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Molecular Endocrinology 17(8):1622–1639 Copyright © 2003 by The Endocrine Society doi: 10.1210/me.2003-0054

Glucocorticoids Regulate the Expression of the Mouse Urocortin II Gene: A Putative Connection between the Corticotropin-Releasing Factor Receptor Pathways ALON CHEN, JOAN VAUGHAN,

AND

WYLIE W. VALE

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037 Peptides encoded by the urocortin II (Ucn II) gene were recently identified as new members of the corticotropin-releasing factor (CRF) family. Ucn II is a specific ligand for the type 2 CRF receptor. Using RT-PCR, DNA sequencing, and immunofluorescence staining, we report the expression of Ucn II mRNA in several human and mouse (m) neuronal cell lines. Using these neuronal cell lines, we provide evidence that exposure to glucocorticoid hormones increases mUcn II mRNA expression and promoter activation. The effect of glucocorticoids on mUcn II mRNA expression was tested in the Ucn II/glucocorticoid receptor-positive cell line NG108-15. The results demonstrate that mUcn II mRNA expression is up-regulated by dexamethasone in a dose- and time-dependent fashion. Computer analysis revealed the presence of 14 putative half-palindrome glucocorticoid response element sequences within 1.2 kb of the mUcn II 5ⴕ flanking region. Transfections with different fragments of the 5ⴕ-flanking region of the mUcn II gene fused to a luciferase reporter gene showed a promoter-dependent expression of the

reporter gene and regulation by dexamethasone. Promoter deletion studies clarify the sufficient putative glucocorticoid response element site mediating this effect. The steroid hormone antagonist RU486 blocked the effect of dexamethasone on mUcn II mRNA expression and promoter activation, suggesting a direct glucocorticoid receptormediated effect of dexamethasone on mUcn II mRNA expression. Ucn II is expressed in vivo in the hypothalamus, brainstem, olfactory bulb, and pituitary. Low levels were also detected in the mouse cortex, hippocampus, and spinal cord. We demonstrated that mUcn II gene transcription was stimulated by glucocorticoid administration in vivo and inhibited by removal of glucocorticoids by adrenalectomy. Administration of dexamethasone to mice resulted in an increase of mUcn II levels in the hypothalamus and brainstem but not in the olfactory bulb region 12 h following ip injection. In light of our present data and the current literature, we propose a putative link between the CRF receptor 1 and CRF receptor 2 pathways. (Molecular Endocrinology 17: 1622–1639, 2003)

P

are more related to Ucn III than to Ucn or CRF (6–8). The effects of CRF-related peptides are mediated through activation of two known receptors, CRF receptor 1 (CRFR1) (9), and CRFR2 (10–13). Receptor binding studies have demonstrated that human and mUcn II bind and activate with high affinity and selectivity to the CRFR2 compared with CRFR1 (1, 6). In addition, Ucn II exhibits high potency to stimulate intracellular cAMP accumulation in cells stably transfected with CRFR2 but not with CRFR1 (6). The distribution of CRFR1 and CRFR2 in the central nervous system (14–16) and periphery (10–13) are distinct and imply diverse physiological functions, as demonstrated by the phenotypes of the CRFR1 or CRFR2 null mice. Although mice deficient for CRFR1 display decreased anxiety-like behavior and have an impaired stress response (17, 18), the CRFR2 mutant mice reveal increased anxiety-like behaviors and an accelerated HPA response to stress (19–20). Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system,

EPTIDES ENCODED BY the urocortin (Ucn) II gene, also known as the stresscopin-related gene, were recently identified as new putative members of the corticotropin-releasing factor (CRF) family (1, 2). CRF, originally isolated from hypothalamus (3), plays an important and well-established role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis and in other endocrine, autonomic, and behavioral responses to stress (4). In addition to CRF and Ucn II peptides, the mammalian CRF peptide family contains Ucn (5) and Ucn III, also known as stresscopin (2, 6). Human (h) and mouse (m) Ucn II cleaved peptides are 76% identical, with the 38 amino acid sequence associated with high biological activity, and Abbreviations: ADX, Adrenalectomy; CRF, corticotropinreleasing factor; CRFR, CRF receptor; DNase, deoxyribonuclease; ␤-gal, ␤-galactosidase; GR, glucocorticoid receptor; GRE, glucocorticoid response element; h, human; HPA, hypothalamus-pituitary-adrenal; m, mouse; PB, phosphate buffer; PVN, paraventricular nucleus; RT, reverse transcriptase; TPA, phorbol-12-myristate-acetate; Ucn, urocortin.

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Chen et al. • Regulation of mUcn II by Glucocorticoids

including the paraventricular and arcuate nuclei in the hypothalamus and locus coeruleus in the brainstem (1). Ucn II injected intracerebroventricularly to rats produced mild motor suppressive and a delayed anxiolytic-like behavior, suggesting an effect of Ucn II in the regulation of stress-related behaviors (21). In contrast, Pelleymounter et al. (22) reported that central administration of Ucn II dose dependently increased anxiety-like behavior in the plus maze task. In addition, administration of the CRFR2 antagonist anti-sauvagine-30 resulted in increased (22, 23) or decreased anxiety-like behavior (20). It is well established that the expression of CRF mRNA and peptide in the hypothalamus is negatively regulated by glucocorticoids, as demonstrated by the response to dexamethasone administration and adrenalectomy (ADX) (24–33). Thymic Ucn mRNA was recently reported to be increased by ACTH and corticosterone injections (34). In addition, Ucn expression in the Edinger-Westphal nucleus was demonstrated to be up-regulated in CRF-deficient mice compared with that in wild-type mice (35). Glucocorticoid treatment of CRF-deficient and wild-type mice did not affect basal mRNA Ucn levels in the Edinger-Westphal nucleus indicating that the up-regulation is not due to a lack of inhibition by glucocorticoids (35). In the present study, we examine the endogenous expression of Ucn II in mouse and human cell lines and use them as a model systems to study the regulation of Ucn II at the mRNA and promoter levels. Furthermore, we present data on the regulation of mUcn II in the whole animal after dexamethasone administration and ADX.

RESULTS Ucn II mRNA and Peptide Expression in Human and Mouse Cell Lines Total RNA preparations derived from the six human and five mouse neuronal cell lines were reverse transcribed to generate cDNA pools. The cDNA products were used as templates for semiquantitative RT-PCR analysis using specific primers for human or mouse Ucn II and for the ribosomal proteins S14 and S16, which served as internal controls. To avoid false positive results caused by DNA contamination, each PCR was performed both on cDNA and RNA obtained from the same sample. Predicted human or mouse Ucn II cDNA fragments were isolated from agarose gels and subcloned into pGEM-T vector, and the nucleotide sequences were determined. Their sequences were compared with those in the GenBank database and were found to be identical with the known sequences of human and mUcn II. The RT-PCR and sequence data demonstrate that mUcn II (Fig. 1A) is expressed in the neuroblastoma cell lines NIE115 and NG108-15 and in the embryonal carcinoma cell line P19, with or without retinoic acid treatment, which cause differentiation into neural or glial-like phenotype. hUcn II is expressed in the neuroblastoma cell line SKNSH, the

