Browsing by Subject "Wnt signaling"
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Item A novel decellularized matrix of Wnt signaling-activated osteocytes accelerates the repair of critical-sized parietal bone defects with osteoclastogenesis, angiogenesis, and neurogenesis(Elsevier, 2022-08-16) Wang, Xiaofang; Ma, Yufei; Chen, Jie; Liu, Yujiao; Liu, Guangliang; Wang, Pengtao; Wang, Bo; Taketo, Makoto M.; Bellido, Teresita; Tu, Xiaolin; Anatomy, Cell Biology and Physiology, School of MedicineCell source is the key to decellularized matrix (DM) strategy. This study compared 3 cell types, osteocytes with/without dominant active Wnt/β-catenin signaling (daCO and WTO) and bone marrow stromal cells (BMSCs) for their DMs in bone repair. Decellularization removes all organelles and >95% DNA, and retained >74% collagen and >71% GAG, maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the BMSC-DM. DM produced higher cell survival rate (90%) and higher proliferative activity. In vitro, daCO-DM induces more and longer stress fibers in BMSCs, conducive to cell adhesion, spreading, and osteogenic differentiation. 8-wk after implantation of the critical-sized parietal bone defect model, daCO-DM formed tight structures, composed of a large number of densely-arranged type-I collagen under polarized light microscope, which is similar to and integrated with host bone. BV/TV (>54%) was 1.5, 2.9, and 3.5 times of WTO-DM, BMSC-DM, and none-DM groups, and N.Ob/T.Ar (3.2 × 102/mm2) was 1.7, 2.9, and 3.3 times. At 4-wk, daCO-DM induced osteoclastogenesis, 2.3 times higher than WTO-DM; but BMSC-DM or none-DM didn't. daCO-DM increased the expression of RANKL and MCSF, Vegfa and Angpt1, and Ngf in BMSCs, which contributes to osteoclastogenesis, angiogenesis, and neurogenesis, respectively. daCO-DM promoted H-type vessel formation and nerve markers β3-tubulin and NeuN expression. Conclusion: daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone defects. These features are expected to achieve the effect of autologous bone transplantation, suitable for transformation application.Item Antagonizing miR-218-5p attenuates Wnt signaling and reduces metastatic bone disease of triple negative breast cancer cells(Impact Journals, 2016-11-29) Taipaleenmäki, Hanna; Farina, Nicholas H.; van Wijnen, Andre J.; Stein, Janet L.; Hesse, Eric; Stein, Gary S.; Lian, Jane B.; Department of Anatomy & Cell Biology, IU School of MedicineWnt signaling is implicated in bone formation and activated in breast cancer cells promoting primary and metastatic tumor growth. A compelling question is whether osteogenic miRNAs that increase Wnt activity for bone formation are aberrantly expressed in breast tumor cells to support metastatic bone disease. Here we report that miR-218-5p is highly expressed in bone metastases from breast cancer patients, but is not detected in normal mammary epithelial cells. Furthermore, inhibition of miR-218-5p impaired the growth of bone metastatic MDA-MB-231 cells in the bone microenvironment in vivo. These findings indicate a positive role for miR-218-5p in bone metastasis. Bioinformatic and biochemical analyses revealed a positive correlation between aberrant miR-218-5p expression and activation of Wnt signaling in breast cancer cells. Mechanistically, miR-218-5p targets the Wnt inhibitors Sclerostin (SOST) and sFRP-2, which highly enhances Wnt signaling. In contrast, delivery of antimiR-218-5p decreased Wnt activity and the expression of metastasis-related genes, including bone sialoprotein (BSP/IBSP), osteopontin (OPN/SPP1) and CXCR-4, implicating a Wnt/miR-218-5p regulatory network in bone metastatic breast cancer. Furthermore, miR-218-5p also mediates the Wnt-dependent up-regulation of PTHrP, a key cytokine promoting cancer-induced osteolysis. Antagonizing miR-218-5p reduced the expression of PTHrP and Rankl, inhibited osteoclast differentiation in vitro and in vivo, and prevented the development of