DKK1 Inhibition Research Articles

Cytokine priming enhances the antifibrotic effects of human adipose derived mesenchymal stromal cells conditioned medium

BackgroundFibrosis is a pathological scarring process characterized by persistent myofibroblast activation with excessive accumulation of extracellular matrix (ECM). Fibrotic disorders represent an increasing burden of disease-associated morbidity and mortality worldwide for which there are limited therapeutic options. Reversing fibrosis requires the elimination of myofibroblasts, remodeling of the ECM, and regeneration of functional tissue. Multipotent mesenchymal stromal cells (MSC) have antifibrotic properties mediated by secreted factors present in their conditioned medium (MSC-CM). However, there are no standardized in vitro assays to predict the antifibrotic effects of human MSC. As a result, we lack evidence on the effect of cytokine priming on MSC’s antifibrotic effects. We hypothesize that the MSC-CM promotes fibrosis resolution in vitro and that this effect is enhanced following MSC cytokine priming.MethodsWe compared the antifibrotic effects of resting versus interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) primed MSC-CM in four in vitro assays: prevention of fibroblast activation, myofibroblasts deactivation, ECM degradation and fibrosis resolution in lung explant cultures. Furthermore, we performed transcriptomic analysis of myofibroblasts treated or not with resting or primed MSC-CM and proteomic characterization of resting and primed MSC-CM.ResultsWe isolated MSC from adipose tissue of 8 donors, generated MSC-CM and tested each MSC-CM independently. We report that MSC-CM treatment prevented TGF-β induced fibroblast activation to a similar extent as nintedanib but, in contrast to nintedanib, MSC-CM reduced fibrogenic myofibroblasts (i.e. transcriptomic upregulation of apoptosis, senescence, and inflammatory pathways). These effects were larger when primed rather than resting MSC-CM were used. Priming increased the ability of MSC-CM to remodel the ECM, reducing its content of collagen I and fibronectin, and reduced the fibrotic load in TGF-β treated lung explant cultures. Priming increased the following antifibrotic proteins in MSC-CM: DKK1, MMP-1, MMP-3, follistatin and cathepsin S. Inhibition of DKK1 reduced the antifibrotic effects of MSC-CM.ConclusionsIn vitro, MSC-CM promote fibrosis resolution, an effect enhanced following MSC cytokine priming. Specifically, MSC-CM reduces fibrogenic myofibroblasts through apoptosis, senescence, and by enhancing ECM degradation. Future studies will establish the in vivo relevance of MSC priming to fibrosis resolution.

Inhibition of both sclerostin and DKK1 results in novel skull findings in the rat and non-human primate that is not observed with inhibition of sclerostin alone

Sclerostin is an extracellular inhibitor of canonical Wnt signaling that inhibits bone formation and stimulates bone resorption. Anti-sclerostin antibodies (Scl-Ab) have been developed as bone-building agents. DKK1, another extracellular inhibitor of the pathway, is upregulated in osteocytes in response to sclerostin inhibition. To further enhance bone-forming effects, a bispecific antibody inhibiting both sclerostin and DKK1 was created (AMG 147). In nonclinical safety studies, AMG 147 resulted in novel skull findings. In the rat, there was increased thickness of skull bones of neural crest origin due to increased subperiosteal compact lamellar and intramembranous woven bone. Externally, subperiosteal fibroblastic/osteoblastic stromal cell proliferation with woven bone and hemorrhage was also observed. Scl-Ab alone resulted in increased skull thickness in the rat, like AMG 147, but without the stromal cell proliferation/woven bone formation. In contrast to embryonic flat bone development, intramembranous bone formed similar to plexiform bone. In the monkey, AMG 147 resulted in macroscopic skull thickening due to a diffuse increase in appositional lamellar bone and increased intramembranous bone on both periosteal surfaces of all skull bones. These data demonstrate that dual inhibition of sclerostin and DDK1 results in unique effects on the skull not observed with sclerostin inhibition alone.

