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Mol. Cells, Vol. 2, pp. 39-42 Induction of y-Gultamyl Transpeptidase by a Thyroid Hormone in Primary Cultures of Rat Hepatocytes and in Rat liver Kang Yeol Yu, Byoung Yul Soh, Sang-Bae Hant, Jin-Ho Kim, Mun-Kuk Park2, Gook Hyun Chung and Tai-Boong Ubm 1* Department of Biology, 'Department of Food Science and Technology, and 2Department of Biological Education, Chonbuk National Univen"ty, Chonju 560-756, Korea (Received on February 1, 1992) We have studied the induction of gamma-g1utamyl trans peptidase (GGT) by a thyroid hormone, triiodothyronine ([3) in primary cultures of rat hepatocytes. When added to cultures for 5-6 days, T3 increased GGT activity to levels 2.5- to 3-times those of control cultures. In rat liver, T3 also causes a 1.8-fold increase in GGT activity when hyperthyroidism in rats was induced. Considering highly related structures between the nuclear receptors for retinoic acid, thyroid and steroid hormones, induction mechanism of GGT by T3 may be similar to those by retinoic acid and steroid hormones, which can also induce GGT activity. Gamma-g1utamyl transpeptidase (GGT) «5-g1utamyl)-peptide:amino acid 5-g1utamyl transferase, EC 2.3.2.2), a plasma membrane associated enzyme, is a key enzyme in the regulation of the level of glutathione via the gamma-glutamyl cycle. It catalyzes transfer of the gamma-glutamyl moiety of glutathione to a variety of acceptors such as amino acids and dipeptides (transpeptidation) and to water (hydrolysis). The highest GGT activity is found in the kidney, but GGT of liver and liver-derived cells has attracted considerable attention, because in human liver GGT activities increase highly during chronic alcoholism (Rosalki and Rau, 1972) or in patients with hepatocellular carcinoma (Gerber and Thung, 1980). Also, in primary culture of rat hepatocytes, the GGT activity is influenced by treatment with various xenobiotics such as dexamethasone (Cotariu et al., 1988) and ste- their receptors, in order to meet cellular needs (Umesono et al., 1988). If this hypothesis is correct, thyroid hormone also may act as a positive regulator for the transcription of GGT gene, because the structure of the thyroid hormone receptor is closely related to those of the retinoic acid and steroid hormone receptors. We now report that thyroid hormone induces the activity of GGT in primary culture of rat hepatocytes and in vivo experiments and propose a possible mechanism of GGT induction in the presence of thyroid hormone. Materials and Methods Animals and materials Male Sprague-Dawley rats weighing 150-200 g were roid hormones (Billon et aI., 1980) and retinoic acid used as sources of liver cells in all experiments. Triio- ([sao and Batist, 1988). It has been known that steroid and thyroid hormones and retinoic acids coordinate complex events involved in development, differentiation, and physiological response to diverse stimuli. Furthermore, recent studies of steroid receptors have led to the identification of a superfamily of ligand-inducible regulatory proteins including receptors for thyroid hormones and retinoic acid (Ham and Parker, 1989). These receptors are homologous to those for the steroid hormones and these appear to function by similar mechanisms. This family of receptors regulates the transcription of certain genes by binding as a hormone-receptor complex to specific DNA sequences termed response elements (RE). Thus, retinoic acid and steroid hormones may also induce GGT gene expression by binding to a common RE as complexes with dothyronine ([3) and dexamethasone were purchased from Sigma Chemical Co. (St. Louis, MO). * To whom correspondence should be addressed Hepatocyte isolation and culture Hepatocytes were isolated by a collagenase perfusion procedure (Whiting and Edwards, 1979). Cultures were established by adding about 5 X 106 freshly isolated hepatocytes to collagen-coated Falcon 3001 tissue culture plates in Williams' medium E (Williams et aI., 1977) with 5% fetal bovine serum. Cells were incubated at 37 °C in a humidified 5% COJ 95% air incubator, and allowed for attachment for 12 h. Hepatocyte integrity and viability were routinely checked by microscopical examination and by lactate dehydrogenase release in the medium as compared to the initial dispersion. The time of seeding was designated The abbreviations used are: GGT, gamma-giutamyl transpeptidase; RE, reponse element; T), 3,5,3-triiodo-L-thyronine. © 1992 The Korean Society of Molecular Biology 40 Mol. Cells Thyroid Honnone Induction of y-Glutamyl Transpeptidase time O. After 24 h the medium was replaced with fresh medium containing 10 nM T3 or 1 ~ dexamethasone. Cultures were maintained for 5 days with daily medium exchanges. Treatment of animals for in vivo induction Chronic hyperthyroidism was induced by a slight modification of the method of Narayan et al. (1984). It was induced by intraperitoneal injection of 35 /lg of T3 per 100 g of