バイヨウ ラット ジカセン デノ ダエキ ブンピツ ソクシンザイ ニヨル オルニチン ダツタンサン コウソ カッセイ ノ チョウセツ ニ カンスル ケンキュウ

種々の唾液分泌促進剤は耳下腺での唾液分泌促進に加え、細胞増殖促進作用がある。 ラット耳下腺cxplantの培養系を用いて、これまでにβ-adrenergic agonistのisoproterenol (IPR)やcholinergic agonistのcarbachol(CC)が、増殖に必須なポリアミンの合成律速酵素 であるオルニチン脱炭酸酵素(ODC)の活性を上昇させ、DNA合成を促進することが報 告されている。一方、ODCはこれらアゴニストのみならず低浸透圧下でも著名な誘導を 受けることが知られている。この低張による誘導はIPRとは相乗効果を示すのに反し、 CCによって強く抑制された。また、CCはIPRによる誘導をも抑制した。そこで本研究 では、このCCの抑制作用を中心に唾液腺でのODC調節機構について検討した。 [方法] 199培地上に浮かべたシリコナイズしたレンズペーパー上でラット耳下腺cxplant (1-2mg)を培養した。低張培地にはNaCl濃度のみを半減した浸透圧が約40%低い199培地 を用いた。分泌能はアゴニスト添加2時間後の培地中のアミラーゼ活性で測定し、ODC 活性は4-6時間後に測定した。ODCのmRNAとタンパク質の定量はそれぞれNorthern blot 法とWestern blot法で行った。また、細胞内Ca2+濃度([Ca²⁺]i)はトリプシンとコラゲナ ーゼを用いて調製したラット耳下腺腺房細胞に、Fura2-AMをとりこませ、ARGUS50 (浜松ホトニクス)にて測定した。cAMPレベルは、cAMP kit「ヤマサ」(ヤマサ醤油)に て測定した。 [結果] 1) ODC活性は等張培地でIPRによって約29倍上昇するが、この上昇はCCで70%も抑制 された。この時、CCはIPRによるcAMPレベルの上昇には影響を及ぼさなかった。ま た低張培地でODC活性は6時間後に約100倍の最大活性に達し、IPRはこれを3倍に増強 したのに反し、CCは1/3に抑制した。 2) アゴニストや低張によるODC誘導では活性とmRNAおよび蛋白質量はほぼ比例し た。ただ、例外として低張でのCCのODC活性の抑制ではmRNAの低下は見られな かった。 3) IPRや低張によるODC誘導でのCCの抑制はODC蛋白質の半減期の短縮によるもので はない。 4) 小胞体のCa²⁺依存性ATPaseを抑制するthapsigarginは、CCと同様にODC活性低下作 用を示すが、[Ca²⁺]iを低下させるBAPTA-AMはCCのODC活性抑制作用を弱めた。 5) 耳下腺腺房細胞の[Ca²⁺]iはCCとthapsigarginでそれぞれ周期的および一過性の上昇を 示した。 6) 腺房細胞の[Ca²⁺]iは低張培地で持続的上昇を示した。 7) ODC誘導と異なりアミラーゼ [結論] 耳下腺での唾液分泌促進とODC誘導は異なる機構によって制御されていることが示唆 された。ODCの誘導は、その誘導因子の種類に関係なく[Ca²⁺]iと関連し、その上昇は ODC誘導を翻訳レベルで抑制すると考えられる

エナメルシツ ケイセイ フゼンショウ ノ シンキ セキニン イデンシ ヘンイ ノ ハッケン ト ソノ イギ : テンシャ インシ Sp6 オ メグル パズル

Tooth is one of the essential organs in our body, and plays the important roles to maintain and improve our quality of life (QOL). Regenerative medicine has been achieving the remarkable progress for several years, and tooth is also one of the hot research targets for regeneration therapy. To regenerate tooth, we need to understand whole tooth developmental program at molecular level. Reverse genetics has brought us many molecular details at the early stage of tooth development, however, the late stage of tooth development is not fully understood. We are now trying to elucidate the molecular paradigm on the development of dental epithelial cells. During these studies, we found the new genetic linkage between amelogenesis imperfecta (AI) and a 2-bp insertional mutation of transcription factor Sp6, and proposed it as a new molecular diagnostic candidate for AI patients. In this review, I would like to summarize about AI based on the responsible genes including current topics in the late stage of tooth development, and to discuss about what we can learn from those for regenerative tooth and future direction

