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A Fukamizu

Publications and source records attributed to A Fukamizu.

At least 19 recordsLinked to original sources
PubMed1997

High human renin hypertension in transgenic rats.

We developed a model of spontaneously high human renin hypertension in the rat by producing two transgenic strains, one for human angiotensinogen with the endogenous promoter and one for human renin with the endogenous promoter. Neither transgenic strain was hypertensive. These strains were then crossed, producing a double transgenic strain. The double transgenic rats, both males and females, developed severe hypertension (mean systolic pressure, 200 mm Hg) and died after a mean of 55 days if untreated. The rats had a human plasma renin concentration of 269 +/- 381 (+/-SD) ng angiotensin I (Ang I)/mL per hour, plasma renin activity of 177 +/- 176 ng Ang I/mL per hour, rat angiotensinogen concentration of 1.49 +/- 1 microgram Ang I/mL, and human angiotensinogen concentration of 78 +/- 39 micrograms Ang I/mL (n = 49). Control rats had plasma renin activity of 3.7 +/- 3.9 ng Ang I/mL per hour and rat angiotensinogen of 1.32 +/- 0.16 micrograms Ang I/mL. Angiotensinogen transgene expression by RNase protection assay was ubiquitously present but most prominent in liver. Renin transgene expression was high in kidney but absent in liver. The rats featured severe cardiac hypertrophy, with increased cross section of cardiomyocytes but little myocardial fibrosis. The kidneys showed atrophic tubules, thickened vessel walls, and increased interstitium. Both the angiotensin-converting enzyme inhibitor lisinopril and the specific human renin inhibitor remikiren lowered blood pressure to normal values. Double transgenic mice have been developed that exhibit features quite similar to those described here; their gene expressions are similar. The specificity of rodent and human renin is similarly documented. Although many elegant physiological studies can now be done in mice, rats nevertheless offer flexibility, particularly in terms of detailed cardiac and renal physiology and pharmacology. We conclude that this double transgenic strain will facilitate simultaneous investigation of genetic and pathophysiological aspects of renin-induced hypertension. The fact that human renin can be studied in the rat is a unique feature of this model.

Angiotensinogen↗
PubMed1997

The neuronal isoform of constitutive nitric oxide synthase is up-regulated in the macula densa of angiotensinogen gene-knockout mice.

Angiotensinogen gene-knockout (Atg -/-) mice lacking angiotensin II exhibit chronic hypotension and an increase in renal renin gene expression. The present study was designed to provide evidence for the possible involvement of neuronal type nitric oxide synthase (N-NOS) at the macula densa in the increased renin production in Atg -/- mice. The enzyme activity of N-NOS was histochemically detected by NADPH diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. N-NOS mRNA expression in the renal cortical tissue was determined using reverse transcription-PCR in a semiquantitative manner. The levels of renal renin mRNA were evaluated by Northern blot analysis. In the kidneys of wild-type (Atg +/+) mice, N-NOS activity was localized to the macula densa as reported previously. On the other hand, N-NOS-positive macula densa cells of Atg -/- mice were distributed beyond the original location of the macula densa. They often occupy the entire cross-sectional profiles of the tubules. In addition, Atg -/- mice showed a stronger signal intensity for the enzyme reaction than Atg +/+ mice. The mean total number of N-NOS-positive cells per 100 glomeruli was 6 times higher in Atg -/- mice than in Atg +/+ mice. Semiquantitative reverse transcription-PCR revealed an increase in the N-NOS mRNA level in renal cortical tissue of Atg -/- mice compared with Atg +/+ mice. Furthermore, the selective inhibition of N-NOS activity by 7-nitrondazole significantly decreased the level of renal renin mRNA in Atg -/- mice. These results suggest that increased N-NOS activity at the macula densa is involved a renal renin overproduction in Atg -/- mice.

Angiotensinogen↗
PubMed1996

Human activin betaA gene. Identification of novel 5' exon, functional promoter, and enhancers.

On the basis of cDNA cloning, primer extension, and transfection experiments, we identified a novel 5' exon of the human activin betaA subunit gene, and found its enhancer and promoter regions as well as multiple transcription start sites. A series of deletion and mutation analyses of the enhancer sequences defined the 45-base pair core region (DR-1 core) containing two short elements with similarity to AP-1 (12-O-tetradecanoylphorbol-13-acetate response element; TRE) and CREB/ATF (cyclic AMP response element; CRE) binding sites, both of which were necessary for full enhancer activity. Gel shift and antibody supershift assays using DR-1 core region revealed the formation of two specific DNA-protein complexes, one of which could be partially dissociated by a competing oligonucleotide containing a single copy of the consensus TRE, but the other of which contained neither CREB/ATF nor AP-1 as major components. Although 12-O-tetradecanoylphorbol-13-acetate and cAMP induced the activin enhancer/promoter-driven CAT activity, such drug induction was obscured when either the TRE- or CRE-like elements were mutated in the native promoter context. Our results demonstrate that the promoter and enhancer regions identified here are essential for maintaining the efficient promoter activity of the human activin betaA subunit gene.

