Michael Desch

Peroxisome Proliferator-Activated Receptor-γ Is Involved in the Control of Renin Gene Expression

Based on the presence of a functional retinoic acid receptor/retinoid X receptor transcription factor binding sequence (hormone-responsive element) in the renin gene enhancer and on the fact that the peroxisome proliferator-activated receptors (PPARs) bind to DNA as heterodimers with retinoid X receptors, we speculated that PPARs are involved in the regulation of renin gene expression. To test this hypothesis, we used the human renin-producing cell line CaLu-6. Endogenous or pharmacological PPAR&ggr; agonists (unsaturated fatty acids and thiazolidinediones, respectively) stimulated renin gene expression. Surprisingly, we found that PPAR&ggr; targets a palindromic repeat with a 3-bp spacer (Pal3) in the proximal human renin promoter. Thus, renin is the first gene described with a functional Pal3 sequence. PPAR&ggr; agonists also stimulated renin gene expression in cultured native juxtaglomerular cells, which are the main source of renin in vivo. In summary, PPAR&ggr; was identified as a novel intracellular mediator involved in the upregulation of renin transcription.

Pituitary Adenylate Cyclase–Activating Polypeptide Stimulates Renin Secretion via Activation of PAC1 Receptors

Besides of its functional role in the nervous system, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of cardiovascular function. Therefore, PACAP is a potent vasodilator in several vascular beds, including the renal vasculature. Because the kidney expresses both PACAP and PACAP-binding sites, it was speculated that PACAP might regulate cardiovascular function by direct vascular effects and indirectly by regulating renin release from the kidneys. PACAP (1-27) stimulated renin secretion from isolated perfused kidneys of rats 4.9-fold with a half-maximum concentration of 1.9 nmol/L. In addition, PACAP stimulated renin release and enhanced membrane capacitance of isolated juxtaglomerular cells, indicating a direct stimulation of exocytotic events. The effect of PACAP on renin release was mediated by the specific PACAP receptors (PAC1), because PACAP (1-27) applied in concentrations in the physiologic range (10 and 100 pmol/L) did not enhance renin release from isolated kidneys of PAC1 receptor knockout mice (PAC1-/-), whereas it stimulated renin release 1.38- and 2.5-fold in kidneys from wild-type mice. Moreover, plasma renin concentration was significantly lower in PAC1-/- compared with their wild-type littermates under control conditions as well as under a low- or high-salt diet and under treatment with the angiotensin-converting enzyme inhibitor ramipril, whereas no differences in plasma renin concentration between the genotypes were detectable after water deprivation. These data show that PACAP acting on PAC1 receptors potently stimulates renin release, serving as a tonic enhancer of the renin system in vivo.

The Pal3 Promoter Sequence Is Critical for the Regulation of Human Renin Gene Transcription by Peroxisome Proliferator-Activated Receptor-γ

We recently reported that human renin gene transcription is stimulated by the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma in the renin-producing cell line Calu-6. The effect of PPARgamma was mapped to two sequences in the renin promoter: a direct repeat hormone response element (HRE), which is related to the classical PPAR response element (PPRE) and a nonconsensus palindromic element with a 3-bp spacer (Pal3). We now find that PPARgamma binds to the renin HRE. Neither the human renin HRE nor the consensus PPRE was sufficient to attain the maximal stimulation of renin promoter activity by the PPARgamma agonist rosiglitazone. In contrast, the human renin Pal3 element mediates both the full PPARgamma-dependent activation of transcription and the PPARgamma-driven basal renin gene transcription. The human renin Pal3 sequence was found to selectively bind PPARgamma and the retinoid X receptor-alpha from Calu-6 nuclear extracts. This is in contrast to the consensus PPRE, which can bind other nuclear proteins. PPARgamma knockdown paradoxically did not attenuate the stimulation of the endogenous renin gene expression by rosiglitazone. Similarly, a deficiency of PPARgamma did not attenuate the activation of the minimal human renin promoter, which contains the endogenous Pal3 motif. However, when the human renin Pal3 site was replaced by the consensus PPRE sequence, PPARgamma knockdown abrogated the effect of rosiglitazone on renin promoter activity. Thus, the human renin Pal3 site appears to be critical for the PPARgamma-dependent regulation of gene expression by mediating maximal transcription activation, particularly at the low cellular level of PPARgamma.

