P fragner, s L lee 1 and s aratan de Leon
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- 170, 91–98 Introduction
- Dispersion of islet cells
| Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets P Fragner, S L Lee 1 and S Aratan de Leon INSERM U-30, Mécanisme d’Action Cellulaire des Hormones, Hôpital Necker-Enfants-Malades, 149 Rue de Sèvres, Paris, France 1 Division of Endocrinology, Diabetes, Metabolism and Molecular Medicine, New England Medical Center Hospitals, Boston, Massachusetts, USA (Requests for offprints should be addressed to S Aratan de Leon, Institut National de Santé et Recherche Médicale, INSERM Unité 30, Hôpital Necker-Enfants-Malades, 149 Rue de Sèvres, 75743 Paris Cedex 15, France; Email: aratan @necker.fr)
TRH was initially found in the hypothalamus and regu- lates TSH secretion. TRH is also produced by insulin- containing -cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T 3
and in vitro. The present study was designed to determine the initial impact of T 3 on rat TRH gene promoter and to compare this e ffect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5 -flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concen- trations in fetal islets in culture, probably due to trans- activating factors, allowed us to explore how T 3 and Dex
regulate the TRH promoter activity in transfected cells and whether the hormone e ffect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T 3
Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets. Journal of Endocrinology (2001) 170, 91–98 Introduction Thyrotropin-releasing hormone (TRH) was originally iso- lated from the hypothalamus (Boler et al. 1969, Burgus
Langerhans and localized in insulin-containing cells (Aratan-Spire et al. 1984a, 1990, Leduque et al. 1987, 1989). Unlike major islet hormones, however, the highest concentrations of TRH and pre-pro-TRH(160–169) (pp-TRH) are detected during the early development of neonatal rat pancreas (Aratan-Spire et al. 1984a, Ebiou
1986). This suggests that TRH gene products are involved in the regulation or growth of fetal islets in an as yet undefined way. Hypothalamic TRH stimulates thyrotropin (TSH) se- cretion (Boler et al. 1969, Burgus et al. 1969). Pancreatic TRH is involved in the stimulation of glucagon secretion (Ebiou et al. 1992b) and the inhibition of exocrine pan- creatic secretion (Fragner et al. 1997). TRH / mice showed obvious hypothyroidism and exhibited hyper- glycemia accompanied by impaired insulin secretion, but thyroid hormone replacement does not correct the deficit in insulin secretion (Yamada et al. 1997). However, despite its biological contribution as a regulatory peptide in the adult pancreas, the physiological significance of TRH in islet development remains an open question. Hence, a clear picture of the hormonal control of TRH gene expression may shed light on this point. The thyroid hormone and the glucocorticoids exert pleiotropic e ffects on the endocrine pancreas. Previous studies have shown that triiodothyronine (T 3 ) selectively inhibits the islet TRH content and secretion. The TRH content (mRNA and peptide) of the pancreas of hypothy- roid rats is elevated and this is reversed by exogenous T 3 replacement (Fragner et al. 1998). Conversely, T 3 pro-
duced a dose-dependent decrease in TRH mRNA and the TRH content in fetal islets in culture (Fragner et al. 1999). In contrast, the synthetic glucocorticoid, dexam- ethasone (Dex), up-regulates TRH mRNA and TRH of the pituitary cells in culture (Bruhn et al. 1994), a thyroid cell line (Tavianini et al. 1989) and in fetal islets in culture (P Fragner & S Aratan de Leon, unpublished observations). Two lines of evidence point to T 3 and Dex having a direct influence on pp-TRH gene expression; the presence of nuclear T 3 receptors in the pancreas (Lee et al. 1989) and T 3 receptor binding site consensus sequences in the TRH promoter (Stevenin & Lee 1995). A glucocorticoid regu- latory element (GRE) is present on the TRH gene promoter (Lee et al. 1988) and the pancreatic -cells are the only islet cells bearing the glucocorticoid receptor (Fischer et al. 1990, Delaunay et al. 1997). The DNA 91
0022–0795/01/0170–091 2001 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org elements mediating the negative regulation of pp-TRH gene promoter by T 3 were localized in the promoter- proximal region between 83 and +46 (Balkan et al. 1998) (Fig. 1). The present study was therefore carried out to demonstrate the impact of two hormones, thyroid hormone and glucocorticoid, on the transcription of TRH gene and to validate previous data (Fragner et al. 1998, 1999), using the transient transfection of several TRH promoter constructs in neoplastic islet cells and fetal islets in culture. The persistence of high TRH gene expression throughout the islet culture period (Scharfmann et al. 1988, Aratan-Spire et al. 1990, Ebiou et al. 1992a) suggests the presence of high levels of transactivating factors. The fetal islet in culture are therefore an appropriate model for investigating the regulation of TRH promoter. The study of the hormonal regulation of TRH gene promoter may help link potentially the regulation of TRH gene expression to islet development and function.