OR19-05 Human Induced Pluripotent Stem Cell-derived Granulosa-like Cells From Ligand-based Differentiation Express FOXL2 After Inhibition Of DKK1

Abstract Disclosure: H. Kubo: None. M.M. Laronda: None. Premature ovarian insufficiency (POI) is characterized by the loss of ovarian function prior to an individual reaching the age of 40. POI can have serious ramifications on health, reducing average life expectancy, and can develop from iatrogenic treatments or genetic variants. POI patients can receive hormone replacement therapies (HRTs), but current methods are not dynamic, responsive, or comprehensive. Contrastingly, cell-based therapies have the potential to produce hormones other than estrogen or progesterone and respond dynamically to each patient. Using human induced pluripotent stem cells (hiPSCs) to generate such cell-based therapies may mitigate rejection upon transplantation and allow for personalized therapies for POI patients. Therefore, we propose to differentiate hiPSCs into granulosa like cells (GLCs) that are functionally the same as granulosa cells (GCs), which are responsible for aromatizing androgens into estrogens. One factor critical to steroidogenesis in GCs is FOXL2, which in mice is suppressed by DKK1, a WNT inhibitor. Therefore, we hypothesized that FOXL2 expression and subsequent steroidogenic functions in our GLCs will be influenced by DKK1. To generate GLCs, we treated the cells with WNT agonist CHIR99021 for two days which has been shown to induce the intermediate mesoderm, and then BMP4 and FST for three days to induce the ovarian somatic lineage. We analyzed our cultures for self-renewal (POU5F1), intermediate mesoderm (PAX2, LHX1, WT1), coelomic epithelium (EMX2), genital ridge (GATA4, NR5A1), and GC (FOXL2) markers using qPCR to assess correct lineage commitment. At day 2, we observed approximately 4000-fold induction of PAX2, 20-fold induction of LHX1, 35-fold increase of WT1, 3000-fold induction of GATA4, and 3.5-fold induction in FOXL2 transcript expression. At day 5, we observed 10-fold induction of NR5A1. We also observed a 7000-fold increase of DKK1 at day 2. Addition of Gallocyanine, a DKK1 inhibitor, from days 2 to 5 resulted in induction of FOXL2 protein expression at day 5, captured by immunofluorescence imaging. Analysis of the images showed that Gallocyanine-treated cells were 42.4% positive for FOXL2, while cells untreated were 0.325% positive for FOXL2. This data suggests that DKK1 does suppress FOXL2 expression in these hiPSC-derived GLCs and demonstrates the potential of these cells as a model to investigate mechanisms important to GC development and maintenance. Future steps include testing the steroidogenic abilities of the GLCs and identification of culture conditions needed to maximize steroidogenesis. This work is supported by the NIH/NICHD R01HD104683 (MML), HuBMAP U01HD110336 (MML), Warren and Eloise Batts Endowment (MML), a Debicki Foundation Grant (MML), the Burroughs Wellcome Fund Career Award at the Scientific Interface (MML), Gesualdo Family Research Scholar (MML), and NIH/NCI training grant T32 CA009560 (HK). Presentation Date: Saturday, June 17, 2023

Upregulation of SOX9 promotes the self-renewal and tumorigenicity of cervical cancer through activating the Wnt/β-catenin signaling pathway.

Sry-box9 (SOX9) maintains stem cell properties and plays crucial roles in many cancers. However, whether SOX9 is correlated with cervical cancer cell stemness and its detailed mechanism remains obscure. We studied the relationship between SOX9 and prognosis of cervical cancer through public database, and SOX9 was related to poor prognosis of cervical cancer. Elevated SOX9 expression enhanced the self-renewal properties and promotes tumorigenicity in cervical cancer. Overexpression of SOX9 could promote the expression of stem cell-related factors in cervical cancer cells and xenografts. Meanwhile, overexpression of SOX9 could also enhance the expressions of FZD10, β-catenin, and c-Myc in cervical cancer cells and xenografts, while inhibiting the expression of DDK1. The activation of Wnt pathway by chir-99 021 raised the tumor spheroid ability of SOX9 knockdown HeLa cells. In addition, SOX9 could transcriptional inhibit DKK1 and activate FZD10 and MYC by binding to their promoters to affect the Wnt/β-catenin pathway. These results demonstrated SOX9 regulated the self-renewal and tumorigenicity of cervical cancer through Wnt/β-catenin pathway by directly transcriptional activation of FZD10, MYC and transcriptional inhibition of DKK1.

