Thyromimetic effect of peroxisome proliferators

Biochimie (1993) 75,257-261 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

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Thyromimetic effect of peroxisome proliferators R Hertz, B Kalderon, J B a r - T a n a * Department of Human Nutrition and Metabolism, Hebrew University, Hadassah Medical School, PO Box 1172, Jerusalem 91010, Israel (Received 1 December 1992; accepted 19 January 1993)

Summary - - Xenobiotic amphipathic carboxylates of varying hydrophobic backbones, known collectively as 'peroxisome proliferators' (PP), affect lipoprotein metabolism, calorigenesis, liver redox and phosphate potentials and adipose conversion. Some biological effects exerted by PP are strikingly similar to those exerted by thyroid hormones (TH). Furthermore, similarly to TH, these compounds have been recently found to induce in euthyroid as well as thyroidectomized rats or in rat hepatocytes cultured in TH-free media, liver activities classically considered as TH-dependent, eg malic enzyme (ME) and S 14. The thyromimetic effect of PP could be accounted for by transcriptional activation of TH-dependent genes as verified by run-on transcription assays. The thyromimetic effect of PP was found not to be mediated by the TH nuclear receptor. Moreover, in contrast to TH, PP were ineffective as thyromimetic agents in the rat heart or pituitary cells, suggesting a tissue specificity different from that of TH. The overall thyromimetic effect of PP appears to involve transcriptional activation of TH-dependent genes, yet being mediated by a novel transduction pathway.

peroxisome proliferators / thyroid hormones Peroxisome proliferators The number of liver peroxisomes is relatively small under normal conditions, but may be markedly increased (10--40-fold) by treatment of rodents and other species with hypolipidemic drugs of the fibrate type (clofibric acid, bezafibrate, fenofibrate, nafenopin and others) [I, 2], substituted long chain dicarboxylic acids (MEDICA compounds [3]), thia acids [4], perfluoro acids [5], phthalate and adipate plasticizers [6] as well as other xenobiotics collectively defined as 'peroxisome proliferators' (PP). The increase in peroxisomes induced by PP is accompanied by a specific and differential induction (20-100-fold) of specific peroxisomal proteins (eg CN-insensitive palmitoylCoA oxidase, enoyl-CoA hydratase, and others) while other peroxisomal proteins are less affected (eg catalase). The induction of peroxisomal activities by PP may be accounted for by transcriptional activation of respective peroxisomal genes [7]. Chronic treatment * Correspondence and reprints

of rodents with PP may result in hepatic transformation. Hepatic transformation by PP does not involve direct DNA damage similar to that observed with genotoxic carcinogenes. It may result from excess peroxisomal H20:, formation leading to DNA damage and tumor initiation, or alternatively, could reflect the capacity of PP to act as liver tumor promoters. Similarly to the in vivo effects of PP, peroxisorne proliferation may be induced in rat hepatocytes cultured with PP [8, 9]. The pattern observed in culture is morphologically and biochemically similar to that generated in vivo. In both systems, the induction of peroxisomal proteins is accounted for by an increase in the rate of transcription of the respective genes with a concomitant increase in respective mRNA species ll0, 11]. In spite of their apparent structural heterogeneity, PP may have a common structural denominator. Indeed, most PP appear to consist of a carboxylic function carried on a hydrophobic backbone to yield an amphipathic carboxylate [3, 9, 12]. The carboxylic function may either be present initially (MEDICA,

258 fibrates, WY-14,6431) or may be derived by metabolic oxidation of respective alcohols or aldehydes (eg tiadenol, phthalate plasticizers). The free carboxylic function or a derivative thereof may thus be directly involved in the inductive process, whereas the nature of the hydrophobic backbone may determine the respective efficacy of PP. Recently, a new member of the steroid/TH receptors superfamily has been cloned using consensus sequences (and some of their variations) for the DNA binding domains of the h-glucocorticoid, estrogen, vitD3, retinoic acid and TH receptors [13]. The cloned receptor (PPAR) was found to mediate PP-induced chloramphenicol transacetylase (CAT) expression in COS-I cells transfected with an expression vector for a chimeric construct consisting of the ligand binding domain of PPAR linked to the DNA binding domain of either the glucocorticoid or the estrogen receptors, together with a CAT reporter plasmid linked to DNA sequences coding for the glucocorticoid (GRE) or the estrogen (ERE) response elements, respectively. This experimental system was further exploited to screen the efficiency of various PP as transcriptional activators of peroxisomal acyI-CoA oxidase [13]. The observed efficiency was correlated with the capacity of PP as inducers of peroxisome proliferation in rodents. Moreover, the PPAR cDNA was observed to hybridize with two major mRNA species of about 2.0 kb detectable in liver, kidney and heart, but not in brain or testis, in line with the tissue specificity of peroxisome proliferation induced by PP. However, PP did not bind to the expressed PPAR, possibly because of their apparent low affinity tbr PPAR or because of their possible indirect action in inducing peroxisomal activities mediated by PPAR. The DNA binding domain of PPAR contains a sequence-specific DNA binding site (Cys(ll9)-GiuGly-Cys-Lys-Gly) expected to recognize the DNA motif TGACC. Indeed, two cis-acting regulatory sequences have been recently characterized by Osumi et al [141 in the peroxisome proliferator responsive enhancer of rat peroxisomal acyl-CoA oxidase. One sequence (-578/-520) was tbund to exert a positive regulation of transcription, whereas the other one (--472/-129) exerted a negative action, The positive enhancer consists of a TGACCTTTGTCC sequence (-570/-559) and may putatively be considered as a peroxisome proliferator response element (PPRE) mediating transcriptional activation of respective peroxisomal genes by PPAR and PP [15].

