Divergent regulation of Wnt-mediated development of the dorsomedial and ventrolateral dermomyotomal lips

The dermomyotome is the dorsal compartment of the somite which gives rise to multiple cell fates including skeletal muscle, connective tissue, and endothelia. It consists of a pseudostratified, roughly rectangular epithelial sheet, the margins of which are called the dermomyotomal lips. The dermomyotomal lips are blastema-like epithelial growth zones, which continuously give rise to resident dermomyotomal cells and emigrating muscle precursor cells, which populate the subjacent myotomal compartment. Wnt signaling has been shown to regulate both dermomyotome formation and maintenance of the dermomyotomal lips. Whereas the epithelialization of the dermomyotome is regulated via canonical, β-catenin-dependent Wnt signaling, the downstream signaling mechanisms suppressing epithelial-mesenchymal transition (EMT) in the mature dermomyotomal lips have been unknown. Here, we present evidence that dermomyotomal lip sustainment is differentially regulated. Whereas the dorsomedial dermomyotomal lip is maintained by canonical Wnt signaling, development of the ventrolateral dermomyotomal lip is regulated by non-canonical, PCP-like Wnt signaling. We discuss our results in the light of the different developmental prerequisites in the dorsomedial and ventrolateral lips, respectively, thus providing a new perspective on the regulation of dermomyotomal EMT.     

The role of TGF‐β1 during skeletal muscle regeneration

The injury of adult skeletal muscle initiates series of well‐coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF‐β family members is TGF‐β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF‐β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function.     

Skeletal muscle cells express the profibrotic cytokine connective tissue growth factor (CTGF/CCN2), which induces their dedifferentiation

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes of different tissues. Connective tissue growth factor (CTGF) seems to be involved in the fibrotic response. Several muscular dystrophies are characterized by a progressive weakness and wasting of the musculature, and by extensive fibrosis. However, the exact role of CTGF in skeletal muscle is unknown. Here we show that myoblasts and myotubes are able to synthesize CTGF in response to transforming growth factor type-beta (TGF-beta) and lysophosphatidic acid (LPA). CTGF induced several ECM constituents such as fibronectin, collagen type I and alpha4, 5, 6, and beta1 integrin subunits in myoblasts and myotubes. CTGF had an important inhibitory effect on muscle differentiation evaluated by the decrease in the nuclear translocation of the early muscle regulatory factor myogenin and myosin. Remarkable, CTGF treatment of myoblasts induced their dedifferentiation, characterized by down regulating MyoD and desmin, two markers of committed myoblasts, together with a strong reorganization of cytoskeletal filaments. These results provide novel evidence for the underlying mechanisms and participation of skeletal muscle cells in the synthesis and role of CTGF inducing fibrosis, inhibiting myogenesis and dedifferentiating myoblasts.     

Roles of Wnt7a in embryo development,tissue homeostasis,and human diseases

Human Wnt family comprises 19 proteins which are critical to embryo development and tissue homeostasis. Binding to different frizzled (FZD) receptor, Wnt7a initiates both β-catenin dependent pathway, and β-catenin independent pathways such as PI3K/Akt, RAC/JNK, and extracellular signal-regulated kinase 5/peroxisome proliferator-activated receptor-γ. In the embryo, Wnt7a plays a crucial role in cerebral cortex development, synapse formation, and central nervous system vasculature formation and maintenance. Wnt7a is also involved in the development of limb and female reproductive system. Wnt7a mutation leads to human limb malformations and animal female reproductive system defects. Wnt7a is implicated in homeostasis maintenance of skeletal muscle, cartilage, cornea and hair follicle, and Wnt7a treatment may be potentially applied in skeletal muscle dystrophy, corneal damage, wound repair, and hair follicle regeneration. Wnt7a plays dual roles in human tumors. Wnt7a is downregulated in lung cancers, functioning as a tumor suppressor, however, it is upregulated in several other malignancies such as ovarian cancer, breast cancer, and glioma, acting as a tumor promoter. Moreover, Wnt7a overexpression is associated with inflammation and fibrosis, but its roles need to be further investigated.     

Initiation of synapse formation by Wnt-induced MuSK endocytosis

In zebrafish, the MuSK receptor initiates neuromuscular synapse formation by restricting presynaptic growth cones and postsynaptic acetylcholine receptors (AChRs) to the center of skeletal muscle cells. Increasing evidence suggests a role for Wnts in this process, yet how muscle cells respond to Wnt signals is unclear. Here, we show that in vivo, wnt11r and wnt4a initiate MuSK translocation from muscle membranes to recycling endosomes and that this transition is crucial for AChR accumulation at future synaptic sites. Moreover, we demonstrate that components of the planar cell polarity pathway colocalize to recycling endosomes and that this localization is MuSK dependent. Knockdown of several core components disrupts MuSK translocation to endosomes, AChR localization and axonal guidance. We propose that Wnt-induced trafficking of the MuSK receptor to endosomes initiates a signaling cascade to align pre- with postsynaptic elements. Collectively, these findings suggest a general mechanism by which Wnt signals shape synaptic connectivity through localized receptor endocytosis.     

Update on Wnt signaling in bone cell biology and bone disease

For more than a decade, Wnt signaling pathways have been the focus of intense research activity in bone biology laboratories because of their importance in skeletal development, bone mass maintenance, and therapeutic potential for regenerative medicine. It is evident that even subtle alterations in the intensity, amplitude, location, and duration of Wnt signaling pathways affects skeletal development, as well as bone remodeling, regeneration, and repair during a lifespan. Here we review recent advances and discrepancies in how Wnt/Lrp5 signaling regulates osteoblasts and osteocytes, introduce new players in Wnt signaling pathways that have important roles in bone development, discuss emerging areas such as the role of Wnt signaling in osteoclastogenesis, and summarize progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists, such as sclerostin, Dkk1 and Sfrp1. Emphasis is placed on the plethora of genetic studies in mouse models and genome wide association studies that reveal the requirement for and crucial roles of Wnt pathway components during skeletal development and disease.     

