Renin Promoter Research Articles

Cis-regulatory elements and trans-acting factors directing basal and cAMP-stimulated human renin gene expression in chorionic cells.

Much knowledge was accumulated in the regulation of plasma renin activity and renin secretion during recent years. However, the mechanisms of renin gene transcription, especially for the human gene, have been poorly studied because of the lack of cell lines expressing renin. Cells derived from chorion tissue were used to study renin gene transcription because these cells express renin and regulate renin secretion in a similar way to JG cells. The present study was performed to determine the cis-regulatory elements and the trans-acting factors involved in human renin gene expression using chorionic cells. Transient DNA transfections were performed with various constructs containing the 5'-flanking region of the human renin gene. 5'-Deletion analysis of the human renin promoter (from -2616 to -67 bp) revealed the presence of two proximal negative cis-regulatory elements between -374 and -273 bp and between -273 and -137 bp. These elements were not present in a non-renin-producing cell line, JEG-3 cells. DNase I footprinting revealed that two sequences located within these regions bind trans-factors present in chorionic cellular nuclear extract: AGE3-like sequence (-293/-273) and apolipoprotein A1 regulatory protein-1-like sequence (-259/-245). The first 110 bp of the renin promoter were sufficient to direct specific expression in chorionic cells and contained two footprints sharing homology with ets (-29/-6) and pituitary-specific factor (Pit-1) (-70/-62) sequences. Furthermore, one footprint (-234/-214) contained the sequence TAGCGTCA, which shares strong homology to the cAMP-responsive element (CRE) binding site. Gel shift analysis showed specific DNA/protein complexes within this region, which were displaced by the somatostatin consensus CRE. Finally, luciferase analysis of 5'-deletion mutant revealed that -273 to +16 bp of the renin promoter was sufficient to confer complete forskolin stimulation, whereas deletion to -130 (deletion of the CRE) decreased cAMP responsiveness by 50% and those to -67 bp (deletion of the CRE and Pit-1-like sequences) suppressed it. Thus, these latter two sequences probably act together to confer complete cAMP responsiveness.

Tissue-Specific trans-Activation of Renin Gene by Targeted Expression of Adenovirus E1A in Transgenic Mice

We generated two transgenic mice carrying the adenovirus type 12 E1 region genes under control of the human renin promoter, in which the E1A and E1B genes were expressed predominantly in the kidney. Interestingly, renal transcription of the gene for mouse renin was shown to be elevated at 28 and 40 folds as compared in those of control animals, but histon mRNA levels were unchanged. Although the transfected mouse renin promoter was promiscuously trans-activated by E1A in HeLa cells where the endogenous renin gene was silent, the transgenic studies suggested that a cellular factor(s), in addition to E1A, was required for the tissue-specific renin gene activation. These findings provide the in vivo evidence that E1A could trans-activate cellular gene transcription in a tissue-specific manner.

SPECIES DIFFERENCES IN BINDING OF SUBMANDIBULAR NUCLEAR PROTEINS TO RENIN PROMOTER DNA

1. Renin is highly expressed in submandibular gland (SMG) of mouse, which has two genes, Ren-1d and Ren-2d, but not at all in rat SMG. Differences in nuclear protein binding to renin promoter DNA were, therefore, explored. 2. Rat -169 to +23 renin DNA formed complexes with both mouse and rat extract, whereas a corresponding fragment of mouse Ren-1d DNA (-121 to +4) bound with rat extract, but much less so with mouse extract. Rat extract bound a -704 to -450 fragment of the Ren-1d promoter. For Ren-2d -578 to -383 and -786 to -718 DNA bound with mouse extract and -383 to +11 and -664 to -578 DNA bound with rat extract. 3. The results support a role for differences in presence or binding of species-specific trans-acting factors in the differential regulation of the renin gene in SMG of mouse and rat. Strong binding near the rat RNA polymerase II binding site could repress transcription in rat SMG, and binding peculiar to the Ren-2d B2 element might contribute to high expression in mouse SMG.

Activation of mouse renin promoter by cAMP and c-Jun in a kidney-derived cell line

We demonstrated that the mouse renin promoter from −365 to +16 can mediate the activation by cAMP and c-Jun in a kidney-cell dominant manner. Deletion analysis indicated that the region from −75 to −48 was responsible for the activation by cAMP. Furthermore, the core promoter region from −47 to +16 was sufficient to confer c-Jun inducibility.

Promoter activity of human renin 5'-flanking DNA sequences is activated by the pituitary-specific transcription factor Pit-1.

