Báo cáo khoa học: Protein kinase Ch activity is involved in the 2,3,7,8tetrachlorodibenzo-p-dioxin-induced signal transduction pathway leading to apoptosis in L-MAT, a human lymphoblastic T-cell line potx

Protein kinase Ch activity is involved in the 2,3,7,8tetrachlorodibenzo-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 Keywords dioxin; apoptosis; PKCh; lymphoblastic T cell; rottlerin Correspondence H Kikuchi, Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Bunkyocho, 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 September 2004, accepted 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 mechanism 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 functionally 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 manner Fourth, both rottlerin and myristoylated PKCh pseudosubstrate peptide 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 kinasedead mutant, K ⁄ R 409) in L-MAT cells conferred significant protection against TCDD-induced apoptosis 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 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 ⁄ Pi, phosphatebuffered saline; nPKC, novel protein kinase C; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; myr-PKCh-PPI, myristoylated-PKCh-pseudosubstrate peptide inhibitor FEBS Journal 272 (2005) 903–915 ª 2005 FEBS 903 PKCh in TCDD-induced signaling for apoptosis some of its congeners by demonstrating the activation of caspase-3, as a sensitive marker, using L-MAT as a model T-cell line [8] The role played by the aromatic hydrocarbon receptor (AhR)-mediated pathway in TCDD immunotoxicity has been well studied [1] However, some of the immunotoxic effects induced by TCDD are known not to be dependent on an AhR gene locus [9,10], and we have already confirmed, by using the human lymphoblastic T-cell line, L-MAT, as the model, that the AhR-mediated 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 polychlorinated 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 TCDDinduced immunotoxicity is not clearly understood, and indeed the lack of a suitable system in which this immunotoxicity 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 transduction 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 cellular 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 activity 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 properties of PKCh led us to believe that it may play a specialized role in many signal transduction events in T cells 904 S Ahmed et al In the present study, we examined the functional participation of the PKC pathway, and in particular attempted to explore the possible role of PKCh in the TCDD-induced AhR-independent signal transduction mechanism involved in L-MAT cell apoptosis Results Inhibition of TCDD-induced L-MAT cell apoptosis 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 participating 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 isoform(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 classical PKC-selective inhibitor, Go6976 [24] (Fig 1B) ă 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 fluorochrome 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 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 isoform-specific antibodies, with purified PKCh and FEBS Journal 272 (2005) 903–915 ª 2005 FEBS S Ahmed et al PKCh in TCDD-induced signaling for apoptosis 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-myristoylation 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 peptideinhibitor myristoylated at its N-terminal site The L-MAT cell apoptosis induced by TCDD was completely 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 Fig 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 (105 cellsỈ100 lL)1 per well in a 96-microwell plate in serum-free RPMI 1640) were preincubated with PKC-selective pharmacological inhibitors (as indicated) for 30 at 37 °C in 95% air and 5% CO2, followed by treatment with TCDD for h Then, a caspase-3 activation assay was performed as described in the Experimental procedures (A) Staurosporine, (B) Go6976, and (C) rottlerin Data are shown as average values SD ă (n ¼ 3) *P < 0.05; **P > 0.01 vs TCDD alone (Student’s t-test) PKCd 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) FEBS Journal 272 (2005) 903–915 ª 2005 FEBS To establish whether TCDD increases nPKC kinase activity, we examined nPKC kinase activity by using PKCh pseudo-substrate, which is preferentially phosphorylated 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 In a previous 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 fractionating 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 905 PKCh in TCDD-induced signaling for apoptosis S