COMBINING SCLEROSTIN AND DKK1 INHIBITORS TO IMPROVE BONE PROPERTIES IN THE AGED SKELETON

Abstract Targeting the secreted Wnt inhibitor sclerostin has been an attractive strategy to improve skeletal health. Sclerostin antibody (romosozumab-aqqg; Evenity) was recently approved by the FDA to treat patients at increased risk of fracture. However, an increased risk of cardiovascular events was reported, resulting in issue a ‘black box warning’ requirement for romosozumab. One potential solution to lower the risk of adverse events is to reduce the medication dose. Previously, we found that dual inhibition of sclerostin and Dkk1 produced extremely potent synergistic bone anabolic effects, in both genetic and pharmacological models. While Dkk1 inhibition alone has no consistent bone-building effects, combining antibodies that target sclerostin (Scl-mAb) and Dkk1 (Dkk1-mAb) at 3:1 ratio resulted in 2-3X more bone gain as Scl-mAb alone. Further, much lower total doses of dual antibody treatment, given at optimized proportions, generated equivalent bone anabolic effects as Scl-mAb alone (at much higher doses), suggesting that a combinational strategy has obvious translational benefits. Finally, we tested whether low-dose combination therapy can maintain the same osteogenic effect as Scl-mAb in adult (6 month) and aged (20 month) mice. Outcome measures derived from radiographic, biomechanical, and histomorphometric assays revealed that a 3:1 ratio of Scl-mAb:Dkk1-mAb at 12.5mg/kg was as efficacious as 25mg/kg of Scl-mAb alone, in both age groups. Moreover, cortical porosity—a significant factor contributing to skeletal fragility in the aged skeleton—was significantly reduced by both Scl-mAb and low-dose combination treatment. In conclusion, our findings suggest that optimized low-dose combinational therapy is viable strategy for improving skeletal fragility.

980 nm Photobiomodulation Promotes Osteo/Odontogenic Differentiation of the Stem Cells from Human Exfoliated Deciduous Teeth Via the Crosstalk Between BMP/Smad and Wnt/β-Catenin Signaling Pathways.

Increasing evidence suggests stem cells from human exfoliated deciduous teeth (SHEDs) serve as desirable sources of dentin regeneration. Photobiomodulation (PBM) has shown great potential in enhancing the proliferation and osteogenesis of human bone marrow mesenchymal stem cells (hBMMSCs). However, the specific role of PBM in odontogenic differentiation of SHEDs is little know, and we further investigated potential mechanism of PBM osteo/odontogenisis. A 980 nm diode laser with different energy densities of (0.5, 5, 10 J cm-2 ) in a 100-mW continuous wave was used for irradiation every 24 h. Osteo/odontogenic differentiation of SHEDs was achieved by performing alkaline phosphatase (ALP) and alizarin red staining (ARS) and osteo/odontogenic markers were also evaluated by qRT-PCR and western blotting. Additionally, western blot and immunohistochemical staining were performed to evaluate the levels of BMP/Smad and Wnt/β-catenin signaling-related proteins. We found that PBM at 5 J cm-1 increased mineral deposition and upregulated the expression of related osteo/odontogenic markers along with the elevated expression of β-catenin and phosphorylation level of Smad1/5/9. Furthermore, Wnt signaling inhibition using DKK1 and BMP signaling inhibition using noggin inhibited PBM-induced osteo/odontogenic marker expression when used individually or jointly. In conclusion, PBM induces the osteo/odontogenic differentiation of SHEDs through cross talk between BMP/Smad and Wnt/β-catenin signaling pathways.

Pharmacological inhibition of DKK1 promotes spine fusion in an ovariectomized rat model.

Osteoporosis is common in patients undergoing spine surgery, and carries a considerable risk of adverse outcomes. New methods to positively influence bone regeneration and spine fusion under osteoporotic conditions would be impactful. Neutralizing anti-Dickkopf-1 (DKK1) antibodies has been used as a bone anabolic agent, and recently reported by our group to aid in stem cell-mediated appendicular bone regeneration. Here, a small molecule designed as a DKK1 inhibitor, WAY-262611, was used to induce posterolateral spine fusion in an ovariectomized rat model. In vitro, pharmacological inhibition of DKK1 enhanced osteogenesis and Wnt signaling activity among rat bone marrow-derived stem/stromal cells (BMSCs). In vivo, systemic treatment with WAY-262611 promoted both chondrogenesis and osteogenesis within the spinal fusion site, and ultimately led to significant improvements in lumbar fusion as assessed by XR, μCT, histology and manual palpation assessments. No significant effect on osteoclast numbers or fusion site angiogenesis was detected, suggesting a primary direct effect on mesenchymal cells of the implantation site. Finally, evidence from human stem/stromal cells further demonstrated that pharmacologic inhibition of DKK1 promoted osteogenic differentiation in vitro. Taken together, our results suggest that targeting DKK1 promotes local bone formation and suggests potential clinical value for osteoporotic bone repair.

