- Email: [email protected]
The transcription of the peroxisome proliferator-activated receptor α gene is regulated by protein kinase C
Toxicology Letters 125 (2001) 133– 141 www.elsevier.com/locate/toxlet
The transcription of the peroxisome proliferator-activated receptor a gene is regulated by protein kinase C Nik-Soriani Yaacob 1, Mohd-Nor Norazmi1, G. Gordon Gibson, George E.N. Kass * School of Biomedical and Life Sciences, Uni6ersity of Surrey, Guildford, Surrey GU2 7XH, UK Received 25 May 2001; received in revised form 21 August 2001; accepted 29 August 2001
Abstract The transcriptional regulation of peroxisome proliferator-activated receptor a (PPARa) by a variety of peroxisome proliferators was investigated. The treatment of primary cultures of rat hepatocytes with Wy14,643 or clofibrate increased mRNA steady state levels of both PPARh and acyl coenzyme A oxidase (ACOX). In contrast, fenofibrate and ciprofibrate increased the expression of ACOX without affecting that of PPARh. Inhibition of protein kinase C (PKC) activity using bisindolylmaleimide or calphostin C abolished the increased PPARh expression by the peroxisome proliferators whereas the expression of the ACOX gene remained unaffected. Phorbol-12-myristate-13acetate increased PPARh mRNA levels without altering ACOX mRNA levels. It can thus be concluded that a number of peroxisome proliferators activate a PKC-dependent signalling pathway in addition to the PPARa pathway. The PKC signal transduction pathway contributes to the regulation of PPARh expression but does not influence the transcriptional activity of PPARa. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peroxisome proliferators; Hypolipidaemic drugs; PPAR; Hepatocytes; Tumours
1. Introduction Peroxisome proliferators (PPs) are a chemically diverse class of non-genotoxic and non-mutagenic compounds that cause extensive increase in the number and size of the peroxisomes in the liver of rodents (Gibson, 1993; Rao and Reddy, 1996). * Corresponding author. Tel.: + 44-1483-686449; fax: + 441483-300374. E-mail address: [email protected] (G.E.N. Kass). 1 Present address: School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
The majority of these compounds belong to the family of clofibrate-like hypolipidaemic agents such as fenofibrate and ciprofibrate. A structurally different hypolipidaemic compound, Wy14,643, is also a potent peroxisome proliferator. Short-term exposure to PPs has been shown to upregulate genes involved in the peroxisomal b-oxidation and microsomal v-oxidation of fatty acids, such as acyl CoA oxidase (ACOX) (Tugwood et al., 1992; Dreyer et al., 1993) and cytochrome P450 4A1 (CYP4A1 ) (Aldridge et al., 1995), respectively. Most importantly, their actions lead to hepatocarcinogenesis in rats and
0378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-4274(01)00433-7
134
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
mice (Cattley et al., 1994; Rao and Reddy, 1996; Peters et al., 1997a). This is thought to be the result of the ability of PPs to promote hepatocyte growth as well as to suppress apoptosis (Marsman et al., 1988; Tanaka et al., 1992; Grasl-Kraupp et al., 1993; Bayly et al., 1994). The pleiotropic effects of PPs have been shown to occur via a nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARa) (Green, 1995; Lee et al., 1995; Peters et al., 1997b) by acting as ligands for this receptor (Forman et al., 1997; Kliewer et al., 1997; Kersten et al., 2000). PPARa is a ligand-dependent transcription factor that belongs to the steroid hormone receptor superfamily (Green, 1995) and binds (as a heterodimer with the retinoic acid receptor (RXR)) to specific peroxisome proliferator response elements (PPRE) located upstream of target genes such as ACOX and CYP4A1 (Tugwood et al., 1992; Dreyer et al., 1993; Aldridge et al., 1995). The direct binding of PPs to PPARa has been demonstrated (Kliewer et al., 1997; Lehmann et al., 1997), and the disruption of the ligand-binding domain of PPARa abolished the induction of ACOX and CYP4A1 in Wy14,643- and clofibrate-treated mice (Lee et al., 1995). Furthermore, in the knockout mouse system (PPARh − / − ), Wy14,643- and clofibratetreated animals did not display hepatomegaly, peroxisome proliferation, transcriptional induction of responsive genes, replicative DNA synthesis or hepatocarcinogenesis (Lee et al., 1995; Peters et al., 1997b), all of which are characteristic effects of these xenobiotics. Little is known about the regulation of PPARh expression in tissues targeted by PPs. Glucocorticoids have been shown to induce PPARh expression in liver cells (Lemberger et al., 1994; Steineger et al., 1994). In addition, the peroxisome proliferator fenofibrate (Gebel et al., 1992) has been reported to increase PPARh mRNA levels in liver following in vivo treatment. A similar induction was also caused by Wy14,643, perfluorodecanoic acid and oleic acid in the rat hepatoma cell line FaO (Sterchele et al., 1996).
It is not clear whether the transcriptional regulation of PPARh by PPs occurs through PPARa itself or whether an additional signalling pathway is involved. In this report the possibility that a member of the protein kinase C (PKC) family regulates PPARh expression was investigated. Several previous studies have demonstrated that the activity of the major classical PKCs was modulated by PPs (Bronfman et al., 1989; Orellana et al., 1990; Bojes and Thurman, 1994; Motojima et al., 1996; Bojes et al., 1997). Here we report that several PPs increased both ACOX and PPARh mRNA levels in rat hepatocytes. The increase in PPARh mRNA levels was blocked by PKC inhibitors whereas ACOX mRNA induction was unaffected by the inhibitors. Likewise, activation of PKC by a phorbol ester led to an increase in PPARh mRNA levels without alterations in ACOX mRNA levels. Therefore, our studies have identified a novel signalling pathway for PPARa expression and have clarified the role of PKC in the mechanism of PP action. 2. Materials and methods
2.1. Chemicals Collagenase A, ultrapure agarose and ultrapure PCR nucleotide mix were purchased from Roche Biochemicals. Phorbol-12-myristate-13acetate (PMA) was obtained from Alexis Corporation Ltd (UK) and bisindolylmaleimide I and calphostin C were obtained from CalbiochemNovabiochem Ltd (UK). The following sources for the peroxisome proliferators were used: Wy 14,643, Chemsyn Science Laboratories, USA; clofibrate, ICN Biomedicals; ciprofibrate, Sterling Winthrop; fenofibrate, Sigma-Aldrich. Cell culture media, supplements and custom made oligonucleotides were purchased from Life Technologies Ltd (UK). Moloney-Murine Leukaemia Virus reverse transcriptase and Red Hot DNA polymerase were obtained from Advanced Biotechnologies Ltd (UK) and recombinant RNase inhibitor, oligo(dT) primer and PCR MIMIC construction kit from Clontech Laboratories. RNeasyTM Total RNA kit was purchased from Qiagen Ltd (UK).
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
2.2. Isolation of primary hepatocytes Male Wistar rats (200–250 g, fed on a commercial chow (Rat and Mouse Diet BK001E, B&K Universal Ltd, UK) ad libitum) were anesthetized by intraperitoneal injection of sodium pentobarbitone at a dose of 100 mg/kg body weight and primary hepatocytes were isolated as previously reported (Kass et al., 1988). The viability of hepatocytes following isolation (determined by trypan blue exclusion) was 90– 95%.
2.3. Culture and treatment of primary hepatocytes Hepatocytes were cultured in 25 cm2 flasks coated with poly-L-lysine at a density of 2×106 cells/4 ml of CL-15 medium (Mitchell et al., 1984) in an humidified incubator maintained at 37 °C. The medium was replaced following an initial attachment period for 4 h before treatment with medium containing Wy14,643 (150 mM), clofibrate (500 mM), fenofibrate (500 mM), ciprofibrate (500 mM) or dexamethasone (1 mM). All chemicals used for the treatment of hepatocytes were dissolved in dimethyl sulphoxide at a 1000× concentration. Cultures to be treated with the PKC inhibitors bisindolylmaleimide I (20 nM) and calphostin C (200 nM), were first pre-incubated with either inhibitor for 1 h (under fluorescent light in the case of calphostin C) prior to the treatment with PPs. The cultures were then incubated at 37 °C for 24 h.