Ahmed et al Fig Morphological alterations in chromatin L-MAT cells were treated with solvent only (Cont), with 20 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 20 lM rottlerin, or with the combination of TCDD + rottlerin Cells were collected after h and fixed in paraformaldehyde, then stained with the DNA-specific fluorochrome, bis-benzimide (Hoechst 33258) comparison 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 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-2KK on 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 separate (on the miniMACS column) transfected cells from the mixture of electroporated cells containing pMACS plasmid DNA, expression of H-2KK on 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-2KK on 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 sup906 press its kinase activity To confirm that PKCh kinase activity really does participate in the signal transduction mechanism involved in TCDD-induced L-MAT cell apoptosis, transfected L-MAT cells expressing H-2KK were separated from the nontransfected cells in the miniMACS column and then tested for caspase-3 activation by treatment with TCDD In this experiment, 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 construct in L-MAT cells was confirmed by immunoprecipitation 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 activationinduced cell death process in T cells, indicating a functional 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 functional involvement of PKCh in TCDD-induced L-MAT cell apoptosis However, rottlerin was originally reported as a novel ATP-competitive protein FEBS Journal 272 (2005) 903–915 ª 2005 FEBS S Ahmed et al A B PKCh in TCDD-induced signaling for apoptosis Fig 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 dehydrogenase (GAPDH) detection was performed as a control Total RNA was extracted from · 107 L-MAT or HepG2 cells Then, 10 lg of the total RNA was reverse transcribed to cDNA and analyzed for PKCh mRNA along with GAPDH (B) Western blotting analysis of endogenous PKCh and PKCd expression Whole cell lysates were prepared from · 107 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 optimized 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 ngỈlg)1 of total L-MAT cell protein The lower panel shows the amount of protein PKCd (in 40 lg of total protein) expressed in L-MAT cells, which seemed to be 500 pgỈlg)1 of total L-MAT cell protein C Fig The effect of myristoylated-PKCh pseudosubstrate peptide inhibitor (myr-PKCh-PPI) on the apoptosis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced L-MAT cells L-MAT cells (105 cellsỈ 100 lL)1 per well in serum-free RPMI 1640 in a 96-microwell plate) were pretreated with myr-PKCh-PPI (as indicated) for 30 at 37 °C in 95% air and 5% CO2, followed by treatment with TCDD for 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.) kinase inhibitor with a very high selectivity for PKCd [25], and it was later used widely as a selective inhibitor for nPKCd, both in vitro and in studies of intact FEBS Journal 272 (2005) 903–915 ª 2005 FEBS 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 concentra907 PKCh in TCDD-induced signaling for apoptosis Fig 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 contained 50 lM ATP, 40 lM of a biotinylated PKCh pseudosubstrate peptide, and lg of phosphatidylserine with or without rottlerin (20 lM) or myr-PKCh-PPI (20 lM) Data are presented as average values ± SD (n ¼ 3) (**P > 0.01 vs PKCh alone; Student’s t-test.) tion 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 particulate fraction from the cytosolic fraction (Fig 7) was observed within of TCDD treatment in L-MAT cells (with the peak being reached at 20 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 expression level ( 500 pgỈlg)1) to be at least 10 times lower than that of PKCh (5 ngỈlg)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 upon TCDD treatment [7] The caspase-3 activation by proteolytic cleavage [the maximal decrease of procaspase-3 at 240 of treatment with TCDD 908 S Ahmed et al Fig 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 · 107 L-MAT cells [treated (j) or not treated (s) with TCDD (20 nM) on ice] by sonication for s with a Branson Sonifier Cell Disruptor (Branson Ultrasonic Corporation, Danbury, CT, USA) equipped with a microtip (output set at 4) in 100 lL of buffer containing 50 mM Tris ⁄ HCl, pH 7.5, 150 mM NaCl, 0.1 mM Na3VO4, 0.1 mM Na4P2O712H2O, mM NaF, 0.1 mM phenylmethanesulfonyl fluoride freshly supplemented with · Complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany) Following centrifugation (16 000 g, 20 min, °C), the supernatant was transferred to fresh