cAMP activates transcription of the human glucocorticoid receptor gene promoter

https://doi.org/10.1016/S0960-0760(98)00097-1Get rights and content

Abstract

Glucocorticoids and cAMP regulate, either in a synergistic or additive fashion, the transcription of multiple genes, although some antagonistic effects of dexamethasone on cAMP-activated transcription have been described. The increased glucocorticoid receptor (GR) mediated response of some cell types, as a result of augmented cAMP, has been considered to be mainly due to an increased stability of GR mRNA, although other plausible explanations should not be ruled out. We studied the possibility that GR transcription itself could be affected by cAMP levels. HeLa cells were transfected with human GR (hGR) promoter constructs and their transcriptional activity determined after inducing a cAMP increase with forskolin. We found that forskolin almost doubled the transcriptional activity of the promoter construct spanning −2995 to +38 of the hGR, whereas no significant variations were observed with shorter chimeras containing sequences downstream −979. Shift mobility showed binding of CREB in vitro to a putative cAMP responsive element located at −1000, suggesting that hGR may be upregulated by cAMP at the transcriptional level, thus adding a new mechanism ascribable to this second messenger, which in conjunction with the cAMP-induced GR mRNA increased stability, would lead to a more precise control of the amount of GR protein within the cell.

Introduction

The ability of steroid hormones to produce diverse and complex effects lies in their distinctive working mechanism: once inside the cell, steroids bind to protein receptors and then are translocated into the nucleus, where the receptor–hormone complex recognizes and binds to specific DNA sequences, thus activating or repressing the transcription of multiple genes.

Regarding the glucocorticoid receptor (GR) the DNA sequence is not the sole determinant for positive or negative regulatory activities of glucocorticosteroids. Phorbol ester induced activation and glucocorticoid mediated repression on plfG sequence linked to a minimal promoter have been observed1, 2. In addition, hormonal regulation was found to be dependent on the cell line used: glucocorticoid activated transcription from plfG in HeLa and S2 Drosophila cells, while repression resulted when CV-1 cells were used under similar conditions[3]. Furthermore, several members of the ATF/CREB (activating transcription factor/ cAMP responsive element binding protein) subfamily elicit similar responses via c-Jun in composite elements such as plfG1, 2, 3. Others[4]have found that AP-1, cAMP response element (CRE) and glucocorticoid response element motifs are required for full cooperative activation either by c-Jun of c-Jun/c-Fos and glucocorticoids.

Glucocorticoids and cAMP seem to act, in an additive or synergistic manner, through the modulation of multiple physiological processes[5]. Albeit mainly acting in a synergistic manner6, 7, 8, 9, some antagonistic effects of dexamethasone, a synthetic glucocorticoid, on cAMP-activated transcription have been described10, 11.

How cAMP affects GR regulation is not yet well established. An increase in the level of glucocorticoid binding in murine lymphoma cell lines in response to cAMP was described long ago[12]and while a significant increase in GR in response to cAMP was found[13], it was considered to be mainly due to some kind of stabilization of the GR messenger RNA.

We have previously located five possible cAMP responsive elements in the human GR promoter sequence[14](Table 1). In transfection experiments with hGR promoter derived constructs linked to a firefly luciferase reporter gene, we tested the possibility that the increase in GR mRNA in response to cAMP could derive not only from an augmented stability of this mRNA, but also from a direct effect on the transcription of hGR.

Section snippets

Plasmid construction

BglII digestion of cosmid cos24[15]rendered three 3 kb fragments that were cloned in pUC19; one of them (pUCGRP6A) contained the sequences between −2945 and +38 of hGR gene. A 300 bp BamHI–HindIII pUCGRP6A fragment was cloned in BglII–HindIII digested pXP2[16]luciferase reporter plasmid producing pGRPLuc16. To obtain pUCGRPLuc14 a 2710 bp EcoRI–NcoI fragment from 24RI-2[15]was cloned in pUC19; a 1031 bp BamHI–SmaI from such primary construct was cloned in BglII–SmaI digested pXP2. Finally, a 406 bp