2.4. Competiti6e RT-PCR and quantitation of steady-state mRNA le6els Total RNA isolation, cDNA synthesis, generation of internal standards (ACOX and PPARa PCR MIMICs) and competitive RT-PCR were carried out as described previously (Yaacob et al., 1997). Briefly, the total RNA was reverse-transcribed with Murine-Moloney Leukemia Virus reverse transcriptase using an oligo(dT) primer. The cDNA synthesised was then mixed with a series of known concentrations of the PCR MIMIC and amplified. The PCR products were electrophoresed on a 1.6% agarose gel and the relative intensities of the bands quantitated using a
135
Shimadzu CS-2000 dual-wavelength flying spot scanner. The steady-state mRNA levels of ACOX and PPARh were quantitated from the ratios of target to MIMIC products plotted against the reciprocal of the concentration of PCR MIMIC used. The concentrations of mRNAs were expressed as attomoles (10 − 18 moles) per mg total RNA.
2.5. Statistical analyses All statistical analyses was carried out using the Bonferroni-Dunn (all means) one-way ANOVA post-hoc test employing the SuperANOVA Software package (Apple Computer, Inc. USA).
3. Results and discussion In a first series of experiments the regulation of ACOX and PPARh mRNA levels in response to a series of PPs was investigated. In agreement with previous studies (Dreyer et al., 1993), the exposure of rat hepatocytes to PPs led to the transcriptional upregulation of the peroxisomal ACOX gene as reflected by the three- to four-fold increases in the steady-state levels of ACOX mRNA levels (Table 1). In contrast, the synthetic glucocorticoid dexamethasone did not alter ACOX mRNA levels. The quantitation of PPARh mRNA levels revealed that Wy14,643 and clofibrate significantly elevated this transcript (Table 1). Likewise, dexamethasone upregulated PPARh mRNA levels over the same period of exposure whereas the treatment of hepatocytes with fenofibrate and ciprofibrate did not result in any significant increase in the steady state PPARh mRNA levels. The average fold-induction of PPARa mRNA expression following treatment with Wy14,643, clofibrate and dexamethasone were 2.9, 2.1 and 8.4, respectively. Thus, PPs upregulated PPARh mRNA levels, although this was not a universal phenomenon. The next step was to investigate the molecular mechanism that is responsible for this transcriptional effect. The possible role of PKC enzymes in the transcriptional upregulation of PPARh mRNA was investigated. Hepatocytes were pretreated with
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
136
bisindolylmaleimide I, a PKC inhibitor that shows broad specificity by inhibiting most PKC isoforms (Toullec et al., 1991) via competition at its ATPbinding site (Wilkinson et al., 1993). As shown in Table 2 this PKC inhibitor had no significant effect on the constitutive expression of PPARh and ACOX in cultured hepatocytes, whereas the induction of PPARh mRNA by both Wy14,643 and clofibrate was completely blocked by bisindolylmaleimide I. In contrast the pre-treatment with bisindolylmaleimide I did not prevent the increase in ACOX mRNA levels by the PPs.
Essentially identical results were obtained with the protein kinase C inhibitor calphostin C (Kobayashi et al., 1989) which interacts with the regulatory domain by competing at the diacylglycerol-binding site (Garcia-Sainz et al., 1992). Like bisindolylmaleimide I, the presence of calphostin C resulted in the complete inhibition of the PP-induced elevation of PPARh mRNA levels without affecting the induction of ACOX mRNA (Table 3). Taken together, our inhibitor studies suggest the involvement of PKC in the pathway of PP-mediated upregulation of PPARh expression but
Table 1 Effect of peroxisome proliferators and dexamethasone on PPARh and ACOX mRNA steady state levels in rat hepatocytes Treatment
Control (Me2SO) Wy14,643 Clofibrate Fenofibrate Ciprofibrate Dexamethasone
n
8 4 4 3 4 4
Attomoles per mg total RNA PPARa mRNA
Fold induction
ACOX mRNA
Fold induction
0.039 0.01 0.09 90.03** 0.07 90.01* 0.03 90.01 0.03 9 0.01 0.209 0.05**
– (2.9) (2.1) (1.4) (1.3) (8.4)
2.23 90.36 7.14 90.97** 5.54 90.82** 6.40 92.00* 6.83 9 1.06** 3.59 9 0.47
– (3.8) (3.3) (3.0) (3.8) (1.6)
Primary hepatocyte cultures were treated with Wy14,643 (150 mM), clofibrate (500 mM), fenofibrate (500 mM), ciprofibrate (500 mM) or with dexamethasone (1 mM) for 24 h. Total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. The data represent the mean values 9 S.E.M. of 3–8 independent experiments. * PB0.05. ** PB0.01. Table 2 Effect of the PKC inhibitor bisindolylmaleimide on Wy14643- and clofibrate-induced PPARh and ACOX steady-state mRNA levels in primary hepatocyte cultures Treatment
Control (Me2SO) Bisindolylmaleimide Wy14,643 Wy14,643+bisindolylmaleimide Clofibrate Clofibrate+bisindolylmaleimide
n
8 7 5 5 5 5
Attomoles per mg total RNA PPARa mRNA
Fold induction
ACOX mRNA
Fold induction
0.049 0.01 0.04 9 0.01 0.1190.03 0.0490.01** 0.0790.02 0.049 0.01*
– (1.0) (2.6) (1.0) (1.8) (1.0)
1.88 90.26 2.62 90.33 6.96 91.01 6.64 91.07 5.16 90.69 5.47 90.76
– (1.5) (4.3) (4.0) (3.3) (3.5)
Primary hepatocyte cultures were treated with Wy14,643 (150 mM) or clofibrate (500 mM) in the absence or presence of bisindolylmaleimide (20 nM) for 24 h. The PKC inhibitor was added 1 h prior to the peroxisome proliferators. Total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. The data represent the mean values 9 S.E.M. of 5–8 independent experiments. * PB0.05; increase in mRNA levels by PPs plus inhibitor being analysed against increase in the presence of PP alone. ** PB0.01; increase in mRNA levels by PPs plus inhibitor being analysed against increase in the presence of PP alone.
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
137
Table 3 Effect of the PKC inhibitor calphostin C on Wy14643- and clofibrate-induced PPARh and ACOX steady-state mRNA levels in primary hepatocyte cultures Treatment
Control (Me2SO) Calphostin C Wy14,643 Wy14,643+Calphostin C Clofibrate Clofibrate+Calphostin C
Attomoles per mg total RNA PPARa mRNA
Fold induction
ACOX mRNA
Fold induction
0.04 90.01 0.0390.01 0.1390.02 0.0390.01* 0.0790.01 0.0390.01*
– (0.7) (3.1) (0.9) (1.7) (0.8)
1.36 90.10 1.59 90.13 6.88 90.98 5.23 90.60 7.01 90.77 7.37 91.27
– (1.2) (5.0) (3.8) (5.1) (5.3)
Primary hepatocyte cultures were treated with Wy14,643 (150 mM) or clofibrate (500 mM) in the absence or presence of calphostin C (200 nM) for 24 h. The PKC inhibitor was added 1 h prior to the peroxisome proliferators. Total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. All data represent the mean values 9S.E.M. of three independent experiments. * PB0.05; increase in mRNA levels by PPs plus inhibitor being analysed against increase in the presence of PP alone.
