Identification of Two Novel ACTH-Responsive Genes Encoding ...

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Molecular Endocrinology 16(6):1417–1427 Copyright © 2002 by The Endocrine Society

Identification of Two Novel ACTH-Responsive Genes Encoding Manganese-Dependent Superoxide Dismutase (SOD2) and the Zinc Finger Protein TIS11b [Tetradecanoyl Phorbol Acetate (TPA)Inducible Sequence 11b] ANNA M. CHINN*†, DELPHINE CIAIS*, SABINE BAILLY, EDMOND CHAMBAZ, JONATHAN LAMARRE‡, AND JEAN-JACQUES FEIGE INSERM EMI 01-05, Department of Molecular and Structural Biology, Commissariat a` l’Energie Atomique, Grenoble, France F-38054 ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nM ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH. Four of these amplicons encoded noninformative repetitive sequences. Of the other two sequenced amplicons, one encoded a partial sequence for mitochondrial manganese-dependent superoxide dismutase (SOD2), an enzyme that is likely to protect adrenocortical cells from the cytotoxic effects of radical oxygen species generated during steroid biosynthesis. The second was identified as TIS11b (phorbol-12-myristate-13-acetate-

inducible sequence 11b)/ERF-1/cMG, a member of the CCCH double-zinc finger protein family. SOD2 induction by ACTH was independent of extracellular steroid concentration or oxidative stress. SOD2 and TIS11b mRNA expressions were rapidly induced by ACTH, reaching a maximal level after 8 h and 3 h of treatment, respectively. These ACTH effects were mimicked by forskolin but appeared independent of cortisol secretion. Upon ACTH treatment, induction of TIS11b expression closely followed the previously characterized peak of vascular endothelial growth factor (VEGF) expression. Transfection of a TIS11b expression plasmid into 3T3 fibroblasts induced a decrease in the expression of a reporter gene placed upstream of the VEGF 3ⴕ-untranslated region, indicating that TIS11b may be an important regulator of VEGF expression through interaction with its 3ⴕuntranslated region. (Molecular Endocrinology 16: 1417–1427, 2002)

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including jun-B, c-jun, and NGFI-B/nur77 (7–10). After more than 6 h, a second wave of gene expression results in the sustained induction of a large panel of genes encoding metabolic and steroidogenic enzymes (11–13) as well as extracellular matrix proteins (14). However, significantly less is known about genes that are expressed between these two waves of gene expression and may regulate adrenocyte function. In the present work, we used differential display RT-PCR, a method that theoretically permits the visualization of every mRNA expressed in a cell or tissue (15), to identify low abundance genes that are induced by ACTH in a 4- to 8-h timeframe. This led to the identification of two previously undescribed ACTHregulated genes: SOD2 (superoxide dismutase 2) encoding an enzyme (mitochondrial manganese-dependent superoxide dismutase) likely to be involved in the protection of endocrine cells against the cytotoxic effects of free radicals generated during steroid biosynthesis, and TIS11b [tetradecanoyl phorbol acetate (TPA)-inducible sequence 11b] encoding a member of a family of CCCH zinc finger proteins that bind to the

HE MAJOR TROPHIC factor responsible for the continued survival and homeostasis of the adrenal cortex is ACTH. This pituitary peptide hormone controls such diverse functions in the adrenal cortex as cellular differentiation, cell shape, mitogenesis, apoptosis, and steroid production (1–5). ACTH-dependent changes in cellular functions are initiated by intracellular signaling cascades that begin with the binding of ACTH to its receptor, continue with the subsequent stimulation of cAMP synthesis, and culminate in changes in the pattern of cellular gene expression (6). Many of the cellular effects of ACTH result from changes in the pattern of cellular gene expression. ACTH is known to rapidly stimulate the transient expression of a first wave of immediate early genes,

Abbreviations: ARE, AU-rich element; BAC, bovine adrenocortical; CMV, cytomegalovirus; DMSO, dimethylsulfoxide; dNTP, deoxynucleoside triphosphate; nt, nucleotide; RACE, rapid amplification of cDNA ends; SOD, superoxide dismutase; TIS11, TPA-inducible sequence 11; TPA, tetradecanoyl phorbol acetate; UTR, untranslated region; VEGF, vascular endothelial growth factor.

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AU-rich elements (AREs) of a number of cytokine mRNAs and regulate their stability (16). We report here that the effects of ACTH on SOD2 and TIS11b expression are mimicked by forskolin but appear independent of cortisol secretion. We also present evidence that TIS11b may be an important regulator of VEGF expression acting through interaction with its 3⬘untranslated region (UTR).