Human glucocorticoid receptor is upregulated by cAMP

None of the five putative cAMP responsive elements identified in the hGR promoter (Table 1 and Fig. 1) corresponded to the canonical CRE sequence[20]. Nevertheless, to test the hypothesis of a direct regulation of hGR expression by cAMP through such motifs, transfections with pGRPLuc plasmids were carried out in HeLa cells, both in the presence and in the absence of 50 mM forskolin. The results are summarized in Fig. 2. Forskolin activated transcription from pGRPLuc16 with respect to vehicle

Discussion

To identify a possible direct effect of cAMP on hGR gene expression we studied the expression of several hGR promoter luciferase gene chimeras in HeLa cells after forskolin treatment. We found a 180% forskolin-induced activation on hGR promoter transcription in the chimera spanning the sequence from −2945 to +38 of hGR, but not in shorter constructs spanning −979 to +38 or −368 to +38. Only two of the five putative CRE found in the hGR promoter are located upstream −979, but neither of them

Acknowledgements

We would like to thank Dr S. Nordeen for the luciferase reporter plasmid pXP2 and Dr S. Detera-Wadleigh for her assistance in the initial transfection experiments. This work was supported in part by grants from the Spanish DGICYT and CICYT. I.P. was recipient of a fellowship from the Government of Navarra.

References (23)

  • R.J. Harrison et al.

    Synergistic activation of neurotensin/neuromedin N gene expression by c-Jun and glucocorticoids: novel effects of Fos family proteins

    Mol. Endocrinol.

    (1995)
  • Cited by (24)

    • Nuclear translocation of the unliganded glucocorticoid receptor is influenced by membrane fluidity, but not A<inf>2A</inf>R agonism

      2020, Steroids
      Citation Excerpt :

      A2ARs have been demonstrated to upregulate certain glucocorticoid response-element (GRE)-mediated genes [16], suggesting a positive genetic crosstalk between the two systems. Increases in cAMP have been shown to increase levels of GR mRNA within HeLa cells, potentially through direct cAMP response-element binding protein (CREB) binding to its promoter sequence [17]. cAMP levels, tightly regulated by A2ARs, have also been shown to be positively correlated with GR mRNA stability [18].

    • Activation of cAMP-protein kinase A abrogates STAT5-mediated inhibition of glucocorticoid receptor signaling by interferon-alpha

      2011, Brain, Behavior, and Immunity
      Citation Excerpt :

      Not addressed in the current set of studies is the possibility that constitutive or stimulated PKA activity may enhance GR phosphorylation, thereby augmenting GR function even in the presence of inflammatory cytokines. PKA has been shown to directly phosphorylate GR (Haske et al., 1994), and treatment with forskolin as well as other PKA agonists such as 8-Br-cAMP can increase the stability and expression of GR mRNA (Dong et al., 1989; Penuelas et al., 1998). Thus, in the current experiments it is possible that treatment with forskolin and therefore activation of PKA may have directly enhanced GR function in addition to independently inhibiting mIFN-alpha-induced STAT5 activity.

    • Involvement of the hypothalamic-pituitary-adrenal axis in the antidepressant-like effects of mild hypoxic preconditioning in rats

      2007, Psychoneuroendocrinology
      Citation Excerpt :

      Possible mechanisms underlying the effects of the PC on GR expression and HPA function might include modifications of glutamatergic and serotoninergic systems since it has been reported that acute intermittent hypoxia or anoxia affects glutamatergic and serotoninergic transmission and function of their receptors (Kinkead et al., 2001; Miller et al., 2003). There is a growing body of evidence that transcriptional regulators such as cAMP response element-binding protein (CREB) and trophic factors such as BDNF, strongly implicated in pathogenesis of depression and HPA regulation both at the level of GR and corticotropin-releasing hormone expression (Penuelas et al., 1998; Malkoski and Dorin, 1999; Givalois et al., 2004; Duman and Monteggia, 2006), are among basic molecular mechanisms by which the hypoxic/ischemic preconditioning can exert its action (Baker-Herman et al., 2004; Brzecka, 2005). In summary, we have demonstrated that PC by means of mild intermittent hypobaric hypoxia, was protective against development of shock-induced depression in rats preventing post-SID HPA hyperactivity and the impairment of negative feedback inhibition of the HPA axis.

    View all citing articles on Scopus
    View full text