DOC2B is a negative regulator of Wnt/β-catenin signaling pathway in cervical cancer

DOC2B is a ubiquitously expressed isoform of the double C-2 protein family that requires Ca2+ for most of its physiological functions. Initial findings have indicated that DOC2B participates in exocytosis, vesicular transport, insulin secretion and regulation, glucose homeostasis, and neurotransmitter release. Aberrant expression of DOC2B has been reported in diabetes, leukemia, and cervical cancer (CC). Our earlier studies have demonstrated the inhibitory effects of DOC2B on CC cell proliferation, migration, invasion, and EMT and suggested the possible role of DOC2B in Wnt signaling inhibition. However, the association between DOC2B downregulation and Wnt/β-catenin signaling activation and the underlying molecular mechanism remain elusive. Herein, we found that DOC2B inhibited Wnt/β-catenin pathway by enhancing the expression of the components of the CTNNB1 destruction complex and by fostering proteasomal degradation of CTNNB1. The translocation of CTNNB1 to the nucleus and its interaction with TCF/LEF family transcription factors was perturbed in the presence of DOC2B in a GSK3β independent manner. Further, we have identified DKK1 as one of the upregulated genes in the presence of DOC2B. DKK1 inhibition in DOC2B expressing cells by WAY262611 reactivated Wnt/β-catenin signaling, relieved DOC2B induced senescence, and alleviated the inhibitory effects of DOC2B on the aforementioned malignant behaviors. We have provided evidence for DOC2B-DKK1-senescence-Wnt/β-catenin-EMT signaling crosstalk to have tumor growth regulatory functions in CC. Thus, targeting DOC2B-DKK1-senescence-Wnt/β-catenin-EMT signaling crosstalk via activation of DOC2B may offer a novel approach to restraint malignant behaviors in CC.

Molecular Insights to the Wnt Signaling During Alzheimer's Disorder: a Potential Target for Therapeutic Interventions.

In the adult brain, Wnt signaling is crucial for neurogenesis, and it also regulates neuronal development, neuronal maturation, neuronal differential, and proliferation. Impaired Wnt signaling pathways are associated with enhanced levels of amyloid-β, reduced β-catenin levels, and increased expression of GSK-3β enzyme, suggesting its direct association with the pathogenesis of Alzheimer's disorder (AD). These findings are consolidated by reports where activation of Wnt signaling by genetic factors and pharmacological intervention has improved the cognitive functions in animals and restored neurogenesis in the adult brain. Various natural and synthetic molecules have been identified that modulate Wnt signaling in the adult brain and promote neurogenesis and alleviate behavioral dysfunction. These molecules include lithium, valproic acid, ethosuximide, selenomethionine, curcumin, andrographolide, xanthoceraside, huperzine A, pyridostigmine, ginkgolide-B, ricinine, cannabidiol, and resveratrol. These molecules are associated with the DKK1 and GSK-3β inhibition and β-catenin stabilization along with their effects on neurogenesis, neuronal proliferation, and differentiation in the hippocampus through modulation of Wnt signaling and thereby could prove beneficial in the management of AD pathogenesis. Although modulation of the Wnt signaling seems to suggest to be promising in the management of AD, unfortunately, most of the literature available for the association of Wnt signaling and AD pathogenesis is either from preclinical studies or post-mortem brain. Therefore, it will be interesting to understand the role of Wnt signaling in AD patients, and a rigorous investigation could provide us with a better understanding of AD pathogenesis and the identification of novel targets for therapeutic interventions.