Mol Endocrinol, August 2003, 17(8):1622–1639

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medulloblastoma cell line TE671, and the neuroepithelioma cell line SKNMC. Other mouse and human cell lines that were studied for the expression of Ucn II mRNA, GT1–7, CATHa, IMR32, A673, and LAN1, did not express detectable levels of Ucn II mRNA. The ribosomal proteins mS16 (Fig. 1A) and hS14 (Fig. 1B), that served as internal controls, were expressed in all cDNA preparations (Fig. 1, A and B, lower panels). The RT-PCR data for expression of Ucn II-mRNA were confirmed by immunofluorescence staining for mUcn II and hUcn II peptides. Figure 1 demonstrates mUcn II-like immunostaining in mouse cell lines (Fig. 1C) and hUcn II-like immunostaining in human cell lines (Fig. 1D). Immunoreactivity in the mouse cell line NG108-15 (Fig. 1C, a–c), NIE115 (Fig. 1C, d–f), P19 (Fig. 1C, g–i) and in human cell lines SKNSH (Fig. 1D, a–c), SKNMC (Fig. 1D, d–f) and TE-671 (Fig. 1D, g–i) is shown. We observed that different levels of immunoreactive intensity could be observed in the same cell line. The immunocytochemistry study did not attempt to compare quantitatively the Ucn II levels between the cell lines, but rather to verify the presence of peptide immunoreactivity in the examined cells. Preabsorption of mUcn II or hUcn II antiserum with an excess of synthetic mUcn II or hUcn II peptides, respectively, abolished the immunoreactive staining [Fig. 1, C (c, f, and i) and D (c, f, and i)]. Glucocorticoid Receptor (GR) Expression in Ucn II Positive Cell Lines and the Effect of Forskolin, Phorbol-12-Myristate-Acetate (TPA), and Dexamethasone on mRNA Expression in NG108-15 Cells Glucocorticoids are regulatory substances in the HPA axis that control the expression of CRF by parvocellular paraventricular cells. To establish a model cell line suitable for the investigation of Ucn II regulation, we used RT-PCR to examine whether the hUcn II and mUcn II-positive cell lines express GR. Our results demonstrate that all three human cell lines, TE671, SKNSH and SKNMC (Fig. 2A), and only one mouse cell line, NG108-15, (Fig. 2B) express GR. We selected the mouse NG108-15 cells as a model system for studying the regulation of mUcn II mRNA expression. A mouse cell line was chosen to investigate Ucn II mRNA and promoter regulation as these studies would be extended to in vivo models of Ucn II regulation. To examine the regulation of mUcn II mRNA, NG108-15 cells were treated with 10 ␮M forskolin (Fig. 2C), 20 nM TPA (Fig. 2D), or 25 nM dexamethasone (Fig. 2E) for 24 h. Expression levels of mUcn II mRNA were determined by quantitative RT-PCR for mUcn II and S16 followed by Southern hybridization for mUcn II and S16. Significant up-regulation of the mUcn II mRNA was observed after forskolin (Fig. 2C) and dexamethasone (Fig. 2E) treatment but not TPA treatment. To further determine the time- and dose-dependent effects of dexamethasone on mUcn II mRNA,

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Fig. 1. Expression of Ucn II Gene in Human and Mouse Neuronal Cell Lines A and B, Semiquantitative RT-PCR for mouse (A) and human (B) Ucn II on RNA isolated from the following cell lines: mouse GT1–7, NIE115, NG108-15, CATHa and P19 (with or without retinoic acid) lines, human IMR32, A673, TE671, SKNMC, SKNSH, and LAN1 lines. This figure demonstrates the expression of Ucn II in three mouse cell lines NIE115, NG108-15 and P19 (A), and in three human cell lines TE671, SKNMC, and SKNSH (B). The ribosomal proteins S16 (A) and S14 (B) that served as internal controls were expressed as expected in all cDNA samples. C, Immunofluorescence microscopy of the mUcn II expressing cells lines. The NG108-15 (a–c), NIE115 (d–f), and P19 (g–i) cells were incubated with a polyclonal anti-mUcn II serum followed by a Cy2-conjugated secondary antibody. Immunoreactive cells were observed using a green filter. c, f, and i, Immunoreactivity after preabsorbtion of the antibody with excess of mUcn II peptide. Scale bar, 10 ␮m. D, Immunofluorescence microscopy of the hUcn II expressing cells lines. The SKNSH (a–c), SKNMC (d–f), and TE671 (g–i) cells were incubated with a polyclonal anti-hUcn II serum followed by a Cy2-conjugated secondary antibody. Immunoreactive (ir) cells were observed with a green filter. c, f, and i, Immunoreactivity after preabsorption of the antibody with excess of hUcn II peptide. Scale bar, 10 ␮m.

NG108-15 cells were cultured in the presence of 1 pM, 100 pM, 10 nM and 1 ␮M dexamethasone for 24 h. Incubation of dexamethasone, 100 pM or greater, produced a significant increase in mUcn II mRNA expression (⬃2-fold over control) (Fig. 3A). The dexamethasone induced increase in Ucn II mRNA was observed as early as 12 h after addition of 10 nM dexamethasone and was continuing to increase at 24 h (Fig. 3B). Therefore, dexamethasone up-regulates mUcn II mRNA levels in a dose- and time-dependent manner. Effect of Dexamethasone on mUcn II mRNA HalfLife and the Effect of Cycloheximide on Dexamethasone-Induced mUcn II mRNA Expression in NG108-15 Cells To examine whether the effect of dexamethasone on mUcn II mRNA levels is due to changes in transcript stability, the half-life of mUcn II mRNA in transcriptionally arrested NG108-15 cells was determined. NG108-15

cells were incubated for 24 h in medium containing vehicle or 10 nM dexamethasone followed by the addition of 1 ␮g/ml actinomycin D for 0, 2, 4, 12, and 24 h (Fig. 4A), which inhibits RNA transcription. There was no consistent effect of dexamethasone on mUcn II mRNA decay in treated cultures (Fig. 4B). These results suggest that dexamethasone does not affect the stability of mUcn II mRNA and therefore that the increases in mUcn II mRNA levels after dexamethasone treatment are not likely due to changes of mUcn II mRNA stability. To determine whether the effect of dexamethasone on mUcn II mRNA expression is dependent on de novo protein synthesis, NG108-15 cells were incubated with 10 ␮g/ml cycloheximide for 24 h in control and dexamethasone-containing medium (Fig. 4, C and D). Cycloheximide alone had no significant effect on mUcn II mRNA levels. Coincubation of 10 nM dexamethasone in the presence of cycloheximide did not affect the dexamethasone induced expression of mUcn II in NG108-15 cells (Fig. 4, C and D).



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Fig. 2. Expression of GR in Ucn II Expressing Cell Lines and Southern Hybridization of Amplified mUcn II and the Ribosomal Protein S16 cDNA Fragment after Forskolin, TPA, and Dexamethasone Treatments in NG108-15 Cells A and B, Semiquantitative RT-PCR for human (A) and mouse (B) GR in Ucn II-expressing cell lines. RNA obtained from human fetal brain (A) or from mouse total brain served as positive control. This figure demonstrates the expression of GR in all the three human cell lines, TE-671, SK-N-SH, SK-N-MC (A), and in the mouse cell line NG108-15 (B). The ribosomal proteins S14 (A) and S16 (B) were expressed as expected in all the cDNA samples. C–E, Amplified mUcn II and S16 cDNA fragments from the mouse cell line NG108-15, untreated, or treated with 10 ␮M forskolin (C), 20 nM TPA (D), and 25 nM dexamethasone (E), were hybridized to 32P-labeled oligonucleotide probes of mUcn II (upper panel) and S16 (lower panel). The hybridizations were performed sequentially on the same membrane. The results represent one of three experiments. The normalized results, corrected against S16 intensity, obtained from three independent experiments is presented by the graph, as fold increase, compared with untreated cells. *, P ⬍ 0.05 vs. untreated cells.

