension to packed gel volume, as described in
Trang 10times with the same cell lysis buffer without the inhibitors,
then mixed with 50 lL of 2· SDS ⁄ PAGE buffer, boiled for
3 min, resolved in a similar way to that described above,
and subjected to immunoblot analysis by incubation with
mouse monoclonal anti-FLAG M2 immunoglobulin
(Sigma) at room temperature for 90 min The membrane
was washed in a similar way to that described above,
followed by a further 90 min incubation with an anti-mouse
IgG⁄ HRP-conjugated antibody (goat polyclonal; Santa
Cruz Biotechnology Inc.) at room temperature Finally, the
signal was detected as described above
In vitro kinase assay for nPKC
A biotinylated PKCh pseudosubstrate peptide
(Biotin-LHQRRGSIKQAKVHHVKC) was used as substrate for an
in vitroPKC kinase assay to evaluate the inhibitory effects
exerted by rottlerin (Calbiochem) and myr-PKCh-PPI
(Calbiochem) on the kinase activity of PKCh The
biotinyl-ated PKCh pseudosubstrate can be phosphorylbiotinyl-ated, because
alanine (A) of real pseudosubstrate is replaced with serine (S)
at position 7 from the biotinylated N terminus The reaction
mixture consisted of 10 ng of purified human recombinant
PKCh (PanVera, Madison, WI, USA) in a reaction volume
of 25 lL containing 50 mm Tris⁄ HCl, pH 7.5, 5 mm MgCl2,
0.1 mm Na3VO4, 0.1 mm Na4P2O712H2O, 1 mm NaF,
0.1 mm phenylmethanesulfonyl fluoride, 50 lm ATP, 5 lCi
[c-32P]dATP[cP], 4 lg of phosphatidyl-l-serine (Sigma), and
40 lm biotinylated-PKCh pseudosubstrate peptide, with or
without rottlerin (20 lm) or myr-PKCh-PPI (20 lm) The
reaction, which took place at 30C in a water bath for
10 min, was terminated by using Protocol A, as previously
described [37] Reactions were centrifuged (in a
microcentri-fuge) at 14 300 g for 10 s Then, 25 lL of the reaction
mix-ture was spotted onto streptavidin-coated square-cut
membrane (Promega, Madison, WI, USA), washed with
95% (v⁄ v) ethanol, dried using a heat-lamp, and counted in
an LSC6000C Scintillation Counter (Beckman Coulter Inc.,
Fullerton, CA, USA), as previously described [37].
Subcellular fractionation for nPKC translocation
assay
L-MAT cells were fractionated, as previously described
[30], with minor modifications, to examine the translocation
son) in the presence of 20 nm TCDD At the end of the incubation, each dish was placed on chilled-ice and taken
to a cold room Cells were collected in a 15 mL precooled centrifuge tube (Nalge Nunc International), spun at
270 g for 10 min at 4C, and washed once with ice-cold NaCl⁄ Pi Finally, the cell pellet was resuspended in 0.5 mL
of buffer B [5 mm Na3VO4, 5 mm Na2P2O7, 5 mm NaF,
5 mm EGTA, 2 mm EDTA, 1 mm dithiothreitol, 20 mm Tris⁄ HCl, pH 7.5, freshly supplemented with 10 mm benz-amidine; Sigma), and 1· Complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics GmbH)] by gentle pipetting, and further sheared by passing 30 times through
a 1.0 mL syringe fitted with a 25-gauge needle Then, 5 lL
of phenylmethanesulfonyl fluoride (10 lgÆlL)1) was added
to the sheared cell suspension on ice After 15 min of incu-bation on ice, the cell suspension was transferred to a 1.5 mL microcentrifuge tube Cell nuclei were removed
by centrifugation (10 min, 1190 g, 4C) Fractionation into cytosolic (cyt) and particulate (pt) fractions was achieved by centrifugation for 30 min at 35 000 r.p.m ( 100 000 g), as previously described [21] After two wash-ing steps with buffer B, 0.25 mL of buffer B containwash-ing 1% NP-40 was added to the particulate fraction (membrane + cytoskeleton), and this was solubilized by pipetting and vig-orous vortexing for 1 min Then, the protein content of the particulate fraction collected at each time-point was estima-ted by using the Bio-Rad Protein Assay Standard Procedure (Bio-Rad Laboratories, Hercules, CA, USA), and 40 lg of the particulate fraction was examined by using the Western blotting method, as described above
Plasmids and subcloning
We used pMACSKK.II (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as the vector for gene expression and functional analysis The kinase-dead mutant of PKCh (K⁄ R 409 mutant), established as a DN mutant [22], was subcloned into pMACSKK.II from a pEF-neo⁄ DN PKCh construct (kindly provided by G Baier, University of Inns-bruck, Austria) First, a SalI-FLAG-Stop oligo with XhoI
at the N terminus and HindIII at the C terminus was sub-cloned into pMACSKK.II Then, PCR was performed to create XhoI at the N terminus, upstream of the start codon, and SalI at the C terminus, just before the stop codon of PKCh, with pEFneo-DN PKCh being used as the template