The cysteine protease legumain is upregulated by vitamin D and regulates vitamin D metabolism

Legumain is a lysosomal asparaginyl endopeptidase involved in different biological processes and pathogenesis of several malignant and non-malignant diseases. Although an increasing number of proteins have been identified as substrates of legumain, the upstream mechanisms regulating the expression and function of legumain are not well-understood. Here, we provide in vitro and in vivo data showing that vitamin D3 (VD3) enhances legumain expression and function. In turn, legumain alters VD3 bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP) and thereby playing a role in the regulation of VD3 metabolism. Active VD3 (1,25(OH)2D3) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Positive regulation of legumain was also observed in vivo, evidenced by increased expression of legumain mRNA in liver and spleen, as well as increased legumain activity in the kidneys, and increased level of circulating legumain in serum from mice treated with 25(OH)D3 (50 µg/kg, subcutaneous) for 8 days, as compared to vehicle-treated mice. We further showed that legumain cleaved VDBP forming a 45 kDa VDBP fragment in vitro when purified VDBP (55 kDa) was incubated with purified active legumain. In vivo, no VDBP cleavage was found in kidneys or liver from legumain deficient mice (Lgmn-/-), whereas VDBP was cleaved in wild type control mice (Lgmn+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D3 and total VD3 and altered expression of key renal enzymes involved in VD3 metabolism (CYP24A1 and CYP27B1). Taken together, our data suggest existence of a regulatory interplay between VD3 and legumain

Bradykinin-Induced Shock Increase Exhaled Nitric Oxide, Complement Activation and Cytokine Production in Pigs

Bradykinin is an important mediator in blood pressure regulation, ischemic precondition and capillary leakage, allergy, anaphylaxis, inflammation, and nociception, at least partly via the generation of nitric oxide (NO). Macrophages are particularly abundant in the porcine lung circulation. Upon bradykinin binding macrophages release cytokines and endothelial cells increase plasma leakage. Both cells produce NO. The complement, hemostatic, fibrinolytic and kinin plasma cascade systems crosstalk and interacts with many inflammatory systems. In the present study we investigated the effect of the shock induced by intravenously infused bradykinin on the cascade systems, cytokines, plasma leakage and exhaled NO in pigs. The metabolite of bradykinin, BK1-5, was measured in plasma by a sensitive, specific and reliable liquid chromatography-mass spectrometry method to verify exposure and in vivo metabolism of bradykinin. We show for the first time in vivo how bradykinin exposure induced shock and increased exhaled NO, activated complement and hemostasis and induced cytokine production and capillary leakage. The results broaden our understanding of how bradykinin activates endothelial cells and macrophages to induce shock and inflammation. This should encourage further studies. This research was first published in the Journal of Cardiology and Clinical Research. © JSciMed Central

Simvastatin Inhibits Glucose Metabolism and Legumain Activity in Human Myotubes

<div><p>Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the <i>Musculus obliquus internus abdominis</i> of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with [¹⁴C]glucose or [³H]deoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of legumain. Dysregulation of glucose metabolism and decreased legumain activity by simvastatin points out new knowledge about the effects of statins on skeletal muscle, and may contribute to the understanding of the myotoxicity observed by statins.</p></div

Legumain activity and expression after treatment with simvastatin, mevalonolactone, geranylgeranyl pyrophosphate and/or farnesyl pyrophosphate.

<p>Differentiated myotubes were incubated for 48(0–40 µM) with or without mevalonolactone (50 or 1000 µM; ML), geranylgeranyl pyrophosphate (3 µM; GGPP) or farnesyl pyrophosphate (3 µM; FPP) prior to harvesting at day 7. <b>A.</b> Dose-dependent effects of simvastatin (0–40 µM) on legumain activity (n = 3–15, student t-test, *p<0.05 vs. 0 µM). <b>B.</b> Effects on legumain activity caused by 30 µM simvastatin (S alone) with or without ML, GGPP or FPP. The data are compared and normalized to untreated myotubes (n = 6–12, student t-test, *p<0.05 vs. S alone; n = 12, paired student t-test, #p<0.01 vs. untreated). <b>C.</b> Effects on legumain expression caused by 30 µM simvastatin (S alone) with or without ML, GGPP or FPP. Equal amounts of total proteins (10 µg) of cell lysates were separated and immunoblot analyzes were performed. One representative immunoblot is shown and band intensity analysis are normalized to 36 or 56 kDa legumain immunobands, respectively, (n = 4–7, student t-test, *p<0.05 vs. S alone).</p

Reduced glucose uptake in myotubes after treatment with simvastatin.

<p><b>A.</b> and <b>B.</b> Differentiated myotubes were incubated for 48 h with simvastatin (S) with or without mevalonolactone (1 mM; ML) and compared to untreated control (C), prior to incubation with radiolabeled substrates at day 7. <b>A.</b> Dose-response of simvastatin on glucose uptake after 4 h incubation with [¹⁴C(U)]glucose (0.2 mM, 21.5 kBq/ml) using a multiwell trapping device. Radioactivity was measured in cell lysates and in trapped CO₂ and corrected for total proteins (n = 3–8, student t-test, *p<0.05 vs. untreated). <b>B.</b> Effects of 5 µM simvastatin with or without ML on uptake of [³H]deoxyglucose (10 µM, 37 kBq/ml, 15 min) (n = 3, student t-test, *p<0.05 vs. S). <b>C.</b> Differentiated myotubes were pre-incubated for 48 h with simvastatin (30 µM) before subcellular fractionation. Equal amount of total proteins from the membrane fraction were loaded to the gel and immunoblot analysis performed. LAMP-2 was used as loading control. Quantification of GLUT1 band intensity is shown, corrected for LAMP-2 and normalized to untreated control (C) (n = 4). <b>D.</b> Myotubes were treated with 0–40 µM simvastatin with or without 50 µM or 1 mM ML for 48 h before cell viability was analyzed at day 7. The cells were incubated with MTS-reagent for 2 h before absorbance at 490 nm was measured (n = 3, student t-test, *p<0.05 vs. untreated control (C)).</p