body weight per day for 6 days. Groups of five animals were fed standard chow diet (Miwon). Six h after the final injection rats were sacrifised under CO 2 anesthesia, and the livers were removed and weighed. Pieces (2 g) of the left median lobes of the liver were then homogenized in 4 rnl of 0.1 M Tris-HCl buffer (PH 7.6 at 4 "c) containing 0.25 M sucrose and 10 mM MgCb. Following centrifugation at 1,000 X g for removal of cell debris, a membrane preparation was made by centrifuging the supernatant fraction at 105,000 X g for 1 h. The pellet was resuspended in buffer containing 0.1 M Tris-HCl and 10 mM MgCb. Assays of GGT and protein At the indicated times plates were rinsed twice with 0.15 M NaCl, and the attached monolayers were scraped off, resuspended in saline and sedimented by centrifugation for 3 min at 100 X g. Cells were homogenized in 50 mM Tris-HCl/250 mM sucrose buffer (pH 7.4) in a glass-glass homogenizer. GGT was assayed in homogenates at 37 "c by the method of Tate and Meister (1974) using L-gamma glutamyl-p-nitroanilide and glycylglycine as substrates. One unit of enzyme activity is that yielding I nmole product per minute at 37 °C. Protein was measured in cell homogenates by the method of Lowry (1951). Statistical analysis for the significance of differences between groups was done by means of Student's t-test. Differences were considered statistically significant when P< 0.05. Results Effect of T3 on hepatocyte GGT Figure 1 shows changes in GGT activity with time in culture when hepatocytes were maintained in defined Williams' medium. Under control culture the activity of GGT increased slowly during the first 5 days. Addition of T3 (final concentration of 10 nM) to mono1ayers increassed activity significantly after 3 days (P<O.OOl) and after 5-6 days with T3, GGT activities were about 2.5-3.0 times those in control cultures. Mode of GGT induction by dexamethasone (1 ~), a well-known GGT inducer, was found to exhibit time-dependence similar to that for induction by T3 (Fig. 1). Microscopic observations showed that control and T 3-treated cells were same in their morphological properties during cultures (data not shown). Also, we did not detect any releae of lactate dehydrogenase in the culture medium from either control or T 3-treated cells 600 .§ 500 e p., bll § 400 E ~ :~ ~ ~ 8 300 200 100 OL---~----~----~----~--~ 1 2 3 5 Time, days Figure 1. Induction of GGT activity by T3 ce) and dexamethasone CO). T3 (10 nM) and dexamethasone (1 f.LM) were added to the medium after 24 h in culture; controls ()). Monolayers were incubated for the periods shown in the figure after which hepatocytes were harvested for GGT and protein assays. The means and standard errors of 5 or 6 plates are shown. (data not shown), indicating that no significant cell damage occurred at the concentration of 10 nM T3. The time-dependent increase in GGT in the presence of T3 appeared to represent enzyme induction since the increase was completely prevented by addition of the protein synthesis inhibitor cycloheximide (100 ~) to 3-day cultures for a 24-h test period and reduced about 50% by adding the RNA synthesis inhibitor, actinomycin D (0.5 ~) for a similar test period. Moreover, incubation of hepatocyte cells in the presence of cycloheximide (100 ~) or actinomycin D (0.5 ~) together with 10 nM T3 for 3 days prevented the hormonal effect. Induction of GGT in hepatocyte primary cultures also depended on the concentration of T 3. As shown in Figure 2, lower concentrations of T3 up to 10 nM caused an increase in GGT activity, which was maximal at 10-50 nM T 3. At the higher T3 concentrations, above 200 nM, the activity rather declined. In vivo GGT induction by T3 Hepatic membrane GGT activity in T 3-treated rats was significantly higher than activity in the controls after 5-day exposure (P<O.Ol), although the inducing effect was small compared to that in primary hepatocyte cell culture (Table 1). Discussion In the present report, T3 as well as dexamethasone specifically promoted an induction of GGT with time- Kang Yeol Yu et al. Vol. 2 (1992) 41 ::::::::::::;::::~i~i;?r: 500 = e Plasma Membrane Nucleus Membrane T .~ ~T)-specific Receptor ~ eo \ E ::l --E 6 .;; 'p u co 20 IThyroid I Promoter Hormone RE f- 0 0 T)-Rec e ptor Complex as a Transcriptional Factor/ GGT gene I Transcription 100 0 0 GGT mRNA 10 20 30 40 50 200 T3, nM Figure 2. The dependence of induction of GGT activity on the concentration of T3 in the culture medium. Primary cell cultures were uniformaly exposed to the various concentration of T3 for 4 days prior to measuring their GGT specific activities. Values represent the means and standard errors of 5 or 6 plates. Table 1. Induction of GGT activity in rat liver after treatment of T3 for 5 days· Treatment GGT activity (mU/mg proteint 84± 19 157 ± 27' See the text for detailed procedure. Means ± S.D. from five animals. ' Significantly greater than control (P<O.OI). a h and dose-dependent mode in