not in the induction of ACOX. To directly test the involvement of PKC in the upregulation of PPARh, the levels of PPARh mRNA were measured in response to the potent PKC activating phorbol ester PMA. A transient and significant increase in PPARh mRNA levels within 3– 9 h of exposure of hepatocytes to PMA that peaked at 6 h was observed (Fig. 1A). In clear contrast, the steady-state levels of ACOX mRNA were not significantly altered in response to the phorbol ester (Fig. 1B). The first part of this study has demonstrated that PPs differentially affect PPARh and ACOX mRNA levels. Wy14,643 and clofibrate increased the expression of both PPARh and ACOX whereas fenofibrate and ciprofibrate increased the expression of ACOX without affecting that of PPARh. It should be noted that fenofibrate and ciprofibrate were more potent than clofibrate in inducing peroxisome proliferation and liver carcinogenesis (Rao and Reddy, 1996). This suggests that the carcinogenic potency of PPs cannot be directly correlated with their ability to induce transcription of the PPARh gene. An increase in the level of PPARh mRNA in FaO hepatoma cells by fenofibrate has been reported (Lemberger et al., 1996). However, here we show that rat hepatocytes in culture did not increase their PPARh mRNA levels in response to fenofibrate. In contrast to PPARh, ACOX expression was
induced by all the PPs used in this study, and there was no correlation between ACOX expression and the extent of PPARh expression. Therefore, it is clear that the constitutive levels of PPARa in hepatocytes are already sufficient to mediate the activation of ACOX gene transcription. However, the additional inducing effect on PPARa itself may play a role in modulating the pleiotropicity and duration of the responses to certain PPs. Although gene knock-out experiments have clearly shown that PPARa accounts for the effects of PPs on lipid metabolism, differentiation, inflammation, apoptosis and carcinogenesis (Lee et al., 1995; Peters et al., 1997b; Torra et al., 1999; Kersten et al., 2000), it is not clear whether all cellular effects elicited by PPs can be attributed to the sole action of PPARa. For instance, the immediate-early gene expression and the high level of proliferation induced by PPs may require other signalling events in addition to the PPARa pathway to explain the rather weak mitogenic effects of PPs in cultured hepatocytes as compared to their in vivo potency (Hong and Glauert, 1998; Vanden Heuvel, 1999). Likewise, the production of Kupffer cell derived reactive oxygen species by Wy14,643 is independent of PPARa (Peters et al., 2000). Finally, di-(2-ethylhexyl)-phthalate-induced reproductive toxicity and teratogenicity are not
138
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
Fig. 1. Effect of PKC activation by PMA on the steady-state PPARh mRNA and ACOX mRNA levels. Primary hepatocyte cultures were exposed to PMA ( , 50 nM) or solvent control (, dimethyl sulphoxide). At the indicated time points total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. The steady-state mRNA levels of PPARh and ACOX were measured and expressed as attomoles PPARh (Panel A) or attomoles ACOX (Panel B) per mg total RNA. Data shown represent the mean values9S.E.M. *P B0.05; **PB 0.01.
mediated by an PPARa-dependent mechanism (Peters et al., 1997b). Several reports have suggested a role for one or more of the PKC isozymes in PP action (Bronfman et al., 1989; Orellana et al., 1990; Bojes and Thurman, 1994; Motojima et al., 1996; Bojes et al., 1997). For example, the inhibition of gapjunctional communication in rat hepatocytes by nafenopin has been linked to the activation of PKC (Elcock et al., 2000). Rats fed with Wy 14,643 or isolated livers perfused with Wy 14,643 were found to have increased liver membranebound PKC activity as assessed by phorbol ester binding (Bojes et al., 1997; Rose et al., 2000). Other PPs of various potencies also elevated PKC activity in rat liver as determined by a radioactive binding assay, in rough proportion to their ability to induce tumours in long-term feeding studies (Bojes and Thurman, 1994). However, unlike the latter study, the lack of effect of ciprofibrate and fenofibrate on PPARh mRNA levels reported here suggests that these PPs failed to activate PKC under our in vitro conditions. Whether this apparent discrepancy may be rationalised by differences in PKC isoforms activated by the PPs or by additional signalling pathways activated by PPs in non-parenchymal cells through the release of cytokines and the induction of conditions of oxidative stress (Bojes et al., 1997; Peters et al., 2000) needs to be investigated. The activation of the ERK1/2 pathway in liver cells by Wy 14,643, ciprofibrate and clofibrate has been reported by at least two groups (Rokos and Ledwith, 1997; Mounho and Thrall, 1999), and it is well established that PKC is an upstream activator of this pathway. Phosphorylation of PPARa at the conserved MAP-kinase sites Ser12 and Ser21 has been linked to the transcriptional activation of human PPARa (Juge-Aubry et al., 1999; Passilly et al., 1999). However, we show here that blocking PKC activity did not affect ACOX mRNA levels suggesting that the ERK1/2 pathway may not have been critical in the regulation of PPARa transcriptional activity under the present experimental conditions. Alternatively, the activation of the ERK1/2 pathway by PPs may be independent of their effect on PKC activity.
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
It is still unclear how PPs interact with PKC and which PKC isoforms are being activated in the process. These compounds do not activate PKC directly (Bojes and Thurman, 1994), and it has been suggested that PKC activation occurs via their acyl-CoA thioesters (Bronfman et al., 1989; Orellana et al., 1990) or by elevated intracellular levels of free fatty acids (Bojes et al., 1997). Previous work from our laboratory has shown that PPs increase cytosolic free Ca2 + levels in rat hepatocytes (Shackleton et al., 1995). Since Ca2 + is an important activator of several PKC isoenzymes, Ca2 + mobilisation may play a role in the activation of PKC reported here. Indeed, we recently observed that the increase in PPARh transcript levels by PPs was abrogated in the absence of extracellular Ca2 + (N.-S.Y. and G.E.N.K., unpublished observations). However, the ultimate identity of the PKC isoenzymes targeted by PPs as well as their role in PP action will need to be addressed by using cells deficient in or with down-regulated individual PKC isoenzymes. In conclusion, we report that PPs can activate at least two independent signal transduction pathways in rat hepatocytes. The first pathway involves activation of responsive genes by PPARa whereas for a number of PPs an additional pathway involving PKC exists. One of the downstream effects of PKC appears to be the transcriptional activation of PPARh. However, further work is necessary to fully understand the implications of PKC activation in the pleiotropic effects of PPs.
References Aldridge, T.C., Tugwood, J.D., Green, S., 1995. Identification and characterization of DNA elements implicated in the regulation of CYP4A1 transcription. Biochem. J. 306, 473– 479. Bayly, A.C., Roberts, R.A., Dive, C., 1994. Suppression of liver cell apoptosis in vitro by the nongenotoxic hepatocarcinogen and peroxisome proliferator nafenopin. J. Cell Biol. 125, 197 – 203. Bojes, H.K., Rose, M.L., Keller, B.J., Germolec, D.R., Simeonova, P., Luster, M.I., Thurman, R.G., 1997. Mitogenic actions of peroxisome proliferators: involvement of protein kinase C and tumor necrosis factor a. Drug Metab. Rev. 29, 235 – 260.
139
Bojes, H.K., Thurman, R.G., 1994. Peroxisomal proliferators inhibit acyl CoA synthetase and stimulate protein kinase C in vivo. Toxicol. Appl. Pharmacol. 126, 233 – 239. Bronfman, M., Orellana, A., Morales, M.N., Bieri, F., Waechter, F., Staubli, W., Bentley, P., 1989. Potentiation of diacylglycerol-activated protein kinase C by acyl coenzyme A thioesters of hypolipemic drugs. Biochem. Biophys. Res. Commun. 159, 1026 – 1031. Cattley, R.C., Kato, M., Popp, J.A., Teets, V.J., Voss, K.S., 1994. Initiator-specific promotion of hepatocarcinogenesis by WY-14,643 and clofibrate. Carcinogenesis 15, 1763 – 1766. Dreyer, C., Keller, H., Mahfoudi, A., Laudet, V., Krey, G., Wahli, W., 1993. Positive regulation of the peroxisomal b-oxidation pathway by fatty acids through activation of peroxisome proliferator activated receptors (PPAR). Biol. Cell 77, 67 – 76. Elcock, F.J., Deag, E., Roberts, R.A., Chipman, J.K., 2000. Nafenopin causes protein kinase C-mediated serine phosphorylation and loss of function of connexin 32 protein in rat hepatocytes without aberrant expression or localization. Toxicol. Sci. 56, 86 – 94. Forman, B.M., Chen, J., Evans, R.M., 1997. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors a and d. Proc. Natl. Acad. Sci. USA 94, 4312 – 4317. Garcia-Sainz, J.A., Lopez Gomez, F.J., Robles Flores, M., 1992. Staurosporine and calphostin C inhibit the phorbol ester-induced decrease of protein kinase C activity in rat hepatocytes. Biochem. Int. 28, 761 – 766. Gebel, T., Arand, M., Oesch, F., 1992. Induction of the peroxisome proliferator activated receptor by fenofibrate in rat liver. FEBS Lett. 309, 37 –40. Gibson, G.G., 1993. Peroxisome proliferators: paradigms and prospects. Toxicol. Lett. 68, 193 – 201. Grasl-Kraupp, B., Huber, W., Timmermanntrosiener, I., Schulte-Hermann, R., 1993. Peroxisomal enzyme induction uncoupled from enhanced DNA-synthesis in putative preneoplastic liver foci of rats treated with a single dose of the peroxisome proliferator nafenopin. Carcinogenesis 14, 2435 – 2437. Green, S., 1995. PPAR: a mediator of peroxisome proliferator action. Mutation Res. 333, 101 – 109. Hong, J.T., Glauert, H.P., 1998. Stimulation of the DNA binding activity of AP-1 by the peroxisome proliferator ciprofibrate and eicosanoids in cultured rat hepatocytes. Toxicology 131, 99 – 107. Juge-Aubry, C.E., Hammar, E., Siegrist-Kaiser, C., Pernin, A., Takeshita, A., Chin, W.W., Burger, A.G., Meier, C.A., 1999. Regulation of the transcriptional activity of the peroxisome proliferator-activated receptor alpha by phophorylation of a ligand-independent trans-activating domain. J. Biol. Chem. 274, 10505 – 10510. Kass, G.E.N., Wright, J.M., Nicotera, P., Orrenius, S., 1988. The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity: role of intracellular calcium. Arch. Biochem. Biophys. 260, 789 – 797.
