Chicken ovalbumin upstream promoter transcription factor II regulates renin gene expression

Abstract

This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression.

FIGURE 1.

COUP-TFII binds to the COUP-TFII recognition sequence proxDR in the renin promoter. Nuclear extracts from As4.1 (top) or Calu-6 (bottom) cells were probed with mouse (top) or human (bottom) proxDR oligonucleotide in EMSAs. The samples in lanes 1 and 5 contained only the corresponding labeled probe. Nonspecific unlabeled oligonucleotide (NS) was added in 100-fold molar excess to the samples in lane 3 to demonstrate the specificity of the binding. Non-labeled probe (WT) was added in 100-fold molar excess to the samples in lane 4 for competition analysis. Anti-COUP-TFII (CII) antibody or isotype control (Iso) antibody (2 μl of each) was added to the samples in lanes 7 and 8, respectively. S, shifted protein complexes; NS bands, nonspecific bands; FP, free probe.

FIGURE 2.

COUP-TFII binds to the proximal renin promoter in its native context. Cross-linked nuclear extracts isolated from As4.1 (top) or Calu-6 cells (bottom) were used in ChIP with COUP-TFII antibody (COUP-TFII Ab) followed by isolation of the precipitated DNA. DNA isolated from non-precipitated samples was used as positive control (Input). The input and the COUP-TFII antibody-precipitated samples were subjected to PCR with a primer pair amplifying a proximal renin promoter fragment containing the COUP-TFII recognition sequence proxDR (promoter) or with a primer pair amplifying a non-related exon sequence that served as a negative control (exon).

FIGURE 3.

COUP-TFII is necessary for the cAMP-induced increase of the renin mRNA expression in As4.1 (A) and Calu-6 cells (B). COUP-TFII was knocked down with sequence-specific siRNA (siCOUP-TFII). Control cells were transfected with nontargeting siRNA (siControl). The top panels in A and B show the efficacy of the knockdown in As4.1 and Calu-6 cells, respectively. Protein extracts isolated from the corresponding cell lines were probed in Western blots with COUP-TFII and β-actin (internal control) antibodies. The bottom panels in A and B show the effect of the COUP-TFII deficiency on the cAMP-stimulated renin mRNA expression in As4.1 and Calu-6 cells, respectively. Renin, human β-actin (internal control), and mouse ribosomal L32 (internal control) mRNA levels were quantified by real-time PCR. The numbers above the columns indicate the -fold stimulation relative to control. IBMX, 3-isobutyl-1-methylxanthine. The data shown are means ± S.E. (error bars).

FIGURE 4.

The COUP-TFII-binding site proxDR in the proximal renin promoter is necessary for the cAMP-induced transactivation of the renin gene in As4.1 (A) and Calu-6 cells (B). Mutation of the COUP-TFII recognition sequence proxDR in a 4.2-kb mouse promoter (construct 4.2kb-proxDRmut; A) or in a 258-bp human proximal promoter proxDR (construct proxDRmut; B) abrogates the cAMP inducibility of the renin promoter. IBMX, 3-isobutyl-1-methylxanthine. The data shown are means ± S.E. (error bars). *, p < 0.05.

FIGURE 5.

COUP-TFII and the proxDR sequence are necessary for the cAMP-induced expression of the renin gene mediated by a CRE site in the proximal promoter. A, COUP-TFII is necessary for the cAMP-induced renin gene transcription in Calu-6 cells. COUP-TFII was knocked down with sequence-specific siRNA (siCOUP-TFII). Control cells were transfected with nontargeting siRNA (siControl). Calu-6 cells were transfected with the 258-bp human proximal promoter construct proxDR containing the COUP-TFII binding site, and the effect of cAMP agonists on its transcriptional activity was tested. B, Calu-6 cells were transfected with the proxDR construct, with construct proxDR-CREmut carrying the mutated cAMP target site CRE, with construct proxDR-CNREmut carrying the mutated cAMP target site CNRE, or with construct proxDRdel in which proxDR was deleted and the effect of cAMP agonists on the transcriptional activity was tested. The data shown are means ± S.E. (error bars). *, p < 0.05.

FIGURE 6.

COUP-TFII cooperates with CREB in the control of renin gene expression. Calu-6 cells were transfected with nontargeting siRNA as control (siControl) or with COUP-TFII sequence-specific siRNA (siCOUP-TFII), CREB sequence-specific siRNA (siCREB), or a combination of COUP-TFII and CREB siRNAs (siCOUP-TFII+siCREB). A, efficacy of the CREB knockdown. Protein extracts were probed in Western blots with CREB and β-actin (internal control) antibodies. See Fig. 3B for the efficacy of the COUP-TFII knockdown. B, effect of the deficiency of COUP-TFII, CREB, or both on renin gene expression. Renin and human β-actin (internal control) mRNA levels were quantified by real-time PCR. C, specificity of the knockdown. COUP-TFI and human β-actin (internal control) mRNA levels were quantified by real-time PCR. D, top, COUP-TFII expression is similarly diminished when COUP-TFII alone or COUP-TFII and CREB are knocked down. Bottom, CREB expression is similarly diminished when CREB alone or COUP-TFII and CREB are knocked down. COUP-TFII, CREB, and human β-actin (internal control) mRNA levels were quantified by real-time PCR. The data shown are means ± S.E. (error bars). *, p < 0.05. NS, not significant.

FIGURE 7.

COUP-TFII knock-out mice have decreased renin expression. A, efficiency of the knockout. Shown are renin (green), COUP-TFII (red), and DAPI (nuclear marker) (blue) co-immunostaining in control (wtCII^FF) and COUP-TFII knock-out (Cre^ERT2CII^FF) mouse kidneys. Arrows, renin immunoreactivity. B and C, renin expression is decreased in the COUP-TFII knock-out mice (Cre^ERT2CII^FF, n = 3) compared with the control mice (wtCII^FF, n = 4). Renin and β-actin (internal control) in mouse kidneys are detected by immunoblotting (B) and quantified by densitometry (C). The data shown are means ± S.E. (error bars).