microcentrifuge tubes Then, 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 was detected by Western blotting, as was the caspase-3 activity (peak activity at 240 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 apoptotic morphology (peak at 180–240 of treatment of TCDD) was observed by fluorescence microscopy (shown in Fig 2, TCDD only at 240 min) Although we 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 described 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 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS S Ahmed et al Fig The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on L-MAT cells Novel protein kinase C (nPKC) activation by translocation L-MAT cells (1 · 107), treated or not treated with TCDD (as indicated in the text), were fractionated Then, 40 lg of the particulate (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 particulate fraction in L-MAT cells The upper panel represents PKCh, the lower panel PKCd Possible events downstream of PKCh in TCDD-induced apoptosis In a previous report, we showed that c-Jun N-terminal kinase (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 activation However, we still not know the details of the signal pathway upstream of PKCh, or the identity of the first molecule that interacts with TCDD and conveys the signal on the downstream side Significance of TCDD-induced L-MAT cell apoptosis Although it is generally considered that the AhRdependent pathway mediates the major part of TCDD FEBS Journal 272 (2005) 903–915 ª 2005 FEBS PKCh in TCDD-induced signaling for apoptosis immunotoxicity [32], not all examples of this immunotoxicity 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 studying 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 apoptosis 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 ImL)1 penicillin, and 0.1% (v ⁄ v) streptomycin at 37 °C 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, supplemented with 10% (v ⁄ v) fetal bovine serum, 100 ImL)1 penicillin, and 0.1% (v ⁄ v) streptomycin at 37 °C in 95% air and 5% CO2 Apoptosis assay by determination of acetylAsp-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 described 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 collected, and fresh medium without serum was added Under these conditions, cells were grown for another 4–6 h at 909 PKCh in TCDD-induced signaling for apoptosis A S Ahmed et al B C Fig 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 transfection, 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 transfected with empty pMACSKK.II or with DN PKCh-FLAG ⁄ pMACSKK.II DNA were collected at 44 h, starved for 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 105 cellsỈ100 lL)1 of serum-free RPMI 1640 per well in a 96-microwell plate, and subsequently treated with TCDD (as indicated) for h Finally, the effect of over-expression of DN-PKCh in L-MAT cells on TCDD-induced apoptosis 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 transfected 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 37 °C 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 105Ỉ100 lL)1 per well in 910 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 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS S Ahmed et al volume of the solvent dimethylsulfoxide [the concentration of which never exceeded the 1% (v ⁄ v) level)] or NaCl ⁄ Pi Three hours later, the cells were centrifuged at 1190 g for 10 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 Next, 50 lL of 100 mm Hepes (pH 7.25), 20% (w ⁄ v) sucrose, 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 monitored against time at 37 °C, 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 guanidium 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-priming 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-phosphate 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 presence 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: initial denaturation for at 94 °C (1 cycle), followed by 35 cycles of amplification, each comprising denaturation for 30 s at 94 °C, annealing for at 55 °C for GAPDH, and elongation for plus a s extension at 72 °C, then finally one more cycle of a elongation at 72 °C, followed by cooling to °C The PCR products (10 lL) were then subjected to electrophoresis in a 1.5% (w ⁄ v) agarose gel supplemented with ethidium bromide (0.5 lgỈmL)1) GAPDH expression was checked as the internal control FEBS Journal 272 (2005) 903–915 ª 2005 FEBS PKCh in TCDD-induced signaling for apoptosis 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 · 107 cells were collected in a 15 mL conical tube (Nalge Nunc International, Rochester, NY, USA) at 154 g for and, after