Although the renal juxtaglomerular cell is the source of circulating renin, the renin gene is also expressed at a number of extrarenal sites including lactotrope cells of human and ovine pituitaries. In the present study, we demonstrate that GC cells, a pituitary lactotrope precursor cell line, efficiently express transiently transfected hybrid genes containing human renin 5'-flanking DNA sequences -148/+11. Gel mobility shift competition analyses show that a highly conserved sequence in human and rodent renin 5'-flanking DNAs (human coordinates: -80/-58) binds a nuclear factor from GC cells, most likely the pituitary-specific factor Pit-1. Deletional and mutational analyses demonstrate that this site is a major determinant of renin promoter activity in GC cells. Transfection of a Pit-1 expression construct into HeLa cells, where activity of the human renin promoter is low, stimulates expression of cotransfected renin-luciferase constructs. Moreover, activation of the human renin promoter by co-expression of Pit-1 is dependent on an intact Pit-1 site. Taken together, these data strongly suggest that Pit-1 activates pituitary renin gene expression. This finding raises the possibility that member(s) of the POU family of transcription factors, of which Pit-1 is an archetypal member, may direct renin expression in other tissues, including the kidney.

Production of transgenic rats using pregnant and pseudopregnant rats prepared at a breeding farm.

We used pregnant and pseudopregnant Wistar-Imamichi rats, prepared at a breeding farm, for production of transgenic rats. The donor and recipient rats were transported to our facility for ova manipulation and embryo implantation, respectively. As a foreign gene, 1.9 kb of a hybrid gene was constructed with the rat renin promoter and chloramphenicol acetyltransferase-coding gene. Fertilized oocytes were collected from the donor and the foreign DNA was microinjected into the pronuclei of 539 oocytes by the method of Hochi. The manipulated oocytes were cultured to the 2-cell stage. One hundred seventeen 2-cell embryos were implanted in the recipient rats. Of 67 newborns, 55 rats grew up to be 3-week-old weanlings. Genomic DNA was extracted from the tail of these weanlings and examined by polymerase chain reaction analysis. Six transgenic rats were found to have been generated by the present method. In this way, transgenic rats were produced by an efficient combination of breeding farm utilization and laboratory research.

Rat renin promoter activity in cultured cells and transgenic mice.

Renin, a key enzyme controlling blood pressure, is mainly synthesized in kidney. To characterize the rat renin promoter, we have constructed a reporter gene containing the 238-bp putative regulatory region linked to a bacterial chloramphenicol acetyltransferase (CAT), and analyzed its promoter activity by in vitro transfection and introduction of the CAT fusion gene into germline of mice. CAT activity was detected in transfected embryonic kidney-derived 293 cells, but not in HeLa cells derived from cervical carcinoma, showing that the putative promoter region of the rat renin gene directs transcription in a cell type-dependent manner. To examine whether the sequence was sufficient to regulate the expression of the CAT chimeric gene in mice, we generated seven transgenic mice carrying the reporter construct. Unexpectedly, the transgene was not expressed in any of the independent transgenic mice examined. These results suggest one possibility that an additional control region may be required for efficient expression of the rat renin promoter in developing mice.

Tissue specificity of renin promoter activity and regulation in mice.

Certain mouse strains (e.g., DBA/2) contain two renin genes (termed Ren-1 and Ren-2) and express higher renin levels in nonkidney tissues than strains with a single renin gene. The 5'-flanking regions of the Ren-1 and Ren-2 genes contain several TATA boxes preceding putative transcriptional start sites. These initiators are termed P1a, P1, P2 (from 5' to 3'), and their function (with the exception of P2) is largely unknown. In this study, we mapped the renin transcriptional start sites in renal and extrarenal tissues [adrenal, brain, testis, heart, and submandibular gland (SMG)] and examined the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on tissue specific promoter usage. Our results showed that, in the unstimulated state, P2 (the predicted initiator) is active in all DBA/2 mouse tissues. Additional transcriptional start sites were detected in the adrenal and testis (originated by P1a and P2) and the SMG (originated by P1a, P1, and P2). The administration of 8-bromoadenosine 3',5'-cyclic monophosphate led to selective stimulation of P1a in the adrenal but did not affect the selective usage of initiation sites in other organs. A locus-specific ddNTP primer extension assay was used to verify which renin gene is induced by cAMP. Results indicated that both Ren-1 and Ren-2 responded to cAMP treatment in identical fashion. Taken together, these data indicate that more than one form of renin transcript is present in several mouse tissues. There is tissue specificity in promoter usage in the unstimulated state and in response to cAMP.

Molecular biology of renin. II: Gene control by messenger RNA, transfection and transgenic studies.

