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C D Sigmund

Publications and source records attributed to C D Sigmund.

At least 73 records · Page 4Linked to original sources
PubMed1995

Endogenous human renin expression and promoter activity in CALU-6, a pulmonary carcinoma cell line.

We have previously reported that transgenic mice containing the human renin gene express high levels of human renin mRNA in the lung. We show in this report that human renin expression in two lines of transgenic mice is developmentally regulated. Human renin expression is not evident in the transgenic mouse lung at 15.5 days of gestation, is detectable at 17.5 days of gestation, peaks around birth, and remains elevated into adulthood. In situ hybridization of mouse fetal lung samples at 18.5 days of gestation revealed that human renin was exclusively expressed in pulmonary type II epithelial cells. A survey of the medical literature revealed a number of clinical cases in which hypertension was caused by renin-secreting pulmonary tumors and a fairly widespread occurrence of immunoreactive renin in banked pulmonary tumors of diverse origin. This prompted us to examine a number of pulmonary tumor cell lines to determine whether they express human renin mRNA. One pulmonary carcinoma cell line, CALU-6, expressed human renin mRNA endogenously. Human renin expression in these cells was induced approximately 100-fold after treatment with forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, or N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate. Transfection analysis of human renin promoter-luciferase fusion constructs revealed the presence of cell-specific positive and negative regulatory elements in the human renin 5'-flanking DNA. This cell line is the only immortalized human cell line that expresses high levels of endogenous human renin mRNA and should provide an excellent tool for studying the regulation of human renin expression in vitro.

Animals↗
PubMed1995

Tissue-specific expression of novel messenger ribonucleic acids cloned from a renin-expressing kidney tumor cell line (As4.1).

As4.1 cells are derived from a renin-expressing kidney tumor induced by tissue-specific oncogene-mediated tumorigenesis in transgenic mice. These cells express high levels of renin messenger RNA (mRNA) and synthesize prorenin and renin; they were therefore used as a model to further investigate the molecular biology of renin-producing kidney cells by cloning and characterizing novel mRNAs expressed in these cells. One clone, designated 1.5, was randomly selected from an As4.1 complementary DNA (cDNA) library, and two other cDNA clones, designated 4.9 and 6.9, were obtained by screening the cDNA library using a strategy to identify As4.1 cell-specific mRNAs. Each clone exhibited a highly restricted tissue-specific expression profile, including high level expression in As4.1 cells and low level expression in kidney. No homology was found between the sequence of the partial 1.5 and 4.9 cDNAs and sequences in Genbank. Southern blot analysis revealed that clone 4.9 is encoded by a single copy gene containing at least two separate exons. A homology search of the sequence of clone 6.9 revealed it to encode a cDNA to serum amyloid A protein; consistent with this identification, expression of 6.9 mRNA was highly induced in both kidney and liver after treatment of mice with Escherichia coli lipopolysaccharide.

Animals↗
PubMed1995

Human renin 5'-flanking DNA to nucleotide-2750.

Renin is one of the most important factors in blood pressure and electrolyte regulation in mammals and the renin locus has been implicated in hypertension. To assist studies of promoter control we therefore determined the 5'-flanking sequence of the human gene (REN) to residue -2750 relative to the transcription start site (+1). Sites of homology to consensus sequences for binding of trans-acting factors involved in transcriptional control of other genes were identified, and functionality for two of these (a CRE and Pit-1 site) have so far been demonstrated.

Base Sequence↗
PubMed1994

Functional expression of the human angiotensinogen gene in transgenic mice.

The renin-angiotensin system is a major determinant of arterial pressure and volume homeostasis in mammals through the actions of angiotensin II, the proteolytic digestion product of angiotensinogen. Molecular genetic studies in several human populations have revealed genetic linkage between the angiotensinogen gene and both hypertension and increased plasma angiotensinogen. Transgenic mice were generated with a human angiotensinogen genomic clone to develop an animal model to examine tissue- and cell-specific expression of the gene and to determine if overexpression of angiotensinogen results in hypertension. Human angiotensinogen mRNA was expressed in transgenic mouse liver, kidney, heart, adrenal gland, ovary, brain, and white and brown adipose tissue and, in kidney, was exclusively localized to epithelial cells of the proximal convoluted tubules. Plasma levels of human angiotensinogen were approximately 150-fold higher in transgenic mice than that found normally in human plasma. The blood pressure of mice bearing the human angiotensinogen gene was normal but infusion of a single bolus dose of purified human renin resulted in a transient increase in blood pressure of approximately 30 mm Hg within 2 min. These results suggest that abnormalities in the angiotensinogen gene resulting in increased circulating levels of angiotensinogen could potentially contribute in part to the pathogenesis of essential hypertension.

