Activation Of Canonical Wnt Signaling Research Articles

Epidermal YAP activity drives canonical WNT16/β-catenin signaling to promote keratinocyte proliferation in vitro and in the murine skin

The skin constantly self-renews throughout adult life. Wnt/β-catenin signaling plays a key role in promoting keratinocyte proliferation in the hair follicles and in the interfollicular epidermis. A recent report demonstrated that epidermal YAP activity drives β-catenin activation to promote keratinocyte proliferation in the murine skin. However, it remains unclear whether this is caused by paracrine activation of canonical Wnt signaling or through other YAP/β-catenin regulatory interactions. In the present study, we found that XAV939-inhibition of canonical WNT signaling in skin of YAP2-5SA-ΔC mice resulted in diminished β-catenin activation, reduced keratinocyte proliferation, and a mitigation of the hyperplastic abnormalities in the interfollicular epidermis, signifying a canonical WNT ligand-dependent mechanism. Our subsequent analyses determined that WNT16 is produced in response to YAP activity in keratinocytes both in vitro and in vivo, and that WNT16 drives HaCaT keratinocyte proliferation via canonical WNT16/β-catenin signaling. We conclude that under normal physiological conditions WNT16 is the paracrine WNT ligand secreted in response to epidermal YAP activity that promotes cell proliferation in the interfollicular epidermis. This study delineates a fundamental YAP-driven mechanism that controls normal skin regeneration, and that may be perturbed in human regenerative disease displaying increased YAP and WNT signaling activity.

The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9, COL2A1, and ACAN, and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5. Inhibition of WNT signaling by PKF increased the expression of SOX9, COL2A1, ACAN, and MMP9, but decreased MMP13 and ADAMTS4/5. In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL, and RUNX2, the dedifferentiation marker COL1A1, and glycolysis genes GULT1 and PGK1. Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were decreased after PKF treatment. All of these chondrogenic effects appeared to be mediated through the canonical WNT signaling pathway, since the target gene Axin2 and other WNT members, such as TCF4 and β-catenin, were upregulated by BIO and downregulated by PKF, respectively, and BIO induced nuclear translocation of β-catenin while PKF inhibited β-catenin translocation into the nucleus. We concluded that addition of BIO to a chondrogenic medium of hMSCs resulted in a loss of cartilage formation, while PKF induced chondrogenic differentiation and cartilage matrix deposition and inhibited hypertrophic differentiation. However, BIO promoted cell survival by inhibiting apoptosis while PKF induced cell apoptosis. This result indicates that either an overexpression or overinhibition of WNT signaling to some extent causes harmful effects on chondrogenic differentiation. Cartilage tissue engineering could benefit from the adjustment of the critical level of WNT signaling during chondrogenesis of hMSC.

Wnt signaling regulates proliferation and differentiation of radial glia in regenerative processes after stab injury in the optic tectum of adult zebrafish.

Zebrafish have superior abilities to generate new neurons in the adult brain and to regenerate brain tissue after brain injury compared with mammals. There exist two types of neural stem cells (NSCs): neuroepithelial-like stem cells (NE) and radial glia (RG) in the optic tectum. We established an optic tectum stab injury model to analyze the function of NSCs in the regenerative condition and confirmed that the injury induced the proliferation of RG, but not NE and that the proliferated RG differentiated into new neurons after the injury. We then analyzed the involvement of Wnt signaling after the injury, using a Wnt reporter line in which canonical Wnt signaling activation induced GFP expression and confirmed that GFP expression was induced specifically in RG after the injury. We also analyzed the expression level of genes related to Wnt signaling, and confirmed that endogenous Wnt antagonist dkk1b expression was significantly decreased after the injury. We observed that Wnt signal inhibitor IWR1 treatment suppressed the proliferation and differentiation of RG after the injury, suggesting that up-regulation of Wnt signaling in RG after the stab injury was required for optic tectum regeneration. We also confirmed that Wnt activation by treatment with GSK3β inhibitor BIO in uninjured zebrafish induced proliferation of RG in the optic tectum. This optic tectum stab injury model is useful for the study of the molecular mechanisms of brain regeneration and analysis of the RG functions in physiological and regenerative conditions.

