Transcriptional regulation of hexokinase I mRNA levels by TSH in cultured rat thyroid FRTL5 cells

Life Sciences, Vol. Printed in the USA

51, pp.

1613-1619

Pergamon

Press

TRANSCRIPTIONAL REGULATION OF HEXOKINASE I mRNA LEVELS BY TSH IN CULTURED RAT THYROID FRTL5 CELLS. Norihiko Yokomori, Masato Tawata, Yoshiyuki Hosaka and Toshimasa Onaya The Third Department of Internal Medicine, University of Yamanashi Medical School, Tamaho 409-38, Japan (Received in final form September 14, 1992)

Summary

We investigated the effect of thyroid stimulating hormone (TSH) on the expression of hexokinase I mRNA by cultured rat thyroid FRTL5 cells. TSH stimulated hexokinase I gene expression in a time- and dose-dependent manner. An increase in hexokinase I mRNA was detected after 3 h of incubation with TSH, and a maximum was reached after 12 h showing about 2.5-fold increase at lmU/ml TSH. A nuclear run-on transcriptional assay showed that the effect of TSH on hexokinase I mRNA was due to an increase in the rate of gene transcription. (Bu)2cAMP and forskolin also increased hexokinase I mRNA expression to almost the same extent as TSH. These findings suggest that TSH stimulates hexokinase I gene expression at the transcriptional level via the cAMP-dependent pathway.

Hexokinase [EC 2.7.1.1] catalyzes the first step of glucose metabolism, the ATP-dependent phosphorylation of glucose to generate glucose 6-phosphate. In mammalian tissues, four isozymes (I-IV) of hexokinase exist, and the regulation of isozyme IV (glucokinase, [EC 2.7.1.2]) has been studied extensively (1-3). Previous in vitro and in vivo studies have shown that its regulation was under multihormonal control. Glucokinase is found only in the liver and pancreas, so glucose metabolism in other tissues appears to be dependent on other hexokinase isozymes. Hexokinase I is the only form that occurs in significant quantities in the other body tissues so far examined (4). And in normal thyroid tissue, hexokinase I is also reported to be the major form (about 90%), while thyroid carcinomas have more hexokinase II and less hexokinase I compared with normal thyroid tissue (5). Recently, the cDNA of rat brain hexokinase I was cloned (6,7), making it possible to study the regulation of hexokinase I gene expression. However, the regulation of hexokinase I gene expression has not been studied well. Therefore, it seemed to be of interest to investigate the regulatory effects of TSH on hexokinase at the gene level in the thyroid tissue. In this study, we examined the effect of TSH on hexokinase I mRNA in cultured rat thyroid FRTL5 cells. Methods Cell cultures: FRTL5 rat thyroid cells were grown at 37°C in a humidified incubator (5% CO2, 95% air), in 100-mm plastic culture dishes containing Ham's 12K medium supplemented with 5% calf serum (Gibco, Grand Island, NY) and a six-hormone mixture (6H medium) including 10 mU/ml TSH (Sigma Chemical Co., St. Louis, MO) (8). When the cells reached subconfluency, they were allowed to become quiescent by changing the medium lacking TSH (5H medium). After 4 days of quiescence, TSH was added again to the dishes without changing the medium. All correspondence should be sent to Masato Tawata, M.D. Copyright