140
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
Kersten, S., Desvergne, B., Wahli, W., 2000. Roles of PPARs in health and disease. Nature 405, 421 –424. Kliewer, S.A., Sundseth, S.S., Jones, S.A., Brown, P.J., Wisely, G.B., Koble, C.S., Devchand, P., Wahli, W., Willson, T.M., Lenhard, J.M., Lehmann, J.M., 1997. Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors a and g. Proc. Natl. Acad. Sci. USA 94, 4318 – 4323. Kobayashi, E., Nakano, H., Morimoto, M., Tamaoki, T., 1989. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 159, 548 – 553. Lee, S.-T., Pineau, T., Drago, J., Lee, E.J., Owens, J.W., Kroetz, D.L., Fernandez-Salguero, P.M., Westphal, H., Gonzalez, F.J., 1995. Targeted disruption of the alpha-isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell. Biol. 15, 3012 – 3022. Lehmann, J.M., Lenhard, J.M., Oliver, B.B., Ringold, G.M., Kliewer, S.A., 1997. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J. Biol. Chem. 272, 3406 – 3410. Lemberger, T., Braissant, O., Juge-Aubry, C., Keller, H., Saladin, R., Staels, B., Auwerx, J., Burger, A.G., Meier, C.A., Wahli, W., 1996. PPAR tissue distribution and interactions with other hormone signaling pathways. Ann. N.Y. Acad. Sci. 804, 231 – 251. Lemberger, T., Staels, B., Saladin, R., Desvergne, B., Auwerx, J., Wahli, W., 1994. Regulation of the peroxisome proliferator-activated receptor-a gene by glucocorticoids. J. Biol. Chem. 269, 24527 – 24530. Marsman, D.S., Cattley, R.C., Conway, J.G., Popp, J.A., 1988. Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643) in rats. Cancer Res. 48, 6739 – 6744. Mitchell, A.M., Bridges, J.W., Elcombe, C.R., 1984. Factors influencing peroxisome proliferation in cultured rat hepatocytes. Arch. Toxicol. 55, 239 –246. Motojima, K., Passilly, P., Jannin, B., Latruffe, N., 1996. Protein phosphorylation by peroxisome proliferators: Species-specific stimulation of protein kinases and its role in PP-induced transcriptional activation. Ann. N.Y. Acad. Sci. 804, 413 – 423. Mounho, B.J., Thrall, B.D., 1999. The extracellular signal-regulated kinase pathway contributes to mitogenic and antiapoptotic effects of peroxisome proliferators in vitro. Toxicol. Appl. Pharmacol. 159, 125 – 133. Orellana, A., Hidalgo, P.C., Morales, M.N., Mezzano, D., Bronfman, M., 1990. Palmitoyl-CoA and the acyl-CoA thioester of the carcinogenic peroxisome-proliferator ciprofibrate potentiate diacylglycerol-activated protein kinase C by decreasing the phosphatidylserine requirement of the enzyme. Eur. J. Biochem. 190, 57 –61.
Passilly, P., Schohn, H., Jannin, B., Malki, M.C., Boscoboinik, D., Dauca, M., Latruffe, N., 1999. Phosphorylation of peroxisome proliferator-activated receptor alpha in Fao cells and stimulation by ciprofibrate. Biochem. Pharmacol. 58, 1001 – 1008. Peters, J.M., Cattley, R.C., Gonzalez, F.J., 1997a. Role of PPARa in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14,643. Carcinogenesis 18, 2029 – 2033. Peters, J.M., Taubeneck, M.W., Keen, C.L., Gonzalez, F.J., 1997b. Di(2-ethylhexyl)phthalate induces a functional zinc deficiency during pregnancy and teratogenesis that is independent of peroxisome proliferator-activated receptor a. Teratology 56, 311 – 316. Peters, J.M., Rusyn, I., Rose, M.L., Gonzalez, F.J., Thurman, R.G., 2000. Peroxisome proliferator-activated receptor a is restricted to hepatic parenchymal cells, not Kupffer cells: implications for the mechanism of action of peroxisome proliferators in hepatocarcinogenesis. Carcinogenesis 21, 823 – 826. Rao, M.S., Reddy, J.K., 1996. Hepatocarcinogenesis of peroxisome proliferators. Ann. N.Y. Acad. Sci. 804, 573 – 587. Rokos, C.L., Ledwith, B.J., 1997. Peroxisome proliferator activate extracellular signal-regulated kinases in immortalized mouse liver cells. J. Biol. Chem. 272, 13452 – 13457. Rose, M.L., Rusyn, I., Bojes, H.K., Belyea, J., Cattley, R.C., Thurman, R.G., 2000. Role of Kupffer cells and oxidants in signaling peroxisome proliferator-induced hepatocyte proliferation. Mutat. Res. 448, 179 – 192. Shackleton, G.L., Gibson, G.G., Sharma, R.K., Howes, D., Orrenius, S., Kass, G.E.N., 1995. Diverse mechanisms of calcium mobilization by peroxisome proliferators in rat hepatocytes. Toxicol. Appl. Pharmacol. 130, 294 – 303. Steineger, H.H., Sorensen, H.N., Tugwood, J.D., Skrede, S., Spydevold, O., Gautvik, K.M., 1994. Dexamethasone and insulin demonstrate marked and opposite regulation of the steady-state mRNA level of the peroxisomal proliferatoractivated receptor (PPAR) in hepatic cells: hormonal modulation of fatty-acid-induced transcription. Eur. J. Biochem. 225, 967 – 974. Sterchele, P.F., Sun, H., Peterson, R.E., Vanden Heuvel, J.P., 1996. Regulation of peroxisome proliferator-activated receptor-a mRNA in rat liver. Arch. Biochem. Biophys. 326, 281 – 289. Tanaka, K., Smith, P.F., Stromberg, P.C., Eydelloth, R.S., Herold, E.G., Grossman, S.J., Frank, J.D., Hertzog, P.R., Soper, K.A., Keenan, K.P., 1992. Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague – Dawley rats treated with tumorigenic doses of clofibrate. Toxicol. Appl. Pharmacol. 116, 71 – 77. Torra, I.P., Gervois, P., Staels, B., 1999. Peroxisome proliferator-activated receptor alpha in metabolic disease, inflammation, atherosclerosis and aging. Curr. Opin. Lipidol. 10, 151 – 159. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grandperret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., Kirilovsky, J.,
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141 1991. The bisindolylmaleimide GF-109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 15771 – 15781. Tugwood, J.D., Issemann, I., Anderson, R.G., Bundell, K.R., Mcpheat, W.L., Green, S., 1992. The mouse peroxisome proliferator activated receptor recognizes a response element in the 5% flanking sequence of the rat acyl CoA oxidase gene. EMBO J. 11, 433 –439. Vanden Heuvel, J.P., 1999. Peroxisome proliferator-activated
141
receptors (PPARS) and carcinogenesis. Toxicol. Sci. 47, 1 – 8. Wilkinson, S.E., Parker, P.J., Nixon, J.S., 1993. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem. J. 294, 335 – 337. Yaacob, N.S., Norazmi, M.N., Kass, G.E.N., Gibson, G.G., 1997. Use of competitive RT-PCR in the molecular analysis of peroxisome proliferation. Eur. J. Drug Metab. Pharmacokin. 22, 321 – 324.