This review addresses the question of renin gene control by examining information from messenger RNA, transgene, transient expression and DNA-protein binding studies. Although information from messenger RNA studies may be reliable for tissues such as kidney and, in mouse, submandibular gland, which have relatively high expression, more sensitive approaches, e.g. polymerase chain reaction technology, are needed for studies of renin gene regulation in other tissues. Control at the molecular level is particularly uncertain, with conflicting data suggesting that renin promoter activity depends either upon a complex interaction of positive and negative regulatory elements or upon the presence of specific trans-acting factor(s) alone, which act upon putative enhancer(s) in the renin-associated DNA. Only after renin-synthesizing cell lines are studied will a clearer picture emerge. Transgenic work has loosely defined the DNA needed as being less than several kilobases of flanking sequences, with the possibility that sequences in the gene itself, particularly in intron 1, are necessary. Such transgenic work has also shown that the renin gene can cause hypertension. Knowledge of DNA and proteins and the interactions that turn on transcription of the renin gene is still rudimentary and currently represents the major challenge in renin research.

A combination of upstream and proximal elements is required for effecient expression of the mouse renin promoter in cultured cells

Renin, a key enzyme controlling blood pressure, is produced mainly in the kidney. To identify the transcriptional regulatory elements of the mouse Ren-1c gene, the promoter regions were fused to the CAT reporter gene and transfected into embryonic kidney-derived 293 cells and four extrarenal cell lines, HeLa, HepG2, HT1080 and NIH3T3 cells. Transient transfection assay showed that sequences from -365 to +16 of the renin gene could direct transcription of the CAT hybrid gene only in 293 cells. Deletion analysis identified two transcriptionally active regions; the renin upstream-promoter element (RU-1 element; position -224 to -138) and the renin proximal-promoter element (RP-2 element; position -75 to -47). Although the RU-1 element functioned as an activator, depending on its orientation, it failed to trans-activate the renin promoter when the RP-2 element was deleted. By contrast, the proximal element alone exhibited a weak trans-activator property. Gel shift assay identified RU-1 element-binding factors in both 293 and HeLa cells, whereas 293 cell-dominant factors were shown to bind only to RP-2 element. Therefore, both RU-1 and RP-2 elements were found to be necessary for efficient CAT expression from the renin promoter in 293 cells, suggesting that activation of the Ren-1c promoter requires combined action between cell type-dominant and ubiquitous nuclear factors.

Transient expression analyses of DNA extending 2.4 kb upstream of the human renin gene

The influence on homologous and heterologous promoter activity of DNA extending 2.4 kb upstream of the human renin gene ( REN) was examined by transient expression assay in JEG-3 cells, using the gene for chloramphenicol acetyl transferase (CAT) as reporter, and cotransfection with pCH110 to control for transfection efficiency. Analyses of constituent subfragments of the region 5′ of residue −144, using the herpes simplex virus thymidine kinase ( tk) promoter to drive transcription, provided no evidence for negative regulatory influences within the −2400 to −144 DNA. That distal 5′-flanking DNA may have little influence on promoter activity is further supported by a sharp decline in nucleotide homology between human, rat and mouse renin genes further upstream than human residue −604. Constructs containing renin DNA to residue + 13, i.e., which retained the REN promoter, all displayed very low CAT activities, consistent with negative cis-acting control within the −149 to + 13 region. This finding contrasts with results of similar studies for mouse, in which renin gene control was suggested to be mediated primarily via cell-specific trans-acting activator(s) acting on yet-to-be identified enhancer(s). Mouse renin genes have, however, a common DNA insertion that could have disrupted the negative element in this region, and which might contain enhancer target(s) for trans-acting factor(s). In conclusion, the present study involving JEG-3 cells has demonstrated that distal human renin 5′-flanking DNA has little cis-acting influence on promoter activity, whereas DNA located within 100 base pairs of the renin promoter may have a negative regulatory effect. Control of the human renin gene may involve conserved cis-acting sequences in the proximal 0.6 kb of 5′-flanking DNA, although strong conservation is also seen in sequences extending into the first intron. Since trans-acting factors that might be present only in cells that express renin could be important in renin gene control, similar studies as these are required in renin-synthesizing cell lines before any conclusions can be drawn concerning the cis-acting influences on renin promoter activity.

Renin promoter SV40 T-antigen transgenic mouse. A model of primary renal vascular hyperplasia.

Transgenic mice containing a ren-2 promoter T-antigen fusion construct (TAG+) develop renal vascular hypertrophy and hyperplasia associated with markedly suppressed renal renin mRNA, renal renin content, and plasma renin concentration. These animals are normotensive. In the present study, the renal and cardiovascular systems are characterized, revealing some surprising findings. Not only are the TAG+ mice normotensive in the face of pronounced renal pathology but also in the presence of an increase in plasma volume. These data raise interesting questions about blood pressure physiology and renal function of the TAG+ mice. Blood nitrogen urea of the TAG+ animal was markedly elevated and plasma creatinine level was in the normal range, indicating prerenal azotemia without renal failure. These findings are consistent with impaired renal perfusion with secondary volume expansion probably as the result of vascular hyperplasia. These transgenic animals provide a unique genetic model for studying the physiology of primarily renal vascular hyperplasia as well as blood pressure control in a low renin state.

