JG cell expression and partial regulation of a human renin genomic transgene driven by a minimal renin promoter

Patrick L. Sinn, Xiaoji Zhang, and Curt D. Sigmund

Departments of Internal Medicine and Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the kidney, renin gene expression is exquisitely localized to the juxtaglomerular (JG) cells lining the afferent arteriole, having the capacity to regulate renin synthesis in response to a variety of physiological cues. We investigated human renin gene expression in transgenic mice containing a genomic construct driven by 149 bp of its proximal promoter to elucidate whether this was sufficient to confer JG-specific expression. Whereas human renin mRNA was permissively expressed in most tissues, the transgene was expressed mainly in JG cells in the kidney. Active human renin and human prorenin were found in the systemic circulation at levels consistent with previous transgenic models. Remarkably, two lines displayed an appropriate upregulation of transgene mRNA in response to angiotensin-converting enzyme inhibition, and two lines exhibited a downregulation of transgene mRNA in response to subpressor and pressor doses of ANG II. Our results suggest that 149 bp of the human renin proximal promoter, in a context of a genomic construct, are sufficient to confer human renin expression in renal JG cells and at least some aspects of appropriate regulation.

angiotensin II; captopril; blood pressure; gene regulation; juxtaglomerular cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RENIN-ANGIOTENSIN system has long been known to be involved in physiological processes regulating blood pressure and electrolyte homeostasis. In most mammals, renin is thought to be the rate-limiting regulator of the enzymatic cascade leading to the production of ANG II. Renin is produced and processed in renal juxtaglomerular (JG) cells, which are primarily responsible for synthesis of blood-borne renin. Transcription of the renin gene and release of renin from these cells are stimulated in response to a variety of physiological and molecular signals resulting from reduced renal perfusion pressure, sodium depletion, and beta -adrenergic traffic from the adrenal gland and sympathetic nerves (11). On the contrary, renin protein release and mRNA production is thought to be downregulated under conditions of elevated pressure or in response to ANG II-mediated feedback (12). In the end, activation of angiotensin receptors results in a wide array of systemic effects, such as vasoconstriction, and tissue-specific effects, such as aldosterone release, enhancement of renal sodium transport, regulation of renal blood flow, increase in thirst, and facilitation of neurotransmitter release (reviewed in Refs. 6 and 22). Clearly, maintaining appropriate regulation of renin production is essential for the maintenance of a variety of homeostatic processes controlled ultimately by ANG II.

Numerous studies aimed at uncovering the DNA sequences required for signaling proper tissue- and cell-specific expression and physiological regulation of the renin gene have been reported (reviewed in Ref. 28). In an attempt to develop model systems to identify regulatory elements controlling renin transcription in vivo, we previously generated transgenic mice (900-hREN) containing a complete human renin (hREN) genomic sequence spanning a region from 896 bp in the 5' flank (promoter) to 400 bp in the 3' flank of the gene (26). The hREN transgene was expressed in the expected spectrum of tissues, including kidney, adrenal gland, testes, and ovary, and was developmentally regulated (24, 26). Moreover, hREN expression and protein release appropriately increased in response to captopril treatment and decreased in response to high dietary salt (32). Both observations are consistent with the renal JG cell-specific expression of the transgene observed by in situ hybridization (26). However, transgene expression was also evident in a number of extrarenal tissues, some of which are not considered classical renin-expressing sites. In addition, an increase in circulating renin was observed after bilateral nephrectomy (37), and a paradoxical increase in hREN mRNA was observed when hypertensive mice were obtained from breeding 900-hREN transgenic mice with mice expressing human angiotensinogen (hAGT) (32). This suggests that there may be a complex arrangement of regulatory elements controlling both cell-specific expression and regulation in response to physiological cues, and that the 900-hREN transgene may contain some but not all signals required for appropriate regulation.

Other attempts to identify specific regulatory sequences controlling hREN expression by fusing various lengths of the hREN promoter (149, 896, 1244, 3000, and 5000 bp) to reporter genes were unsuccessful. Although we obtained and bred a total of 46 transgenic founders, there was no evidence of significant transgene expression in any construct examined (Sigmund, unpublished observations), suggesting that important regulatory elements may exist either further upstream or within the structural gene itself. Both possibilities are supported by in vitro data (17, 36, 38). Other recent data demonstrating a marked increase in the stability of renin mRNA in response to cAMP treatment in both primary mouse JG cells (4) and hREN expressing Calu-6 cells (27) suggest that fusion transgenes may fail to express in transgenic mice because they lack the hREN mRNA sequence itself.