Towards a ‘hot’ tumor phenotype: DKN-01 sensitizes the tumor micro-environment via pro-immune cell cytokine release in vitro and ex vivo

<h3>Objectives:</h3> The Wnt/-catenin pathway has been associated with a ‘cold' tumor microenvironment (TME) in ovarian cancer that allows for unregulated tumor progression. We characterized the impact of inhibiting DKK1 - a Wnt pathway target gene - on immune-related molecular responses in ovarian cancer. <h3>Methods:</h3> Data was collected from human cell lines, ascites collected from patients with HGSOC, and tissue from patient tumor samples treated with a DKK-1 inhibitor (DKN-01.) Ascites from 10 high grade serous ovarian cancer (HGSOC) patients were isolated, cultured <i>in vitro,</i> and treated with control or 0.5uL/mL DKN-01 for 48 hours. Cell lysates were collected for multiplex cytokine/chemokine array. Human ES2 epithelial ovarian cancer (EOC) cells were cultured <i>in vitro</i> and treated with control or DKN-01; RNA-sequencing was performed to characterize differential gene expression changes in response to treatment. We also performed H&E staining of human tumor tissue from six patients with recurrent gynecologic malignancies (EEC, MMMT, EOC), pre- and post-DKN-01 monotherapy. <h3>Results:</h3> DKK1 blockade via DKN-01 was associated with an enhanced pro-inflammatory response as exhibited by enhanced inflammatory cytokine/chemokine gene expression and secretion; these findings were associated with improved local immune cell presence. In human ascites, there was a >20% increase in IL-8, CCL5 and CCL4 cytokines in response to DKN-01; these cytokines are associated with enhanced leukocyte, T cell, NK cell, and monocyte recruitment. Additionally, there was a >20% increase in CXCL9 and EGF cytokines (both associated with T cell activation), and GM-CSF (associated with pro-inflammatory/anti-tumor M1 macrophage polarization) in response to DKN-01. RNA-seq analysis of human ovarian tissue from patients with DKN-01 treatment showed a statistically significant increases in gene expression of pro-inflammatory chemokines CXCL8 (p<0.001) and IL-18BP (p<0.05) compared to matched controls. <h3>Conclusions:</h3> DKK1 blockade facilitates a pro-inflammatory TME via increased local immunostimulatory cytokine production and release, providing a mechanism of action for improved clinical outcomes for gynecologic cancer patients by creating a ‘hot' TME. Based on these findings, further studies are needed to investigate how DKK-1 inhibition could prime the TME to sensitize ovarian cancer to immune checkpoint blockade.

Abstract 1039: Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1

Abstract Background: Colorectal cancer (CRC) ranks as the third leading cause of cancer-related deaths in the US. In this malignancy, 5-fluorouracil (5FU)-based treatment remains the mainstay as first-line chemotherapy. Unfortunately, ~50-60% of patients treated with 5FU eventually develop resistance, leading to increased mortality. Hence, a better understanding of the molecular mechanisms underlying 5FU-resistance will lead to development of more effective therapeutic strategies. Our previous studies have shown that, andrographis, a botanical with diverse biological activities, synergistically enhanced 5FU-induced pro-apoptotic effects by mediating Wnt/β-catenin pathways. However, a more comprehensive knowledge of andrographis-mediated synergism with 5-FU remains largely unclear. Methods: We first established 5FU resistant isogenic cell lines of HCT116 and SW480 CRC cells (HCT116 5FUR and SW480 5FUR). To determine the synergistic anti-tumor effects of andrographis and 5FU, we performed cell viability, proliferation and colony formation assays by treating parental and 5FUR cells with andrographis, 5FU and their combination. To explore the molecular mechanisms responsible for the synergistic anti-tumor efficacy, we next performed gene expression microarrays to identify candidate pathways and genes that mediated anti-tumor efficacy of andrographis in 5FUR cells. We analyzed an expression profiling dataset from CRC patients (GSE52735) who either responded or did not respond to 5FU. We also studied the andrographis-mediated synergism with 5FU in a xenograft animal model, and in 3D patient-derived tumor organoids. Results: Combined treatment with andrographis and 5FU exhibited significantly superior effects on cell viability (P&amp;lt;0.01), proliferation (P&amp;lt;0.001) and colony formation capacity (P&amp;lt;0.001), compared to treatment with individual treatments. In line with our in vitro findings, the combined treatment also significantly reduced tumor growth in mice xenografts (P&amp;lt;0.0001) as well as patient-derived tumoroids (P&amp;lt;0.001). By analyzing gene expression profiling data from cell lines and CRC patients, we identified that overexpression of DKK1 gene was a critical event for 5FU resistance in CRC. Moreover, qRT-PCR and western blotting data revealed that andrographis resulted in downregulation of 5FU-induced DKK1 overexpression accompanied with the synergistic anti-tumor effects in animal xenografts (P&amp;lt;0.001) and patient-derived tumor organoids (P&amp;lt;0.001), which were reversed following siRNA-mediated silencing of DKK1 (P&amp;lt;0.01). Conclusions: Our data provide novel evidence for andrographis-mediated anti-tumor synergism with 5-FU through DKK1, highlighting that in addition to its preventive efficacy, andrographis may serve as a potential adjunctive treatment to conventional chemotherapeutic drugs in patients with CRC. Citation Format: Yinghui Zhao, Chuanxin Wang, Ajay Goel. Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1039.