The transcription of the peroxisome proliferator-activated receptor a gene is regulated by protein kinase C Nik-Soriani Yaacob 1, Mohd-Nor Norazmi1, G. Gordon Gibson, George E.N. Kass * School of Biomedical and Life Sciences, Uni6ersity of Surrey, Guildford, Surrey GU2 7XH, UK Received 25 May 2001; received in revised form 21 August 2001; accepted 29 August 2001
Abstract The transcriptional regulation of peroxisome proliferator-activated receptor a (PPARa) by a variety of peroxisome proliferators was investigated. The treatment of primary cultures of rat hepatocytes with Wy14,643 or clofibrate increased mRNA steady state levels of both PPARh and acyl coenzyme A oxidase (ACOX). In contrast, fenofibrate and ciprofibrate increased the expression of ACOX without affecting that of PPARh. Inhibition of protein kinase C (PKC) activity using bisindolylmaleimide or calphostin C abolished the increased PPARh expression by the peroxisome proliferators whereas the expression of the ACOX gene remained unaffected. Phorbol-12-myristate-13acetate increased PPARh mRNA levels without altering ACOX mRNA levels. It can thus be concluded that a number of peroxisome proliferators activate a PKC-dependent signalling pathway in addition to the PPARa pathway. The PKC signal transduction pathway contributes to the regulation of PPARh expression but does not influence the transcriptional activity of PPARa. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peroxisome proliferators; Hypolipidaemic drugs; PPAR; Hepatocytes; Tumours
1. Introduction Peroxisome proliferators (PPs) are a chemically diverse class of non-genotoxic and non-mutagenic compounds that cause extensive increase in the number and size of the peroxisomes in the liver of rodents (Gibson, 1993; Rao and Reddy, 1996). * Corresponding author. Tel.: + 44-1483-686449; fax: + 441483-300374. E-mail address: [email protected] (G.E.N. Kass). 1 Present address: School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
The majority of these compounds belong to the family of clofibrate-like hypolipidaemic agents such as fenofibrate and ciprofibrate. A structurally different hypolipidaemic compound, Wy14,643, is also a potent peroxisome proliferator. Short-term exposure to PPs has been shown to upregulate genes involved in the peroxisomal b-oxidation and microsomal v-oxidation of fatty acids, such as acyl CoA oxidase (ACOX) (Tugwood et al., 1992; Dreyer et al., 1993) and cytochrome P450 4A1 (CYP4A1 ) (Aldridge et al., 1995), respectively. Most importantly, their actions lead to hepatocarcinogenesis in rats and
0378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-4274(01)00433-7
134
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
mice (Cattley et al., 1994; Rao and Reddy, 1996; Peters et al., 1997a). This is thought to be the result of the ability of PPs to promote hepatocyte growth as well as to suppress apoptosis (Marsman et al., 1988; Tanaka et al., 1992; Grasl-Kraupp et al., 1993; Bayly et al., 1994). The pleiotropic effects of PPs have been shown to occur via a nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARa) (Green, 1995; Lee et al., 1995; Peters et al., 1997b) by acting as ligands for this receptor (Forman et al., 1997; Kliewer et al., 1997; Kersten et al., 2000). PPARa is a ligand-dependent transcription factor that belongs to the steroid hormone receptor superfamily (Green, 1995) and binds (as a heterodimer with the retinoic acid receptor (RXR)) to specific peroxisome proliferator response elements (PPRE) located upstream of target genes such as ACOX and CYP4A1 (Tugwood et al., 1992; Dreyer et al., 1993; Aldridge et al., 1995). The direct binding of PPs to PPARa has been demonstrated (Kliewer et al., 1997; Lehmann et al., 1997), and the disruption of the ligand-binding domain of PPARa abolished the induction of ACOX and CYP4A1 in Wy14,643- and clofibrate-treated mice (Lee et al., 1995). Furthermore, in the knockout mouse system (PPARh − / − ), Wy14,643- and clofibratetreated animals did not display hepatomegaly, peroxisome proliferation, transcriptional induction of responsive genes, replicative DNA synthesis or hepatocarcinogenesis (Lee et al., 1995; Peters et al., 1997b), all of which are characteristic effects of these xenobiotics. Little is known about the regulation of PPARh expression in tissues targeted by PPs. Glucocorticoids have been shown to induce PPARh expression in liver cells (Lemberger et al., 1994; Steineger et al., 1994). In addition, the peroxisome proliferator fenofibrate (Gebel et al., 1992) has been reported to increase PPARh mRNA levels in liver following in vivo treatment. A similar induction was also caused by Wy14,643, perfluorodecanoic acid and oleic acid in the rat hepatoma cell line FaO (Sterchele et al., 1996).
It is not clear whether the transcriptional regulation of PPARh by PPs occurs through PPARa itself or whether an additional signalling pathway is involved. In this report the possibility that a member of the protein kinase C (PKC) family regulates PPARh expression was investigated. Several previous studies have demonstrated that the activity of the major classical PKCs was modulated by PPs (Bronfman et al., 1989; Orellana et al., 1990; Bojes and Thurman, 1994; Motojima et al., 1996; Bojes et al., 1997). Here we report that several PPs increased both ACOX and PPARh mRNA levels in rat hepatocytes. The increase in PPARh mRNA levels was blocked by PKC inhibitors whereas ACOX mRNA induction was unaffected by the inhibitors. Likewise, activation of PKC by a phorbol ester led to an increase in PPARh mRNA levels without alterations in ACOX mRNA levels. Therefore, our studies have identified a novel signalling pathway for PPARa expression and have clarified the role of PKC in the mechanism of PP action. 2. Materials and methods
2.1. Chemicals Collagenase A, ultrapure agarose and ultrapure PCR nucleotide mix were purchased from Roche Biochemicals. Phorbol-12-myristate-13acetate (PMA) was obtained from Alexis Corporation Ltd (UK) and bisindolylmaleimide I and calphostin C were obtained from CalbiochemNovabiochem Ltd (UK). The following sources for the peroxisome proliferators were used: Wy 14,643, Chemsyn Science Laboratories, USA; clofibrate, ICN Biomedicals; ciprofibrate, Sterling Winthrop; fenofibrate, Sigma-Aldrich. Cell culture media, supplements and custom made oligonucleotides were purchased from Life Technologies Ltd (UK). Moloney-Murine Leukaemia Virus reverse transcriptase and Red Hot DNA polymerase were obtained from Advanced Biotechnologies Ltd (UK) and recombinant RNase inhibitor, oligo(dT) primer and PCR MIMIC construction kit from Clontech Laboratories. RNeasyTM Total RNA kit was purchased from Qiagen Ltd (UK).
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
2.2. Isolation of primary hepatocytes Male Wistar rats (200–250 g, fed on a commercial chow (Rat and Mouse Diet BK001E, B&K Universal Ltd, UK) ad libitum) were anesthetized by intraperitoneal injection of sodium pentobarbitone at a dose of 100 mg/kg body weight and primary hepatocytes were isolated as previously reported (Kass et al., 1988). The viability of hepatocytes following isolation (determined by trypan blue exclusion) was 90– 95%.
2.3. Culture and treatment of primary hepatocytes Hepatocytes were cultured in 25 cm2 flasks coated with poly-L-lysine at a density of 2×106 cells/4 ml of CL-15 medium (Mitchell et al., 1984) in an humidified incubator maintained at 37 °C. The medium was replaced following an initial attachment period for 4 h before treatment with medium containing Wy14,643 (150 mM), clofibrate (500 mM), fenofibrate (500 mM), ciprofibrate (500 mM) or dexamethasone (1 mM). All chemicals used for the treatment of hepatocytes were dissolved in dimethyl sulphoxide at a 1000× concentration. Cultures to be treated with the PKC inhibitors bisindolylmaleimide I (20 nM) and calphostin C (200 nM), were first pre-incubated with either inhibitor for 1 h (under fluorescent light in the case of calphostin C) prior to the treatment with PPs. The cultures were then incubated at 37 °C for 24 h.
