cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

Abstract

Background: The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE).

Results: The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP) family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2), known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene.

Conclusions: Using a constitutively active CREB2/CREB fusion protein and a mutant of the PKA catalytic subunit that is targeted to the nucleus, we have shown that the glucose-6-phosphatase gene has two distinct genetic elements that function as bona fide CRE. This study further shows that the expression vectors encoding C2/CREB and catalytic subunit of PKA are valuable tools for the study of CREB-mediated gene transcription and the biological functions of CREB.

Figure 1

The human glucose-6-phosphatase gene promoter. (A) Sequence of a portion of the human glucose-6-phosphatase gene promoter including the CRE-like sequences CRE1 and CRE2. (B) The reporter plasmid pG6PCRE1/CRE2luc contains a minimal promoter consisting of the human immunodeficiency virus TATA box, the adenovirus major late promoter initiator element, the luciferase open reading frame, and glucose-6-phosphatase promoter sequences encompassing both CRE-like sequences CRE1 and CRE2. The reporter plasmids pG6PCRE1mut/CRE2luc and pG6PCRE1/CRE2mutluc carry mutations in either the CRE1 or the CRE2, to inactivate these sites. (C) Reporter plasmids pG6PCRE1luc and pG6PCRE2luc contain glucose-6-phosphatase promoter sequences encompassing either the CRE1 or the CRE2. (D) The reporter plasmid pTNFα(CRE/AP1)²luc contains two identical copies of the composite CRE/AP1 sequence derived from the human TNFα gene, upstream of a minimal promoter. The sequence of one of these motifs is depicted.

Figure 2

Schematic representation and expression of the constitutively active CREB2/CREB fusion protein C2/CREB. (A) Schematic representation of the modular structure of CREB, CREB2, and C2/CREB. The basic region leucine zipper domain (bZIP) is indicated. The chimeric bZIP protein C2/CREB consists of the constitutively active transcriptional activation domain of CREB2 and the bZIP domain of CREB, responsible for dimerization and DNA-binding. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding C2/CREB. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag.

Figure 3

Biological activity of C2/CREB, a constitutively active CREB2/CREB fusion protein. One of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (A), pG6PCRE1luc, pG6PCRE2luc (B), or pTNFα(CRE/AP1)²luc (C) (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate), encoding β-galactosidase under the control of the Rous sarcoma virus long terminal repeat, and either the "empty" expression vector pCMV5 or an expression vector encoding C2/CREB (20 ng plasmid/plate). The data are presented as the ratio of luciferase activity (light units) to β-galactosidase units (OD units) measured in the cell extracts. The mean +/- SD is depicted.

Figure 4

Schematic representation and biological activity of the nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. (A) Schematic representation of the modular structure of the catalytic subunit of cAMP-dependent protein kinase (Cα) and the mutant NLSCα. The wild-type enzyme is myristylated as indicated (Myr). The sequence of the nuclear localization signal derived from the SV40 large T antigen and the triple FLAG epitope in NLSCα are shown. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding NLSCα. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag. (C) Modular structure of the GAL4-CREB fusion protein. The protein consists of the DNA-binding domain of GAL4 (amino acids 1–147) and the activation domain of CREB (amino acids 1–281). The reporter plasmid pUAS⁵luc contains a transcription unit encompassing the luciferase open reading frame and a minimal promoter that consists of five copies of the upstream activating sequence (UAS), a TATA box derived from the HIV long terminal repeat and the initiator element from the adenovirus major late promoter. The reporter plasmid pUAS⁵luc (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and either an expression vector encoding the DNA binding domain of GAL4 (plasmid pM1) or the GAL4-CREB fusion protein (1 μg plasmid/plate). In addition, cells were transfected with an expression vector encoding either the wild-type (Cα) or mutated (NLSCα) form of cAMP-dependent protein kinase (100 ng/plate). Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined.

Figure 5

Transcriptional activity of CREB and CREB mutants in the presence of a nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. One of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (A), pG6PCRE1luc, pG6PCRE2luc (B), or pTNFα(CRE/AP1)²luc (C) (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and the "empty" expression vector pCMV5 or an expression vector encoding either CREB, CREBS133A, or K-CREB (20 ng plasmid/plate). In addition, an expression vector encoding NLSCα (100 ng/plate) was transfected. Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined. The data are presented as the ratio of luciferase activity (light units) to β-galactosidase units (OD units) measured in the cell extracts. The mean +/- SD is depicted.

Figure 6

Biological activity of constitutively active ATF2 fusion protein towards glucose-6-phosphatase promoter-containing reporter genes. (A) Schematic representation of the modular structure of C2/ATF2. This chimeric bZIP protein consists of the constitutively active transcriptional activation domain of CREB2 and the bZIP domain of ATF2, responsible for dimerization and DNA-binding. (B, C, D) HepG2 cells were transfected with one of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (B), pG6PCRE1luc, pG6PCRE2luc (C), or pTNFα(CRE/AP1)²luc (D), the pRSVβ internal reference plasmid, and either the "empty" expression vector pCMV5 or an expression vector encoding C2/ATF2 (100 ng expression plasmid/plate). Lysates were prepared forty-eight hours post-transfection and β-galactosidase and luciferase activities were measured. The mean +/- SD is depicted.