Angiotensinogen↗
PubMed1994

Chromosome 8 alterations accompany tumorigenesis in renin-SV40 T antigen transgenic mice.

s.c. and abdominal tumors from interspecific hybrid, transgenic mice containing the SV40 early region linked to a renin enhancer/promoter were analyzed for loss of heterozygosity to identify chromosomal regions involved in tumorigenesis. A very high frequency of loss of heterozygosity/partial loss of heterozygosity or allelic imbalance involving the distal regions of chromosome 8 was observed in the s.c. tumors (76%) with frequent amplification (3-6 times) of the corresponding proximal regions including Junb, suggesting that chromosome 8 breakage and amplification promotes tumorigenesis in these mice.

Animals↗
PubMed1994

Differential expression of angiotensin receptor 1A and 1B in mouse.

At least two distinct genes (AT1A and AT1B) encode type 1 angiotensin II (AT1) receptors in rodents. Receptor binding and Northern blot analysis have clearly demonstrated the presence of AT1 receptors and AT1-receptor mRNA in many tissues but fail to differentiate which type 1 receptor subtype is expressed. A reverse-transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) assay was developed to differentiate the expressed mRNA by subtype. Expression of AT1A was clearly evident in kidney, liver, adrenal gland, ovary, brain, testes, adipose tissue, lung, and heart of adult mice. AT1B was absent from most of these tissues but was detectable in brain, testes, and adrenal gland. No significant differences in expression were evident in kidney, liver, brain, lung, or heart from 16.5- or 18.5-gestation-day fetuses, and only AT1A was evident in placenta. Expression of AT1B was confirmed in adrenal gland, brain, and testes, using a primer set that specifically amplifies only AT1B mRNA. Expression of AT1A and AT1B was also examined in As4.1 cells, a renin-expressing mouse kidney tumoral cell line. Receptor binding and competition assays using AT1- and AT2-receptor antagonists revealed that only AT1 receptors are present on the cell surface. Extremely low levels of AT1-receptor mRNA was detected by Northern blot, and RT-PCR-RFLP analysis revealed that only the AT1A subtype is expressed in this cell line. Despite the high homology between the coding sequence of the AT1A and AT1B genes, they exhibit disparate tissue-specific expression profiles.

Animals↗
PubMed1993

Expression of the human renin gene in transgenic mice throughout ontogeny.

Expression of a human renin genomic DNA clone extending 900 base pairs upstream and 400 base pairs downstream of the gene has been previously examined in adult transgenic mice. In adults, expression of human renin was evident in kidney, reproductive tissues, adrenal gland and lung. Previous studies of mouse and rat renin have demonstrated that kidney renin becomes evident at approximately 15 days of gestation and that expression is localized first to smooth muscle cells of the developing renal arterial tree and becomes progressively restricted to juxtaglomerular cells. As a prelude to performing cell specificity studies to elucidate the pattern of human renin gene expression in the developing kidney, 15.5 and 17.5 days of gestation fetuses and newborns were obtained for expression analysis. Tissues were pooled and expression was examined in kidney, liver, gastrointestinal (GI) tract, lung, heart and brain. The number of transgenic fetuses in each pool was determined by human renin-specific polymerase chain reaction of DNA purified from placenta or tail biopsies. Renal human renin expression was abundant at all three time points. Expression was also evident in the GI tract at 15.5 and 17.5 days of gestation. Interestingly, although no human renin mRNA was evident in lung at 15.5 or 17.5 days of gestation, extremely high levels of human renin mRNA were detected in the newborn lung. Expression of the human renin gene in these tissues was further confirmed by differential primer extension analysis which is capable of differentiating the closely related human and mouse renin messages.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Major approaches for generating and analyzing transgenic mice. An overview.

