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Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2 cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment with trichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(-2018)-luc, that contains nt -2018 through nt -35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited for studies examining the transcriptional regulation of hepcidin.

We previously reported the presence of brown/beige adipocytes in the white fat depots of mature cattle. The present study examined the effects of dietary vitamin A on the expression of brown/beige adipocyte-related genes in the white fat depots of fattening cattle. No significant differences were observed in the expression of Ucp1 between vitamin A-deficient cattle and control cattle. However, the expression of the other brown/beige adipocyte-related genes was slightly higher in the mesenteric fat depots of vitamin A-deficient cattle. The present results suggest that a vitamin A deficiency does not markedly affect the expression of Ucp1 in white fat depots, but imply that it may stimulate the emergence of beige adipocytes in the mesenteric fat depots of fattening cattle.

Activin B is induced in response to inflammation in the liver and enhances hepcidin expression, but the source of activin B and the molecular mechanism underlying hepcidin induction are not clear yet. Lipopolysaccharide (LPS)-induced inflammation induced inhibin βB but not inhibin α or inhibin βA expression in the liver, implicating activin B induction. Immunoreactive inhibin βB was detected in endothelial cells and Kupffer cells in LPS-treated liver. Activin B, but not activin A or activin AB, directly increased hepcidin expression. Activin B induced phosphorylation and activation of Smad1/5/8, the BMP-regulated (BR)-Smads. The stimulation of hepcidin transcription by activin B was mediated by ALK2 and ActRIIA, receptors for the TGF-β family. Unexpectedly, activin B-induced hepcidin expression and BR-Smad phosphorylation were resistant to the effects of LDN-193189, an ALK2/3/6 inhibitor. ALK2 and ActRIIA complex formation in response to activin B may prevent the approach of LDN-193189 to ALK2 to inhibit its activity. Activin B also induced phosphorylation of Smad2/3, the TGF-β/activin-regulated (AR)-Smad, and increased expression of connective tissue growth factor, a gene related to liver fibrogenesis, through ALK4 and ActRIIA/B. Activin B-induced activation of the BR-Smad pathway was also detected in non-liver-derived cells. The present study reveals the broad signaling of activin B, which is induced in non-parenchymal cells in response to hepatic inflammation, in hepatocytes.

We previously revealed that endogenous bone morphogenetic protein (Bmp) activity is required for lipid accumulation in 3T3-L1 adipocytes. The present study characterized the role of endogenous Bmp activity in preadipocytes. Endogenous Bmp activity was monitored by analyzing the level of phosphorylation of Smad1/5/8, downstream molecules in the Bmp pathway. Higher levels of phosphorylated Smad1/5/8 were detected in adipogenic cells but not in non-adipogenic cells prior to differentiation induction. The inhibition of the Bmp pathway during this period decreased the expression of Pparγ2 and C/ebpα, which are transcription factors responsible for adipocyte differentiation. The expression of these transcription factors were also down-regulated by Bmp4 knockdown. In addition, endogenous Bmp4 was required for the repression of Intrleukin-11 expression. Endogenous Bmp4 in preadipocytes is indispensable for the onset of the adipogenic program, and may help to maintain the preadipocytic state during adipocyte differentiation.

Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle fibre types. As a first step in using diet to manipulate the proportion of muscle fibre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (−2553 to +78 bp) was prepared. A screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examined the effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription. Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis of myosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle fibre types. Copyright © 2013 John Wiley & Sons, Ltd.

There are two types of brown adipocytes: classical brown adipocytes that form the brown fat depots and beige adipocytes that emerge in the white fat depots. Beige adipocytes have a low level of uncoupling protein 1 (Ucp1) expression in the basal state, but Ucp1 expression is increased in response to β adrenergic receptor activation. The present study explored the factors responsible for the differentiation of 3T3-L1 white preadipocytes to beige adipocytes. Significant expression of Ucp1 was not detected under any tested conditions in the absence of isoproterenol (Iso), an agonist of β adrenergic receptor. Iso-induced Ucp1 expression was significantly higher in the cells treated with a mixture of triiodothyronine (T3) and 3-isobutyl-1-methylxanthine (IBMX) for days 0–8 than in the control cells. Chronic IBMX treatment was indispensable for the enhanced Iso-induced Ucp1 expression, and treatment with additional rosiglitazone (Rosi) for days 0–8 further increased the Ucp1 expression. Recently, genes were identified that are predominantly expressed in beige adipocytes, which were induced from stromal vascular cells in white fat depots. However, the expression levels of the beige adipocyte-selective genes in the adipocytes induced by the mixture of T3, IBMX and Rosi did not differ from those in the control adipocytes. The present study indicates that 3T3-L1 cells can differentiate to beige-like adipocytes by prolonged treatment with the mixture of T3, IBMX and Rosi and that the gene expression profile of the adipocytes is distinct from those previously induced from white fat depots.

Brown adipocytes, which regulate non-shivering thermogenesis, have been believed to exist in a limited number of mammalian species, and only under limited physiological conditions. Recent discoveries indicate that adult humans possess a significant number of functional brown adipocytes. This study explores the regulatory emergence of brown adipocytes in white adipose tissue (WAT) depots of fattening cattle. RT-PCR analyses indicated significant expression of Ucp1, a brown adipocyte-specific gene, in the WAT of 31-month-old Japanese Black steers. Immunohistochemical analysis revealed that Ucp1-positive small adipocytes were dispersed in the subcutaneous WAT. Next, we examined expression level of Ucp1 and other brown adipocyte-selective genes such as Pgc1α, Cidea, Dio2, Cox1, Cox7a1 and Cox8b in WAT of 30-month-old steers fed either diet with low protein/energy content (roughage diet) or that with high protein/energy content (concentrate diet) for 20months. Ucp1 expression in the subcutaneous WAT was significantly higher in the concentrate diet group than in the roughage diet group. Furthermore, the higher Ucp1 expression levels were limited to the subcutaneous WAT, and no differences between groups were detected in the mesenteric, perirenal, intermuscular or intramuscular WAT. Expression of Dio2, Cox1 and Cox8b was higher in the subcutaneous WAT but not in the mesenteric WAT of the concentrate diet group. Furthermore, expression of Prdm16, a positive regulator of differentiation toward brown adipocyte-lineage cells, and expression of leptin, a molecule that enhances activity of brown adipocytes, were significantly higher in the subcutaneous WAT of the concentrate diet group. This study demonstrates the presence of brown adipocytes in WAT depots of fattening cattle, and suggests the diet-related modulation of expression of genes predominantly expressed in brown adipocytes.

Mast cells, multifunctional effector cells of the immune system, are implicated in the pathogenesis of hepatic steatosis and fibrosis. Magnesium (Mg) deficiency was reported to increase triglyceride concentration in the liver, and to exacerbate the collagen deposition induced by carbon tetrachloride in the liver. Although Mg deficiency increases mast cells in the small intestine, the kidney and bone marrow, the effect of Mg deficiency on mast cells has not been clarified in the liver. We examined the emergence of mast cells in the liver of Sprague-Dawley rats given an Mg-deficient diet. Rats were fed a control diet or an Mg-deficient diet for 4 wk. Mg deficiency increased the levels of mRNA known to be expressed by mast cells in the liver; the mRNA of α- and β-chain high-affinity immunoglobulin E receptor (FcεR1α, FcεR1β), and the mRNA of mast cell protease 1 (Mcpt1), and mast cell protease 2 (Mcpt2). Histological observation showed that some mast cells were locally distributed around portal triads in the Mg-deficient group but mast cells were scarcely found in the control group. These results clearly indicate that Mg deficiency induces the emergence of mast cells around portal triads of the liver in Sprague-Dawley rats.