Activins↗
PubMed1996

Hypertension induced in pregnant mice by placental renin and maternal angiotensinogen.

Maternal hypertension is a common complication of pregnancy and its pathophysiology is poorly understood. This phenomenon was studied in an animal model by mating transgenic mice expressing components of the human renin-angiotensin system. When transgenic females expressing angiotensinogen were mated with transgenic males expressing renin, the pregnant females displayed a transient elevation of blood pressure in late pregnancy, due to secretion of placental human renin into the maternal circulation. Blood pressure returned to normal levels after delivery of the pups. Histopathologic examination revealed uniform enlargement of glomeruli associated with an increase in urinary protein excretion, myocardial hypertrophy, and necrosis and edema in the placenta. These mice may provide molecular insights into pregnancy-associated hypertension in humans.

Angiotensin II↗
PubMed1996

The signal-dependent coactivator CBP is a nuclear target for pp90RSK.

We have examined the mechanism by which growth factor-mediated induction of the Ras pathway interferes with signaling via the second messenger cAMP. Activation of cellular Ras with insulin or NGF stimulated recruitment of the S6 kinase pp90RSK to the signal-dependent coactivator CBP. Formation of the pp90RSK-CBP complex occurred with high stoichiometry and persisted for 6-8 hr following growth factor addition. pp90RSK specifically recognized the E1A-binding domain of the coactivator CBP. In addition, like E1A, binding of pp90RSK to CBP was sufficient to repress transcription of cAMP-responsive genes via the cAMP-inducible factor CREB. By contrast with its effects on the cAMP pathway, formation of the pp90RSK-CBP complex was required for induction of Ras-responsive genes. These results provide a demonstration of cross-coupling between two signaling pathways that occurs at the level of a signal-dependent coactivator.

Adenovirus E1A Proteins↗
PubMed1996

A cis-acting DNA element located between TATA box and transcription initiation site is critical in response to regulatory sequences in human angiotensinogen gene.

The promoter of the human angiotensinogen (hAG) gene functioned in its own core promoter context but not when replaced with simian virus 40 (SV40) core promoter, suggesting the presence of a transcriptionally important cis-acting sequence. Electrophoretic mobility shift assays demonstrated that a ubiquitously expressed nuclear factor, AGCF1, bound to AGCE1 (hAG core promoter element 1; positions -25 to -1) located between the TATA box and transcription initiation site. Substitution mutation in AGCE1 which disrupted AGCF1 binding affected the promoter activity more severely than a nonsense mutation of the hAG TATA sequences did. When AGCE1 was placed at the downstream of SV40 core promoter, the responsiveness to hAG upstream region was significantly restored. Furthermore, mutation and in vivo competition experiments suggested that AGCF1 acts as a critical regulator of hAG transcription by mediating the activity of the hAG upstream and downstream enhancer elements. DNase I footprinting and UV cross-linking analyses showed that AGCF1 with apparent molecular masses of 31, 33, and 43 kDa as the components protected the region from -26 to -9 which partially overlapped with the TATA box consensus sequences. These findings indicate that AGCE1 in addition to the TATA box plays a key role in mediating the hAG regulatory elements.

Angiotensinogen↗
PubMed1996

Detection of coagulating gland renin by hybridohistochemistry.

To obtain evidence of renin-synthesizing cells in the murine coagulating gland (CG), CG renin mRNA was detected by hybridohistochemistry, as well as in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) in intact, castrated and testosterone-treated C57BL/6 mice. Hybridohistochemistry using paraffin sections of the kidneys and the CGs for the detection of renin mRNA was performed with digoxigenin-labelled probes. Some paraffin sections were immunohistochemically stained for renin by the peroxidase-anti-peroxidase method. Total RNA was extracted, incubated by reverse transcriptase, and amplified by PCR. In the kidneys, the immunoreactivity and the positive signals of hybridohistochemistry using an antisense probe were restricted to the same juxtaglomerular cells. In the control and at 7 days after testosterone administration to castrated mice, both renin-immunoreactivity and -hybridoreactivity were expressed by the epithelial cells in the CGs, while, in the CGs of the castrated mice and 3 days after testosterone injection of castrated animals, neither renin-immunoreactivity nor -hybridoreactivity was detected in the epithelial cells. Using RT-PCR, renin mRNA from the mice in the control and 7 days after testosterone injection of castrated was amplified, whereas, in the castrated and the 3 days after testosterone injection of castrated groups, it was not detected. The data presented here provide additional evidence that CG renin is regulated by testosterone.