5ⴕ-Flanking Regions of the mUcn II Gene and Promoter Activity of Reporter Gene Constructs in NG108-15 and NIE115 Cells Three fragments of the 5⬘-flanking region of the mUcn II gene (Fig. 5A) were amplified by PCR using a mouse genomic DNA clone isolated previously in our laboratory, and were subcloned into a luciferase reporter plasmid. The basal activity of the three constructs (⫺670, ⫺1064, and ⫺2173 bp) transfected into NG108-15 cells was determined (Fig. 5B) and found to be strong for the two shorter constructs, suggesting that positive regulatory elements are located within the

first 1.2 kb. The ⫺2173-bp construct exhibited a modest activity and may indicate a negative regulatory control element in that fragment. To determine whether the mUcn II promoter contained a glucocorticoid response element (GRE) consensus site, we screened the promoter sequence for a GRE using Transcription Element Search Software (http://www. cbil.upenn.edu/tess/index.html). The sequence analysis revealed the presence of 14 putative half-palindrome GRE sequences, within the 1.2 kb of the mUcn II 5⬘ flanking region at positions ⫺86, ⫺93, ⫺187, ⫺336, ⫺391, ⫺485, ⫺580, ⫺724, ⫺904, ⫺923, ⫺942, ⫺962, ⫺1083, and ⫺1145.



Fig. 3. Quantitative RT-PCR for mUcn II mRNA Levels in NG108-15 Cells Treated with Dexamethasone A and B, Expression of mUcn II and S16 genes in NG108-15 cells treated with increasing doses of dexamethasone (1 pM to 1 ␮M) for 24 h (A) or with 10 nM dexamethasone for different periods of time (2–48 h) (B). Southern blot hybridization of amplified mUcn II (middle panels, A and B) and the ribosomal protein S16 (lower panels, A and B) cDNA fragments were performed. The radioactive bands were quantified by PhosphorImager and the normalized values (relative to the control S16 expression) are presented (mean ⫾ SD of three independent experiments, graphs A and B) as relative densitometry units. The mRNA levels of the mUcn II were significantly up-regulated after 24 h treatment of 100 pM, 10 nM, and 1 ␮M dexamethasone (A) or by 10 nM dexamethasone treatment for 12–48 h (B). *, P ⬍ 0.05 vs. untreated cells.

To demonstrate mUcn II promoter activity after dexamethasone treatment, NG108-15 cells were transfected with 2.5 ␮g of the mUcn II reporter plasmid construct ⫺670 bp (Fig. 5C), ⫺1064 bp (Fig. 5D) or ⫺2173 bp (Fig. 5E) and 50 ng of ␤-galactosidase (␤gal) construct, and were treated with different concentrations of dexamethasone (1 pM to 1 ␮M) for 24 h. Dexamethasone (10 nM) for 24 h was sufficient to induce a significant luciferase reporter gene activity in all three constructs compared with untreated NG108-15 cells (Fig. 5, C–E). A similar procedure performed using the ⫺670-bp construct transfected into NIE115 cells, which express Ucn II (Fig. 1A) but do not express GR (Fig. 2B), showed no significant changes after dexamethasone treatment (Fig. 5F). These results may imply that the effect of dexamethasone on mUcn II promoter activity is mediated through GR. To clarify the putative half palindromic GRE sites necessary or sufficient to mediate the effects of glucocorticoids, we performed additional promoter deletion experiments (Fig. 6). Seven promoter deletion constructs have been generated deleting sequentially all the seven putative half palindromic GRE sites in the

(⫺670)-(⫹206) fragment (Fig. 6, A and B). Luciferase assays of NG108-15 cells transfected with the promoter deleted constructs and treated with 10 nM dexamethasone for 24 h indicate that the 5⬘ deletion of domains that contain putative GREs results in sequential loss of glucocorticoid mediated transactivation, suggesting a role of multiple GREs in the activation of the Ucn II promoter observed after dexamethasone treatment. (Fig. 6C). Interestingly, promoter fragment (⫺85)-(⫹206), which does not contain any putative GRE sites, demonstrates relatively high basal activity but no response to dexamethasone. In addition to the evidence that the GR negative cell line, NIE-115 expresses Ucn II (Figs. 1A and 2B), and the strong basal activity of the (⫺670)(⫹260) luciferase construct in this cell line (Fig. 5F) indicates that glucocorticoids are not necessary for Ucn II expression. Effect of RU486 on Dexamethasone-Induced mUcn II mRNA Expression in NG108-15 Cells To further demonstrate that the positive effect of dexamethasone on mUcn II mRNA expression is mediated



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Fig. 4. Effect of 10 nM Dexamethasone on mUcn II mRNA Decay in Transcriptionally Blocked NG108-15 Cells by Actinomycin D and the Effect of Cycloheximide on mUcn II mRNA Regulation in NG108-15 Cells Treated with Dexamethasone A, NG108-15 cells were treated for 24 h with 10 nM dexamethasone before the addition of actinomycin D (1 ␮g/ml). Total RNA was extracted from control and treated cultures after 0, 2, 4, 12, and 24 h of 1 ␮g/ml actinomycin D treatment. RNA was analyzed by RT-PCR (upper panel) and Southern hybridization using specific 32P-labeled mUcn II probe (lower panel). Data are representative of three experiments. B, The bands were analyzed by densitometry and 0, 2, and 4 h mUcn II mRNA levels were plotted with linear regression as a percentage of the 0 h value against time. C, NG108-15 cells were treated for 24 h with vehicle, 10 ␮g/ml cycloheximide, 10 nM dexamethasone, or 10 nM dexamethasone in the presence of 10 ␮g/ml cycloheximide. Total RNA was extracted and RT-PCR (upper panel) and Southern hybridization using specific 32P-labeled mUcn II probe (lower panel) were performed. D, The corrected mUcn II mRNA signal is plotted as relative densitometry units. The presented data are representative of three identical experiments. *, P ⬍ 0.05 vs. untreated cells.

by a mechanism involving GR, the GR binding sites were blocked with the nonspecific steroid hormone antagonist RU486. RU486 (100 nM) showed no positive effect on mUcn II mRNA level when given alone but completely blocked the induction of mUcn II mRNA expression by 10 nM dexamethasone (Fig. 7A). There was no difference between the mUcn II mRNA levels of the dexamethasone plus RU486-treated group and the control group (Fig. 7A). To study whether the effect of dexamethasone on mUcn II mRNA expression is also reflected at the promoter level, a luciferase assay was performed on NG108-15 cells transfected with the ⫺670-bp mUcn II promoter construct and treated with or without RU486, in the presence of 10 nM dexamethasone (Fig. 7B). RU486 completely abolished the induction of the mUcn II promoter construct by 10 nM dexamethasone (Fig. 7B). Regulation of Mouse Brain Ucn II Expression by Dexamethasone Administration and Adrenalectomy The distribution of mUcn II mRNA in the mouse brain was determined by quantitative RT-PCR (Fig. 8A). Ucn

