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Biomedical subjects

A Fukamizu

Publications and source records attributed to A Fukamizu.

At least 37 records · Page 2Linked to original sources
PubMed2000

Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice.

Renin plays a key role in controlling blood pressure through its specific cleavage of angiotensinogen to generate angiotensin I (AI). Although possible existence of the other angiotensin forming enzymes has been discussed to date, its in vivo function remains to be elucidated. To address the contribution of renin, we generated renin knockout mice. Homozygous mutant mice show neither detectable levels of plasma renin activity nor plasma AI, lowered blood pressure 20-30 mm Hg less than normal, increased urine and drinking volume, and altered renal morphology as those observed in angiotensinogen-deficient mice. We recently found the decreased density in granular layer cells of hippocampus and the impaired blood-brain barrier function in angiotensinogen-deficient mice. Surprisingly, however, such brain phenotypes were not observed in renin-deficient mice. Our results demonstrate an indispensable role for renin in the circulating angiotensin generation and in the maintenance of blood pressure, but suggest a dispensable role for renin in the blood-brain barrier function.

Angiotensin I↗
PubMed2000

R127W-HNF-4alpha is a loss of function mutation but not a rare polymorphism and causes Type II diabetes in a Japanese family with MODY1.

AIMS/HYPOTHESIS: Mutations in the hepatocyte nuclear factor (HNF)-4alpha gene cause the type 1 form of maturity-onset diabetes of the young (MODY1). The R127W mutation is a missense mutation located in the T-box region of HNF-4alpha that was first identified in a Japanese family with MODY. We have examined the functional properties of this mutation in order to clarify the molecular basis of MODY1. METHODS: The intracellular localisation, DNA binding ability, transactivation activity and functional synergism with the coactivator CREB-binding protein (CBP) of R127W-HNF-4alpha were investigated. RESULTS: The nuclear import and functional synergy with CBP of R127W-HNF-4alpha were normal. The DNA binding ability of the mutant was decreased as was its transcriptional activation of the HNF-1alpha and L-type pyruvate kinase (PKL) genes. CONCLUSION/INTERPRETATION: The R127W mutation seems to be a loss-of-function mutation.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2000

Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin.

A family of basic-helix-loop-helix (bHLH) nuclear factors play important roles in controlling cell growth and differentiation as critical regulatory components in transcription. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Transactivation property of POD-1/Capsulin was analyzed by the Gal4 fusion system in six mammalian cell lines. The results indicated that an activation property was shown in HT1080 and HeLa cells, but a repression activity in HepG2 cells. Mapping analysis for the transactivation and repression activities revealed that the C-terminal domain of POD-1/Capsulin is essential for the transactivation and both the N-terminal and C-terminal domains are contributed to the repression activities. Furthermore, in order to identify possible interactants of the POD-1/Capsulin, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified a class A bHLH protein, ITF-2 as potential heterodimeric partner of the bHLH protein.

3T3 Cells↗
PubMed2000

Angiotensinogen, angiotensin II and adipose tissue development.

Adipose tissue is an important source of angiotensinogen (AGT). Recent evidence shows that a local renin-angiotensinogen system (RAS) is present in human adipose tissue and may act as a distinct system from plasma RAS. In obese patients, the involvement of angiotensin II (angII) as a consequence of increased plasma AGT secreted from adipose tissue has been proposed in the development of hypertension. Another role of AGT via angII in the development of adipose tissue is supported by the following: (i) in vitro, angII stimulates the production and release of prostacyclin from adipocytes, which in turn promotes the differentiation of precursor cells into adipocytes; (ii) ex vivo and in vivo, both angII and (carba)prostacyclin promote the formation of new fat cells; and (iii) AGT -/- mice exhibit a slowing down of adipose tissue development, as compared to wild-type mice. Altogether the data are consistent with an autocrine/paracrine mechanism implicating AGT, angII and prostacyclin in adipose tissue development.

Adipocytes↗
PubMed2000

Genomic expression systems on hierarchy and network leading to hypertension: long on history, short on facts.

