Expression of Type 2 Iodothyronine Deiodinase in Corticotropin ...

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AtT-20 mouse pituitary tumor cells that secrete corticotropin. Iodothyronine ..... not show iodothyronine deiodination, although thyrotropic tumors showed ...
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Endocrinology 144(10):4459 – 4465 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0419

Expression of Type 2 Iodothyronine Deiodinase in Corticotropin-Secreting Mouse Pituitary Tumor Cells Is Stimulated by Glucocorticoid and CorticotropinReleasing Hormone OSAMU ARAKI, TADASHI MORIMURA, TAKAYUKI OGIWARA, HARUO MIZUMA, MASATOMO MORI, AND MASAMI MURAKAMI First Department of Internal Medicine (O.A., T.M., T.O., H.M., M.Mo.) and Department of Laboratory Medicine (M.Mu.), Gunma University School of Medicine, Maebashi 371-8511, Japan We identified the presence of iodothyronine deiodinase in AtT-20 mouse pituitary tumor cells that secrete corticotropin. Iodothyronine deiodinating activity in AtT-20 cells fulfills all the characteristics of type 2 iodothyronine deiodinase (D2), including the inhibition by thyroid hormones, the insensitivity to inhibition by 6-propyl-2-thiouracil, and the low Michaelis-Menten constant value for T4. Northern analysis using mouse D2 cRNA probe demonstrated the hybridization signal of approximately

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O EXERT ITS biological activity, T4, which is a major secretory product of thyroid gland, needs to be converted to T3 by iodothyronine deiodinase (1). Iodothyronine deiodinase, which converts T4 to T3, has two main subgroups (2). Type 1 iodothyronine deiodinase (D1) is present in thyroid gland, liver, kidney, and many other tissues, whereas D2 is present in a limited number of tissues, including central nervous system, anterior pituitary, and brown fat in the rat. Michaelis-Menten constant (Km) of D2 is approximately 1–10 nm for T4, which is 100 times lower than that of D1. D2 is relatively insensitive to inhibition by 6-propyl-2-thiouracil (PTU), which inhibits D1. D1 activity is known to decrease in the hypothyroid state, and D1 is believed to have a primary role in maintaining circulating T3 levels. D2 activity, in contrast, increases in the hypothyroid state, and D2 is considered to play a critical role in providing local T3 to regulate intracellular T3 concentration (1). Though the source of T3 mainly depends on circulating T3 in most tissues, local intracellular conversion of T4 to T3 is an important source of T3 in certain tissues where D2 exists. The local conversion of T4 to T3 by D2 is considered of physiological importance in regulating anterior pituitary function, especially the inhibition of TSH secretion by thyroid hormones (1, 3). ACTH is secreted from pituitary corticotrophs to regulate adrenocortical functions. ACTH secretion is stimulated by CRH secreted from hypothalamus and negatively regulated by glucocorticoid secreted from adrenal glands (4, 5). The physiological role of thyroid hormones in the regulation of Abbreviations: D1 and D2, Type 1 and type 2 iodothyronine deiodinase; DEX, dexamethasone; DTT, dithiothreitol; PC, prohormone convertase; PTU, 6-propyl-2-thiouracil; SSC, saline sodium citrate.

7.0 kb in size in AtT-20 cells. D2 activity and D2 mRNA were stimulated by glucocorticoid in a dose-dependent manner but were not stimulated by testosterone or ␤-estradiol. D2 expression was stimulated by (Bu)2cAMP, and CRH in a dosedependent manner in the presence of dexamethasone. These results suggest the previously unrecognized role of local thyroid hormone activation by D2 in the regulation of pituitary corticotrophs. (Endocrinology 144: 4459 – 4465, 2003)

functions of pituitary corticotrophs, including the secretion of ACTH, is not fully understood. Recently, it has been reported that thyroid hormones regulate prohormone convertase (PC)1 and PC2, members of the mammalian family of the substilisin-like endoproteases, which are responsible for processing prohormone, proopiomelanocortin, to generate ACTH (6 – 8). Therefore, it is of considerable importance to study the presence and the regulation of thyroid hormone activation mechanism in pituitary corticotrophs. In the present study, we have characterized iodothyronine deiodinating activity and identified D2 expression in ACTHsecreting AtT-20 mouse pituitary tumor cells, which is a well accepted model of pituitary corticotrophs (6, 9). Materials and Methods Materials We purchased [125I]T4, [125I]rT3, and [␣-32P]uridine 5⬘-triphosphate from NEN Life Science Products Corp. (Boston, MA). LH-20 was obtained from Pharmacia (Uppsala, Sweden). AG 50W-X2 resin and a protein assay kit were from Bio-Rad Laboratories, Inc. (Hercules, CA). All other chemicals, obtained at the highest quality, were from Sigma Chemical Company (St. Louis, MO) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise indicated.