osteolytic lesions in a preclinical metastasis model. We conclude that pathological elevation of miR-218-5p in breast cancer cells activates Wnt signaling to enhance metastatic properties of breast cancer cells and cancer-induced osteolytic disease, suggesting that miR-218-5p could be an attractive therapeutic target for preventing disease progression.Item Characterization of Ethanol-induced Effects on Zebrafish Retinal Development: Mechanistic Perspective and Therapeutic Strategies(2016) Muralidharan, Pooja; Marrs, James A.; Leung, Yuk Fai; Belecky-Adams, Teri; Meyer, Jason; Anderson, Ryan M.; Randall, Stephen K.Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.Item Chemically-defined generation of human hemogenic endothelium and definitive hematopoietic progenitor cells(Elsevier, 2022) Chang, Yun; Syahirah, Ramizah; Oprescu, Stephanie N.; Wang, Xuepeng; Jung, Juhyung; Cooper, Scott H.; Torregrosa-Allen, Sandra; Elzey, Bennett D.; Hsu, Alan Y.; Randolph, Lauren N.; Sun, Yufei; Kuang, Shihuan; Broxmeyer, Hal E.; Deng, Qing; Lian, Xiaojun; Bao, Xiaoping; Microbiology and Immunology, School of MedicineHuman hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFβ inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.Item Conversion of Osteoclasts into Bone-Protective, Tumor-Suppressing Cells(MDPI, 2021-11-09) Li, Ke-Xin; Sun, Xun; Li, Bai-Yan; Yokota, Hiroki; Biomedical Engineering, School of Engineering and TechnologyOsteoclasts are a driver of a vicious bone-destructive cycle with breast cancer cells. Here, we examined whether this vicious cycle can be altered into a beneficial one by activating Wnt signaling with its activating agent, BML284. The conditioned medium, derived from Wnt-activated RAW264.7 pre-osteoclast cells (BM CM), reduced the proliferation, migration, and invasion of EO771 mammary tumor cells. The same inhibitory effect was obtained with BML284-treated primary human macrophages. In a mouse model, BM CM reduced the progression of mammary tumors and tumor-induced osteolysis and suppressed the tumor invasion to the lung. It also inhibited the differentiation of RANKL-stimulated osteoclasts and enhanced osteoblast differentiation. BM CM was enriched with atypical tumor-suppressing proteins such as Hsp90ab1 and enolase 1 (Eno1). Immunoprecipitation revealed that extracellular Hsp90ab1 interacted with latent TGFβ (LAP-TGFβ) as an inhibitor of TGFβ activation, while Hsp90ab1 and Eno1 interacted and suppressed tumor progression via CD44, a cell-adhesion receptor and a cancer stem cell marker. This study demonstrated that osteoclast-derived CM can be converted into a bone-protective, tumor-suppressing agent by activating Wnt signaling. The results shed a novel insight on the unexplored function of osteoclasts as a potential bone protector that may develop an unconventional strategy to combat bone metastasis.Item Generation of the tumor-suppressive secretome from tumor cells(Ivyspring International, 2021-07-25) Liu, Shengzhi; Sun, Xun; Li, Kexin; Zha, Rongrong; Feng, Yan; Sano, Tomohiko; Dong, Chuanpeng; Liu, Yunlong; Aryal, Uma K.; Sudo, Akihiro; Li, Bai-Yan; Yokota, Hiroki; Biomedical Engineering, School of Engineering and TechnologyRationale: The progression of cancer cells depends on the soil and building an inhibitory soil might be a therapeutic option. We previously created tumor-suppressive secretomes by activating Wnt signaling in MSCs. Here, we examined whether the anti-tumor secretomes can be produced from tumor cells. Methods: Wnt signaling was activated in tumor cells by overexpressing β-catenin or administering BML284, a Wnt activator. Their conditioned medium (CM) was applied to cancer cells or tissues, and the effects of CM were evaluated. Tumor growth in the mammary fat pad and tibia in C57BL/6 female mice was also evaluated through μCT imaging and histology. Whole-genome proteomics analysis was conducted to determine and characterize novel tumor-suppressing proteins, which