Mg deficiency induces various metabolic disturbances including glucose metabolism in the liver. However, no comprehensive information is currently available on the metabolic pathways affected by Mg deficiency. The present study examined metabolite content in the liver of Mg-deficient rats using a metabolomic analysis. In this study, 4-week-old, male Sprague–Dawley rats were fed a control diet or a Mg-deficient diet for 8 weeks. The metabolomic analysis identified 105 metabolites in the liver, and significant differences were observed in the hepatic contents for thirty-three metabolites between the two groups. An analysis by MetaboAnalyst, a web-based metabolome data analysis tool, indicated that the Mg deficiency affected taurine/hypotaurine metabolism, methionine metabolism and glycine/serine/threonine metabolism; taurine, hypotaurine, glycine, serine and threonine contents were increased by Mg deficiency, whereas the amounts of 2-ketobutyric acid (a metabolite produced by the catabolism of cystathionine or threonine) and 5'-methylthioadenosine (a metabolite involved in spermidine synthesis) were decreased. The amount of glucose 6-phosphate, a hub metabolite of glycolysis/gluconeogenesis and the pentose phosphate pathway, was significantly decreased in Mg-deficient rats. Mg deficiency also decreased metabolite contents from the citric acid cycle, including citric acid, fumaric acid and malic acid. Aberrant metabolism may be related to the allosteric regulation of enzymes; the mRNA levels of enzymes were generally similar between the two groups. The present study suggests that the Mg deficiency-mediated modulation of hepatic metabolism is as yet uncharacterised.

We demonstrated that castration enhanced the expression of uncoupling protein 1 (Ucp1), a thermogenic protein, in brown adipose tissue (BAT) and subcutaneous (sc) white adipose tissue (WAT) in male mice. Castration of male mice increased body temperature and reduced body weight gain compared with those of sham-operated mice. BAT Ucp1 mRNA expression in castrated male mice was significantly higher than that in sham-operated mice. Histologically, cells with multilocular fat droplets were observed in the castrated inguinal scWAT. Immunohistochemical staining revealed that these cells positively reacted with the anti-Ucp1 antibody. The Ucp1-positive area near the inguinal lymph node in the castrated WAT was extensive compared with that of the sham-operated WAT. Castration-induced Ucp1 up-regulation in scWAT was suppressed by high-fat diet feeding. These findings suggest that thermogenesis by BAT activation and scWAT browning contribute to castration-induced inhibition of body weight gain. However, considering that the effect of castration was blunted by high-fat diet consumption, thermogenesis stimulation in response to castration is inhibited by chronic over-nutrition.

Brown adipocytes dissipate chemical energy in the form of heat through the expression of mitochondrial uncoupling protein 1 (Ucp1); Ucp1 expression is further upregulated by the stimulation of β-adrenergic receptors in brown adipocytes. An increase in energy expenditure by activated brown adipocytes potentially contributes to the prevention of or therapeutics for obesity. The present study examined the effects of milk by-products, buttermilk and butter oil, on brown adipogenesis and the function of brown adipocytes. The treatment with buttermilk modulated brown adipogenesis, depending on the product tested; during brown adipogenesis, buttermilk 1 inhibited the differentiation of HB2 brown preadipocytes. In contrast, buttermilk 3 and 5 increased the expression of Ucp1 in the absence of isoproterenol (Iso), a β-adrenergic receptor agonist, suggesting the stimulation of brown adipogenesis. In addition, the Iso-induced expression of Ucp1 was enhanced by buttermilk 2 and 3. The treatment with buttermilk did not affect the basal or induced expression of Ucp1 by Iso in HB2 brown adipocytes, except for buttermilk 5, which increased the basal expression of Ucp1. Conversely, butter oil did not significantly affect the expression of Ucp1, irrespective of the cell phase of HB2 cells, ie, treatment during brown adipogenesis or of brown adipocytes. The results of the present study indicate that buttermilk is a regulator of brown adipogenesis and suggest its usefulness as a potential food material for antiobesity.

Our previous report indicated that magnesium (Mg) deficiency increased molybdenum (Mo) concentration in the rat liver, suggesting the possibility that Mg deficiency affects Mo metabolism. Growing male rats were given a control diet or a Mg-deficient diet for 4 weeks. Urine and feces were collected during the second and fourth weeks of the feeding trial. The liver, kidney, spleen, skeletal muscle, and blood were collected at the end of the feeding trial. Mg deficiency did not affect the apparent absorption of Mo, but it reduced urinary excretion of Mo. The retention of Mo tended to be higher in the Mg-deficient group than in the control group. Hepatic Mo concentration was higher in the Mg-deficient group than in the control group, but Mg deficiency did not affect Mo concentration in other tissues and plasma. Mg deficiency downregulated the mRNA expression of Mo transporter 2 (MOT2) in the liver, but not in the kidney. These results suggest that Mg deficiency decreases urinary Mo excretion, which is too slight to affect plasma Mo concentration, and that Mg deficiency selectively disturbs the homeostatic mechanism of Mo in the liver, which is not related to the mRNA expression of MOT2 in the liver.

To date, minerals of interest have been analyzed individually to understand mineral dynamics and metabolism. Our recent development of metallomic analyses enabled us to evaluate minerals in an unbiased and global manner. Here, we evaluated the effects of ingestion of excess zinc to plasma and tissue concentrations of minerals in growing rats. A total of 26 minerals were simultaneously evaluated by metallomic analyses using inductively coupled plasma-mass spectrometry (ICP-MS) in semi-quantification mode; the concentrations of several minerals exhibited consistent changes in response to the concentrations of dietary zinc. Manganese concentrations in plasma and femur increased, while concentrations in the liver and pancreas decreased with increasing dietary zinc concentrations. Because the interaction between zinc and manganese is not known, we further focused our analysis on liver manganese. Quantitative analyses also indicated that the hepatic concentration of manganese decreased in response to the ingestion of diets containing excess zinc, a result that is partly explained by the decreased expression of hepatic Zip8, a manganese transporter. The present study reveals mineral interaction by using metallomic analyses and proposes a possible mechanism that underlies this novel interaction.

The growth of weaning piglets is effectively improved by feeding a high-Zn diet (3000 mg Zn/kg of diet). The present study examined whether feeding a diet supplemented with Zn (1016–3000 mg/kg) for 10 d induces growth benefits in rats. In addition, tissue weight, Zn content of tissues and expression of Zn transporters were examined in these rats. Zn supplementation did not significantly increase body weight. Breaking line model analyses indicated that the weight of the pancreas, the organ most sensitive to excess Zn, significantly decreased with increasing Zn intake beyond 15·2 mg/d. Excess Zn has been suggested to accumulate in the liver, kidney and bone in order to protect the pancreas. Zn concentrations in the plasma, liver, kidney and femur increased with increasing Zn intake up to approximately 30 mg/d, whereas those in the pancreas increased up to 8·4 mg/d and decreased by Zn intake beyond 8·4 mg/d. The expression levels of the Zn transporters Zip4 and ZnT1 in the intestinal epithelium were significantly lower in rats fed a diet supplemented with 1016 mg/kg Zn compared to those fed the basal diet. The present study reveals that (1) excess Zn intake does not accelerate growth in rats, but is detrimental to the pancreas, (2) the excess Zn is effectively accumulated in the liver, kidney and bone, without sufficient protection of the pancreas and (3) expression of Zn transporters is down-regulated in response to excess Zn intake.

