Histochemical and Cytochemical Studies on Processing Protease PACE4 (SPC4)

インスリンに代表されるペプチドホルモン, 神経ペプチドや増殖因子といった生理活性ペプチドや蛋白質 の大部分はまず，不活性な前駆体蛋白質として合成され，特異的酵素(プロセシング酵素)による前駆体蛋白 質中の塩基性アミノ酸対部位(LysArg，ArgArgのカルボキシ末端側)での限定切断を受けて生理活性を有す る成熟体となる.従って， この限定切断は生理活性ペプチド・蛋白質生合成において必須の過程であり，そ のようなプロセシングに関与する酵素の生理機能の解明は生物学的に極めて重要である. 1984年に酵母のプロセシング酵素であるKexin(EC3.4.21.61)が同定されて以後，哺乳類においても Kexinの相同体であるfurin，PC2，PC1/PC3，PACE4，PC4，PC5/PC6及びLPC/PC7/PC8からなる一群 のKexinファミリープロテアーゼが同定されている.これらのKexinファミリープロテアーゼはCa²⁺依存性の セリンプロテアーゼで，サチライシンに類似の触媒領域を持つことから順にサチライシン様プロ蛋白質変換 酵素(Subtilisin-like Proprotein Convertase; SPC) SPC1・SPC7とも呼ばれる. 本論文はファミリーの1つPACE4(SPC4)の生理機能の解明を目的として，その組織・細胞特異的発現を 主として組織化学的手法により詳細に解析した研究成果である. PACE4は1991年にKieferらにより同定さ れたが，その後Tsujiら(1994)及びMoriら(1997)により複数のアイソフォームの存在が明らかにされた.個々 のPACE4アイソフォームの生理機能を解明するためにはまずアイソフォーム各々の組織・細胞特異的発現を 詳細に解析することが必要不可欠であるにもかかわらず.これまで全く明らかにされていなかった.本研究 では特に，プロセシング活性を有し，C末端構造の違いから生体内で異なる機能を果たすと考えられる2つ のアイソフォーム， PACE4AとPACE4Eの中枢神経系における発現を，各々に特異的なジゴキシゲニン標識 化アンチセンスcRNAプローブを用いたin situハイブリダイゼーション法により，成獣ラット脳及びその発 生段階において詳細に解析し，アイソフォーム個々の発現部位をはじめて特異的に検出した. 成獣ラット脳では，2つのアイソフォームは広範囲に発現し，その発現は非常に類似していた.また，神 経ペプチドの豊富な領域・細胞など特定の部位で特に強く発現しており，PACE4EとPACE4Aの一方または 両方の神経ぺプチド生合成への関与が示唆された. 更に，種々の発生段階においてもPACE4EとPACE4Aは よく似た発現パターンを示し，発生過程が進み脳の層構造が形成されるにつれ明らかに特定の領域・細胞に 局在化が認められた.このことはPACE4EとPACE4Aが増殖因子，神経栄養因子あるいは細胞接着分子など のプロセシングを介して中枢神経系の形成に深く関与していると考えられる.実際にPACE4アイソフォーム がどの様な生理活性ぺプチド・蛋白質と共存しているかも全く不明であったが，本研究において初めてニュ ーロペプチドY，副腎皮質刺激ホルモンや接着分子L1などとの共存が明らかになった.以上より，PACE4ア イソフォームが種々の生理活性ぺプチド・蛋白質の活性化により中枢神経系の構造と機能の形成・維持に重 要な役割を果たしていることが示唆され，PACE4の生理機能の解明において極めて重要な知見が得られた

ダエキセン ノ コウゾウ ト キノウ

The salivary gland is developed under the epithelial-mesenchymal interaction and formed by repeating branching morphogenesis, which is a common process in the development of the glandular tissues. Although the differentiation and maturation of salivary gland is continued into early postnatal life, the fundamental ability of saliva secretion is already expressed after birth. These development, differentiation, and maturation of salivary gland are regulated by many growth/ differentiation factors, which are initially synthesized as inactive precursors and activated by the limited proteolysis. On the other hand, saliva secretion is one of the important physiological functions of the salivary gland and occurs dependently on the increase of the osmolality in the lumen through two pathways, paracellular and transcellular pathways. It is revealed that a water channel aquaporin 5 (AQP5) is involved in saliva secretion through the latter pathway. Saliva contains various components, which express various physiological functions of saliva. Because the oral cavity is confronted with hazards of various allergens from the outside continuously, the salivary gland produces and secretes various factors as the defense system. Saliva is, therefore, one of an important body fluid to maintain the oral health, and the decrease of saliva secretion causes xerostomia/dry mouth, which affects not only oral disease and dysfunction but also systemic disease. We previously reported the involvement of a subtilisin-like proprotein convertase PACE4 in the development and differentiation of salivary gland, lipopolysaccharide-mediated induction of inflammatory cytokines in the salivary gland, degradation of salivary AQP5 by parasympathetic denervation, and so on. This review will describe the structure and function of the salivary gland, from its development to functional expression and regulation