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Northern blot analysis: The total RNA was extracted from FRTL5 cells with guanidine and isothiocyanate using the standard method (9), and poly(A) mRNA was selected on oligo(dT)cellulose as described (10). Ten microgram amounts of the poly(A)-containing RNA were denatured with glyoxal, subjected to 1.4% agarose gel electrophoresis, and transferred to nitrocellulose filters. Filters were prehybridized, and then hybridized with riP-labeled oligonucleotides at 37 ° C in a solution containing 6 x SSPE (20 x SSPE is 3 M NaC1, 0.2 M sodium phosphate, and 25 mM EDTA, pH 7.4), 1 x Denhardt's solution, 1% SDS, and 0.1 mg/ml heatdenatured salmon sperm DNA. The filters were washed three times in 6 x SSC (20 x SSC is 3 M NaC1 and 0.3 M sodium citrate) at 37 ° C for 15 min and then 6 x SSC at 47 ° C for 10 min. Air-dried filters were exposed to Kodak XAR-5 films at -80 ° C using a fluorescence-intensifying screen. Quantitation of mRNA: m R N A was quantitated by reverse transcription-polymerase chain reaction (RT-PCR) method. Total RNA was reverse transcribed into cDNA as described previously (11, 12). Total cellular RNA (5 ~g) was heated at 70°C for 10 min, cooled slowly to 42°C, and then reverse transcribed at 42°C for 90 min in a 20 lal reaction mixture containing 50 mM Tris-HC1 (pH 8.3), 100 mM KC1, 10 mM MgCh, 10 mM dithiothreitol (D'VF), 0.5 mM dNTPs, 0.5 mg oligo(dT), 300 units of RNasin (Takara Shuzo Co., Kyoto, Japan), and 11 units of Rous-associated virus 2 (RAV-2) reverse transcriptase (Takara). Ten microliters of 1:10 diluted cDNA was used as a template for amplification. The polymerase chain reaction (PCR) was performed in a total volume of 50 I.tl containing PCR buffer (50 mM Tris-HCl (pH 8.3), 2 mM MgC12, 30 mM KC1, and 10 mM DTT, 0.25 mM dNTPs, 0.1 mM each 5' and 3' primers, riP-end-labeled 5' primers (1 x 106cpm), and 2.5 units of Thermus aquaticus DNA polymerase (Taq polymerase) (Perkin-Elmer/Cetus). Amplification involved 25 cycles of denaturation at 93°C for 60 sec, primer annealing at 55°C for 120 sec, and extension at 72°C for 120 sec. Ten microliters of each PCR reaction mixture was subjected to electrophoresis on 2% agarose gels in Tris acetate/EDTA buffer containing ethidium bromide. Appropriate bands were cut out from the gels and the radioactivity was determined by scintillation counting. The results were expressed as a ratio between hexokinase I mRNA and actin mRNA. Oligonucleotides used for amplification and hybridization: Oligonucleotides were chemically synthesized using a DNA synthesizer (DNA Synthesizer, model 381 A, Applied Biosystems, Foster). The 5' hexokinase primer (5'-ACTGAACACGGTGAC'Iq'CCT-3') was identical to nucleotides 1656-1675 of rat brain hexokinase I cDNA, and the 3' primer (5'-CATGTTGATGCACATCTGGC3') was complementary to nucleotides 2191-2210 (6, 7). As an internal standard, we also synthesized [~-actin primers. The 5' ~-actin primer (5'-TGT'ITGAGACCTTCAACACC-3') was identical to nucleotides 368-387 of rat [~-actin cDNA, and the 3' primer (5'-CGCTCATTGCCCATAGTGAT-3') was complementary to nucleotides 739-758 (13). These 3' primers were used for hybridization. Nuclear run-on transcription assay: An in vitro nuclear run-on transcription assay was performed by the method of Greenberg and Ziff (14). Briefly, FRTL5 cells cultured with or without TSH were washed three times with ice-cold phosphate-buffered saline (PBS), scraped in PBS and centrifuged at 500g for 5 rain. The pellet was resuspended in NP40 lysis buffer (10 mM Tris-HC1 (pH 7.4), 10 mM NaC1, 3 mM MgCh, 0.5% (v/v) NP40), incubated for 5 rain on ice and centrifuged at 500g for 5 min. The nuclear pellet was wahsed again with NP40 lysis buffer and centrifuged again, then resuspended in 100 mM HEPES (pH 8.3), 40% (v/v) glycerol, 5 mM MgCh, 0.1 mM EDTA and stored in liquid N2. The nuclei were incubated at 26°C for 45 min in a 0.1 ml reaction mixture containing 16% glycerol, 20 mM HEPES (pH 8), 0.04 mM EDTA, 0.5 mM MnC12. 90 mM NH4CI, 5 mM M g C h , 2 mM DTT, 0.4 mM each of ATP, CTP and GTP, and 0.1 mCi of [ct-32P]UTP. Radiolabeled RNA was then purified and hybridized to 2 ~tg of DNA immobilized on nitrocellulose filters. After hybridization, the filters were washed and treated with RNase A and RNase T1. Airdried filters were then exposed to Kodak XAR-5 film. For immobilizing DNA on the filters, we used 2 ktg each of the partial hexokinase I and the [~-actin cDNAs amplified by the PCR as described above. Data are normalized to the densitometric reading for a nontranscriptionally regulated gene on the same nitrocellulose strip (~-actin) (15).