Cell culture AtT-20/D16v-F2 cells and GH3 cells were obtained from American Type Culture Collection (Manassas, VA). AtT-20/D16v-F2 cells were cultured in DMEM containing 10% FBS, and GH3 cells were cultured in Ham’s F10 medium containing 15% horse serum and 2.5% FBS at 37 C in the humidified atmosphere of 5% CO2–95% air, and the culture medium was changed every 3 d. Cells were grown to semiconfluence in the culture medium in 6-well or 12-well culture plates (Becton Dickinson and Co., Franklin Lakes, NJ), and then incubated in the same medium containing the compounds to be tested for the periods indicated. The

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culture medium with thyroid hormone-stripped FBS (10) was used for 24 h before harvest, when the effects of thyroid hormones were tested.

Measurement of iodothyronine deiodinase activity Iodothyronine deiodinase activity was measured as previously described (11), with minor modifications (12). Briefly, AtT-20 cells or GH3 cells per each well were washed twice with PBS, scraped off, and transferred into 1.5 ml ice-cold assay buffer [100 mm potassium phosphate (pH 7.0), containing 1 mm EDTA and 20 mm dithiothreitol (DTT)]. After centrifugation at 3,000 rpm for 10 min at 4 C, the resultant precipitates were sonicated in 100 ␮l of the assay buffer per tube and were incubated in a total vol of 50 ␮l with 2 nm or indicated amount of [125I]T4 or [125I]rT3, which was purified using LH-20 column chromatography on the day of the experiment, in the presence or absence of 1 mm PTU or in the presence of 1 mm iopanoic acid, for indicated periods at indicated temperatures, in duplicate. After the characterization of the deiodinating activity in AtT-20 cells, the sonicates were routinely incubated with 2 nm [125I]T4 in the presence of 1 mm PTU at 37 C for 1 h. The reaction was terminated by adding 100 ␮l ice-cold 2% BSA and 800 ␮l ice-cold 10% trichloroacetic acid. After centrifugation at 3,000 rpm for 10 min at 4 C, the supernatant was applied onto a small column packed with AG 50W-X2 resin (bed vol ⫽ 1 ml) and then eluted with 2 ml of 10% glacial acetic acid (column method). Separated 125I was counted with a ␥-counter. Nonenzymatic deiodination was corrected by subtracting I⫺ released in control tubes without cell sonicates. The protein concentration was determined by Bradford’s method using BSA as a standard (13). The deiodinating activity was calculated as femtomoles of I⫺ released/mg protein䡠h, after multiplication by a factor of 2 to correct random labeling at the equivalent 3⬘ and 5⬘ positions. In some experiments, the incubation mixtures were extracted with 2 vol of absolute ethanol after the addition of 5 ␮l of 10 ␮m T4 and T3, and analyzed by descending paper chromatography (hexane:tertiary amyl alcohol:2 n ammonia, 1:5:6) (14, 15).

Araki et al. • Deiodinase in Corticotrophs

scribed above, and the membrane was exposed for 1 h. mRNA levels were quantitated by densitometry using NIH Image, version 1.61, and the OD of the D2 band was corrected for ␤-actin. RNA samples for comparison were analyzed on the same blot (18).

Statistics Statistical differences were evaluated by Student’s t test or ANOVA with the Newman-Keuls test for multiple comparisons.

Results Analysis of iodothyronine deiodinase activity in AtT-20 cells

The deiodinating activity was measured by the release of I⫺ from [125I]T4 in the presence of 20 mm DTT using the column method. Significant deiodinating activity was detectable in the sonicate of AtT-20/D16v-F2 cells. The T4 deiodination was dependent on the incubation period up to 2 h and the protein concentration of AtT-20 cells as shown in Fig. 1. Incubation at 4 C or preheating the cell sonicate at 56 C for 30 min completely abolished the deiodination. The deiodinating activity was not influenced by 1 mm PTU but was completely inhibited by 1 mm iopanoic acid. From the double reciprocal plot, kinetic constants were calculated to be: Km ⫽3.07 nm, Vmax ⫽ 88.5 fmol I⫺ released/mg protein䡠h in AtT-20/D16v-F2 cells. When [125I]rT3 was used as the substrate, Vmax was less than that from [125I]T4 (Km ⫽ 3.15 nm,