II was found to be expressed in all examined brain regions and in the pituitary. The three main regions in the mouse brain expressing the highest amount of Ucn II mRNA are the olfactory bulb, brainstem and hypothalamus (Fig. 8A, graph). To permit reliable quantitation of mUcn II mRNA by competitive PCR, it was necessary to generate a mUcn II cRNA bearing a short internal deletion for use as an internal standard to determine whether dexamethasone regulates mUcn II expression in vivo (Fig. 8B). We examined the effect of dexamethasone injections on mUcn II mRNA levels in three regions of the mouse brain, olfactory bulb, brainstem and hypothalamus. All three doses of dexamethasone significantly increased the expression of mUcn II in the hypothalamus (Fig. 9) and brainstem (Fig. 10) in animals killed 12 h following injection of the steroid (Figs. 9, B and C, and 10, B and C). No changes in mUcn II mRNA levels were observed in animals killed 2 h following dexamethasone injection (Figs. 9, A and C, and 10, A and C). The mUcn II mRNA expression levels in the olfactory bulb were not changed in animals killed 2 or 12 h following dexamethasone injection (data not shown).



Fig. 5. Dexamethasone Responsiveness and Basal Activity of mUcn II Promoter Constructs A, Schematic demonstration of the mUcn II 5⬘-flanking region constructs fused to luciferase gene in PGL3 Basic vector. B, Luciferase reporter gene assay of mUcn II promoters basal activity in NG108-15 cells. The cells were cultured and transfected with various reporter plasmid DNA, as described in Materials and Methods. After 24 h, the luciferase basal activity of the promoters was determined and a strong basal activity was observed for the ⫺670-bp and ⫺1064-bp gene fragment as compared with the low activity of the ⫺2173 gene fragment. C–E, NG108-15 cells were transfected with of the mUcn II reporter plasmids constructs, ⫺670 bp (C), ⫺1064 bp (D) ⫺2173 (E) and parallel plates were treated with different concentrations of dexamethasone (1 pM to 1 ␮M) for 24 h. The cells of all groups were harvested at the same time and luciferase activity was measured. The results, corrected to ␤-gal values, (mean ⫾ SD of six independent experiments) are presented as relative activity compared with the basal activity of the specific promoter construct. Significant activation of the all three-mUcn II promoter constructs is observed after dexamethasone treatment. *, P ⬍ 0.05 vs. basal promoter activity. F, NIE115 cells (mUcn II positive cells and GR negative cells) transfected with the ⫺670-bp-mUcn II reporter construct were treated with different concentrations of dexamethasone (1 pM to 1 ␮M) for 24 h. The cells were harvested, and luciferase activity was measured. The results, corrected to ␤-gal values (mean ⫾ SD of six independent experiments) are presented as relative activity, compared with the basal activity of the specific promoter construct.



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Fig. 6. Dexamethasone Responsiveness and Basal Activity of mUcn II Deleted Promoter Constructs A and B, DNA constructs (A) and schematic demonstration (B) of the deleted mUcn II 5⬘-flanking region constructs fused to luciferase gene, and the location of the putative half-palindromic GRE sites. C, NG108-15 cells were transfected with the deleted mUcn II reporter plasmids constructs, and parallel plates were treated with 10 nM dexamethasone for 24 h. The cells of all groups were harvested at the same time and luciferase activity was measured. The results, corrected to ␤-gal values (mean ⫾ SD of three independent experiments) are presented as relative activity. *, P ⬍ 0.05 vs. basal promoter activity.



Two weeks after ADX, with or without corticosterone replacement, mUcn II mRNA levels in the whole hypothalamus (Fig. 11, A–D), brainstem (Fig. 11, E–H) and olfactory bulb (Fig. 11, I–L) were determined using competitive RT-PCR. Levels of mUcn II mRNA in the whole hypothalamus (Fig. 11, A, B, and D) and brainstem (Fig. 11, E, F, and H) of ADX mice were decreased to 62% and 68% of control values, respectively. The effect was reversed by corticosterone replacement both in the hypothalamus (Fig. 11, C and D) and the brainstem (Fig. 11, G and H). In contrast to the hypothalamus and brainstem regions, neither ADX or corticosterone replacement affected mUcn II mRNA levels in the olfactory bulb (Fig. 11, I–L).

DISCUSSION

Fig. 7. Effect of the Steroid Hormone Receptor Antagonist RU486 on mUcn II mRNA Expression A, NG108-15 cells were treated for 24 h with vehicle (control), 10 nM dexamethasone, 100 nM RU486, or 10 nM dexamethasone/100 nM RU486. Quantitative RT-PCR analysis for mUcn II and S16 were performed (upper panel), followed by Southern hybridization with 32P-labeled mUcn II (middle panel) and S16 (lower panel) probes. The graph shows corrected mUcn II mRNA signals in relative densitometry units of three independent experiments. *, P ⬍ 0.05 vs. untreated cells. B, NG108-15 cells were transfected with the ⫺670 bp-mUcn II reporter construct and parallel plates were treated for 24 h with vehicle (control), 10 nM dexamethasone, 100 nM RU486, and 10 nM dexamethasone/100 nM RU486 coincubation. The cells were harvested at the same time and luciferase activity was measured. The results, corrected to ␤-gal values (mean ⫾ SD of six independent experiments) are presented as relative activity compared with the basal activity of the promoter construct. *, P ⬍ 0.05 vs. basal promoter activity. The blocking of the gloucocorticoid receptors binding site with the nonspecific steroid hormone antagonist RU486 showed inhibition of dexamethasone activation both in the mRNA (A) and promoter (B) levels.

By using human and mouse Ucn II-specific oligunucleotide primers for RT-PCR, DNA sequencing and immunofluorescence staining, we report the endogenous expression of Ucn II in several human and mouse neuronal cell lines. These human and mouse cell lines are proving to be useful tools for studying the regulation of hUcn II and mUcn II expression and for transfection studies with Ucn II promoter constructs to investigate the regulation of hUcn II or mUcn II gene expression. Ideally the host cell line should express the endogenous gene of interest, as regulatory factors that affect the endogenous promoter will also control the transfected construct. Future studies can use these cell lines for studying hUcn II or mUcn II prohormone processing. The present study provides evidence that exposure to glucocorticoid hormones increases mUcn II mRNA expression and promoter activation. The effect of glucocorticoids on mUcn II mRNA expression was tested in the Ucn II/GR-positive cell line, NG108-15. The results demonstrate that mUcn II mRNA expression is up-regulated by dexamethasone in a dose- and timedependent fashion. Experiments with actinomycin D revealed that increased mUcn II mRNA levels are not due to increased stability of transcripts. Moreover, the increase of mUcn II mRNA by dexamethasone does not require de novo protein synthesis, indicating that the effect of dexamethasone on mUcn II mRNA expression does not require de novo synthesis of protein mediators such as transcription factors (36). Computer-aided sequence analysis revealed the presence of 14 putative half-palindrome GRE sequences, within the 1.2 kb of the mUcn II 5⬘ flanking region. Transient transfections of NG108-15 cells with chimeric constructs of three different fragments of the 5⬘-flanking region of the mUcn II gene fused to a luciferase reporter gene showed a promoter-dependent expression of the reporter gene and regulation by dexamethasone. More detailed promoter deletion studies indicate that the 5⬘ deletion of domains that contain putative GREs results in sequential loss of