Dynamics of Salivary Gland AQP5 under Normal and Pathologic Conditions

Aquaporin 5 (AQP5) plays an important role in the salivary gland function. The mRNA and protein for AQP5 are expressed in the acini from embryonic days E13-16 and E17-18, respectively and for entire postnatal days. Ligation-reopening of main excretory duct induces changes in the AQP5 level which would give an insight for mechanism of regeneration/self-duplication of acinar cells. The AQP5 level in the submandibular gland (SMG) decreases by chorda tympani denervation (CTD) via activation autophagosome, suggesting that its level in the SMG under normal condition is maintained by parasympathetic nerve. Isoproterenol (IPR), a β-adrenergic agonist, raised the levels of membrane AQP5 protein and its mRNA in the parotid gland (PG), suggesting coupling of the AQP5 dynamic and amylase secretion-restoration cycle. In the PG, lipopolysaccharide (LPS) is shown to activate mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalings and potentially downregulate AQP5 expression via cross coupling of activator protein-1 (AP-1) and NF-κB. In most species, Ser-156 and Thr-259 of AQP5 are experimentally phosphorylated, which is enhanced by cAMP analogues and forskolin. cAMP-dependent phosphorylation of AQP5 does not seem to be markedly involved in regulation of its intracellular traffcking but seems to play a role in its constitutive expression and lateral diffusion in the cell membrane. Additionally, Ser-156 phosphorylation may be important for cancer development

Relationships between feeding behaviors and emotions : an electroencephalogram (EEG) frequency analysis study

Feeding behaviors may be easily affected by emotions, both being based on brain activity; however, the relationships between them have not been explicitly defined. In this study, we investigated how emotional environments modulate subjective feelings, brain activity, and feeding behaviors. Electroencephalogram (EEG) recordings were obtained from healthy participants in conditions of virtual comfortable space (CS) and uncomfortable space (UCS) while eating chocolate, and the times required for eating it were measured. We found that the more participants tended to feel comfortable under the CS, the more it took time to eat in the UCS. However, the EEG emergence patterns in the two virtual spaces varied across the individuals. Upon focusing on the theta and low-beta bands, the strength of the mental condition and eating times were found to be guided by these frequency bands. The results determined that the theta and low-beta bands are likely important and relevant waves for feeding behaviors under emotional circumstances, following alterations in mental conditions

Biological interaction of alginetin

Alginetin is the major product formed from pentoses and hexurionic acids. Alginetin is producted by cooking process of food including pection, a naturally-occurring polysacharride found in many plants. However, the biological interaction and toxicity of alginetin are not known at all. The aim of the present study was to investigate the cellular actions of alginetin on rat thymic lymphocytes. The effects of alginetin on the cell were examined using flow cytometry with fluorescent probes. Alginetin increased cellular content of non-protein thiols ([NPT]i) and elevated intracellular Zn2+ levels ([Zn2+]i). Chelation of intracellular Zn2+ reduced the effect of alginetin on [NPT]i, and chelation of external Zn2+ almost completely diminished alginetin-induced elevation of [Zn2+]i, indicating that alginetin treatment increased Zn2+ influx. Increased [NPT]i and [Zn2+]i levels in response to alginetin were positively correlated. Alginetin protected cells against oxidative stress induced by hydrogen peroxide and Ca2+ overload by calcium ionophore. It is considered that the increases in [NPT]i and [Zn2+]i are responsible for the cytoprotective activity of alginetin because NPT attenuates oxidative stress and Zn2+ competes with Ca2+. Alginetin may be produced during manufacturing of jam, which may provide additional health benefits of jam