Animals↗
PubMed1996

Hypertensive and hypotensive mice produced by the introduction and disruption of genes on the renin-angiotensin system.

We constructed in mice the chimeric renin-angiotensin cascade comprising human renin, human angiotensinogen, and the endogenous angiotensin-converting enzyme and angiotensin II (A II) receptors by crossmating separate lines of transgenic mice carrying either gene. Although hypertension did not develop in the single-gene carriers despite the observed tissue-specific expression of the transgenes, dual-gene strains exhibited a chronically sustained increase in blood pressure (BP). The systolic BP was 130 +/- 7 mmHg, which is about 30 mmHg higher than that of control mice. Administration of a selective antagonist directed at the A II receptor decreased the basal BP in both single-gene and dual-gene mice. In addition, we generated two lines of knockout mice by homologous recombination in mouse embryonic stem cells. One line consisted of angiotensionogen-deficient mice, which failed to produce angiotensinogen in the liver, resulting in the complete loss of plasma angiotensin 1. The systolic BP of the mice was 66.9 +/- 4.1 mmHg, significantly lower than that of the controls (100.4 +/- 4.4 mmHg). The second line consisted of A II-receptor type la-deficient mice, which also exhibited hypotension (24 mmHg lower than that of controls). These results demonstrated that the renin-angiotensin system plays an indispensable role in maintaining normal BP in vivo, and cannot be replaced by other hormonal and neuronal systems.

Animals↗
PubMed1996

Nephrogenesis and renovascular development in angiotensinogen-deficient mice.

Angiotensinogen-deficient mice provide a model to examine the roles of angiotensin II as a renal growth factor in vivo. We monitored nephrogenesis and renovascular development in angiotensinogen-deficient mice from Embryonic Day 13 (E13) to 4 weeks after birth. Northern analysis of homozygote (Atg-/-) mice confirmed the absence of angiotensinogen mRNA in the liver and the kidneys. Embryonic kidneys in Atg-/- mice from E13 to E18 exhibited active nephrogenesis, as also observed in Atg+/- mice and Atg+/+ mice. Furthermore, metanephroi harvested at E12 from Atg-/- embryos showed branching morphogenesis of ureteric bud and tubulogenesis similar to metanephrol from Atg-/- embryos grown with exogenous angiotensin II in serum-free culture. In newborn Atg-/- mice, we observed uniform dilation of the pelvis accompanied by a coarse medulla, which was not noted in Atg+/- or Atg+/+ mice. Hydronephrosis in Atg-/- mice continued, and renal papillae underwent atrophy for the 4 weeks after birth. Another characteristic aspect of the morphology of Atg-/- mice was the thickening of vascular walls as little as 2 weeks after birth. Immunohistochemistry revealed recruitment of renin in hyperplastic vascular smooth muscle cells (VSMC) in Atg-/- mice after 2 weeks. Electron microscopy confirmed that the majority of hyperplastic VSMC contained various sized renin granules with abundant endoplasmic reticulum. In situ hybridization demonstrated that expression of renin mRNA became prominent in parallel with hyperplasia of VSMC, as well as recruitment of renin protein. Furthermore, at 4 weeks, Atg-/- mice expressed alpha-smooth muscle actin in the mesangium, whereas none was ever found in that of Atg+/- mice and Atg+/+ mice. In conclusion, the renin-angiotensin system seems not be essential for nephrogenesis in vivo. Furthermore, hyperplasia of VSMC and expression of the smooth-muscle phenotype in the mesangium are inducible even in the absence of angiotensin II, with hypotension, in vivo.

Angiotensinogen↗
PubMed1995

Heritable formation of neuroectodermal tumor in transgenic mice carrying the combined E1 region gene of adenovirus type 12 with the deregulated human renin promoter.

Adenovirus early 1 (E1) region gene products, including E1A and E1B, are required for transcriptional regulation of viral and cellular promoters in infected and transfected culture cells and for transformation of primary rodent cells. Here, we established a line of transgenic mice carrying the E1 region gene of human adenovirus type 12 under the control of the human renin promoter, in which a neuroectodermal tumor derived from retroperitoneal, olfactory, and/or pelvic regions was heritably developed with varying degrees of incidence and the phenotype was successfully passed through six generations. The transgenes were located in the region E2-E3 bands of chromosome 7 with which no genetic linkage to neuroectodermal tumors was previously demonstrated, and expressed only in the tumors but not in another tissue examined. Notably, in addition to the expression of a neural marker gene N-CAM, the three nuclear oncogenes, c-, L-, and N-myc, were coexpressed in the tumors. These results suggest that E1A and E1B are cooperatively involved in the heritable formation of neuroectodermal tumors associated with co-expression of the three sets of myc family genes.

