LXRalpha functions as a cAMP-responsive transcriptional regulator of gene expression

Abstract

LXRalpha is a member of a nuclear receptor superfamily that regulates transcription. LXRalpha forms a heterodimer with RXRalpha, another member of this family, to regulate the expression of cholesterol 7alpha-hydroxylase by means of binding to the DR4-type cis-element. Here, we describe a function for LXRalpha as a cAMP-responsive regulator of renin and c-myc gene transcriptions by the interaction with a specific cis-acting DNA element, CNRE (an overlapping cAMP response element and a negative response element). Our previous studies showed that renin gene expression is regulated by cAMP, at least partly, through the CNRE sequence in its 5'-flanking region. This sequence is also found in c-myc and several other genes. Based on our cloning results using the yeast one-hybrid system, we discovered that the mouse homologue of human LXRalpha binds to the CNRE and demonstrated that it binds as a monomer. To define the function of LXRalpha on gene expression, we transfected the renin-producing renal As4.1 cells with LXRalpha expression plasmid. Overexpression of LXRalpha in As4.1 cells confers cAMP inducibility to reporter constructs containing the renin CNRE. After stable transfection of LXRalpha, As4.1 cells show a cAMP-inducible up-regulation of renin mRNA expression. In parallel experiments, we demonstrated that LXRalpha can also bind to the homologous CNRE in the c-myc promoter. cAMP promotes transcription through c-myc/CNRE:LXRalpha interaction in LXRalpha transiently transfected cells and increases c-myc mRNA expression in stably transfected cells. Identification of LXRalpha as a cAMP-responsive nuclear modulator of renin and c-myc expression not only has cardiovascular significance but may have generalized implication in the regulation of gene transcription.

Figure 1

Specific and preferential binding of mLXRα to CNRE as a monomer. (A) EMSA with mLXRα produced by in vitro transcription/translation and ³²P-labeled CNRE probe. Anti-LXRα monoclonal antibody abolished the complexes formed by mLXRα with CNRE. (B) EMSA using mLXRα or CREB with or without the monoclonal antibodies to LXRα or CREB. CNRE and the classical CRE (FN/CRE) were used as probes. mLXRα preferentially bound to CNRE and the binding affinity of CREB to CNRE was very weak. (C) EMSA using mLXRα and ³²P-labeled full CNRE probe with different fragments of the full CNRE as cold competitors. The whole consensus sequence (del/CNRE-6) is required for the efficient binding of mLXRα to the CNRE. (D) EMSA with mLXRα and N-terminal deletion mutant of mLXRα (delN-mLXRα). DR4/LXRE and del/CNRE-6 were used as probes. Although DR4/LXRE:mLXRα interaction is homodimeric, mLXRα binds to CNRE as a monomer.

Figure 2

Stably expressed mLXRα increases nuclear binding activity to CNRE and renin mRNA levels. (A) Western blot analysis with nuclear extracts from GFP/As4.1 and LXRα/As4.1 cells. LXRα protein was detected only in LXRα/As4.1 cells. (B) EMSA with nuclear extracts (NE) from GFP/As4.1 and LXRα/As4.1 cells, using CNRE as the probe. The unlabeled competitors and antibodies (5 μg) are indicated at the top of the gel. Stable transfection of mLXRα significantly increased nuclear binding activity to CNRE, which was inhibited by unlabeled CNRE probes and by anti-LXRα antibody. (C) Northern blot analysis with total RNA from GFP/As4.1 and LXRα/As4.1 cells. Basal renin mRNA expression is higher in LXRα/As4.1 cells than in GFP/As4.1 cells. (D) Northern blot analysis with total RNA from GFP/As4.1 and LXRα/As4.1 cells treated with 1 mM 8-Br-cAMP for 0, 1, 6, 12, and 24 h. The relative renin mRNA levels were measured by using a densitometer, normalized by 18S expression, and expressed as mean ± SE (n = 3, GFP/As4.1 without cAMP as 100%). cAMP increased renin mRNA levels in LXRα/As4.1 cells but not in GFP/As4.1 cells. (E) Northern blot analysis with total RNA from six independent LXRα-stably transfected As4.1 cell lines (from LA-1 to LA-6) treated with 1 mM 8-Br-cAMP for 12 h. These isolated clones show a similar up-regulation of renin mRNA in response to cAMP.