Cardiogenic programming of human pluripotent stem cells by dose-controlled activation of EOMES

Master cell fate determinants are thought to induce specific cell lineages in gastrulation by orchestrating entire gene programs. The T-box transcription factor EOMES (eomesodermin) is crucially required for the development of the heart—yet it is equally important for endoderm specification suggesting that it may act in a context-dependent manner. Here, we define an unrecognized interplay between EOMES and the WNT signaling pathway in controlling cardiac induction by using loss and gain-of-function approaches in human embryonic stem cells. Dose-dependent EOMES induction alone can fully replace a cocktail of signaling molecules otherwise essential for the specification of cardiogenic mesoderm. Highly efficient cardiomyocyte programming by EOMES mechanistically involves autocrine activation of canonical WNT signaling via the WNT3 ligand, which necessitates a shutdown of this axis at a subsequent stage. Our findings provide insights into human germ layer induction and bear biotechnological potential for the robust production of cardiomyocytes from engineered stem cells.

Amphibian Zic Genes.

Studies in Xenopus laevis have greatly contributed to understanding the roles that the Zic family of zinc finger transcription factors play as essential drivers of early development. Explant systems that are not readily available in other organisms give Xenopus embryos a unique place in these studies, facilitated by the recent sequencing of the Xenopus laevis genome. A number of upstream regulators of zic gene expression have been identified, such as inhibition of BMP signaling, as well as calcium, FGF, and canonical Wnt signaling. Screens using induced ectodermal explants have identified genes that are direct targets of Zic proteins during early neural development and neural crest specification. These direct targets include Xfeb (also called glipr2; hindbrain development), aqp3b (dorsal marginal zone in gastrula embryos and neural folds), snail family members (premigratory neural crest), genes that play roles in retinoic acid signaling, noncanonical Wnt signaling, and mesoderm development, in addition to a variety of genes some with and many without known roles during neural or neural crest development. Functional experiments in Xenopus embryos demonstrated the involvement of Zic family members in left-right determination, early neural patterning, formation of the midbrain-hindbrain boundary, and neural crest specification. The role of zic genes in cell proliferation vs. differentiation remains unclear, and the activities of Zic factors as inhibitors or activators of canonical Wnt signaling may be dependent on developmental context. Overall, Xenopus has contributed much to our understanding of how Zic transcriptional activities shape the development of the embryo and contribute to disease.

LGR4 modulates breast cancer initiation, metastasis, and cancer stem cells.

The fourth member of the leucine-rich repeat-containing GPCR family (LGR4, frequently referred to as GPR48) and its cognate ligands, R-spondins (RSPOs) play crucial roles in the development of multiple organs as well as the survival of adult stem cells by activation of canonical Wnt signaling. Wnt/β-catenin signaling acts to regulate breast cancer; however, the molecular mechanisms determining its spatiotemporal regulation are largely unknown. In this study, we identified LGR4 as a master controller of Wnt/β-catenin signaling-mediated breast cancer tumorigenesis, metastasis, and cancer stem cell (CSC) maintenance. LGR4 expression in breast tumors correlated with poor prognosis. Either Lgr4 haploinsufficiency or mammary-specific deletion inhibited mouse mammary tumor virus (MMTV)- PyMT- and MMTV- Wnt1-driven mammary tumorigenesis and metastasis. Moreover, LGR4 down-regulation decreased in vitro migration and in vivo xenograft tumor growth and lung metastasis. Furthermore, Lgr4 deletion in MMTV- Wnt1 tumor cells or knockdown in human breast cancer cells decreased the number of functional CSCs by ∼90%. Canonical Wnt signaling was impaired in LGR4-deficient breast cancer cells, and LGR4 knockdown resulted in increased E-cadherin and decreased expression of N-cadherin and snail transcription factor -2 ( SNAI2) (also called SLUG), implicating LGR4 in regulation of epithelial-mesenchymal transition. Our findings support a crucial role of the Wnt signaling component LGR4 in breast cancer initiation, metastasis, and breast CSCs.-Yue, Z., Yuan, Z., Zeng, L., Wang, Y., Lai, L., Li, J., Sun, P., Xue, X., Qi, J., Yang, Z., Zheng, Y., Fang, Y., Li, D., Siwko, S., Li, Y., Luo, J., Liu, M. LGR4 modulates breast cancer initiation, metastasis, and cancer stem cells.