Genomic research that is part of current DNA sequencing projects is having a great impact in many fields of study. With the discovery of sequence information for human and mouse genomes, an explosion of genetical and functional data will be revolutionizing mechanistic studies of hypertension syndrome. Assuming the availability of genome sequences, the challenge includes identifying genes, predicting the proteins they encode, determining when and where genes are expressed and how they interact, and learning how these expression and interaction profiles change in response to environmental signals. As the hypertension research community enters the post-genome era, the study of gene expression patterns and phenotypes in model animals will be a part of analyzing the role of genes involved in the pathogenesis of hypertension. The molecular dissection of hypertension genetics is a complex multidisciplinary challenge and a medical imperative. The understanding of complex disorders such as hypertension has advanced considerably through the use of genetically engineered mice. Studies of hypertension using transgenic and knockout mice have uncovered multiple potential functions of the regulatory systems controlling blood pressure. In this review, I discuss the molecular basis of the hierarchy and network of genomic expression cascades of hypertension by focusing on the functional significance of the renin-angiotensin system.

Animals↗
PubMed2000

[A role of angiotensin II found in knockout mice with angiotensin signal breakdown].

The renin-angiotensin system, composed of enzymatic and signal-transduction cascades, plays a key role in the regulation of arterial blood pressure and in the development of certain forms of experimental and human hypertension. The products of this system, angiotensin peptides, exert a wide range of physiologically important effects on many tissues, including those of the cardiovascular systems, through their actions on angiotensin receptors. Molecular genetic and transgenic studies have begun to implicate some of the genes encoding components of the renin-angiotensin system in the development of cardiovascular diseases. Here, I review new developments related to the functional role of angiotensin II, an important product of the renin-angiotensin system, by focusing on knockout approaches including cardiovascular and central nervous systems.

Aging↗
PubMed2000

Molecular characterization of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin.

Members of the basic helix-loop-helix (bHLH) family of transcription factors have been implicated in the regulation of cell proliferation and differentiation. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Northern blot analysis showed that POD-1/Capsulin are preferentially expressed in human vascular smooth muscle cells, and are enriched in mouse heart, kidney, testis, spleen, and lung. Ectopic expression of the HA-tagged POD-1/Capsulin in 293T cells revealed that this protein has an apparent molecular mass of 26 kDa and localized in the nucleus. Furthermore, in order to identify possible interactants of the bHLH protein, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified alternative-splicing form of HEB, as potential heterodimeric partner of POD-1/Capsulin. Our results provide the molecular basis for cell type-specific transcriptional regulation by POD-1/Capsulin.

3T3 Cells↗
PubMed2000

RGS domain in the amino-terminus of G protein-coupled receptor kinase 2 inhibits Gq-mediated signaling.

We have previously shown that not only G protein-coupled receptor kinase (GRK) 2, but also a catalytically inactive Lys220Trp GRK2 decreases endothelin (ET)-1-induced inositol 1,4,5-trisphosphate (IP3) formation, and demonstrated the presence of phosphorylation-independent desensitization mechanism. To clarify the role of GRK2 other than that as a kinase, we characterized an RGS (regulator of G protein signaling)-like domain in the amino-terminus of GRK2. Both GRK2(1-181) and GRK2(54-174) suppressed Ca2+ responses induced by angiotensin II (Ang II) and ET-1, and bound directly with Galphaq but not Galphas nor Galphai3 in the presence of GDP and AlF4-. These results demonstrate that GRK2 regulates Gq-mediated signaling negatively by direct interaction between its RGS domain and the transitional state of Galphaq, as well as through phosphorylation of activated receptors by its kinase domain.

Aluminum Compounds↗
PubMed1999

Hypernuclear acetylation in atherosclerotic lesions and activated vascular smooth muscle cells.