2.4. Competiti6e RT-PCR and quantitation of steady-state mRNA le6els Total RNA isolation, cDNA synthesis, generation of internal standards (ACOX and PPARa PCR MIMICs) and competitive RT-PCR were carried out as described previously (Yaacob et al., 1997). Briefly, the total RNA was reverse-transcribed with Murine-Moloney Leukemia Virus reverse transcriptase using an oligo(dT) primer. The cDNA synthesised was then mixed with a series of known concentrations of the PCR MIMIC and amplified. The PCR products were electrophoresed on a 1.6% agarose gel and the relative intensities of the bands quantitated using a
135
Shimadzu CS-2000 dual-wavelength flying spot scanner. The steady-state mRNA levels of ACOX and PPARh were quantitated from the ratios of target to MIMIC products plotted against the reciprocal of the concentration of PCR MIMIC used. The concentrations of mRNAs were expressed as attomoles (10 − 18 moles) per mg total RNA.
2.5. Statistical analyses All statistical analyses was carried out using the Bonferroni-Dunn (all means) one-way ANOVA post-hoc test employing the SuperANOVA Software package (Apple Computer, Inc. USA).
3. Results and discussion In a first series of experiments the regulation of ACOX and PPARh mRNA levels in response to a series of PPs was investigated. In agreement with previous studies (Dreyer et al., 1993), the exposure of rat hepatocytes to PPs led to the transcriptional upregulation of the peroxisomal ACOX gene as reflected by the three- to four-fold increases in the steady-state levels of ACOX mRNA levels (Table 1). In contrast, the synthetic glucocorticoid dexamethasone did not alter ACOX mRNA levels. The quantitation of PPARh mRNA levels revealed that Wy14,643 and clofibrate significantly elevated this transcript (Table 1). Likewise, dexamethasone upregulated PPARh mRNA levels over the same period of exposure whereas the treatment of hepatocytes with fenofibrate and ciprofibrate did not result in any significant increase in the steady state PPARh mRNA levels. The average fold-induction of PPARa mRNA expression following treatment with Wy14,643, clofibrate and dexamethasone were 2.9, 2.1 and 8.4, respectively. Thus, PPs upregulated PPARh mRNA levels, although this was not a universal phenomenon. The next step was to investigate the molecular mechanism that is responsible for this transcriptional effect. The possible role of PKC enzymes in the transcriptional upregulation of PPARh mRNA was investigated. Hepatocytes were pretreated with
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
136
bisindolylmaleimide I, a PKC inhibitor that shows broad specificity by inhibiting most PKC isoforms (Toullec et al., 1991) via competition at its ATPbinding site (Wilkinson et al., 1993). As shown in Table 2 this PKC inhibitor had no significant effect on the constitutive expression of PPARh and ACOX in cultured hepatocytes, whereas the induction of PPARh mRNA by both Wy14,643 and clofibrate was completely blocked by bisindolylmaleimide I. In contrast the pre-treatment with bisindolylmaleimide I did not prevent the increase in ACOX mRNA levels by the PPs.
Essentially identical results were obtained with the protein kinase C inhibitor calphostin C (Kobayashi et al., 1989) which interacts with the regulatory domain by competing at the diacylglycerol-binding site (Garcia-Sainz et al., 1992). Like bisindolylmaleimide I, the presence of calphostin C resulted in the complete inhibition of the PP-induced elevation of PPARh mRNA levels without affecting the induction of ACOX mRNA (Table 3). Taken together, our inhibitor studies suggest the involvement of PKC in the pathway of PP-mediated upregulation of PPARh expression but
Table 1 Effect of peroxisome proliferators and dexamethasone on PPARh and ACOX mRNA steady state levels in rat hepatocytes Treatment
Control (Me2SO) Wy14,643 Clofibrate Fenofibrate Ciprofibrate Dexamethasone
n
8 4 4 3 4 4
Attomoles per mg total RNA PPARa mRNA
Fold induction
ACOX mRNA
Fold induction
0.039 0.01 0.09 90.03** 0.07 90.01* 0.03 90.01 0.03 9 0.01 0.209 0.05**
– (2.9) (2.1) (1.4) (1.3) (8.4)
2.23 90.36 7.14 90.97** 5.54 90.82** 6.40 92.00* 6.83 9 1.06** 3.59 9 0.47
– (3.8) (3.3) (3.0) (3.8) (1.6)
Primary hepatocyte cultures were treated with Wy14,643 (150 mM), clofibrate (500 mM), fenofibrate (500 mM), ciprofibrate (500 mM) or with dexamethasone (1 mM) for 24 h. Total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. The data represent the mean values 9 S.E.M. of 3–8 independent experiments. * PB0.05. ** PB0.01. Table 2 Effect of the PKC inhibitor bisindolylmaleimide on Wy14643- and clofibrate-induced PPARh and ACOX steady-state mRNA levels in primary hepatocyte cultures Treatment
Control (Me2SO) Bisindolylmaleimide Wy14,643 Wy14,643+bisindolylmaleimide Clofibrate Clofibrate+bisindolylmaleimide
n
8 7 5 5 5 5
Attomoles per mg total RNA PPARa mRNA
Fold induction
ACOX mRNA
Fold induction
0.049 0.01 0.04 9 0.01 0.1190.03 0.0490.01** 0.0790.02 0.049 0.01*
– (1.0) (2.6) (1.0) (1.8) (1.0)
1.88 90.26 2.62 90.33 6.96 91.01 6.64 91.07 5.16 90.69 5.47 90.76
– (1.5) (4.3) (4.0) (3.3) (3.5)
Primary hepatocyte cultures were treated with Wy14,643 (150 mM) or clofibrate (500 mM) in the absence or presence of bisindolylmaleimide (20 nM) for 24 h. The PKC inhibitor was added 1 h prior to the peroxisome proliferators. Total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. The data represent the mean values 9 S.E.M. of 5–8 independent experiments. * PB0.05; increase in mRNA levels by PPs plus inhibitor being analysed against increase in the presence of PP alone. ** PB0.01; increase in mRNA levels by PPs plus inhibitor being analysed against increase in the presence of PP alone.
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
137
Table 3 Effect of the PKC inhibitor calphostin C on Wy14643- and clofibrate-induced PPARh and ACOX steady-state mRNA levels in primary hepatocyte cultures Treatment
Control (Me2SO) Calphostin C Wy14,643 Wy14,643+Calphostin C Clofibrate Clofibrate+Calphostin C
Attomoles per mg total RNA PPARa mRNA
Fold induction
ACOX mRNA
Fold induction
0.04 90.01 0.0390.01 0.1390.02 0.0390.01* 0.0790.01 0.0390.01*
– (0.7) (3.1) (0.9) (1.7) (0.8)
1.36 90.10 1.59 90.13 6.88 90.98 5.23 90.60 7.01 90.77 7.37 91.27
– (1.2) (5.0) (3.8) (5.1) (5.3)
Primary hepatocyte cultures were treated with Wy14,643 (150 mM) or clofibrate (500 mM) in the absence or presence of calphostin C (200 nM) for 24 h. The PKC inhibitor was added 1 h prior to the peroxisome proliferators. Total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. All data represent the mean values 9S.E.M. of three independent experiments. * PB0.05; increase in mRNA levels by PPs plus inhibitor being analysed against increase in the presence of PP alone.