Increased Renin Production in Mice With Deletion of Peroxisome Proliferator-Activated Receptor-γ in Juxtaglomerular Cells

We recently found that endogenous (free fatty acids) and pharmacological (thiazolidinediones) agonists of nuclear receptor Peroxisome proliferator-activated receptor (PPAR)&ggr; stimulate renin transcription. In addition, the renin gene was identified as a direct target of PPAR&ggr;. The mouse renin gene is regulated by PPAR&ggr; through a distal enhancer direct repeat closely related to consensus PPAR response element (PPRE). In vitro studies demonstrated that PPAR&ggr; knockdown stimulated PPRE-driven transcription. These data predicted that deficiency of PPAR&ggr; would upregulate mouse renin expression. Consistent with these observations knockdown of PPAR&ggr; increased the transcription of a reporter gene driven by the mouse renin PPRE-like motif in vitro. To study the impact of PPAR&ggr; on renin production in vivo, we used a cre/lox system to generate double-transgenic mice with disrupted PPAR&ggr; locus in renin-producing juxtaglomerular (JG) cells of the kidney (RC-PPAR&ggr;fl/fl mice). We provide evidence that PPAR&ggr; expression was effectively reduced in JG cells of RC-PPAR&ggr;fl/fl mice. Fluorescent immunohistochemistry showed stronger renin signal in RC-PPAR&ggr;fl/fl than in littermate control RC-PPAR&ggr;wt/wt mice. Renin mRNA levels and plasma renin concentration in RC-PPAR&ggr;fl/fl mice were almost 2-fold higher than in littermate controls. Arterial blood pressure and pressure control of renal vascular resistance, which play decisive roles in the regulation of renin production were indistinguishable between RC-PPAR&ggr;wt/wt and RC-PPAR&ggr;fl/fl mice. These data demonstrate that the JG-specific PPAR&ggr; deficiency results in increased mouse renin expression in vivo thus corroborating earlier in vitro results. PPAR&ggr; appears to be a relevant transcription factor for the control of renin gene in JG cells.

Regulation of the renin expression in the retinal pigment epithelium by systemic stimuli

The retina expresses a local renin-angiotensin system (RAS). This study aimed to investigate the influence of systemic modulation of renin synthesis on the expression of renin in the retinal pigment epithelium (RPE), which forms part of the blood/retina barrier. Freshly isolated RPE cells showed expression of renin 1A, which is the secreted isoform of renin. Systemic administration of the angiotensin-converting enzyme inhibitor enalapril in mice increased the renin expression in both the kidney and the retina. Systemic infusion of ANG II led to a decrease in the renin expression in the kidney and in the retina and RPE. The ANG II-dependent down-regulation of renin expression in the RPE was prevented by systemic application of the AT(1) receptor blocker losartan. However, water deprivation lead to an increase of the renin expression in the kidney but unexpectedly to a decrease of the renin expression in the retina. In sections of the mouse retina, the ANG II receptor AT(1) was found in the RPE and localized at the blood side of the epithelium. Short-time cultured RPE cells showed increases in intracellular free Ca(2+) in response to stimulation by ANG II that were sensitive to losartan. In summary, we conclude that the renin expression in cells of the blood/retina barrier is influenced by the systemic RAS. ANG II circulating in the plasma is likely a mediator of this influence.

Chicken Ovalbumin Upstream Promoter Transcription Factor II Regulates Renin Gene Expression

Background: The production of the hormone renin is transcriptionally regulated. Results: The nuclear receptor COUP-TFII binds to the renin gene promoter and is necessary for the cAMP-induced renin gene expression. Conclusion: COUP-TFII stimulates renin gene transcription. Significance: The molecular mechanisms controlling the expression of renin are crucial for the understanding of its role in blood pressure regulation and nephrogenesis. 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.

Identification of ATF2 as a transcriptional regulator of renin gene.

Abstract The cAMP response element (enhCRE) in the distal enhancer regulatory region of renin gene is believed to play a major role in the control of renin transcription. enhCRE binds the CRE-binding protein (CREB), which is the main transcription factor target of cAMP signaling. Using the mouse renin-producing cell line As4.1 we found that activating transcription factor-2 (ATF2) also binds to enhCRE. N-terminal phosphorylation of ATF2, which controls its transactivation, is associated with downregulation of renin gene expression by the cytokine tumor necrosis factor-α (TNFα). The ubiquitin proteasome inhibitor MG132 also phosphorylates ATF2 and inhibits renin expression. Knockdown of ATF2 attenuated the suppression of renin gene expression by MG132, thus demonstrating that ATF2 mediates the inhibitory effect of MG132. In addition, MG132 increased the DNA-binding of ATF2 as well as the ratio of bound ATF2 to CREB. Using ATF2- and CREB-Gal4 fusion protein constructs coupled with luciferase reporter system we showed that ATF2 has a weaker transactivating capacity than CREB. These data suggest that ATF2 represses renin expression by drifting the transcriptional control of renin gene away from CREB. Accordingly, TNFα completely abrogated the cAMP-dependent stimulation of renin gene expression.