Non-Canonical (RANKL-Independent) Pathways of Osteoclast Differentiation and Their Role in Musculoskeletal Diseases.

Osteoclasts are multinucleated cells derived from mononuclear phagocyte precursors (monocytes, macrophages); in the canonical pathway of osteoclastogenesis, these cells fuse and differentiate to form specialised bone-resorbing osteoclasts in the presence of receptor activator for nuclear factor kappa B ligand (RANKL). Non-canonical pathways of osteoclastogenesis have been described in which several cytokines and growth factors are able to substitute for RANKL. These humoral factors can generally be divided into those which, like RANKL, are tumour necrosis family (TNF) superfamily members and those which are not; the former include TNFα lymphotoxin exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes (LIGHT), a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF); the latter include transforming growth factor beta (TGF-ß), interleukin-6 (IL-6), IL-8, IL-11, nerve growth factor (NGF), insulin-like growth factor-I (IGF-I) and IGF-II. This review summarises the evidence for these RANKL substitutes in inducing osteoclast differentiation from tissue-derived and circulating mononuclear phagocytes. It also assesses the role these factors are likely to play in promoting the pathological bone resorption seen in many inflammatory and neoplastic lesions of bone and joint including rheumatoid arthritis, aseptic implant loosening and primary and secondary tumours of bone.

Functional genomic screening to enhance oncolytic virotherapy.

Functional genomic screening has emerged as a powerful approach for understanding complex biological phenomena. Of the available tools, genome-wide RNA interference (RNAi) technology is unquestionably the most incisive, as it directly probes gene function. Recent applications of RNAi screening have been impressive. Notable amongst these are its use in elucidated mechanism(s) for signal transduction, various aspects of cell biology, tumourigenesis and metastasis, resistance to cancer therapeutics, and the host's response to a pathogen. Herein we discuss how recent RNAi screening efforts have helped turn our attention to the targetability of non-oncogene support pathways for cancer treatment, with a particular focus on a recent study that identified a non-oncogene addiction to the ER stress response as a synergist target for oncolytic virus therapy (OVT). Moreover, we give our thoughts on the future of RNAi screening as a tool to enhance OVT and describe recent technical improvements that are poised to make genome-scale RNAi experiments more sensitive, less noisy, more applicable in vivo, and more easily validated in clinically relevant animal models.

A call to arms: using RNAi screening to improve oncolytic viral therapy.

Replicating virus-based therapeutics for cancer, or oncolytic virus therapy (OVT), is rapidly emerging as a promising treatment modality for a wide range of cancers. In pre-clinical studies, oncolytic viruses have produced remarkable results in a variety of experimental animal models, and several viruses have entered phase I/II clinical trials. However, OVT is not effective against all tumours, with major treatment bottlenecks being the inability to infect, replicate within, or kill certain cancer cells. Unfortunately, the underlying molecular mechanisms governing these limitations are largely unknown. Recently, RNAi technology has been adapted for systematic interrogation of entire eukaryotic genomes. Since then, several groups have conducted genome-wide RNAi screens to study host/virus interactions. Herein we briefly summarize RNAi screening and its recent application to virology, and propose its use in overcoming key barriers to successful OVT.

IAP-targeted therapies for cancer.