the removal of culture medium, were washed once with NaCl ⁄ Pi and 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, mm EDTA, mm Na4P2O712H2O, mm Na3VO4, and 1% NP-40) freshly supplemented with 1· Complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany) Then, 10 lL of 10 mgỈmL)1 phenylmethanesulfonyl 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 maintained on ice for 30 min, transferred to microcentrifuge tubes, and centrifuged (4 °C, 20 min, 16 000 g) The supernatant was collected by filtration through a 0.45 lm Acrodisk Syringe Filter (Pall Corporation, East Hills, NY, USA), and total protein was estimated by using the Bradford 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 min, resolved by electrophoresis 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 · TTBS [50 mm Tris ⁄ HCl, pH 7.5, 0.15 m NaCl, 0.1% (v ⁄ v) Tween 20] for 30 at room temperature on a shaker, then subjected to immunoblot analysis by incubation overnight with antihuman PKCh immunoglobulin (goat polyclonal, sc-1875; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at °C The membrane was washed four times (15 each wash) with · 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 at room temperature Finally, the signal was detected 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 °C 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 : ratio of suspension to packed gel volume, as described in 911 PKCh in TCDD-induced signaling for apoptosis the attached protocol The resin was washed once with 0.5 mL of 0.1 m glycine HCl, pH 3.5, for min, while thoroughly tapping and inverting to remove traces of unbound anti-FLAG immunoglobulin from the resin The supernatant was aspirated out using a Hamilton syringe after centrifugation (4 °C, s, 7300 g) The resin was immediately washed twice with 0.5 mL of NaCl ⁄ Tris (TBS) buffer and once with 0.5 mL of cell lysis buffer, as described in the attached protocol, without inhibitors After immunoprecipitation overnight, the affinity gel was washed three times with the same cell lysis buffer without the inhibitors, then mixed with 50 lL of 2· SDS ⁄ PAGE buffer, boiled for 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 The membrane was washed in a similar way to that described above, followed by a further 90 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 (BiotinLHQRRGSIKQAKVHHVKC) was used as substrate for an in vitro PKC kinase assay to evaluate the inhibitory effects exerted by rottlerin (Calbiochem) and myr-PKCh-PPI (Calbiochem) on the kinase activity of PKCh The biotinylated PKCh pseudosubstrate can be phosphorylated, because alanine (A) of real pseudosubstrate is replaced with serine (S) at position 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, mm MgCl2, 0.1 mm Na3VO4, 0.1 mm Na4P2O712H2O, mm NaF, 0.1 mm phenylmethanesulfonyl fluoride, 50 lm ATP, lCi [c-32P]dATP[cP], 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 30 °C in a water bath for 10 min, was terminated by using Protocol A, as previously described [37] Reactions were centrifuged (in a microcentrifuge) at 14 300 g for 10 s Then, 25 lL of the reaction mixture 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 912 S Ahmed et al of PKCh and PKCd during the TCDD-induced apoptosis mechanism In brief, L-MAT cells growing exponentially in RPMI 1640 containing 5% (v ⁄ v) fetal bovine serum were collected, resuspended in the same serum-free medium, and cultured for another h at 37 °C in 95% air and 5% CO2 Then, the cells were collected and washed once with NaCl ⁄ Pi First, · 107 cells without any TCDD treatment were collected as a control Then, at each time-point, · 107 cells were incubated in mL of serum-free RPMI in a 60 mm cell-culture dish (Falcon 3002; Becton Dickinson) 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 at °C, and washed once with ice-cold NaCl ⁄ Pi Finally, the cell pellet was resuspended in 0.5 mL of buffer B [5 mm Na3VO4, mm Na2P2O7, mm NaF, mm EGTA, mm EDTA, mm dithiothreitol, 20 mm Tris ⁄ HCl, pH 7.5, freshly supplemented with 10 mm benzamidine; 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, lL of phenylmethanesulfonyl fluoride (10 lgỈlL)1) was added to the sheared cell suspension on ice After 15 of incubation on ice, the cell suspension was transferred to a 1.5 mL microcentrifuge tube Cell nuclei were removed by centrifugation (10 min, 1190 g, °C) Fractionation into cytosolic (cyt) and particulate (pt) fractions was achieved by centrifugation for 30 at 35 000 r.p.m ( 100 000 g), as previously described [21] After two washing steps with buffer B, 0.25 mL of buffer B containing 1% NP-40 was added to the particulate fraction (membrane + cytoskeleton), and