RESULTS Differential Display of Products Amplified from ACTH-Treated Bovine Adrenocortical (BAC) Cells A number of genes induced by ACTH have been previously described, most notably those involved in steroidogenesis (12, 17). We were interested in identifying genes induced by ACTH in a time period preceding the induction of steroidogenic enzymes, particularly those genes that might be involved in relaying the physiological effects of ACTH. Following the protocol schematized in Fig. 1, we performed two independent rounds of differential display RT-PCR on total RNA

Chinn et al. • Identification of ACTH-Induced Genes

isolated from BAC cells treated with ACTH for either 4, 5.5, 7, or 8.5 h vs. untreated cells. From the electrophoretic analysis of the amplification products on DNA sequencing gels, we identified 42 differentially amplified products. These products were extracted, reamplified, and used to probe Northern blots of control and ACTH-treated BAC cell RNAs. Thirty six amplicons gave either no hybridization signal or a signal whose intensity was not affected by ACTH treatment (false positives), and only six products gave signals that were induced (between 3 and 5 times) in ACTHtreated cells (true positives). The sequencing of these validated amplicons generated four noninformative sequences highly homologous to repetitive sequences present in the 3⬘-UTR of numerous genes (Alu, MER, SINE) and two informative sequences partially identical with the 3⬘-ends of two known mRNAs: SOD2 and TIS11b/ERF-1/cMG. We used rapid amplification of cDNA ends (RACE) PCR to obtain additional nucleotide sequence from the mRNA recognized by the differential amplicon HTAa1. Primers were designed from the sequence of HTAa1, and a 690-bp fragment was RT-PCR amplified and sequenced. The BLAST algorithm (18) revealed

Fig. 1. Strategy and Overall Results of the Identification of ACTH-Induced Genes by Differential Display RT-PCR


Mol Endocrinol, June 2002, 16(6):1417–1427

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Fig. 3. Specificity of SOD2 Induction by ACTH Primary cultures of bovine fasciculata cells were treated for 5 h with the indicated factors: none (Ctl); 0.1 ng/ml FGF-2 (FGF); 10 ␮M forskolin (Fsk); 15 nM angiotensin II (AII); 10 nM ACTH 1–24 (ACTH); and RNA was prepared for Northern blot analysis. The blots were sequentially hybridized with bovine SOD2 (upper panel) and bovine ribosomal protein L27 (RPL27, lower panel) 32P-labeled cDNA probes. This experiment is representative of two independent experiments.

ACTH Regulation of SOD2 Expression

Fig. 2. Expression of SOD2 in the Adrenal Cortex Primary cultures of bovine fasciculata cells were treated for the indicated periods of time with 10 nM ACTH, and RNA was prepared for Northern blot analysis. The blots were sequentially hybridized with bovine SOD2 (upper panel) and bovine ribosomal protein L27 (RPL27, lower panel) 32P-labeled cDNA probes. The size of the three SOD2 transcripts is indicated on the right. RNA was prepared from the glomerulosa (G) and the fasciculata (F) zones of bovine adrenal cortex and from bovine adrenal medulla (M) tissues. Northern blots were sequentially hybridized with bovine SOD2 (upper panel) and 18S rRNA (lower panel) 32P-labeled cDNA probes. This experiment is representative of two independent experiments.

similarity between the sequences of the amplified fragment and the human gene for manganese-dependent superoxide dismutase in the 3⬘-untranslated region of the longest of the three SOD2 transcripts (19). We designed primers within the coding sequence of the bovine gene and amplified a 321-bp PCR product from bovine adrenal cortex RNA. This was used to probe Northern blots, and the same 3.4-kb transcript recognized by the HTAa1 probe was also recognized by the bovine SOD2 probe (data not shown). Using the BLAST algorithm, we observed similarities between the sequences of the second clone (HTG-A2) and the 3⬘-UTRs of human ERF-1 and its ortholog in rat, cMG (20–22). We designed primers in a well conserved region of the coding sequence of these genes and RT-PCR amplified a 262-bp fragment from bovine adrenal cortex RNA. Sequence comparison showed greater than 95% identity with both human and rat TIS11b/ERF-1/cMG nucleotide sequences in this region. This probe hybridized to the same transcripts recognized by the HTG-A2 amplicon (data not shown).