Preparation of cRNA probes Because the expression of D2 mRNA has been demonstrated in mouse cerebral tissue, total RNA was extracted from mouse cerebral tissue obtained by a modified acid guanidinium thiocyanate-phenol-chloroform method according to Chomczynski and Sacchi (16). Total RNA was reverse transcribed, and a mouse D2 cDNA fragment containing residues 180 – 614 (numbering of residues as GenBank accession no. AF096875) (17) was amplified by PCR (Perkin-Elmer Applied Biosystems, Foster City, CA). The PCR product was fractionated on a 1% agarose gel and subsequently cloned into pCRII TA-cloning vector (Invitrogen, San Diego, CA). Sequencing analysis (Perkin-Elmer Applied Biosystems) confirmed the identity of the amplified DNA. The cRNA probe for mouse D2 was synthesized with [␣-32P]uridine 5⬘-triphosphate and T7 RNA polymerase. Rat D2 cRNA probe was prepared as previously described (18).

RNA preparation and Northern analysis Total RNA was isolated from each well, and Northern analysis was performed as previously described (18). Twenty micrograms of total RNA per each lane was electrophoresed on a 1.4% agarose gel containing 0.66 m formaldehyde and transferred overnight in 20⫻ saline sodium citrate (SSC) (1⫻ SSC: 150 mm sodium chloride and 15 mm trisodium citrate) to a nylon membrane (Biodyne; Pall BioSupport Corp., East Hills, NY). RNA was cross-linked to the nylon membrane with a UV Stratalinker (Stratagene, San Diego, CA). The membrane was prehybridized with the hybridization buffer (50% formamide, 0.2% SDS, 5% dextran sulfate, 50 mm HEPES, 5⫻ SSC, 5⫻ Denhardt’s solution, and 100 ␮g/ml denatured salmon sperm DNA) at 68 C for 2 h. Subsequently, the membrane was hybridized at 68 C overnight with the hybridization buffer containing a mouse or a rat D2 cRNA probe. The membrane was washed twice in 2⫻ SSC, 0.1% SDS at 25 C for 15 min and twice in 0.1⫻ SSC, 0.1% SDS at 68 C for 1 h. Autoradiography was established by exposing the filters for 4 –24 h to x-ray film (Kodak XAR-2; Eastman Kodak Co., Rochester, NY) at ⫺70 C. After the detection of D2 mRNA, the probe was stripped off and blots rehybridized with ␤-actin cRNA probe as a control. Hybridization and washing were performed as de-

FIG. 1. Characterization of deiodinating activity in AtT-20 cells. A, T4 deiodination in the cell sonicate of AtT-20 cells for various incubation periods up to 2 h. B, T4 deiodination in various protein concentrations of the cell sonicate of AtT-20 cells. Incubation was performed for 1 h. T4 deiodinating activity was measured as described in Materials and Methods.

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Vmax ⫽ 62.8 fmol I⫺ released/mg protein䡠h). These results indicate that iodothyronine deiodinating activity in AtT-20/ D16v-F2 cells has low Km and prefers T4 as the substrate. When sonicates of AtT-20/D16v-F2 cells were incubated with 2 nm [125I]T4 in the presence of 20 mm DTT and 1 mm PTU and subsequently the reaction products were analyzed by descending paper chromatography, there were only three definable peaks corresponding to I⫺, T4, and T3, and radioactivity in the I⫺ peak was comparable with that in the T3 peak. These results indicate that all the characteristics of the deiodinating activity in AtT-20/D16v-F2 cells are compatible with D2. Effects of CRH, cAMP, and dexamethasone (DEX) on D2 expression in AtT-20 cells

