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ABSTRACT: We previously demonstrated that the activation of prostaglandin E-prostanoid-3 (EP3) receptor sensitized the canine nociceptor response to bradykinin (BK). To elucidate the molecular mechanism for this sensitization, we cloned two cDNAs encoding EP3s with different C-terminals, from canine dorsal root ganglia, and established the transformed cell lines stably expressing them. In both transformants, EP3 agonist did not increase intracellular cAMP levels, but it attenuated forskolin-dependent cAMP accumulation in a pertussis toxin (PTX)-sensitive manner and increased intracellular calcium levels in a PTX-resistant manner, indicating that both EP3s can couple with Gi and Gq, but not with Gs proteins. As the nociceptor response to BK is mediated by BK B2 receptor, it was transfected into the transformants and the effects of EP3 agonist on BK-dependent calcium mobilization were investigated. When BK was applied twice with a 6-min interval, the second response was markedly attenuated. Pre-treatment with EP3 agonist had no effect on the initial response, but restored the second response in a PTX-sensitive manner. A protein kinase A inhibitor mimicked the effect of EP3 agonist. These results demonstrate that the activation of EP3 restores the response to BK by attenuating the desensitization of BK B2 receptor activity via Gi protein.
Journal of Neurochemistry 04/2007; 100(6):1636-47. · 4.06 Impact Factor
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ABSTRACT: The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24(-/-) mice. Both DHCR24(-/-) and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24(-/-) MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.
Endocrinology 07/2006; 147(6):3123-32. · 4.46 Impact Factor
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ABSTRACT: An important role for the cyclin-dependent kinase inhibitors (CDKIs), p27Kip1 and p57Kip2, in the proliferation and differentiation of adrenal cells has been suggested by their knockout mice, which display adrenal hyperplasia. Adrenal development and function are primarily regulated by ACTH. In the present study, we investigated the effects of ACTH on the expression of p27Kip1, p57Kip2 and proliferating cell nuclear antigen (PCNA) in rat adrenals. Male Wistar rats were treated with dexamethasone (Dex) to inhibit endogenous ACTH secretion. ACTH was then administered to the rats, and the adrenals were examined by Western blot and immunohistochemical analyses. Dex treatment induced shrinkage of adrenals where no PCNA-expressing cells were detected, but most of the cells expressed p27Kip1. Subsequent ACTH treatment resulted in the marked suppression of p27Kip1 expression, specifically in adrenocortical cells at 12 h after the stimulus. At 48 h, the p27Kip1 suppression still continued in the cortex, while the PCNA-expressing cells appeared mainly around the zona glomerulosa and increased at 72 h. At this time, the p27Kip1-expressing cells also appeared in the same zone. In contrast to p27Kip1, the expression of p57Kip2 was not detected in the Dex-treated adrenal. However, its expression was markedly induced by ACTH in the zona glomerulosa at 48 and 72 h. The results demonstrate that the primary site for mitogenic action of ACTH in rat adrenocortex is the zona glomerulosa, and that ACTH modulates proliferation of adrenocortical cells by regulating p27Kip1 and p57Kip2 expression in a time- and site-specific manner.
Journal of Endocrinology 07/2006; 189(3):671-9. · 3.55 Impact Factor
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Rusella Mirza,
Shizu Hayasaka,
Yoshiko Takagishi,
Fukushi Kambe, Sachiko Ohmori,
Kazuko Maki,
Michiyo Yamamoto,
Kohji Murakami,
Takahide Kaji,
David Zadworny,
Yoshiharu Murata,
Hisao Seo
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ABSTRACT: Desmosterolosis is an autosomal recessive disorder due to mutations in the 3beta-hydroxysterol-Delta24 reductase (DHCR24) gene that encodes an enzyme catalyzing the conversion of desmosterol to cholesterol. To date, only two patients have been reported with severe developmental defects including craniofacial abnormalities and limb malformations. We employed mice with targeted disruption of DHCR24 to understand the pathophysiology of desmosterolosis. All DHCR24-/- mice died within a few hours after birth. Their skin was wrinkleless and less pliant, leading to restricted movement and inability to suck (empty stomach). DHCR24 gene was expressed abundantly in the epidermis of control but not of DHCR24-/- mice. Accordingly, cholesterol was not detected whereas desmosterol was abundant in the epidermis of DHCR24-/- mice. Skin histology revealed thickened epidermis with few and smaller keratohyaline granules. Aberrant expression of keratins such as keratins 6 and 14 suggested hyperproliferative hyperkeratosis with undifferentiated keratinocytes throughout the epidermis. Altered expression of filaggrin, loricrin, and involcrin were also observed in the epidermis of DHCR24-/-. These findings suggested impaired skin barrier function. Indeed, increased trans-epidermal water loss and permeability of Lucifer yellow were observed in DHCR24-/- mice. DHCR24 thus plays crucial role for skin development and its proper function.