We studied the time course of the induction of SOD2 expression by ACTH. BAC fasciculata cells were treated with 10 nM ACTH for different periods of time, and the abundance of SOD2 mRNA was determined by Northern blot analysis. As shown in Fig. 2A, all three SOD2 transcripts at 3.4, 1.7, and 1.4 kb were induced by ACTH to a similar extent. This induction, between 3- and 10-fold in different experiments, was apparent by 4 h and reached a plateau after 8–12 h of treatment. We analyzed the expression of SOD2 in the different zones of fresh adrenal gland tissue. The Northern blot shown in Fig. 2B revealed expression of SOD2 in the cortical fasciculata zone, which is the most ACTHsensitive in vivo, but no detectable expression was observed in the cortical glomerulosa zone (which is mostly angiotensin II sensitive in vivo) or in the adrenal medulla. We then examined the specificity of the ACTH effect on SOD2 expression. BAC cells (from the fasciculata zone) were treated for 5 h with either 0.1 ng/ml FGF-2 (FGF-2 is the most potent mitogenic factor active on BAC cells), 10 ␮M forskolin (a direct activator of adenylate cyclase), 15 nM angiotensin II (a steroidogenic hormone active on BAC cells), or 10 nM ACTH and the abundance of SOD2 mRNA was quantitated by Northern blot analysis. The results shown in Fig. 3 indicate that forskolin mimicked the stimulatory effect of ACTH and that angiotensin II also significantly increased SOD2 levels, whereas FGF-2 had no effect. This observation prompted us to check whether the induction of SOD2 expression could result from an indirect stimulation by corticosteroids produced in response to these hormones. The incubation of BAC cells for 5 h with 500 ng/ml cortisol (the concentration found in BAC cell medium after 5 h of ACTH stimulation) did not modify the SOD2 mRNA levels, as determined by Northern blot analysis (Fig. 4, lanes 1 and 3). Further-

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Fig. 4. ACTH Induction of SOD2 Expression in the Presence of the Antioxidant DMSO Primary cultures of bovine fasciculata cells were incubated for 5 h with or without ACTH (10 nM) or cortisol (500 ng/ml), in the presence or absence of 10 mM DMSO, as indicated. RNA was prepared for Northern blot analysis. The blots were sequentially hybridized with bovine SOD2 (upper panel), bovine SOD1 (middle panel), and 18S rRNA (lower panel) 32 P-labeled cDNA probes. This experiment is representative of two independent experiments.

more, inhibition of ACTH-stimulated cortisol production using aminogluthetimide, an inhibitor of P450scc, or metyrapone, an inhibitor of P45011␤, did not prevent ACTH induction of SOD2 mRNA expression (Fig. 5), confirming that this ACTH effect is not mediated by corticosteroids. SOD2 expression can also be induced by free radicals (23). To determine whether the effect of ACTH on MnSOD expression was secondary to an increase in free radicals generated as byproducts of steroid synthesis (24, 25), we preincubated BAC cells overnight in the presence of dimethylsulfoxide (DMSO) (10 mM), an efficient antioxidant for these cells (26), and then stimulated them for 5 h in the absence or the presence of either cortisol (500 ng/ml) or ACTH (10 nM). As shown in Fig. 4, DMSO had a very limited effect on both basal and ACTH-induced SOD2 mRNA expression, indicating that free radicals do not mediate the effect of ACTH on SOD2 expression. The same blot was subsequently probed with a bovine SOD1 cDNA probe to determine whether the effect of ACTH was specific to SOD2, or whether it was a general effect on antioxidants. No significant modification of SOD1 expression was observed in response to either ACTH, cortisol, or


Fig. 5. ACTH Induction of SOD2 Expression in the Presence of Steroidogenesis Inhibitors Primary cultures of bovine fasciculata cells were preincubated for 15 min in the absence (Ctl) or the presence of 0.5 mM aminogluthetimide (AMG) or 0.5 ␮g/ml metyrapone (Met) and then incubated for 5 h in the same media containing 0 or 10 nM ACTH, as indicated. RNA was prepared for Northern blot analysis. The blots were sequentially hybridized with bovine SOD2 (upper panel) and 18S rRNA (lower panel) 32Plabeled cDNA probes.

DMSO, suggesting that the mitochondrial SOD2 enzyme is a specific target of ACTH. ACTH Regulation of TIS11b Expression BAC cells were treated with 10 nM ACTH for varying lengths of time, and TIS11b mRNA levels were determined by Northern blot. As shown in Fig. 6A, bovine TIS11b is expressed at a moderate level in unstimulated bovine fasciculata cells in culture and is induced 4-fold by ACTH. This induction was apparent as early as 30 min after stimulation and peaked after 3–4 h. TIS11b expression remained slightly elevated 24 h after ACTH treatment. We next examined the expression of TIS11b in adrenocortical tissue. As shown in Fig. 6B, TIS11b was undetectable in adrenal medulla, weakly expressed in glomerulosa tissue, and strongly expressed in fasciculata tissue, the most ACTH-sensitive zone in vivo. As observed for SOD-2, forskolin (10 ␮M) mimicked the effect of ACTH on TIS11b expression whereas cortisol did not (Fig. 7), indicating that the effects of ACTH on SOD2 and TIS11b expression are cAMP-mediated and glucocorticoid-independent. TIS11b Regulation of VEGF3ⴕ-UTR We recently observed that the expression of the angiogenic cytokine VEGF is very rapidly but transiently induced by ACTH in adrenocortical cells (reaching a maximum after 2–3 h of ACTH treatment) (27). A simultaneous analysis of TIS11b and VEGF expression in ACTH-treated BAC cells was performed by RT-PCR to compare the kinetics of induction of these two