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Fig. 8. mUcn II Expression in Different Brain Regions and the Design of mUcn II Competitor Internal Control Construct for Competitive RT-PCR A, Total RNA was extracted from mouse brain regions and pituitary. Quantitative RT-PCR analysis for mUcn II and S16 were performed (upper panel), followed by Southern hybridization with 32P-labeled mUcn II (middle panel) and S16 (lower panel) probes. The graph shows corrected mUcn II mRNA signals in relative densitometry units of three independent experiments. B, Schematic demonstration of the mUcn II gene and the location of the oligonucleotide primers to generate endogenous and competitive fragments. The isolation of the mUcn II competitor internal control fragment was performed using primers A and BC for the PCR. The 279-bp fragment was isolated, subcloned and a cRNA was transcribed as described in Materials and Methods. After RT reactions containing 2-fold serial dilutions of mUcn II 279-bp internal standard (competitor mUcn II), in addition to the sample RNA, PCR were performed using oligonucleotide primers A and C. The PCR products (402 bp for endogenous mUcn II and 279 bp for mUcn II internal standard) were separated on 2.2% agarose gel (B, gel), visualized with ethidium bromide, photographed and quantified using ImageQuant software program 1.2. The ratio of internal standard to endogenous area was plotted as a function of the competitor concentration added to each PCR (B, graph). The concentration of mUcn II mRNA was determined where the ratio of the internal standard mUcn II and endogenous mUcn II area was equal to 1 (i.e. the equivalence point).



Fig. 9. Competitive RT-PCR Assay for Hypothalamic mUcn II mRNA after Dexamethasone Injections A–C, Saline or dexamethasone (75, 150, or 300 ␮g/kg) were injected ip to C57BL/6 mice. The mice were killed 2 h (A), or 12 h (B) after injection and the hypothalami were dissected. Total RNA was extracted followed by DNase treatment and the mUcn II mRNA levels were determined using competitive RT-PCR as described in Materials and Methods. Each experimental group includes at least three animals. The data shown are representative of three identical experiments (C). This figure demonstrates a significant increase in expression of hypothalamic mUcn II mRNA 12 h after dexamethasone injections. *, P ⬍ 0.05 vs. saline injections.

glucocorticoid mediated transactivation, suggesting a role of multiple GREs in the activation of the Ucn II promoter observed after dexamethasone treatment. RU486, a nonspecific steroid hormone antagonist, blocked the effect of dexamethasone on mUcn II mRNA expression and promoter activation, suggesting a direct glucocorticoid receptor-mediated effect of the dexamethasone on mUcn II mRNA expression. Using hybridization histochemistry, Ucn II has been reported to be located in restricted regions of the mouse and rat brain (1). Ucn II mRNA was seen to be predominantly subcortical, with major sites of expression including neuroendocrine cell groups such as the paraventricular, supraoptic, and arcuate nuclei of the hypothalamus and the locus coeruleus of the rostral pons. The trigeminal, facial, and hypoglossal motor nuclei of the brainstem, as well as the spinal ventral horn, were also identified as sites of Ucn II mRNA expression. In the present study, using a more sensitive method for mUcn II mRNA detection (RT-PCR), we demonstrate the expression of a significant amount of Ucn II in the hypothalamus and brainstem and also in the olfactory bulb and pituitary. Low levels of Ucn II mRNA were detected in the mouse cortex, hippocampus and spinal cord. We show that mUcn II gene transcription is stimulated by glucocorticoid administration in vivo and inhibited by removal of glucocorticoids by ADX in a tissue-selective manner. Administration of dexamethasone to mice resulted in an increase of mUcn II levels

in the whole hypothalamus and brainstem but not in the olfactory-bulb region 12 h following ip injections. The potent inhibitory effect of adrenal glucocorticoids on CRF biosynthesis and release from the hypothalamus have been well documented (24–33). Acute administration of dexamethasone has been found to significantly inhibit CRF mRNA accumulation in the paraventricular nucleus after stress (30). Dexamethasone has also been found to reduce CRF mRNA levels in a human primary liver carcinoma cell line, NPLC, which endogenously expresses CRF (37, 38) and in mouse anterior pituitary tumor cells, AtT-20, that were stably transfected with the human CRF gene (39). ADX induced a significant increase in CRF mRNA levels in whole hypothalamus, specifically in the PVN, which were normalized after dexamethasone replacement (32). In contrast to the hypothalamus, no effects of ADX or dexamethasone replacement were detected in the olfactory bulb, midbrain, cerebral cortex, or brainstem, indicating regional differences in the regulation of CRF mRNA by glucocorticoids (32). In addition, it has been reported that ADX is accompanied by significant elevations in CRF peptide levels in the hypothalamus and that CRF heteronuclear RNA in the PVN is augmented after ADX and inhibited after dexamethasone treatment (33). By contrast, corticosterone delivered to the amygdala increases CRF mRNA in the central amygdaloid nucleus (40). In addition, CRF from amnion, chorion, and decidual placental cells is positively regulated by cortisol and dexamethasone (41).



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Fig. 10. Competitive RT-PCR Assay for Brainstem mUcn II mRNA after Dexamethasone Injection A–C, Saline or dexamethasone (75 ␮g/kg, 150 ␮g/kg, or 300 ␮g/kg) were injected ip into C57BL/6 mice. The mice were killed 2 h (A) or 12 h (B) after injection and the brainstem was dissected. Total RNA was extracted followed by DNase treatment and the mUcn II mRNA levels were determined using competitive RT-PCR as described in Materials and Methods. Each experimental group includes at least three animals. The data shown are representative of three identical experiments (C). This figure demonstrates a significant increase in brainstem mUcn II mRNA 12 h after dexamethasone injection. *, P ⬍ 0.05 vs. saline injections.

We have recently reported the increase in thymic Ucn mRNA after corticosterone or ACTH injection, in the absence of immune stimulation, indicating that the increased Ucn mRNA by endotoxin is induced by HPA axis activation (34). In CRF-deficient mice, Ucn expression is elevated in the basal state compared with that in WT mice, and this up-regulation is not due to glucocorticoid deficiency (35). Ucn II is expressed in brain regions known to be involved in neuroendocrine, visceral, and appetitive behavioral responses. In addition, Ucn II is regulated by glucocorticoids and Ucn II peptide modulates anxiety-like behaviors after intracerebroventricular injections into rats (21, 22). Therefore, it is reasonable to assume that Ucn II can mediate stress-induced behaviors. Deletion of the CRFR1 gene during development results in an impaired stress response due to the absence of CRFR1 in the anterior pituitary corticotrophs and an increase in CRF levels in the PVN, indicating a diminished negative feedback response due to decreased corticosterone levels and a decreased anxiety-like behavior (17, 18). In addition, administration of CRFR1 antagonists are active in a variety of tests for anxiety-like behavior, such as elevated plus maze, defensive withdrawal, and conditioned fear (42–45). In our hands, the CRFR2-mutant mice display an opposite phenotype with increased anxiety-like behavior and hypersensitivity to stress (19–20). In addition, CRF and Ucn III were found to be elevated in the PVN and in the lateral perifornical region of CRFR2mutant mice, respectively (46). Coste et al. (47) did not