580 Hair growth stimulation effects of b-catenin stimulating peptides through DKK-1 inhibition

580 Hair growth stimulation effects of b-catenin stimulating peptides through DKK-1 inhibition

Utilizing porcupine (PORCN) and DKK1 inhibition to improve anti-tumor immunity in a murine model of ovarian cancer.

e18041 Background: Epithelial ovarian cancers (EOC) are immunologically “cold” tumors and consequently have seen limited success with immunotherapy. Wnt-beta/catenin signaling is dysregulated in multiple cancers, including EOC, and is characterized by immune exclusion. Porcupine (PORCN) is the enzyme necessary for secretion of Wnt ligands, which is required for activation of Wnt/beta-catenin signaling. Up-regulation of the Wnt/beta-catenin pathway leads to increased levels of the target gene DKK1, which plays a role in immune exclusion. Our goal is to improve the tumor immune response via targeting Wnt-signaling in EOC. Methods: We utilized a syngeneic murine model (ID8 cell line) deficient in p53 (ID8p53−/−) with and without the addition of a Luciferase tag (Luc+/-) to assess the effect of treatment with a PORCN inhibitor, CGX-1321, with and without DKN-01, a monoclonal antibody targeting DKK1. Mice were treated with vehicle/control, CGX-1321, DKN-01, or CGX-1321/DKN-01. Treatment was given for either 14 or 31 days. In Luc+ mice, bioluminescence imaging was performed weekly. Following mouse sacrifice, omental weights and ascites volume were measured. Omental tumor was used for flow cytometry and representative samples were sent for NanoString analysis. Results: Treatment with CGX-1321 for 14 days reduced omental weight and ascites volume in Luc- mice only (p = 0.02); however, earlier and extended treatment with CGX-1321 and CGX-1321/DKN-01 in Luc+ mice decreased omental weight (p = 0.008 and 0.0025); mice treated with CGX did not develop any ascites with the same treatment schedule. While treatment with DKN-01 alone did not decrease omental weight, treatment with DKN-01 and combination CGX-1321/DKN-01 increased total percentage of NK cells (p = 0.0192 and 0.0070). CGX-1321 and DKN-01 alone and in combination increased the total percentage of CD8+ T cells in omental tumors (p = 0.0238, 0.037, and 0.0127). Conclusions: CGX-1321 decreased omental weight and ascites volume, while DKN-01 increased the cellular percentage of NK cells. Both DKN-01 and CGX-1321 increased CD8+ T cells. Wnt-inhibition is therapeutically promising in EOC, and the combination of a PORCN inhibitor with DKN-01 could improve the response to immune checkpoint blockage and further investigation is warranted.

Long noncoding RNA MAGI1-IT1 regulates cardiac hypertrophy by modulating miR-302e/DKK1/Wnt/beta-catenin signaling pathway.