Magnesium (Mg) deficiency is well known to affect metabolism of some trace minerals. We investigated the effect of Mg deficiency on hepatic concentration of various minerals in rats. Twelve 5-week-old male rats were divided into the groups given a control diet and an Mg-deficient diet. After 4 weeks, liver sample was collected from each rat. The concentrations of 36 minerals were simultaneously determined by a semiquantitative method of inductively coupled plasma-mass spectrometry (ICP-MS). The semiquantitative analysis showed that Mg deficiency significantly increased iron (Fe), copper (Cu), zinc (Zn), gallium (Ga), yttrium (Y), zirconium (Zr), molybdenum (Mo), rhodium (Rh), silver (Ag), and barium (Ba) concentrations, and significantly decreased scandium (Sc) and niobium (Nb) concentrations in rat liver. Then, hepatic Fe, Cu, Zn, Sc, Zr, and Mo concentrations were quantitatively measured, which indicated the similar effects as observed by the semiquantitative analysis. Additionally, the semiquantitative measurements of these minerals were highly correlated to these measurements with the quantitative method, but the measurements were not completely consistent between these analyses. The present study is the first research indicating the changes of hepatic Ga, Y, Zr, Mo, Rh, Ag, Ba, Sc, and Nb concentrations in Mg-deficient rats. The present study also indicates that the semiquantitative analysis with ICP-MS is a valid method for screening analysis to investigate various mineral concentrations in animal tissues.

Magnesium (Mg) deficiency induces the production of free radicals, increases cytosolic ionized calcium concentration, and modulates the function of skeletal muscle in rats. The present study examined the effects of Mg deficiency on the gene expression of molecules related to myogenesis in the gastrocnemius muscle as well as in C2C12 myogenic cells. Ingestion of an Mg-deficient diet resulted in a lower weight of the gastrocnemius muscle and higher concentration of muscular TBARSs, an index of oxidative stress. Mg deficiency also enhanced the expression of Myod and myogenin. In vivo effects of Mg deficiency on myogenic gene expression were partially reproduced in in vitro C2C12 cells; expression of Myod was up-regulated by a mixed culture of myoblasts and myotubes with Mg-deficient medium, which related to the simultaneous up-regulation of Myhc IIb, a myotube-specific protein. The culture with Mg-deficient medium did not increase the gene transcript level of HO-1, another marker of oxidative stress, suggesting that Mg deficiency-induced Myod expression does not result from oxidative stress. Furthermore, oxidative stress induced by hydrogen peroxide did not increase Myod expression, whereas the expression of Myod, myogenin and Myhc IIb was decreased by oxidative stress from the initial phase of differentiation. The effects of Mg deficiency depended on the stages of myogenesis; myoblast culture in Mg-deficient differentiation medium did not affect the expression of Myod and Myhc IIb. The present study revealed stage-dependent effects of Mg deficiency on myogenesis.

We determined the true absorption and endogenous fecal loss of zinc (Zn) in goats using its stable isotope. Three goats were fed with the diet containing 50 mg/kg Zn twice a day for 17 days. In the morning of day 11, the goats were given a meal labeled by (67) Zn as the tracer with dysprosium as the unabsorbed marker. Then the goats were given unlabeled diet as the rest of the morning feed. We measured dietary and fecal Zn concentration, (67) Zn abundance and dysprosium concentration in feces. The excretion pattern of the tracer Zn into feces differed from that of dysprosium. Therefore, we directly calculated the true absorption of Zn from Zn concentration and (67) Zn abundance in fecal samples collected after the labeled diet was given. The apparent absorption of Zn was -0.009 ± 0.016 mg/kg bodyweight (fractional absorption, -1.07 ± 1.85%). The true absorption of Zn was 0.162 ± 0.018 mg/kg bodyweight (fractional absorption, 18.25 ± 2.01%). The endogenous fecal loss of Zn was 0.172 ± 0.004 mg/kg bodyweight and the intestinal secretion of Zn was 0.210 ± 0.009 mg/kg bodyweight. The present experiment indicates that stable isotopic Zn is a powerful tool for examining Zn metabolism in ruminants.

The whole genome sequence of a Boxer dog suggested that the amino acid sequence of the carboxyl terminus of a putative Smad3 is SSVF-(COOH), not SSVS-(COOH) as in all Smad3 sequences identified in many species. Because phosphorylation of the last two serines at the carboxyl terminus is generally indispensable for Smad3-mediated signaling, the role of Smad3 may be unique in dogs. The present study determines the nucleotide sequence of the coding region of canine Smad3 and deduces the carboxyl terminal amino acids of Smad3 in several breeds. Except for the Boxer, the deduced amino acid sequence was SSVS-(COOH) in all dogs examined. In addition, the nucleotide at position 1204 in the Boxer was different from that of the other dogs. Furthermore, there was a SNP at nt 240. The present study indicates that the carboxyl terminal amino acid of canine Smad3 is not unique, although it is unknown in the Boxer breed.

Microbes such as yeast and Aspergillus are known to produce phytase, and Aspergillus phytase has been used as a feed additive for improving phytate-phosphorus bioavailability in monogastric animals. We measured phytase activity in some by-products from fermented food and beverage productions by yeast and Aspergillus. The phytase activity was as high as 3577 and 2225 PU/kg DM in raw and dried brewer's yeasts, respectively. On the other hand, the phytase activity was approximately 400 PU/kg DM in white-wine yeast and red-wine yeast. The phytase activity was further low in natto (fermented soybean) residue, soy sauce cake, rice brewer's grain and the activity was not detected in dried corn-barley distiller's grain with soluble and sweet-potato distiller's residue. The stability of phytase against pepsin was much lower in the brewer's yeast than in an Aspergillus phytase preparation. On the other hand, the addition of raw brewer's yeast effectively degraded phytate phosphorus in a corn-soybean meal diet during soaking. These results suggest that phytase in the examined by-products is not suitable for the phytase source of conventional diets, but that the soaking treatment with a raw brewer's yeast is an alternative method for improving phytate-phosphorus bioavailability in corn-soybean meal diets for pigs.

The CRNP5 variant of Borna disease virus (BDV) has stronger pathogenesis than the CRP3 variant in which only 4 nucleotides in the whole genome are different. The CRP3 is produced by 3 passages in rat brains of BDV, whereas the CRNP5 is produced by 5 passages in mouse brains after 2 passages in rat brains of the BDV. Thymidylic acids at nt 3608 and 3673 were replaced by cytidylic acids during 3 passages in mice. Three passages in mice caused replacement of adenylic acid at nt 7936 by guanylic acid. No replacement at nt 8742 occurred during passages in mice.

In current models of transforming growth factor-beta (TGF-beta) family signaling, type II receptors activate specific activin receptor-like kinase (ALK) type I receptors. These serine/threonine kinases activate ligand-dependent receptor regulated (R)-Smad by phosphorylating carboxy-terminal serines. We found that the receptor expression levels affected the phosphorylation and activation of the two R-Smad subclasses, activin/TGF-beta-specific (AR-Smad) and bone morphogenetic protein (BMP)-specific (BR-Smad). Co-expressing constitutively active type I and type II receptors in COS7 cells resulted in the phosphorylation of both R-Smad subclasses in a ligand-independent manner. This was verified using in vitro kinase assays. In untransfected B16 melanoma cells, TGF-beta1 and BMP-2 induced phosphorylation of both R-Smad subclasses, and TGF-beta1 up-regulated the inhibitor of differentiation (Id) gene, which is usually regulated by BMP. By contrast, BMP-2 up-regulated plasminogen activator inhibitor-1 (PAI-1), which is an AR-Smad-regulated gene. Except for ALK4 and ALK6, levels of type I and type II receptor mRNAs were higher in B16 cells than in HeLa and HepG2 cells, in which TGF-beta1 and BMP-2 induced phosphorylation of only the expected R-Smad. These results help to explain the diverse effects of this ligand family.

