Syndecan-3 enhances anabolic bone formation through WNT signaling.

Osteoporosis is the most common age-related metabolic bone disorder, which is characterized by low bone mass and deterioration in bone architecture, with a propensity to fragility fractures. The best treatment for osteoporosis relies on stimulation of osteoblasts to form new bone and restore bone structure, however, anabolic therapeutics are few and their use is time restricted. Here, we report that Syndecan-3 increases new bone formation through enhancement of WNT signaling in osteoblasts. Young adult Sdc3-/- mice have low bone volume, reduced bone formation, increased bone marrow adipose tissue, increased bone fragility, and a blunted anabolic bone formation response to mechanical loading. This premature osteoporosis-like phenotype of Sdc3-/- mice is due to delayed osteoblast maturation and impaired osteoblast function, with contributing increased osteoclast-mediated bone resorption. Indeed, overexpressing Sdc3 in osteoblasts using the Col1a1 promoter rescues the low bone volume phenotype of the Sdc3-/- mice, and also increases bone volume in WT mice. Mechanistically, SDC3 enhances canonical WNT signaling in osteoblasts through stabilization of Frizzled 1, making SDC3 an attractive target for novel bone anabolic drug development.

The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/.

Characterization of wooden breast myopathy: a focus on syndecans and ECM remodeling.

Introduction: The skeletal muscle deformity of commercial chickens (Gallus gallus), known as the wooden breast (WB), is associated with fibrotic myopathy of unknown etiology. For future breeding strategies and genetic improvements, it is essential to identify the molecular mechanisms underlying the phenotype. The pathophysiological hallmarks of WB include severe skeletal muscle fibrosis, inflammation, myofiber necrosis, and multifocal degeneration of muscle tissue. The transmembrane proteoglycans syndecans have a wide spectrum of biological functions and are master regulators of tissue homeostasis. They are upregulated and shed (cleaved) as a regulatory mechanism during tissue repair and regeneration. During the last decades, it has become clear that the syndecan family also has critical functions in skeletal muscle growth, however, their potential involvement in WB pathogenesis is unknown. Methods: In this study, we have categorized four groups of WB myopathy in broiler chickens and performed a comprehensive characterization of the molecular and histological profiles of two of them, with a special focus on the role of the syndecans and remodeling of the extracellular matrix (ECM). Results and discussion: Our findings reveal differential expression and shedding of the four syndecan family members and increased matrix metalloproteinase activity. Additionally, we identified alterations in key signaling pathways such as MAPK, AKT, and Wnt. Our work provides novel insights into a deeper understanding of WB pathogenesis and suggests potential therapeutic targets for this condition.

Marking the tempo for myogenesis: Pax7 and the regulation of muscle stem cell fate decisions.

Post-natal growth and regeneration of skeletal muscle is highly dependent on a population of resident myogenic precursors known as satellite cells. Transcription factors from the Pax gene family, Pax3 and Pax7, are critical for satellite cell biogenesis, survival and potentially self-renewal; however, the underlying molecular mechanisms remain unsolved. This is particularly true in the case of Pax7, which appears to regulate myogenesis at multiple levels. Accordingly, recent data have highlighted the importance of a functional relationship between Pax7 and the MyoD family of muscle regulatory transcription factors during normal muscle formation and disease. Here we will critically review key findings suggesting that Pax7 may play a dual role by promoting resident muscle progenitors to commit to the skeletal muscle lineage while preventing terminal differentiation, thus keeping muscle progenitors poised to differentiate upon environmental cues. In addition, potential regulatory mechanisms for the control of Pax7 activity will be proposed.

The INSR/AKT/mTOR pathway regulates the pace of myogenesis in a syndecan-3-dependent manner.

Muscle stem cells (MuSCs) are indispensable for muscle regeneration. A multitude of extracellular stimuli direct MuSC fate decisions from quiescent progenitors to differentiated myocytes. The activity of these signals is modulated by coreceptors such as syndecan-3 (SDC3). We investigated the global landscape of SDC3-mediated regulation of myogenesis using a phosphoproteomics approach which revealed, with the precision level of individual phosphosites, the large-scale extent of SDC3-mediated regulation of signal transduction in MuSCs. We then focused on INSR/AKT/mTOR as a key pathway regulated by SDC3 during myogenesis and mechanistically dissected SDC3-mediated inhibition of insulin receptor signaling in MuSCs. SDC3 interacts with INSR ultimately limiting signal transduction via AKT/mTOR. Both knockdown of INSR and inhibition of AKT restore Sdc3-/- MuSC differentiation to wild type levels. Since SDC3 is rapidly downregulated at the onset of differentiation, our study suggests that SDC3 acts a timekeeper to restrain proliferating MuSC response and prevent premature differentiation.

Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated signaling pathway that controls myoblast fusion.

The mechanism of skeletal myoblast fusion is not well understood. We show that endogenous nitric oxide (NO) generation is required for myoblast fusion both in embryonic myoblasts and in satellite cells. The effect of NO is concentration and time dependent, being evident only at the onset of differentiation, and direct on the fusion process itself. The action of NO is mediated through a tightly regulated activation of guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP), so much so that deregulation of cGMP signaling leads to a fusion-induced hypertrophy of satellite-derived myotubes and embryonic muscles, and to the acquisition of fusion competence by myogenic precursors in the presomitic mesoderm. NO and cGMP induce expression of follistatin, and this secreted protein mediates their action in myogenesis. These results establish a hitherto unappreciated role of NO and cGMP in regulating myoblast fusion and elucidate their mechanism of action, providing a direct link with follistatin, which is a key player in myogenesis.

Syndecan-3 regulates MSC adhesion, ERK and AKT signalling in vitro and its deletion enhances MSC efficacy in a model of inflammatory arthritis in vivo.

Rheumatoid arthritis (RA) is a debilitating and painful inflammatory autoimmune disease characterised by the accumulation of leukocytes in the synovium, cartilage destruction and bone erosion. The immunomodulatory effects of bone marrow derived mesenchymal stem cells (MSCs) has been widely studied and the recent observations that syndecan-3 (SDC3) is selectively pro-inflammatory in the joint led us to hypothesise that SDC3 might play an important role in MSC biology. MSCs isolated from bone marrow of wild type and Sdc3-/- mice were used to assess immunophenotype, differentiation, adhesion and migration properties and cell signalling pathways. While both cell types show similar differentiation potential and forward scatter values, the cell complexity in wild type MSCs was significantly higher than in Sdc3-/- cells and was accompanied by lower spread surface area. Moreover, Sdc3-/- MSCs adhered more rapidly to collagen type I and showed a dramatic increase in AKT phosphorylation, accompanied by a decrease in ERK1/2 phosphorylation compared with control cells. In a mouse model of antigen-induced inflammatory arthritis, intraarticular injection of Sdc3-/- MSCs yielded enhanced efficacy compared to injection of wild type MSCs. In conclusion, our data suggest that syndecan-3 regulates MSC adhesion and efficacy in inflammatory arthritis, likely via induction of the AKT pathway.

Syndecan-4-/- Mice Have Smaller Muscle Fibers, Increased Akt/mTOR/S6K1 and Notch/HES-1 Pathways, and Alterations in Extracellular Matrix Components.

BACKGROUND: Extracellular matrix (ECM) remodeling is essential for skeletal muscle development and adaption in response to environmental cues such as exercise and injury. The cell surface proteoglycan syndecan-4 has been reported to be essential for muscle differentiation, but few molecular mechanisms are known. Syndecan-4-/- mice are unable to regenerate damaged muscle, and display deficient satellite cell activation, proliferation, and differentiation. A reduced myofiber basal lamina has also been reported in syndecan-4-/- muscle, indicating possible defects in ECM production. To get a better understanding of the underlying molecular mechanisms, we have here investigated the effects of syndecan-4 genetic ablation on molecules involved in ECM remodeling and muscle growth, both under steady state conditions and in response to exercise. METHODS: Tibialis anterior (TA) muscles from sedentary and exercised syndecan-4-/- and WT mice were analyzed by immunohistochemistry, real-time PCR and western blotting. RESULTS: Compared to WT, we found that syndecan-4-/- mice had reduced body weight, reduced muscle weight, muscle fibers with a smaller cross-sectional area, and reduced expression of myogenic regulatory transcription factors. Sedentary syndecan-4-/- had also increased mRNA levels of syndecan-2, decorin, collagens, fibromodulin, biglycan, and LOX. Some of these latter ECM components were reduced at protein level, suggesting them to be more susceptible to degradation or less efficiently translated when syndecan-4 is absent. At the protein level, TRPC7 was reduced, whereas activation of the Akt/mTOR/S6K1 and Notch/HES-1 pathways were increased. Finally, although exercise induced upregulation of several of these components in WT, a further upregulation of these molecules was not observed in exercised syndecan-4-/- mice. CONCLUSION: Altogether our data suggest an important role of syndecan-4 in muscle development.