were enriched in CM. Results: The overexpression of β-catenin or the administration of BML284 generated tumor-suppressive secretomes from breast, prostate and pancreatic cancer cells. In the mouse model, β-catenin-overexpressing CM reduced tumor growth and tumor-driven bone destruction. This inhibition was also observed with BML284-treated CM. Besides p53 and Trail, proteomics analysis revealed that CM was enriched with enolase 1 (Eno1) and ubiquitin C (Ubc) that presented notable tumor-suppressing actions. Importantly, Eno1 immunoprecipitated CD44, a cell-surface adhesion receptor, and its silencing suppressed Eno1-driven tumor inhibition. A pan-cancer survival analysis revealed that the downregulation of MMP9, Runx2 and Snail by CM had a significant impact on survival outcomes (p < 0.00001). CM presented a selective inhibition of tumor cells compared to non-tumor cells, and it downregulated PD-L1, an immune escape modulator. Conclusions: The tumor-suppressive secretome can be generated from tumor cells, in which β-catenin presented two opposing roles, as an intracellular tumor promoter in tumor cells and a generator of extracellular tumor suppressor in CM. Eno1 was enriched in CM and its interaction with CD44 was involved in Eno1's anti-tumor action. Besides presenting a potential option for treating primary cancers and metastases, the result indicates that aggressive tumors may inhibit the growth of less aggressive tumors via tumor-suppressive secretomes.Item In-Vivo Nucleus Pulposus-Specific Regulation of Adult Murine Intervertebral Disc Degeneration via Wnt/Beta-Catenin Signaling(Nature Publishing Group, 2018-07-25) Holguin, Nilsson; Silva, Matthew J.; Mechanical and Energy Engineering, School of Engineering and TechnologyB-Catenin, transcription factor of Wnt signaling, is promoted in patients with intervertebral disc (IVD) degeneration, but Wnt signaling decreases with aging. We hypothesize that IVD degeneration is associated with decreased Wnt signaling despite more b-Catenin. Chronic compression of tail IVDs of young-adult and aged Wnt-reporter (TOPGAL) animals initiated an age-related cascade of degenerative-like changes, which included reduced Wnt ligand expression and Wnt signaling in nucleus pulposus cells, despite elevation of b-Catenin protein and gene expression. To determine the effect of upregulated and downregulated Wnt signaling in adult discs, b-Catenin in the nucleus pulposus was stabilized (Shh-CreErT2/b-Cateninfl(Ex3)/fl(Ex3), cACT) or knocked out (Shh-CreErT2/b-Cateninfl/fl, cKO). cACT discs had promoted expression of Wnt-targets and -ligands, brachyury, extracellular matrix production and 34% greater compressive stiffness than WT (b-Cateninfl(Ex3)/fl(Ex3)) discs, but 50% less tensile stiffness. By contrast, knockout reversed the cACT phenotype: less protein expression of b-catenin in the nucleus pulposus, less expression of brachyury, heightened expression of extracellular matrix breakdown and 46% less compressive stiffness than wild-type (b-Cateninfl/fl,WT) discs. These data suggest that intervertebral disc degeneration is associated with loss of Wnt signaling and that the concomitant increase in b-catenin is a regenerative response, potentially offering a therapeutic approach to degeneration.Item Knee loading repairs osteoporotic osteoarthritis by relieving abnormal remodeling of subchondral bone via Wnt/β-catenin signaling(Wiley, 2020-02) Zheng, Weiwei; Ding, Beibei; Li, Xinle; Liu, Daquan; Yokota, Hiroki; Zhang, Ping; Biomedical Engineering, School of Engineering and TechnologyOsteoporotic osteoarthritis (OPOA) is a common bone disease mostly in the elderly, but the relationship between Osteoporotic (OP) and osteoarthritis (OA) is complex. It has been shown that knee loading can mitigate OA symptoms. However, its effects on OPOA remain unclear. In this study, we characterized pathological linkage of OP to OA, and evaluated the effect of knee loading on OPOA. We