Recent studies have implicated acetylation of several nuclear proteins such as histones and p53 on their epsilon-portion of lysine residues in eukaryotic transcription. Here we raised a specific polyclonal antibody against epsilon-acetylated lysine. Using the antibody, we detected hypernuclear acetylation (HNA) in atherosclerotic vascular smooth muscle cells (VSMCs). Thrombin, a humoral factor known to cause activation and proliferation of VSMCs, strongly potentiated HNA in cultured VSMCs. MAP kinase pathway and a signal coactivator CREB binding protein (CBP) were involved in thrombin-induced HNA of VSMCs. Our results suggest that coactivators cooperating with signal-dependent transcription activators play an important role in atherosclerogenesis via HNA in VSMCs.

Acetylation↗
PubMed1999

Regulated expression of human angiotensinogen gene by hepatocyte nuclear factor 4 and chicken ovalbumin upstream promoter-transcription factor.

We previously identified various upstream and downstream regulatory elements and factors important for hepatic expression of the human angiotensinogen (ANG) gene, the precursor of vasoactive octapeptide angiotensin II. In the present study, to further investigate the molecular mechanism of human ANG transcriptional regulation, we generated transgenic mice carrying the fusion gene composed of the 1. 3-kilobase promoter of the human ANG gene, its downstream enhancer, and the chloramphenicol acetyltransferase reporter gene. Because expression of the chloramphenicol acetyltransferase gene was observed strongly in the liver and weakly in the kidney, we suspected that hepatocyte nuclear factor (HNF) 4 with a tissue expression pattern similar to that of the reporter gene would regulate ANG transcription. In vitro assays indicated that HNF4 bound to the promoter elements and strongly activated the ANG transcription, but that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a transcriptional repressor, dramatically repressed human ANG transcription through the promoter elements and the downstream enhancer core elements. Furthermore, COUP-TF dramatically decreased the human ANG transcription in the mouse liver by the Helios Gene Gun system in vivo. These results suggest that an interplay between HNF4 and COUP-TF could be important in hepatic human ANG transcription.

Angiotensins↗
PubMed1999

Transgenic and knockout models in renin-angiotensin system.

The renin-angiotensin system, composed of enzymatic and signal-transduction cascades, plays a key role in the regulation of arterial blood pressure and in the development of certain forms of experimental and human hypertension. The products of this system, angiotensin peptides, exert a wide range of physiologically important effects on many tissues, including those of the cardiovascular systems, through their actions on angiotensin receptors. Molecular genetic and transgenic studies have begun to implicate some of the genes encoding components of the renin-angiotensin system in the development of cardiovascular diseases. Recently, we succeeded in generating transgenic mice with chronic hypertension and with inducible hypertension during pregnancy and in creating mice homozygous for a targeted disruption of the angiotensinogen gene (the only known precursor of angiotensins), resulting in the complete loss of angiotensin signals in vivo. Here, we will review recent advances related to the functional analysis of the renin-angiotensin system, in particular by focusing on transgenic approaches including gene targeting.

Animals↗
PubMed1999

CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mice.

The nonstructural protein 2 (NS2) from parvovirus minute virus of mice (MVMp) is a 25-kDa polypeptide which localizes preferentially to the cytoplasm and associates with cellular proteins in cytoplasm. These lines of evidence suggest that NS2 is positively exported from the nucleus to cytoplasm and functions in cytoplasm. We report here that nuclear export of NS2 is inhibited by leptomycin B (LMB), a drug that specifically blocks nuclear export signal (NES)-chromosomal region maintenance 1 (CRM1) interactions. CRM1 binds specifically to the 81- to 106-amino-acid (aa) region of NS2, and the region of NS2 actually functions as a NES. Interestingly, this region appears to be distinct from a typical NES sequence, which consists of leucine-rich sequences. These results indicate that NS2 protein is continuously exported from the nucleus by a CRM1-dependent mechanism and suggest that CRM1 also exports to distinct type of NESs.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and expression of human neurochondrin-1 and -2.

Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucine-rich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain.

Amino Acid Sequence↗
PubMed1999

Angiotensinogen gene-activating elements regulate blood pressure in the brain.