Fig. 6. Expression of TIS11b in the Adrenal Cortex Primary cultures of bovine fasciculata cells were treated for the indicated periods of time with 10 nM ACTH, and RNA was prepared for Northern blot analysis. The blots were sequentially hybridized with bovine TIS11b (upper panel) and bovine ribosomal protein L27 (RPL27, lower panel) 32P-labeled cDNA probes. RNA was prepared from the glomerulosa (G) and the fasciculata (F) zones of bovine adrenal cortex and from bovine adrenal medulla (M) tissues. Northern blots were sequentially hybridized with bovine TIS11b (upper panel) and 18S rRNA (lower panel) 32P-labeled cDNA probes. This experiment is representative of three independent experiments.

genes. As shown in Fig. 8, TIS11b expression appeared to follow the induction of VEGF expression by 1 h and to remain elevated when VEGF mRNA levels dropped (between 3 and 5 h). This could suggest that TIS11b might play a role in the rapid decay of VEGF mRNA observed after 3 h of ACTH treatment. Indeed, recent observations demonstrated that members of the CCCH double zinc finger protein family TIS11/ tristetraprolin negatively regulate TNF␣, granulocyte macrophage-colony stimulating factor, and IL-3 mRNA stability through binding to the AREs present in the 3⬘-UTR of these mRNAs (28–31). Moreover, several AREs are present in the 3⬘-UTR of the VEGF gene (32). These observations prompted us to investigate whether TIS11b could similarly interfere with the 3⬘UTR of VEGF mRNA. To this end, we cotransfected 3T3 fibroblasts with increasing amounts of a TIS11b expression plasmid (or a ␤-galactosidase expression plasmid, as a control) and fixed amounts of two thymidine kinase promoterdriven reporter plasmids encoding, respectively, firefly luciferase cloned upstream of the VEGF 3⬘-UTR and renilla luciferase. Although transfection of TIS11b did not modify the renilla luciferase enzymatic activity (Fig. 9A), it did result in a dose-dependent inhibition of firefly luciferase activity (Fig. 9B). This suggested that, in contrast to the results of one recent study (33), TIS11b did not interfere with the thymidine kinase promoter. It appeared to alter gene expression in a manner dependent on the 3⬘-UTR of the VEGF mRNA,


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Fig. 7. Specificity of TIS11b Induction by ACTH Primary cultures of bovine fasciculata cells were treated for 5 h with the indicated factors: none (Ctl); 10 mM DMSO; 500 ng/ml cortisol; 500 ng/ml cortisol ⫹ 10 mM DMSO; 10 nM ACTH 1–24 (ACTH); 10 nM ACTH 1–24 (ACTH) ⫹ 10 mM DMSO; 10 ␮M forskolin, as indicated. RNA was prepared for Northern blot analysis. The blot was hybridized with a 32Plabeled bovine TIS11b cDNA probe (upper panel). 18S ribosomal RNA is shown as a loading control (lower panel). This experiment is representative of two independent experiments.

resulting in increased activity of the reporter gene product. Whether this occurs through regulation of mRNA stability or translation still remains to be evaluated and will be addressed in future studies. It should be noted, however, that several attempts to visualize a physical interaction between TIS11b and the VEGF 3⬘-UTR by UV cross-linking or EMSA were unsuccessful. The mechanism of TIS11b action may thus be considerably more complex than the direct interaction with the AREs that was initially anticipated.

DISCUSSION Several experimental approaches have been developed in the past to identify ACTH-regulated genes. Because of the well characterized effect of ACTH on steroidogenesis, genes encoding steroidogenic enzymes and accessory proteins essential to steroidogenesis such as adrenodoxin and steroidogenic acute regulatory protein were among the first to be identified as ACTH-responsive genes (5, 12, 13, 34, 35). An additional group consisting of mitochondrial genomeencoded RNAs, including several subunits of oxidative phosphorylation enzymes (cytochrome oxydase, ATPase, reduced nicotinamide adenine dinucleotide dehydrogenase) was later identified by differential



Fig. 8. Differential Kinetics of VEGF and TIS11b Induction by ACTH Primary cultures of bovine fasciculata cells were treated with 10 nM ACTH 1–24 for the indicated periods of time, and total RNA was prepared after each time point. VEGF, TIS11b, and HPRT expression was then measured by RT-PCR as indicated in Materials and Methods.