Northern analysis of total RNA prepared from AtT-20/ D16v-F2 cells using mouse D2 cRNA probe clearly demonstrated the single hybridization signal of approximately 7 kb in size as shown in Fig. 2A, indicating the presence of D2 mRNA, as well as D2 activity, in AtT-20/D16v-F2 cells. Although 10⫺8 m CRH or 10⫺3 m (Bu)2cAMP slightly stimulated D2 activity (1.4-fold and 2.3-fold, respectively) and D2 mRNA (1.4-fold and 1.7-fold, respectively) in AtT-20 cells, 10⫺6 m DEX significantly stimulated D2 activity (3.6-fold) and D2 mRNA (3.9-fold). In the presence of 10-6 M DEX, 10⫺8 m CRH or 10⫺3 m (Bu)2cAMP markedly stimulated D2 activity (5.8-fold and 12-fold, respectively) and D2 mRNA (6.3fold and 10.6-fold, respectively) as shown in Fig. 2, A and B. As shown in Fig. 2C, 10⫺6 m DEX or 10⫺6 m DEX plus 10⫺8 m CRH did not change Km but increased Vmax of T4 deiodination, indicating D2 activity per se was stimulated by DEX and CRH. In the next experiment, the time course of stimulation of D2 expression by 10⫺6 m DEX or 10⫺6 m DEX plus 10⫺8 m CRH was studied. By 6 h of incubation, 10⫺6 m DEX or 10⫺6 m DEX plus 10⫺8 m CRH significantly stimulated D2 expression in AtT-20 cells as shown in Fig. 3. In these experiments, medium change itself slightly increased D2 expression, as reported in cultured mouse neuroblastoma cells (19). Effects of graded doses of DEX or CRH were studied in the next experiment. CRH itself did not stimulate D2 expression up to 10⫺8 m, but CRH stimulated D2 expression in a dosedependent manner in the presence of 10⫺6 m DEX (data not shown). In addition, DEX stimulated D2 expression in a dose-dependent manner, and this increase was augmented by the addition of 10⫺8 m CRH (data not shown).

FIG. 2. Effects of CRH, (Bu)2cAMP, and/or DEX on D2 mRNA and D2 activity in AtT-20 cells. A, Northern analysis of D2 mRNA in AtT-20 cells incubated with medium only (control), or medium containing CRH (10⫺8 M), (Bu)2cAMP (10⫺3 M), DEX (10⫺6 M), DEX (10⫺6 M) and CRH (10⫺8 M), or DEX (10⫺6 M) and (Bu)2cAMP (10⫺3 M) for 6 h. Each lane represents 20 ␮g total RNA obtained from cells in an individual well. B, D2 mRNA (D2 mRNA/␤-actin mRNA ratio) and D2 activity in AtT-20 cells incubated with medium only (control), or medium containing CRH (10⫺3 M), (Bu)2cAMP (10⫺3 M), DEX (10⫺6 M), DEX (10⫺6 M) and CRH (10⫺8 M), or DEX (10⫺6 M) and (Bu)2cAMP (10⫺3 M) for 6 h. The optical density of the D2 band was corrected for ␤-actin,

and D2 mRNA was expressed as a percentage of control D2 mRNA. D2 activity shown represents the mean ⫾ SE of three wells. Where SE is too small to depict, SE bars are not shown. *, P ⬍ 0.05; **, P ⬍ 0.01 (Newman-Keuls test). C, Double reciprocal plot of T4 deiodination by AtT-20 cells incubated with DEX or DEX plus CRH. AtT-20 cells were incubated with medium only (control), or medium containing DEX (10⫺6 M) or DEX (10⫺6 M) and CRH (10⫺8 M) for 6 h. Incubations were performed for 1 h at 37 C with various concentrations of [125I]T4 in the presence of 20 mM DTT. Kinetic constants were calculated to be: control (open circle), km ⫽ 3.07 nM, Vmax ⫽ 88.5 fmol I⫺ released/mg protein䡠h; DEX (closed circle), km ⫽ 2.86 nM, Vmax ⫽ 277.8 fmol I⫺ released/mg protein䡠h; DEX⫹CRH (closed square), km ⫽ 2.50 nM, Vmax ⫽ 500.0 fmol I⫺ released/mg protein䡠h.

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FIG. 3. Time course of stimulation of D2 mRNA and D2 activity in AtT-20 cells. A, D2 mRNA (D2 mRNA/␤-actin mRNA ratio) in AtT-20 cells incubated with medium only (control), CRH (10⫺8 M), DEX (10⫺6 ⫺6 ⫺8 M), or DEX (10 M) and CRH (10 M) for various hours. The OD of the D2 mRNA band was corrected for ␤-actin, and the results were expressed as a percentage of the value obtained for control (0 h). B, D2 activity in AtT-20 cells incubated with medium only (control), CRH (10⫺8 M), DEX (10⫺6 M), or DEX (10⫺6 M) and CRH (10⫺8 M) for various hours. The D2 activity shown represents the mean ⫾ SE of three wells. Open circle, control; open square, CRH (10⫺8 M); closed circle, DEX (10⫺6 M); closed square, DEX (10⫺6 M)⫹CRH (10⫺8 M). Where SE is too small to depict, SE bars are not shown. *, P ⬍ 0.01, compared with control at each time point (t test).