TGF-beta induces epithelial-mesenchymal transition, which occurs during tumor cell invasiveness in pathological state, in limited cells. As a first step to understand the role of TGF-beta and the structurally related activin during melanoma metastasis, expression of metastasis-related genes was examined in murine melanoma cells. Treatment with TGF-beta1 or activin A down-regulated E-cadherin in B16 cells in a dose-dependent manner. In epithelial cells, TGF-beta-induced high mobility group A2 (HMGA2) gene product is suggested to down-regulate E-cadherin through up-regulation of zinc-finger transcription factors Slug and Snail, and basic helix-loop-helix transcription factor Twist. Unlike the regulation in epithelial cells, TGF-beta1 treatment rather decreased mRNA expression of HMGA2, indicating a distinct mechanism on TGF-beta/activin-induced down-regulation. Transfection of double-stranded interfering RNA (dsRNAi) for activin receptor-like kinase (ALK) type I receptors revealed that ALK5, a prototype of TGF-beta receptor, mainly transmits TGF-beta signals on the E-cadherin down-regulation at the mRNA level, and that a prototype receptor ALK4 elicited the activin effect. TGF-beta/activin potentiated down-regulation of E-cadherin and HMGA2 also in B16 sublines that are susceptible to metastasis. However, the extent of down-regulation tended to be smaller, and less Smad2, a signal mediator for TGF-beta/activin, was phosphorylated in response to the ligand, resulting from less expression of type I receptors in the B16 sublines. These results suggest that the receptor expression level determines strength of the signals for TGF-beta/activin through phosphorylation of Smad2, which explains pluripotency of the ligand family partly.

マスト細胞は,自然免疫および適応免疫において様々なメディーターを放出することにより細菌感染免疫応答関与している。PCB126のマスト細胞免疫応答に対する影響を調べるため,PCB126前処理マスト細胞におけるLPS刺激によるサイトカイン誘導能とその関連分子について検討した。LPS刺激によりマスト細胞は前炎症性サイトカイン,ケモカインの遺伝子および蛋白発現が上昇するが,PCB126で前処理されたマスト細胞ではその発現が阻害された。cq1はPCB126特異的にマスト細胞内で強く誘導された。cq1蛋白の作用綴字がNFkBを利用することから,PCB126によるLPS刺激後のマスト細胞サイトカイン産生抑制はNFk-Bにおける競合による可能性が示唆された。, Mast cell plays central regulators of innate and acquired immunities through production of various chemical mediators and cytokines. It has been mentioned that Co-PCB influence host immune systems, however the mechanism remains unclear. We have previously reported that LPS stimulates mast cells to produce various cytokines. In the present study, we have studied the effect of PCS 126 on LPS-mediated cytokine and chemokine production from mast cell. Administration of PCB126 reduced production of pro-inflammatory cytokines and chemokines. Interestingly, PCB126 specifically enhanced Cql mRNA expression which is regulated by NFkB signaling pathway. Pro-inflammatory cytokine and clq induction are mediated by the same NFkB signaling pathway, therefore the reduction of LPS-mediated cytokine production from mast cell pretreated with PCB126 might be competed NFkB signaling pathway.

TGF-βは,細胞の分化・増殖を調節する分泌性蛋白質である。TGF-βはII型受容体に結合した後,I型受容体(Alk5)と会合し,情報を細胞内に伝達する。ラット血管平滑筋細胞では,細胞外領域をコードする12塩基の選択的スプライシングの結果生じる,2つのAlk5 isoform-short form: Alk5-Sおよびlong form: Alk5-L-の存在が知られているものの不明な点が多い。本研究は,マウス組織ならびに各種細胞株におけるAlk5 isoformの遺伝子発現ならびにAlk5 isoformを介した情報伝達を検討した。マウスの臓器(肝臓,腎臓,脾臓,心臓,肺,脳,精巣,筋肉)におけるAlk5 isoformの発現をRT-PCRならびにPAGEにより比較したところ,Alk5-SとAlk5-Lは調べた臓器全てにおいて発現が見られ,発現比は臓器間で違いはなかった。また,ES細胞様細胞(P19),色素細胞(B16),マスト細胞(P-815, BMMC)とマクロファージ(Raw264)においてもAlk5 isoformの発現が見られ,発現比に違いはなかった。次に, Alk5 isoform間での転写活性能を比較した。Alk5を介して情報伝達を行うリガンドとして,TGF-βだけでなく,GDF-8も知られている。そこで,TGF-β_1ならびにGDF-8の情報伝達に関して,機能的なAlk5を欠損しているL17細胞を用いて検討した。Alk5の活性化によりFoxH2依存的に転写が促進されるAR3-lucをreporterとしてluciferase-based reporter assayを行ったところ,いずれのAlk5 isoformを発現させた場合でも,TGF-β_1とGDF-8はともに用量依存的にAR3-lucの転写を促進した。Alk5発現時の各リガンドに対するEC_濃度は,TGF-β_1をリガンドとした場合もGDF-8をリガンドとした場合もAlk5 isoform間で違いはなかった。以上のことから,Alk5の2つのアイソフォームはマウスの様々な臓器および細胞で発現しており,今回調べた活性に関して,これらの両方が同等にTGF-β/GDF-8の受容体として機能していると考えられた。, Mouse TGF-β type I receptor, Alk5, encodes two types of mRNA generated by alternative splicing of four amino acids in the extracellular region. In the present study, the expression and function of alternative splice variants of Alk5, i.e., Alk5-L (Alk5 with the four amino acids) and Alk5-S (Alk5 without the four amino acids), were examined. Expression of Alk5 was detected in all examined tissues, and no significant differences in the ratio of Alk5-S to Alk5-L were detected between tissues. Expression of Alk5 was also detected in various cells, with Alk5-L being especially abundant in A-6 ES cells. No clear difference in Smad2 phosphorylation in response to treatment with TGF-β_1 was detected between L17 cells expressing Alk5-S and those expressing Alk5-L. Consistent with these results, transcription mediated by Alk5-S in L17 cells treated with TGF-β_1 was comparable to that mediated by Alk5-L. In addition, alternative splicing had no effect on transcriptional activation induced by GDF-8, a member of the TGF-β family. The present results indicate ubiquitous expression of Alk5 isoforms in mouse tissues and cells, and insignificant effects of alternative splicing on signaling induced by TGF-β_1 and GDF-8.

Factors affecting struvite, a magnesium-ammonium-phosphate complex (MgNH(4)PO(4).6H(2)O), in feline urine were evaluated. Incubation of just "urine mineral (UM)" solution, in which mineral concentrations are compatible with those in feline urine, for 4 h at 37 degrees C did not induce the formation of crystals. Similarly, incubation of urine alone did not produce crystals. However, struvite crystals were formed by the addition of urine to UM solution. Mg, NH(3) and P were all required for urine-induced struvite crystallization. The lower molecular weight (LMW) fraction of urine was essential for struvite crystal formation, and the higher molecular weight (HMW) fraction enhanced formation of LMW-induced struvite crystals. The effects of urine proteins further fractionated by column chromatography were examined. A protein at >250 kDa and cauxin, a major urine protein recently identified as a regulator of felinine production, potentiated struvite crystal formation induced by the LMW fraction. In contrast, Tamm-Horsfall glycoprotein, a urine protein thought to promote struvite crystallization, did not have this activity. The present study reveals a novel mechanism of feline struvite crystallization.

In this study, we have characterized the negative regulation of the IL-12 p40 expression by TGF-beta in macrophages. Although murine IL-12 p40 promoter contains a putative TGF-beta inhibitory element (TIE), neither mutation nor deletion of the TIE had any effect on the inhibitory activity of TGF-beta. The NF-kappaB p65 and interferon regulatory factor (IRF)-1 induced promoter activity was suppressed by the expression of a constitutively active TGF-beta type I receptor in the presence of Smad3 and Smad4, which was abrogated by expression of an inhibitory Smad, Smad7. Transcription of a reporter gene containing three copies of both NF-kappaB and IRF-1 elements from the IL-12 p40 promoter was significantly repressed by activation of Smad-dependent TGF-beta pathway. In contrast, reporter containing three copies of either the NF-kappaB or IRF-1 sites was not affected by TGF-beta-Smad pathway. These findings indicated that both the NF-kappaB and IRF-1 sites are required for the repression of promoter activity of IL-12 p40 by TGF-beta.

Our previous study has revealed that providing dry feeds increased the plasma concentration of antidiuretic hormone (ADH) in suckling calves, leading to altered water balance. To examine whether ketone bodies formed from ruminal fermentation-derived butyrate induced ADH secretion in suckling calves, the effects of intraruminal administration of butyrate on plasma concentration of ADH and ketone bodies, plasma and urine osmolality, and urine volume were examined. Six male Holstein calves aged 4 wk were used. Three levels of butyrate (0 g, 22 g and 44 g) were intraruminally administrated in a 3 × 3 Latin square design, and blood plasma and urine were analyzed. Plasma concentration of ketone bodies was increased by intraruminal administration of butyrate within 15 min in a dose-dependent manner, and the elevation of plasma levels continued until 4 h. Plasma concentration of ADH was also increased by the butyrate treatment, and it was higher in the 44 g butyrate group than in the 22 g butyrate group from 15 min to 2 h. The duration of the elevated plasma concentration of ADH was shorter than that of plasma concentration of ketone bodies. The relationship between plasma concentrations of ADH and those of ketone bodies was statistically significant, although the relationship was weaker. In accordance with the elevation of plasma ADH levels, the butyrate treatment resulted in the decreases in urine volume and increases in urine osmolality. Plasma osmolality was not different among the groups. The present results suggest that ruminal butyrate-derived ketone bodies are at least partly responsible for ADH secretion in suckling calves fed with dry feeds, and that the secreted ADH decreases urine volume through the increase in urine osmolality.

Microphthalmia-associated transcription factor (Mitf) is a tissue-specific transcription factor. At least nine distinct mouse isoform mRNAs are encoded by alternative splicing of the first exon of Mitf (Mitf-A, -B, -C, -D, -E, -H, -J, -M, and -mc), while exons 2-9 of all Mitf isoforms examined to date are identical. In addition, alternative splice variants of exon 6a encoding 6 amino acid proximal to the basic region of the protein are known in Mitf-A, -H, and -M. In this study, we identified alternative splice variants of exon 6a in other Mitf isoforms (Mitf-E, -J, and -mc) in melanocytes, mast cells, macrophages, and heart. We also compared the transcriptional activity of Mitf variants containing exon 6a to that of Mitf variants that did not contain exon 6a. PCR-RFLP analysis revealed that expression of Mitf with exon 6a was comparable with that of Mitf without exon 6a, irrespective of the specificity of the first exon, or cell type, although Mitf isoforms with different first exons were expressed in a cell type-dependent manner. Luciferase-based reporter assays revealed that transcription of Tyrosinase, which is known Mitf-regulated gene, was elicited more efficiently by expression of Mitf isoforms containing exon 6a, compared to isoforms that did not contain exon 6a. However, when transcription of Tyrp-1, Mmcp-6, and PAI-1 was examined, no significant differences were detected between Mitf isoforms with exon 6a and those without exon 6a, except for Tyrp-1 transcription by Mitf-D/E isoform. These results reveal a diverse pattern of gene expression and different transcriptional activities of Mitf isoforms, suggesting discrete regulation of gene transcription in specific tissues by Mitf.

ボルナ病ウイルス(borna disease virus;BDV)は,馬や羊に髄膜炎あるいは脳脊髄炎をおこし,神経疾患や時に致死的な経過を引き起こすボルナ病の原因ウイルスである。ところが,ラットにおいて高病原性であるCRNP5株は,免疫応答の未熟な新生仔ラットに感染させると脳炎は認められないものの重篤なボルナ病を発症する。従って,CRNP5株は免疫応答非依存的に感染ラットを発症させること,すなわち直接的/間接的な中枢神経障害がある可能性が示唆されていた。そこで,本研究では,ボルナ病発症における宿主の免疫応答の必要性について調べるために,T細胞機能欠如ラット(ヌードラット)における病原性を解析した。4週令のヌードラットにCRNP5株とCRP3株を感染したところ,CRNP5株感染群は全頭重篤なボルナ病を発症したが,CRP3株感染ラットは7週間の観察期間中に発症は認められなかった。CRNP5株感染ラットでは,海馬の神経細胞層が消失し,特に錐体細胞層における神経細胞変性が顕著だった。以上の結果から,CRP3株はT細胞性免疫応答介在性にボルナ病を発症すること,一方,CRNP5株は発症には宿主のT細胞性免疫は必要としていないことが強く示唆された。, Borna disease virus (BDV) causes classical Borna disease (BD), a fatal mononuclear inflammatory encephalomyelitis with severe signs of neurological disease in horses and sheep. Classical BD in adult rats is in large part due to immunopathogenic damage to the nervous system by blood-borne inflammatory cells. However, newborn rats inoculated with mouse-adapted BDV variant (CRNP5) develop severe neurological disease without encephalitis. Thus, it is suggested that rats infected with CRNP5 develop BD without immune response, and CRNP5 is directly and/or indirectly due to damage to the central nervous system. In order to gain a better understanding of immune responses contributions to BD outcomes, we studied the neuropathogenesis of BDV in athymic nude rats (rnu/rnu), naturally deficient in the T-lymphocyte system. Four-week old female nude rats were either inoculated intracranially with 2×10^3 FFU of rat-adapted prototype variant (CRP3) (n=7) or CRNP5 (n=7) or an equal volume of uninfected mouse brain material (n=7). All CRNP5-infected nude rats developed fatal neurological disease although all CRP3-infected rats survived to 7 week post inoculation without obvious neurological signs of disease. No evidence of inflammatory cell infiltration was observed in rats infected with CRP3 or CRNP5, but an intensive gliosis was present. The brains of CRNP5-infected rats showed severe neuronal degeneration in pyramidal neurons in hippocampus. These results suggest that the cellular immune response to virus infection contributes to BD outcome in CRP3-infected rats but CRNP5-infected rats.

TGF-βファミリーに属する分子群は,生命を維持する上で重要と考えられる生物現象の多くに関与しているが,現在知られている情報伝達経路は極めて単純である。したがって,多様な生物活性を惹起するためには,標的細胞の種類に関わらない普遍的な情報伝達機構と標的細胞特異的な情報伝達機構があるものと推測される。そこで,TGF-βファミリーが如何にして多機能を発揮するかを分子レベルで明らかにすることを目的として本研究を行った。本年度は,免疫担当細胞であるマスト細胞を用いて,TGF-βファミリーによる標的細胞特異的な情報伝達についての解析を試みた。マウスの骨髄より調製した前駆マスト細胞(BMMC)においてactivin A(4 nM)刺激2時間後に誘導される遺伝子群を網羅的に解析したところ,CRAL/TRIO並びにGOLDドメインを有するtocopherol-associated protein(Tap) がactivin Aによって誘導されることが明らかになった。activin A刺激によるTap発現の増加は,マスト細胞株であるMC/9においても認められたが,マクロファージ細胞株であるRAW264,メラノーマ細胞株であるB16や胚性腫瘍細胞株であるP19ではactivin A処理によってTapの増加は認められなかった。また,BMMCにおけるTap誘導は,TGF-β_1(200pM)刺激後にも認められたのに対して,BMP-2(4nM)刺激時には認められなかった。一方,activin/TGF-βのI型受容体キナーゼの阻害剤であるSB431542の前処理によってこの遺伝子誘導は減少したが,その程度は,activin/TGF-β誘導性の別の遺伝子群(mmcp-1とmmcp-7)のSB431542による抑制に比べて小さいものであった。Tapがactivin/TGF-β刺激によって誘導されることの意義を調べるため,activin/TGF-β responsive reporterを用いて解析したところ,Tap存在下によってactivin/TGF-β シグナルは増強された。Tap発現がactivin/TGF-βによって誘導されるのはマスト細胞に特異的であり,誘導されたTapはactivin/TGF-βシグナルの増強因子であると考えられた。, In order to gain better understanding on multifunction of pluripotent TGF-β family members at the molecular level, the genes that were up-regulated in response to treatment with members of the TGF-β family were investigated in mouse bone marrow-derived cultured mast cells (BMMC). The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4nM activin A, a member of the TGF-β family, for 2h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by competitive RT-PCR and real-time RT-PCR analyses. Treatment with TGF-β_1 at 200pM but not BMP-2 at 4nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC and MC/9 mast cells but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with > 1 μM SB431542, an inhibitor of activin and TGF-β type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-β_1, whereas < 0.5 μM SB431542 effectively reduced TGF-β_1-induced expression of mmcp-1 and mmcp-7. Induction of Tap was also detected in BMMC from Smad3-null mice, although the levels were lower than observed with wild-type mice. These results suggest that Tap is induced not only through an activated ALK4/5- and Smad3-mediated mechanism but also through an ALK4/5-independent mechanism. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-β pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-β occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-β signaling.

The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activation of brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator-activated receptor (Ppar) γ coactivator-1α (Pgc-1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown preadipocytes, brown adipocyte function, or both, through its significant absorption. We herein demonstrated that Trpv1, a capsaicin receptor, is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium influx in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte-selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc-1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1 induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis.

A previous study revealed that the expression of the Borna disease virus (BDV)-encoding phosphoprotein in glial cells was sufficient to induce neurobehavioral abnormalities resembling Borna disease. To evaluate the involvement of the TGF-β family in BDV-induced changes in cell responses by C6 glial cells, we examined the expression levels of the TGF-β family and effects of inhibiting the TGF-β family pathway in BDV-infected C6 (C6BV) cells. The expression of activin βA and BMP7 was markedly increased in BDV-infected cells. Expression of Smad7, a TGF-β family-inducible gene, was increased by BDV infection, and the expression was decreased by treatment with A-83-01 or LDN-193189, inhibitors of the TGF-β/activin or BMP pathway, respectively. These results suggest autocrine effects of activin A and BMP7 in C6BV cells. IGFBP-3 expression was also induced by BDV infection; it was below the detection limit in C6 cells. The expression level of IGFBP-3 was decreased by LDN-193189 in C6BV cells, suggesting that endogenous BMP activity is responsible for IGFBP-3 gene induction. Our results reveal the regulatory expression of genes related to the TGF-β family, and the role of the enhanced BMP pathway in modulating cell responses in BDV-infected glial cells.

Members of the transforming growth factor-β (TGF-β) family function through Smad-dependent and Smad-independent pathways. The Smad-dependent pathway is stimulated through the phosphorylation of receptor-regulated Smad (R-Smad) and inhibited through the dephosphorylation of R-Smad or the gene induction of inhibitory Smad (I-Smad). Little information is available on the regulation of R-Smad gene expression. BMP4 potentiated the up-regulation of Smad8/9 expression in C2C12, H9c2, 3T3-L1, HepG2, B16, and primary fibroblasts. BMP4-induced Smad8/9 expression was cycloheximide-insensitive and LDN-193189-sensitive, suggesting a direct event mediated through BMP type I receptors. BMP4 transcriptionally stimulated the Smad8/9 gene, and BMP-responsive elements (BREs) spanning nt -121 to nt -44 are involved in the up-regulation of Smad8/9 expression in response to BMP4. Phosphorylated Smad1/5/8/9 specifically bound to the BREs of Smad8/9 gene. The present study reveals that Smad8/9 is a unique R-Smad regulated through the BMP pathway at the mRNA level. J. Cell. Biochem. 117: 1788-1796, 2016. © 2016 Wiley Periodicals, Inc.

Four Japanese Black steers (16 months of age) were assigned to a 4 × 4 Latin square design to investigate the effect of graded levels of sweet-potato condensed distillers solubles (SCDS) in their diets on intake and urinary excretion of minerals. The four diets consisted of 0%, 10%, 20% and 30% (dry matter (DM) basis) SCDS, with SCDS replacing commercial concentrate (CC). Intake of K, Cl, S, P and Mg increased linearly with increasing SCDS content. Urinary pH increased linearly with increasing dietary SCDS content. SCDS feeding increased urinary K concentrations (linear and quadratic effects). Urinary concentrations of Cl increased linearly with increasing SCDS content. In contrast, urinary concentrations of Mg decreased with increasing SCDS content. Feeding of SCDS did not apparently affect urinary NH

細菌感染におけるマスト細胞の役割はほとんど注目されていない。しかしながら,近年の多くの研究からマスト細胞が多様な腸内細菌による感染症に対する感染防御の重要な細胞であることが示されてきた。本研究ではマスト細胞欠損マウス(WBBF1-W/W^v)を用いて,細菌感染におけるマスト細胞感染防御能検討した。その結果,(1)マスト細胞はアドヒジョンタンパク質により活性化される。(2)細菌は活性化されたマスト細胞によって殺菌されること,(3)活性化されたマスト細胞はIL-3産生し,それがオートクラインによってマスト細胞数の増殖をもたらすことがわかった。これらのことから,細菌感染において,マスト細胞は重要な役割を果たしていることが示唆された。, The role of mast cells in infections is invariably thought of as a detrimental one. However, recent studies have shown that mast cell play a key role in host defense against various enterobacterial infections. In this study, using mast cell-dificient and mast cell-sufficient mice we have investigated the protective role of mast cells in the infections. Our findings indicate that i. the mast cells are activated by adhesion protein, ii. Killing bacteria is dependent on the activated mast cells, and iii.activated mast cell secret IL-3 and the IL-3 promote mast cell proliferation by autocrine. Taken together, these findings suggest a beneficial role of mast cells in infections in mice.

Activinシグナル伝達の組織特異的制御を調べた。前駆マスト細胞をactivin AあるいはTGF-β_1刺激するとmMCP-7 mRNAレベルは上昇した。リポーターアッセイによるプロモーター領域の解析から,この遺伝子発現の上昇は転写レベルの制御であり,activin A/TGF-β_1の細胞内シグナル伝達分子の一つであるSmad3が関与していることが分かった。マスト細胞の分化に組織特異的転写因子であるMITFが関わっていることが知られているのでmMCP-7転写とMITFとの関係を検討したところ,Smad3によるmMCP-7転写はMITFの共発現によって抑制された。MITFとSmad3は細胞内で複合体を形成し,Smad3のMH1+linker領域とMITFのC末端がこの複合体形成に必要であった。しかしながら,複合体形成は,MITFによるSmad3シグナル抑制に対して必要条件でも十分条件でもなかった。Smad3のMH2領域によるmMCP-7転写上昇はMITFの共発現によって抑制され,N末端を欠いたMITFはSmad3シグナルを抑制しなかった。MITFの共発現によってSmad3の蛋白発現量は減少し,Smad3蛋白発現量とシグナル活性は相関した。以上の結果,MITFはactivin//TGF-βシグナル伝達を組織特異的に抑制することが明らかになった。, Tissue-specific regulation of activin signaling was examined in mast cells. Gene expression of mouse mast cell protease-7 (mMCP-7) was up-regulated by activin A and TGF-β_1 in bone marrow-derived cultured mast cell progenitors. Smad3, a signal mediator of the activin/TGF-β pathway, transcriptionally activated mMCP-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, heart and skeletal muscle, inhibited Smad3-mediated mMCP-7 transcription. MITF associated with Smad3, and the C-terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF truncated N-terminal amino acids could associate with Smad3, but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-β signaling in a tissue-specific manner.