find significant changes in anxiety levels in CRFR2 mutant mice, whereas Kishimoto et al. (20) detected anxiety-like behavior only in male mutant mice. Differences in breeding schemes and mouse strains have been proposed to explain the variation in results obtained for mutant mice and in these pharmacological experiments. In addition, administration of the CRFR2 antagonist anti-sauvagine-30 resulted in increased (22, 23) or decreased anxiety-like behavior (20). Thus, in contrast to the unanimity regarding the role of the CRFR1 pathway in physiological and behavioral responses to stress, the function of CRFR2 and its ligands are unclear at this time. In light of our present data and the current literature, we propose a putative link between the CRFR1 and CRFR2 pathways (Fig. 12). The well-established activation of the HPA axis, mediated by the CRFR1 pathway, results in an increase in plasma glucocorticoids, which in response will modulate centrally the expression of CRF and at least two of the urocortins, respectively. Overexpression of the Ucn II peptide, in parallel to reduction in the CRF expression, would lead to activation of the CRFR2 pathway and to inhibition of the CRFR1 pathway, resulting in the same endocrine or behavioral response such as reduced anxiety-like behavior. Further examining the regulation of CRFR2 and its specific ligands, Ucn II and Ucn III, may reveal the manner by which the CRFR2 pathway is involved in physiological responses to stress.



Fig. 11. Competitive RT-PCR Assay for Hypothalamus (A, upper panel), Brainstem (B, middle panel), and Olfactory Bulb (C, lower panel) mUcn II mRNA Levels after ADX or ADX with Corticosterone (Cort.) Replacement A–C, Hypothalamus, brainstem, and olfactory bulb were isolated 2 wk after ADX or ADX ⫹Cort treatment of C57BL/6 mice. The mice were killed and the hypothalamus, brainstem, and olfactory bulb were dissected. Total RNA was extracted followed by DNase treatment and the mUcn II mRNA levels were determined using competitive RT-PCR as described in Materials and Methods. Each experimental group includes at least three animals. The data shown are representative of three identical experiments (D, H, and I). This figure demonstrates a significant inhibition of hypothalamic and brainstem but not olfactory bulb mUcn II mRNA expression levels after ADX. The mUcn II mRNA level returned to normal levels after corticosterone administration. *, P ⬍ 0.05 vs. control mice.

MATERIALS AND METHODS

Invitrogen Corp.). The cells were cultured at 37 C in a humidified atmosphere of 5% CO2/95% air.

Cell Culture

RNA Preparation and Semiquantitative RT-PCR

The following human and mouse neuronal cell lines were used: human neuroblastoma cell lines, SK-N-SH (48), IMR32 (49), and LAN-1 (50); human medulloblastoma cell line TE671 (51); human neuroepithelioma cell lines, A673 (52), and SK-N-MC (48); mouse neuroblastoma cell lines NIE115 (53) and NG108-15 (54); mouse embryonal carcinoma cell line P19 (55) (the P19 cells were differentiated into neural and glial like cells by 500 nM retinoic acid) (56); and immortalized mouse neuronal cell lines, GT1–7 (57) and CATHa (58). The NIE-115, GT1–7, NG108-15, TE-671, and A673 cell lines were maintained in DMEM (Gibco Invitrogen Corp., Grand Island, NY). The IMR32, SK-N-SH, SK-N-MC and P19 cell lines were maintained in MEM (Gibco Invitrogen Corp.), whereas the LAN-1 and CATHa cell lines were maintained in RPMI 1640 medium (Gibco Invitrogen Corp.). The media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin (100 IU/ml), and streptomycin (100 ␮g/ml) (Gibco

Total RNA was extracted from human and mouse cell lines using the RNeasy Mini Kit (QIAGEN Inc., Valencia, CA), according to the manufacturer’s recommendations. Total RNA was also extracted from different regions of the mouse brain using the Trizol RNA isolation reagent (Molecular Research Center, Cincinnati, OH) based on the acid guanidinium thiocyanate-phenol-chloroform extraction method according to the manufacturer’s recommendations. To avoid false positive results caused by DNA contamination, we performed a deoxyribonuclease (DNase) treatment for 30 min at 37 C using the RQ1 ribonuclease-free DNase (catalog no. M6101, Promega Corp., Madison, WI). We used semiquantitative RTPCR to amplify the levels of endogenous Ucn II that may be present in the human and mouse cell lines studied. Total RNA extracted from mouse brain or total human fetal brain RNA (catalog no. 738005, Stratagene, La Jolla, CA) served as positive controls. The expression of the mRNA of ribosomal



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Fig. 12. Schematic Presentation of a Putative Feedback Connection between CRFR1 and CRFR2 Central Pathways Activation of the HPA axis (mediated by the CRFR1 pathway), manifested by CRF release to the portal blood system followed by ACTH release from the pituitary to the general circulation, will lead to accumulation of plasma glucocorticoids. The circulating glucocorticoids will down-regulate or up-regulate centrally the expression of CRF or Ucn II, respectively. The overexpression of Ucn II peptide in parallel to the reduction in CRF expression will lead to activation of the CRFR2 pathway and to inhibition of the CRFR1 pathway, resulting in the same endocrine or behavioral response such as reduced anxiety-like behavior.

proteins S16 (59) for mouse cell lines and S14 (60) for human cell lines served as internal controls. The PCR conditions were as follows: the cDNA equivalent to 200 ng of total RNA was amplified by PCR for 35 cycles at an annealing temperature of 60 C. The final MgCl2concentration was 3 mM, and each reaction contained 2.5 U of Taq DNA polymerase (BIOX-ACT DNA polymerase, Bioline UK Ltd., London, UK). Antiserum and Controls Anti-hUcn II serum (PBL no. 6488, 12/07/00 bleed) was raised in rabbit using the synthetic peptide GlyTyr-hUcn II; antimUcn II serum (PBL no. 6555, 09/12/01 bleed) was raised in rabbit using the synthetic peptide GlyTyr-mUcn II. Synthetic peptides were conjugated to human ␣-globulins via bisdiazotized benzidine using methods previously described for inhibin subunits (61). To test the specificity of the antisera, we included control sections in which the antisera (1:2000–1: 4000 final) were preabsorbed with an excess (2–100 ␮g) of synthetic mUcn II or hUcn II for 24 h at 4 C. Additional control sections were incubated without the first antibody or with normal rabbit serum or with an irrelevant antibody. Fluorescence Immunocytochemical Analysis Human and mouse cell lines that were found to express Ucn II were subjected to immunofluorescence staining using specific Ucn II antibodies developed in our laboratory (rabbit anti-mUcn II and rabbit anti-hUcn II). NIE-115, NG108-15, P19 mouse cells, and TE671, SK-N-MC, SK-N-SH human