Effects of various steroids on D2 expression in AtT-20 cells

Effects of 10⫺6 m of various steroids on D2 expression were studied. As shown in Fig. 4, testosterone or ␤-estradiol did not increase D2 expression, although corticosterone stimulated D2 activity (2.4-fold) and D2 mRNA (3-fold) in AtT-20 cells. The potency of stimulation of D2 expression by corticosterone was less than that of DEX. These results indicate that glucocorticoid, but not sex steroids, stimulates D2 expression in AtT-20 cells.

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FIG. 4. Effects of various steroids on D2 mRNA and D2 activity in AtT-20 cells. A, Northern analysis of D2 mRNA in AtT-20 cells incubated with 10⫺6 M of corticosterone, DEX, testosterone, or ␤-estradiol for 6 h. Each lane represents 20 ␮g total RNA obtained from cells in an individual well. B, D2 mRNA (D2 mRNA/␤-actin mRNA ratio) and D2 activity in AtT-20 cells incubated with 10⫺6 M of various steroids for 6 h. The OD of the D2 band was corrected for ␤-actin, and D2 mRNA was expressed as a percentage of D2 mRNA in control (incubated with medium only). D2 activity shown represents the mean ⫾ SE of three wells. *, P ⬍ 0.01 (Newman-Keuls test).

T3, T4, or rT3 did not alter D2 mRNA levels up to 6 h (data not shown). These results suggest that the thyroid hormones inhibit D2 expression mainly at the posttranslational level in AtT-20 cells in these experimental conditions.

Effects of thyroid hormones on D2 activity in AtT-20 cells

One of the important features of D2 is the negative regulation of the enzyme activity by thyroid hormones. Addition of thyroid hormones to the incubation medium for 6 h decreased the deiodinating activity in AtT-20 cells, and the potency of the inhibitory effect was T4⬎rT3⬎T3 as shown in Fig. 5. The inhibition of deiodinating activity in AtT-20 cells by thyroid hormones further supports the presence of authentic D2 activity in cultured AtT-20 cells. When the effects of thyroid hormones on D2 mRNA were studied, 10⫺7 m of

Effects of cAMP and DEX on D2 expression in GH3 cells

Because it was reported that D2 activity was present in GH3 rat pituitary tumor cells (20, 21), effects of DEX and cAMP on D2 expression in GH3 cells were also studied. As shown in Fig. 6, 10⫺6 m DEX or 10⫺3 m (Bu)2cAMP slightly stimulated D2 activity (1.5-fold) and D2 mRNA (1.6-fold and 1.8-fold, respectively), and 10⫺6 m DEX plus 10⫺3 m (Bu)2cAMP stimulated D2 activity (2.8-fold) and D2 mRNA (2.6-fold) in GH3 cells.

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FIG. 5. Effects of thyroid hormones on D2 activity in AtT-20 cells. AtT-20 cells were cultured with thyroid-hormone-depleted medium for 24 h, and then incubated with thyroid hormones for 6 h. Closed circle, T3; open circle, T4; closed square, rT3. D2 activity shown represents the mean ⫾ SE of three wells. Where SE is too small to depict, SE bars are not shown. *, P ⬍ 0.05; **, P ⬍ 0.01, compared with thyroid-hormone-depleted medium only (Newman-Keuls test).