Citrus sudachi Peel Extract Suppresses Cell Proliferation and Promotes the Differentiation of Keratinocytes through Inhibition of the EGFR–ERK Signaling Pathway

Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma

Salivary gland development : its mediation by a subtilisinlike proprotein convertase, PACE4

The submandibular gland (SMG) develops under the epithelial-mesenchymal interaction. Its process is regulated by various growth/differentiation factors, which are synthesized as inactive precursors and activated via the limited proteolysis at their multi basic amino acid site(s) such as Arg-X-Lys/Arg-Arg. Although many of these processing steps are elucidated to be catalyzed by subtilisin-like proprotein convertases (SPCs), little is known about the role of SPCs in the SMG development. Here, we focused upon the physiological role of PACE4 (SPC4), a member of SPC family, in the SMG development. In the organ culture system of rat embryonic SMG (E15), Dec-RVKR-CMK, a potent inhibitor for SPCs, inhibited the salivary branching and the expression of an exocrine gland type water channel, AQP5. However, other peptidyl-CMKs and inhibitors for trypsin-like serine proteases including leupeptin did not affect the salivary branching and AQP5 expression. Dec-RVKR-CMK also suppressed the expression of PACE4, but not furin, another member of the family. The specific antibody for the catalytic domain of PACE4 suppressed the salivary branching and AQP5 expression similarly. These inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2 whose precursor is a candidate for the physiological substrates of PACE4. Further, the transcriptional silencing of PACE4 by its specific siRNAs caused the suppression of both the salivary branching and AQP5 expression in the present organ culture system. These observations strongly support the idea that PACE4 mediates the SMG development

Down-regulation of submandibular gland AQP5 following parasympathetic denervation in rats

Following chorda tympani denervation (CTD, parasympathetomy), the protein levels of aquaporin5 (AQP5) as well as AQP1 and Na+K+ATPase α-subunit in the rat submandibular gland (SMG) were found to be decreased significantly. However, the level of another membrane protein, dipeptidyl peptidase IV was not affected by CTD, suggesting a selective reduction of AQP5, AQP1, and Na+K+ATPase1α-subunit proteins by CTD. However, the AQP5 mRNA level was scarcely affected by CTD, which suggested that transcription process of AQP5 was unaffected by this operation. AQP5 protein was shown to be degraded in vitro by the extract of the SMG obtained from normal rat ; inhibitor experiments in vitro suggested cathepsin B was a responsible enzyme. Co-localization of AQP5 and LAMP-2, a lysosomal marker, implicated AQP5 is degraded in lysosomes. A significant increase in the protein levels of LC3-II, an autophagy marker, at day 1 after CTD, and co-localization of the LC3 protein and AQP5, suggested that CTD activated autophagy of SMG, leading to AQP5 degradation

Involvement of the IL-6/STAT3/Sca-1 system in proliferation of duct cells following duct ligation in the submandibular gland of mice

Ligation of the main excretory duct (MED) of the mouse submandibular gland (SMG) induced the expression of Sca-1, a stem cell marker. Sca-1 expression increased prominently in almost all of cells in the duct system, except the acinar cells. Sca-1 induction was accompanied with phosphorylated-STAT3 (Y705) elevation, which was localized in the nuclei of all duct cells. Electrophoretic mobility shift assay (EMSA) confirmed the specific binding of STAT3 to the GAS sequence, a biding site of gamma interferon activating site. Present study suggested one of the initial steps of the tissue regeneration after injury includes STAT3 pathway

Trafficking of GFP-AQP5 chimeric proteins conferred with unphosphorylated amino acids at their PKA-target motif (152SRRTS) in MDCK-II cells

Three constructs having mutated PKA-target motif at 152SRRTS of AQP5, an exocrine type water channel, were prepared and fused to C-terminus of green fluorescence protein cDNA to examine the effects of blocking of phosphorylation at 152SRRTS (a consensus PKA-target motif of AQP5) on translocation or trafficking of the chimeric proteins expressed in the Madin-Darby canine kidney-II (MDCK-II) cells. H-89 treatment increased translocation of wild-type GFP-AQP5 to the apical membrane. All 3 mutant molecules translocated 1.5 to 2 times more than the control wild-type GFP-AQP5. Colchicine but not cytochalasin B inhibited the translocation of wild-type GFP-AQP5. Present results suggest dephosphorylation of this consensus sequence increase GFP-AQP5 translocation, and that microtubules but not microfilaments are involved in this event