Journal of Investigative Dermatology 04/2006; 126(3):638-47. · 6.31 Impact Factor
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ABSTRACT: We reported that the first two cysteine residues out of three present in paired domain (PD), a DNA-binding domain, are responsible for redox regulation of Pax-8 DNA binding activity. We show that glutathionylation of these cysteines has a regulatory role in PD binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC, CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing various ratios of GSH/GSSG and subjected to gel shift assay. Binding of CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas that of SSC and SSS was not affected. Because [3H]glutathione was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the binding impairment was ascribed to glutathionylation of the redox-reactive cysteines. This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol and by redox factor (Ref)-1 in vitro. To explore the glutathionylation in cells, Chinese hamster ovary cells overexpressing CSS and SCS were labeled with [35S]cysteine in the presence of cycloheximide. Immunoprecipitation with an antibody against PD revealed that treatment of the cells with an oxidant diamide induced the 35S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational modification of PD would provide a new insight into molecular basis for modulation of Pax function.
Journal of Biological Chemistry 08/2005; 280(27):25901-6. · 4.77 Impact Factor
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ABSTRACT: We identified a thyroid hormone [3,5,3'-tri-iodothyronine (T(3))]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, alpha, beta1 and beta2, in human brain by 5'- and 3'-RACE (rapid amplification of cDNA ends). The alpha transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that beta1 and beta2 encode an identical protein product, beta, which differs from alpha in its N-terminus. Since alpha and beta contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the alpha transcript is expressed exclusively in the brain, whereas beta transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both alpha and beta transcripts, raising the question of which transcript is up-regulated by T(3). It was revealed that T(3) markedly induced the expression of alpha isoform but not of beta. This T(3)-mediated increase in the alpha isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting a complex signalling network through inhibition of calcineurin.
Biochemical Journal 11/2002; 367(Pt 2):459-66. · 4.90 Impact Factor
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ABSTRACT: We have reported reversible oxidoreductive regulation of DNA-binding activity of Pax-8: oxidation inhibits its binding and subsequent reduction restores the binding. Here, we show that Cys-45 and Cys-57 in the paired domain of rat Pax-8, which are conserved in all Pax members, are responsible for the redox regulation of its binding. Electrophoretic mobility shift assay using deletion mutants and mutants with substitution of cysteine with serine revealed that oxidation by diamide of either Cys-45 or Cys-57 loses the DNA binding of Pax-8. An intracellular oxidoreductive enzyme redox factor-1 (Ref-1) could reduce the oxidized Cys-45 or Cys-57 and restored the binding. Furthermore, reporter gene assay showed that transcriptional activity of wild-type Pax-8 was enhanced by co-expression of Ref-1. When the mutant with double substitutions of Cys-45 and Cys-57, which was insensitive to oxidation, was transfected, the basal transactivation level was much higher than that of wild-type Pax-8, while it was not enhanced by Ref-1. These results demonstrated that oxidoreductive modification of Cys-45 and Cys-57 via Ref-1 plays a role in redox regulation of Pax-8 in living cells.
Biochemical and Biophysical Research Communications 10/2002; 297(2):288-93. · 2.48 Impact Factor
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ABSTRACT: Mutations of the myosin-Va gene (Myo5a) cause diluted coat color in mice and are occasionally associated with severe neurological disorders. Dilute-opisthotonus
(dop) is a spontaneous gene mutation in the rat, and phenotypes of the homozygote (dop/dop) are similar to those of the Myo5a-deficient mouse, suggesting that the mutation resides in the rat Myo5a gene. To elucidate the molecular basis of the dop mutation, we cloned the rat Myo5a cDNA from the wild type and the dop/dop. The wild-type rat Myo5a cDNA contained a 5487-bp ORF and showed higher homology with Myo5a of the other species than Myr6 (Myo5b) in the rat. A 141-bp in-frame deletion was detected in the head region in the dop cDNA. An intragenic rearrangement consisting of a 306-bp inversion associated with 17-bp and 217-bp deletions were identified
in the Myo5a gene of the dop genome. This rearrangement involved a 141-bp exon, which was skipped in the dop transcript. The MyoVA protein expression was severely impaired in the dop/dop brain. This is the first report to define the dop mutation as the Myo5a gene abnormality in the rat.