screening of BAC cell cDNA libraries (11, 36). Biochemical purification of ACTH-induced secreted proteins allowed our group to identify several ACTHstimulated extracellular proteins including thrombospondin-2, tissue inhibitor of metalloproteinases type 2, and laminin (37–40). The kinetics of ACTH induction of these three families of genes appears to be similar, beginning after 6 h of ACTH treatment and reaching a plateau after 12–24 h. On the other hand, the expression of the oncogenes c-fos, jun-B, and NGFI-B/nur77 has been shown to be rapidly (within 1–2 h) and transiently stimulated by ACTH in both ovine and BAC cells in vitro (7, 8, 10) and even more rapidly (within 30 min) in the rat adrenal in vivo (9, 13), defining a group of immediate-early genes. In the present study, we aimed to characterize genes whose expression would be induced by ACTH between the peak of immediate-early genes and the sustained activation of the families of genes induced between 6 and 12 h. We therefore designed a differential display RT-PCR assay that would allow us to selectively identify genes induced between 4 and 8 h of ACTH treatment. Due to the use of a poly Aanchored primer for the reverse-transcription step, all the amplified sequences characterized were found to lie within the 3⬘-UTRs of the genes. This may explain why several differentially amplified sequences were noninformative due to their homology to polyA-retrotransposon sequences such as Alu, MER, or SINE (41). Only two differential amplicons were confirmed by Northern blot analysis to be induced by ACTH and provided sequence homology to known genes. These amplicons encoded the manganese-dependent SOD (SOD2) and the zinc finger protein TIS11b. SODs (EC 1.15.1.1) are metalloenzymes that act as scavengers of the free oxygen radical superoxide. Three mammalian forms of SOD have been characterized: a cytoplasmic copper, zinc SOD (CuZn-SOD, SOD1), a mitochondrial manganese SOD (Mn-SOD, SOD2) and an extracellular copper, zinc SOD

(EC-SOD, SOD3) (42, 43). All three enzymes have been previously characterized in the adrenal cortex (44–46). We report for the first time that SOD2 expression is induced 3- to 10-fold by ACTH in BAC cells. This induction is cAMP mediated but is independent of the synthesis of steroids, suggesting that it is directly triggered by ACTH and does not result from a feedback mechanism switched on by steroid end products. Induction of SOD2 by oxidants such as reactive oxygen species has been reported in renal glomerular cells (23). In the adrenal cortex, the abundant consumption of molecular oxygen by the steroid hydroxylase enzymes results in the sustained physiological production of potentially cytotoxic oxygen-derived free radicals (47), which may trigger induction of SOD2. However, neutralization of free radicals by DMSO does not prevent ACTH induction of SOD2 expression. These radicals, including superoxide ions, may initiate lipid peroxidation and loss of cytochrome P45011␤ activity (24–26). Together with ascorbate, which is present in high concentrations in the adrenal cortex, SODs participate in the protection of adrenocortical steroidogenic cells against the cytotoxic effects of these deleterious byproducts (48, 49). The induction of SOD2 and the absence of induction of SOD1 by ACTH thus suggest that SOD2 may be the most important enzyme in this process. Given its subcellular mitochondrial colocalization with a number of steroid hydroxylases, SOD2 is likely to eliminate superoxide radicals at their site of production. Induction of SOD2 by ACTH thus represents a coordinated mechanism of prevention against the toxic effects that could result from increased steroidogenesis. In line with this conclusion, the recent generation of SOD1-deficient and SOD2-deficient mice has revealed that homozygous mutant mice lacking SOD1 can survive to the adult stage, whereas homozygous mutant mice lacking SOD2 die perinatally due to oxidative mitochondrial injury in several tissues (50, 51).



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Fig. 9. Effect of TIS11b Expression on VEGF 3⬘-UTR NIH-3T3 fibroblasts were cotransfected with 1, 10, or 50 ng of either a bovine TIS11b (hatched bars) or a control ␤-galactosidase (gray bars) expression plasmid, with 500 ng of the pFLuc-V3⬘ plasmid, 10 ng of pRL-TK, and a complementary quantity of pUC19 up to a total of 1 ␮g of plasmid DNA. After 48 h of expression, cells were lysed and both renilla and firefly luciferase activities were measured in a luminometer as described in Materials and Methods. Panel A represents the renilla luciferase activity. Panel B represents the ratios between firefly luciferase and renilla luciferase activities. This is a representative experiment of three independent experiments. Each point was performed in triplicate, and the mean values ⫾ SEM obtained in TIS11b- and ␤-galactosidase-expressing cells were compared using the unpaired t test. *, P ⬍ 0.01.