To address these issues, we have developed new hREN transgenic models that retain their overall genomic structure (all exons and introns) but differ in the amount of 5' flanking sequence present. In the present study, we examined whether a genomic construct containing only a minimal promoter (149 bp) would be sufficient to direct JG cell-specific expression of hREN in transgenic mice. Here we report the surprising finding that, although the transgene was ubiquitously and permissively expressed in most tissues, its expression in kidney was generally restricted to JG cells. Moreover, hREN expression in some transgenic lines was appropriately upregulated after angiotensin-converting enzyme ACE inhibition and downregulated after ANG II infusion.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Creation of transgenic mice and animal husbandry. The 140-hREN transgene construct was generated by first subcloning the downstream KpnI to BglII fragment containing exon IX (Fig. 1) from 900-hREN (26) into the pSL301 vector (Invitrogen). The large KpnI fragment from 900-hREN was then inserted upstream of the smaller fragment, and the orientation was verified by sequencing. The 140-hREN construct was prepared for microinjection by freeing it from the vector by digestion with NotI and BglII, followed by gel purification as previously described (26). Generation of transgenic mice was as previously described (26). All mice were fed standard mouse chow and water ad libitum unless otherwise indicated. Care of mice met or exceeded the standards set forth by the National Research Council in the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the University of Iowa Animal Care and Use Committee. Experimental mice were killed by CO2 asphyxiation.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 1.   Transgene maps: a schematic representation of 900-hREN and 140-hREN transgenes. Diagonal hatch, 5' flanking region; solid bars, exons; horizontal black line, intron sequences; vertical stripes, 3' flanking DNA. Localization of restriction sites used in cloning (KpnI and BglII), for excision of transgene from plasmid backbone (NotI and BglII), and for Southern blotting (BamHI) are indicated. hREN, human renin.

Transgenic mice were initially identified by PCR of DNA isolated from tail biopsy samples with the use of the same primer set routinely used to identify 900-hREN transgenics (26). Transgenic mice were then confirmed by Southern blot analysis after digestion with either BamHI, HindIII, or EcoRI and probing with an entire hREN cDNA as previously described (26). The expected pattern was observed with all three enzymes. Fifty live offspring were obtained from the microinjection, and twelve transgenic founders were obtained. Six of the founders were bred to establish lines for the studies described herein.

Administration of drugs and blood-pressure measurements. Captopril (Sigma) was dissolved in the drinking water (0.5 mg/ml) to give ~100 mg · kg-1 · day-1 of captopril for 10 days, as previously described (26). Microosmotic pumps (Alzet model 1007D) were utilized to administer vehicle (saline), subpressor (200 ng · kg-1 · min-1), and pressor (1,000 ng · kg-1 · min-1) doses of ANG II. Systolic blood pressure was determined with the use of the Visitech Systems BP-2000 blood pressure monitoring system, an automated device for measuring systolic blood pressure by tail cuff. Mice were trained for 7 consecutive days, during which time blood-pressure measurements were discarded; baseline systolic pressure was then measured for the next 3 days. The Alzet minipumps were then implanted under Metofane anesthesia, and measurements were repeated daily for the next 5 days.

RNase protection assay. hREN and mouse ren-1c mRNA levels were determined with an RNase protection assay (RPA). hREN and ren-1c probes were partial cDNA sequences cloned into pCRII (Invitrogen) and pGEM-4 (Promega), respectively, and the mouse beta -actin cDNA probe template was obtained from Ambion. The ren-1c probe was the kind gift of Dr. David C. Merrill (Bowman Gray School of Medicine). Tissues were homogenized, and RNA purification was conducted with the use of TriReagent (MRC) and the manufacturer's protocol. Total RNA (10-20 µg) was hybridized to a cRNA antisense probe labeled with the use of an in vitro transcription reaction containing [alpha -32P]UTP and purified with the use of a Sephadex G-50 quick spin column (Boehringer Mannheim). RPAs were performed with the use of a Hyb-Speed RPA kit (Ambion) and the manufacturer's protocol. Protection products were visualized by electrophoresis through a 7% polyacrylamide-5 M urea gel. The length of the full-length probe for hREN, mouse renin (mREN), and mAct is ~410, 430, and 315 nucleotides, respectively, and the expected protected fragment size is 300, 326, and 245 nucleotides, respectively. Quantification of RPAs were performed with the use of a Molecular Dynamics Storm 620 phosphorimager system and the ImageQuant software provided by the manufacturer. hREN and mREN mRNA were normalized for mouse actin expression in each RPA reaction by dividing the renin mRNA signal by the actin signal. All comparisons for the captopril and ANG II infusion experiments were run on one to two RPA gels run simultaneously.

Plasma renin activity and concentration. Plasma renin activity and plasma renin concentration were determined with the use of an ANG I 125I-radioimmunoassay kit (NEN) and purified hAGT substrate (Scripps Laboratories), using the protocol and calculations previously described (32). The trypsin-based prorenin activation protocol was conducted as previously described (23). Briefly, 100 µl of plasma were combined with activation buffer and 75 µg trypsin in a final volume of 300 µl. The reaction was incubated overnight at 4°C with gentle agitation. Activation buffer consisted of 50 mmol/l Tris (pH 7.4), 0.1 mol/l NaCl, 0.5% BSA, 0.1% EDTA, and 0.1% sodium azide.

