Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading.

Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine(608)-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.


Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes.

Oxidative stress is a key factor in stimulating the expression of atrogenes, which are muscle atrophy-related ubiquitin ligases, in skeletal muscle, and it induces muscle atrophy during unloading. However, the effects of antioxidative nutrients on atrogene expression have not been demonstrated. We report on the inhibitory effects of polyphenols, such as epicatechin (EC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) and quercetin, on atrogene expression up-regulated by three dimensional (3D)-clinorotation or glucocorticoid. These treatments markedly elevated the expression of atrogenes, including atrogin-1 and MuRF-1, in mouse C2C12 myoblasts and myotubes. Interestingly, EC, ECg, EGCg and quercetin significantly decreased the expression of atrogin-1 and MuRF-1 up-regulated by 3D-clinorotation, whereas they hardly affected atrogene expression induced by dexamethasone. ERK signaling is a well known MAPK pathway to mediate oxidative stress. Therefore, we also investigated the effect of these polyphenols on phosphorylation of ERK in C2C12 myotubes. As expected, EC, ECg, EGCg, and quercetin significantly suppressed phosphorylation of ERK, corresponding to the up-regulation of atrogenes induced by 3D-clinorotation. These results suggest that antioxidative nutrients, such as catechins and quercetin, suppress atrogene expression in skeletal muscle cells, possibly through the inhibition of ERK signaling. Thus, catechins and quercetin may prevent unloading-mediated muscle atrophy.

The Journal of Medical Investigation : JMI


Glucose Infusion Suppresses Surgery-induced Muscle Protein Breakdown by Inhibiting Ubiquitin-proteasome Pathway in Rats

It appears to have been well established that after surgery, protein catabolism is accelerated and glucose infusion suppresses the catabolic reactions. However, in the early postoperative period, the effects of surgical stress and glucose infusion on muscle protein catabolism and the related mechanisms remain unclear. Rats undergoing laparotomy were infused with acetated Ringer's solution (10 ml x kg(-1) x h(-1)) without glucose (control) or containing 1% or 5% glucose. The infusion was continued for a further 4 h after the surgical treatment. The catabolic index, excretion of urinary nitrogen and 3-methylhistidine, and release of tyrosine and 3-methylhistidine from isolated muscle were determined. Furthermore, muscular mRNA expression of proteolytic-related genes (atrogin-1/MAFbx, muscle ring finger-1, mu- and m-calpain, and cathepsin L and H) and phosphorylation of components of insulin signaling (forkhead box O3 and protein kinase B) were evaluated. Surgery increased the catabolic index, and this increase was suppressed by glucose infusion (both 1% and 5%). In the control group, mRNA expression of atrogin-1/MAFbx and muscle ring finger-1 was increased, and they were suppressed in the two glucose groups. Furthermore, insulin signaling (phosphorylation of protein kinase B and forkhead box O3) in muscles was stimulated by glucose infusion. The present study indicates that glucose infusion, even at a relatively low rate, suppresses muscle protein breakdown in the early postoperative period. The mechanism of this effect is related to the suppression of the ubiquitin-proteasome pathway, accompanied by activation of insulin signaling.


Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.


Peroxidized cholesterol-induced matrix metalloproteinase-9 activation and its suppression by dietary beta-carotene in photoaging of hairless mouse skin.

The activation of matrix metalloproteinase (MMP)-9 leading to the formation of wrinkle and sagging of skin is an essential step in the skin photoaging on exposure to ultraviolet A (UVA). This study attempted to elucidate the role of peroxidized cholesterol including cholesterol hydroperoxides (Chol-OOHs), primary products of lipid peroxidation in biomembranes, in MMP-9 activation and the effect of dietary beta-carotene in MMP-9 activation. Hairless mice were subjected to periodic UVA irradiation for 8 weeks. The amount of peroxidized cholesterol detected as total hydroxycholesterol in the skin was increased significantly by the exposure. The activity and protein level of MMP-9 were elevated with wrinkling and sagging formation. MMP-9 activity was also enhanced by the intracutaneous injection of Chol-OOHs into the mouse skin. Adding beta-carotene to the diet of the mice during the period of irradiation suppressed the activity and expression of MMP-9 as well as the wrinkling and sagging formation. The amount of cholesterol 5alpha-hydroperoxide, a singlet molecular oxygen oxygenation-specific peroxidized cholesterol, was significantly lowered by the addition of beta-carotene to the diet. These results strongly suggest that Chol-OOHs formed on exposure to UVA contribute to the expression of MMP-9, resulting in photoaging. Dietary beta-carotene prevents the expression of MMP-9, at least partly, by inhibiting photodynamic action involved in the formation of Chol-OOHs.


Subcutaneous injection, from birth, of epigallocatechin-3-gallate, a component of green tea, limits the onset of muscular dystrophy in mdx mice: a quantitative histological, immunohistochemical and electrophysiological study.

Dystrophic muscles suffer from enhanced oxidative stress. We have investigated whether administration of an antioxidant, epigallocatechin-3-gallate (EGCG), a component of green tea, reduces their oxidative stress and pathophysiology in mdx mice, a mild phenotype model of human Duchenne-type muscular dystrophy. EGCG (5 mg/kg body weight in saline) was injected subcutaneously 4x a week into the backs of C57 normal and dystrophin-deficient mdx mice for 8 weeks after birth. Saline was injected into normal and mdx controls. EGCG had almost no observable effects on normal mice or on the body weights of mdx mice. In contrast, it produced the following improvements in the blood chemistry, muscle histology, and electrophysiology of the treated mdx mice. First, the activities of serum creatine kinase were reduced to normal levels. Second, the numbers of fluorescent lipofuscin granules per unit volume of soleus and diaphragm muscles were significantly decreased by about 50% compared to the numbers in the corresponding saline-treated controls. Third, in sections of diaphragm and soleus muscles, the relative area occupied by histologically normal muscle fibres increased significantly 1.5- to 2-fold whereas the relative areas of connective tissue and necrotic muscle fibres were substantially reduced. Fourth, the times for the maximum tetanic force of soleus muscles to fall by a half increased to almost normal values. Fifth, the amount of utrophin in diaphragm muscles increased significantly by 17%, partially compensating for the lack of dystrophin expression.