TNF-alpha downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents.

Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-alpha. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-alpha downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-alpha. Our findings demonstrate that TNF-alpha impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.

Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis.

Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass spectrometry. Thus, effective methods to enrich for phosphopeptides from protein extracts have been developed. Here, we discuss and present in detail two such methods that we routinely employ. These methods are based on a sample enrichment step performed on titanium dioxide matrices followed by label-free tandem mass spectrometry and semi-quantitation.

The Muscle Stem Cell Niche in Health and Disease.

The regulation of stem cells that maintain and regenerate postnatal tissues depends on extrinsic signals originating from their microenvironment, commonly referred to as the stem cell niche. Complex higher-order regulatory interrelationships with the tissue and factors in the systemic circulation are integrated and propagated to the stem cells through the niche. The stem cell niche in skeletal muscle tissue is both a paradigm for a structurally and functionally relatively static niche that maintains stem cell quiescence during tissue homeostasis, and a highly dynamic regenerative niche that is subject to extensive structural remodeling and a flux of different support cell populations. Conditions ranging from aging to chronically degenerative skeletal muscle diseases affect the composition of the niche and thereby impair the regenerative potential of muscle stem cells. A holistic and integrative understanding of the extrinsic mechanisms regulating muscle stem cells in health and disease in a broad systemic context will be imperative for the identification of regulatory hubs in the niche interactome that can be targeted to maintain, restore, or enhance the regenerative capacity of muscle tissue. Here, we review the microenvironmental regulation of muscle stem cells, summarize how niche dysfunction can contribute to disease, and discuss emerging therapeutic implications.

The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53.

Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence.

The Role of Eif6 in Skeletal Muscle Homeostasis Revealed by Endurance Training Co-expression Networks.

Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.

Impaired interleukin-12-dependent T-cell functions during aging: role of signal transducer and activator of transcription 4 (STAT4) and suppressor of cytokine signaling 3 (SOCS3).

Interleukin (IL)-12 is the major inducer of T helper cell (Th) 1-type responses. Despite a higher IL-12 production, phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC), as well as CD4(+) or CD8(+) T cells from elderly donors released interferon (IFN)-gamma amounts similar to those observed in young controls, and underwent only a slight increase in IFN-gamma production after IL-12 costimulation. These findings were not due to an age-related reduction in IL-12 receptor expression. Interestingly, no difference in PHA-triggered signal transducer and activator of transcription 4 (STAT4) phosphorylation between young and elderly donors was found, and a significant IL-12-induced STAT4 activation occurred only in PHA-preactivated cells from the younger group. The age-related defect in IL-12 signaling was STAT4-restricted as it did not involve the p38 mitogen-activated protein kinase (MAPK) pathway. Finally, suppressor of cytokine signaling 3 (SOCS3) expression was significantly higher in unstimulated cells from elderly individuals, and it did not diminish after cell stimulation. These results indicate that a defective STAT4 activation, likely dependent on elevated SOCS3 levels, is involved in the impaired IL-12-dependent T-cell functions with aging.

Nitric oxide confers therapeutic activity to dendritic cells in a mouse model of melanoma.

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell homeostasis improving muscle regeneration.

BACKGROUND: The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells, required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressed in satellite cells, supports communication with the niche, providing cell interactions and signals to maintain quiescent satellite cells. RESULTS: Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophic mice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscle aging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulate Pax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellite cell homeostasis and satellite cell homing to the niche. CONCLUSIONS: Manipulating syndecan-3 provides a promising target for development of therapies to enhance muscle regeneration in muscular dystrophies and in aged muscle.

APC-dependent impairment of T cell proliferation in aging: role of CD28- and IL-12/IL-15-mediated signaling.