Over the past decade, the development of gene-transfer technology in whole animals has afforded unprecedented opportunities for investigators to probe complex regulatory systems in vivo. Important advances in our understanding of the mechanisms of gene expression and regulation and the development of animal models of human diseases are but two examples of how this technology has affected medical science. Transgenic animals are defined as animals in which a segment of DNA has been physically integrated into the genome of all cells, including the germ line, so that it can be transmitted to offspring as a simple Mendelian trait. The DNA segment generally consists of a whole cloned gene, cDNA, or a novel gene modified by recombinant DNA methodologies. Whole genomic clones of genes are often used to study tissue- and cell-specific expression and regulation or can be used to overexpress a gene product. Alternatively, the coding region of one gene can be fused to the transcriptional regulatory region of another gene, causing it to be expressed in a new spectrum of tissues and cell types. A number of methods can be used to introduce the DNA segment, including direct microinjection of one-cell fertilized embryos, retroviral-mediated transfer, or gene transfer in embryonic stem cells. The technique most often used to generate transgenic animals and perform "gene addition" experiments is direct microinjection. Alternatively, gene deletions or "knockouts" are performed by gene transfer in embryonic stem cells by specifically targeting the site of integration in the genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products.

Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of translation termination and the sites of nucleolytic cleavage.

Alleles↗
PubMed1992

Regulated tissue- and cell-specific expression of the human renin gene in transgenic mice.

Transgenic mice containing the human renin gene were constructed with the aim of examining the tissue- and cell-specific expression of human renin. The human renin transgene used consisted of a genomic sequence extending approximately 900 bp upstream and 400 bp downstream of the coding region and included all exon and intron sequences. Two assays were developed to differentiate human renin transcripts from endogenous mouse renin transcripts at the whole-tissue level. High level human renin expression was evident in the kidney, adrenal gland, ovary, testis, lung, and adipose tissue of all four transgenic lines examined. Human renin mRNA could also be detected at lower levels in the submandibular gland and heart of two different individual lines. No expression was evident in the liver or brain of any line tested. In situ hybridization revealed the human renin mRNA to be localized and exquisitely restricted to renal juxtaglomerular cells. Treatment of transgenic mice with captopril resulted in an increase in the accumulation of renal renin mRNAs derived from both the mouse and human renin genes. Plasma renin activity assays using synthetic human renin substrate clearly demonstrated the elaboration of active human renin into the systemic circulation of transgenic mice. These data strongly suggest that the human renin transgene exhibits both tissue- and cell-specific expression in transgenic mice. Its expression is entrained to the same regulatory signals as the endogenous renin gene in kidney, and active human renin is released into the plasma of the transgenic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Pathophysiology of vascular smooth muscle in renin promoter-T-antigen transgenic mice.

The pathophysiological consequence of targeted production of SV-40 T-antigen to renin-expressing cells in the kidney of transgenic mice is reported. A histopathologic analysis of the kidney from adult transgenic mice (12-16 wk old) revealed the presence of severe vascular lesions manifested by marked atypical hyperplasia of vascular smooth muscle. The levels of plasma renin, kidney renin, and kidney renin mRNA were examined in 6- and 9-wk-old transgenic mice and were found to be significantly lower than their age-matched non-transgenic littermates and were nonresponsive to captopril treatment. However, there was no significant difference in conscious mean arterial pressure between transgenic and non-transgenic mice. The levels of renal renin mRNA in transgenics and nontransgenic littermates were compared throughout ontogeny and were found to be equal in newborns, elevated 3- to 5-fold in 1-wk-old transgenics, and yet decreased 10-fold by 6 wk of age in transgenic mice. Expression of the transgene in the kidney exhibited the proper developmental pattern and was properly restricted to juxtaglomerular cells in neonatal mice. Nevertheless, in adult mice, T-antigen-containing cells were found throughout the entire renal arterial tree. The observed ability of renal vascular cells to be recruited to express both renin and T-antigen suggests a mechanism that can explain the development of the renal pathology in these mice.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Structure, expression, and regulation of the murine renin genes.

It has long been known that the renin-angiotensin system plays an integral role in the regulation of blood pressure and electrolyte and fluid balance in mammals. The advent of molecular biologic techniques has afforded new insights into the genes regulating blood pressure. Laboratory mice and rats have been used as experimental models to examine the structural organization and expression of the renin gene. It is now well established that some mice, unlike rats and humans, contain a duplicated copy of the renin locus, which accounts for the high level of renin activity long known to be found in the submandibular gland of some mice. Indeed it is this fortuitous observation that facilitated the isolation of the first complementary DNA clones for renin and ultimately the many species-specific probes now available to analyze mammalian tissues for evidence of primary renin expression. The use of complementary DNAs as probes for primary renin expression helped confirm and further clarify earlier studies demonstrating the presence of renin activity in a number of extrarenal tissues. Although expression in some of these tissues is evolutionarily conserved, their significance has still been elusive. In this report we review the impact of molecular biology on our current understanding of renin gene structure and organization, tissue- and cell-specific expression and regulation, and the changes in renin expression throughout ontogeny. In addition, we describe how new developments in gene transfer technology have added important tools to our arsenal for examining renin gene regulation and how these technologies can be used to develop new tools for renin and hypertension research.