cultured rat hepatocytes. Also, induction of GGT in the liver in vivo was caused by the treatment of T 3. A structurally diverse group of xenobiotics and steroids have been shown to elevate GGT activity in hepatocytes in primary culture. Also, retinoic acid increased the level of the GGT mRNA in transformed cell lines. This effect apparently involves time-dependent induction of the enzyme, requiring continuing RNA and protein synthesis. These findings suggest that GGT induction by T3 may also be modulated via a similar mechanism. Consistent with this view, thyroid hormone such as T 3, retinoic acid, and steroid hormones and their derivatives which induce GGT have been reported to be inducers for growth and differentiation of the cell. The metabolic turnover of glutathione is closely geared to the activity of GGT. Also, GGT functions in cellular transport of amino acids, detoxication and cellular + GGT Figure 3. A hypothetical model for GGT induction by TJ • Binding of T3 to a T3-specific nuclear receptor induces an activated T3-nuclear receptor complex. This leads to the binding to the thyroid hormone response element, which can also act as a common response element both for the binding of retinoic acid- and steroid hormone-nuclear receptor complexes. neutralization by ammonia production (Tate, 1980). Therefore, the induction of GGT activity may represent a necessary physiological adaptation in the cell differentiation, growth, and the increase of metabolic rates by upregulating the synthesis of cellular glutathione. Recently, it was found that the family of steroid hormone receptors also includes receptors for thyroid hormone, a number of vitamines and several other ligands yet to be identified (Evans, 1988). Because of the highly related structures among the above receptors, a human retinoic acid receptor expressed from cloned complementary DNA or the endogenous retinoic acid receptor present in teratocarcinoma cells could activate gene expression from promoters fused to a natural or synthetic thyroid hormone respone element (Umesono et aI., 1988). The implication of these findings is that the thyroid and steroid hormones (or their derivatives) and retinoic acid, acting through their respective receptors, could control overlapping gene networks involved in the regulation of vertebrate morphogenesis and homeostasis. We here propose a posssible mechanism of GGT induction by T3 as follows, based on our results and others. T 3, like steroid hormones, diffuses freely across the plasma membrane and binds specifically with high affinity to receptors in the nucleus of liver. Next, the Trnuclear receptor complex binds to a thyroid hormone RE with high affinity. Or the Trreceptor complex may bind to a steroid and/or retinoic acid RE to activate GGT expression as a common respo- 42 Thyroid Hormone Induction of y-Glutamyl Transpeptidase nse element. This binding can then stimulate promoter activity of GGT gene (Fig. 3). Major concern in this hypothesis is that a common response element for binding of T r , steroid hormone-, and retinoic acid-receptor complexes may exist near the promoter site of the GGT gene. This should be solved by analyzing the regulation sites of GGT gene. However, other mechanisms can be proposed as well. One of these could be the induction of a factor which in tum directly activates the target enzyme. That is, a Tr induced intermediate affects the enzyme conformation without changing the mRNA level. However, cycloheximide and actinomycin D prevented the induction, suggesting that protein and mRNA synthesis were necessary for this process. Specific mRNA assays will be necessary for further studies. Acknowledgment This work was supported by a Genetic Engineering Research grant to T. B. Uhm from Ministry of Education in 1990. References Billon, M. c., Dupre, G., and Hanoune, J. (1980) Mol. Cell. Endocr. 18, 99-108 Mol. Cells Cotariu, D., Barr-Nea, L., Papo, N., and Zaidman, J. L. (1988) Enzyme 40, 212-216 Evans, R. M. (1988) Science 240, 889-895 Gerber, M. A., and Thung, S. N. (1980) Am. J Pathol. 98, 395-400 Ham, J., and Parker, M. G. (1989) in Current Opinion in Cell Biology (Laskey, R. A., and Gerace, L., eds) Vol. 1, pp. 503-511 , Current Science, London Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J Bioi. Chem. 193, 265-275 Narayan, P., Liew, C. W., Towle, H. C. (1984) Proc. Nat!. Acad. Sci. USA 81, 4687-4691 Rosalki, S. B., and Rau, D. (1972) Clin. Chim. Acta. 39, 41-47 Tate, S. S., and Meister, A. (1975) J Bioi. Chem. 250, 4619-4627 Tate, S. S. (1980) in Enzymatic Basis of Detoxication (Jakoby, W.B., ed) Vol. 2, pp 95-120, Academic Press, New York Tsao, M., and Batist, G. (1988) Biochem. Biophys. Res. Commun. 157, 1039-1045 Umesono, K , Giguere, V., Glass, C. K, Rosenfeld, M. G., and Evans, R. M. (1988) Nature 336, 262-264 Whiting, M. J., and Edwards, A. M. (1979) J Lipid Res. 20, 914-918 Williams, G . M., Bermudez, E., and Scaranuzzino, D. (1977) In Vitro 13, 809-817