The age-related impairment of phytohaemagglutinin (PHA)-triggered peripheral blood mononuclear cell (PBMC) proliferation was paralleled by an expansion of CD28 (-) T lymphocytes with a poor capacity to undergo lectin-induced blastogenesis. However, both CD28 (-) and CD28 (+) T cells isolated from aged individuals exhibited a significant reduction of proliferative response to PHA in comparison with young controls, this implies that the CD28-mediated signaling is not the only defective pathway in the elderly. Thus, PBMC or T cell subsets plus monocytes from aged donors were stimulated with PHA and assayed for the production of, or the response to cytokines known to regulate T cell functions. Results can be so summarized: (i). interleukin (IL)-2 as well as IL-10 release was unaffected by age; (ii). in both groups of subjects, IL-15 concentrations were similar to those spontaneously released by PBMC; (iii). surprisingly, IL-12 p70 and IL-12 p40 production by PBMC was markedly increased in the aged group; (iv) in spite of this finding and of the experimental outcome that IFN-gamma synthesis was almost completely dependent on IL-12. PBMC from old individuals did not release higher amounts of IFN-gamma in comparison with young controls; (v). moreover, only a slight increase in IFN-gamma production was observed in PBMC cultures from the aged group as a result of IL-12 and/or IL-15 costimulation; (vi) at the same time, even though IL-12 as well as IL-15 were necessary for an efficient T cell proliferation, the addition of exceeding doses of cytokines proved to be ineffective in enhancing the proliferative outcome of PBMC or of both CD28 (+) and CD28 (-) T cells in the aged group. Taken together, the data outline the role of CD28 and IL-12/IL-15 signaling impairment in T cell proliferative deficiency during senescence.

Syndecans in skeletal muscle development, regeneration and homeostasis.

Skeletal muscle is a highly dynamic tissue that can change in size in response to physiological demands and undergo successful regeneration even upon extensive injury. A population of resident stem cells, termed satellite cells, accounts for skeletal muscle plasticity, maintenance and regeneration. Mammalian satellite cells, generated from muscle precursor cells during development, are maintained quiescent in the musculature throughout a lifespan, but ready to activate, proliferate and differentiate into myocytes upon demand. Syndecans are transmembrane heparan sulfate proteoglycans expressed in muscle precursors during embryonic development and in satellite cells during postnatal life. In the last decades a number of crucial functions for syndecans in myogenesis and muscle disease have been described. Here we review the current knowledge of the multiple roles played by syndecans in the skeletal muscle of several animal models and explore future perspectives for human muscle health, with a focus on muscle aging and muscular dystrophy.

Abcg2 labels multiple cell types in skeletal muscle and participates in muscle regeneration.

Skeletal muscle contains progenitor cells (satellite cells) that maintain and repair muscle. It also contains muscle side population (SP) cells, which express Abcg2 and may participate in muscle regeneration or may represent a source of satellite cell replenishment. In Abcg2-null mice, the SP fraction is lost in skeletal muscle, although the significance of this loss was previously unknown. We show that cells expressing Abcg2 increased upon injury and that muscle regeneration was impaired in Abcg2-null mice, resulting in fewer centrally nucleated myofibers, reduced myofiber size, and fewer satellite cells. Additionally, using genetic lineage tracing, we demonstrate that the progeny of Abcg2-expressing cells contributed to multiple cell types within the muscle interstitium, primarily endothelial cells. After injury, Abcg2 progeny made a minor contribution to regenerated myofibers. Furthermore, Abcg2-labeled cells increased significantly upon injury and appeared to traffic to muscle from peripheral blood. Together, these data suggest an important role for Abcg2 in positively regulating skeletal muscle regeneration.

Syndecan-3 and Notch cooperate in regulating adult myogenesis.

Skeletal muscle postnatal growth and repair depend on satellite cells and are regulated by molecular signals within the satellite cell niche. We investigated the molecular and cellular events that lead to altered myogenesis upon genetic ablation of Syndecan-3, a component of the satellite cell niche. In the absence of Syndecan-3, satellite cells stall in S phase, leading to reduced proliferation, increased cell death, delayed onset of differentiation, and markedly reduced numbers of Pax7(+) satellite cells accompanied by myofiber hypertrophy and an increased number of centrally nucleated myofibers. We show that the aberrant cell cycle and impaired self-renewal of explanted Syndecan-3-null satellite cells are rescued by ectopic expression of the constitutively active Notch intracellular domain. Furthermore, we show that Syndecan-3 interacts with Notch and is required for Notch processing by ADAM17/tumor necrosis factor-alpha-converting enzyme (TACE) and signal transduction. Together, our data support the conclusion that Syndecan-3 and Notch cooperate in regulating homeostasis of the satellite cell population and myofiber size.