cells were analyzed by fluorescence immunocytochemistry. The cells were plated on round glass coverslips (13 mm) coated with poly-L-lysine (15 ␮g/ml), in 24-well culture plates. Two days later, the cells were fixed by the addition of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 30 min, washed (three times for 5 min each) with PB, and permeabilized for 3 min with 0.5% Triton X-100. After washing (three times), the cells were incubated for 2 h at room temperature in a blocking medium (PBS containing 10% normal donkey serum, 2% BSA, 1% glycine, and 0.5% Triton X-100) to saturate nonspecific binding sites for IgG. The human and mouse specific Ucn II primary antiserum (1:2000– 1:4000 final) were added for 12–15 h at 4 C, and the cells were washed (three times for 5 min each) with 0.1 M PB. The cells were then incubated for 2 h at room temperature with Cy2-conjugated affinipure donkey antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). The cells were washed with PB and coverslipped with fluorescence mounting medium. Cells were visualized under a fluorescence microscope (ECLIPSE E600, Nikon Corp., Tokyo, Japan) equipped with filters for Cy2 epifluorescence. Quantitative RT-PCR and Southern Analysis We used quantitative RT-PCR (62) to amplify the levels of endogenous mUcn II mRNA present in the mouse cell line NG108-15 after 24-h treatment with 10 ␮M forskolin (Calbiochem-Novabiochem Corp., La Jolla, CA), 20 nM TPA (Calbiochem-Novabiochem), and 25 nM dexamethasone (Sigma Chemical Co., St. Louis, MO) (Fig. 2, C–E). mUcn II mRNA levels were also determined in NG108-15 cells treated with


different doses of dexamethasone and at different time points (Fig. 3). The expression of the ribosomal protein, S16 (59), served as internal control. Each reaction contained four oligonucleotide primers, two for the mUcn II and two for the S16 internal control. The PCR conditions were: cDNA equivalent to 200 ng of RNA was amplified by PCR for 32–35 cycles; the annealing temperature was 60 C; the final MgCl2 concentration was 3 mM; and each reaction contained a 2.5 U of Taq DNA polymerase (BIO-X-ACT DNA polymerase, Bioline UK Ltd.). The PCR products were transferred onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Buckinghamshire, UK) by overnight capillary blotting in 20⫻ sodium chloride/sodium citrate solution and the nylon was baked in a vacuum oven at 80 C for 2 h. Overnight hybridizations were performed sequentially on the same membrane, using Super Hyb Kit (Molecular Research Center Inc., Cincinnati, OH) in the presence of a 32P-labeled probe, specific to the mUcn II or S16 cDNA. Hybridizations were performed at 60 C for mUcn II and at 62 C for the S16 probe. The corresponding bands could be seen after exposure of the membranes to PhosphorImager plates (445 SI, Molecular Dynamics, Inc., Jersey City, NJ). Gels were also exposed to x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) for 2–16 h at ⫺80 C and were developed in CURIX 60 processor (AGFA, Ko¨ ln, Germany). Oligonucleotide Primers Sense and antisense primers were selected, when possible, to be located on different exons to avoid false positive results caused by DNA contamination. The specific human and mouse oligonucleotide primers for Ucn II, GR, and the internal control ribosomal proteins S14 and S16 are summarized in Table 1. DNA Sequencing The appropriate cDNA fragments of human and mouse Ucn II, obtained from the Ucn II expressing cell lines, were extracted from the gels using the QIAquick Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany) and subcloned into pGEM-T vector by using the pGEM-T Easy Vector System I (Promega Corp.). Nucleotide sequencing of the specific PCR bands was performed by automated direct DNA sequencing,


according to the manufacturer’s recommendations (model 377, PE Applied Biosystems, Perkin-Elmer Corp., Foster City, CA).

Construction of Luciferase Reporter Gene Plasmids The mUcn II 5⬘ flanking region constructs were cloned by PCR using mouse genomic DNA clone isolated previously in our laboratory. The primers that were used for the construct were designed to include an artificial restriction endonuclease site for XhoI and HindIII. The mUcn II transcription start site has been predicted using the Genome Exploring and Modeling Software and determined to be located 209 bp upstream to the ATG. Nine fragments of the mUcn II 5⬘ flanking region (⫹1 is the transcription start site) have been isolated, (⫺85)-(⫹206), (⫺186)-(⫹206), (⫺335)-(⫹206), (⫺390)-(⫹206), (⫺482)-(⫹206), (⫺579)-(⫹206), (⫺670)-(⫹206), (⫺1064)-(⫹206), and (⫺2173)-(⫹206). The primers sequence were as follows. Antisense primer for all the constructs 5⬘-CCCAAGCTTTGTGGGAAGGCTGTAAGGAGAAAA-3⬘ (2 bp upstream to the ATG). Sense primer for the ⫺85-bp fragment: 5⬘-CCGCTCGAGTTTTGAGACAGTTTCCTGGAACTC-3⬘. Sense primer for the ⫺186-bp fragment: 5⬘-CCGCTCGAGGTCTCTGGTCCTGTTTGGCCTTCT-3⬘. Sense primer for the ⫺335-bp fragment: 5⬘CCGCTCGAGTAGCTCCTTCTGTTATCTCTGGAG-3⬘. Sense primer for the ⫺390-bp fragment: 5⬘-CCGCTCGAGGCTCCTCCCCCTACCAATCCCCT-3⬘. Sense primer for the ⫺482-bp fragment: 5⬘-CCGCTCGAGCTGGATGGCTGCTCTCTTTCTGCC-3⬘. Sense primer for the ⫺579-bp fragment: 5⬘CCGCTCGAGTCCTCAAGCAGCCCTGTGCTCAG-3⬘. Sense primer for the ⫺670-bp fragment: 5⬘-CCGCTCGAGGGGGGCCTGGGGTTGGTA. Sense primer for the ⫺1064-bp fragment: 5⬘-CCGCTCGAGGGGGGCATCAGGAGGCAAACT. Sense primer for the ⫺2173-bp fragment: 5⬘-CCGCTCGAGGTGCCCCCTGAAGATGACGATGAC. The PCR products were analyzed by agarose gel electrophoresis in 1⫻ TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA), and eluted from the gel. After digestion by the appropriate restriction enzymes, HindIII and XhoI, the DNA fragments were cloned into the luciferase reporter plasmid pGL3 (catalog no. E1751, Promega Corp.) and sequence was verified using automated direct DNA sequencing.

Table 1. Primers Used in PCR Amplification Neuropeptide and Proteins

Primers

Location on Gene

Exon no.

Product Size (bp)

mUcn II

F: 5⬘-GGCCGCCGCTGAGACTGAA-3⬘ (“A”) R: 5⬘-GGCCTGTGGACCTTAGATGGACTT-3⬘ (“C”) H: 5⬘-CTCTGTGACTGCCAGACTCAG-3⬘

329–347 707–730 560–583

1 1 1

402

AF33151

hUcn II

F: 5⬘-GTGTCGGCCACTGCTGAGCCTGAGAGA-3⬘ R: 5⬘-ATCTGATATGACCTGCATGACAGTGGCT-3⬘

401–427 569–596

1 1

196

XM–030113

S16

F: 5⬘-TGCGGTGTGGAGCTCGTGCTTGT-3⬘ R: 5⬘-GCTACCAGGCCTTTGAGATGGA-3⬘ H: 5⬘-AAGGAGCGATTTGCTGGTGTGGA-3⬘

369–391 1968–1990 1798–1920

1 4 3

309

M11408

S14

F: 5⬘-GGCAGACCGAGATGAATCCTCA-3⬘ R: 5⬘-CAGGTCCAGGGGTCTTGGTCC-3⬘ H: 5⬘-ATATGCTGCTATGTTGGCTGC-3⬘

2941–2962 4166–4186 2965–2985

3 4 3

143

M13934

mGR

F: 5⬘-GCAGCAGCCGCAGCCAGATT-3⬘ R: 5⬘-CCCGCCAAAGGAGAAAGCAAGTT-3⬘

258–277 709–731

1 1

474

X04435

hGR

F: 5⬘-TATTGAATTCCCCGAGATGTTAGC-3⬘ R: 5⬘-ACTTTGGGCACTGGTGGTTTAGGT-3⬘

2370–2393 2767–2790

8 8

421

M10901

F, Forward; R, reverse; H, hybridization.