this was solubilized by pipetting and vigorous vortexing for Then, the protein content of the particulate fraction collected at each time-point was estimated 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 Innsbruck, Austria) First, a SalI-FLAG-Stop oligo with XhoI at the N terminus and HindIII at the C terminus was subcloned 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 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS S Ahmed et al Next, the XhoI-DN PKCh-SalI was subcloned into pMACSKK.II at the XhoI ⁄ SalI site in-frame with the SalIFLAG-Stop, and finally a DN PKCh-FLAG construct was made in the pMACSKK.II vector In a similar manner, a DN PKCh)3·FLAG construct was also made The sequence was confirmed by restriction enzyme digestion and sequencing analyses at all the steps involved in subcloning by using standard procedures Transient transfection assay Plasmid DNAs were transfected into L-MAT cells by using the electroporation method Exponentially growing L-MAT cells were collected and resuspended at · 106 cells per 10 mL of RPMI [containing 5% (v ⁄ v) fetal bovine serum] in each 100 mm dish, then cultured overnight at 37 °C in 95% air and 5% CO2 for transfection the next day L-MAT cells were collected, resuspended at · 106 cells in 200 lL of intracellular buffer CYTOMIX, pH 7.6, prepared as previously described [38], then transfected with 10 lg of empty pMACSKK.II or DN PKCh-FLAG ⁄ pMACSKK.II DNA at 280 V, 72 ohm, 1050 lF in each mm gap electroporation cuvette (Molecular BioProducts Inc., San Diego, CA, USA) using the BTX Electroporation System (BTX Inc., San Diego, CA, USA) Transfected L-MAT cells were collected at optimal time-points for further analysis Immunofluorescence microscopy for transient gene expression analysis Transfected L-MAT cells were collected at 44 h and immediately prepared for immunofluorescence staining with antimouse H-2KK-fluorescein isothiocyanate (FITC) antibody (clone H100-27.R55; Miltenyi Biotec), as described in the attached protocol Immunostained cells were mounted and examined by using a Leica DMLB (Leica, Mikroskopie und Systeme GmbH, Wetzlar, Germany) fluorescent microscope Magnetic separation of transfected L-MAT cells for functional assay Transfected L-MAT cells were collected at 44 h, resuspended in RPMI 1640 without fetal bovine serum, and incubated for another h at 37 °C in 95% air and 5% CO2 Cells were collected again, washed once with NaCl ⁄ Pi, and labeled with MACSelect KK magnetic MicroBeads for separation by the MiniMACS Separation System, as described in the attached protocol (Miltenyi Biotec) The only modification to this process was that we used serum-free RPMI 1640 instead of PBE (phosphate-buffered saline supplemented with mm EDTA) Then, the transfected L-MAT cells retained within the MiniMACS Column were eluted with serum-free RPMI 1640 and counted for viability by using a Trypan Blue exclusion assay Finally, the effect of TCDD on caspase-3 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS PKCh in TCDD-induced signaling for apoptosis activation was examined, as described above in the apoptosis assay section Acknowledgements We thank Dr Gottfried Baier (University of Innsbruck, Austria) for generously providing the dominant negative PKCh construct of plasmid DNA This work was supported, in part, by Grants-in-Aid for Scientific Research (B) [Nos 11558068 and 12480153 from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Monbu Kagakusho)] and supported by a Scholarship for foreign students from Monbu Kagakusho (S.A.) 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(2000) A novel functional interaction between Vav and PKCtheta is required for TCR-induced T cell activation Immunity 12, 151–160 37 Schaefer EM & Guimond S (1998) Detection of protein tyrosine kinase activity using a high-capacity streptavidin-coated membrane and optimized biotinylated peptide substrates Anal Biochem 261, 100–112 38 van den Hoff MJ, Moorman AF & Lamers WH (1992) Electroporation in ‘intracellular’ buffer increases cell survival Nucleic Acids Res 20, 2902 915 ... explained by the single AhR pathway The molecular mechanism involved in the AhR-independent pathway( s) leading to TCDDinduced immunotoxicity is not clearly understood, and indeed the lack of a. .. PKCh and specifically sup906 press its kinase activity To confirm that PKCh kinase activity really does participate in the signal transduction mechanism involved in TCDD-induced L-MAT cell apoptosis, ... dependent on an AhR gene locus [9,10], and we have already confirmed, by using the human lymphoblastic T-cell line, L-MAT, as the model, that the AhR-mediated pathway is in no way involved in TCDD-induced