Thyroid hormones and thyroid hormone dependent genes The pleiotropic actions of thyroid hormones (TH) consist of modulating differentiation and develop-

ment, calorie metabolism, lipoprotein metabolism, cardiac and skeletal muscle functions and others (reviewed in [16-19]). The pleiotropic actions of TH may be partly accounted for by tissue-specific transcriptional activation/inhibition of TH dependent genes (reviewed in [20]), such as those coding for pituitary growth hormone (GH) and tx-/~-TSH, liver malic enzyme (ME), mitochondrial glycerol-3-phosphate dehydrogenase, S 14, fatty acid synthetase, HMG-CoA reductase, glucokinase, PFK-2 and LDL-receptor, cardiac o~-/~l-myosin heavy chain kinase (MHC) and CaATPase, Na-K ATPase (liver, kidney, muscle), and others. In addition to transcriptional regulation of THdependent genes, TH may affect mRNAs stability (eg GH, ME, S14, apolipoprotein AI), mRNA editing (eg apolipoprotein B 100/48) or degradation of respective proteins (eg tadpole liver carbamyl phosphate synthetase, brain tubulin). It is unclear whether post-transcriptional effects of TH on specific gene expression are secondarily mediated by factors which are transcriptionally regulated by TH. The 'nuclear pathway' of TH action is initiated by TH binding to TH nuclear receptors (reviewed in [20, 21]). Transcriptional activation results from interaction of the TH nuclear receptors with DNA sequences that act as thyroid hormone response elements (TRE) of TH-dependent genes. Using v-erbA sequences as probes, a number of highly related c-erbA clones were identified from avian, mammalian and Xenopus cDNA libraries which encode proteins with properties of TH receptors, namely, high affinity TH binding, binding to TREs of TH-dependent genes as well as conferring TH-dependent regulation of transcription in TH receptor-negative cells. The ic-erbA gene products were found to be highly related to other members of the steroid/TH super-family of nuclear receptors [22], all being characterized by a highly conserved cysteine-rich Zn-finger domain that functions in sequence-specific DNA binding together with a ligand binding domain which binds the respective specific ligands. Two mammalian c-erbA genes have been identified. The tx-form is localized (in humans) on chromosome 17 and gives rise through alternative splicing to the 0q, o~2 and other (rev erb/ear 7) TH receptor subtypes [23]. The ~l-fonn is localized in humans on chromosome 3 and gives rise to the ~-TH receptor subtypes [241. The o~2 variant is not capable of TH binding but still may act as a modulator of TH receptors function. Transcriptional activation by TH receptors is mediated by their binding to respective TREs having specific sequence determinants. A core binding consensus motif based on comparing TRE sequences of various TH dependent genes (rGH, rTSH, rotMHC, MoMLV, rME) appears to consist of two half site (A/G)GG(T/A)(C/G)(A/G) sequences flanked by a

259 cluster of G residues (ME) [21, 25]. A somewhat different sequence ((G/A)GGGCCTG) has been recently suggested based on defining the S14 TREs [26]. The core binding motif usually is a direct tandem repeat or an inverse repeat or a palindrome, suggesting dimerization of receptors upon their binding to respective TREs. Dimerization is believed to be governed by amino acids constituting the D box of the 2nd Znfinger [27] as well as dimerization motifs within the ligand binding domain [28]. Moreover, the interaction of TH receptors with their TREs may be modulated by TH receptor-auxiliary proteins (TRAP) [29], other receptors related to the superfamily (eg RXR, c-erbAtt2) [30] or other transcriptional factors (eg AP-1).