DNA damage, chromosomal abnormalities, oncogene activation, viral infection, substrate detachment and hypoxia can all trigger apoptosis in normal cells. However, cancer cells acquire mutations that allow them to survive these threats that are part and parcel of the transformation process or that may affect the growth and dissemination of the tumor. Eventually, cancer cells accumulate further mutations that make them resistant to apoptosis mediated by standard cytotoxic chemotherapy or radiotherapy. The inhibitor of apoptosis (IAP) family members, defined by the presence of a baculovirus IAP repeat (BIR) protein domain, are key regulators of cytokinesis, apoptosis and signal transduction. Specific IAPs regulate either cell division, caspase activity or survival pathways mediated through binding to their BIR domains, and/or through their ubiquitin-ligase RING domain activity. These protein-protein interactions and post-translational modifications are the subject of intense investigations that shed light on how these proteins contribute to oncogenesis and resistance to therapy. In the past several years, we have seen multiple approaches of IAP antagonism enter the clinic, and the rewards of such strategies are about to reap benefit. Significantly, small molecule pan-IAP antagonists that mimic an endogenous inhibitor of the IAPs, called Smac, have demonstrated an unexpected ability to sensitize cancer cells to tumor necrosis factor-alpha and to promote autocrine or paracrine production of this cytokine by the tumor cell and possibly, other cells too. This review will focus on these and other developmental therapeutics that target the IAPs in cancer.

Both cIAP1 and cIAP2 regulate TNFalpha-mediated NF-kappaB activation.

The cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2) proteins have been implicated in the activation of NF-kappaB by TNFalpha; however, genetic deletion of either cIAP1 or 2 did not support a physiologically relevant role, perhaps because of functional redundancy. To address this, we used combined genetic and siRNA knockdown approaches and report that cIAP1 and 2 are indeed critical, yet redundant, regulators of NF-kappaB activation upon TNFalpha treatment. Whereas NF-kappaB was properly activated by TNFalpha in cultured and primary cells deficient in either cIAP1 or 2, removal of both cIAPs severely blunted its activation. After treatment with TNFalpha, cIAP1 and 2 were rapidly recruited to the TNF receptor 1, along with the adapter protein TNF receptor associated factor 2. Importantly, either cIAP1 or 2 was required for proper TNF receptor 1 signalosome function. In their combined absence, polyubiquitination of receptor interacting protein 1, an upstream event necessary for NF-kappaB signaling, was attenuated. As a result, phosphorylation of the inhibitor of kappaB kinase beta was diminished, and signal transduction was severely blunted. Consequently, cells missing both cIAP1 and 2 were sensitized to TNFalpha-mediated apoptosis. Collectively, these data demonstrate that either cIAP1 or 2 is required for proper Rip1 polyubiquitination and NF-kappaB activation upon TNFalpha treatment.

Gene expression profiling in human skeletal muscle during recovery from eccentric exercise.

We used cDNA microarrays to screen for differentially expressed genes during recovery from exercise-induced muscle damage in humans. Male subjects (n = 4) performed 300 maximal eccentric contractions, and skeletal muscle biopsy samples were analyzed at 3 h and 48 h after exercise. In total, 113 genes increased 3 h postexercise, and 34 decreased. At 48 h postexercise, 59 genes increased and 29 decreased. On the basis of these data, we chose 19 gene changes and conducted secondary analyses using real-time RT-PCR from muscle biopsy samples taken from 11 additional subjects who performed an identical bout of exercise. Real-time RT-PCR analyses confirmed that exercise-induced muscle damage led to a rapid (3 h) increase in sterol response element binding protein 2 (SREBP-2), followed by a delayed (48 h) increase in the SREBP-2 gene targets Acyl CoA:cholesterol acyltransferase (ACAT)-2 and insulin-induced gene 1 (insig-1). The expression of the IL-1 receptor, a known regulator of SREBP-2, was also elevated after exercise. Taken together, these expression changes suggest a transcriptional program for increasing cholesterol and lipid synthesis and/or modification. Additionally, damaging exercise induced the expression of protein kinase H11, capping protein Z alpha (capZalpha), and modulatory calcineurin-interacting protein 1 (MCIP1), as well as cardiac ankryin repeat protein 1 (CARP1), DNAJB2, c-myc, and junD, each of which are likely involved in skeletal muscle growth, remodeling, and stress management. In summary, using DNA microarrays and RT-PCR, we have identified novel genes that respond to skeletal muscle damage, which, given the known biological functions, are likely involved in recovery from and/or adaptation to damaging exercise.