Recombinant frizzled1 protein attenuated cardiac hypertrophy after myocardial infarction via the canonical Wnt signaling pathway.

Postinfarct cardiac hypertrophy is an independent risk factor for heart failure and sudden death. Regression of cardiac hypertrophy has emerged as a promising strategy in the treatment of myocardial infarction (MI). Here we hypothesized that frizzled1 (FZD1), a receptor of the canonical Wnt signaling pathway, is a novel mediator of ischemia-associated cardiac hypertrophy. MI was induced in mice by left anterior descending (LAD) coronary occlusion. One week after MI, the expression of FZD1 was found to be notably increased in the left ventricles (LVs) of the MI-mice compared to shams. Mouse recombinant FZD1 protein (RFP) was subcutaneously injected in the mice to provoke autoimmunization response. Anti-FZD1 antibody titer was significantly increased in the plasma of RFP-treated mice. RFP significantly mitigated the MI-induced cardiac hypertrophy and improved cardiac function in the MI mouse hearts. Moreover, increased heart and LV weights, myocardial size and the expression of β-myosin heavy chain in the MI-mice were also found to be attenuated by RFP. FZD1 was found to be significantly up-regulated in hypoxia-treated neonatal rat cardiomyocytes (NRCMs). Silencing FZD1 by siRNA transfection notably repressed the hypoxia-induced myocardial hypertrophy in NRCMs. Mechanistically, activation of canonical Wnt signaling induced by MI, e.g., β-catenin and glycogen synthase kinase-3β, was restrained in the LVs of the MI-mice treated by RFP, these inhibition on canonical Wnt signaling was further confirmed in hypoxic NRCMs transfected with FZD1 siRNA. In conclusion, immunization of RFP attenuated cardiac hypertrophy and improved cardiac function in the MI mice via blocking the canonical Wnt signaling pathway.

Efficient Induction of Syncytiotrophoblast Layer II Cells from Trophoblast Stem Cells by Canonical Wnt Signaling Activation.

SummaryThe syncytiotrophoblast layer is the most critical and prominent tissue in placenta. SynT cells are differentiated from trophoblast stem cells (TSCs) during early embryogenesis. Mouse TSCs can spontaneously differentiate into cells of mixed lineages in vitro upon withdrawal of stemness-maintaining factors. However, differentiation into defined placental cell lineages remains challenging. We report here that canonical Wnt signaling activation robustly induces expression of SynT-II lineage-specific genes Gcm1 and SynB and suppresses markers of other placental lineages. In contrast to mouse TSCs, the induced SynT-II cells are migratory. More importantly, the migration depends on hepatocyte growth factor (HGF) and the c-MET signaling axis. Furthermore, HGF-expressing cells lie adjacent to SynT-II cells in developing murine placenta, suggesting that HGF/c-MET signaling plays a critical role in SynT-II cell morphogenesis during the labyrinth branching process. The availability of SynT-II cells in vitro will facilitate molecular understanding of labyrinth layer development.

Fibrin-Enhanced Canonical Wnt Signaling Directs Plasminogen Expression in Cementoblasts.

Cementum is a mineralized layer on the tooth’s root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of β-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/β-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or β-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/β-catenin complex in the plasminogen proximal promoter regions (−900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation.

Primary Osteoporosis in Young Adults: Genetic Basis and Identification of Novel Variants in Causal Genes.