These contain the Pst-Pvu II fragment of rat TRH genomic DNA.
Promoter truncations ( 554/
+84, 242/+84 and 113/+84) were created using restriction endonucleases. The promoter fragments of di fferent lengths were cloned upstream of the luciferase gene in the vector pA3 LUC (Fig. 1). This vector contains a trimerized SV40 poly(A) termination site that prevents transcription readthrough (Wood et al. 1989).
RIN m5F and HIT T-15 are two pancreatic islet cell lines and 3T3 is a fibroblastic cell line. RIN m5F and 3T3 cells were cultured in RPMI-1640 and HIT T-15 cells in DMEM. All the standard media contained 10% fetal calf serum (FCS) unless otherwise indicated. Cells were routinely cultured in 5% CO 2 and 95% humidified air at 37 C. Preparation of islets Fetal islets were prepared according to Hellerström et al. (1979). Briefly, fetuses were removed from pregnant Wistar rats at 21 days of gestation. The day of mating was considered to be day 0. The fetal pancreases were removed aseptically, placed in cold Hanks’ balanced salt solution (HBSS) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, and minced. HBSS (4 ml) con- taining 6 mg/ml collagenase CLS 4 (Worthington Bio- chemical, Freehold, NJ, USA) were added to each of four centrifuge tubes containing 10–12 pancreases each. The tissue was digested in a shaking water bath at 37 C for 8 min. The resulting digests were washed three times with cold HBSS; the pellets were pooled and resuspended in 500 µl HBSS. Aliquots of this suspension (100 µl) were finally distributed in 50 mm plastic culture dishes. The islets were cultured for 5 days in 5 ml RPMI-1640 medium, containing 11 mM glucose, 10% heat inactivated FCS, 100 U/ml penicillin and 100 µg/ml streptomycin, at 37 C in a humidified atmosphere of 5% CO 2 . The
medium was changed every day. At the end of the preculture period, the islets attached to the bottom of the culture dishes were gently blown free using a sterilized Pasteur pipette under a stereomicroscope. The fibroblast layer remaining on the bottom of the culture dishes was used as a primary non-islet cell (negative) control. The detached islets were cultured free-floating in 50 mm Petri dishes which did not permit cell attachment (Falcon 1007; Falcon Plastics, San Diego, CA, USA), in complete
positions of a putative TATA box, a GRE (Lee et al. 1989) and a T 3 -response element half-site (TRE) (Stevenin & Lee 1995) are indicated. Promoter fragments of indicated lengths were cloned upstream of the luciferase gene in the vector pA3 LUC. P FRAGNER and others · TRH promoter is inhibited by T 3 and stimulated by Dex in pancreatic islets 92 www.endocrinology.org Journal of Endocrinology (2001) 170, 91–98 RPMI-1640 medium supplemented with 10% FCS changed every other day. About 1000 to 1500 islets were obtained from 10–15 fetal pancreases. The islets were distributed as 300–400 islets per dish. The precise number of islets per batch was determined from the total insulin content per batch and the insulin content per islet (Ebiou et al. 1992a, Fragner
Islet cells were dispersed by placing washed islets (1000–1500) in 500 µl calcium- and magnesium-free HBSS containing trypsin (0·05%) and EDTA (0·02%) for 5 min. An equal volume of culture medium was then added and the remaining intact islets were mechanically dispersed by mild trituration. The dispersed islets were washed and suspended in culture medium (RPMI-1640 containing 10% FCS) before being used for transfection experiments. Transfection The day before transfection, neoplastic cells and the primary fibroblasts were subcultured in 35 mm culture dishes (5 10 5
the primary fibroblasts were rinsed once with 1 ml Opti-MEM (Gibco-BRL, Gaithersburg, MD, USA) and incubated for 5 h in Opti-MEM medium (1 ml/dish). This medium contained no serum or antibiotics, but did contain plasmid DNA (1 µg TRH-LUC reporter gene DNA/dish) and lipofectamine reagent (Gibco-BRL) (5 µg/dish) Dispersed islets were treated and transfected for 5 h exactly as the cell lines except that higher doses of lipofectamine were used (30 µg/dish). The medium was then carefully removed, and the cells were incubated overnight in complete medium containing 10% FCS. The islets were rinsed twice with PBS and kept in serum- free RPMI-1640 with 0·1% (w/v) BSA. T 3 or Dex was added as concentrated sterile solutions. The medium was not changed during the experiments to avoid repeated exposure to hormone. Intra-experiment controls were included in all experiments because the nutritional con- ditions might change during the 48 h in culture. This allowed comparison between islets exposed to hormone (T 3
(controls). Cells were harvested, lysed and assayed for luciferase and -galactosidase activities and protein content.
Luciferase activity was detected with the Promega Luciferase assay system (Promega, Madison, WI, USA). Light units were measured for 10 s with a standard luminometer (Berthold Cliniluminometer, Germany). Luciferase activity was normalized to the protein concentration (light units/protein).
Transfection e fficiency was monitored in neoplastic cells (HIT T-15, RIN m5F, 3T3) by cotransfection with CMV- -Gal ( -galactosidase ex- pression vector linked to CMV promoter) and in primary cells (islets and fibroblasts), by transfection of parallel dishes with CMV- -Gal. -Galactosidase activity
was assayed
using 2- nitrophenyl- - -galactopyranoside as substrate. Typically, 20 µl lysate was used in a 30 min assay. Protein measurements The protein concentrations were measured by Bradford’s method (Bradford 1976).
Because protein concentrations of all extractions in previous experiments have been found to be correlated with
cotransfected -galactosidase activity, relative
light units were monitored by quantification of protein concentration, in lieu of -galactosidase for simplicity. Histochemical staining for -galactosidase assay Dispersed islets (groups of 200) transfected with the pCMV- -Gal were harvested after 72 h, and the percent- age of
-Gal-expressing cells was determined by -galactosidase histochemical staining. Intact islets were used in parallel to allow comparison (Saldeen et al. 1996). The islets were washed twice in PBS, fixed in 2% formaldehyde and 0·2% glutaraldehyde and washed again. The fixed islets were incubated in a chromogenic solution (5 mmol/l potassium ferricyanide, 5 mmol/l potassium ferrocyanite, 2 mmol/l MgCl 2 and 1 mg/ml 5-bromo-4- chloro-3-indolyl- - -galactopyranoside) at 37 C for 24 h after which they were washed twice and resuspended in PBS. Cells staining deep blue-green were regarded as positive and photographed.
The e
ffect of the hormones on luciferase activity was compared by ANOVA using a one-factor model for repeated measures. Di fferences were considered signifi- cant if P
Three cell lines and primary fibroblasts were tested for the basal activity of TRH-LUC expression vectors. The luciferase activity of the reporter gene was expressed with respect to protein concentration. Figure 2 shows the relative luciferase activity of three TRH promoter con- structs, normalized to the protein concentration. Each TRH-LUC construct was transfected into HIT T-15, RIN m5F and 3T3 cell lines and primary fibroblasts in culture. To allow comparison of basal activities across cell TRH promoter is inhibited by T 3 and stimulated by Dex in pancreatic islets · P FRAGNER and others 93 www.endocrinology.org Journal of Endocrinology (2001) 170, 91–98 lines, the relative luciferase activity of each TRH-LUC construct has been compared with that of CMV-LUC as maximum activity (i.e. to 100 in all cases). The luciferase activity was about 8-fold higher in HIT T-15 and 3T3 cell lines than in RIN m5F cells and primary fibroblasts. Among the islet cell lines, the highest activity was always found in HIT T-15 cells transfected with the TRH-LUC ( 242/+84) construct. In pilot experiments HIT T-15 cells were transfected with TRH-LUC constructs containing the fragments 554/+84, 242/+84 or 113/+84, to determine the length of TRH promoter fragment needed to mediate hormone regulation. Hormone (T 3 or Dex) was added to the medium 24 h after the transfection for another 24 h. The basal activity of all three TRH-LUC constructs remained unchanged following exposure of the transfected cells to T 3 (10
8 M, 24 h). In contrast, Dex (10 7 M)
554/ +84) and TRH-LUC ( 242/+84) by 100%, while that of TRH-LUC ( 113/+84) remained unchanged (Fig. 3). Similar results were obtained with RIN m5F cells transfected with TRH-LUC constructs of various lengths. The transfection e fficiency of dispersed islet cells was compared with that of intact islets by histochemical staining. Dispersed islet cells and intact islets were trans- fected with the CMV- -Gal vector. Cells were harvested 3 days after transfection and -Gal-positive cells assayed. In contrast
to intact
islets, very
poorly stained,
lipofectamine-mediated transfection yielded an average of 29 8% -Gal-positive islet cells. Figure 4 shows a typical histochemical staining of 20–25% -Gal-positive cells. The e
ffects of T 3 and Dex on primary islet cells and fibroblasts transfected with TRH-LUC ( 242/+84)
construct were then tested. T 3 (24 h, 10 8 M) inhibited the basal luciferase activities in the islet cells and primary fibroblasts by 50%. In contrast, Dex (24 h, 10 7 M)
100% but only in primary islet cells. Dex was without e ffect on the luciferase activity of transfected fibroblasts (Fig. 5). The specificity of the hormone regulation was deter- mined by comparison with CMV-LUC vector activity following normalization by protein concentration. T 3 and Dex did not a ffect the basal luciferase activity of CMV-LUC vector transfected into the primary islet cells and fibroblasts (Fig. 5). Likewise, the cells transfected with
cells (HIT T-15, RIN m5F and 3T3 cells) and primary fibroblasts. Neoplastic cells and primary fibroblasts (5 10 5 cells/dish) were transfected with the indicated TRH-LUC constructs (1 g/dish) using lipofectamine (5 g/dish) for 5 h, in OPTI-MEM medium without FCS or antibiotics. Cell lysates were assayed for luciferase activity and protein content. The luciferase activity was normalized to the protein content. The relative luciferase activity of each TRH-LUC construct was compared with that of CMV-LUC. The values are means S.E. of triplicate transfections in three independent experiments. Results are similar following the normalization of the data to the -Gal activity in cotransfected neoplastic cells.
regulation. The HIT T-15 cells (5 10 5
incubation for 5 h with the indicated TRH-LUC constructs (1 g/dish) using lipofectamine (5 g/dish) in serum- and antibiotic-free Opti-MEM medium. The medium was removed and complete medium containing 10% FCS was added for 24 h. The medium was again replaced with fresh serum-free medium containing 0·1% BSA with or without hormone (T 3 (10
8 M), Dex
(10 7 M) or control (C)) for a further 24 h. Cells were then harvested and cell lysates were assayed for luciferase activity and protein content. The relative basal activity of each TRH-LUC construct was normalized to the protein content (arbitrary light units/protein content). Data are means S.E. of at least three independent experiments. P FRAGNER and others · TRH promoter is inhibited by T
94 www.endocrinology.org Journal of Endocrinology (2001) 170, 91–98 the promoterless LUC vector displayed no activity in the absence or presence of hormone (T 3 or Dex).
Discussion This description of the pp-TRH promoter activity and its hormonal regulation in the pancreatic islet cells compares the e
ffects of T 3 and Dex on the basal activities of the TRH promoter following the transient transfection of neoplastic and primary islet cells with di fferent promoter sequences. The promoter constructs used do not possess islet-cell specificity, since they can be expressed in islet and non-islet (fibroblasts, 3T3) cells, as well as TRH + (fetal islets) and TRH (HIT T-15 and RIN m5F) cells Figure 4 -Galactosidase histochemical staining assay. Dispersed islet cells (equivalent of 200 islets) were transfected with 1 g pCMV- -Gal, using 30 g lipofectamine. In parallel, intact islets were used in the same experimental conditions, to allow comparison. Cells were harvested 3 days later and stained for -galactosidase activity. (a, b) Dispersed islets (magnification 200); (c) dispersed islets (magnification 400); (d) intact islets. -Gal-positive cells are dark green-blue, whereas untransfected cells are sometimes light green. Staining appears dark in the figure. -Gal-positive cells represent 20–25% of the dispersed islet cells. TRH promoter is inhibited by T 3 and stimulated by Dex in pancreatic islets · P FRAGNER and others 95 www.endocrinology.org Journal of Endocrinology (2001) 170, 91–98 derived from pancreatic islets. The basal activity in all cell types tested suggests the presence of ubiquitous transcrip- tion factors interacting with common basal promoter elements. The data also suggest that there are suppressor sequences in the TRH-LUC ( 554/+84) construct, which has less basal activity than TRH-LUC ( 242/
+84). The promoter-proximal region was reported to contain the DNA elements necessary for the inhibitory regulation of the pp-TRH gene promoter by T 3 : deletion analysis localized the element mediating the negative regulation in the region between 83 and +46 and the sequence between +12 and +46 was found to be essential for inhibitory regulation by thyroid hormone (Balkan et al. 1998). However, because of low basal activity found in the cell types used in the present study, the TRH-LUC ( 113/+84) construct was not appropriate to show the negative regulation by T 3 . The basal activity of the TRH promoter was greatest in HIT T-15 cells, while RIN m5F cells had the lowest basal activity. HIT T-15 cells transfected with TRH-LUC constructs of di fferent lengths were exposed to T 3 and Dex to compare the hormonal regulation of the TRH promoter. As shown in Fig. 3, only TRH-LUC ( 554/ +84) and TRH-LUC ( 242/+84) were hormone- sensitive, while TRH-LUC ( 113/+84) was not. Moreover, TRH-LUC ( 242/+84) displays higher level of expression than TRH-LUC ( 554/+84); there- fore it was selected as the most appropriate construct for showing the hormonal regulation of the TRH promoter. The comparison of the transfection e fficiency in primary and neoplastic islet cells was concluded following normal- ization of the luciferase activity to the cell protein content. For a given amount of protein, the luciferase activity was 4- to 5-fold lower in primary islet cells than in primary fibroblasts or in neoplastic cells. This lower activity is due, at least in part, to the percent of cells which are e ffectively transfected. As visualized by immunostaining in Fig. 4, only 25% of dispersed islet cells are -Gal-positive. Compared with neoplastic cells, islet cells were thus transfected with higher doses of lipofectamine. DNA/ lipofectamine ratio was 1:30 in all transfections of dis- persed islet cells with approximately 200 islets (equivalent of 3–5 10
cells/2 ml per dish). This appeared to give the optimal transfection e fficiency without any significant cytotoxic e ffect as evidenced by the unaffected cell protein contents 3 days after transfection. It can not be excluded, however, that part of the islet cells were lost before the transfection, following the trypsination of the intact islets. Nevertheless, the dispersed islet cells are more easily transfected than the cells of intact islets. This is probably because more cells are exposed to the transfection agents after dispersion. The principal finding of this study is that the promoter is influenced di fferently by T 3 and Dex. T 3 inhibits and Dex stimulates the luciferase activity of TRH-LUC ( 242/+84). To rule out any potential vector-mediated artifact (Lopez et al. 1993, Maia et al. 1996) and to demonstrate the specificity of the regulation of the pp-TRH promoter construct, we also used the CMV promoter in a CMV-LUC construct as well as a pro- moterless vector. In contrast to the promoterless vector, CMV-LUC displays high luciferase activity in primary and neoplastic islet and non-islet cells, but both are insensitive to T 3 and Dex. Our data demonstrate that the negative regulation by T 3 is restricted to primary cells (islets and fibroblasts). There was no change in the basal Figure 5 Changes in rat TRH promoter activity in transfected islet cells by T 3 and Dex. Fetal islets were prepared, cultured and dispersed. Dispersed islets (equivalent of 200 islets or 3–5 10 5 cells/dish) were transferred to serum- and antibiotic-free Opti-MEM medium containing lipofectamine (30 g/dish) and transfected by incubation for 5 h with plasmid TRH-LUC ( 242/ +84) (1 g/dish). Pancreatic fibroblasts (5 10 5 cells/dish) were also transfected with the same construct (1 g/dish), and lipofectamine (5 g/dish). The medium was removed and complete medium containing 10% FCS was added for 18 h. The medium was again replaced with fresh serum-free medium containing 0·1% BSA, with or without T 3 (10
8 M) or
Dex (10 7 M) for a further 24 h. Islet cells and fibroblasts were harvested, lysed and assayed for luciferase activity (arbitrary light units) under basal conditions (no hormone) or after incubation with T 3
was also measured under basal conditions and after T 3 or Dex treatment. The luciferase activities were normalized to the protein concentrations. The changes in luciferase activity following T 3 and
Dex treatment are reported as the ratio to basal luciferase activity (100%).
P FRAGNER and others · TRH promoter is inhibited by T 3 and stimulated by Dex in pancreatic islets 96 www.endocrinology.org Journal of Endocrinology (2001) 170, 91–98 activities of any TRH-LUC construct following transfec- tion with neoplastic cells (HIT T-15, RIN m5F or 3T3). In contrast, Dex increases TRH-LUC ( 242/+84)
activity in primary islet cells and neoplastic cells of islets. The synthetic glucocorticoid has no e ffect on non-islet cells (primary fibroblasts or 3T3 cells). The pp-TRH gene belongs to the family of T 3 - responsive genes that includes - and
-subunits of the TSH gene and epidermal growth factor receptor gene, whose expression is negatively regulated by T 3 at the level of gene transcription (Lezoualc’h et al. 1992). T 3 inhibition of pp-TRH gene promoter requires the thyroid receptor (TR)–T
3 complex and an additional co-suppressor protein, which may form a bridge between the TR–T 3 complex and the transcription initiation complex (Feng et al. 1994, Li et al. 1996, Satoh et al. 1996). T 3 receptor is present at very low levels, even in highly responsive cells. Interactions of the receptor with specific DNA sequences can only be examined using partially purified and concen- trated receptor. It has been reported that a thyroid hormone-response element of the insulin gene enhancer responds to thyroid hormone in COS-7 cells bearing the nuclear TR, but not in HIT T-15 cells (Clark et al. 1995). Cotransfection with TR was often a prerequisite for T 3
constructs (Höllenberg et al. 1995). The activity of the sequence also depends on the relative concentrations of other unknown transcription factors. Our results indicate that T
3 may inhibit TRH promoter activity, at least in primary islets and fibroblasts that possess T 3 receptor sites (Lee et al. 1989). Our previous results already indicated changes in the steady-state concentrations of pp-TRH mRNA and TRH. However, these changes may be due to changes in pp-TRH mRNA stability or in transcrip- tional activity. The present results mirror the changes in pp-TRH mRNA: the T 3 -dependent inhibition of TRH gene expression in cultured islets is of the same magnitude (50%) as the decrease in the promoter activity in the presence of T 3 . Our data are also consistent with the results obtained in vivo on hypothyroid rats where the steady-state concentrations of islets pp-TRH mRNA and TRH contents markedly increase, and T 3 replacement restores the euthyroid levels. Taken together, the consist- ency of the changes obtained by di fferent approaches makes it highly unlikely that these are artifacts produced by transfection experiments and indicates that the hor- mone first regulates pp-TRH transcription. The initial impact should produce a cascade of inhibition of the messenger, content, and secretion of TRH (Wolf et al. 1984, Aratan-Spire et al. 1984b, Fragner et al. 1998, 1999). The regulation by Dex of the pp-TRH promoter activity contrasts with the action of T 3 . The glucocorticoid receptor is present in the pancreatic -cells (Fischer et al. 1990, Delaunay et al. 1997). Dex stimulates the luciferase activity of TRH-LUC ( 242/+84) in primary islet cells and cell lines derived from pancreatic islets. It is without e ffect on the same construct in the primary fibroblasts and 3T3 cells. As for the T 3 e
assayed on TRH-LUC ( 242/+84) construct, which contains GRE sequence. Previous studies examining glu- cocorticoid regulation of the rat TRH promoter demon- strated that the sequence at 200 of the rat TRH gene can bind to glucocorticoid receptors in vitro. Also, transient transfection studies in a cell line containing endogenous glucocorticoid receptors demonstrated a 4- to 6-fold stimulation by Dex (Lee et al. 1989, Stevenin & Lee 1995). As expected, TRH-LUC ( 113/+84) construct lacking GRE sequence was not sensitive to the Dex e ffect.
Taken together these data suggest that as for other glucocorticoid-responsive genes in the islets (Jamal et al. 1991, Hamamdzic et al. 1995, Khan et al. 1995), Dex regulates pp-TRH promoter activity after binding to islet glucocorticoid receptors which then interact with the GRE sequence. The hormonal regulation of pp-TRH promoter appears not to be islet cell-specific, since the pp-TRH promoter in hypothalamic neurons is also negatively regulated by T 3 (Lezoualc’h et al. 1992); like- wise, T 3 inhibits while Dex stimulates pp-TRH promoter activity in rat embryonic cardiomyocytes (Shi et al. 1997). In summary, data obtained using fetal islets raise the possibility that age-dependent decrease in TRH gene expression is due, at least in part, to extra-insular (hormo- nal) events. The molecular mechanism of how transcrip- tion is regulated and factors involved in this event remain to be elucidated.
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