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Results and Discussion Initially, to determine the amounts of the hexokinase I and ~-actin mRNAs, we performed Northern blot analysis using the poly(A)-containing RNA obtained from FRTL5 cells incubated with or without TSH (1 mU/ml) for 12 h. This analysis showed that the hexokinase I probe identified a single band of RNA with a size of about 3.8-kb, which was present in an increased amount in the TSH-treated cells (Fig. 1, lane a vs. lane b). The size of hexokinase I mRNA in FRTL5 was slightly smaller than the one reported by Schwab and Wilson (7). The 13-actin probe also identified a single band of RNA, which was present in equal quantities in control and the TSHtreated cells (lane c vs. lane d). Next, we determined the amounts and sizes of the PCR products of reverse-transcribed mRNA of hexokinase I and I~-actin mRNA. As shown in Fig. 2 A, the product of hexokinase I mRNA was a single band and migrated to the predicted size of 555 base pairs (bp). The product of 13-actin mRNA was also a single band and migrated to the predicted size of 391 bp. We sequenced both PCR products, and the results coincided with those reported for hexokinase I cDNA (6, 7) and 13-actin cDNA (13). The time course of the accumulation of hexokinase I and 13-actin mRNA after exposure to TSH (1 mU/ml) was examined. As shown in Fig. 2 A, B, hexokinase I mRNA increased in a time-dependent manner to reach a maximum after 12 h showing a 2.5-fold increase over the basal value, and thereafter, decreased gradually. The 13-actin mRNA level was not changed at any time, as reported previously (16, 17), so we used 13-actin mRNA as the control in this present experiment. We also examined the effect of varying the concentration of TSH (0-10 mU/ml), using RT-PCR analysis of RNA obtained from cells at 12 h after stimulation. As shown in Fig. 3, TSH increased the expression of hexokinase I mRNA in a dose-dependent manner up to 10 mU/ml. The mechanism of hexokinase I mRNA accumulation in TSH-treated FRTL5 cells was further examined using an in vitro nuclear run-on transcription assay. When nuclei were prepared from FRTL5 cells incubated with TSH (1 mU/ml) for various periods of time, hexokinase I gene transcription was found to increase in a time-dependent manner and to reach a maximum at 12 h after the addition of TSH (Fig. 4). To investigate the role of mRNA stability in the hexokinase I mRNA level, RNA synthesis was blocked by adding actinomycin D (5 I.tg/ml) to the culture medium at 12 h after incubation with and without TSH (1 mU/ml), and the relative rates of mRNA disappearance were compared. The halflife of hexokinase I mRNA was calculated to be about 6 h, either in the presence or in the absence of TSH. So, TSH did not increase the stability of hexokinase I mRNA (data not shown). The majority of the effects of TSH on thyroid cells are mediated by a cAMP-dependent pathway (18). To examine whether the effect of TSH on hexokinase I gene expression was also cAMPdependent, we examined the effect of (Bu)2cAMP (1 mM) and forskolin (50 I.tM), two agents which increase the intracellular cAMP level or mimic the actions of cAMP in various cell types. These agents stimulated the expression of hexokinase I mRNA after 12 h of incubation to almost the same extent as TSH itself, showing a 2.2-fold increase by (Bu)2cAMP and a 2.6-fold increase by forskolin (Fig. 5). Although the structure of the 5'-flanking region of the hexokinase I gene is not known at present, these findings suggest the existence of cAMp-responsive elements. The present study showed that TSH stimulates hexokinase I gene expression at the transcriptional level and does not influence mRNA stability. However it is also important to determine whether TSH stimulates the amount of hexokinase at the protein level and increases its activity. Such experiments are now underway.

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Fig. 1 Northern blot analysis. Poly(A)-containing RNA was extracted from FRTL5 cells incubated with (lanes b and d) or without (lanes a and c) TSH (1 mU/ml) for 12 h. Tenmicrogram amounts o f the poly(A)-containing RNA were hybridized with a riP-labeled hexokinase I probe (lanes a and b) and a riP-labeled [3-actin probe (lanes c and d), as in the methods. The positions of 28S and 18S ribosomal RNA are shown at the left. The size of hexokinase I mRNA was calculated to be about 3.8-kb.

A

B 3.

.<

=;55 bp---~ 391 bp --~ 0

3

6

12

18

24

36

T i m e after T S H (h) 0

0

3

6

12

18

24

36

Time after T S H (h)

Fig.2. Time course of the accumulation of hexokinase I mRNA in response to TSH. Total RNA was extracted from FRTL5 cells treated with 1 mU/ml TSH for various periods as indicated. Five micrograms of total RNA was used for the RT-PCR analysis, as described in Methods. Panel A shows the ethidium bromide-stained PCR products and Panel B shows the relative abundance of hexokinase I mRNA in relation to 13-actin mRNA. Values are the mean + SE of three independent experiments. Control (0 h) values are normalized to 1.0 arbitrary units.