not in the induction of ACOX. To directly test the involvement of PKC in the upregulation of PPARh, the levels of PPARh mRNA were measured in response to the potent PKC activating phorbol ester PMA. A transient and significant increase in PPARh mRNA levels within 3– 9 h of exposure of hepatocytes to PMA that peaked at 6 h was observed (Fig. 1A). In clear contrast, the steady-state levels of ACOX mRNA were not significantly altered in response to the phorbol ester (Fig. 1B). The first part of this study has demonstrated that PPs differentially affect PPARh and ACOX mRNA levels. Wy14,643 and clofibrate increased the expression of both PPARh and ACOX whereas fenofibrate and ciprofibrate increased the expression of ACOX without affecting that of PPARh. It should be noted that fenofibrate and ciprofibrate were more potent than clofibrate in inducing peroxisome proliferation and liver carcinogenesis (Rao and Reddy, 1996). This suggests that the carcinogenic potency of PPs cannot be directly correlated with their ability to induce transcription of the PPARh gene. An increase in the level of PPARh mRNA in FaO hepatoma cells by fenofibrate has been reported (Lemberger et al., 1996). However, here we show that rat hepatocytes in culture did not increase their PPARh mRNA levels in response to fenofibrate. In contrast to PPARh, ACOX expression was
induced by all the PPs used in this study, and there was no correlation between ACOX expression and the extent of PPARh expression. Therefore, it is clear that the constitutive levels of PPARa in hepatocytes are already sufficient to mediate the activation of ACOX gene transcription. However, the additional inducing effect on PPARa itself may play a role in modulating the pleiotropicity and duration of the responses to certain PPs. Although gene knock-out experiments have clearly shown that PPARa accounts for the effects of PPs on lipid metabolism, differentiation, inflammation, apoptosis and carcinogenesis (Lee et al., 1995; Peters et al., 1997b; Torra et al., 1999; Kersten et al., 2000), it is not clear whether all cellular effects elicited by PPs can be attributed to the sole action of PPARa. For instance, the immediate-early gene expression and the high level of proliferation induced by PPs may require other signalling events in addition to the PPARa pathway to explain the rather weak mitogenic effects of PPs in cultured hepatocytes as compared to their in vivo potency (Hong and Glauert, 1998; Vanden Heuvel, 1999). Likewise, the production of Kupffer cell derived reactive oxygen species by Wy14,643 is independent of PPARa (Peters et al., 2000). Finally, di-(2-ethylhexyl)-phthalate-induced reproductive toxicity and teratogenicity are not
138
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
Fig. 1. Effect of PKC activation by PMA on the steady-state PPARh mRNA and ACOX mRNA levels. Primary hepatocyte cultures were exposed to PMA ( , 50 nM) or solvent control (, dimethyl sulphoxide). At the indicated time points total RNA was extracted and a series of competitive RT-PCR carried out as described under Section 2. The steady-state mRNA levels of PPARh and ACOX were measured and expressed as attomoles PPARh (Panel A) or attomoles ACOX (Panel B) per mg total RNA. Data shown represent the mean values9S.E.M. *P B0.05; **PB 0.01.
mediated by an PPARa-dependent mechanism (Peters et al., 1997b). Several reports have suggested a role for one or more of the PKC isozymes in PP action (Bronfman et al., 1989; Orellana et al., 1990; Bojes and Thurman, 1994; Motojima et al., 1996; Bojes et al., 1997). For example, the inhibition of gapjunctional communication in rat hepatocytes by nafenopin has been linked to the activation of PKC (Elcock et al., 2000). Rats fed with Wy 14,643 or isolated livers perfused with Wy 14,643 were found to have increased liver membranebound PKC activity as assessed by phorbol ester binding (Bojes et al., 1997; Rose et al., 2000). Other PPs of various potencies also elevated PKC activity in rat liver as determined by a radioactive binding assay, in rough proportion to their ability to induce tumours in long-term feeding studies (Bojes and Thurman, 1994). However, unlike the latter study, the lack of effect of ciprofibrate and fenofibrate on PPARh mRNA levels reported here suggests that these PPs failed to activate PKC under our in vitro conditions. Whether this apparent discrepancy may be rationalised by differences in PKC isoforms activated by the PPs or by additional signalling pathways activated by PPs in non-parenchymal cells through the release of cytokines and the induction of conditions of oxidative stress (Bojes et al., 1997; Peters et al., 2000) needs to be investigated. The activation of the ERK1/2 pathway in liver cells by Wy 14,643, ciprofibrate and clofibrate has been reported by at least two groups (Rokos and Ledwith, 1997; Mounho and Thrall, 1999), and it is well established that PKC is an upstream activator of this pathway. Phosphorylation of PPARa at the conserved MAP-kinase sites Ser12 and Ser21 has been linked to the transcriptional activation of human PPARa (Juge-Aubry et al., 1999; Passilly et al., 1999). However, we show here that blocking PKC activity did not affect ACOX mRNA levels suggesting that the ERK1/2 pathway may not have been critical in the regulation of PPARa transcriptional activity under the present experimental conditions. Alternatively, the activation of the ERK1/2 pathway by PPs may be independent of their effect on PKC activity.
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
It is still unclear how PPs interact with PKC and which PKC isoforms are being activated in the process. These compounds do not activate PKC directly (Bojes and Thurman, 1994), and it has been suggested that PKC activation occurs via their acyl-CoA thioesters (Bronfman et al., 1989; Orellana et al., 1990) or by elevated intracellular levels of free fatty acids (Bojes et al., 1997). Previous work from our laboratory has shown that PPs increase cytosolic free Ca2 + levels in rat hepatocytes (Shackleton et al., 1995). Since Ca2 + is an important activator of several PKC isoenzymes, Ca2 + mobilisation may play a role in the activation of PKC reported here. Indeed, we recently observed that the increase in PPARh transcript levels by PPs was abrogated in the absence of extracellular Ca2 + (N.-S.Y. and G.E.N.K., unpublished observations). However, the ultimate identity of the PKC isoenzymes targeted by PPs as well as their role in PP action will need to be addressed by using cells deficient in or with down-regulated individual PKC isoenzymes. In conclusion, we report that PPs can activate at least two independent signal transduction pathways in rat hepatocytes. The first pathway involves activation of responsive genes by PPARa whereas for a number of PPs an additional pathway involving PKC exists. One of the downstream effects of PKC appears to be the transcriptional activation of PPARh. However, further work is necessary to fully understand the implications of PKC activation in the pleiotropic effects of PPs.
References Aldridge, T.C., Tugwood, J.D., Green, S., 1995. Identification and characterization of DNA elements implicated in the regulation of CYP4A1 transcription. Biochem. J. 306, 473– 479. Bayly, A.C., Roberts, R.A., Dive, C., 1994. Suppression of liver cell apoptosis in vitro by the nongenotoxic hepatocarcinogen and peroxisome proliferator nafenopin. J. Cell Biol. 125, 197 – 203. Bojes, H.K., Rose, M.L., Keller, B.J., Germolec, D.R., Simeonova, P., Luster, M.I., Thurman, R.G., 1997. Mitogenic actions of peroxisome proliferators: involvement of protein kinase C and tumor necrosis factor a. Drug Metab. Rev. 29, 235 – 260.
139
Bojes, H.K., Thurman, R.G., 1994. Peroxisomal proliferators inhibit acyl CoA synthetase and stimulate protein kinase C in vivo. Toxicol. Appl. Pharmacol. 126, 233 – 239. Bronfman, M., Orellana, A., Morales, M.N., Bieri, F., Waechter, F., Staubli, W., Bentley, P., 1989. Potentiation of diacylglycerol-activated protein kinase C by acyl coenzyme A thioesters of hypolipemic drugs. Biochem. Biophys. Res. Commun. 159, 1026 – 1031. Cattley, R.C., Kato, M., Popp, J.A., Teets, V.J., Voss, K.S., 1994. Initiator-specific promotion of hepatocarcinogenesis by WY-14,643 and clofibrate. Carcinogenesis 15, 1763 – 1766. Dreyer, C., Keller, H., Mahfoudi, A., Laudet, V., Krey, G., Wahli, W., 1993. Positive regulation of the peroxisomal b-oxidation pathway by fatty acids through activation of peroxisome proliferator activated receptors (PPAR). Biol. Cell 77, 67 – 76. Elcock, F.J., Deag, E., Roberts, R.A., Chipman, J.K., 2000. Nafenopin causes protein kinase C-mediated serine phosphorylation and loss of function of connexin 32 protein in rat hepatocytes without aberrant expression or localization. Toxicol. Sci. 56, 86 – 94. Forman, B.M., Chen, J., Evans, R.M., 1997. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors a and d. Proc. Natl. Acad. Sci. USA 94, 4312 – 4317. Garcia-Sainz, J.A., Lopez Gomez, F.J., Robles Flores, M., 1992. Staurosporine and calphostin C inhibit the phorbol ester-induced decrease of protein kinase C activity in rat hepatocytes. Biochem. Int. 28, 761 – 766. Gebel, T., Arand, M., Oesch, F., 1992. Induction of the peroxisome proliferator activated receptor by fenofibrate in rat liver. FEBS Lett. 309, 37 –40. Gibson, G.G., 1993. Peroxisome proliferators: paradigms and prospects. Toxicol. Lett. 68, 193 – 201. Grasl-Kraupp, B., Huber, W., Timmermanntrosiener, I., Schulte-Hermann, R., 1993. Peroxisomal enzyme induction uncoupled from enhanced DNA-synthesis in putative preneoplastic liver foci of rats treated with a single dose of the peroxisome proliferator nafenopin. Carcinogenesis 14, 2435 – 2437. Green, S., 1995. PPAR: a mediator of peroxisome proliferator action. Mutation Res. 333, 101 – 109. Hong, J.T., Glauert, H.P., 1998. Stimulation of the DNA binding activity of AP-1 by the peroxisome proliferator ciprofibrate and eicosanoids in cultured rat hepatocytes. Toxicology 131, 99 – 107. Juge-Aubry, C.E., Hammar, E., Siegrist-Kaiser, C., Pernin, A., Takeshita, A., Chin, W.W., Burger, A.G., Meier, C.A., 1999. Regulation of the transcriptional activity of the peroxisome proliferator-activated receptor alpha by phophorylation of a ligand-independent trans-activating domain. J. Biol. Chem. 274, 10505 – 10510. Kass, G.E.N., Wright, J.M., Nicotera, P., Orrenius, S., 1988. The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity: role of intracellular calcium. Arch. Biochem. Biophys. 260, 789 – 797.