Immunohistochemistry. Excised kidneys were immediately immersed in 15% dextrose followed by 30% dextrose until they were saturated. Kidneys were then snap frozen on dry ice and sectioned (6- to 14-µm thick). Sections were fixed in 4% paraformaldehyde for 5-10 min. The sections were incubated overnight at 4°C with a polyclonal hREN primary antibody (R12) diluted 1:35 in DMEM with 5% fetal bovine serum. The hREN antibody was a generous gift from Professors Pierre Corvol and Florence Pinet (INSERM U36, College de France, Paris, France) and was described previously (7). The sections were washed with 1× PBS and incubated with an FITC-conjugated anti-rabbit secondary antibody (Pierce) for 30 min at 37°C. After the secondary incubation, slides were rinsed briefly with 1× PBS before being mounted with a cover slip. Sections were examined from five mice from line 2957/1, six mice from line 2814/2, and three mice from 900-hREN line 9 (26). At least eight sections per kidney were examined. The images were captured with a Bio-Rad MRC-1024 Hercules laser scanning confocal microscope equipped with a Kr/Ar laser.

Statistics. All numerical data are presented as means ± SE. Statistical analysis was performed with the use of Student's t-test, using the SigmaPlot or Systat software package.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic mice were constructed with the hREN gene driven by 149 bp of native promoter region (Fig. 1). This construct is identical to the 900-hREN transgenic model we previously described except for employing a shorter promoter (26). Multiple transgenic lines were established, and the presence of the transgene in each line was confirmed by Southern blot analysis (Fig. 2). These results confirmed the presence of an internal 2.0-kb BamHI fragment (containing exons III-V; solid arrow) and a larger genomic fragment derived from the 3' end of one transgene (exons VI-IX; hatched arrow) and the 5' end of the next transgene (exons I-II) along the contiguous array made by the insertion of multiple copies of the transgene. The large fragment allows us to distinguish 900-hREN (open arrow) from 140-hREN (hatched arrow) transgenics at the DNA level.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 2.   Southern blot analysis. A Southern blot was performed with BamHI digested genomic DNA isolated from tail biopsies. Blot was probed with a full-length hREN cDNA. Molecular-weight marker (M) is a mixture of phage lambda  DNA digested with HindIII and EcoRI/HindIII (sizes are indicated in kb) and was probed with total phage lambda  DNA. Identification of transgenic lines is indicated. Solid arrow, internal BamHI fragment containing exons III-V; hatched arrow, junctional BamHI fragment detected in concatemer and containing 3' exons VI-IX fused to 5' exons I-II in 140-hREN construct; open arrow, junctional BamHI fragment detected in concatemer and containing 3' exons VI-IX fused to 5' exons I-II in 900-hREN construct. All 3 sections of figure were cut from same Southern blot.

An RPA was used to assess the tissue-specific expression of the transgene. Figure 3 shows a representative RPA from line 2813/4 probed for hREN (solid arrow) and mouse beta -actin (open arrow) as a loading control. High-level expression of the hREN gene in the lung and adipose tissue is readily apparent after an overnight exposure (Fig. 3, top), whereas a longer exposure (Fig. 3, bottom) was required to observe expression in the kidney and other extrarenal tissues. The results of 16 RPA experiments from an analysis of six lines of 140-hREN and three lines of 900-hREN mice is shown in Table 1. Extremely high levels of hREN mRNA were routinely found in the lung and adipose tissue across all lines of both constructs. hREN expression was evident in the kidney of five of six 140-hREN lines examined, and its expression was generally less than that observed in 900-hREN mice. Expression of the transgene was also detected in testes, spleen, and skeletal muscle in most of the lines and was occasionally detected in liver, heart, brain, and salivary gland. This pattern of expression is reminiscent of the tissue distribution reported in the 900-hREN animals; however, the expression of hREN in "ectopic" extrarenal sites was much more pronounced and variable among lines of 140-hREN mice.


View larger version (81K):
[in this window]
[in a new window]
 
Fig. 3.   Representative RNase protection assay (RPA) of RNA from a 140-hREN mouse is shown. Top: a 24-h exposure. Bottom: a 7-day exposure. Solid arrow, hREN-specific transcript; open arrow, beta -actin-specific transcript; Lg, lung; KL, left kidney; KR, right kidney; Lv, liver; H, heart; Sp, spleen; T, testes; B, brain; M, skeletal muscle; A, adipose tissue; Sg, submandibular gland.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Summary of tissue-specific expression of hREN in transgenic mice

Immunohistochemistry was conducted with the use of the hREN specific antibody R12 (7) and an FITC-conjugated anti-rabbit secondary antibody to determine whether renin expression was properly restricted to renal JG cells. JG cell localization was evident in the two independent lines (2814/2 and 2957/1) of 140-hREN mice tested (Fig. 4) and was similarly detected in the kidney of five different mice from each line. In addition, however, low-level staining was occasionally observed in epithelial and vascular cells in line 2957/1. The antibody was specific for hREN, as no significant FITC staining was observed in the nontransgenic control sections. It is interesting to note that fewer JG apparatuses demonstrated evidence of hREN expression (judged to be ~10%) in the kidney of 140-hREN mice than in 900-hREN mice (data not shown). Moreover, captopril treatment did not significantly increase the number of JG apparatuses expressing hREN in these mice (data not shown).


