Prolonged submaximal exercise induces isoform-specific Na+-K+-ATPase mRNA and protein responses in human skeletal muscle

This study investigated effects of prolonged submaximal exercise on Na⁺-K⁺-ATPase mRNA and protein expression, maximal activity, and content in human skeletal muscle. We also investigated the effects on mRNA expression of the transcription initiator gene, RNA polymerase II (RNAP II), and key genes involved in protein translation, eukaryotic initiation factor-4E (eIF-4E) and 4E-binding protein 1 (4E-BP1). Eleven subjects (6 men, 5 women) cycled at 75.5% (SD 4.8%) peak O₂ uptake and continued until fatigue. A vastus lateralis muscle biopsy was taken at rest, fatigue, and 3 and 24 h postexercise. We analyzed muscle for Na⁺-K⁺-ATPase α1, α2, α3, β1, β2, and β3, as well for RNAP II, eIF-4E, and 4E-BP1 mRNA expression by real-time RT-PCR and Na⁺-K⁺-ATPase isoform protein abundance using immunoblotting. Muscle homogenate maximal Na⁺-K⁺-ATPase activity was determined by 3-O-methylfluorescein phosphatase activity and Na⁺-K⁺-ATPase content by [³H]ouabain binding. Cycling to fatigue [54.5 (SD 20.6) min] immediately increased {alpha}3 (P = 0.044) and {beta}2 mRNA (P = 0.042) by 2.2- and 1.9-fold, respectively, whereas {alpha}1 mRNA was elevated by 2.0-fold at 24 h postexercise (P = 0.036). A significant time main effect was found for α3 protein abundance (P = 0.046). Exercise transiently depressed maximal Na⁺-K⁺-ATPase activity (P = 0.004), but Na⁺-K⁺-ATPase content was unaltered throughout recovery. Exercise immediately increased RNAP II mRNA by 2.6-fold (P = 0.011) but had no effect on eIF-4E and 4E-BP1 mRNA. Thus a single bout of prolonged submaximal exercise induced isoform-specific Na⁺-K⁺-ATPase responses, increasing α1, α3, and β2 mRNA but only α3 protein expression. Exercise also increased mRNA expression of RNAP II, a gene initiating transcription, but not of eIF-4E and 4E-BP1, key genes initiating protein translation.

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10–12 month lactation period, female fur seals suckle pups on shore for 2–3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 μm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (αS2-casein, β-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell–matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Induction of propranolol metabolism by Ginkgo biloba extract EGb 761 in rats

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. When used in the elderly, Ginkgo has a high potential for interactions with cardiovascular drugs. This study aimed to investigate the effects of the standard Ginkgo biloba extract (EGB 761) treatment on the pharmacokinetics of propranolol and its metabolism to form Ndesisopropylpropranolol (NDP) in rats. We also examined the activity and expression of cytochrome P450 (CYP) 1A and other CYPs in rats treated with EGb 761 at 10 and 100 mg/kg/day for 10 days. A single oral dose of propranolol (10 mg/kg) was administered on day 11 and the concentrations of both propranolol and NDP were determined using validated liquid chromatography-mass spectrometry (LC-MS) methods. The levels of mRNA and protein of various CYPs were determined by RT-PCR and Western blotting analysis, respectively. Pretreatment of EGb 761 at 100 mg/kg, but not 10 mg/kg, for 10 days significantly reduced the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (C max) of propranolol, whereas those values of NDP were significantly increased. CYP1A1, 1A2, 2B1/2, and 3A1 activities and gene expression in the rat liver were significantly increased in a dose-dependent manner by pretreatment with EGb 761. The ex-vivo formation of NDP in liver microsomes from rats pretreated with EGb 761 was markedly enhanced. The formation of NDP from propranolol in liver microsomes was significantly inhibited by α- naphthoflavone (ANF, a selective CYP1A2 inhibitor), but not by quinidine (a CYP2D inhibitor). These results indicated that EGb 761 pretreatment decreased the plasma concentrations of propranolol by accelerated conversion of parental drug to NDP due to induction of CYP1A2. EGb 761 pretreatment also significantly induced CYP2B1/2 and CYP3A1, suggesting potential interactions with substrate drugs for these two enzymes. Further study is needed to explore the potential for gingko-drug interactions and the clinical impact.