Discussion

The present results have clearly demonstrated that iodothyronine deiodinating activity is present in the ACTHsecreting AtT-20/D16v-F2 mouse pituitary tumor cell line, which is a well-accepted model of anterior pituitary lobe corticotrophs (6, 9). Iodothyronine deiodinating activity in AtT-20 cells was dependent on protein concentrations, incubation period, and temperature. These characteristics clearly indicate its enzymatic nature. Iodothyronine deiodinating activity was not inhibited by 1 mm PTU and showed low Km for T4 or rT3. In addition, the deiodinating activity was decreased by the addition of thyroid hormones to the culture medium. Iodothyronine deiodinating activity in AtT-20 cells, therefore, has characteristics compatible with D2 (1, 2). Northern analysis using mouse D2 cRNA probe clearly demonstrated the hybridization signals of approximately 7 kb in size in AtT-20 cells. The size of D2 mRNA demonstrated in AtT-20 cells was in agreement with the previous observations (17). The present results seem to be the first demonstration of the expression of D2, which plays a pivotal role in providing local intracellular T3, in AtT-20 mouse pituitary tumor cells that secrete ACTH. The presence of iodothyronine deiodinase in pituitary corticotrophs has been controversial. In old literature, it was demonstrated that mouse adrenocorticotropic tumors did not show iodothyronine deiodination, although thyrotropic tumors showed deiodination (22). However, recent studies showed that D2 activity and D2 mRNA were detected in human pituitary tumors, including Cushing’s disease (23, 24), suggesting the expression of D2 in human pituitary corticotrophs. Because D2 activity in AtT-20 cells was inhibited by thyroid hormones, mainly at the posttranslational level, in the present study, D2 expression in pituitary corticotrophs could be increased in the hypothyroid state, as demonstrated in the rat central nervous system, pituitary, and brown adipose tissue in vivo (1). It is, therefore, possible that D2 in pituitary corticotrophs may play a role in protecting corticotrophs from local T3 deficiency in hypothyroidism.

FIG. 6. Effects of (Bu)2cAMP and/or DEX on D2 mRNA and D2 activity in GH3 cells. A, Northern analysis of D2 mRNA in GH3 cells incubated with medium only (control), or medium containing (Bu)2cAMP (10⫺3 M), DEX (10⫺6 M), or DEX (10⫺6 M) and (Bu)2cAMP (10⫺3 M) for 6 h. Each lane represents 20 ␮g total RNA obtained from cells in an individual well. B, D2 mRNA (D2 mRNA/␤-actin mRNA ratio) and D2 activity in GH3 cells incubated with medium only (control), or medium containing (Bu)2cAMP (10⫺3 M), DEX (10⫺6 M), or DEX (10⫺6 M) and (Bu)2cAMP (10⫺3 M) for 6 h. The OD of the D2 band was corrected for ␤-actin, and D2 mRNA was expressed as a percentage of control D2 mRNA. D2 activity shown represents the mean ⫾ SE of three wells. *, P ⬍ 0.05; **, P ⬍ 0.01 (Newman-Keuls test).

In the present study, both D2 activities and D2 mRNA levels were rapidly stimulated by (Bu)2cAMP in AtT-20 cells. These results indicate that D2 expression in AtT-20 cells is regulated by a cAMP-dependent mechanism at the pretranslational level, as previously reported in human skeletal muscle cells, human thyroid follicular cells, rat astrocytes, and rat pineal glands (15, 18, 25, 26). Recently, cAMP response element was reported to be present in the human D2 promoter region (26, 27, 28), suggesting that the transcriptional regu-

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lation of D2 expression by cAMP regulatory mechanism might exist in AtT-20 cells. The present results demonstrated that glucocorticoid significantly stimulated D2 expression in AtT-20 cells. Furthermore, CRH markedly stimulated D2 expression in the presence of DEX, presumably via a cAMP-dependent mechanism. These results suggest the permissive role of glucocorticoid in the stimulation of D2 expression by CRH in AtT-20 cells. In the present study, cAMP stimulated D2 expression more potently than CRH in AtT-20 cells. CRH is also known to stimulate phospholipase C-diacylglycerol-inositolposphate-Ca2⫹ signaling cascade (29), which may modulate the stimulatory effect of cAMP on D2 expression. It is of interest that D2 expression is increased by factors that either stimulate or inhibit ACTH secretion, namely CRH and glucocorticoid, respectively. D2 activity was reported to be present in GH3 rat pituitary tumor cells (20, 21), and DEX or cAMP was demonstrated to cause modest increases in D2 mRNA in GH4C1 rat pituitary tumor cells (30). In the present study, DEX or cAMP slightly stimulated D2 expression, and DEX plus cAMP modestly stimulated D2 expression in GH3 cells. Thus, the magnitude of stimulation of D2 expression by DEX and cAMP in GH3 cells was less than that in AtT-20 cells. It was also reported that glucocorticoid showed a permissive role in the stimulation of D2 activity by TPA in rat astroglial cells (31). In contrast, DEX was reported to decrease D2 activity in cultured mouse neuroblastoma cells (32). Therefore, the effect of glucocorticoid on D2 expression could be different among tissues where D2 exists. The physiological importance of intracellular thyroid hormone activation by D2 has been clearly demonstrated in certain tissues. Adenohypophyseal T3 production by D2 plays an important role in feedback regulation of TSH secretion by thyroid hormones (1, 3). In rat brown adipose tissue, the expression of uncoupling protein is regulated by locally generated T3, which is provided by D2 (33, 34). Recently, it has been reported that thyroid hormone regulates PC1 and PC2, members of the mammalian family of the substilisin-like endoproteases, which are responsible for processing prohormone, proopiomelanocortin, to generate ACTH (6). It has been shown that T3 negatively regulates PC1 and PC2 expression at the transcriptional level and that functional negative thyroid hormone response elements exist in human PC1 and PC2 promoter (7, 8). Furthermore, T4 treatment in vivo has been shown to decrease PC2 mRNA (6). Therefore, D2 in pituitary corticotrophs may play a role in the regulation of PCs by thyroid hormones. It is also of interest to study the possible role of locally produced T3 by D2 in the regulation of other corticotroph-specific genes. In summary, the present results demonstrate the expression of functional D2 in ACTH-secreting AtT-20 mouse pituitary tumor cells, and its activity is stimulated by glucocorticoid and CRH and inhibited by thyroid hormones, which may open novel perspectives on the roles of thyroid hormone metabolism in the physiological regulation of pituitary corticotrophs.