Analysis of global mRNA expression in human skeletal muscle during recovery from endurance exercise.

To search for novel transcriptional pathways that are activated in skeletal muscle after endurance exercise, we used cDNA microarrays to measure global mRNA expression after an exhaustive bout of high-intensity cycling (approximately 75 min). Healthy, young, sedentary males performed the cycling bout, and skeletal muscle biopsies were taken from the vastus lateralis before, and at 3 and 48 h after exercise. We examined mRNA expression in individual muscle samples from four subjects using cDNA microarrays, used repeated-measures significance analysis of microarray (SAM) to determine statistically significant expression changes, and confirmed selected results using real-time RT-PCR. In total, the expression of 118 genes significantly increased 3 h postcycling and 8 decreased. At 48 h, the expression of 29 genes significantly increased and 5 decreased. Many of these are potentially important novel genes involved in exercise recovery and adaptation, including several involved in 1) metabolism and mitochondrial biogenesis (FOXO1, PPARdelta, PPARgamma, nuclear receptor binding protein 2, IL-6 receptor, ribosomal protein L2, aminolevulinate delta-synthase 2); 2) the oxidant stress response (metalothioneins 1B, 1F, 1G, 1H, 1L, 2A, 3, interferon regulatory factor 1); and 3) electrolyte transport across membranes [Na+-K+-ATPase (beta3), SERCA3, chloride channel 4]. Others include genes involved in cell stress, proteolysis, apoptosis, growth, differentiation, and transcriptional activation, as well as all three nuclear receptor subfamily 4A family members (Nur77, Nurr1, and Nor1). This study is the first to characterize global mRNA expression during recovery from endurance exercise, and the results provide potential insight into 1) the transcriptional contributions to homeostatic recovery in human skeletal muscle after endurance exercise, and 2) the transcriptional contributions from a single bout of endurance exercise to the adaptive processes that occur after a period of endurance exercise training.

Creatine monohydrate enhances strength and body composition in Duchenne muscular dystrophy.

OBJECTIVE: To determine whether creatine monohydrate (CrM) supplementation increases strength and fat-free mass (FFM) in boys with Duchenne muscular dystrophy (DD). METHODS: Thirty boys with DD (50% were taking corticosteroids) completed a double-blind, randomized, cross-over trial with 4 months of CrM (about 0.10 g/kg/day), 6-week wash-out, and 4 months of placebo. Measurements were completed of pulmonary function, compound manual muscle and handgrip strength, functional tasks, activity of daily living, body composition, serum creatine kinase and gamma-glutamyl transferase activity and creatinine, urinary markers of myofibrillar protein breakdown (3-methylhistidine), DNA oxidative stress (8-hydroxy-2-deoxyguanosine [8-OH-2-dG]), and bone degradation (N-telopeptides). RESULTS: During the CrM treatment phase, there was an increase in handgrip strength in the dominant hand and FFM (p < 0.05), with a trend toward a loss of global muscle strength (p = 0.056) only for the placebo phase, with no improvements in functional tasks or activities of daily living. Corticosteroid use, but not CrM treatment, was associated with a lower 8-OH-2-dG/creatinine (p < 0.05), and CrM treatment was associated with a reduction in N-telopeptides (p < 0.05). CONCLUSIONS: Four months of CrM supplementation led to increases in FFM and handgrip strength in the dominant hand and a reduction in a marker of bone breakdown and was well tolerated in children with DD.

Mapping the hyaluronan-binding site on the link module from human tumor necrosis factor-stimulated gene-6 by site-directed mutagenesis.