Squalene supplementation alters genes associated with liver cholesterol metabolism

Background – Squalene is a component of shark liver oil and has been speculated to have cholesterol reducing properties. High levels of total and LDL cholesterol have been shown to contribute to the development of chronic heart disease. The liver is central to the regulation of cholesterol metabolism and dietary intervention has long been recognized as a primary means to reduce the risks of chronic heart disease and related ailments.
Objectives – To determine the effect of dietary squalene supplementation on gene transcripts associated with liver cholesterol metabolism. Specifically the effect of squalene supplementation on mRNA levels for proteins that
regulate cholesterol biosynthesis (HMDH & ERG1), cholesterol elimination (SRB1), bile synthesis (CP7A1 & CP27A) and cholesterol excretion by the liver into bile (ABCG5 & ABCG8) was investigated.
Design – Rats (n=32) were divided into four groups and supplemented for 12 weeks. Groups one and two were fed a cholesterol rich diet for six weeks followed by six weeks of a cholesterol rich diet plus 1.75mg/day of squalene or 3.5 mg/day. Group three was fed a cholesterol rich diet for 12 weeks and group four was fed standard rat chow for 12 weeks. Blood lipid levels were monitored during the study and liver gene expression was determined at the
conclusion of the feeding trial via RT-PCR.
Outcomes – 3.5 mg/day of squalene lowered total and LDL cholesterol in rats consuming a cholesterol rich diet. This dose of squalene also resulted in constant levels of HMDH and ERG1 whereas the cholesterol rich diet halved mRNA levels of these enzymes. Furthermore 3.5 mg/day of squalene caused a greater than 3.0 fold increase in mRNA levels of the proteins SRB1, CP7A1, CP27A and ABCG5.
Conclusion – Dietary squalene supplementation at a dose of 3.5 mg/day lowers total and LDL cholesterol in rats consuming a cholesterol rich diet. These reductions in cholesterol levels may be due to increased cholesterol
elimination, bile synthesis and cholesterol excretion by the liver into bile mediated by changes in gene expression of key enzymes involved in these metabolic pathways

Lignin production and monolignol pathway gene expression is suppressed by abscisic acid during defence reactions of Arabidopsis

The phytohormone, abscisic acid (ABA) has been shown to influence the outcome of the interactions between various hosts with biotrophic and hemibiotrophic pathogens. Susceptibility to avirulent isolates can be induced by addition of low physiological concentrations of ABA to plants. In contrast, addition of ABA biosynthesis inhibitors induced resistance following challenge of plants by virulent isolates. ABA deficient mutants of Arabidopsis, such as aba1-1, were resistant to virulent isolates of Peronospora parasitica. In interactions of Arabidopsis with avirulent isolates of Pseudomonas syringae pv. tomato, susceptibility was induced following addition of ABA or imposition of drought stress. These results indicate a pivotal, albiet undefined, role for ABA in determining either susceptibility or resistance to pathogen attack. We have found that the production of the cell wall strengthening compound, lignin, is increased during resistant interactions of aba1-1 but suppressed in ABA induced susceptible interactions. Using RT-PCR and microarray analysis we have found down-regulation by ABA of key genes of the phenylpropanoid pathway especially of those genes involved directly in lignin biosynthesis. ABA also down-regulates a number of genes in other functional classes including those involved in defence and cell signalling.

Suppression by abscisic acid of lignin production and monolignol pathway gene expression in interactions of Arabidopsis with oomycete and bacterial pathogens

The phytohormone, abscisic acid (ABA) has been shown to influence the outcome of the interactions between various hosts with biotrophic and hemibiotrophic pathogens. Susceptibility to avirulent isolates can be induced in plants by addition of low physiological concentrations of ABA. In contrast, addition of ABA biosynthesis inhibitors induced resistance following challenge of plants by virulent isolates. ABA deficient mutants of Arabidopsis, such as aba1-1, were resistant to virulent isolates of Peronospora parasitica. In interactions of Arabidopsis with avirulent isolates of Pseudomonas syringae pv. tomato, susceptibility was induced following addition of ABA or imposition of drought stress. These results indicate a pivotal, albiet undefined, role for ABA in determining either susceptibility or resistance to pathogen attack. We have found that the production of the cell wall strengthening compound, lignin, is increased during resistant interactions of aba1-1 but suppressed in ABA-induced susceptible interactions. Using RT-PCR and microarray analysis we have found down-regulation by ABA of key genes of the phenylpropanoid pathway especially of those genes involved directly in lignin biosynthesis. ABA also down-regulates a number of genes in other functional classes including those involved in defence and cell signalling.

Identification of secreted proteins associated with obesity and type 2 diabetes in Psammomys obesus

Objective: Skeletal muscle produces a variety of secreted proteins that have important roles in intercellular communication and affects processes such as glucose homoeostasis. The objective of this study was to develop a novel Signal Sequence Trap (SST) in conjunction with cDNA microarray technology to identify proteins secreted from skeletal muscle of Psammomys obesus that were associated with obesity and type 2 diabetes (T2D).