Mouse mast cell protease-7 (mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-beta (TGF-beta) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-beta. Luciferase-based reporter assays using deletion and point mutations of mmcp-7 promoter showed a critical region spanning nt -126 to -122 relative to the transcriptional start site, a Smad-binding element, for transcriptional activation by the TGF-beta pathway. In addition, a region from nt -104 to -98, a TPA-responsive element, was also necessary for the transactivation. Consistent with the current model for the TGF-beta signaling, Smad4 was required for the transcription of mmcp-7 by Smad3, a signal mediator of TGF-beta. Treatment with TGF-beta in mast cells resulted in the differential gene induction of the AP-1 components, i.e., transient induction of c-fos but not of c-jun and junB. Expression of c-fos further enhanced Smad3 and Smad4-induced transcription of mmcp-7, whereas c-jun expression inhibited the transcription. Our results suggest that TGF-beta stimulates mmcp-7 transcription through the Smad3-Smad4 pathway as well as c-fos induction, and that the AP-1 components distinctly related with the TGF-beta pathway.

Murine bone marrow-derived cultured mast cells (BMMCs) are most widely used in in vitro experiments for evaluation of mast cell functions. The present study has shown that cell preparation procedure, i.e., cell collection by centrifugation and the subsequent adjustment and culture of cell density at the desired concentrations, transiently induced gene expression of plasminogen activator inhibitor-1 (PAI-1) and the AP-1 components (c-fos, c-jun, and junB). The level of PAI-1 gene transcript was closely related to the cell density and the gene expression was enhanced by pretreatment with okadaic acid, an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A). The cell preparation procedure also caused dephosphorylation of MAP kinases, i.e., ERK, p38, and JNK, resulting from PP1/PP2A activation. In view of the cell responses to the cell preparation procedure itself, care is needed in the interpretation of in vitro data using BMMCs.

Previous studies have revealed that members of the transforming growth factor-beta (TGF-beta) including TGF-beta1 and activin A modulate the function of mast cells. Here we show the up-regulation of mouse mast cell protease-6 (mMCP-6), which is expressed in differentiated mast cells, by TGF-beta1 and activin A in bone marrow-derived cultured mast cell progenitors (BMCMCs). Quantitative real time RT-PCR analyses revealed that the mRNA level of mMCP-6 was slightly but reproducibly increased by treatment with TGF-beta1 or activin A, which was regulated at the transcription level. Reporter assays showed that Smad3, a signal mediator of the TGF-beta/activin pathway, was responsible for the transcription. The TGF-beta response element is located at -153 bp relative to the transcription initiation site, CAGA. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, the heart and skeletal muscle, also stimulated the transcription of mMCP-6. The region at -166 bp, GACCTG, was responsible for MITF-induced transcription. Mutations of the CAGA motif and the MITF responsive site indicated that the MITF site of mMCP-6 promoter is indispensable for the transcriptional activation by a constitutively active TGF-beta receptor (ALK5-TD), whereas the CAGA motif is dispensable for transcription by MITF. Transcriptional activation of mMCP-6 by the TGF-beta pathway was differently interacted with that by MITF isoform; ALK5-TD further enhanced MITF-E-induced transcription, whereas MITF-M-induced transcription abolished responsiveness to ALK5-TD. The positive regulation of mMCP-6 by the TGF-beta/activin pathway and the differential regulation by the MITF isoform suggest a rigorous regulation of mast cell function as effector cells of immune response.

Three methods to quantify gene transcript levels in mast cells, real-time RT-PCR, competitive RT-PCR and conventional RT-PCR analyses, were compared. Linear regression analysis on five gene transcripts revealed that the mRNA levels measured by real-time RT-PCR analysis were minimally correlated with those by conventional RT-PCR analysis. In addition, differences in the mRNA level between samples measured by conventional RT-PCR analysis were smaller than those by real-time RT-PCR analysis, suggesting that conventional RT-PCR analysis is less sensitive at measuring mRNA levels. Results from competitive RT-PCR analysis correlated closely with those from real-time RT-PCR analysis. When the differences in mRNA level between samples are relatively smaller, however, the correlation tended to be weaker. Real-time RT-PCR analysis has higher reliability, but is expensive. In contrast, competitive RT-PCR analysis is inexpensive, but is weaker at detecting smaller differences in gene transcript level between samples. Therefore, the most appropriate analytical method to measure mRNA levels should be chosen, depending on the experimental conditions.

Objective—To evaluate effects of dietary carbohydrate on urine volume; struvite crystal formation; and calcium, phosphorus, and magnesium balance in clinically normal cats. Animals—21 healthy adult cats (15 sexually intact males and 6 sexually intact females). Procedure—Diets containing no carbohydrate source (control diet), control plus starch, or control plus fiber were given in a 3 × 3 Latin-square design. The diets were available ad libitum in study 1 (n = 12) and given under restrictions in study 2 (9) to equalize daily intakes of crude protein among the 3 groups. Formation of struvite crystals and balance of calcium, phosphorus, and magnesium were measured. Results—Urine volume was lower in the starch group and fiber group in study 1, whereas no differences were detected among the groups in study 2. Urinary pH and struvite activity product were higher in the starch group in both studies, and the fiber group also had higher struvite activity product in study 2. In both studies, urinary concentrations of HCl-insoluble sediment were higher in the starch group and fiber group. In the fiber group, a net loss of body calcium, phosphorus, and magnesium was detected in study 2. Conclusions and Clinical Relevance—Starch and fiber in diets potentially stimulate formation of struvite crystals. Hence, reducing dietary carbohydrate is desirable to prevent struvite urolith formation. In addition, a net loss of body calcium, phosphorus, and magnesium during feeding of the fiber diet suggests that dietary inclusion of insoluble fiber could increase macromineral requirements of cats. (Am J Vet Res 2004;65:138–142)

We explored transcriptional regulation of mouse mast cell protease-9 (mMCP-9), which is implicated in inflammation of the jejunum during helminth infections and tissue remodeling of the uterus during pregnancy. Transcription was positively regulated by microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix-leucine zipper family that binds to the E-box, a CANNTG sequence. The most significant segment for positive regulation by MITF was nt -183 to -177 of the mMCP-9 promoter, CATCATG, which bound MITF-M. In addition, not only other MITF isoforms but also TFE3, another member of the family, activated mMCP-9 transcription through this nucleotide sequence inserted one base within the E-box.