Adenovirus E1 Proteins↗
PubMed1995

Activation of the nuclear oncogenes N-myc and c-jun in carcinoid [correction of cartinoid] tumors of transgenic mice carrying the human adenovirus type 12 E1 region gene.

The adenovirus (Ad) E1 region genes, E1A and E1B, are well known cooperatively to transform primary rodent cells and activate a number of cellular promoters, including nuclear oncogenes such as N-myc and c-jun, in transfected cell lines. However, there is still less information available on the in vivo mechanism(s) by which the E1 region gene, when chromosomally integrated in the living animals, exerts its effect on nuclear oncogene activation coupled with transformation. To investigate such in vivo activity of E1A we have used a series of microinjection experiments into fertilized eggs to generate three transgenic mice carrying the Ad12-type E1A/E1B genes under the control of the human renin gene. This transgene caused an early onset of bowel cartinoid tumors that express neural cell adhesion molecules, but do not metastasize to any region. Northern blot analysis revealed that the transgenes were considerably expressed in the tumors, but not in other tissues at detectable levels. Interestingly, the levels of N-myc and c-jun mRNAs in the cartinoid tumors were elevated 19- and 8-fold, respectively, as compared with those found in the control intestine. In contrast, the major histocompatibility complex (MHC) class I mRNA level was not altered between the tumor and control intestines, suggesting that this unchanged expression may reflect the loss of tumor metastasis. These findings provide the first in vivo evidence that the expression of the Ad12 E1 region gene induces cartinoid tumors associated with the activation of the nuclear oncogenes N-myc and c-jun.

Adenovirus E1 Proteins↗
PubMed1995

Transient decrease in high blood pressure by in vivo transfer of antisense oligodeoxynucleotides against rat angiotensinogen.

The renin-angiotensin system plays an important role in blood pressure regulation. Angiotensinogen, which is mainly produced in the liver, is a unique component of the renin-angiotensin system, because angiotensinogen is only known as a substrate for angiotensin I generation. It is unclear whether circulating angiotensinogen is a rate-limiting step in blood pressure regulation. Recent findings of genetic studies and analyses suggest that the angiotensinogen gene may be a candidate as a determinant of hypertension. To test the hypothesis that angiotensinogen may modulate blood pressure, we transfected antisense oligonucleotides against rat angiotensinogen into the rat liver via the portal vein using liposomes that contain viral agglutinins to promote fusion with target cells, a technique that has been reported to be highly efficient. Transfection of antisense oligonucleotides resulted in a transient decrease in plasma angiotensinogen levels in spontaneously hypertensive rats from day 1 to day 7 after the injection, consistent with the reduction of hepatic angiotensinogen mRNA. Plasma angiotensin II concentration was also decreased in rats transfected with antisense oligonucleotides. Moreover, a transient decrease in blood pressure from day 1 to day 4 was observed, whereas transfection of sense and scrambled oligonucleotides did not result in any changes in plasma angiotensinogen level, blood pressure, or angiotensinogen mRNA level. Overall, our results demonstrate that transfection of antisense oligonucleotides against rat angiotensinogen resulted in a transient decrease in the high blood pressure of spontaneously hypertensive rats, accompanied by a decrease in angiotensinogen and angiotensin II levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1995

Recent advances in the study of renin and angiotensinogen genes: from molecules to the whole body.

The renin-angiotensin system (RAS) plays a key role in the regulation of the circulation and is critically involved in the pathogenesis of several diseases, including hypertension. Renin is synthesized mainly in the kidney and is secreted into the bloodstream. It catalyzes the rate-limiting cleavage of substrate angiotensinogen, which is derived mainly from the liver, to generate angiotensin I. Renin and angiotensinogen genes have been isolated and their structure has been determined by the methods of molecular biology. Renin and angiotensinogen genes are expressed in many tissues, and the tissue-specific regulation of these genes has been studied. The existence of local RASs in contrast to the classical circulating RAS has been suggested, although their exact functional role remains to be determined. Recent molecular analyses have led to a detailed description of the transcriptional mechanism of the renin and angiotensinogen genes, and have made it possible to study the regulation of the expression of these genes in several physiological and pathological states. In addition, several types of transgenic animals have been developed to study the functional importance of the RAS in vivo. Transgenic mice with human renin and human angiotensinogen genes may be a good model of human hypertension. In such mice, the human genes are expressed in the normal tissue-specific pattern, the circulating RAS is activated, and blood pressure is high. Finally, angiotensinogen-deficient mice have also been developed by homologous recombination in mouse embryonic stem cells. These mice do not produce angiotensinogen in the liver. As a result, they have no plasma immunoreactive angiotensin I and are hypotensive. The profound hypotension in these mice indicates the importance of the RAS in maintaining pressure.