140
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141
Kersten, S., Desvergne, B., Wahli, W., 2000. Roles of PPARs in health and disease. Nature 405, 421 –424. Kliewer, S.A., Sundseth, S.S., Jones, S.A., Brown, P.J., Wisely, G.B., Koble, C.S., Devchand, P., Wahli, W., Willson, T.M., Lenhard, J.M., Lehmann, J.M., 1997. Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors a and g. Proc. Natl. Acad. Sci. USA 94, 4318 – 4323. Kobayashi, E., Nakano, H., Morimoto, M., Tamaoki, T., 1989. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 159, 548 – 553. Lee, S.-T., Pineau, T., Drago, J., Lee, E.J., Owens, J.W., Kroetz, D.L., Fernandez-Salguero, P.M., Westphal, H., Gonzalez, F.J., 1995. Targeted disruption of the alpha-isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell. Biol. 15, 3012 – 3022. Lehmann, J.M., Lenhard, J.M., Oliver, B.B., Ringold, G.M., Kliewer, S.A., 1997. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J. Biol. Chem. 272, 3406 – 3410. Lemberger, T., Braissant, O., Juge-Aubry, C., Keller, H., Saladin, R., Staels, B., Auwerx, J., Burger, A.G., Meier, C.A., Wahli, W., 1996. PPAR tissue distribution and interactions with other hormone signaling pathways. Ann. N.Y. Acad. Sci. 804, 231 – 251. Lemberger, T., Staels, B., Saladin, R., Desvergne, B., Auwerx, J., Wahli, W., 1994. Regulation of the peroxisome proliferator-activated receptor-a gene by glucocorticoids. J. Biol. Chem. 269, 24527 – 24530. Marsman, D.S., Cattley, R.C., Conway, J.G., Popp, J.A., 1988. Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643) in rats. Cancer Res. 48, 6739 – 6744. Mitchell, A.M., Bridges, J.W., Elcombe, C.R., 1984. Factors influencing peroxisome proliferation in cultured rat hepatocytes. Arch. Toxicol. 55, 239 –246. Motojima, K., Passilly, P., Jannin, B., Latruffe, N., 1996. Protein phosphorylation by peroxisome proliferators: Species-specific stimulation of protein kinases and its role in PP-induced transcriptional activation. Ann. N.Y. Acad. Sci. 804, 413 – 423. Mounho, B.J., Thrall, B.D., 1999. The extracellular signal-regulated kinase pathway contributes to mitogenic and antiapoptotic effects of peroxisome proliferators in vitro. Toxicol. Appl. Pharmacol. 159, 125 – 133. Orellana, A., Hidalgo, P.C., Morales, M.N., Mezzano, D., Bronfman, M., 1990. Palmitoyl-CoA and the acyl-CoA thioester of the carcinogenic peroxisome-proliferator ciprofibrate potentiate diacylglycerol-activated protein kinase C by decreasing the phosphatidylserine requirement of the enzyme. Eur. J. Biochem. 190, 57 –61.
Passilly, P., Schohn, H., Jannin, B., Malki, M.C., Boscoboinik, D., Dauca, M., Latruffe, N., 1999. Phosphorylation of peroxisome proliferator-activated receptor alpha in Fao cells and stimulation by ciprofibrate. Biochem. Pharmacol. 58, 1001 – 1008. Peters, J.M., Cattley, R.C., Gonzalez, F.J., 1997a. Role of PPARa in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14,643. Carcinogenesis 18, 2029 – 2033. Peters, J.M., Taubeneck, M.W., Keen, C.L., Gonzalez, F.J., 1997b. Di(2-ethylhexyl)phthalate induces a functional zinc deficiency during pregnancy and teratogenesis that is independent of peroxisome proliferator-activated receptor a. Teratology 56, 311 – 316. Peters, J.M., Rusyn, I., Rose, M.L., Gonzalez, F.J., Thurman, R.G., 2000. Peroxisome proliferator-activated receptor a is restricted to hepatic parenchymal cells, not Kupffer cells: implications for the mechanism of action of peroxisome proliferators in hepatocarcinogenesis. Carcinogenesis 21, 823 – 826. Rao, M.S., Reddy, J.K., 1996. Hepatocarcinogenesis of peroxisome proliferators. Ann. N.Y. Acad. Sci. 804, 573 – 587. Rokos, C.L., Ledwith, B.J., 1997. Peroxisome proliferator activate extracellular signal-regulated kinases in immortalized mouse liver cells. J. Biol. Chem. 272, 13452 – 13457. Rose, M.L., Rusyn, I., Bojes, H.K., Belyea, J., Cattley, R.C., Thurman, R.G., 2000. Role of Kupffer cells and oxidants in signaling peroxisome proliferator-induced hepatocyte proliferation. Mutat. Res. 448, 179 – 192. Shackleton, G.L., Gibson, G.G., Sharma, R.K., Howes, D., Orrenius, S., Kass, G.E.N., 1995. Diverse mechanisms of calcium mobilization by peroxisome proliferators in rat hepatocytes. Toxicol. Appl. Pharmacol. 130, 294 – 303. Steineger, H.H., Sorensen, H.N., Tugwood, J.D., Skrede, S., Spydevold, O., Gautvik, K.M., 1994. Dexamethasone and insulin demonstrate marked and opposite regulation of the steady-state mRNA level of the peroxisomal proliferatoractivated receptor (PPAR) in hepatic cells: hormonal modulation of fatty-acid-induced transcription. Eur. J. Biochem. 225, 967 – 974. Sterchele, P.F., Sun, H., Peterson, R.E., Vanden Heuvel, J.P., 1996. Regulation of peroxisome proliferator-activated receptor-a mRNA in rat liver. Arch. Biochem. Biophys. 326, 281 – 289. Tanaka, K., Smith, P.F., Stromberg, P.C., Eydelloth, R.S., Herold, E.G., Grossman, S.J., Frank, J.D., Hertzog, P.R., Soper, K.A., Keenan, K.P., 1992. Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague – Dawley rats treated with tumorigenic doses of clofibrate. Toxicol. Appl. Pharmacol. 116, 71 – 77. Torra, I.P., Gervois, P., Staels, B., 1999. Peroxisome proliferator-activated receptor alpha in metabolic disease, inflammation, atherosclerosis and aging. Curr. Opin. Lipidol. 10, 151 – 159. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grandperret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., Kirilovsky, J.,
N.-S. Yaacob et al. / Toxicology Letters 125 (2001) 133–141 1991. The bisindolylmaleimide GF-109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 15771 – 15781. Tugwood, J.D., Issemann, I., Anderson, R.G., Bundell, K.R., Mcpheat, W.L., Green, S., 1992. The mouse peroxisome proliferator activated receptor recognizes a response element in the 5% flanking sequence of the rat acyl CoA oxidase gene. EMBO J. 11, 433 –439. Vanden Heuvel, J.P., 1999. Peroxisome proliferator-activated
141
receptors (PPARS) and carcinogenesis. Toxicol. Sci. 47, 1 – 8. Wilkinson, S.E., Parker, P.J., Nixon, J.S., 1993. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem. J. 294, 335 – 337. Yaacob, N.S., Norazmi, M.N., Kass, G.E.N., Gibson, G.G., 1997. Use of competitive RT-PCR in the molecular analysis of peroxisome proliferation. Eur. J. Drug Metab. Pharmacokin. 22, 321 – 324.