Design: Secreted proteins that were differentially expressed between lean, normal glucose tolerant (NGT), overweight and impaired glucose tolerant (IGT) and obese, T2D P. obesus were isolated using SST in conjunction with cDNA microarray technology. Subsequent gene expression was measured in tissues from P. obesus by real-time PCR (RT-PCR).

Results: The SST yielded 1600 positive clones, which were screened for differential expression. A total of 91 (B6%) clones were identified by microarray to be differentially expressed between NGT, IGT and T2D P. obesus. These clones were sequenced to identify 51 genes, of which only 27 were previously known to encode secreted proteins. Three candidate genes not previously associated with obesity or type 2 diabetes, sushi domain containing 2, collagen and calcium-binding EGF domains 1 and periostin (Postn), as well as one gene known to be associated, complement component 1, were shown by RT-PCR to be differentially expressed in  skeletal muscle of P. obesus. Further characterization of the secreted protein Postn revealed it to be predominantly expressed in adipose tissue, with higher expression in visceral compared with subcutaneous adipose depots.

Conclusion: SST in conjunction with cDNA microarray technology is a powerful tool to identify differentially expressed secreted proteins involved in complex diseases such as obesity and type 2 diabetes. Furthermore, a number of candidate genes were identified, in particular, Postn, which may have a role in the development of obesity and type 2 diabetes.

Effect of carbohydrate ingestion on exercise-induced alterations in metabolic gene expression

Skeletal muscle possesses a high degree of plasticity and can adapt to both the physical and metabolic challenges that it faces. An acute bout of exercise is sufficient to induce the expression of a variety of metabolic genes, such as GLUT4, pyruvate dehydrogenase kinase 4 (PDK-4), uncoupling protein-3 (UCP3), and peroxisome proliferator-activated receptor-? coactivator 1 (PGC-1). Reducing muscle glycogen levels before exercise potentiates the effect of exercise on many genes. Similarly, altered substrate availability induces transcription of many of these genes. The purpose of this study was to determine whether glucose ingestion attenuates the exercise-induced increase in a variety of exercise-responsive genes. Six male subjects (28 ± 7 yr; 83 ± 3 kg; peak pulmonary oxygen uptake = 46 ± 6 ml·kg–1·min–1) performed 60 min of cycling at 74 ± 2% of peak pulmonary oxygen uptake on two separate occasions. On one occasion, subjects ingested a 6% carbohydrate drink. On the other occasion, subjects ingested an equal volume of a sweet placebo. Muscle samples were obtained from vastus lateralis at rest, immediately after exercise, and 3 h after exercise. PDK-4, UCP3, PGC-1, and GLUT4 mRNA levels were measured on these samples using real-time RT-PCR. Glucose ingestion attenuated (P < 0.05) the exercise-induced increase in PDK-4 and UCP3 mRNA. A similar trend (P = 0.09) was observed for GLUT4 mRNA. In contrast, PGC-1 mRNA increased following exercise to the same extent in both conditions. These data suggest that glucose availability can modulate the effect of exercise on metabolic gene expression.

Virulence determinants of infectious bursal disease virus

The very virulent (vv) pathotype of infectious bursal disease virus (IBDV) has spread rapidly throughout Europe, Asia, and the Middle East. Although Australia is currently unaffected, there remains the potential for incursion of an exotic isolate. The aim of this study was to identify putative virulence determinants of IBDV to facilitate the development of improved diagnostic assays for detection and characterisation of vvIBDV isolates. Sequencing of Indonesian vvIBDV Tasik94 revealed a unique substitution [ A�¨S222] in the hypervariable region (HVR) of viral protein (VP) VP2, which did not appear to impinge on virulence or antigenicity. Phylogenetic analyses indicated that Tasik94 was closely related to Asian and European vvIBDV strains. Extensive alignment of deduced protein sequences across the HVR of VP2 identified residuesI242 I256 and I294 as putative markers of the vv phcnotype. Comparison of the pathology induced by mildly-virulent Australian IBDV 002/73 and Indonesian vvIBDV Tasik94, revealed that histological lesions in the spleen, thymus and bone marrow were restricted to Tasik94-infected birds, suggesting the enhanced pathogenicity of vvIBDV might be attributed to replication in non-bursal lymphoid organs. The biological significance of the VP2 HVR in virulence was assessed using recombinant viruses generated by reverse genetics. Both genomic segments of Australian IBDV 002/73, and recombinant segment A constructs in which the HVR of 002/73 was replaced with the corresponding region of either tissue culture-adapted virus or vvIBDV (Tasik94), were cloned behind T7 RNA polymerase promoter sequences. In vitro transcription/translation of each construct resulted in expression of viral proteins. Co-transfection of synthetic RNA transcripts initiated replication of both tissue culture-adapted parental and recombinant viruses, however attempts to rescue non-adapted viruses in specific-pathogen-free (SPF) chickens were unsuccessful. Nucleotide sequence variation in the HVR of VP2 was exploited for the development of a new diagnostic assay to rapidly detect exotic IBDV isolates, including vvIBDV, using reverse transcription polymerase chain reaction (RT-PCR) amplification and Bmrl restriction enzyme digestion. The assay was capable of differentiating between endemic and exotic IBDV in 96% of 105 isolates sequenced to date.