Cardiac hypertrophy (CH) is an adaptive cardiac response to overload whose decompensation eventually leads to heart failure or sudden death. Recently, accumulating studies have indicated the implication of long noncoding RNAs (lncRNAs) in CH progression. MAGI1-IT1 is a newly-identified lncRNA that is highly associated with CH, while its specific role in CH progression remains masked. In this study, we uncovered that MAGI1-IT1 was distinctly downregulated in angiotensin (Ang) II-induced hypertrophic H9c2 cells. Also, MAGI1-IT1 overexpression in Ang II-treated H9c2 cells strikingly abolished the enlarged surface area and the enhanced levels of hypertrophic markers such as ANP, BNP, and β-MHC. Mechanically, we found MAGI1-IT1 sponged miR-302e which was identified as a hypertrophy-facilitator here, and that miR-302e upregulation countervailed the inhibition of MAGI1-IT1 overexpression on hypertrophic cells. Moreover, it was confirmed that MAGI1-IT1 boosted DKK1 expression by absorbing miR-302e. Subsequently, we also illustrated that MAGI1-IT1 inactivated Wnt/beta-catenin signaling through a DKK1-dependent pathway. Finally, both the DKK1 inhibition and LiCI (Wnt activator) supplement abrogated the hypertrophy-suppressive impact of MAGI1-IT1 on Ang II-simulated hypertrophic H9c2 cells. Jointly, our findings disclosed that MAGI1-IT1 functioned as a negative regulator in CH through inactivating Wnt/beta-catenin pathway via targeting miR-302e/DKK1 axis, revealing a novel road for CH treatment.

Hypoxic postconditioning activates the Wnt/β-catenin pathway and protects against transient global cerebral ischemia through Dkk1 Inhibition and GSK-3β inactivation.

The Wingless/Int (Wnt)/β-catenin pathway plays an essential role in cell survival. Although postconditioning with 8% oxygen can alleviate transient global cerebral ischemia (tGCI)-induced neuronal damage in hippocampal CA1 subregion in adult rats as demonstrated by our previous studies, little is understood about the role of Wnt/β-catenin pathway in hypoxic postconditioning (HPC)-induced neuroprotection. This study tried to investigate the involvement of Wnt/β-catenin pathway in HPC-induced neuroprotection against tGCI and explore the underlying molecular mechanism thereof. We observed that HPC elevated nuclear β-catenin level as well as increased Wnt3a and decreased Dickkopf-1 (Dkk1) expression in CA1 after tGCI. Accordingly, HPC enhanced the expression of survivin and reduced the ratio of B-cell lymphoma/lewkmia-2 (Bcl-2)-associated X protein (Bax) to Bcl-2 following reperfusion. Moreover, our study has shown that these effects of HPC were abolished by lentivirus-mediated overexpression of Dkk1, and that the overexpression of Dkk1 completely reversed HPC-induced neuroprotection. Furthermore, HPC suppressed the activity of glycogen synthase kinase-3β (GSK-3β) in CA1 after tGCI, and the inhibition of GSK-3β activity with SB216763 increased the nuclear accumulation of β-catenin, up-regulated the expression of survivin, and reduced the ratio of Bax to Bcl-2, thus preventing the delayed neuronal death after tGCI. Finally, the administration of LY294002, an inhibitor of PI3K, increased GSK-3β activity and blocked nuclear β-catenin accumulation, thereby decreasing survivin expression and elevating the Bax-to-Bcl-2 ratio after HPC. These results suggest that activation of the Wnt/β-catenin pathway through Dkk1 inhibition and PI3K/protein kinase B pathway-mediated GSK-3β inactivation contributes to the neuroprotection of HPC against tGCI.-Zhan, L., Liu, D., Wen, H., Hu, J., Pang, T., Sun, W., Xu, E. Hypoxic postconditioning activates the Wnt/β-catenin pathway and protects against transient global cerebral ischemia through Dkk1 inhibition and GSK-3β inactivation.

Systemic Dysfunction of Osteoblast Differentiation in Adipose-Derived Stem Cells from Patients with Multiple Myeloma.