Regulation of human renin expression in chorion cell primary cultures.

The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promotor linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3- to 6-fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. The transfected renin promoter (584 or 100 base pairs of 5'-flanking DNA) initiated at the correct start site in these cells and forskolin increased its expression 2.5- to 4-fold. Constructs containing renin 5'-flanking DNA linked to a heterologous promoter cotransfected into HeLa cells with either glucocorticoid or estrogen receptor expression vectors were not regulated by dexamethasone or 17 beta-estradiol. These results suggest that (i) the first 584 base pairs of the renin gene 5'-flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'-flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter.

The activity of the mouse renin promoter in cells that do not normally produce renin is dependent upon the presence of a functional enhancer

The expression of a hybrid gene containing the promoter region of the mouse Ren-1 or Ren-2 genes and the chloramphenicol acetyl transferase (CAT) gene coding region was analysed in five cell lines that do not normally express the renin gene. The renin promoter is inactive in each of these cell lines unless the SV40 enhancer is also present in the construct. In the latter case, transcription initiates at the normal renin start site. This is in contrast to the situation observed in lymphoid cells where the renin promoter is inactive even when coupled to a functional enhancer [(1987) EMBO J. 6, 1685–1690]. Furthermore, 5′-flanking sequences of Ren-2, placed upstream of the thymidine kinase or SV40 early region promoters do not alter the activity of these promoters in three different cell lines. The results suggest that, except for B cells, the renin promoter is not tissue-specific but that its lack of activity in cells that do not express the gene is not the result of repression but may be due to the absence in these cells of one or several trans-acting positive factors.

Functional human renin promoter in transfected cells: evidence for cell-specific expression.

Expression of the human renin gene is regulated in a tissue-specific manner, but study of this regulation has been limited by a lack of suitable cell lines that simulate endogenous control. In order to characterize the regulation of renin gene expression, the 5' flanking region (892 base pairs) from the human renin gene was linked to the chloramphenicol acetyltransferase gene and was introduced into multiple human cell lines by calcium phosphate precipitation or electroporation methods to assess transcriptional control. The human renin promoter was active when transfected into cultured human choriocarcinoma cells (JEG-3) and rat vascular smooth muscle cells, but it was not active in many other cloned cell types. These results suggest that selective cell lines contain the specific trans-acting factors necessary for human renin gene expression, and support the concept of cell-specific expression of this gene.

A mouse renin promoter containing the conserved decanucleotide element binds the same B-cell factors as an authentic immunoglobulin heavy chain promoter

A mouse renin-1 gene promoter fragment, normally inactive in B-cells, becomes a potent promoter in these cells after insertion of the highly conserved decanucleotide (dc/cd sequence) of immunoglobulin heavy and light chain promoters [(1987) EMBO J. 6, 1685–1690]. We observe retarded complexes of the same electrophoretic mobility when the cd-containing renin promoter fragment or an authentic immunoglobulin heavy chain promoter fragment is incubated with a nuclear extract from myeloma cells, suggesting that the renin promoter is activated due to its acquired ability to bind a B-cell-specific positive factor. No retarded complexes are observed with the original renin promoter fragment thus questioning the presence of a repressor as an explanation for its lack of activity in B-cells.

The conserved decanucleotide from the immunoglobulin heavy chain promoter induces a very high transcriptional activity in B-cells when introduced into an heterologous promoter.

A conserved decanucleotide (ATGCAAATNA) is present 45-60 nucleotides upstream from the transcription startpoint in all immunoglobulin heavy chain promoters (VH promoters). We have introduced this decanucleotide (cd sequence) at a similar position into the upstream flanking sequence of the mouse Renin-1 gene. This gene is only transcribed in highly specialized tissues, and the fragment used here (-449 to +30 with respect to the main transcription startpoint) has little promoter activity in fibroblastic or myeloma cell lines, even if coupled to a functional enhancer. In contrast, after insertion of the decanucleotide, this fragment, while still inactive in non-lymphoid cells, becomes a potent promoter in B-cells when associated with SV40 or immunoglobulin heavy chain enhancer. In all respects, the engineered fragment behaves like an authentic VH promoter isolated in this laboratory, except that it is even more active in B-cells. Deletion experiments show that all renin sequences are dispensable for the activity of the chimaeric promoter, except probably for the renin TATA box which defines the precise transcription startpoint. We conclude that the decanucleotide is sufficient to activate a promoter in B-cells but not in non-B-cells, and therefore that no other element is needed to account for the B-cell specificity of the VH promoter. In addition, our results suggest that the lack of activity of the renin promoter in non cognate cells is not due to the binding of a repressor.