Angiotensin II receptor imbalance associated with neonatal cardiac growth restriction is a prelude to adult cardiac hypertrophy

The Hypertrophic Heart Rat (HHR) displays spontaneous cardiomyocyte hypertrophy in association with an apparent reduction in myocyte number in adulthood. This suggests the possibility of reduced hyperplasia or increased apoptosis during early cardiac development. The angiotensin AT1 and AT2 receptor subtypes have been implicated in both cellular growth and apoptosis, but the precise mechanisms are unclear. The aim of this study was to determine the relationship between cardiac AngII receptor expression levels and neonatal cardiomyocyte growth and apoptotic responses in the HHR compared with the Normal Heart Rat (NHR) control strain. Cardiac tissues were freshly harvested from male HHR and NHR at several developmental stages (p2 and 4, 6, 8, 12wks). HHR cardiac weight indices were considerably smaller than NHR at day 2 (4.330.19 vs 5.010.08 mg/g), but ‘caught-up’ to NHR by 4 weeks (5.100.15 vs 5.160.11 mg/g). By 12 weeks, HHR hearts were 27% larger than NHR. Tissue AT1A and AT2 mRNA expression levels were quantified by real-time RT-PCR. Relative to NHR, HHR neonatal hearts exhibited a 4.6-fold higher AT2/AT1 mRNA expression ratio. Cultured neonatal cardiomyocytes were infected with AT1A and/or AT2 receptor-expressing adenoviruses to achieve a physiological level of receptor expression (150 fmol receptor protein/mg total cell protein). In addition, to emulate receptor expression in neonatal HHR hearts, cells were co-infected with AT1A and AT2 receptors at a 4:1 ratio. Apoptosis incidence was studied by morphological analysis after 72 hours exposure to 0.1 M AngII. When infected with the AT1A receptor alone, a higher proportion of HHR myocytes appeared apoptotic than NHR (22.7 4.1% vs 1.1 0.6%, P 0.001). This implies that intrinsic differences predispose HHR cells to accentuated AT1-mediated apoptosis. Interestingly, the bax-1/bcl-2 mRNA expression ratio was significantly higher (50%) in HHR neonatal hearts. When cells were co-infected with AT1A and AT2 receptors, evidence of apoptosis in HHR cells virtually disappeared (0.4 0.1%). These findings suggest a novel capacity of AT2 receptors to counteract accentuated AT1A receptor-induced apoptosis in the HHR in early cardiac growth.

Elevated dietary sodium intake exacerbates myocardial hypertrophy associated with cardiac-specific overproduction of angiotensin II

Introduction/hypothesis
Cardiac hypertrophy is an independent risk factor predictive of cardiovascular disease and is significantly associated with morbidity and mortality. The mechanism by which angiotensin II (Ang II) and dietary sodium exert additive effects on the development of cardiac hypertrophy is unclear. The goal of this study was to evaluate the hypothesis that, where there is a genetic predisposition to Ang II-dependent hypertrophy, there is also an increased susceptibility to sodium-induced hypertrophy mediated by AT₁-receptor expression.

Methods
Diets of low sodium (LS, 0.3% w:w) and high sodium (HS, 4.0% w:w) content were fed to adult (age 25 weeks) control wild-type mice (WT) and to weeks) control wild-type mice (WT) and to transgenic mice exhibiting cardiac specific overexpression of angiotensinogen (TG). At the conclusion of a 40-day dietary treatment period, cardiac tissue weights were compared and the relative expression levels of Ang II receptor subtypes (AT_1A and AT₂) were evaluated using RT-PCR.