Araki et al. • Deiodinase in Corticotrophs

Acknowledgments The authors are indebted to Dr. Takayuki Kasahara and Dr. Katsuhiko Tsunekawa for useful discussion and encouragement. Received April 3, 2003. Accepted July 7, 2003. Address all correspondence and requests for reprints to: Masami Murakami, M.D., Department of Laboratory Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan. E-mail: mmurakam@ showa.gunma-u.ac.jp. This work was supported, in part, by Grant-in-Aid no.14572175 (to M.Mu.) for scientific research, from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Results were presented, in part, at the 12th International Thyroid Congress, Kyoto, Japan, 2000.

References 1. Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23:38 – 89 2. Visser TJ, Leonard JL, Kaplan MM, Larsen PR 1982 Kinetic evidence suggesting two mechanisms for iodothyronine 5⬘-deiodination in rat cerebral cortex. Proc Natl Acad Sci USA 79:5080 –5084 3. Schneider MJ, Fiering SN, Pallud SE, Parlow AF, St Germain DL, Galton VA 2001 Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol 15:2137–2148 4. Keller-Wood ME, Dallman MF 1984 Corticosteroid inhibition of ACTH secretion. Endocr Rev 5:1–24 5. Antoni FA 1986 Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endocr Rev 7:351–378 6. Day R, Schafer MK, Watson SJ, Chretien M, Seidah NG 1992 Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol Endocrinol 6:485– 497 7. Li QL, Jansen E, Brent GA, Naqvi S, Wilber JF, Friedman TC 2000 Interactions between the prohormone convertase 2 promoter and the thyroid hormone receptor. Endocrinology 141:3256 –3266 8. Li QL, Jansen E, Brent GA, Friedman TC 2001 Regulation of prohormone convertase 1 (PC1) by thyroid hormone. Am J Physiol 280:E160 –170 9. Axelrod J, Reisine TD 1984 Stress hormones: their interaction and regulation. Science 224:452– 459 10. Samuels HH, Stanley F, Casanova J 1979 Depletion of L-3,5,3⬘-triiodothyronine and l-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105:80 – 85 11. Leonard JL, Rosenberg IN 1980 Iodothyronine 5⬘-deiodinase from rat kidney: substrate specificity and the 5⬘-deiodination of reverse triiodothyronine. Endocrinology 107:1376 –1383 12. Murakami M, Tanaka K, Greer MA, Mori M 1988 Anterior pituitary type II thyroxine 5⬘-deiodinase activity is not affected by lesions of the hypothalamic paraventricular nucleus which profoundly depress pituitary thyrotropin secretion. Endocrinology 123:1676 –1681 13. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 –254 14. Bellabarba D, Peterson RE, Sterling K 1968 An improved method for chromatography of iodothyronines. J Clin Endocrinol Metab 28:305–307 15. Hosoi Y, Murakami M, Mizuma H, Ogiwara T, Imamura M, Mori M 1999 Expression and regulation of type II iodothyronine deiodinase in cultured human skeletal muscle cells. J Clin Endocrinol Metab 84:3293–3300 16. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156 –159 17. Davey JC, Schneider MJ, Becker KB, Galton VA 1999 Cloning of a 5.8-kb cDNA for a mouse type 2 deiodinase. Endocrinology 140:1022–1025 18. Kamiya Y, Murakami M, Araki O, Hosoi Y, Ogiwara T, Mizuma H, Mori M 1999 Pretranslational regulation of rhythmic type II iodothyronine deiodinase expression by beta-adrenergic mechanism in the rat pineal gland. Endocrinology 140:1272–1278 19. Gavin LA, Moller M, McMahon F, Gulli R, Cavalieri RR 1989 Carbohydrate reactivation of thyroxine 5⬘-deiodinase (type II) in cultured mouse neuroblastoma cells is dependent upon new protein synthesis. Endocrinology 124: 635– 641 20. Melmed S, Nelson M, Kaplowitz N, Yamada T, Hershman JM 1981 Glutathione-dependent thyroxine 5⬘-monodeiodination modulates growth hormone production by cultured nonthyrotropic rat pituitary cells. Endocrinology 108:970 –976 21. St Germain DL 1985 Metabolic effect of 3,3⬘,5⬘-triiodothyronine in cultured growth hormone-producing rat pituitary tumor cells. Evidence for a unique mechanism of thyroid hormone action. J Clin Invest 76:890 – 893 22. Werner SC, Volpert EM, Grinberg R 1961 Difference in metabolism of labelled