Multiple myeloma is characterized by bone lesions linked to increased osteoclast and decreased osteoblast activities. In particular, the osteoblast differentiation of bone marrow-derived stem cells (MSC) is impaired. Among the potential therapeutic tools for counteracting bone lesions, adipose-derived stem cells (ASC) could represent an appealing source for regenerative medicine due to their similar characteristics with MSC. Our study is among the first giving detailed insights into the osteoblastogenic capacities of ASC isolated by fat aspiration from myeloma patients (MM-ASC) compared to healthy subjects (HD-ASC). We showed that MM-ASC and HD-ASC exhibited comparable morphology, proliferative capacity, and immunophenotype. Unexpectedly, although normal in adipocyte differentiation, MM-ASC present a defective osteoblast differentiation, as indicated by less calcium deposition, decreased alkaline phosphatase activity, and downregulation of RUNX2 and osteocalcin. Furthermore, these ASC-derived osteoblasts displayed enhanced senescence, as shown by an increased β-galactosidase activity and cell cycle inhibitors expression (p16INK4A, p21WAF1/CIP1.), associated with a markedly increased expression of DKK1, a major inhibitor of osteoblastogenesis in multiple myeloma. Interestingly, inhibition of DKK1 attenuated senescence and rescued osteoblast differentiation, highlighting its key role. Our findings show, for the first time, that multiple myeloma is a systemic disease and suggest that ASC from patients would be unsuitable for tissue engineering designed to treat myeloma-associated bone disease.

Fluid shear stress improves morphology, cytoskeleton architecture, viability, and regulates cytokine expression in a time-dependent manner in MLO-Y4 cells.

The effects of load-induced interstitial fluid shear stress (FSS) on instantaneous signaling response of osteocytes (e.g., calcium signaling) have been well documented. FSS can also initiate the release of many important messenger molecules of osteocytes (e.g., ATP and PGE2 ). However, the effects of FSS on cellular function and bone metabolism-modulating cytokine expression of osteocytes have not been fully identified (some inconsistent/conflicting results have been documented). Herein, osteocyte-like MLO-Y4 cells were stimulated with 1 Pa, 2-h FSS, and the effects of FSS on cellular morphology, cytoskeletal microstructure, biological activity, and gene and protein expression of important cytokines were investigated. SEM and cytoskeleton staining revealed that FSS induced well-organized cytoskeleton and increased filopodia processes. The osteocytic viability was sustained and apoptosis was inhibited via flow cytometry. FSS promoted Wnt3a and β-catenin gene and protein expression in 0-, 3-, and 6-h (sample collection time post FSS) groups. The FSS-stimulated cells in the 3-h group exhibited more significant effects on the promotion of OCN and Cx43 and inhibition of DKK1 and SOST expression than the 0- and 6-h groups. The 3-h group with FSS stimulation also showed the most prominent effects on suppressing RANKL and RANKL/OPG gene and protein expression. This study revealed a direct regulatory effect of FSS on osteocytic morphology and apoptotic characteristics, and showed that osteocyte-secreted bone metabolism-modulating molecule expression was regulated by FSS in a time-dependent manner. This study not only enriches our basic knowledge for understanding osteocytic mechanotransduction, but also provides important evidence for more scientific experimental design.

Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition.

The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.

Sclerostin and DKK1 Inhibition Preserves and Augments Alveolar Bone Volume and Architecture in Rats with Alveolar Bone Loss

Alveolar bone is a mechanosensitive tissue that provides structural support for teeth. Alveolar bone loss is common with aging, menopause, tooth loss, and periodontitis and can lead to additional tooth loss, reduced denture fixation, and challenges in placing dental implants. The current studies suggest that sclerostin and DKK1, which are established osteocyte-derived inhibitors of bone formation, contribute to alveolar bone loss associated with estrogen ablation and edentulism in rats. Estrogen-deficient ovariectomized rats showed significant mandibular bone loss that was reversed by systemic administration of sclerostin antibody (SAB) alone and in combination with DKK1 antibody (DAB). Osteocytes in the dentate and edentulous rat maxilla expressed Sost (sclerostin) and Dkk1 (DKK1) mRNA, and molar extraction appeared to acutely increase DKK1 expression. In a chronic rat maxillary molar extraction model, systemic SAB administration augmented the volume and height of atrophic alveolar ridges, effects that were enhanced by coadministering DAB. SAB and SAB+DAB also fully reversed bone loss that developed in the opposing mandible as a result of hypo-occlusion. In both treatment studies, alveolar bone augmentation with SAB or SAB+DAB was accompanied by increased bone mass in the postcranial skeleton. Jaw bone biomechanics showed that intact sclerostin-deficient mice exhibited stronger and denser mandibles as compared with wild-type controls. These studies show that sclerostin inhibition, with and without DKK1 coinhibition, augmented alveolar bone volume and architecture in rats with alveolar bone loss. These noninvasive approaches may have utility for the conservative augmentation of alveolar bone.

Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted.