Creb and Sp/Krüppel response elements cooperate to control rat TRH gene transcription in response to cAMP

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Abstract

Expression of hypophysiotropic TRH, that controls thyroid axis activity, is increased by cold exposure; this effect is mimicked in rat hypothalamic cells incubated with norepinephrine or cAMP analogs. TRH proximal promoter contains three putative CRE: Site-4 or CRE-1 that overlaps an element recognized by thyroid hormone receptors, CRE-2 with adjacent sequences GC box or CACCC recognized by Sp/Krüppel factors (extended CRE-2), and AP-1 sites flanking a GRE1/2. To evaluate the role of each element in the cAMP response, these sites were mutated or deleted in rat TRH promoter linked to luciferase gene (TRH-luc) and co-transfected with β-gal expression vector in various cell lines; C6 cells gave the highest response to forskolin. Basal activity was most affected by mutations or deletion of CRE-2 site, or CACCC (50–75% of wild type—WT). Forskolin-induced 3× stimulation in WT which decreased 25% with CRE-1 or AP-1 deletions, but 50% when CRE-2 or its 5′ adjacent GC box was altered. SH-SY5Y cells co-transfected with CREB-expression vector increased dB-cAMP response in the wild type but not in the CRE-2 mutated plasmid; cotransfecting CREB-A (a dominant negative expression vector) strongly diminished basal or cAMP response. Primary cultures of hypothalamic cells transfected with plasmids containing deletions of CRE-1, CRE-2, or extended CRE-2 failed to respond to forskolin when CRE-2 was modified. These results corroborate the CRE-2 site as the main cAMP-response element of rat TRH promoter, not exclusive of transcription factors of hypothalamic cells, and stress the relevance of adjacent Sp-1 sites, important mediators of some metabolic hormones.

Research Highlights

►Forskolin or cAMP stimulates TRH transcription in various cell types. ►Mutations in CRE-1 (Site-4) had little effect on basal or cAMP-induced transcription. ►CRE-2 (TGCCGTCA) of TRH promoter is the most important site for cAMP response. ►Sp/KFL sites adjacent to CRE-2 participate in basal or forskolin-induced activity. ►Transcription was reduced by expression of a dominant negative CREB.

Introduction

Thyrotropin releasing hormone (TRH) was originally identified (and named) as the hypophysiotropic hormone controlling the adenohypophysial release of thyrotropin [1]. TRH and its receptors were later detected in several brain regions where it functions as a neuromodulator [2]. Biosynthesis of TRH in the parvocellular neurons of the hypothalamic paraventricular nuclei (PVN) is tightly modulated by hormonal influences including thyroid hormones, glucocorticoids, and leptin, and in a fast and transient manner by neuronal inputs that respond to metabolic changes or to environmental stimuli such as cold [1], [3], [4], [5]. TRH mRNA levels increase in a fast and transient manner in rats exposed to cold (1 h) or in dams after 30 min of suckling, conditions that stimulate the release of TRH and in turn, that of thyrotropin or prolactin (respectively) [6], [7], [8]. In vitro, TRH biosynthesis has been studied in primary cultures of hypothalamic or pancreatic islet cells, as well as in various cell lines co-transfected with the TRH promoter linked to a reporter gene and expression vectors of thyroid hormone receptors or CREB [5], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. These systems have provided information of transcriptional down-regulation by thyroid hormones [5], [14], [16], [17] as well as the stimulatory effect of neuromodulators, or cAMP analogs that activate protein kinase A (PKA) [3], [10], [11], [12], [13], [17], [18]. Stimulation of hypothalamic cells by cAMP analogs or by norepinephrine mimics the fast increase in TRH mRNA levels seen in vivo, after cold exposure [6], [7], [8], [10], [11], [13], [17], [18]. Primary cultures of hypothalamic cells increase TRH mRNA levels and TRH release within 1 h of noradrenaline or 8Br-cAMP treatment [13], while it takes over 2 h by protein kinase C (PKC) stimulation [10], [11], [19].

The multifactorial modulation of TRH gene expression involves several transcription factors interacting with their cognate recognition elements, or with other transducing molecules that affect their binding to DNA. TRH gene promoter contains response elements to various transcription factors whose binding has been demonstrated (CREB [12], [13], [17], [18], thyroid hormone receptor (TR) [5], [9], [17], GR [13], [18], STAT [12], c-Jun [13], [18], c-Fos [18], Sp-1[15], [18]). The CREB response element (CRE) of the TRH promoter was first suggested [12] to be the one overlapping the thyroid hormone response element, named Site-4 [5], [9] (TGACCTCA; at −59/−52 of rat TRH promoter [20]). Site-4 is a functional thyroid response element (THRE) that binds TR and mediates T3-induced repression of TRH transcription [4], [5], [9], [16], [21]. Its role as a CRE was characterized in 283T cells transfected with −900/+55 human TRH promoter linked to a reporter gene and CREB expression vectors [12]. However, binding of nuclear extracts of primary cultures of embryonic rat hypothalamic cells stimulated with cAMP analogs to labeled oligonucleotides containing Site-4 (or CRE-1) is lower than to oligonucleotides containing the CRE-2 sequence (TGCCGTCA: at −101/−94 bp of rat TRH gene) [13]; only the latter region is protected in DNase footprinting assays [17]. The sequences of both response elements are conserved in various eutherian species including human, mouse and some primates [22]. DNA-footprinting analyses, using rat TRH promoter and nuclear extracts of hypothalamic cultures incubated with 8Br-cAMP, revealed a protected region extending at both sides of CRE-2 (extended CRE-2) that contains the sequences of a GC box (GGGCGGG at −119/−113 bp) and a CACCC motif (at −92/−88 bp) [17], [18], [20]; both are response elements of Sp/Krüppel-like factors (Sp/KLF) [23]. Another element located upstream is also protected, the 3′-AP-1 site (TTAGTCA) of a composite GRE (cGRE: −218/−197 bp) that contains a GRE half site flanked by two AP-1 recognition sites; binding to both AP-1 sites is increased with PKC activation [13], [18], [19]. The preferential binding of phospho CREB (pCREB) to the region containing the extended CRE-2 was demonstrated by chromatin immunoprecipitation (ChIP) assays [17], [18]. ChIP analyses of cAMP-stimulated hypothalamic cells revealed pCREB, c-Fos, c-Jun and Sp1 binding to the extended CRE-2 region and to that containing cGRE, c-Fos, c-Jun and in a lower proportion, pCREB; a negligible signal was detected in the pCREB-immunoprecipitate of short chromatin fragments selectively amplified for the CRE-1 region [18].

The differences observed in the involvement of CRE-1 between transfected heterologous and hypothalamic cells could involve either cell specificity of CREB proteins, the intrinsic difference of the nucleosome-free DNA template vs. organized chromatin, species, cell type, or the use of endogeneous transcription factors vs. transfected expression vectors. A parallel comparison of the importance of the CRE-1 and CRE-2 sites on TRH transcription has not been performed. We therefore studied the contribution of each of these two proposed CRE sequences, as well as the AP-1 of cGRE, in the response to PKA stimulation on TRH transcription. Since protection of the extended CRE-2 region included the GC box where Sp1 binding has been demonstrated by EMSA [15], or by ChIP assays using nuclear extracts from stimulated hypothalamic cultured cells [18], the role of this and of the CACCC site was also studied. Different cell types were initially tested to identify a cell line with a robust transcriptional response to cAMP, measured with a plasmid containing the rat TRH promoter linked to luciferase (pNASS-TRH-Luc plasmid, −776/+85 pb of rat promoter) [24]. The transcriptional activity (basal and forskolin-stimulated) of site-directed mutants and deletion constructs was compared to that of the wild type plasmid. Finally, plasmids containing deletions in the CRE-1 or CRE-2 sites were transfected into primary cultures of hypothalamic cells to verify the results obtained in the cell lines.

Section snippets

Plasmids

Plasmids were amplified in E. coli-DH5-α. Source of plasmids: pCH110 containing β-galactosidase under SV40 promoter (GE Healthcare, Piscataway, NJ); pUC18 (Fermentas, Life Sci., MD,); pNASS-TRH-Luc (−776/+85 pb of rat TRH gene promoter linked to luciferase reporter, kindly donated by Dr. Wayne Balkan, U. Miami [24]). CREB [25] or CREB-A [26] expression vectors were kindly donated by Dr. R. Goodman (Institut Vollum, Oregon University). Mutants of the GC box (m-GC: cGGGCTAG) or CACCC site

Results

Cells from different origins were transfected with pNASS-TRH-Luc and assayed for their response to PKA activation by incubating each with 1, 10 or 100 μM forskolin for 3 h. β-galactosidase activity was similar in the different cell lines but luciferase activity varied considerably, suggesting differences in modulating TRH promoter. The lowest basal luc/β-gal activity was observed in NIH-3T3 cells (967 ± 215 relative light units/μg protein) increasing over 4000 fold in COS-7 cells and even more, in

Discussion

Our results demonstrate that the various sequences of the rat TRH proximal promoter that bind transcription factors in response to cAMP activation are functional and contribute to regulate TRH promoter activity in C6 cells. They support the crucial role of CRE-2 and the contribution of the adjacent sites, Sp/KLF-response elements, to the basal activity (site CACCC) and to the response to forskolin (mainly the GC box). Although CRE-1 or cGRE affected only slightly basal activity, both diminished

Conclusion

The present work demonstrated the important role of the extended CRE that provides a platform for a cluster of transcription factors able to bind and affect basal and PKA-stimulated transcription of the rat TRH gene. The Sp/KLF recognition elements flanking the CRE site proved essential for maintaining basal and forskolin stimulated transcription. The Sp-1 cooperativity with pCREB may influence the response of TRH transcription to diverse environmental and hormonal signals able to activate or

Acknowledgements

We acknowledge Q. Fidelia Romero for technical support, P. Gaytan and E. López of the Macromolecule synthesis and sequencing Unit (IBT-UNAM) for synthesizing the oligonucleotides used in this work, and Sergio González for providing pregnant rats. This work was partially supported by grants from CONACYT (83363) and DGAPA-UNAM (PJB: IN221710, JO: IN206509).

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