Link modules are hyaluronan-binding domains found in extracellular proteins involved in matrix assembly, development, and immune cell migration. Previously we have expressed the Link module from the inflammation-associated protein tumor necrosis factor-stimulated gene-6 (TSG-6) and determined its tertiary structure in solution. Here we generated 21 Link module mutants, and these were analyzed by nuclear magnetic resonance spectroscopy and a hyaluronan-binding assay. The individual mutation of five amino acids, which form a cluster on one face of the Link module, caused large reductions in functional activity but did not affect the Link module fold. This ligand-binding site in TSG-6 is similar to that determined previously for the hyaluronan receptor, CD44, suggesting that the location of the interaction surfaces may also be conserved in other Link module-containing proteins. Analysis of the sequences of TSG-6 and CD44 indicates that the molecular details of their association with hyaluronan are likely to be significantly different. This comparison identifies key sequence positions that may be important in mediating hyaluronan binding, across the Link module superfamily. The use of multiple sequence alignment and molecular modeling allowed the prediction of functional residues in link protein, and this approach can be extended to all members of the superfamily.

Short-term 17beta-estradiol decreases glucose R(a) but not whole body metabolism during endurance exercise.

The female sex hormone 17beta-estradiol (E(2)) has been shown to increase lipid and decrease carbohydrate utilization in animals. We administrated oral E(2) and placebo (randomized, double blind, crossover) to eight human male subjects for 8 days ( approximately 3 mg/day) and measured respiratory variables, plasma substrates, hormones (E(2), testosterone, leptin, cortisol, insulin, and catecholamines), and substrate utilization during 90 min of endurance exercise. [6,6-(2)H]glucose and [1,1,2,3,3-(2)H]glycerol tracers were used to calculate substrate flux. E(2) administration increased serum E(2) (0.22 to 2.44 nmol/l, P < 0.05) and decreased serum testosterone (19.4 to 11.5 nmol/l, P < 0.05) concentrations, yet there were no treatment effects on any of the other hormones. Glucose rates of appearance (R(a)) and disappearance (R(d)) were lower, and glycerol R(a)-to-R(d) ratio was not affected by E(2) administration. O(2) uptake, CO(2) production, and respiratory exchange ratio were not affected by E(2); however, there was a decrease in heart rate (P < 0.05). Plasma lactate and glycerol were unaffected by E(2); however, glucose was significantly higher (P < 0. 05) during exercise after E(2) administration. We concluded that short-term oral E(2) administration decreased glucose R(a) and R(d), maintained plasma glucose homeostasis, but had no effect on substrate oxidation during exercise in men.

Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length.

Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.

Mutations in the HML E silencer of Saccharomyces cerevisiae yield metastable inheritance of transcriptional repression.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, mediated independently through two cis-acting sites, termed the E and I silencers. We have found that in the absence of the I silencer, deletion of any one of three distinct elements within E yields partial derepression of the mating-type genes resident at HML, whereas deletion of any two yields full derepression. These elements correspond to a binding site for the abundant DNA-binding protein RAP1, an autonomous replicating sequence (ARS), and an as yet undistinguished region. From detailed deletion analysis of the E site we conclude that the ARS element contributes to silencer function in a capacity distinct from its role as an initiator of DNA replication. In addition, we find that strains deleted for any one of these elements comprise two genetically identical but phenotypically distinct types of cells: Those with HML apparently fully derepressed, and those with HML apparently completely repressed. These results reinforce the notion that epigenetic inheritance is an intrinsic characteristic of silencer action.

Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase beta genes.

The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.

The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, most likely mediated through two cis-acting sites located on opposite sides of the locus. We showed that deletion of either of these two cis-acting sites from the chromosome did not yield any detectable derepression of HML, while deletion of both sites yielded full expression of the locus. In addition, each of these sites was capable of exerting repression of heterologous genes inserted in their vicinity. Thus, HML expression is regulated by two independent silencers, each fully competent for maintaining repression. This situation was distinct from the organization of the other silent locus, HMR, at which a single silencer served as the predominant repressor of expression. Examination of identifiable domains and binding sites within the HML silencers suggested that silencing activity can be achieved by a variety of combinations of various functional domains.