Results
WT and TG mice fed HS and LS diets maintained comparable weight gains during the treatment period. The normalised heart weights of TG mice were elevated compared to WT, and the extent of the increase was greater for mice maintained on the HS diet treatments (WT 12% vs. TG 41% increase in cardiac weight index). While a similar pattern of growth was observed for ventricular tissues, the atrial weight parameters demonstrated an additional significant effect of dietary sodium intake on tissue weight, independent of animal genetic type. No differences in the relative (GAPDH normalised) expression levels of AT_1A- and AT₂-receptor mRNA were observed between diet or animal genetic groups.

Conclusion
This study demonstrates that, where there is a pre-existing genetic condition of Ang II-dependent cardiac hypertrophy, the pro-growth effect of elevated dietary sodium intake is selectively augmented. In TG and WT mice, this effect was evident with a relatively short dietary treatment intervention (40 days). Evaluation of the levels of Ang II receptor mRNA further demonstrated that this differential growth response was not associated with an altered relative expression of either AT_1A- or AT₂-receptor subtypes. The cellular mechanistic bases for this specific Ang II-dietary sodium interaction remain to be elucidated.

Changes in the composition of the extracellular matrix in patellar tendinopathy

Objective: To compare the chemical levels and mRNA expression of proteoglycan and collagen in normal human patellar tendons and tendons exhibiting chronic overuse tendinopathy.

Methods: Sulfated glycosaminoglycan and hydroxyproline content were investigated by spectrophotometric measurement using papain-digested samples. Deglycosylated proteoglycan core proteins were analysed by Western blot using specific antibodies. Total mRNA isolated from samples of frozen tendons was assayed by relative quantitative RT-PCR for decorin, biglycan, fibromodulin, versican, aggrecan, and collagens Type I, II and III and normalised to glyceraldehyde-3-phosphate dehydrogenase.

Results: There was a significant increase in sulfated glycosaminoglycan content in pathologic tendons compared to normal. This was attributed to an increased deposition of the large aggregating proteoglycans versican and aggrecan and the small proteoglycans biglycan and fibromodulin, but not decorin. Aggrecan and versican were extensively degraded in both normal and pathologic tendons, biglycan was more fragmented in the pathologic tendons while predominantly intact fibromodulin and decorin were present in normal and pathologic tendons. There was a greater range in total collagen content but no change in the level of total collagen in pathologic tendons. There were no significant differences between the pathologic and normal tendon for all genes, however p values close to 0.05 indicated a trend in downregulation of Type I collagen and fibromodulin, and upregulation in versican and Type III genes in pathologic tissue.

Conclusion: The changes in proteoglycan and collagen levels observed in patellar tendinopathy appear to be primarily due to changes in the metabolic turnover of these macromolecules. Changes in the expression of these macromolecules may not play a major role in this process.

Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone

In molluscs, the neurotransmitter serotonin (5-HT) has been linked to a variety of biological roles including gamete maturation and spawning. The possible involvement of 5-HT in abalone gamete release was demonstrated by a dose-dependent increase in Haliotis rubra gonad contractile bioactivity following 5-HT stimulation. Physiological functions associated with 5-HT, are mediated through binding to 5-HT receptors. A cDNA encoding a putative 5-HT receptor consisting of 359 amino acids was isolated from the tropical abalone H. asinina, termed 5-HT_{1 ha}. The 5-HT_{1 ha} shares G-protein-coupled receptor motifs with metazoan 5-HT receptors, including predicted transmembrane domains, active sites for protein kinase action, and N-linked glycosylation sites. However, the third intracellular loop of 5-HT_{1 ha} is relatively short, and only six transmembrane domains are predicted, implying a truncated receptor. Phylogenetic analysis with known 5-HT receptor genes suggests that 5-HT_{1 ha} belongs to the type 1 5-HT receptor family. Expression analysis by RT-PCR showed that 5-HT_{1 ha}  mRNA was present in all tissues examined, including the neural ganglia and gonad tissues. Immunocytochemistry revealed the presence of 5-HT_{1 ha} specifically within the soma of neuronal cells located in the outer cortex of both cerebral and pleuropedal ganglia. In ovarian and testicular tissues, 5-HT_{1 ha} immunoreactivity was observed in epithelial cells of the outer capsule and connective tissue of the trabeculae to which the gamete follicles adhere. Whether this receptor transcript is translated to a functional protein needs to be verified, but if so, it could play a role in reproduction.

Timing of immune escape linked to success or failure of vaccination

Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIV_mac251 challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIV_mac251 stock had high levels of viral replication and rapid CTL escape. Unvaccinated naïve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of naïve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.

10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

Objective
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.

Design
HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.

Results
Ten mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.

Conclusions
Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.