Thyromimetic effect of PP The biological activity of PP is not limited to induction of peroxisomal activities. Activities induced by PP appear to be very similar to those induced by TH. a) TH [18] and PP [31, 32] are potent hypolipidemic agents. Their hypo-lipidemic effect is due to inhibition of liver VLDL production together with activation of plasma lipoproteins clearance [33, 34]. b) TH [16. 35] and PP [36-38] act as potent effectors of adipose mass. Lipolysis is initiated by a left shift in the response curve to lipolytic hormones with a concomitant decrease in fatty acid re-esterification. The enhanced iipolytic response is accompanied by an increase in mitochondrial liver uptake of free fatty acids (due to a decrease in mitochondrial malonyl-CoA [39, 40]), with a concomitant increase in mitochondrial oxidation of long chain fatty acids and ketogenesis [ 18, 41 ]. c) The calorigenic-hypolipidemic effects of TH and PP may be partially reduced to basic principles related to liver redox and phosphate potentials [42, 43]. Treatment of rats with either TH or MEDICA resulted in decreases of liver cytosolic redox potential (2.0- and 3.5-fold, respectively) concomitant with an increase in the liver capacity of handling an ethanol or xylitol load. The apparent liver cytosolic phosphate potential was found to remain unaffected by TH or MEDICA while the apparent mitochondrial phosphate potential was substantially decreased by both treatments. d) The similarity of effects exerted by PP and TH on hypolipidemia, calorigenesis and liver redox/phosphate potentials may indicate that the thyromimetic effect of PP reflects their capacity to act as transcriptional activators in general and of TH dependent genes in particular. Several PP have been recently found by Hertz et al [ 11] to induce in euthyroid, thyroidectomized rats or in rats made hypothyroid by methimazole liver activi-

ties classically considered as TH-dependent, eg malic enzyme (ME) mitochondrial glycerol-3-phosphate dehydrogenase, glucose 6-phosphate dehydrogenase and S14. The maximal inductive potential of PP with respect to the above activities was similar to that of TH. However, the doses required for inducing the thyromimetic response in vivo were in the mg/kg-range for PP and the ~tg/kg-range for TH. e) The thyromimetic effect of PP with respect to ME, S14 and mitochondrial glycerol-3-phosphate dehydrogenase was similarly observed in rat hepatocytes cultured in TH-free media and in the presence of PP added to the culture medium [11]. The concentrations of PP required in culture were in 104-105 fold higher (l.tM range) than those of TH (pM range). However, the maximal activities of ME and S14 induced by PP were similar to or higher than those induced by TH. f) As for TH, the thyromimetic inductive effects of PP with respect to liver malic enzyme and S14 were found to result from increases in the corresponding mRNA contents, and the increases in liver ME and S14 mRNAs induced by PP were accounted for by transcriptional activation of the S14 genes as verified by run-on transcription assays [ 11]. g) The thyromimetic effect of PP is not accounted for by modulating plasma TH levels. No significant differences in plasma T4, T3, TSH and T3-uptake were noted in PP-treated euthyroid rats [43]. Furthermore, the thyromimetic effect of PP was observed in thyroidectomized rats, in methimazole-induced hypothyroid rats and in rat hepatocytes cultured in TH-free media. h) In contrast to TH, the thyromimetic effect of PP is not mediated by the TH nuclear receptor. TH binding to isolated liver nuclei or to liver nuclear extract was competitively displaced by some, but not all, PP capable of inducing TH-dependent liver activities [11]. The thyromimetic effect o1: PP is mediated by a transduction pathway different from that involved in the thyromimetic effect induced by TH. i) The tissue specificity of the thyromimetic effect of PP is different from that of TH. No increase in growth honnone (GH) mRNA was observed in cultured GHI pituitary cells incubated in the presence of PP under conditions where TH was found to induce GH mRNA [11]. Heart high energy intermediates were observed to be dramatically affected by TH but not by MEDICA treatment, in contrast to liver where both TH and MEDICA were found to affect cellular high energy adenine nucleotides and phosphate content [43]. Similarly, mitochondrial glycerol-3phosphate dehydrogenase and ME could be induced by both treatment modes in liver, whereas the two cardiac activities (including ME mRNA) 'were induced by TH only while MEDICA was without effect. This

260 tissue selectivity of PP could reflect selectivity in transport, or tissue specific metabolic conversion into an active immediate effector (eg CoA thioester) or a transduction pathway other than that mediating the effect exerted by TH. Conclusions

The scope of the effects induced by xenobiotic amphipathic carboxylates collectively defined as 'peroxisomal proliferators' may indicate that these compounds should be realized as eminent biological effectors capable of modulating transcriptional activation of genes other than those related to peroxisomes. Some of the major therapeutic/toxic effects induced by PP should be considered within the framework of their thyromimetic activity. Searching for a transduction pathway mediating the thyromimetic activity of PP may result in novel mechanisms involved in their pleiotropic action. References

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