Although the angiotensinogen gene is a possible candidate as a determinant of hypertension, the molecular mechanisms of tissue angiotensinogen gene regulation have yet to be clarified. We identified essential transcription regulators of angiotensinogen production in the central nervous system using synthetic double-stranded oligodeoxynucleotides (ODNs) as "decoy" cis elements to block the binding of nuclear factors to promoter regions of the targeted gene. Using a gel mobility shift assay, angiotensinogen gene-activating element (AGE) 2 binding protein was detected in the brain nuclear extracts of both spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKYs). Importantly, the binding activity of AGE 2 and angiotensinogen mRNA level were significantly higher in the brain of SHRs than in that of WKYs. Using the decoy approach, we demonstrated a significant decrease in the blood pressure of SHRs by transfection of AGE 2 decoy, but not mismatched, ODNs into the lateral cerebroventricle, accompanied by a significant decrease in brain angiotensinogen concentration and mRNA, and angiotensin II level. That these effects, demonstrated herein, are due to central effects is confirmed by the fact that no changes in circulating levels of angiotensinogen or angiotensin II concentrations were observed. Notably, AGE 2 decoy ODNs did not decrease the blood pressure of WKYs. We conclude that the abnormal expression of AGE 2 binding protein in the central nervous system plays a crucial role in high blood pressure of a genetically hypertensive rat model.

Angiotensinogen↗
PubMed1999

Male sterility in transgenic mice expressing activin betaA subunit gene in testis.

Activins and inhibins, which are endocrine regulators of anterior pituitary function, have also been reported to participate in the paracrine and autocrine regulation of reproductive function. To determine the in vivo effects of overexpressed activin/inhibin, we generated transgenic mice carrying the human activin/inhibin betaA subunit mini gene under the regulatory control of the mouse methallothionein promoter. In one of the transgenic line analyzed, the betaA subunit gene was preferentially expressed in the testis. Ectopic and allochronic expression of the betaA gene started at 3 weeks after birth and transgenic male mice became sterile in the ensuing several weeks. Histological analysis revealed testicular degeneration in these mice. The results from this transgenic line strongly support the in vivo activity of activin/inhibin in male reproductive functions.

Animals↗
PubMed1999

Induction of hydroxyapatite resorptive activity in bone marrow cell populations resistant to bafilomycin A1 by a factor with restricted expression to bone and brain, neurochondrin.

Bone, one of the favored sites for tumor metastasis, is a dynamic organ undergoing formation and resorption. We found bone metastasis with osteolytic lesion in the bone marrow of the femur by injecting BW5147 T-lymphoma cells into the tail vein of AKR mice. To understand this bone destruction, we constructed a cDNA library from BW5147 with a cloning vector that allowed in vitro synthesis of mRNAs, and then identified a particular cDNA clone by adding the conditioned medium from Xenopus oocytes following injection of the mRNA synthesized in vitro to primary bone marrow heterogeneous cell populations on hydroxyapatite thin films. By means of this method, we isolated a factor with 16% leucine residues, termed neurochondrin, that induces hydroxyapatite resorptive activity in bone marrow cells resistant to bafilomycin A1, an inhibitor of macrophage- and osteoclast-mediated resorption. Expression of the gene was localized to chondrocyte, osteoblast, and osteocyte in the bone and to the hippocampus and Purkinje cell layer of cerebellum in the brain. This may provide insights into the molecular mechanisms underlying bone resorption with potential implications for the activation of cells other than macrophages and osteoclasts in bone marrow cells.

Amino Acid Sequence↗
PubMed1999

Reduction of depressive-like behavior in mice lacking angiotensinogen.

We made use of targeted disruption of the mouse angiotensinogen (ATN) gene to examine the functional role of the ATN in the central nervous system. Both male and female ATN-deficient mice displayed the reduction of depressive-like behavior in the behavioral despair swim tests and spontaneous locomotor activity diminished. However, both male and female ATN-deficient mice showed no anxiogenic-like or memory-deficit behavior and there was no change in the pain threshold. We propose that endogenous ATN in the central nervous system may regulate the depressant state in the brain.

Angiotensin-Converting Enzyme Inhibitors↗