An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

Rat cytosolic sialidase is expressed at elevated levels in skeletal muscle and is believed to play a role in the myogenic differentiation of muscle cells. Here, we observed varying levels of enhancement of sialidase activity in the presence a range of divalent cations. In particular, a significant enhancement of activity was observed in the presence of Ca2+. Conversely, inhibition of the sialidase activity was found when the enzyme was incubated in the presence of Cu2+, EDTA, and a range of carbohydrate-based inhibitors. Finally, an investigation of the enzymatic hydrolysis of a synthetic substrate, 4-methylumbelliferyl N-acetyl-alpha-D-neuraminide, by 1H NMR spectroscopy revealed that the reaction catalysed by rat skeletal muscle cytosolic sialidase proceeds with overall retention of anomeric configuration. This result further supports the notion that all sialidases appear to be retaining enzymes.     

Novel aspects of osteoclast activation and osteoblast inhibition in myeloma bone disease

Increased bone resorption is a major characteristic of multiple myeloma and is caused by osteoclast activation and osteoblast inhibition (uncoupling). Myeloma cells alter the local regulation of bone metabolism by increasing the receptor activator of NF-kappaB ligand (RANKL) and decreasing osteoprotegerin expression within the bone marrow microenvironment, thereby stimulating the central pathway for osteoclast formation and activation. In addition, they produce the chemokines MIP-1alpha, MIP-1beta, and SDF-1alpha, which also increase osteoclast activity. On the other hand, myeloma cells suppress osteoblast function by the secretion of osteoblast inhibiting factors, e.g., the Wnt inhibitors DKK-1 and sFRP-2. Moreover, they inhibit differentiation of osteoblast precursors and induce apoptosis in osteoblasts. The resulting bone destruction releases several cytokines, which in turn promote myeloma cell growth. Therefore, the inhibition of bone resorption could stop this vicious circle and not only decrease myeloma bone disease, but also the tumor progression.     

TCR engagement increases hypoxia-inducible factor-1 alpha protein synthesis via rapamycin-sensitive pathway under hypoxic conditions in human peripheral T cells

Peripheral T cells encounter rapid decrease in oxygen tension because they are activated by Ag recognition and migrate into inflammatory sites or tumors. Activated T cells, therefore, are thought to have such machineries that enable them to adapt to hypoxic conditions and execute immune regulation in situ. We have recently shown that survival of CD3-engaged human peripheral blood T cells is prolonged under hypoxic conditions and hypoxia-inducible factor-1 (HIF-1) and its target gene product adrenomedullin play a critical role for the process. It is also shown that hypoxia alone is not sufficient, but TCR-mediated signal is required for accumulation of HIF-1alpha in human peripheral T cells. In the present study, we showed that TCR engagement does not influence hypoxia-dependent stabilization but stimulates protein synthesis of HIF-1alpha, most possibly via PI3K/mammalian target of rapamycin system, and that expression of HIF-1alpha and its target genes is blocked by treatment with rapamycin. Since some of those gene products, e.g., glucose transporters and phosphoglycerokinase, are considered to be essential for glycolysis and energy production under hypoxic conditions and adequate immune reaction in T cells, this TCR-mediated synthesis of HIF-1alpha may play a pivotal role in peripheral immune response. Taken together, our results may highlight a novel aspect of downstream signal from Ag recognition by TCR and a unique pharmacological role of rapamycin as well.     

Localization of Src homology 2 domain-containing phosphatase 1 (SHP-1) to lipid rafts in T lymphocytes: functional implications and a role for the SHP-1 carboxyl terminus

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.     

Zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses TNF-alpha and IL-1 beta production in monocytes by elevation of guanosine 3',5'-cyclic monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.     

ABCG5 and ABCG8 require MDR2 for secretion of cholesterol into bile

The major pathway for the removal of cholesterol from the body is via secretion into the bile. Three members of the ATP binding cassette (ABC) family, ABCG5 (G5), ABCG8 (G8), and ABCB4 (MDR2), are required for the efficient biliary export of sterols. Here, we examined the interdependence of these three ABC transporters for biliary sterol secretion. Biliary lipid levels in mice expressing no MDR2 (Mdr2-/- mice) were compared with those of Mdr2-/- mice expressing 14 copies of a human G5 (hG5) and hG8 transgene (Mdr2-/-;hG5G8Tg mice). Mdr2-/- mice had only trace amounts of biliary cholesterol and phospholipids. The Mdr2-/-;hG5G8Tg mice had biliary cholesterol levels as low as those of Mdr2-/- mice. Thus, MDR2 expression is required for G5G8-mediated biliary sterol secretion. To determine whether the reduction in fractional absorption of dietary sterols associated with G5G8 overexpression is secondary to the associated increase in biliary cholesterol, we compared the fractional absorption of sterols in Mdr2-/-;hG5G8Tg and hG5G8Tg animals. Inactivation of MDR2 markedly attenuated the reduction in fractional sterol absorption associated with G5G8 overexpression. These results are consistent with the notion that increased biliary cholesterol secretion contributes to the reduction in fractional sterol absorption associated with G5G8 overexpression.     

Inhibition of cholesterol absorption by the combination of dietary plant sterols and ezetimibe: effects on plasma lipid levels

Consumption of plant sterols and treatment with ezetimibe both reduce cholesterol absorption in the intestine. However, the mechanism of action differs between the two treatments, and the consequences of combination treatment are unknown. Therefore, we performed a double-blind, placebo-controlled, crossover study for the plant sterol component with open-label ezetimibe treatment. Forty mildly hypercholesterolemic subjects were randomized to the following treatments for 4 weeks each: 10 mg/day ezetimibe combined with 25 g/day control spread; 10 mg/day ezetimibe combined with 25 g/day spread containing 2.0 g of plant sterols; 25 g/day spread containing 2.0 g of plant sterols; and placebo treatment consisting of 25 g/day control spread. Combination treatment of plant sterols and ezetimibe reduced low density lipoprotein cholesterol (LDL-C) by 1.06 mmol/l (25.2%; P < 0.001) compared with 0.23 mmol/l (4.7%; P = 0.006) with plant sterols and 0.94 mmol/l (22.2%; P < 0.001) with ezetimibe monotherapy. LDL-C reduction conferred by the combination treatment did not differ significantly from ezetimibe monotherapy (-0.12 mmol/l or -3.5%; P = 0.13). Additionally, the plasma lathosterol-to-cholesterol ratio increased with all treatments. Sitosterol and campesterol ratios increased after plant sterol treatment and decreased upon ezetimibe and combination therapy. Our results indicate that the combination of plant sterols and ezetimibe has no therapeutic benefit over ezetimibe monotherapy in subjects with mild hypercholesterolemia.     

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.     

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.