Cathepsin C propeptide interacts with intestinal alkaline phosphatase and heat shock cognate protein 70 in human Caco-2 cells

The oligomeric structure and the residual propeptide are distinct characteristics of cathepsin C from other members in the papain superfamily. In this study, we examined the physiological role of the cathepsin C propeptide. The stable overexpression of cathepsin C propeptide significantly decreased the activities of intestinal alkaline phosphatase (IAP) and sucrase in human Caco-2 intestinal epithelial cells, whereas it did not change the proliferation and cathepsin C activity. The overexpression of cathepsin C propeptide significantly decreased the amounts of IAP protein in differentiated Caco-2 cells, compared with the transfection of mock vector, whereas the amounts of IAP transcripts were not changed. Pulse-chase analysis confirmed that the reduction in IAP activity was due to an increase in IAP degradation, but not a decrease in IAP expression. For the mechanism of the enhanced IAP degradation, we identified proteins interacting with cathepsin C propeptide in Caco-2 cells by immunoprecipitation and mass spectrometry. Cathepsin C propeptide interacted with proteins with a molecular mass of approximately 70 kDa, including IAP and heat shock cognate protein 70. Our present results suggest that the propeptide of cathepsin C may stimulate the sorting to the lysosome, at least in part, contributing to the degradation of IAP in Caco-2 cells.


Effects of dietary fish oil on lipid peroxidation and serum triacylglycerol levels in psychologically stressed mice

The intake of omega-3 polyunsaturated fatty acids and psychological stress can each induce tissue lipid peroxidation. In our present study, we investigated their combined effects on the oxidative status of mouse tissues. Mice were group-housed (four mice/cage) and fed a diet containing fish oil (as a source of omega-3 polyunsaturated fatty acids), soybean oil, or olive oil for 3 wk. These animals were then 1) housed under the same conditions (four per cage, control group) or 2) individually housed to generate psychological stress conditions (isolation stress). After 2 wk of isolation stress, the levels of thiobarbituric acid-reactive substances (an index of lipid peroxidation) and antioxidants in the liver and kidney and the serum levels of triacylglycerol were measured. Fish oil-fed mice showed increased levels of thiobarbituric acid-reactive substances in their livers and kidneys compared with soybean oil- or olive oil-fed mice. These increases in thiobarbituric acid-reactive substance levels in the fish oil-fed mice were less profound under isolation stress conditions when compared with the group-housed animals on the same diet. In the fish oil-fed mice, isolation stress led to an increase in liver vitamin E levels when compared with their group-housed counterparts. The fish oil-fed mice exhibited lower serum triacylglycerol levels compared with the soybean oil- or olive oil-fed mice, and this decrease was more profound under conditions of isolation stress when compared with group-housing conditions. Dietary fish oil combined with isolation stress results in lower levels of lipid peroxidation in the liver and kidney compared with dietary fish oil alone.


Deficiency of Cbl-b gene enhances infiltration and activation of macrophages in adipose tissue and causes peripheral insulin resistance in mice.

c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. C57BL/6 (Cbl-b(+/+)) or Cbl-b-deficient (Cbl-b(-/-)) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. Elderly Cbl-b(-/-) mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b(-/-) mice than age-matched Cbl-b(+/+) mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b(-/-) mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b(-/-) macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b(-/-) macrophages compared with Cbl-b(+/+) macrophages. Treatment with a neutralizing anti-MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b(-/-) mice. Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.


Distinct expression of mast cell tryptase and protease activated receptor-2 in synovia of rheumatoid arthritis and osteoarthritis.

The objective of this study is to examine the differential expression of mast cell tryptase and its receptor, protease-activated receptor-2 (PAR-2), in the synovium and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Biochemical and immunohistochemical analyses were performed to determine whether the trypsin-like protease in the synovium is identical to mast cell tryptase. The effects of mast cell tryptase on the proliferation of synovial fibroblast-like cells (SFCs) and the release of IL-8 thereof were evaluated by the [3H]-thymidine incorporation and ELISA, respectively. The trypsin-like protease in the synovium of RA patients was identical to human mast cell tryptase, which was composed of two subunits: 33 and 34 kDa. The 33- and 34-kDa proteins are different glycosylated forms of the 31-kDa protein, which was unglycosylated. Mast cell tryptase activity in RA synovial fluid was significantly higher than that in OA synovial fluid, while their activities and expression in the synovium were similar. Expression of PAR-2 mRNA in the synovium was higher in RA than in OA. Mast cell tryptase containing the unglycosylated 31-kDa subunit was the predominant form in synovial fluid. RA patients had higher amounts of this subunit in their synovial fluid than OA patients. Mast cell tryptase and PAR-2 activating peptide stimulated the proliferation of SFCs and release of IL-8 from these cells. Mast cell tryptase secretion into RA synovial fluid is higher than OA synovial fluid. Mast cell tryptase in synovial fluid stimulates the proliferation of SFCs and the release of pro-inflammatory cytokines via PAR-2, which may contribute to exacerbation of synovitis in RA.


Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest.

In animal models, several ubiquitin ligases play an important role in skeletal muscle atrophy caused by unloading. In this study we examined protein ubiquitination and ubiquitin ligase gene expression in quadriceps femoris muscle from healthy volunteers after 20-day bedrest to clarify ubiquitin-dependent proteolysis in human muscles after unloading. During bedrest, thickness and cross-sectional area of the quadriceps femoris muscle decreased significantly by 4.6% and 3.7%, respectively. Ubiquitinated proteins accumulated in these atrophied human muscles. A real-time reverse transcription-polymerase chain reaction system showed that bedrest significantly upregulated expression of two ubiquitin ligase genes, Cbl-b and atrogin-1. We also performed DNA microarray analysis to examine comprehensive gene expression in the atrophied muscle. Bedrest mainly suppressed the expression of muscle genes associated with control of gene expression in skeletal muscle. Our results suggest that, in humans, Cbl-b- or atrogin-1-mediated ubiquitination plays an important role in unloading-induced muscle atrophy, and that unloading stress may preferentially inhibit transcriptional responses in skeletal muscle.