Angiotensinogen↗
PubMed1994

Angiotensinogen-deficient mice with hypotension.

The renin-angiotensin system is an enzymatic cascade that produces a potent vasoconstrictor octapeptide angiotensin II, through its physiologically inactive intermediate decapeptide angiotensin I, from their precursor angiotensinogen. In the present study, we generated angiotensinogen-deficient mice by homologous recombination in mouse embryonic stem cells. These mice do not produce angiotensinogen in the liver, resulting in the complete loss of plasma immunoreactive angiotensin I. The systolic blood pressure of the homozygous mutant mice was 66.9 +/- 4.1 mm Hg, significantly lower than that of wild-type mice (100.4 +/- 4.4 mm Hg). This profound hypotension in angiotensinogen-deficient mice demonstrates an indispensable role for the renin-angiotensin system in maintaining blood pressure.

Angiotensin I↗
PubMed1994

A cell type-dependent enhancer core element is located in exon 5 of the human angiotensinogen gene.

We have recently characterized a cell type-dependent downstream enhancer that is contained within an 832-bp B2 sequences spanning the exon 5 and 3'-flanking region of the human angiotensinogen gene and have localized one of core elements to a 24-bp region in the 3'-flanking region. In the present study, we functionally dissected the 5'-half of B2 region and identified an additional cell type-dependent enhancer core element composed of the 80-bp sequences (+1399 to +1478) in the exon 5. Electrophoretic mobility shift assays demonstrated that the element interacts with two HepG2 cell-specific and several ubiquitous nuclear factors. These results, together with our previous results, suggest that the 80-bp exon 5 enhancer core element as well as 24-bp region plays an important role in the downstream enhancer effect.

Angiotensinogen↗
PubMed1994

Neuroectodermal tumors expressing c-, L-, and N-myc in transgenic mice that carry the E1A/E1B gene of human adenovirus type 12.

Adenovirus early 1 (E1) region gene products, including E1A and E1B, are required for transformation of primary cultured rodent cells. In order to investigate in vivo action of the E1 region, we established a line of transgenic mice carrying the oncogenic E1A and E1B genes of human adenovirus type 12 under control of the human angiotensinogen promoter. Histopathological analyses indicated that transgenic mice heritably develop neuroectodermal tumors arising from the pelvic region with varying degrees of incidence. The transgene was expressed in the neuroectodermal tumors as well as in TNT-1 cells, a cell line established from the tumors, where the human angiotensinogen promoter was constitutively active. The high level expression of c-, L-, and N-myc without gene amplification was notable in the original tumors and TNT-1 cells, but not in another tissue examined. The co-expression of the three sets of myc family genes in both the original tumors and the established cell line provided the possibility that the target cells for transformation may belong to a specific cell type that expresses all these oncogenes during development.

3T3 Cells↗
PubMed1994

Identification of cell type-dependent enhancer core element located in the 3'-downstream region of the human angiotensinogen gene.

We previously demonstrated that the 3.8-kilobase DNA fragment containing exons 4 and 5 of the human angiotensinogen (hAG) gene enhances the expression of chloramphenicol acetyltransferase gene, under control of the hAG promoter, in human hepatoma HepG2 cells. In the present study, to define regulatory elements of the hAG gene, we have functionally dissected this downstream region and localized a cell type-dependent enhancer to the 832-base pair sequence containing the exon 5 and 3'-flanking region. Further deletion analyses revealed that the 24-base pair core DNA fragment present in the 3'-flanking region was responsible for this enhancement. Electrophoretic mobility shift assay demonstrated that the 3'-enhancer core element interacts specifically with two nuclear factors from the HepG2 cells, one of which is an uncharacterized factor (human angiotensinogen enhancer factor-1: hAEF-1), the other is an AP-3-related factor. Mutation analyses indicated that the disruption of hAEF-1 binding alone completely impaired the enhancer activity of the core element. These results suggested that the downstream enhancer core element interacting with hAEF-1 plays an important role in activating the angiotensinogen promoter in a cell type-dependent manner.

Angiotensinogen↗