Genbank Accession no.


Transient Transfections and Luciferase Assay NG108-15 and NIE115 cells mouse neuroblastoma cell lines were grown in phenol red-free DMEM (Gibco Invitrogen Corp.), containing 10% charcoal-treated fetal bovine serum supplemented with 100 ␮g/ml of penicillin/streptomycin (Gibco Invitrogen Corp.). For transfection, NG108-15 and NIE115 cells (3 ⫻ 104) were plated on six-well plates. On the following day, the cells were transfected with 0.5–4.0 ␮g of the reporter gene construct and 50 ng ␤-gal expression plasmid using the Gene PORTER 2 Transfection Reagent (Gene Therapy Systems Inc., San Diego, CA) according to manufacturer’s recommendations. Each experiment contained a transfection with a positive control reporter plasmid containing the cytomegalovirus promoter to permit estimation of transfection efficiencies and comparison of results obtained from different experiments. Cells were treated with different concentrations of dexamethasone (Sigma) for 24 h and harvested 48 or 72 h after transfection and luciferase reporter activity was assayed as follows. Cells were washed three times with PBS and lysed by resuspending in 100 ␮l of 100 mM KPO4 buffer (pH 7.8) containing 1 mM dithiothreitol and 0.5% (vol/vol) Triton X-100. The luciferase assay buffer contains 100 mM Tris acetate (pH 7.8), 10 mM MgOAc, 100 mM EDTA, 2 mM ATP (pH 7.0), and 74 ␮M luciferin. Activity tests were performed and luminescence was measured in a Biocounter M2500 luminometer (Lumac, Landgraaf, The Netherlands) for 20 sec immediately after addition of luciferin assay buffer. Transfections were performed at least six times (in triplicates) for each construct or treatment tested. To correct for variations in transfection efficiencies, luciferase activities were normalized on the basis of ␤-gal activity. RNA Competitor Construction Homologous RNA competitive internal standards that shared the same primer binding sites but contained a shortened internal sequence with respect to the endogenous target RNA for mUcn II, were prepared as follows (Fig. 8B, diagram). The sense primer was primer A, which is a conventional PCR primer and corresponds to nucleotides 329–347 (1) (Fig. 8B). The antisense primer was 48 nucleotides in length in which 24 nucleotides at the 3⬘ end correspond to primer B (Table 1) and 24 nucleotides at the 5⬘ end (123 nucleotides upstream from primer B) correspond to nucleotides 707–730 (primer C) (Table 1). Primer A and BC (primer BC: 5⬘-GGCCTGTGGACCTTAGATGGACTTAACATACATGCTAAGGTCCAAGAT-3⬘ corresponding to nucleotides 560–583 ⫹ 707– 730). were introduced to the PCR, and the 279-bp product, which is 123 bp smaller than the product resulting from PCR with primer A and C, was subcloned into pGEM-T vector (Promega) and sequenced. The pGEM-T-279 bp mUcn II vector was linearized by NcoI restriction digestion and transcribed into cRNA template by SP6 RNA Polymerase (Riboprobe System SP6, catalog no. M6101, Promega Corp.). The DNA template after transcription was removed, and the cRNA product was quantified and used as internal standard in RT-PCR for mUcn II gene expression.


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added to separate PCR mixtures containing 100 pmol of specific mUcn II oligonucleotide primers A and C, 100 ␮M of deoxynucleotide triphosphate, 3 mM MgCl2៮, 1⫻ Taq buffer, and 2 U Taq DNA Polymerase (BIO-X-ACT DNA polymerase, Bioline UK Ltd.) in a total volume of 50 ␮l. The PCRs were amplified for 35 cycles using a programmable thermal controller (PTC-200, MJ Research Inc., Watertown, MA). Each cycle included denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and primer extension at 72 C for 1 min. PCR products (402 bp for endogenous mUcn II and 279 bp for mUcn II internal standard) (Fig. 8B) were separated on 2.2% agarose gel, visualized with ethidium bromide, photographed and quantified using ImageQuant software program (version 1.2, Molecular Dynamics Inc.). The ratio of internal standard to endogenous area was plotted as a function of the competitor concentration added to each PCR. The concentration of mUcn II mRNA was determined where the ratio of the internal standard mUcn II and endogenous mUcn II area was equal to 1 (i.e. the equivalence point). Animals Adult C57BL/6 male mice (27–30 g) were used in all experiments. Animals were housed three per cage in a room with controlled temperature and a fixed lighting schedule (lights on from 0600–1800 h). Food and water were given ad libitum. All experimental protocols were approved by the animal committee of The Salk Institute for Biological Studies. ADX, Corticosterone Replacement, and Dexamethasone Administration ADX was performed through a dorsal incision under isoflurane anesthesia. Sham operation was performed by manipulating the animal in the same manner but without removing the adrenal gland. One group of ADX mice received a replacement of corticosterone (catalog no. C-2502, Sigma) in the drinking water at final concentration of 25 ␮g/ml, immediately after ADX surgery. All mice were killed 2 wk after ADX surgery, the brains were isolated and the hypothalamus, brainstem, and the olfactory bulbs were dissected and subjected to RNA isolation. Blood collected from control, ADX, and corticosterone replacement mice was measured for plasma corticosterone using the ImmuChem Double Antibody Corticosterone 125I RIA Kit (ICN Biomedicals, Inc., Costa Mesa, CA). Dexamethasone (75, 150, 300 mg/kg) or saline were injected ip to mice. The mice were killed 2 or 12 h post injection, the brains were isolated and the hypothalamus, brainstem, and the olfactory bulbs were dissected and used for RNA isolation.

Acknowledgments We thank Y. Haas for corticosterone RIAs; L. Bilezikjian, T. Bale, B. Brar, and C. Li for critically reading the manuscript; and S. Guerra for assistance in the preparation of this manuscript.

Competitive RT-PCR Ucn II mRNA levels in the mouse hypothalamus, brainstem, and olfactory bulb after ADX or dexamethasone administration were quantified by a competitive RT-PCR assay. After DNase treatment, a constant amount of RNA (100 ng) was added to a reverse transcriptase (RT) mixture (SuperScript II RNase H-Reverse Trascriptase, catalog no. 18064-014, Gibco Invitrogen Corp.) containing 2-fold serial dilutions of mUcn II 279-bp internal standard (500–7.8 fg). To test for possible pseudo gene or genomic DNA contamination, either the RT enzyme or RNA was omitted from the reaction tube. Upon completion of the RT, 25% of the RT mixture was

Received February 14, 2003. Accepted May 15, 2003. Address all correspondence and requests for reprints to: Wylie Vale, Ph.D., Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: [email protected]. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant DK 26741, The Robert J. and Helen C. Kleberg Foundation, The Adler Foundation, and the Foundation for Research (to W.V.). W.V. is a Senior Foundation for Research Investigator.

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