Restraint stress alters the duodenal expression of genes important for lipid metabolism in rat.

Stress, such as trauma and injury, is known to cause transcriptional changes in various tissues; however, there is little information on tissue-specific gene expression in response to stress. Here, we have examined duodenal gene expression in rats subjected to whole-body immobilization in order to elucidate the mechanism underlying the stress response in the duodenum--one of the tissues that is most sensitive to external stress. DNA microarray analysis revealed that the immobilization for 2 weeks caused great changes in gene expression in the rat duodenum: 165 genes exhibited more than a two-fold change in expression level (103 up-regulated; 62 down-regulated). In addition, functional classification of these genes showed that immobilization preferentially stimulated the expression of genes related to lipid metabolism, including genes encoding mitochondrial HMG-CoA synthase, a key enzyme in ketogenesis; solute carrier 27A2, a fatty acid transporter; and dienoyl CoA reductase, a key enzyme in beta-oxidation. To elucidate the factors mediating these immobilization-induced changes, we treated rats and small intestinal IEC-6 cells with dexamethasone and hydrogen peroxide. In both rats and IEC-6 cells, treatment with dexamethasone induced changes in gene expression that mimicked the immobilization-mediated increase in expression of the mitochondrial HMG-CoA synthase and dienoyl CoA reductase transcripts, suggesting that stress-induced synthesis of glucocorticoid hormones mediates, at least in part, the stress response in the duodenum. These results suggest that immobilization may alter lipid metabolism in the small intestine by modifying the expression of specific genes through which the small intestine may seek to protect itself from stress-induced damage.


Soy protein diet prevents hypermethioninemia caused by portacaval shunt in rats.

In hepatic disorders, abnormal plasma amino acid profiles are observed. In this study, we examined whether soy protein isolate (SPI) improved plasma methionine concentration in the model animals. Portacaval shunt (PCS) increased alanine aminotransferase (ALT) activity and methionine concentration in blood of rats fed a 40% casein diet supplemented with 0.6% methionine (casein-M diet). A 40% SPI diet supplemented with 1.28% methionine (SPI-M diet), which contained the same amount of methionine as that in 40% casein-M diet, normalized plasma ALT activity and methionine level in PCS rats. These effects of a SPI diet may be due to its amino acid composition, since an amino acid mixture diet mimicking a 40% SPI-M diet was also effective to hypermethioninemia of PCS rats. To find key enzymes for the beneficial effect of soy protein, we examined effects of a 40% SPI-M or casein-M diet on the activities of three methionine-metabolizing enzymes in liver of PCS rats. A SPI-M diet stimulated only the activity of cystathionine gamma-lyase, compared with a casein-M diet. A SPI diet has a preventive effect on hypermethioninemia, at least in part, by stimulating cystathionine gamma-lyase activity in liver and may be used for nutritional management of liver disorders with hypermethioninemia.

The Journal of Medical Investigation : JMI


Immobilization decreases duodenal calcium absorption through a 1,25-dihydroxyvitamin D-dependent pathway.

Immobilization induces significant and progressive bone loss, with an increase in urinary excretion and a decrease in intestinal absorption of calcium. These actions lead to negative calcium balance and the development of disuse osteoporosis. The aims of this study were to evaluate the molecular mechanisms of decreased intestinal calcium absorption and to determine the effect of dietary 1,25-dihydroxyvitamin D [1,25(OH)2D] and a high-calcium diet on bone loss due to immobilization. The immobilized rat model was developed in the Bollman cage III to induce systemic disuse osteoporosis in the animals. There was a significant decrease in lumbar bone mineral density (BMD) and intestinal calcium absorption in the immobilized group compared with the controls. Serum 25-hydroxyvitamin D concentration did not change, but 1,25(OH)2D concentration decreased significantly. The mRNA levels of renal 25-hydoxyvitamin D 24-hydroxylase (24OHase) increased, whereas those of renal 25-hydroxyvitamin D 1-alpha hydroxylase (1alpha-hydroxylase), duodenal transient receptor potential cation channel, subfamily V, member 6 (TRPV6), TRPV5, and calbindin-D9k were all decreased. A high-calcium diet did not prevent the reduction in lumbar BMD or affect the mRNA expression of proteins related to calcium transport. Dietary administration of 1,25(OH)2D increased the intestinal calcium absorption that had been downregulated by immobilization. TRPV6, TRPV5, and calbindin-D9k mRNA levels were also upregulated, resulting in prevention of the reduction in lumbar BMD. Therefore, it is concluded that dietary 1,25(OH)2D prevented decreases in intestinal calcium absorption and simultaneously prevented bone loss in immobilized rats. However, it remains unclear that calcium absorption and expression of calcium transport proteins are essential for the regulation of lumbar BMD.