AK2 in Neutrophil Differentiation

Adenine nucleotide dynamics in the mitochondrial intermembrane space (IMS) play a key role in oxidative phosphorylation. In a previous study, Drosophila adenylate kinase isozyme 2 (Dak2) knockout was reported to cause developmental lethality at the larval stage in Drosophila melanogaster. In addition, two other studies reported that AK2 is a responsible gene for reticular dysgenesis (RD), a human disease that is characterized by severe combined immunodeficiency and deafness. Therefore, mitochondrial AK2 may play an important role in hematopoietic differentiation and ontogenesis. Three additional adenine nucleotide metabolizing enzymes, including mitochondrial creatine kinases (CKMT1 and CKMT2) and nucleoside diphosphate kinase isoform D (NDPK-D), have been found in IMS. Although these kinases generate ADP for ATP synthesis, their involvement in RD remains unclear and still an open question. In this study, mRNA and protein expressions of these mitochondrial kinases were firstly examined in mouse ES cells, day 8 embryos, and 7-week-old adult mice. It was found that their expressions are spatiotemporally regulated, and Ak2 is exclusively expressed in bone marrow, which is a major hematopoietic tissue in adults. In subsequent experiments, we identified increased expression of both AK2 and CKMT1 during macrophage differentiation and exclusive production of AK2 during neutrophil differentiation using HL-60 cells as an in vitro model of hematopoietic differentiation. Furthermore, AK2 knockdown specifically inhibited neutrophil differentiation without affecting macrophage differentiation. These data suggest that AK2 is indispensable for neutrophil differentiation and indicate a possible causative link between AK2 deficiency and neutropenia in RD

Novel genetic linkage of rat Sp6 mutation to Amelogenesis imperfecta

Background: Amelogenesis imperfecta (AI) is an inherited disorder characterized by abnormal formation of tooth enamel. Although several genes responsible for AI have been reported, not all causative genes for human AI have been identified to date. AMI rat has been reported as an autosomal recessive mutant with hypoplastic AI isolated from a colony of stroke-prone spontaneously hypertensive rat strain, but the causative gene has not yet been clarified. Through a genetic screen, we identified the causative gene of autosomal recessive AI in AMI and analyzed its role in amelogenesis. Methods: cDNA sequencing of possible AI-candidate genes so far identified using total RNA of day 6 AMI rat molars identified a novel responsible mutation in specificity protein 6 (Sp6). Genetic linkage analysis was performed between Sp6 and AI phenotype in AMI. To understand a role of SP6 in AI, we generated the transgenic rats harboring Sp6 transgene in AMI (Ami/Ami + Tg). Histological analyses were performed using the thin sections of control rats, AMI, and Ami/Ami + Tg incisors in maxillae, respectively. Results: We found the novel genetic linkage between a 2-bp insertional mutation of Sp6 gene and the AI phenotype in AMI rats. The position of mutation was located in the coding region of Sp6, which caused frameshift mutation and disruption of the third zinc finger domain of SP6 with 11 cryptic amino acid residues and a stop codon. Transfection studies showed that the mutant protein can be translated and localized in the nucleus in the same manner as the wild-type SP6 protein. When we introduced the CMV promoter-driven wild-type Sp6 transgene into AMI rats, the SP6 protein was ectopically expressed in the maturation stage of ameloblasts associated with the extended maturation stage and the shortened reduced stage without any other phenotypical changes. Conclusion: We propose the addition of Sp6 mutation as a new molecular diagnostic criterion for the autosomal recessive AI patients. Our findings expand the spectrum of genetic causes of autosomal recessive AI and sheds light on the molecular diagnosis for the classification of AI. Furthermore, tight regulation of the temporospatial expression of SP6 may have critical roles in completing amelogenesis

Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII) cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cellmorphology andmarker gene expression. Two clones (B7 and H12)were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC), an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5), an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1) expression levels were enhanced. After treatment with dexamethasone (DEX), 8-bromoadenosine 3’5’-cyclic monophosphate (8-Br-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and keratinocyte growth factor (KGF), surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation

Putrescine-dependent Tumor Invasion

Our previous study showed that treatment of highly invasive rat ascites hepatoma (LC-AH) cells with α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, decreased both their intracellular level of putrescine and their in vitro invasion of a monolayer of calf pulmonary arterial endothelial (CPAE) cells, and that both these decreases were completely reversed by exogenous putrescine, but not spermidine or spermine. Here we show that all adhering control (DFMO-untreated) cells migrated beneath CPAE monolayer with morphological change from round to cauliflower-shaped cells (migratory cells). DFMO treatment increased the number of cells that remained round without migration (nonmigratory cells). Exogenous putrescine, but not spermidine or spermine, induced transformation of all nonmigratory cells to migratory cells with a concomitant increase in their intracellular Ca2+ level, [Ca2+]i. The putrescine-induced increase in their [Ca2+]i preceded their transformation and these effects of putrescine were not affected by antagonists of the voltage-gated Ca2+ channel, but were completely suppressed by ryanodine, which also suppressed the invasiveness of the control cells. The DFMO-induced decreases in both [Ca2+]i and the invasiveness of the cells were restored by thapsigargin, which elevated [Ca2+]i by inhibiting endoplasmic Ca2+-ATPase, indicating that thapsigargin mimics the effects of putrescine. These results support the idea that putrescine is a cofactor for Ca2+ release through the Ca2+ channel in the endoplasmic reticulum that is inhibited by ryanodine, this release being initiated by cell adhesion and being a prerequisite for tumor cell invasion

Gene Signature of Human Oral Mucosa Fibroblasts : Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells

Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity.These data may provide useful insight into application of oral fibroblasts for direct reprograming

Redefining GBA gene structure unveils the ability of Cap-independent, IRES-dependent gene regulation

Glucosylceramide is the primary molecule of glycosphingolipids, and its metabolic regulation is crucial for life. Defects in the catabolizing enzyme, glucocerebrosidase (GCase), cause a lysosomal storage disorder known as Gaucher disease. However, the genetic regulation of GCase has not been fully understood. Here we show the redefined structure of the GCase coding gene (GBA), and clarify the regulatory mechanisms of its transcription and translation. First, alternative uses of the two GBA gene promoters were identified in fibroblasts and HL60-derived macrophages. Intriguingly, both GBA transcripts and GCase activities were induced in macrophages but not in neutrophils. Second, we observed cap-independent translation occurs via unique internal ribosome entry site activities in first promoter-driven GBA transcripts. Third, the reciprocal expression was observed in GBA and miR22-3p versus GBAP1 transcripts before and after HL60-induced macrophage differentiation. Nevertheless, these findings clearly demonstrate novel cell-type-specific GBA gene expression regulatory mechanisms, providing new insights into GCase biology

Localization of Adenylate Kinase 4 in Mouse Tissues

Adenylate kinase (AK) is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. Of its isoforms, AK4 has a similar sequence and subcellular localization to that of AK3 in the mitochondrial matrix. However, unlike AK3, AK4 lacks the guanosine triphosphate: adenosine monophosphate phosphotransferase activity. To elucidate the physiological role of AK4, we explored the protein localization of AK4 in various mouse tissues by immunohistochemical analysis. AK4 protein was detected in the kidney, liver, brain, heart, stomach, intestine, and gonads but not in the lung and spleen. Interestingly, cell-type specific expression was evident in the brain, gastrointestinal tract, and gonads. In the cerebellum, AK4 was detected in granular cells but not in Purkinje cell bodies. In the gastrointestinal tract, AK4 was highly expressed in epithelia. In the ovary, AK4 was detected in oocytes and corpora lutea. In the testis, AK4 was detected in spermatocytes but not in spermatogonia. Our findings demonstrate that AK4 localizes uniquely in a cell-type and tissue-specific manner in mouse tissues