Objective—To evaluate the effects of a high-protein diet versus dietary supplementation with ammonium chloride (NH4Cl) on struvite crystal formation in the urine of clinically normal cats by measuring the urine concentration of hydrochloric acid (HCl)-insoluble sediment, urine pH, struvite activity product (SAP), number of struvite crystals in urine, and urine volume. Animals—23 healthy adult cats. Procedure—Urine was fractionated by centrifugation with subsequent extraction of the sediment with 1 N HCl (study 1). Diets containing either 29% crude protein or 55% crude protein were fed to cats in a crossover trial of 3 weeks/period (study 2). Diets supplemented with either sodium chloride (NaCl) or NH4Cl were fed, by use of a 3 X 3 Latin-square design with 3 wk/period (study 3). In studies 2 and 3, urine samples were collected for the last 7 days of each period. Results—The HCl-insoluble sediment contained Tamm-Horsfall glycoprotein (THP; study 1). The highprotein diet (study 2) and dietary supplementation with NH4Cl (study 3) resulted in a decrease in urine pH, SAP, and the number of struvite crystals in urine. However, the high-protein diet decreased urine concentrations of HCl-insoluble sediment containing THP (study 2), in contrast to the NH4Cl supplementation that increased urine volume without a significant effect on the urine concentration of the HCl-insoluble sediment (study 3). Conclusions and Clinical Relevance—Our results indicate that compared with dietary supplementation with NH4Cl, the high-protein diet is preferable as a urine acidifier for the prevention of struvite crystal formation in clinically normal cats. (Am J Vet Res 2003;64:1059–1064)

Activins, members of the transforming growth factor-β (TGF-β) superfamily, are potent growth and differentiation factors. Our previous studies revealed that activin A, a homodimer of inhibin/activin βA, was induced in mast cells and peritoneal macrophages in response to their activation. In the present study, we examined the roles of activin A in murine bone marrow-derived, cultured mast cell progenitors (BMCMCs), which expressed gene transcripts for molecules involved in activin signaling, suggesting that BMCMCs could be target cells of activin A. Treatment of activin A inhibited 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide uptake into BMCMCs in a dose-dependent manner. The IC50 concentration was 2.1 nM, which was less potent than 185 pM TGF-β1. Activin A treatment caused morphological changes toward the differentiated cells at 2 nM and up-regulated mRNA of mouse mast cell protease-1 (mMCP-1), a marker enzyme of mature mucosal mast cells, at 1 nM. Activin A also showed activity in inducing migration of BMCMCs; the optimal concentration for maximal migration was 10 pM, which was much lower than the concentrations to inhibit cell growth and to activate the mMCP-1 gene. Taking the present results together with our previous results, it is suggested that activin A secreted from activated immune cells recruits mast cell progenitors to sites of inflammation and that with increasing activin A concentration, the progenitors differentiate into mature mast cells. Thus, activin A may positively regulate the functions of mast cells as effector cells of the immune system.

The present study examined the regulatory expression of activin A, a potent growth and differentiation factor, in rat basophilic leukemia (RBL-2H3) mast cells. Treatment of RBL-2H3 cells sensitized with anti-dinitrophenyl IgE with multivalent dinitrophenyl led to a clear increase in RT-PCR products of inhibin/activin beta(A). The steady-state mRNA of inhibin/activin beta(A) was also induced by increasing cytosolic Ca(2+) concentration with ionomycin, which required de novo protein synthesis, and was regulated at the transcriptional level. Pretreatment of RBL-2H3 cells with antagonists or inhibitors for the calmodulin pathway blocked ionomycin-dependent inhibin/activin beta(A) transcription and mRNA induction, suggesting the involvement of calmodulin-dependent kinase (CaMK) and calcineurin. The ionomycin-dependent inhibin/activin beta(A) induction was also partially blocked by preincubation with c-Jun NH(2)-terminal kinase (JNK) and p38 kinase inhibitors, but not with MEK1 inhibitor. These results suggest that inhibin/activin beta(A) gene activation is achieved by the JNK and p38 kinase activation through the calmodulin pathway in mast cells.

Members of the transforming growth factor-β (TGF-β) family are known to regulate the commitment of mesenchymal stem cells to an adipocyte lineage and the differentiation of preadipocytes to adipocytes. However, the roles of TGF-β family members in mature adipocytes are not known. The present study explored the modulation of cell function in 3T3-L1 adipocytes treated with the TGF-β family members TGF-β1, activin A, activin B, bone morphogenetic protein (BMP) 4, and BMP7. These TGF-β family members modulated the expression of some adipokines; activin A upregulated leptin expression, and activin B downregulated the expression of plasminogen activator inhibitor-1. All examined TGF-β family members, except for activin B, increased the expression of resistin. The TGF-β family members also modulated the free amino acid content in cells. Insulin-induced reductions in the cellular content of arginine, glutamic acid, and phenylalanine were exaggerated by TGF-β1, activin A, and activin B. In contrast, the cellular glutamine content decreased with BMP4 or BMP7 treatment, inhibiting the response to insulin. In addition, although the cellular citric acid cycle metabolite contents were not generally affected by TGF-β family members alone, activin A, activin B, BMP4, and BMP7 potentiated the insulin-induced accumulation of the metabolites. The present results suggest that TGF-β family members regulate adipocyte function by modulating adipokine expression and cell metabolism in differentiated adipocytes.

The expression of uncoupling protein (UCP1) is up-regulated in mammalian brown adipocytes during cold exposure. However, a previous study revealed that UCP1 was highly expressed in the liver of common carps, and that the hepatic expression of UCP1 was down-regulated during cold exposure. The present study examined the effects of temperature on the recovery of UCP1 expression levels and the expression of genes involved in UCP1 transcription in the livers and kidneys of common carps. The hepatic and renal expressions of UCP1 were decreased by acclimation from 22 °C to 8 °C, and a subsequent increase in the water temperature from 8 °C to 28 °C recovered the renal, but not hepatic expression of UCP1. Changes in the expression of peroxisome proliferator-activator receptor (PPAR) γ, retinoid X receptor (RXR) α and PPARγ co-activator (PGC)-1α, genes that are involved in the expression of UCP1 in mammals, with ambient temperature indicated that the expressions of PPARγ and RXRα, but not expression of PGC-1α was decreased in response to cold exposure; the hepatic and renal expressions of PPARγ and RXRα recovered to basal levels with the cessation of cold exposure, although this was not complete for hepatic expression of PPARγ. The results of the present study indicate that a unique regulatory mechanism is responsible for the hepatic and renal expressions of carp UCP1 during cold exposure and subsequent reacclimation, and is distinct from that in murine brown adipocytes.

Borna disease virus (BDV) infection causes neurological disease in cats. Here, we report BDV infection in 199 hospitalized domestic cats in the Tokyo area. BDV infection was evaluated by detection of plasma antibodies against BDV-p24 or -p40. BDV-specific antibodies were detected in 54 cats (27.1%). Interestingly, the percentage of seropositive cats was not significantly different among the three clinical groups, i.e., healthy (29.8%), neurologically asymptomatic disease (22.2%) and neurological disease (33.3%). The specific antibodies were present even in cats aged below one year. The seropositive ratio was constant, irrespective of age and sampling season. The present study suggests that additional factors are required for onset of Borna disease in naturally infected cats and that BDV is transmitted through vertical routes in cats.

Microphthalmia-associated transcription factor (MITF) is a transcription factor that is expressed in limited types of cells, including osteoclasts, but the expression and role of MITF during osteoclastogenesis have not been fully elucidated. The expression of the MITF-E isoform but not that of the MITF-A isoform was induced in response to differentiation stimulation towards osteoclasts by receptor activator of NF-κB ligand (RANKL) in both RAW264.7 cells and primary bone marrow cells. The RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was inhibited in RAW264.7 cells expressing siRNA for MITF-E. Transforming growth factor-β (TGF-β) enhanced RANKL-induced MITF-E expression and -TRAP positive multinucleated cell formation. In particular, TGF-β potentiated the formation of larger osteoclasts. The expression levels of NFATc1, TRAP and CtsK, genes related to osteoclast development and activity, were concurrently enhanced by TGF-β in the presence of RANKL. Furthermore, the expression of dendritic cell-specific transmembrane protein (DC-STAMP), Itgav, Itga2, Itga5, Itgb1, Itgb3 and Itgb5, genes related to cell adhesion and fusion, were up-regulated by co-treatment with TGF-β. In particular, the regulatory expression of Itgav and Itgb5 in response to RANKL with or without TGF-β resembled that of MITF-E. Because MITF is involved in cell fusion in some cell systems, these results imply a role for MITF-E as an enhancer of osteoclastogenesis and that RANKL-induced levels of both MITF-E mRNA and of MITF-dependent gene expression are enhanced by treatment with TGF-β.