Mammalian Genome 07/2000; 11(8):649-655. · 2.89 Impact Factor
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ABSTRACT: 国立情報学研究所で電子化したコンテンツを使用している。 Disuse atrophy of skeletal muscle is a serious complication for a number of bed-ridden patients. We previously demonstrated that unloading of hindlimb muscles by rat tail-suspension for 7 days resulted in disuse atrophy. This is characterized by the change from slow-twitch-oxidative to fast-twitch-glycolytic muscle fibers. Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) is an important glycolytic enzyme in muscles. On the other hand calcineurin (Cn) plays important roles to change fiber subtypes of skeletal muscle. Since we have recently demonstrated that ZAKI-4β, an inhibitor of Cn, is expressed in skeletal muscles, we investigated the changes in the expression of GAPDH and ZAKI-4β mRNA after 7 days of tail-suspension in rats. It reduced the body weight gain (20g) which was about one third of that of control group (60g). Thus the weights of muscles were corrected by body weight. The weight of gastrocnemius and soleus decreased significantly by the tail suspension. In the control group, abundance of GAPDH mRNA in the gastrocnemius was ten times higher than that in soleus. Tail-suspension significantly increased the abundance of GAPDH mRNA in solues. The same tendency was observed in gastrocnemius without statistical significance. In the control group, the levels of ZAKI-4β mRNA in the soleus was about five times higher than that in gastrocnemius. In the suspended group, ZAKI-4β mRNA in the gastrocnemius decreased significantly but did not change in the soleus. The results of GAPDH mRNA suggest that fast-glycolytic fibers are more abundant in gastrocnemius than that in soleus before suspension and tail-suspension increased the ratio of this fiber type in soleus. On the other hand, ZAKI-4β mRNA seems to be expressed selectively in slow-twitch-oxidative fibers, which is compatible with the decreased ratio of slow-twitch fiber in gastrocnemius after the tail-suspension. Environmental Medicine : annual report of the Research Institute of Environmental Medicine, Nagoya University. v.47, 2003, p.15-19
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ABSTRACT: 国立情報学研究所で電子化したコンテンツを使用している。 We identified several mutations in the intron 3 of human growth hormone gene I (hGH-I) in patients with isolated GH deficiency (IGHD) type II characterized by an autosomal dominant trait. The mutations result in exon 3 skipping and generation of 17 Kd mutant GH. To elucidate how the mutation causes dominant trait, transgenic mice expressing a mutant hGH gene (the first guanine to adenine transversion in intron 3: GH-I; IVS3+1: G-A) were produced in C57BL/6 strain. Genotypes of mice were identified by PCR-amplified products of tail snip DNAs. Delivery of the mutant hGH transgene into 76 fertilized eggs resulted in production of two male heterozygous transgenic mice (hGH^<+/->, the zero filial generation, FO). Since the mating of the transgenic mice with the same strain was unsuccessful, they were outcrossed with CD-I (ICR) strain. Only one mouse gave birth, producing 4 male and 7 female (F1) harboring the mutant hGH gene in one allele (hGH^<+/->). F1 mice were mated again with the wild type ICR strain, generating 82 hGH+/" mice (F2 : 51 males and 31 females). To study whether somatotrophs in F2 mice express the mutant hGH gene, RNA extracted from the pituitary was subjected to RT-PCR. It was demonstrated that the F2, hGH^<+/-> mice express the mutant hGH gene, lacking exon 3. Thus, these heterozygous mice were sib-mated to generate homozygous mice (F3). The mating resulted in 27% hGH^<-/->, 64% hGH+/ and 9% hGH^<+/+> mice, indicating that the transgene was carried stably to the descendants and did not interfere with the reproduction. These mice will be a valuable model to study how type II IGHD develops during the course of development. Environmental medicine : annual report of the Research Institute of Environmental Medicine, Nagoya University. v.46, n.1/2, 2002, p.36-39
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ABSTRACT: 国立情報学研究所で電子化したコンテンツを使用している。 Regulation of osteocalcin (OC) gene expression in osteoblasts by estrogen has been controversial. We thus investigated the effect of 17β-estradiol (Ea) on 1α,25-dihydroxyvitamin D_3 (VD_3)-mediated increase in the transcription of OC gene in rat osteoblastic ROS 17/2.8 cells. Treatment of the cells with E_2 or VD_3 alone did not increase OC mRNA. However, combined treatment with E_2 and VD_3 significantly increased the OC mRNA levels, suggesting that VD_3-dependent up-regulation of OC expression requires E_2 action. Since it was demonstrated that treatment with E_2 alone increased the expression of VD_3 receptor mRNA, it was suggested that E_2- mediated increase in the receptor number is contributing to VD_3-mediated increase in OC gene transcription. Environmental medicine : annual report of the Research Institute of Environmental Medicine, Nagoya University. v.46, n.1/2, 2002, p.43-44
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ABSTRACT: A G – C transversion at the fifth nucleotide of intron 3 of GH-I gene was identified in a sporadic case of isolated growth hormone deficiency (IGHD). The mutation was absent in both of the parents, indicating that the mutation occurred de novo. An abnormal hGH mRNA lacking a region encoded by exon 3 was spliced when the mutantGH-I gene was expressed in cultured cells. Since skipping of exon 3 is a common feature for four different mutantGH-I genes identified in patients with autosomal dominantly inherited IGHD, we conclude that the mutation causes IGHD in this case.
Growth Hormone & IGF Research.
