SLIT3 promotes myogenic differentiation as a novel therapeutic factor against muscle loss.

BACKGROUND: Sarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently discovered as a novel therapeutic factor against osteoporosis, and a SLIT3 fragment containing the second leucine-rich repeat domain (LRRD2) had a therapeutic efficacy against osteoporosis. However, a role of SLIT3 in the skeletal muscle is unknown. METHODS: Skeletal muscle mass, strength, and/or physical activity were evaluated in Slit3-/- , ovariectomized, and aged mice, based on the measurements of muscle weight and grip strength, Kondziella's inverted hanging test, and/or wheel-running test. Skeletal muscles were also histologically evaluated by haematoxylin and eosin staining and/or immunofluorescence. The ovariectomized and aged mice were intravenously injected with recombinant SLIT3 LRRD2 for 4 weeks. C2C12 cells were used to know cellular effects of SLIT3, such as in vitro myogenesis, fusion, cell viability, and proliferation, and also used to evaluate its molecular mechanisms by immunocytochemistry, immunoprecipitation, western blotting, real-time PCR, siRNA transfection, and receptor-ligand binding ELISA. RESULTS: Slit3-deficient mice exhibited decreased skeletal muscle mass, muscle strength, and physical activity. The relative masses of gastrocnemius and soleus were lower in the Slit3-/- mice (0.580 ± 0.039% and 0.033 ± 0.003%, respectively) than those in the WT littermates (0.622 ± 0.043% and 0.038 ± 0.003%, respectively) (all, P < 0.05). Gastrocnemius of Slit3-/- mice showed the reduced number of Type I and Type IIa fibres (all, P < 0.05), but not of Type IIb and Type IIx fibres. SLIT3 activated ß-catenin signalling by promoting its release from M-cadherin, thereby increasing myogenin expression to stimulate myoblast differentiation. In vitro experiments involving ROBO2 expression, knockdown, and interaction with SLIT3 indicated that ROBO2 functions as a SLIT3 receptor to aid myoblast differentiation. SLIT3 LRRD2 dissociated M-cadherin-bound ß-catenin and up-regulated myogenin expression to increase myoblast differentiation, in a manner similar to full-length SLIT3. Systemic treatment with SLIT3 LRRD2 increased skeletal muscle mass in both ovariectomized and aged mice (all, P < 0.05). The relative masses of gastrocnemius and soleus were higher in the treated aged mice (0.548 ± 0.045% and 0.033 ± 0.005%, respectively) than in the untreated aged mice (0.508 ± 0.016% and 0.028 ± 0.003%, respectively) (all, P < 0.05). SLIT3 LRRD2 treatment increased the hanging duration of the aged mice by approximately 1.7-fold (P < 0.05). CONCLUSIONS: SLIT3 plays a sarcoprotective role by activating ß-catenin signalling. SLIT3 LRRD2 can potentially be used as a therapeutic agent against muscle loss.

DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

Effect of rice cell-derived human granulocyte-macrophage colony-stimulating factor on 5-fluorouracil-induced mucositis in hamsters.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.

Purification and characterization of a cytosolic Ca(2+)-independent phospholipase A(2) from bovine brain.

The Ca(2+)-independent phospholipase A(2) (iPLA(2)) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA(2) from bovine brain. iPLA(2) was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA(2) activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on two-dimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)), Triton X-100, iron, and Ca(2+). However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA(2), and adenosine triphosphate (ATP). The spot with the iPLA(2) activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA(2).

C(6)-ceramide enhances phagocytic activity of Kupffer cells through the production of endogenous ceramides.

Ceramide has been suggested to be not only a tumor-suppressive lipid but also a regulator of phagocytosis. We examined whether exogenous cell-permeable C(6)-ceramide enhances the phagocytic activity of Kupffer cells (KCs) and affects the level of cellular ceramides. Rat KCs were isolated by collagenase digestion and differential centrifugation, using Percoll system. Phagocytic activity was measured by FACS analysis after incubating KCs with fluorescence-conjugated latex beads, and the level of cellular ceramide was analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). In this study we found that permeable C(6)-ceramide increases the cellular levels of endogenous ceramides via a sphingosine-recycling pathway leading to enhanced phagocytosis by KCs.

Neutral sphingomyelinase 2 induces dopamine uptake through regulation of intracellular calcium.

Ceramide serves as a second messenger produced from sphingomyelin by the activation of sphingomyelinase (SMase). Here, we suggest that neutral SMase 2 (nSMase2) may regulate dopamine (DA) uptake. nSMase2 siRNA-transfected PC12 cells showed lower levels of nSMase activity and ceramide than scramble siRNA-transfected and control cells. Interestingly, transfection of nSMase2 siRNA or pretreatment with the nSMase2-specific inhibitor GW4869 resulted in decreased DA uptake. Reciprocally, exposure of PC12 cells to cell-permeable C(6)-ceramide induced a concentration-dependent increase in DA uptake. Removal of extracellular calcium by EGTA increased DA uptake in scramble-transfected and control cells, but not in nSMase2 siRNA-transfected or GW4869-pretreated cells. Moreover, siRNA-transfected cells showed higher levels of intracellular calcium than scramble cells, while C(6)-ceramide treatment resulted in decreased intracellular calcium compared to vehicle treatment alone. Taken together, these data suggest that nSMase2 may increase DA uptake through inducing ceramide production and thereby decreasing intracellular calcium levels.

Hypoxia-induced neuronal apoptosis is mediated by de novo synthesis of ceramide through activation of serine palmitoyltransferase.

Cellular hypoxia can lead to cell death or adaptation and has important effects on development, physiology, and pathology. Here, we investigated the role and regulation of ceramide in hypoxia-induced apoptosis of SH-SY5Y neuroblastoma cells. Hypoxia increased the ceramide concentration; subsequently, we observed biochemical changes indicative of apoptosis, such as DNA fragmentation, nuclear staining, and poly ADP-ribose polymerase (PARP) cleavage. The hypoxic cell death was potently inhibited by a caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone). l-Cycloserine, a serine palmitoyltransferase (SPT) inhibitor, and fumonisin B(1) (FB(1)), a ceramide synthase inhibitor, inhibited the hypoxia-induced increase in ceramide, indicating that the increase occurred via the de novo pathway. Hypoxia increased the activity and protein levels of SPT2, suggesting that the hypoxia-induced increase in ceramide is due to the transcriptional up-regulation of SPT2. Specific siRNA of SPT2 prevented hypoxia-induced cell death and ceramide production. However, hypoxia also increased the cellular level of glucosylceramide, which was inhibited by a glucosylceramide synthase (GCS) inhibitor and specific siRNA, but not a ceramidase inhibitor. The increase in glucosylceramide was accompanied by increases in both PARP cleavage and DNA fragmentation. Together, the current results suggest that both SPT and GCS may regulate the cellular level of ceramide, and thus may be critical enzymes for deciding the fate of the cells exposed to hypoxia.

Subcutaneous Four-Week Repeated Dose Toxicity Studies of Rice Cell-Derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor in Rats.

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 µg/kg subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.

Identification of three competitive inhibitors for membrane-associated, Mg2+-dependent and neutral 60 kDa sphingomyelinase activity.

Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purified N-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purified N-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on the in vitro assay, three plant extracts significantly inhibited the N-SMase activity in a time- and concentration-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts of Abies nephrolepis, Acer tegmentosum, and Ginkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of 11.9 microg/ mL, 9.4 microg/mL, and 12.9 microg/mL, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.