ABSTRACTGenetic determinants contribute to osteoporosis and enhance the risk of fracture. Genomewide association studies of unselected population‐based individuals or families have identified polymorphisms in several genes related to low bone density, but not in osteoporotic patients with Z‐score < −2.0 SD with fragility fracture(s). The aim of this study was to determine the causal genes of idiopathic osteoporosis in the adulthood. Also, we used next‐generation sequencing of candidate genes in a cohort of 123 young or middle‐aged adults with idiopathic osteoporosis. All patients were included if they had a low bone mineral density (Z‐score < −2 SD), a diagnosis before age 55 years (mean ± SD, 48.4 ± 10.6 years; mean ± SD age at first fracture, 30.4 ± 17.4 years) and fracture or not. We found that 11 patients carried rare or novel variants in COL1A2 (n = 4), PLS3 (n = 2), WNT1 (n = 4), or DKK1 (n = 1). We showed a high prevalence of pathogenic variants in LRP5: 22 patients (17.8%) had the p.Val667Met variant, including three at the homozygous level and 16 (13%) carrying a novel or very rare variant. Functional analysis revealed that the LRP5 missense variants resulted in reduced luciferase activity, which indicates reduced activation of canonical WNT signaling. The clinical phenotype of patients carrying causal gene variants was indistinguishable. In conclusion, molecular screening of young osteoporotic adults revealed several variants and could be useful to characterize susceptibility genes for personalizing treatment, in particular for the new anabolic drugs.© 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Inhibition of human mesenchymal stem cell proliferation via Wnt signaling activation.

Human mesenchymal stem cells (hMSCs), characterized by rapid in vitro expandability and multi-differentiation potential, have been widely used in the clinical field of tissue engineering. Recent studies have shown that various signaling networks are involved in the growth and differentiation of hMSCs. Although Wnts and their downstream signaling components have been implicated in the regulation of hMSCs, the role of Wnt signaling in hMSC self-renewal is still controversial. Here, it was observed that activation of endogenous canonical Wnt signaling with LiCl, which decreased β-catenin phosphorylation, leads to a decrease in hMSC proliferation. The fact that this growth arrest is not linked to apoptosis was verified by annexin V-FITC/propidium iodide assay. It was associated with sealing off of the cells in the G1 phase of the cell cycle accompanied by changes in expression of cell cycle-associated genes such as cyclin A and D. In addition, activation of Wnt signaling during hMSC proliferation seemed to reduce their clonogenic potential. On the contrary, Wnt signaling activation during hMSC proliferation had little effect on the osteogenic differentiation capability of cells. These findings show that canonical Wnt signaling is a critical regulator of hMSC proliferation and clonogenicity.

Blockage of Wnt/β-Catenin Signaling by Nanoparticles Reduces Survival and Proliferation of CLL Cells In Vitro-Preliminary Study.

The Wnt/β-catenin signaling pathway is shown to play a significant role in the control of the survival, proliferation, and differentiation of hematopoietic cells. Studies have confirmed that aberrant activation of canonical Wnt signaling occurs in various forms of leukemia, and is crucial for chronic lymphocytic leukemia (CLL) pathogenesis. The aim of the study is to evaluate the influence of maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) on Wnt/β-catenin pathway gene expression in CLL (MEC-1) cells and to compare these findings with those obtained with fludarabine (FA). Microarray data analysis reveals seven of 19 Wnt/β-catenin pathway genes whose expression changes significantly during dendrimer and FA treatment: WNT10A, WNT6, and CDH1 among others. PPI-G4-M3 is already known to influence MEC-1 cell apoptosis and proliferation. The obtained results suggest that the reduction in cell survival under the influence of glycodendrimers and FA may be due to loss of Wnt signaling.

Identification and characterization of a new source of adult human neural progenitors

Adult neural progenitor cells (aNPCs) are a potential source for cell based therapy for neurodegenerative diseases and traumatic brain injuries. These cells have been traditionally isolated from hippocampus, subventricular zone and white matter. However, there is still a need for an easily accessible source with better yield to counter the limitations of small surgical samples of previously characterized aNPCs. Here we show that ultrasonic aspirate (UA) samples currently considered as ‘biological waste after surgery,' offer a good source for aNPCs. Furthermore, we show that culture conditions dictated the phenotype of cells across patients. The neurosphere-enriched cells were more similar to freshly isolated brain cells, while cells expanded adherently in serum conditions were similar to mesenchymal stem cells. However, cells expanded in these adherent conditions expressed some NPC and glial markers in addition to active canonical Wnt signaling. This suggests a mesenchymal-neuroectodermal hybrid nature of these cells. Finally, we show that UA-NPCs are comparable to those from neurogenic regions. Our findings suggest that UA samples can be used as a source for fresh and in vitro propagated aNPCs that could have various clinical applications.