Ubiqitin Ligase Cbl-b Downregulates Bone Formation Through Suppression of IGF-I Signaling in Osteoblasts During Denervation

Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl-b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl-b may mediate denervation-induced osteopenia by inhibiting IGF-I signaling in osteoblasts. Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading-mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING-type ubiquitin ligase, Cbl-b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl-b played an important role in the resistance of osteoblasts to IGF-I. Cbl-b-deficient (Cbl-b(-/-)) or wildtype (Cbl-b(+/+)) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF-I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl-b-overexpressing or -deficient osteoblastic cells to various growth factors. In Cbl-b(+/+) mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl-b protein in osteoprogenitor cells and in osteocalcin-positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl-b(-/-) mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF-I. Therefore, we focused on Cbl-b-mediated modification of IGF-I signaling. Denervation decreased the amounts of insulin receptor substrate-1 (IRS-1), phosphatidly inositol 3-phosphate kinase (PI3K), and Akt-1 proteins in femora of Cbl-b(+/+) mice, whereas the amounts of these IGF-I signaling molecules in femora of Cbl-b(-/-) mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl-b(-/-) mice were more stimulated to proliferate by IGF-I treatment compared with those from Cbl-b(+/+) mice. Furthermore, overexpression of Cbl-b increased ubiquitination and degradation of IRS-1 in primary Cbl-b(-/-) osteoblastic cells, leading to their impaired mitogenic response to IGF-I. These results suggest that Cbl-b induces resistance of osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.


Distinct gene expression profiles in the femora of rats exposed to spaceflight, tail-suspension and dnervation.


Identification of mono-ubiquitinated LDH-A in skeletal muscle cells exposed to oxidative stress

We previously reported that oxidative stress is associated with unloading-mediated ubiquitination of muscle proteins. To further elucidate the involvement of oxidative stress in ubiquitination, we examined the ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after the treatment. Microsequencing revealed that the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. Under unloading conditions, such as tail-suspension and spaceflight, mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, while they did not affect the amount of poly-ubiquitinated LDH. In contrast, epoxomicin, a potent proteasome inhibitor, did not change the amount of mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it significantly increased the amount of poly-ubiquitinated LDH. Our results suggest that oxidative stress induces not only poly-ubiquitination but also mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation during unloading.

Biochemical and Biophysical Research Communications


Osteoactivin upregulates expression of MMP-3 and MMP-9 in fibroblasts infiltrated into denervated skeletal muscle in mice

In this study, we examined pathophysiological roles of osteoactivin, a functionally unknown type I membrane glycoprotein, in mouse skeletal muscle atrophied by denervation (sciatic neurectomy). Denervation increased the amounts of osteoactivin, vimentin, matrix metalloproteinase-3 (MMP-3), and MMP-9 in mouse gastrocnemius muscle. Interestingly, immunohistochemical analysis revealed that vimentin, MMP-3, and MMP-9 were mainly present in fibroblast-like cells infiltrated into denervated mouse gastrocnemius muscle, whereas osteoactivin was expressed in the sarcolemma of myofibers adjacent to the fibroblast-like cells. On the basis of these findings, we reasoned that osteoactivin in myocytes was involved in activation of the infiltrated fibroblasts. To address this issue, we examined effects of osteoactivin on expression of MMPs in fibroblasts in vitro and in vivo. Overexpression of osteoactivin in NIH-3T3 fibroblasts induced expression of MMP-3, but not in mouse C(2)C(12) myoblasts, indicating that osteoactivin might functionally target fibroblasts. Treatment with recombinant mouse osteoactivin increased the amounts of collagen type I, MMP-3, and MMP-9 in mouse NIH-3T3 fibroblasts. The upregulated expression of these fibroblast marker proteins was significantly inhibited by heparin, but not by an integrin inhibitor, indicating that a heparin-binding motif in the extracellular domain might be an active site of osteoactivin. In osteoactivin-transgenic mice, denervation further enhanced expression of MMP-3 and MMP-9 in fibroblasts infiltrated into gastrocnemius muscle, compared with wild-type mice. Our present results suggest that osteoactivin might function as an activator for fibroblasts infiltrated into denervated skeletal muscles and play an important role in regulating degeneration/regeneration of extracellular matrix.

American Journal of Physiology, Cell Physiology


Clinorotation prevents differentiation of rat myoblastic L6 cells in association with reduced NF-κB signaling

In this study, we examined effects of the three-dimensional (3D)-clinorotation, a simulated-model of microgravity, on proliferation/differentiation of rat myoblastic L6 cells. Differentiation of L6 cells into myotubes was significantly disturbed in the 3D-clinorotation culture system, although the 3D-clinorotation had no effect on the proliferation. The 3D-clinorotation also suppressed the expression of myogenesis marker proteins, such as myogenin and myosin heavy chain (MHC), at the mRNA level. In association with this reduced differentiation, we found that the 3D-clinorotation prevented accumulation of ubiquitinated proteins, compared with non-rotation control cells. Based on these findings, we focused on the ubiquitin-dependent degradation of I kappa B, a myogenesis inhibitory protein, to clarify the mechanism of this impaired differentiation. A decline in the amount of I kappa B protein in L6 cells was significantly prevented by the rotation, while the amount of the protein in the non-rotated cells decreased along with the differentiation. Furthermore, the 3D-clinorotation reduced the NF-kappaB-binding activity in L6 cells and prevented the ubiquitination of I kappa B proteins in the I kappa B- and ubiquitin-expressing Cos7 cells. Other myogenic regulatory factors, such as deubiquitinases, cyclin E and oxygen, were not associated with the differentiation impaired by the clinorotation. Our present results suggest that simulated microgravity such as the 3D-clinorotation may disturb skeletal muscle cell differentiation, at least in part, by inhibiting the NF-kappa B pathway.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research


Short-term hypergravity does not affect protein-ubiquitination and proliferation in rat L6 myoblastic cells

We previously reported that spaceflight (STS-90) and tail-suspension stimulated muscle protein ubiquitination and accumulated the degradation fragments. However, in space experiments the side-effects of hypergravity on samples are inevitable during the launch of a space shuttle into space or the reentry. To examine whether hypergravity also caused protein-ubiquitination in skeletal muscle cells, we exposed rat myoblastic L6 cells to various hypergravity conditions. Immunoblot analysis showed that the centrifugation at 2, 3, 30 or 100 G for 10 min did not increase the amount of ubiquitinated proteins in L6 cells, whereas the centrifugation at 100 G for 1 or 2 hrs significantly induced the protein-ubiquitination. In contrast, heat shock protein 70 (HSP70), another stress-responsive protein, in L6 cells was accumulated only by centrifugation at 100 G for more than 10 min. Short-term (10 min) hypergravity including 3 or 100 G did not affect the proliferation and morphological changes in L6 cells. Our present results suggest that the ubiquitination of muscle proteins is less sensitive to hypergravity than the induction of HSP70, and that the effect of hypergravity on protein-ubiquitination and proliferation of skeletal muscle cells may be negligible, as far as its duration is short-term.