Araki et al. • Deiodinase in Corticotrophs

23.

24.

25.

26.

27.

28.

thyroxine between thyrotropic and adrenotropic mouse pituitary tumors. Nature 192:1193–1194 Itagaki Y, Yoshida K, Ikeda H, Kaise K, Kaise N, Yamamoto M, Sakurada T, Yoshinaga K 1990 Thyroxine 5⬘-deiodinase in human anterior pituitary tumors. J Clin Endocrinol Metab 71:340 –344 Tannahill LA, Visser TJ, McCabe CJ, Kachilele S, Boelaert K, Sheppard MC, Franklyn JA, Gittoes NJ 2002 Dysregulation of iodothyronine deiodinase enzyme expression and function in human pituitary tumours. Clin Endocrinol (Oxf) 56:735–743 Courtin F, Chantoux F, Pierre M, Francon J 1988 Induction of type II 5⬘deiodinase activity by cyclic adenosine 3⬘,5⬘-monophosphate in cultured rat astroglial cells. Endocrinology 123:1577–1581 Murakami M, Araki O, Hosoi Y, Kamiya Y, Morimura T, Ogiwara T, Mizuma H, Mori M 2001 Expression and regulation of type II iodothyronine deiodinase in human thyroid gland. Endocrinology 142:2961–2967 Bartha T, Kim SW, Salvatore D, Gereben B, Tu HM, Harney JW, Rudas P, Larsen PR 2000 Characterization of the 5⬘-flanking and 5⬘-untranslated regions of the cyclic adenosine 3⬘,5⬘-monophosphate-responsive human type 2 iodothyronine deiodinase gene. Endocrinology 141:229 –237 Canettieri G, Celi FS, Baccheschi G, Salvatori L, Andreoli M, Centanni M 2000 Isolation of human type 2 deiodinase gene promoter and characterization

Endocrinology, October 2003, 144(10):4459 – 4465

29. 30.

31.

32. 33. 34.

4465

of a functional cyclic adenosine monophosphate response element. Endocrinology 141:1804 –1813 Dautzenberg FM, Hauger RL 2002 The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol Sci 23:71–77 Kim S-W, Harney JW, Larsen PR 1998 Studies of the hormonal regulation of type 2 5⬘-iodothyronine deiodinase messenger ribonucleic acid in pituitary tumor cells using semiquantitative reverse transcription-polymerase chain reaction. Endocrinology 139:4895– 4905 Courtin F, Chantoux F, Gavaret JM, Toru-Delbauffe D, Jacquemin C, Pierre M 1989 Induction of type II 5⬘-deiodinase activity in cultured rat astroglial cells by 12-O-tetradecanoylphorbol-13-acetate: dependence on glucocorticoids. Endocrinology 125:1277–1281 St Germain DL 1986 Hormonal control of a low Km (type II) iodothyronine 5⬘-deiodinase in cultured NB41A3 mouse neuroblastoma cells. Endocrinology 119:840 – 846 Bianco AC, Silva JE 1987 Intracellular conversion of thyroxine to triiodothyronine is required for the optimal thermogenic function of brown adipose tissue. J Clin Invest 79:295–300 de Jesus LA, Carvalho SD, Ribeiro MO, Schneider M, Kim S-O, Harney JW, Larsen PR, Bianco AC 2001 The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue. J Clin Invest 108:1379 – 1385