TIS11b belongs to a relatively poorly studied class of zinc finger proteins containing two tandem fingers of the CCCH type (i.e. a CX8CX5CX3H motif). The prototype of this family is TIS11 (TPA-inducible sequence 11), also known as tristetraprolin and Nup475, which was initially identified in fibroblasts as the product of an immediate early response gene induced by insulin, serum, or the phorbol ester TPA (21, 52, 53). TIS11b (also known as cMG1, ERF1, and Berg-36) and TIS11d (ERF2) are the other two mammalian members of this family. Both are constitutively expressed at a low level, and are inducible by a number of factors

(mostly mitogenic) in different cell types (21, 22, 54). After remaining elusive for several years, the biological function of the TIS11 family has begun to be better characterized by the work of Blackshear and colleagues (16, 28–30, 55–57). TIS11 was shown to be rapidly phosphorylated and translocated from nucleus to cytoplasm in response to mitogens (55, 57). More recently, it was reported that TIS11, TIS11b, and TIS11d all bind to the AREs of TNF␣, granulocyte macrophage-colony stimulating factor, and IL-3 mRNAs in vitro and induce the destabilization and breakdown of these mRNAs (16, 28–31). These effects

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are relevant to the cachectic and arthritic phenotype of TIS11-deficient mice and its correction by treatment with antibodies to TNF␣ (56). In the present study, we report for the first time that ACTH, a trophic hormone for the adrenal cortex but not a growth factor per se, strongly and rapidly induces TIS11b expression in adrenocortical glandular cells. This induction, starting after 30 min and peaking between 3 and 5 h after ACTH treatment, positioned the expression of this gene between that of immediate early genes (c-fos, jun-B, and NGFI-B) (7, 10) and that of stably induced genes such as those encoding steroidogenic enzymes or extracellular matrix proteins. We recently observed that ACTH also transiently induces the expression of VEGF, an angiogenic cytokine, reaching a maximum after 2–3 h of ACTH treatment and then decreasing rapidly (27). This was further characterized in the present study in which we observed that induction of TIS11b expression closely followed the induction of VEGF expression by 1 h and remained elevated when VEGF mRNA levels started to drop. A functional interaction between TIS11b and the 3⬘-UTR of VEGF was also clearly demonstrated, although we were unable to demonstrate a direct physical interaction between this zinc finger protein and sequences within the mRNA. As the regulation of VEGF expression by several factors (including mitogens, insulin, advanced glycation end-products, and hypoxia) occurs through a complex combination of transcriptional and posttranscriptional mechanisms (32, 58–63), the mechanisms of action of TIS11b are also likely to be complex and will require further work to be deciphered. In conclusion, this study, identifying for the first time both SOD2 and TIS11b as novel ACTH-regulated genes, provides several potentially novel directions in our understanding of the signaling cascade triggered by ACTH in steroidogenic adrenocortical cells.

MATERIALS AND METHODS


mary culture, as described in the text. Microdissection of glomerulosa and fasciculata layers was realized using a Steadie-Riggs microtome. The glands were sagitally midsectioned and the medulla was detached with a scalpel. The cortex was then serially sectioned starting from reticularis layer up to the capsule. The first two or three slices containing reticularis cells were eliminated. The middle slices from the prominent fasciculata zone were collected, and the last two slices before the capsule were collected as glomerulosa tissue. RNA from these samples was then prepared for Northern blot analysis. Differential Display RT-PCR Differential display RT-PCR was performed by a variation of the method of Liang et al. (Ref. 65 and H. Ernoe, personal communication). RNA (200–400 ng) from control or ACTH-treated cells was reverse transcribed using 1 ␮M of a HindIII-heeled T10 primer with a single base anchor (AAGCTTTTTTTTTTX, where X ⫽ A, C, or G), 20 ␮M deoxynucleoside triphosphates (dNTPs), and 200 U SuperScript II (Life Technologies, Inc., Cergy Pontoise, France) in a 30-␮l reaction volume. PCR with the same T10 primer and a HindIII-heeled arbitrary 13 mer primer (e.g. RandomA primer: AAGCTTAACGAGG) were carried out using 2 ␮l of the reverse transcription reaction in 1.5 mM MgCl2, 1 ␮M T10 primer, 0.2 ␮M arbitrary primer, 4 ␮M dNTPs, and 1 ␮Ci 33 P-dATP, in a 20 ␮l reaction volume. Reactions were overlaid with 15 ␮l mineral oil and 1 U Taq polymerase (Promega Corp., Charbonnieres, France) was added as a hot start. The following PCR program was run in a Biometra Trioblock thermal cycler (Go¨ ttingen, Germany): 93 C for 30 sec, 39 C for 60 sec, 72 C for 60 sec for 3 cycles; 93 C for 30 sec, 43 C for 60 sec, 72 C for 60 sec for 37 cycles; 72 C for 5 min. Reactions were stopped with formamide loading buffer. Aliquots were run in duplicate on a 6% sequencing gel and the gel was dried. Amplicons were visualized by autoradiography. Differentially amplified products were identified by aligning the gel with the film and then were excised from the gel and recovered by boiling for 5 min in 100 ␮l H2O. The supernatant was extracted with phenol/CHCl3, ethanol precipitated, and resuspended in 20–40 ␮l H2O. Ten microliters were used to reamplify products with 1.5 mM MgCl2, 160 ␮M dNTPs, 1 ␮M T10 primer, 0.6 ␮M arbitrary primer, and 5 U Taq polymerase in a 50-␮l reaction volume using the same PCR program as above. Bands were gel purified and used to probe Northern blots. Amplicons giving a positive signal were cloned into pGEMT-Easy (Promega Corp.) or pUC18 (Amersham Pharmacia Biotech, Les Ulis, France) and sequenced by Genome Express (Grenoble, France) on an automated sequenator (ABI Prism model 377, Applied Biosystems, Foster City, CA).