Figure 3

Transient transfection assays confirming the CNRE:LXRα interaction. (A) Luciferase assay using As4.1 cells transiently cotransfected with pcDNA3.1, pc-mLXRα, or pc-mRXRα in combination with pCNRE-TK-luc, pCNRE-mREN-luc, phuREN-luc, and phuREN/mCNRE-luc plasmids. After DNA transfection, the cells were incubated with vehicle or 1 mM 8 Br-cAMP for 12 h. The luciferase activities were expressed in relative units (mean ± SE, n = 6, pcDNA3.1 without cAMP as 100%) normalized by β-gal activity as an internal standard for the transfection efficiency. Expression of mLXRα increased basal luciferase activity and conferred cAMP inducibility only to constructs containing the CNRE. mLXRα conferred the cAMP-inducible transactivation property to CNRE both in the native renin promoter and in the heterologous TK promoter contexts. (B) Luciferase assay using As4.1 cells cotransfected with either wild-type pc-mLXRα or mutant LXRα expression plasmids in combination with pTK-luc and pCNRE-TK-luc plasmids. Altered amino acids of mLXRα mutants (M1, M2, M3, and M4) were designated by underline. DBD, DNA-binding domain; LBD, ligand-binding domain; pK_a, potential pK_a phosphorylation sites; AF-2, AF-2 domain. The luciferase activities were expressed as in A. Mutations in the AF-2 domain of mLXRα abolished cAMP-inducible transactivation by CNRE:mLXRα interaction.

Figure 4

Additive effect of CREB and LXRα on cAMP-mediated transactivation of human renin promoter containing the classical CRE and CNRE. Luciferase assay using As4.1 cells cotransfected with pcDNA3.1, pc-CREB, or pc-mLXRα in combination with pFN/CRE-TK-luc, pCNRE-TK-luc (A); and huREN-luc plasmids (B). The luciferase activities were expressed as in Fig. 3A. The classical CRE:CREB and CNRE:LXRα interactions could confer cAMP-inducible transactivation property to human renin promoter containing the consensus CRE and CNRE in an additive manner.

Figure 5

Potential role of LXRα on c-myc expression through CNRE. (A) EMSA with mLXRα and ³²P-labeled myc/CNRE probe. mLXRα specifically binds to the myc/CNRE as well as the renin CNRE. (B) Luciferase assay using As4.1 cells cotransfected with either pcDNA3.1 or pc-mLXRα in combination with pMYC/CNRE-TK-luc and phuMYC-luc plasmids. The luciferase activities were expressed as in Fig. 3A. mLXRα conferred the cAMP-inducible transactivation property to myc/CNRE. (C) Northern blot analysis with total RNA from GFP/As4.1 and LXRα/As4.1 cells. The cells were cultured in low serum medium (1% FBS) for 24 h, and then treated with 5% FBS or 1 mM 8-Br-cAMP for 2 h. cAMP increased c-myc mRNA levels in LXRα/As4.1 cells but not in GFP/As4.1 cells.

Figure 6

Effect of cAMP on DR4/LXRE:LXRα/RXRα interaction. Luciferase assay using As4.1 cells cotransfected with pc-mLXRα and pc-mRXRα in combination with the pDR4/LXRE-TK-luc plasmid. The cells were treated with 1 mM 8-Br-cAMP, 5 μM 22(R)-hydroxycholesterol (22-R-CHO), or vehicle for 12 h, and luciferase activities were expressed as in Fig. 3A. Note that cAMP also activates DR4/LXRE:LXRα/RXRα interaction.