Membrane Targeting of Disheveled Can Bypass the Need for Arrow/LRP5

The highly conserved Wnt signaling pathway regulates cell proliferation and differentiation in vertebrates and invertebrates. Upon binding of a Wnt ligand to a receptor of the Fz family, Disheveled (Dsh/Dvl) transduces the signal during canonical and non-canonical Wnt signaling. The specific details of how this process occurs have proven difficult to study, especially as Dsh appears to function as a switch between different branches of Wnt signaling. Here we focus on the membrane-proximal events that occur once Dsh is recruited to the membrane. We show that membrane-tethering of the Dsh protein is sufficient to induce canonical Wnt signaling activation even in the absence of the Wnt co-receptor Arrow/LRP5/6. We map the protein domains required for pathway activation in membrane tethered constructs finding that both the DEP and PDZ domains are dispensable for canonical signaling only in membrane-tethered Dsh, but not in untethered/normal Dsh. These data lead to a signal activation model, where Arrow is required to localize Dsh to the membrane during canonical Wnt signaling placing Dsh downstream of Arrow.

Identification of CD146 as a novel molecular actor involved in systemic sclerosis

We highlight for the first time that CD146/sCD146 is involved in fibrotic process during SSc. sCD146 could thus constitute a new biomarker to assess disease activity, and potentially a new target for therapeutic applications.

Effects of WNT3A and WNT16 on the Osteogenic and Adipogenic Differentiation of Perivascular Stem/Stromal Cells

Human perivascular stem/stromal cells (hPSC) are a multipotent mesenchymogenic stromal cell population defined by their perivascular locale. Recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting (FACS)-derived cell population for autologous bone tissue engineering. However, the mechanisms underlying the osteogenic differentiation of PSC are incompletely understood. The current study investigates the roles of canonical and noncanonical Wnt signaling in the osteogenic and adipogenic differentiation of PSC. Results showed that both canonical and noncanonical Wnt signaling activity transiently increased during PSC osteogenic differentiation in vitro. Sustained WNT3A treatment significantly decreased PSC osteogenic differentiation. Conversely, sustained treatment with Wnt family member 16 (WNT16), a mixed canonical and noncanonical ligand, increased osteogenic differentiation in a c-Jun N-terminal kinase (JNK) pathway-dependent manner. Conversely, WNT16 knockdown significantly diminished PSC osteogenic differentiation. Finally, WNT16 but not WNT3A increased the adipogenic differentiation of PSC. These results indicate the importance of regulation of canonical and noncanonical Wnt signaling for PSC fate and differentiation. Moreover, these data suggest that WNT16 plays a functional and necessary role in PSC osteogenesis.

Galectin‑3 induces the phenotype transformation of human vascular smooth muscle cells via the canonical Wnt signaling.

Galectin‑3, a galactoside‑binding protein, is highly expressed in carotid plaques and plays an important role in the atherosclerotic lesions. The phenotype transformation of vascular smooth muscle cells is the basic pathological change of atherosclerosis. This study investigated the effects of exogenous galectin‑3 on the function and phenotype transformation of human umbilical vascular smooth muscle cells (HUSMC). In this study, we treated vascular smooth muscle cells with recombinant galectin‑3 and tested its effect on cell proliferation, migration, and phenotype transformation. Our results showed that exogenous galectin‑3 promoted human umbilical vascular smooth muscle cells (HUSMC) proliferation and migration. Exogenous galectin‑3 enhanced the expression of the smooth muscle synthetic protein osteopontin, smooth muscle contractile proteins calponin and smooth muscle α‑actin. The galectin‑3‑induced change in cell phenotype was associated with the activation of canonical Wnt signaling, as measured by β‑catenin axin2 and cyclinD1 expression. β‑catenin inhibition by small interfering RNA reduced cell proliferation, decreased cell motility, and blocked galectin‑3‑induced phenotype transformation of human umbilical vascular smooth muscle cells (HUSMC). Our data suggest galectin‑3 promotes the phenotype transformation of human umbilical vascular smooth muscle cells (HUSMC) by activating Wnt/β‑catenin signaling pathway.