Impaired Skeletal Muscle Regeneration in the Absence of Fibrosis during Hibernation in 13-Lined Ground Squirrels

Skeletal muscle atrophy can occur as a consequence of immobilization and/or starvation in the majority of vertebrates studied. In contrast, hibernating mammals are protected against the loss of muscle mass despite long periods of inactivity and lack of food intake. Resident muscle-specific stem cells (satellite cells) are known to be activated by muscle injury and their activation contributes to the regeneration of muscle, but whether satellite cells play a role in hibernation is unknown. In the hibernating 13-lined ground squirrel we show that muscles ablated of satellite cells were still protected against atrophy, demonstrating that satellite cells are not involved in the maintenance of skeletal muscle during hibernation. Additionally, hibernating skeletal muscle showed extremely slow regeneration in response to injury, due to repression of satellite cell activation and myoblast differentiation caused by a fine-tuned interplay of p21, myostatin, MAPK, and Wnt signaling pathways. Interestingly, despite long periods of inflammation and lack of efficient regeneration, injured skeletal muscle from hibernating animals did not develop fibrosis and was capable of complete recovery when animals emerged naturally from hibernation. We propose that hibernating squirrels represent a new model system that permits evaluation of impaired skeletal muscle remodeling in the absence of formation of tissue fibrosis.     

The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFbeta and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.     

Activation of Wnt11 by transforming growth factor-β drives mesenchymal gene expression through non-canonical Wnt protein signaling in renal epithelial cells

Transforming growth factor β1 (TGF-β) promotes renal interstitial fibrosis in vivo and the expression of mesenchymal genes in vitro; however, most of its direct targets in epithelial cells are still elusive. In a screen for genes directly activated by TGF-β, we found that components of the Wnt signaling pathway, especially Wnt11, were targets of activation by TGF-β and Smad3 in primary renal epithelial cells. In gain and loss of function experiments, Wnt11 mediated the actions of TGF-β through enhanced activation of mesenchymal marker genes, such as Zeb1, Snail1, Pai1, and αSMA, without affecting Smad3 phosphorylation. Inhibition of Wnt11 by receptor knockdown or treatment with Wnt inhibitors limited the effects of TGF-β on gene expression. We found no evidence that Wnt11 activated the canonical Wnt signaling pathway in renal epithelial cells; rather, the function of Wnt11 was mediated by the c-Jun N-terminal kinase (JNK) pathway. Consistent with the in vitro results, all the TGF-β, Wnt11, and JNK targets were activated in a unilateral ureteral obstruction (UUO) model of renal fibrosis in vivo. Our findings demonstrated cooperativity among the TGF-β, Wnt11, and JNK signaling pathways and suggest new targets for anti-fibrotic therapy in renal tissue.     

The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFβ and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.     

Wnt signaling in development and disease

ABSTRACT: Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.     

The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells

Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.     

Role of hypoxia in skeletal muscle fibrosis: Synergism between hypoxia and TGF-β signaling upregulates CCN2/CTGF expression specifically in muscle fibers

Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-β (TGF-β) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle.We evaluated the role of hypoxia and TGF-β on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-β1 under hypoxic conditions. Hypoxia and TGF-β1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-β signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-β1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-β signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.     

Developmental roles of heparan sulfate proteoglycans in Drosophila

The formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens. Published in 2003.     

Fibrotic response induced by angiotensin-II requires NAD(P)H oxidase-induced reactive oxygen species (ROS) in skeletal muscle cells

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes in different tissues. Angiotensin-II (Ang-II) is involved in the fibrotic response. Several muscular dystrophies are characterized by extensive fibrosis. However, the exact role of Ang-II in skeletal muscle fibrosis is unknown. Here we show that myoblasts responded to Ang-II by increasing protein levels of connective tissue growth factor (CTGF/CCN2), collagen-III and fibronectin. These Ang-II-induced pro-fibrotic effects were mediated by AT-1 receptors. Remarkably, Ang-II induced reactive oxygen species (ROS) via a NAD(P)H oxidase-dependent mechanism, as shown by inhibition of ROS production via the NAD(P)H oxidase inhibitors diphenylene iodonium (DPI) and apocynin. This increase in ROS is critical for Ang-II-induced fibrotic effects, as indicated by the decrease in Ang-II-induced CTGF and fibronectin levels by DPI and apocynin. We also show that Ang-II-induced ROS production and fibrosis require PKC activity as indicated by the generic PKC inhibitor chelerythrine.These results strongly suggest that the fibrotic response induced by Ang-II is mediated by AT-1 receptor and requires NAD(P)H-induced ROS in skeletal muscle cells.     

New aspects of Wnt signaling pathways in higher vertebrates

The development of tissues and organs in embryos is controlled by an interplay of several signaling pathways that cross-talk to provide positional information and induce cell fate specification. One of the major signaling systems is the Wnt pathway which was recently shown to split into several intracellular branches which regulate multiple cellular functions. In the present review, we discuss novel members and their role in the diversification of the Wnt pathway. Many of these components were studied in model organisms such as C.elegans, Drosophila and Xenopus. Here we focus on recent studies of mutant phenotypes in Mouse and Zebrafish which implicate members of the Wnt pathway in processes such as axis and mesoderm formation, initiation of organ development and stem cell differentiation.