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3 .¢ ~;~

2

0

0

0.001

0:1

0.01

1.0

10.0

TSH (mU/ml)

Fig. 3. Concentration dependency of the effect of TSH on hexokinase I mRNA. FRTL5 cells were cultured for 12 h with various concentration of TSH as indicated. Five micrograms of total RNA was used for the RT-PCR analysis, as described in Methods. The relative abundance o f hexokinase I mRNA in relation to ~-actin mRNA is shown. Values are the mean + SE o f three i n d e p e n d e n t e x p e r i m e n t s . Control (without TSH) values are normalized to 1.0 arbitrary units.

A

B 3-

Hexokinase I f~-Actin

0

12

24 li o

Time after TSH (h) 0

~4 Time after TSH 011)

Fi N. 4. Effect of TSH on hexokinase I gene transcription in nuclei isolated from FRTL5 cells. A. FRTL5 cells were incubated with TSH (1 mU/ml) for the indicated periods o f time, nuclei were isolated, and a nuclear run-on assay was performed as described in Methods. L a b e l e d transcripts produced in vitro were hybridized for 48 h to immobilized D N A (hexokinase I and [3-actin c D N A s ) , and autoradiography was then performed. B. S u m m a r y o f the densitometric reading. Values are the mean of two independent experiments. Control (0 h) values are normalized to 1.0 arbitrary units.

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Fig. 5. Effect of (Bu)2cAMP and forskolin on hexokinase I mRNA accumulation. FRTL5 cells were cultured for 12 h with (Bu)2cAMP or forskolin. Five micrograms of total RNA was used for the RT-PCR analysis, as described in Methods. Values are the mean + SE of three independent experiments. Control (without TSH) values are normalized to 1.0 arbitrary units. References 1. R. H. MINDEROP, W. HOEPPNER and H. J. SEITZ, Eur. J. Biochem. 164181-187 (1987). 2. P. B. IYNEDJIAN, P. R. PILOT, T. NOUSPIKE, J. L. MILBURN, C. OUAADE, S. HUGHES, C. UCLA and C. B. NEWGAND, Proc. Natl. Acad. Sci. USA 8_._667838-7842(1989). 3. P. B. IYNEDJIAN, D. JOTFERAND, T. NOUSPIKEL, M. ASFARI and P. R. PILOT, J. Biol. Chem. 26421824-21829 (1989). 4. M. B.ALLEN, J. L. BROCKELBANK and D. G. WALKER, Biochim. Biophys. Acta 614357366 (1980). 5. J. N. VERHAGEN andG. E. J, STAAL, Cancer 5_551519-1524 (1985). 6. D. A. SCHWAB and J. E. WILSON, J. Biol. Chem. 2633220-3224 (1988). 7. D. A. SCHWAB and J. E. WILSON, Proc. Natl. Acad. Sci. USA 8_.662563-2567 (1989). 8. T. ENDO, H. SHIMURA, T. SAITO and T. ONAYA, Endocrinology 1261492-1497 (1990). 9. T. MANIATIS, E. F. FRITHCH and J. SAMBROOK, Molecular Cloning, A Laboratory Manual, p. 187-209. Cold Spring Harbor Laboratory, Cold Spring Horbor, N.Y. (1982) 10. S. J. PITI'LER, L. P. KOZAK and J. E. WILSON, Biochem. Biophys. Acta 843186-192 (1985). 11. A. M. WANG, M. U. DOYLE and D. F. MARK, Proc. Natl. Acad. Sci. USA 8__6_69717-9721. (1989) 12. Y. HOSAKA, M. TAWATA and T. ONAYA, Endocrinology 131159-165 (1992). 13. U. NUDEL, R. ZAKUT, S. NEUMAN, Z. LEVY and D. YAFFE, Nucleic Acids Res. 1_! 17591771 (1983). 14. M. E. GREENBERG and E. B. ZIFF, Nature 31 ! 433-438 (1984). 15. G. D. CHAZENBALK, H. L. WADSWORTH and B. RAPOPORT, J. Biol. Chem. 265666670 (1990). 16. M. IKEDA, T. SAITO, T. ENDO, K. TSURUGI and T. ONAYA, Endocrinology 1 2 8 2 5 4 0 2547 (1992).

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17. Y. IWASA, K. AIDA, N. YOKOMORI, M. INOUE and T. ONAYA, Biochem. Int. 21 473-480 (1990). 18. J. E. DUMONT and G. VASSART, Thyroid gland metabolism and the action of TSH, Textbook of Endocrinology. p.311-329. Grune and Stratton, New York (1979).