Reagents Culture media and sera were purchased from Life Technologies, Inc. (Gaithersburg, MD). ACTH 1–24 and metyrapone were provided by Ciba (Basel, Switzerland). Angiotensin II, forskolin, aminogluthetimide, and dimethylsulfoxide were obtained from Sigma (St. Louis, MO). Adrenocortical Cell Culture and Adrenal Cortex Microdissection Bovine adrenal glands were collected on ice at the local slaughterhouse and primary cultures of BAC fasciculata cells were prepared as previously described (64). BAC cells were cultured at 37 C in Ham’s F12 medium supplemented with 10% horse serum, 2.5% FCS, 100 U/ml penicillin G, and 100 ␮g/ml streptomycin under a 5%CO2-95%air atmosphere. On d 4, cells were stimulated for 0, 4, 5.5, 6, 7, 8.5, or 12 h with 10 nM ACTH before RNA isolation. For Northern blots, RNA was prepared from BAC cells that were serum starved for 24 h before stimulation with various inducers on d 4 of pri-

Northern Blot Analysis Total RNA was isolated from primary cultures or slices of fresh tissue using the RNAgents kit (Promega Corp.). RNA (20 ␮g) was run overnight at 25 V on a 1% agarose, 1.9% formaldehyde gel, as described by Fourney et al. (66), then vacuum blotted onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech). Membranes were hybridized for 2 h at 65 C in RapidHyb Buffer (Amersham Pharmacia Biotech) with 32P-labeled, random-primed probes and then washed 20 min in 2⫻ SSC, 0.1% SDS at room temperature, 10 min in 1⫻ SSC, 0.1% SDS at 65 C and 10 min in 0.1⫻ SSC, 0.1% SDS at 65 C. Approximately 300-bp cDNA fragments from the coding sequences of bovine SOD1, SOD2, TIS11b, and RPL27 were generated by RT-PCR from BAC cell RNA as described below and used as probes. Hybrids were visualized and quantitated using a ␤ imager (Phosphor Imager; Molecular Dynamics, Inc., Sunnyvale, CA). Probes were stripped by incubating the membrane for 15 min in hot 0.1% SDS and hybridized under the same conditions with a


cDNA probe for the ribosomal large subunit protein RPL27 or the 18S rRNA as a loading control.


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cycles. The PCR products were cloned into pGEMT-Easy and sequenced for confirmation of their identity (Genome Express, Grenoble, France).