Relationship Between Cathepsin B and Thrombin in Rheumatoid Arthritis

To investigate the pathophysiological significance of cathepsin B and thrombin in synovial fluid (SF) from patients with rheumatoid arthritis (RA). Thrombin and cathepsin B activities of samples from patients with RA and osteoarthritis (OA) were measured using fluorogenic synthetic substrates. The concentration of interleukin 8 (IL-8) in SF was measured by ELISA. The effect of thrombin on the proliferation of synovial fibroblast-like cells (SFC) was examined by measuring 3H-thymidine incorporation. The effect of thrombin on the release of IL-8 and cathepsin B from SFC was investigated. The expression of IL-8 mRNA in SFC after stimulation with thrombin was evaluated using real-time quantitative RT-PCR. The effect of recombinant IL-8 on the activation of cathepsin B was examined using the knee joints of rabbits. In SF supernatants, cathepsin B and thrombin-like activity was significantly higher in RA than in OA, and there was a significant correlation between them. Cathepsin B activity was also significantly higher in SF cells and synovial tissue extracts from RA patients than in those from OA patients. There was a significant correlation between cathepsin B activity and the concentration of IL-8 in RA SF. Thrombin enhanced the proliferation of SFC in a dose-dependent manner. Thrombin significantly enhanced the release of IL-8 from SFC as well as the expression of IL-8 mRNA in SFC. IL-8 induced activation of cathepsin B in the knee joints of rabbits. However, thrombin did not directly increase cathepsin B activity in SFC. In RA, thrombin was found to be related to the enhanced growth of SFC and the release of IL-8 from these cells; thus thrombin is probably related to worsening of inflammation through the recruitment of leukocytes (neutrophils), which release cathepsin B into the SF. Thrombin can induce activation of cathepsin B in SFC via increased expression of IL-8.


Skeletal muscle gene expression in space-flown rats

Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of approximately 26,000 gastrocnemius muscle genes in space-flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail-suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight-specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria-anchoring proteins, A-kinase anchoring protein, and cytoplasmic dynein, and 2) up-regulated expression of ubiquitin ligase genes, MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress-inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.


Physical stress by magnetic force accelerates differentiation of human osteoblasts

We examined the effect of magnetic force on differentiation of cultured human osteoblasts. Magnetic microparticles (MPs) were introduced into the cytoplasm of a human osteoblast cell line and the cells were cultured in a magnetic field (MF) in group MP-MF. Three groups of controls were used: cells without MPs were cultured out of MF (group C), cells without MPs were cultured in MF (group MF), and cells with MPs were cultured out of MF (group MP). The cells in group MP-MF became larger and were elongated along the axis of the magnetic poles. Appearance of alkaline phosphatase (AlPase) activity, formation of bone nodules, and calcium deposition were accelerated depending on the intensity of the magnetic field. It takes longer culture in the other three groups to exhibit these changes. Core-binding factor A1 (Cbfa1: transcription factor for osteoblast differentiation) and osteocalcin (a bone-matrix protein involved in controlling osteogenesis) were expressed earlier or stronger in group MP-MF than the other groups. Then we compared phosphorylation of mitogen-activated protein kinase (MAPK) between group MP-MF and group C. Phosphorylation of p38(MAPK) (p38) was increased in group MP-MF, while total p38 as well as total and phosphorylated forms of MAPK/ERK 1/2 and SAPK/JNK were not changed between the two groups. When a p38 inhibitor, SB 203580, was added to the culture medium in group C, AlPase activity, formation of bone nodules, and calcium deposits were completely inhibited. On the other hand, they were inhibited only partially by a MAPK/ERK 1/2 inhibitor, U-0126. Based on these results, it is concluded that (1) osteoblast differentiation is accelerated by a magnetic force, (2) this acceleration is mainly attributed to the activation of p38 phosphorylation, and (3) the stimulus induced by a magnetic field offers a new approach to osteoblast differentiation.


Dietary supplementation with docosahexaenoic acid, but not with eicosapentaenoic acid, reduces host resistance to fungal infection in mice

The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated. Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA. Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound. This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil. These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance. DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver. However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation. Greater infection-induced increases in spleen and serum interferon-gamma concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.

Biochimica et Biophysica Acta (BBA) - General Subjects


Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90)

We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle.

The Journal of Medical Investigation : JMI


A Relative High Dose of Vitamin E Does Not Attenuate Unweighting-Induced Oxidative Stress and Ubiquitination in Rat Skeletal Muscle

We previously reported that intragastric administration of cysteine could be beneficial to prevent unweighting-induced ubiquitination and degradation of muscle protein in association with redox regulation [Ikemoto et al., Biol. Chem., 383 (2002), 715-721]. In this study, we investigated whether vitamin E, another potent antioxidative nutrient, also had beneficial effects on the muscle protein catabolism. However, daily intragastric supplementation of 1.5 or 15 mg/rat of alpha-tocopherol did not prevent weight loss of hindlimb skeletal muscle in tail-suspended rats. To elucidate the reason for the non-effectiveness of vitamin E, we further examined concentrations of oxidative stress markers, ubiquitination of muscle proteins and fragmentation of myosin heavy chain in gastrocnemius muscle of rats daily treated with 15 mg of alpha-tocopherol. Unexpectedly, vitamin E increased concentrations of glutathione disulfide and thiobarbituric acid-reactive substance and decreased glutathione level in the muscle, compared with those of vehicle treatment, indicating that vitamin E enhanced unweighting-induced oxidative stress in skeletal muscle. The vitamin E supplementation did not suppress the ubiquitination of muscle proteins and fragmentation of myosin heavy chain caused by tail-suspension. Our results suggest that supplementation of a relative high dose of vitamin E could not inhibit ubiquitin-dependent degradation of muscle protein in tail-suspended rats possibly due to its prooxidant action.