Epigenetic‐Modification of Adult Fibroblast Cells into OCT4+ and VEGFR‐2+ Angiogenic Cells

RationaleThe epigenome of fibroblast cells is plastic. Here, we address the hypothesis that fibroblast cells can be epigenetically modified into blood vessel forming cells.Methods and ResultsHuman adult dermal fibroblast cells (hADFCs) were treated with optimal concentrations of chromatin‐modifying agents 5‐aza‐2′‐deoxycytidine (5‐Aza) and trichostatin A (TSA), thereafter subjected to activation of canonical Wnt signaling. We provide evidence that these epigenetically modified hADFCs increasingly expressed β‐catenin, pluripotency factor octamer‐binding transcription factor‐4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor‐2 (VEGFR‐2) and Lipid Phosphate Phosphatase‐3 (LPP3). Staining and microscopic analysis showed differential localization of β‐catenin and OCT4 proteins in epigenetically modified hADFCs. Accordingly, in a chromatin immunoprecipitation (ChIP) assay, OCT4 bound to the VEGFR‐2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a three‐dimensional (3D) Matrigel, a subset of modified cells formed branching point network‐like structures in VEGF‐dependent manner.SummaryThe epigenome of adult fibroblast cells is amenable to epigenetic modification, and has significant implications for the use of epigenetically modified cells to promote angiogenesis in vivo.Support or Funding InformationStudies were supported by the RO1‐HL079356 and (GRNT25710129) and by the University of Illinois at Chicago Center for Clinical and Translation Science Award (UL1RR029879) to K.K.W. J.B was supported by AHA pre‐doctoral fellowship Mid‐West affiliate (15PRE22760004) and CCTS‐PECTs (1UL1TR002003). The UIC, RRC Core Imaging Facility, at the University of Illinois Chicago.

Agrin inhibits canonical WNT signalling through LRP4

Purpose: Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. We recently identified Agrin as an essential molecule in articular cartilage homeostasis, uniquely requiring both α-dystroglycan and LRP4 to support SOX9 expression and chondrocyte differentiation in vitro and in vivo. Despite identifying α-dystroglycan and LRP4 as necessary for the chondrogenic effects of Agrin in chondrocytes, the signalling pathway(s) by which this occurs remains unclear. LRP4 signalling is known to negatively regulate WNT signalling. Therefore, we tested whether Agrin is required for the WNT-inhibitory properties of LRP4 and whether this is the mechanism by which Agrin signals in chondrocytes. Methods: Mammalian Agrin or GFP was overexpressed in bovine primary chondrocytes and cultured in micromass for 5 days in the presence or absence of 100ng/ml WNT3a. Chondrogenic potential was assessed by qPCR for SOX9 mRNA expression. Agrin or GFP stably expressing COS7 cells were transfected with the SUPER8TOPFLASH reporter plasmid in combination with Vehicle or LRP4 plasmids. COS7 monolayer cultures were treated with vehicle or increasing doses of WNT3a (50, 100, 200ng/ml). WNT signalling activation was determined by luciferase expression. Results: LRP4 overexpression was sufficient to induce SOX9 upregulation in chondrocytes, however this effect was abolished following Agrin knockdown demonstrating the requirement of Agrin for the chondrogenic effect of LRP4 signalling. In addition, downregulation of SOX9 mRNA expression induced by WNT3a treatment was rescued by Agrin overexpression in micromass cultures. Using the WNT reporter assay, activation of canonical WNT signalling was inhibited in COS7 cells transduced with human Agrin compared to GFP-transduced control COS7 cells; and this effect was exacerbated by overexpression of LRP4. Conclusions: Agrin inhibits canonical WNT signalling through LRP4 and enhance chondrocyte differentiation. However, it remains unclear how Agrin-LRP4 signalling achieves its specificity in promoting chondrogenesis compared to other LRP4 ligands.

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/β-catenin signaling

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/β-catenin pathway assessed by phosphorylation of GSK-3β. Over-expressing of constitutively active GSK-3β (caGSK-3β) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3β over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.