RACE PCR DNA Transfection and Dual Luciferase Assay Primers were chosen from the sequence of the clone HTAa1 and 5⬘-RACE PCR was performed from a cDNA library previously prepared from ACTH-stimulated (24 h) BAC cells (38) using the Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Montigny le Bretonneux, France). RACE PCR was performed according to the manufacturer’s directions, using the primer 5⬘-GATGGGATTGTGAGAGCGTGACTG-3⬘ and the AP1 primer included in the kit. A 690-bp fragment was isolated, cloned into pGEMT-Easy, and sequenced (Genome Express). Plasmid Construction pCMV-TIS11b was obtained by cloning TIS11b cDNA obtained by PCR from H295R cells into the pTarget vector (Promega Corp.), which carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region. The pFLucV3⬘-plasmid that contains the firefly luciferase cDNA cloned upstream of the VEGF 3⬘-UTR and downstream of the thymidine kinase promoter was provided by Dr. Edurne Berra (CNRS UMR 6543, Nice, France) and prepared from the pSP64 clone containing nucleotides 1–2,201 of the rat VEGF 3⬘-UTR (GenBank accession no. U22372) described by Levy et al. (67). RT-PCR Analysis For RT-PCR analysis of gene expression, total RNA was prepared from BAC cells and reverse transcribed into cDNAs as previously described (27). Differential RT-PCR amplification of VEGF mRNA isoforms was performed using the same primers and amplification conditions described by Gaillard et al. (27). For TIS11b amplification, we used the following pair of primers: 5⬘-CGAAGAAAACGGTGCCTGTAAG-3⬘ and 5⬘AGTAGGTGAGCCCAAGAGGTCATC-3⬘ and the following conditions: 94 C for 1 min, 55 C for 1 min, 72 C for 1 min for 30 cycles. This allowed the amplification of a 354-bp fragment. For HPRT amplification, we used the following pair of primers: 5⬘-GCCATCACATTGTAGCCTCT-3⬘ and 5⬘-TGCGACCTTGACCATCTTTGG-3⬘ and the following conditions: 94 C for 1 min, 55 C for 1 min, 72 C for 1 min for 30 cycles. This allowed the amplification of a 305-bp fragment. Amplification products were then analyzed by 1.5% agarose gel electrophoresis and visualized under UV light. For the preparation of cDNA probes to be used in Northern blot analysis, PCR primers for the bovine SOD2 and SOD1 coding regions were chosen from the published sequences using the Mac Vector program (Genetics Computer Group, Madison, WI). A 321-bp PCR product corresponding to SOD2 nucleotide (nt) 372–692 was generated by RT-PCR of BAC cell total RNA using primers 5⬘-GGAATTGCTGGAAGCCATCAAACG-3⬘ and 5⬘-TTGCTGCAAGCCGTGTATCGTG-3⬘ with the following program: 94 C for 30 sec, 59 C for 45 sec, 72 C for 30 sec for 30 cycles. A 437-bp PCR product corresponding to SOD1 nt 4–440 was generated by RT-PCR of BAC cell total RNA using primers 5⬘-TGTTCTGCGGCGTCGTTTTCTC-3⬘ and 5⬘-TTTCATGGACCACCATCGTGCG-3⬘ with the following program: 94 C for 30 sec, 61 C for 45 sec, 72 C for 30 sec for 30 cycles. Primers for the bovine TIS11b sequence were chosen by inspection from highly conserved regions in the rat and human homologs. A 262-bp amplification product corresponding to nt 1,131–1,393 of the human sequence (called ERF-1) was generated by RT-PCR using the primers 5⬘-ATGGACGTGGGGCTGTCCAG-3⬘ and 5⬘-CACGGCATCCACGAGTCC-3⬘ with the following program: 94 C for 30 sec, 62 C for 45 sec, 72 C for 45 sec for 35

NIH-3T3 fibroblasts were grown in monolayers in DMEM supplemented with 10% FCS, 2% glutamine, 100 U/ml penicillin G, and 100 ␮g/ml streptomycin. Cells were transfected with lipofectamine (Life Technologies, Inc.) according to the manufacturer’s recommendations. Transfections were performed in six-well plates (3 ⫻ 105 cells per well) with 0–50 ng of either pCMV-TIS11b or PRK7␤gal (a control plasmid in which ␤-galactosidase expression is driven by the CMV immediate-early enhancer/promoter region), 500 ng pLuc-V3⬘, 10 ng of pRL-TK (Promega Corp.), and varying quantities of pUC19 up to a total of 1 ␮g of plasmids. Forty-eight hours after transfection, cells were lysed and luciferase activities were measured with the Dual-Luciferase reporter assay system (Promega Corp.) on a LUMAT LB 9507 luminometer (EGG-Berthold, Bad Wildbad, Germany). Results are expressed as relative light units of luciferase activity in the presence of CMV-TIS11b plasmid over relative light units of luciferase activity in the presence of the same amount of the PRK7␤gal plasmid. Renilla and firefly luciferase activities were measured sequentially. The results are shown as the mean ⫾ SEM of triplicate experiments. The unpaired t test was employed to assess differences between two groups.

Acknowledgments We are indebted to Dr. Edurne Berra (CNRS UMR 6543, Nice, France) and Dr. Andrew Levy (Technion Israel Institute of Technology, Haifa, Israel) for providing us the pFLuc-V3⬘ reporter plasmid. We thank Dr. Edurne Berra and Dr. Herve´ Prats (INSERM U397, Toulouse, France) for helpful discussions and experimental suggestions. We would like to thank Isabelle Gaillard and Dr. Ce´ line Brand for preparations of primary cultures of adrenocortical cells.

Received March 14, 2001. Accepted February 4, 2002. Address all correspondence and requests for reprints to: Dr. Jean-Jacques Feige, INSERM EMI 01-05, DBMS/BRCE, CEA/G, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9, France. E-mail: [email protected]. This work was supported by the Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM) and the Commissariat a` l’Energie Atomique. A.M.C. and J.L. were recipients of postdoctoral fellowships (poste vert and poste orange, respectively) from INSERM. * These authors contributed equally to this report. † Present address: Incyte Genomics, Palo Alto, California 94304. ‡ Present address: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada.

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