View larger version (87K):
[in this window]
[in a new window]
 
Fig. 4.   Confocal immunofluorescence detection of hREN in kidney. Confocal images of frozen kidney sections from a 2814/2 (A) and 2957/1 (B) and a control nontransgenic mouse (C) are shown. Position of juxtaglomerular apparatus labeled with antibody is indicated by white arrows. Magnification, photographed at ×20.

We next assayed for the presence of both active hREN and prorenin in the plasma, using purified hAGT as the substrate and a radioimmunoassay designed to measure plasma ANG I. Plasma hREN was detectable in two (of three) lines tested, at a level comparable with that previously reported for 900-hREN (Table 2) (32). Moreover, a majority of the circulating hREN appeared to be in the form of prorenin. It is likely that expression of hREN in the extrarenal sites discussed above may contribute a significant amount of prorenin to the plasma. There was no significant difference in the level of mREN in either transgenic or nontransgenic control mice.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Plasma renin content in 140-hREN transgenic mice

Given the JG cell-specific expression described above, we next investigated whether this transgene responded appropriately to physiological stimuli that are known to regulate expression of the renin gene. In the first set of experiments, three lines of 140-hREN transgenic and nontransgenic mice were treated with the ACE inhibitor captopril for 10 days. Captopril prevents the conversion of ANG I to ANG II and thus relieves the negative feedback normally exerted on renin production, resulting in an increase of renin mRNA. Indeed, endogenous mREN mRNA was increased approximately fourfold in all three transgenic lines and nontransgenic littermates (see Fig. 5B). Renal hREN mRNA was significantly increased in one line (2846/3; P < 0.02), was increased but did not reach statistical significance in another line (2957/1; P = 0.06), and did not increase at all in third line (2814/2; Fig. 5A). The increase in hREN mRNA in lines 2846/3 and 2957/1 was approximately twofold, whereas mREN mRNA increased approximately fourfold. Although captopril caused an induction in hREN mRNA in kidney, no changes in hREN mRNA abundance were observed in lung or adipose tissue of line 2846/3 in response to captopril (data not shown).


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 5.   Renal renin mRNA response to captopril. hREN (A) and mouse renin (mREN; B) response to captopril treatment is shown for three independent transgenic lines. hREN and mREN mRNA was normalized to beta -actin signal, and quantification was performed as described in METHODS. Units, although normalized to same arbitrary scale, should not be used to compare hREN mRNA abundance between independent lines. A representative RPA is shown at bottom of each column. Solid arrow, hREN-specific or Ren-1c-specific transcript; open arrow, beta -actin-specific transcript. Presence (+) of captopril (Cap) in drinking water is indicated. Presence (+) or absence (-) of transgene (Tg) in genome is indicated. Individual P values (above hatched bars) are from a comparison of untreated vs. captopril-treated mice; n = 3 for 2814/2 (4 in captopril-treated group), 3 for 2846/3, and 4 for 2957/1. Three or 4 nontransgenic littermates were tested in each line.

In the second set of experiments, two lines of 140-hREN transgenic and nontransgenic mice were administered either a nonpressor or pressor dose of ANG II with the use of a subcutaneous microosmotic pump. Systolic blood pressure was measured in each mouse before and after ANG II administration to verify the efficacy of each dose of ANG II (Fig. 6). There was no significant difference between the systolic blood pressure of the saline (vehicle) and subpressor ANG II infusion groups. However, the pressor dose of ANG II caused a 25- to 30-mmHg increase in systolic blood pressure that generally peaked 3 days after implantation of the pump. Endogenous renal mREN mRNA was significantly downregulated in response to both the nonpressor (P < 0.04) and pressor (P < 0.01) dose of ANG II (Fig. 7). Interestingly, a significant decrease in renal hREN mRNA was observed in line 2814/2 that was similar in magnitude and significance to endogenous mREN mRNA. As in the captopril experiment, although there was a trend toward reduced hREN mRNA in the kidney in line 2957/1, it did not reach statistical significance.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6.   Blood pressure responses to ANG II. Systolic blood pressure (SBP) was measured in saline-infused (S), nonpressor dose ANG II-infused (N), and pressor dose ANG II-infused (P) transgenic mice from 2 independent lines and in control mice. A: SBP after 5 days of infusion. B: maximal change in SBP (Delta SBP) in response to ANG II from baseline pressure recorded before implantation of minipump. * P < 0.05 vs. saline; n in each group was 4 for 2814/2, 4 for 2957/1, and 3 for nontransgenic controls.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 7.   Renal renin mRNA response to ANG II infusion. mREN (A) and hREN (B and C ) mRNA levels were quantified by RPA in mice from saline-infused (S), nonpressor dose ANG II-infused (N), and pressor dose ANG II-infused (P) transgenic mice from 2814/2 (B) and 2957/1 (C ) lines. hREN and mREN mRNA was normalized to beta -actin signal, and quantification was performed as described in methods. A representative RPA is shown at bottom of each column. Solid arrow, hREN-specific or Ren-1c-specific transcript; open arrow, beta -actin-specific transcript. Individual P values (above hatched and filled bars) are from a comparison of ANG II-treated vs. saline-treated mice; n in each group was 4 for 2814/2, 4 for 2957/1, and 3 for nontransgenic controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We report the surprising finding that an hREN genomic construct driven by only a 149-bp minimal promoter is sufficient to target JG cell-specific expression despite permissive expression in most other tissues. Moreover, several lines of mice exhibited appropriate hREN responses to ACE inhibition and ANG II infusion, suggesting that the transgene exhibits at least some aspects of appropriate regulation. When compared with 900-hREN transgenic mice, expression in the kidney was reduced, with fewer JG cells exhibiting renin immunostaining, and expression in ectopic sites was more abundant and variable among transgenic lines.

Among these ectopic sites are lung and adipose tissue, where abundant hREN mRNA was observed. It remains unclear whether the lung and adipose tissue are true ectopic sites. Indeed, low levels of hREN expression have previously been detected in human fetal lung, in tumors derived from the lung, and in Calu-6 cells derived from a human pulmonary carcinoma (16, 30, 31). In addition, hREN mRNA was reported in human adipose tissue and isolated adipocytes (14). It is interesting to note that adipose tissue is a known site of angiotensinogen expression, suggesting the potential for local synthesis of ANG II in fat (14). Moreover, ANG II has been reported to increase prostacyclin synthesis in fat, which can induce the differentiation of preadipocytes to adipocytes and stimulate lipid synthesis and storage (5, 13).

Independent lines were tested for their ability to upregulate and downregulate hREN mRNA in response to ACE inhibition and ANG II treatment, respectively. In lines 2846/3 and 2957/1, captopril caused a 2-fold increase in hREN mRNA, less than the 3.9- and 3.0-fold increases in endogenous mREN mRNA, respectively. As in the captopril experiment, ANG II caused only a modest but not statistically significant decrease in hREN mRNA in line 2957/1. There was no hREN response to captopril in line 2814/2, although mREN mRNA increased 4.3-fold. It is therefore surprising that this same line, which did not respond to captopril, responded appropriately and with the same magnitude as the mREN gene to both nonpressor and pressor doses of ANG II. Moreover, we previously reported that the 900-hREN construct is upregulated both in response to captopril and, paradoxically, in hypertensive hREN/hAGT double transgenic mice (32). We were therefore surprised that two lines of 140-hREN mice exhibited a decrease in hREN mRNA in response to ANG II infusion. Preliminary studies suggest that the mechanism for the upregulation observed in the 900-hREN mice may be due to an increase in pressure and not ANG II per se (H. Keen and C. D. Sigmund, unpublished observation), suggesting the possibility that there is a complicated arrangement of regulatory elements controlling hREN transcriptional responses to physiological cues such as elevated pressure, ANG II, and ACE inhibition. The exact reasons for the discrepant results between constructs remain unclear. It is important to recognize that captopril not only relieves feedback caused by the loss of ANG II but also reduces blood pressure when administered chronically (18). Therefore, the induction of hREN mRNA in the 140-hREN mice in response to captopril may be due to both mechanisms.

Our data suggest that the 140-hREN transgene may have the intrinsic ability to be appropriately regulated by both ACE inhibition and ANG II but that other factors may be strongly influencing the response in the different transgenic lines. The factors influencing regulation of the transgene by physiological cues may also be effecting variable expression of hREN in ectopic sites. Of the factors that may be responsible, transgene position effects are most likely to have the greatest effect. The site of transgene integration has been repeatedly documented to have a strong influence on transgene expression, and these effects will be different among lines because of the utilization of different insertion sites (1). Transgenes immune to position effects are generally very large (encoded on BAC or YAC vectors) and contain dominant control regions that are thought to insulate the transgene from effects of neighboring DNA or to act as transcriptional enhancers (10). In our construct, it is likely that the juxtaposition of basal transcription factor-binding sites present in the hREN promoter (Fig. 8) next to anonymous DNA at or near the insertion site may impart a new or altered set of transcriptional instructions to the gene. These instructions may include directing expression to ectopic sites such as the liver, heart, skeletal muscle, and submandibular gland and may differ from line to line. Recent results suggest that sequences in the renin promoter normally interact with an enhancer of transcription located ~2.6 kb upstream of the mREN gene (21) and 13 kb upstream of the hREN gene (38). Enhancers of transcription are known to be able to increase transcription in an orientation- and position-independent manner and may impart influences on the transgene even if located far upstream or downstream from the transgene. Therefore, it is possible that the hREN minimal promoter, which normally responds to signals from the renin enhancer (which is missing in 140-hREN) may therefore be quite susceptible to the influence of transcription factors binding near the integration site.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   Potential transcription factor-binding sites in 149-bp hREN promoter. Potential transcription factor-binding sites were identified in 149-bp promoter with use of TFSEARCH (available online at http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html). Only sites with 85% homology in mammalian database were scored. In addition, any site detected in 149-bp sequence that contained bases different from invariant bases in consensus were discarded. Sites detected on sense strand are in solid colors and those on antisense strand are hatched. Sites: green hatch, transcriptional activator Pbx-1 (34); solid green, nuclear zinc-finger factor Gfi-1 (40); solid red, POU-domain transcription factor Oct-1 (35); blue hatch, GATA-1 (19); solid blue, stress-induced factor CHOP (33); solid pink, helix-loop-helix factor SREBP1 (15); yellow, TATA Box (2); light purple, myeloid zinc finger factor MZF-1 (20). In addition, sequence identified as a Pit-1 binding site and shown to be important for response to cAMP is indicated by thin black bar (3, 29).

Until recently, most studies examining the transcriptional regulation of the renin gene focused on the renin promoter and employed transfection of cultured cells. In nearly all studies reported thus far, the 149-bp promoter, because of the localization of a convenient KpnI restriction site at -149, has been considered the basal promoter. Extensions of this sequence with DNA further 5' were used to identify sequences acting as enhancers or repressors of transcription (reviewed in Ref. 28). In vitro data have suggested that the hREN minimal promoter is extremely weak, driving expression of reporter genes at only a small fraction of the level of strong viral promoters (17). The 149-bp promoter contains a TATA box located at -29 and a binding site homology to the POU-domain transcription factor Pit-1 located at -67. Interestingly, the Pit-1 site has been reported to be required in conjunction with a cAMP response element (located at -218 and absent from 140-hREN) for the transactivation of the hREN promoter by cAMP (8, 9, 39). In addition, a search of the 149-bp promoter revealed potential binding sites for a number of other transcription factors that are reported to act as transcriptional activators and repressors in other genes (Fig. 8). It is therefore tempting to speculate that Pit-1, or some other transcription factor-binding sites, may be necessary for directing JG cell-specific expression, although further experimentation will be required before a specific site can be implicated.

Similar to in vitro studies, in vivo studies initially focused on the creation of transgenic mice containing a fusion between the hREN promoter and a reporter gene. Transgenic mice containing constructs consisting of variable lengths of the hREN promoter (from -149 to -5000) fused to either SV40 T-antigen or luciferase were generated and either failed to demonstrate any appreciable transgene expression or required RT-PCR followed by Southern blot for detection (Sigmund, unpublished observations). These data suggest that the important regulatory elements may lie either further 5', within the body of the gene, or in the 3' flanking region. Such a candidate may be an enhancer of transcription identified upstream of the mouse and hREN promoters (21, 38). A 4.6-kb segment of the mouse Ren-2 promoter containing the mREN enhancer confers tissue-specific expression on an SV40 T-antigen reporter gene (25). Interestingly, however, a fusion construct consisting of the mouse enhancer upstream from the hREN promoter (896 bp) and the T-antigen reporter gene failed to be expressed in transgenic mice (Q. Shi and C.D. Sigmund, unpublished observation). When these data are taken together, it suggests that the mechanism controlling tissue- and cell-specific expression of hREN may be very complicated. That the distance between the mouse and hREN enhancer sequences and their respective promoters differ substantially suggests the provocative possibility that the mechanisms controlling their regulation may be somewhat divergent.

Finally, data supporting a posttranscriptional mechanism regulating renin mRNA stability may further complicate this picture (4, 27). It is tempting to speculate that the process of transcription, controlled by sequences in the 5' flanking region of the gene, in conjunction with mRNA processing signals present at the exon/intron boundaries and signals regulating mRNA stability, located within the mRNA itself, are all intimately coupled. Consequently, disruption of these processes by removal of either 1) regulatory sequences in the promoter through truncation, 2) exons and introns in the hREN structural gene, or 3) the structure of the hREN mRNA may result in loss of renin expression and may explain our previous inability to map regulatory elements controlling expression of hREN in vivo. It is therefore likely that future transgenic studies of the hREN gene will need to incorporate the entire 13-kb exon/intron region of the gene to be appropriately expressed in mice. We are currently examining the expression of two large hREN transgenes (140 kb and 160 kb) encoded on P1 artificial chromosomes to assess whether they are immune to position artifacts and exhibit appropriate regulation.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Pierre Corvol and Florence Pinet for the gift of the R12 hREN antibody and Dr. David Merrill for the gift of a new ren-1c probe. We also acknowledge the excellent technical assistance of Lisa Hancox, Lucy Robbins, and Norma Sinclair for generation of transgenic mice; Debbie Davis for assistance with blood pressure recordings; Haley Williams for assistance with the fluorescence studies; Henry Keen for assistance with implantation of osmotic minipumps; and Lihong Zhao for the Southern blot.


    FOOTNOTES

Funds in support of this work were obtained from National Heart, Lung, and Blood Institute Grants HL-55006 and HL-48058 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52617 and the American Heart Association.

C. D. Sigmund was an established investigator of the American Heart Association.

Transgenic mice were generated and maintained at the University of Iowa Transgenic Animal Facility, which is supported in part by the College of Medicine and the Diabetes and Endocrinology Research Center. DNA sequencing was performed at the University of Iowa DNA Core Facility. Confocal microscopy was performed at the University of Iowa Central Microscopy Facility.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. D. Sigmund, Molecular Biology Interdisciplinary Program, Transgenic and Gene Targeting Facility, Dept. of Internal Medicine and Physiology and Biophysics, 2191 Medical Laboratory, The Univ. of Iowa College of Medicine, Iowa City, Iowa 52242 (E-mail: curt-sigmund{at}uiowa.edu).

Received 16 March 1999; accepted in final form 21 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Al Shawi, R., J. Kinnaird, J. Burke, and J. O. Bishop. Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect. Mol. Cell. Biol. 10: 1192-1198, 1990[Medline].

2.   Bucher, P. Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. J. Mol. Biol. 212: 563-578, 1990[Medline].

3.   Catanzaro, D. F., J. Sun, M. T. Gilbert, Y. Yan, T. Black, C. D. Sigmund, and K. W. Gross. A Pit-1 binding site in the human renin gene promoter stimulates activity in pituitary, placental and juxtaglomerular cells. Kidney Int. 46: 1513-1515, 1994[Medline].

4.   Chen, M., J. Schnermann, A. M. Smart, F. C. Brosius, P. D. Killen, and J. P. Briggs. Cyclic AMP selectively increases renin mRNA stability in cultured juxtaglomerular granular cells. J. Biol. Chem. 268: 24138-24144, 1993[Abstract/Free Full Text].

5.   Darimont, C., G. Vassaux, G. Ailhaud, and R. Negrel. Differentiation of preadipose cells: paracrine role of prostacyclin upon stimulation of adipose cells by angiotensin-II. Endocrinology 135: 2030-2036, 1994[Abstract].

6.   Fitzsimons, J. T. Angiotensin, thirst, and sodium appetite. Physiol. Rev. 78: 583-686, 1998[Abstract/Free Full Text].

7.   Galen, F. X., T. T. Guyenne, C. Devaux, C. Auzan, P. Corvol, and J. Menard. Direct radioimmunoassay of human renin. J. Clin. Endocrinol. Metab. 48: 1041-1043, 1979[Abstract].

8.   Germain, S., T. Konoshita, J. Philippe, P. Corvol, and F. Pinet. Transcriptional induction of the human renin gene by cyclic AMP requires CREB and a factor binding to pituitary-specific trans-acting factor (Pit-1) motif. Biochem. J. 316: 107-113, 1996[Medline].

9.   Gilbert, M. T., J. Sun, Y. Yan, C. Oddoux, A. Lazarus, W. P. Tansey, T. N. Lavin, and D. F. Catanzaro. Renin gene promoter activity in GC cells is regulated by cAMP and thyroid hormone through Pit-1-dependent mechanisms. J. Biol. Chem. 269: 1-6, 1994[Free Full Text].

10.   Grosveld, F., G. B. van Assendelft, D. R. Greaves, and G. Kollias. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell 51: 975-985, 1987[Medline].

11.   Hackenthal, E., M. Paul, D. Ganten, and R. Taugner. Morphology, physiology, and molecular biology of renin secretion. Physiol. Rev. 70: 1067-1116, 1990[Free Full Text].

12.   Johns, D. W., M. J. Peach, R. A. Gomez, T. Inagami, and R. M. Carey. Angiotensin II regulates renin gene expression. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F882-F887, 1990[Abstract/Free Full Text].

13.   Jones, B. H., M. K. Standridge, and N. Moustaid. Angiotensin II increases lipogenesis in 3T3-L1 and human adipose cells. Endocrinology 138: 1512-1519, 1997[Abstract/Free Full Text].

14.   Karlsson, C., K. Lindell, M. Ottosson, L. Sjostrom, B. Carlsson, and L. M. Carlsson. Human adipose tissue expresses angiotensinogen and enzymes required for its conversion to angiotensin II. J. Clin. Endocrinol. Metab. 83: 3925-3929, 1998[Abstract/Free Full Text].

15.   Kim, J. B., G. D. Spotts, Y. D. Halvorsen, H. M. Shih, T. Ellenberger, H. C. Towle, and B. M. Spiegelman. Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain. Mol. Cell. Biol. 15: 2582-2588, 1995[Abstract].

16.   Lang, J. A., G. Yang, J. A. Kern, and C. D. Sigmund. Endogenous human renin expression and promoter activity in a pulmonary carcinoma cell line (Calu-6). Hypertension 25: 704-710, 1995[Abstract/Free Full Text].

17.   Lang, J. A., L.-H. Ying, B. J. Morris, and C. D. Sigmund. Transcription and posttranscriptional mechanisms regulate expression of the human renin gene in Calu-6 cells. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F94-F100, 1996[Abstract/Free Full Text].

18.   Mattson, D. L., and K. R. Krauski. Chronic sodium balance and blood pressure response to captopril in conscious mice. Hypertension 32: 923-928, 1998[Abstract/Free Full Text].

19.   Merika, M., and S. H. Orkin. DNA-binding specificity of GATA family transcription factors. Mol. Cell. Biol. 13: 3999-4010, 1993[Abstract].

20.   Morris, J. F., R. Hromas, and F. J. Rauscher. Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core. Mol. Cell. Biol. 14: 1786-1795, 1994[Abstract].

21.   Petrovic, N., T. A. Black, J. R. Fabian, C. M. Kane, C. A. Jones, J. A. Loudon, J. P. Abonia, C. D. Sigmund, and K. W. Gross. Role of proximal promoter elements in regulation of renin gene transcription. J. Biol. Chem. 271: 22499-22505, 1996[Abstract/Free Full Text].

22.   Schnermann, J. Juxtaglomerular cell complex in the regulation of renal salt excretion. Am. J. Physiol. 274 (Regulatory Integrative Comp. Physiol. 43): R263-R279, 1998[Abstract/Free Full Text].

23.   Sealey, J. E. Plasma renin activity and plasma prorenin assays. Clin. Chem. 37: 1811-1819, 1991[Abstract].

24.   Sigmund, C. D. Expression of the human renin gene in transgenic mice throughout ontogeny. Pediatr. Nephrol. 7: 639-645, 1993[Medline].

25.   Sigmund, C. D., C. A. Jones, J. R. Fabian, J. J. Mullins, and K. W. Gross. Tissue and cell-specific expression of a renin promoter-T antigen reporter gene construct in transgenic mice. Biochem. Biophys. Res. Commun. 170: 344-350, 1990[Medline].

26.   Sigmund, C. D., C. A. Jones, C. M. Kane, C. Wu, J. A. Lang, and K. W. Gross. Regulated tissue- and cell-specific expression of the human renin gene in transgenic mice. Circ. Res. 70: 1070-1079, 1992[Abstract].

27.   Sinn, P. L., and C. D. Sigmund. Human renin mRNA stability is increased in response to cAMP in Calu-6 cells. Hypertension 33: 900-905, 1999[Abstract/Free Full Text].

28.   Sinn, P. L., and C. D. Sigmund. Understanding the regulation of renin gene expression through in vitro and in vivo models. In: Drugs, Enzymes and Receptors of the Renin Angiotensin System. A Century of Discovery, edited by A. Husain, and R. M. Graham. Sydney: Harwood Academic/Gordon and Breach Scientific International, 1999.

29.   Sun, J., C. Oddoux, M. T. Gilbert, Y. Yan, A. Lazarus, W. G. Campbell, and D. F. Catanzaro. Pituitary-specific transcription factor (Pit-1) binding site in the human renin gene 5'-flanking DNA stimulates promoter activity in placental cell primary cultures and pituitary lactosomatotropic cell lines. Circ. Res. 75: 624-629, 1994[Abstract].

30.   Taylor, G. M., H. T. Cook, C. Hanson, W. S. Peart, T. Zondek, and L. H. Zondek. Renin in human fetal lung---a biochemical and immunohistochemical study. J. Hypertens. 6: 845-851, 1988[Medline].

31.   Taylor, G. M., H. T. Cook, E. A. Sheffield, C. Hanson, and W. S. Peart. Renin in blood vessels in human pulmonary tumors: an immunohistochemical and biochemical study. Am. J. Pathol. 130: 543-551, 1988[Abstract].

32.   Thompson, M. W., S. B. Smith, and C. D. Sigmund. Regulation of human renin mRNA expression and protein release in transgenic mice. Hypertension 28: 290-296, 1996[Abstract/Free Full Text].

33.   Ubeda, M., X. Z. Wang, H. Zinszner, I. Wu, J. F. Habener, and D. Ron. Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element. Mol. Cell. Biol. 16: 1479-1489, 1996[Abstract].

34.   Van Dijk, M. A., P. M. Voorhoeve, and C. Murre. Pbx1 is converted into a transcriptional activator upon acquiring the N-terminal region of E2A in pre-B-cell acute lymphoblastoid leukemia. Proc. Natl. Acad. Sci. USA 90: 6061-6065, 1993[Abstract].

35.   Verrijzer, C. P., M. J. Alkema, W. W. van Weperen, H. C. Van Leeuwen, M. J. Strating, and P. C. van der Vliet. The DNA binding specificity of the bipartite POU domain and its subdomains. EMBO J. 11: 4993-5003, 1992[Abstract].

36.   Voigtlander, T., D. Ganten, and M. Bader. Transcriptional regulation of the rat renin gene by regulatory elements in intron I. Hypertension 33: 303-311, 1999[Abstract/Free Full Text].

37.   Yan, Y., R. Chen, T. Pitarresi, C. D. Sigmund, K. W. Gross, J. E. Sealey, J. H. Laragh, and D. F. Catanzaro. Kidney is the only source of human plasma renin in 45-kb human renin transgenic mice. Circ. Res. 83: 1279-1288, 1998[Abstract/Free Full Text].

38.   Yan, Y., C. A. Jones, C. D. Sigmund, K. W. Gross, and D. F. Catanzaro. Conserved enhancer elements in human and mouse renin genes have different transcriptional effects in As4.1 cells. Circ. Res. 81: 558-566, 1997[Abstract/Free Full Text].

39.   Ying, L., B. J. Morris, and C. D. Sigmund. Transactivation of the human renin promoter by the cyclic AMP/protein kinase A pathway is mediated by both CREB-dependent and CREB-independent mechanisms in Calu-6 cells. J. Biol. Chem. 272: 2412-2420, 1997[Abstract/Free Full Text].

40.   Zweidler-Mckay, P. A., H. L. Grimes, M. M. Flubacher, and P. N. Tsichlis. Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor. Mol. Cell. Biol. 16: 4024-4034, 1996[Abstract].


Am J Physiol Renal Physiol 277(4):F634-F642
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society