Animals↗
PubMed1990

Differential expression of the murine and rat renin genes in peripheral subcutaneous tissue.

We have previously identified peripheral subcutaneous tissue as a bonafide site of primary renin expression in the mouse fetus by virtue of oncogene mediated tumorigenesis in transgenic mice. In this report we demonstrate that the murine renin genes are differentially expressed in this tissue. Through selective breeding and differential primer extension we demonstrate that Ren-1d and Ren-1c transcripts were several fold more abundant than Ren-2. Renin transcripts were also identified in fetal subcutaneous tissues of Spontaneously Hypertensive (SHR) and Wistar Kyoto (WKY) rats. We conclude from these studies that expression of renin during fetal development may be widespread in rodents with its temporal and spatial localization consistent with a role in fetal development.

Animals↗
PubMed1990

Isolation and characterization of renin-expressing cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct.

We constructed transgenic mice containing a renin-promoter SV40 T antigen fusion transgene with the intention of inducing neoplasia in renin-expressing cells and isolating renin-expressing cell lines in vitro. We examined six kidney tumors from mice representing three different transgenic lines and found they expressed their endogenous renin gene. Initially, five nonclonal kidney tumor-derived cell lines were established which expressed their endogenous renin gene in addition to the transgene. They retained active renin intracellularly and constitutively secreted an inactive form of renin (prorenin). One of these cell lines was cloned to homogeneity. This line maintained high level expression of renin mRNA throughout 3 months of continuous culture. Although the cells contained an equal proportion of active and inactive renin, the species constitutively secreted into the media was predominantly (95%) prorenin. However, active renin secretion was stimulated 2.3- and 4.6-fold by treatment with 8-bromo-cAMP after 4 and 15 h, respectively. In addition, the presence of multiple secretory granules was confirmed by ultrastructural analysis. These cells, which express renin mRNA and can regulate secretion of active renin, should provide an excellent tool for studying renin gene regulation and secretion. Furthermore, these mice should provide a useful source for the establishment of renin-expressing cell lines from a variety of renin-expressing tissues.

Animals↗
PubMed1990

Tissue and cell specific expression of a renin promoter-reporter gene construct in transgenic mice.

The tissue specific expression of a fusion transgene consisting of 4.6 kb of Ren-2 5' flanking sequence and the SV40 T antigen viral oncogene was analyzed by northern hybridization and immunocytochemistry. Among the 10 tissues examined, endogenous renin transcripts were identified in and restricted to kidney, submandibular gland, testes and ovary consistent with the expression pattern of the Ren-1c gene. In addition to these tissues, significant levels of transgene mRNA were detectable in the brain. Expression of the transgene was restricted to juxtaglomerular cells in the kidney and the granular convoluted tubule cells of submandibular gland. These results suggest that 4.6 Kb of Ren-2 5' flanking sequence is sufficient to confer tissue and cell specific expression upon an exogenous reporter gene.

Animals↗
PubMed1990

Expression of murine renin genes in subcutaneous connective tissue.

A renin promoter-large tumor antigen (T antigen) fusion gene was constructed to provide a reporter function for renin expression in transgenic mice. These transgenic mice gave rise to tumors in subcutaneous soft tissue, which was attributed to transgene expression at this site. An immunohistochemical analysis of transgenic fetuses from several independent lines revealed scattered T-antigen-containing mesenchymal cells and fibroblasts in the subcutaneous layer of the skin between the panniculus carnosus muscle of the skin and the skeletal muscle of the body wall. This localization is consistent with the location of overt tumorigenesis in adult mice. This pattern was specific for the renin-T antigen fusion gene as no immunohistochemical staining was observed in transgenic fetuses containing a heterologous promoter-T antigen fusion gene. Northern blot analysis of tumor RNA indicated that most of the tumors expressed both T antigen and the endogenous renin gene Ren-1c. In addition, when multiple renin genes were introduced by crossing transgenic mice with nontransgenic DBA/2J mice, which contain another allele of the Ren-1 locus as well as the duplicated locus Ren-2, the resultant tumors expressed the Ren-2 gene. Northern blots were then used to analyze renin expression in the subcutaneous tissue of normal mice. Fully processed renin mRNA was detected in eviscerated 15.5-day postcoitus fetal and newborn carcasses and in newborn skin. Our data indicate that there is a renin-expressing cell population in fetal and newborn subcutaneous tissue.

Animals↗