Journal of Physiological Anthropology and Applied Human Science


Effect of timing of food deprivation on host resistance to fungal infection in mice

Mice were deprived of food for a period of 72 h at varying times relative to the time of infection with Paracoccidioides brasiliensis. Host resistance was diminished profoundly when the period of food deprivation was from 48 h before to 24 h after infection (group B). When food deprivation was initiated immediately after infection (group C), host resistance was reduced less profoundly. When food deprivation was initiated at 24 and 48 h post-infection, reductions in host resistance were only moderate or not observed respectively. These results suggest that the earlier in the course of infection starvation occurs, the more profoundly host resistance is impaired. When food deprivation was initiated 72 h before infection, finishing at the time of infection (group A), the reduction in host resistance was considerably less profound compared with group B mice, suggesting that refeeding initiated immediately after infection is responsible for rapid restoration of the antifungal resistance in starved mice. Infection-induced responses of corticosterone and interferon-gamma were changed according to the timing of food deprivation. Group A mice, similar to non-fasted controls, showed an infection-induced increase in serum corticosterone concentration, while groups B and C did not. Group C mice showed a substantially greater infection-induced increase in serum interferon-gamma compared with the other fasted and non-fasted control groups.


Effects of a Soy Protein Diet on Exercise-Induced Muscle Protein Catabolism in Rats

We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise. In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of mu- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma (P < 0.05). Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet (P < 0.05), indicating that this increase inhibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise. Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.


A Cysteine Protease Inhibitor Prevents Suspension-Induced Declines in Bone Weight and Strength in Rats

In this study, we examined the effects of a potent cysteine protease inhibitor, N-(L-3-trans-carboxyoxirane-2-cabonyl)-L-leucine-4-aminobutylamide (E-64a), on bone weight and strength in tail-suspended rats. We first administered a vehicle or 4 or 8 mg/rat of E-64a to rats fed with a low calcium diet for 7 wks to determine effective doses of E-64a on bone resorption in vivo. Femoral cathepsin K-like activity and serum hydroxyproline level in rats fed with a low calcium diet were significantly higher than those in rats fed with a standard diet. The intraperitoneal injection of 8 mg/rat of E-64a to rats decreased their serum calcium and hydroxyproline concentrations after 3 to 6 hrs in parallel with changes in femoral cathepsin K-like activity, while 4 mg/rat of E-64a had weaker effects on these parameters. Based on these results, we injected 8 mg/rat of E-64a to tail-suspended rats twice a day for 2 wks and compared the results with those of treatment with 1 mg/rat of etidronate, a bisphosphonate, twice a week. In tail-suspended rats, femoral weight and strength, assessed by three-point bending test, significantly decreased from Day 5 to 21, while femoral cathepsin K-like activity and serum calcium and hydroxyproline concentrations did not change. E-64a inhibited femoral cathepsin K-like activity in tail-suspended rats, but etidronate did not. E-64a as well as etidronate significantly prevented the suspension-induced declines in bone weight and strength. However, more frequent injection and higher doses were required for E-64a to exhibit significant efficacy of antiresorption, compared with those of etidronate. Our results suggest that a cysteine protease inhibitor could improve suspension-induced osteopenia by inhibiting cathepsin K-like activity in bone; however, it needs several improvements in the effect as a clinical drug.


Toll-like receptor 4 regulates gastric pit cell responses to Helicobacter pylori infection

Gastric pit cells express mitogen oxidase1 (Mox1) and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phoxes). Helicobacter pylori (Hp) lipopolysaccharide (LPS) is a potent up-regulator of the Mox 1 oxidase. In this study, we examined the expression levels of several key members of the Toll-like receptor (TLR) family in primary cultures of guinea pig gastric pit cells. These cells expressed the TLR4 mRNA. Immunoblot analysis and immunofluorescence histochemistry with an anti-TLR4 antibody showed that gastric pit cells possessed significant amounts of TLR4 protein preferentially on the plasma membrane. In contrast, the cells did not express the TLR2 and TLR9 transcripts and did not contain detectable amounts of TLR2 protein. Neither peptidoglycan from Staphylococcus aureus nor Hp DNA with the CpG motif up-regulated Mox1 oxidase activity. Hp LPS activated nuclear factor-kappa B in association with the expression of cyclooxygenase II and tumor necrosis factor alpha transcripts. These findings suggest that TLR4 may play a crucial role in the initiation of inflammatory responses of gastric pit cells against Hp infection.


Helicobacter pylori lipopolysaccharide from type 1, but not type 2 strains, stimulates apoptosis of cultured gastric mucosal cells

The cag pathogenicity island (cag PAI) genes are a major determinant of virulence of Helicobacter pylori (Hp). Lipopolysaccharide (LPS) purified from the cag PAI-positive (type I) strains induced apoptosis of primary cultures of guinea pig gastric mucosal cells. Lipid A catalyzed this apoptosis. These cells expressed the Toll-like receptor 4 (TLR4) mRNA and its protein, and type I Hp LPS phosphorylated transforming growth factor beta-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in association with up-regulation of the TLR4 expressions, suggesting that type I Hp LPS evoked distinct TLR4 signaling. In contrast, Hp LPS from type II strains with complete or partial deletion of the cag PAI genes did not phosphorylate TAK1 and TAB1 and failed to induce apoptosis. Accelerated apoptosis of gastric epithelial cells is one of the important events relevant to chronic, atrophic gastritis caused by Hp infection. The difference in proapoptotic action of LPS between the type I and II strains may support an important role of the cag PAI genes in the pathogenesis of gastric lesions caused by Hp infection.


Impaired Vitamin A-Mediated Mucosal IgA Response in IL-5 Receptor-Knockout Mice

To clarify actions of vitamin A on mucosal immunity associated with interleukin-5 (IL-5), we examined effects of vitamin A on mucosal IgA level in IL-5 receptor alpha-chain-knockout (IL-5Ralpha(-/-)) mice. Daily supplementation of retinyl acetate (1 mg/mouse) increased Th2 cytokine levels and a number of their positive cells in the small intestinal mucosa of IL-5Ralpha(-/-) mice, as observed in wild-type or IL-5Ralpha(+/-) mice. Wild-type and heterozygous mice increased the IgA level and a number of IgA-containing cells in the mucosa in response to the vitamin A treatment, but not in IL-5Ralpha(-/-) mice. Retinyl acetate increased anti-cholera toxin (CT) IgA level in the mucosa of wild-type mice, improving their survival rate after an exposure to 0.4 mg of CT. However, retinyl acetate failed to induce resistance to CT toxicity in IL-5Ralpha(-/-) mice. Our results suggest that IL-5 may play an important role in an action of vitamin A on mucosal IgA system.

Biochemical and Biophysical Research Communications


Space Shuttle flight (STS-90) enhances dagradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway


Interleukin-1β enhances retinoic acid-mediated expression of bone-type alkaline phosphatase in rat IEC-6 cells

We previously showed that vitamin A upregulated the expression of bone-type alkaline phosphatase (ALP) in fetal rat small intestine and rat intestinal IEC-6 cells. In this study, we examined interactions between retinoic acid (RA) and several growth factors/cytokines on the isozyme expression in IEC-6 cells. Epidermal growth factor and interleukins (ILs)-2, -4, -5, and -6 completely blocked the RA-mediated increase in ALP activity. In contrast, IL-1beta markedly increased the activity, protein, and mRNA of the bone-type ALP only when RA was present. IL-1beta and/or RA did not change the type 1 IL-1 receptor transcript level, whereas IL-1beta enhanced the RA-induced expressions of retinoic acid receptor-beta (RAR-beta) and retinoid X receptor-beta (RXR-beta) mRNAs and RA-mediated RXR response element binding. The synergism of IL-1beta and RA on ALP activity was completely blocked by protein kinase C (PKC) inhibitors. Our results suggest that IL-1beta may modify the ALP isozyme expression in small intestinal epithelial cells by stimulating PKC-dependent, RAR-beta- and/or RXR-beta-mediated signaling pathways.

American Journal of Physiology, Gastrointestinal and Liver Physiology


Vitamin A Prevents the Decline in Immunoglobulin A and Th2 Cytokine Levels in Small Intestinal Mucosa of Protein-Malnourished Mice1

Biochemical and Molecular Action of Nutrients


Macrophage Colony-Stimulating Factor Stimulates Synthesis and Secretion of a Mouse Homolog of a Human IgE-Dependent Histamine-Releasing Factor by Macrophages In Vitro and In Vivo


Macrophage colony-stimulating factor stimulates synthesis and secretion of a mouse homolog of a human IgE-dependent histamine-releasing factor by macrophages in vitro and in vivo.

Treatment of murine resident peritoneal macrophages with macrophage-CSF (M-CSF) up-regulated the synthesis of a discrete set of proteins, including a 26-kDa protein (p26). The sequence of 20 NH2-terminal amino acids of the purified p26 was identical with the mouse homolog of a human IgE-dependent histamine-releasing factor (HRF). Among macrophage activators tested (M-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, IFN-gamma, and LPS), only M-CSF could up-regulate the p26 HRF synthesis by cultured macrophages. M-CSF not only increased the levels of p26 HRF mRNA and protein, but also stimulated the secretion of an N-glycosylated p26 HRF with a m.w. of 30 kDa. Repeated injections of M-CSF into mouse peritoneal cavity for 4 days elicited macrophages expressing abundant p26 HRF. A single i.p. injection of M-CSF failed to increase the p26 HRF level in peritoneal macrophages of thioglycollate-, LPS-, or adjuvant-treated mice, while M-CSF challenge to OVA-immunized mice caused macrophage infiltration and overproduction of p26 HRF, similarly as did OVA challenge. The Ag-specific priming for enhanced synthesis and secretion of p26 HRF by M-CSF was also demonstrated in cultured macrophages prepared from OVA-immunized mice. An i.p. injection of M-CSF or recombinant p26 HRF triggered eosinophil recruitment, even in the absence of the Ag, in the sensitized mice, but not in normal mice. Furthermore, recombinant p26 HRF could induce eosinophilia without marked macrophage and lymphocyte infiltrations. Our results suggest that p26 HRF secreted by M-CSF-stimulated macrophages may be an important mediator for the late phase allergic inflammation.


Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system

Superoxide anion (O2-) plays an important role in gastric pathophysiology. The aims of this study were to identify O2--producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells. The amount of O2- was measured by the reduction of cytochrome c, and O2--producing cells were visualized by nitroblue tetrazolium reaction. Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox. Gastric pit cells, but not parietal cells, spontaneously released O2- at 50 nmol . mg protein-1 . h-1. NADPH or guanosine 5'-O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it. A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity. p47-phox and p67-phox were expressed in the cells. Live Helicobacter pylori organisms and their culture supernatants significantly increased the O2- release. Furthermore, H. pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide. The O2--dependent activation of nuclear factor κB occurred in these primed cells. Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase-like activity.


Glutathione depletion inhibits oxidant-induced activation of nuclear factor-kappa B,AP-1,and c-Jun/ATF-2 in cultured guinea-pig gastric epithelial cells

The aim of this study was to reveal the role of intracellular glutathione in the oxidative stress responses of gastric epithelial cells. Metabolic radiolabeling with L-[35S]methionine and analysis of synthesized proteins by gel electrophoresis and fluorography showed that upon exposure to hydrogen peroxide (H2O2) or diamide, primary cultures of guinea-pig gastric epithelial cells rapidly induced several undefined proteins, as well as heat shock proteins. When intracellular glutathione was depleted to less than 10% of the control value by treatment with buthionine-[S,R]-sulfoximine, these inductions were completely inhibited. Gel mobility shift assay demonstrated that H2O2 and diamide rapidly activated nuclear factor-kappa B (NF-kappaB), and diamide activated activator protein (AP)-1, and c-Jun/activating transcription factor (ATF)-2, suggesting that the response may be coupled to these reduction-oxidation (redox)-sensitive transcription factors, as well as heat shock transcription factor 1. The activations of NF-kappaB, AP-1, and c-Jun/ATF-2 by the oxidants did not occur in glutathione-depleted cells. Northern blot analysis showed that glutathione depletion markedly or completely suppressed the diamide-induced expression of c-fos and c-jun mRNAs. These results suggest that intracellular glutathione redox may participate in the initiation of oxidative stress responses; thereby, it plays an important role in gastric mucosal defense.


Cultured guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system.


Vitamin A Up-Regulates Expression of Bone-Type Alkaline Phosphatase in a Rat Small Intestinal Crypt Cell Line and Fetal Rat Small Intestine

Biochemical and Molecular Roles of Nutrients


Oxidant-lnduced Activation of Nuclear Factor-Kappa B in Cultured Guinea Pig Gastric Epithelial Cells


Geranylgeranylacetone induces heat shock proteins. in cultured guinea pig gastric mucosal cells and rat gastric mucosa


Induction of heat shock proteins and their possible roles in macrophages during activation by macrophage colony-stimulating factor


Amino acids and glucose differentially increased extracellular 5-hydroxyindoleacetic acid of the rat brain.

Journal of Nutritional Science and Vitaminology


Efficacy of All-trans-β-Carotene, Canthaxanthin, and All-trans, 9-cis-, and 4-Oxoretinoic Acids in Inducing Differentiation of an F9 Embryonal Carcinoma RARβ-lacZ Reporter Cell Line


Alteration of the Respiratory Burst and Phagocytosis of Macrophages under Protein Malnutrition


Interaction of ebselen with glutathione S-transferase and papain in vitro

The interaction of ebselen(2-phenyl-1,2-benzisoselenazol-3(2H)-one) with rat liver cytosolic glutathione S-transferases (GSTs) and the plant cysteine protease, papain, was studied as cysteine residues are important for the activity of these enzymes. The capacity of GST 1-2 and 3-4 for ebselen binding is similar (1.5 mol ebselen/mol GST isozyme), while GST 2-2 and GST 7-7 bind 0.3 and more than 2.0 mol ebselen/mol GST isozyme, respectively. Ebselen does not bind to N-ethylmaleimide-treated GST, and its binding to GST is prevented by 5 mM thiols. Ebselen irreversibly inactivates the different GST isozymes with a second order rate constant ranging from 20 to 2250 M-1 sec-1 for the different subunits. GST inhibition by ebselen is partially restored by 5 mM thiols. Ebselen binds to untreated papain and to cysteine-treated papain at a ratio of about 0.1 and 0.75 mol ebselen/mol papain, respectively. Ebselen does not bind to N-ethylmaleimide-treated papain, and its binding to papain is interfered with by added thiols. Papain is inactivated by ebselen with a second order rate constant of 1800 M-1 sec-1 in the absence of thiols. However, in the presence of GSH, 2-mercaptoethanol or sodium borohydride, ebselen exerts an activating effect on papain. The binding of ebselen by a seleno-sulfide bond to cysteine residues of GSTs and papain leads to their inactivation.


Interaction of albumin-bound ebselen with rat liver glutathione S-transferase and microsomal proteins

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a selenium-containing heterocyclic compound, transported bound to albumin in blood. In this report, we demonstrate the transfer of ebselen from its albumin complex to rat liver cytosolic glutathione-S-transferase (GST) and microsomal proteins, as compared to papain. Free ebselen binds to the sulfhydryl groups of proteins, such as GST and papain [Nikawa, T., Schuch, G., Wagner, G. & Sies, H. Biochem. Pharmacol. (in press)]. 2-Mercaptoethanol and N-ethylmaleimide interfere with ebselen binding to microsomal proteins, suggesting that ebselen also binds to the sulfhydryl groups of microsomal proteins. Ebselen is transferred from its albumin complex to rat liver cytosolic GST and microsomal proteins, but not to papain. These results suggest the possibility that ebselen is transferred from albumin to other proteins using their sulfhydryl groups in vivo.

Biochemistry and Molecular Biology International


Participation of cathepsin L on bone resorption


Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro


システインプロテアーゼインヒビターによる骨粗鬆症治療の可能性

システインプロテアーゼインヒビターによる骨粗鬆症治療の可能性


Purification and characterization of cathepsin J from rat liver


Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo

エポキシサクシニル構造を有するE-64は協力なシステインプロテアーゼインヒビターである．この側鎖を修飾することによりin vitroでシステインプロテアーゼの中でカテプシンBのみを特異的に阻害する新規E-64誘導体を開発した．


Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vivo


Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants

For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well-conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxynucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G-50 and HPLC DEAE-5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N-terminal amino acid sequences and elution positions on ion-exchange chromatography of the products. The Ki values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N-terminal region of cystatin for inhibitory activities on cysteine proteinases.

内在性のシステインプロテアーゼインヒビターであるシスタチンの阻害活性部位を遺伝子工学的手法で決定した．シスタチンファミリーでよく保存されているQVVAG配列をほかのアミノ酸に置換した変異体を3種類作成し大腸菌に発現させた．いずれの変異体も阻害活性を失わなかったことから，QVVAG配列は直接的にはプロテアーゼの阻害に関与しないことを明らかにした．