employed two mouse models (OA and OPOA), and conducted histology, cytology, and molecular analyses. In the OA and OPOA groups, articular cartilage was degenerated and Osteoarthritis Research Society International score was increased. Subchondral bone underwent abnormal remodeling, the differentiation of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts and chondrocytes was reduced, and migration and adhesion of pre-osteoclasts were enhanced. Compared to the OA group, the pathological changes of OA in the OPOA group were considerably aggravated. After knee loading, however, cartilage degradation was effectively prevented, and the abnormal remodeling of subchondral bone was significantly inhibited. The differentiation of BMSCs was also improved, and the expression of Wnt/β-catenin was elevated. Collectively, this study demonstrates that osteoporosis aggravates OA symptoms. Knee loading restores OPOA by regulating subchondral bone remodeling, and may provide an effective method for repairing OPOA.Item Mathematical modeling of the effects of Wnt-10b on bone metabolism(Wiley, 2022) Cook, Carley V.; Islam, Mohammad Aminul; Smith, Brenda J.; Ford Versypt, Ashlee N.; Obstetrics and Gynecology, School of MedicineBone health is determined by factors including bone metabolism or remodeling. Wnt-10b alters osteoblastogenesis through pre-osteoblast proliferation and differentiation and osteoblast apoptosis rate, which collectively lead to the increase of bone density. To model this, we adapted a previously published model of bone remodeling. The resulting model for the bone compartment includes differential equations for active osteoclasts, pre-osteoblasts, osteoblasts, osteocytes, and the amount of bone present at the remodeling site. Our alterations to the original model consist of extending it past a single remodeling cycle and implementing a direct relationship to Wnt-10b. Four new parameters were estimated and validated using normalized data from mice. The model connects Wnt-10b to bone metabolism and predicts the change in trabecular bone volume caused by a change in Wnt-10b input. We find that this model predicts the expected increase in pre-osteoblasts and osteoblasts while also pointing to a decrease in osteoclasts when Wnt-10b is increased.Item Mutational Mechanisms That Activate Wnt Signaling and Predict Outcomes in Colorectal Cancer Patients(AACR, 2018-02) Hankey, William; McIlhatton, Michael A.; Ebede, Kenechi; Hancioglu, Baris; Zhang, Jie; Brock, Guy N.; Huang, Kun; Groden, Joanna; Medical and Molecular Genetics, School of MedicineAPC biallelic loss-of-function mutations are the most prevalent genetic changes in colorectal tumors, but it is unknown whether these mutations phenocopy gain-of-function mutations in the CTNNB1 gene encoding β-catenin that also activate canonical WNT signaling. Here we demonstrate that these two mutational mechanisms are not equivalent. Furthermore, we show how differences in gene expression produced by these different mechanisms can stratify outcomes in more advanced human colorectal cancers. Gene expression profiling in Apc-mutant and Ctnnb1-mutant mouse colon adenomas identified candidate genes for subsequent evaluation of human TCGA (The Cancer Genome Atlas) data for colorectal cancer outcomes. Transcriptional patterns exhibited evidence of activated canonical Wnt signaling in both types of adenomas, with Apc-mutant adenomas also exhibiting unique changes in pathways related to proliferation, cytoskeletal organization, and apoptosis. Apc-mutant adenomas were characterized by increased expression of the glial nexin Serpine2, the human ortholog, which was increased in advanced human colorectal tumors. Our results support the hypothesis that APC-mutant colorectal tumors are transcriptionally distinct from APC-wild-type colorectal tumors with canonical WNT signaling activated by other mechanisms, with possible implications for stratification and prognosis.