DKK1 Expression Research Articles

Biological and molecular profile of fracture non-union tissue: A systematic review and an update on current insights.

Fracture non‐union represents a common complication, seen in 5%–10% of all acute fractures. Despite the enhancement in scientific understanding and treatment methods, rates of fracture non‐union remain largely unchanged over the years. This systematic review investigates the biological, molecular and genetic profiles of both (i) non‐union tissue and (ii) non–union‐related tissues, and the genetic predisposition to fracture non‐union. This is crucially important as it could facilitate earlier identification and targeted treatment of high‐risk patients, along with improving our understanding on pathophysiology of fracture non‐union. Since this is an update on our previous systematic review, we searched the literature indexed in PubMed Medline; Ovid Medline; Embase; Scopus; Google Scholar; and the Cochrane Library using Medical Subject Heading (MeSH) or Title/ words (non‐union(s), non‐union(s), human, tissue, bone morphogenic protein(s) (BMPs) and MSCs) from August 2014 (date of our previous publication) to 2 October 2021 for non‐union tissue studies, whereas no date restrictions imposed on non–union‐related tissue studies. Inclusion criteria of this systematic review are human studies investigating the characteristics and properties of non‐union tissue and non–union‐related tissues, available in full‐text English language. Limitations of this systematic review are exclusion of animal studies, the heterogeneity in the definition of non‐union and timing of tissue harvest seen in the included studies, and the search term MSC which may result in the exclusion of studies using historical terms such as ‘osteoprogenitors’ and ‘skeletal stem cells’. A total of 24 studies (non‐union tissue: n = 10; non–union‐related tissues: n = 14) met the inclusion criteria. Soft tissue interposition, bony sclerosis of fracture ends and complete obliteration of medullary canal are commonest macroscopic appearances of non‐unions. Non‐union tissue colour and surrounding fluid are two important characteristics that could be used clinically to distinguish between septic and aseptic non‐unions. Atrophic non‐unions had a predominance of endochondral bone formation and lower cellular density, when compared against hypertrophic non‐unions. Vascular tissues were present in both atrophic and hypertrophic non‐unions, with no difference in vessel density between the two. Studies have found non‐union tissue to contain biologically active MSCs with potential for osteoblastic, chondrogenic and adipogenic differentiation. Proliferative capacity of non‐union tissue MSCs was comparable to that of bone marrow MSCs. Rates of cell senescence of non‐union tissue remain inconclusive and require further investigation. There was a lower BMP expression in non‐union site and absent in the extracellular matrix, with no difference observed between atrophic and hypertrophic non‐unions. The reduced BMP‐7 gene expression and elevated levels of its inhibitors (Chordin, Noggin and Gremlin) could potentially explain impaired bone healing observed in non‐union MSCs. Expression of Dkk‐1 in osteogenic medium was higher in non‐union MSCs. Numerous genetic polymorphisms associated with fracture non‐union have been identified, with some involving the BMP and MMP pathways. Further research is required on determining the sensitivity and specificity of molecular and genetic profiling of relevant tissues as a potential screening biomarker for fracture non‐unions.

Chlorogenic acid alleviates thioacetamide-induced toxicity and promotes liver development in zebrafish (Danio rerio) through the Wnt signaling pathway.

Chlorogenic acid (CGA) is a phenylpropanoid compound that is well known to improve the antioxidant capacity and other biological activities. However, the roles of CGA in the liver development of organisms are unclear. In the present study, we aimed to investigate the function of CGA in the hepatic development in thioacetamide (TAA)-induced zebrafish embryos. We found that CGA exerted certain beneficial effects on zebrafish larvae from TAA-exposed zebrafish embryos, such as increasing the liver size, body length, heart rate, acetylcholinesterase activity, and motor ability. In addition, CGA displayed an antioxidant effect on TAA-induced zebrafish embryos by enhancing the activities of superoxide dismutase (SOD), catalase (CAT), and glucose-6-phosphate dehydrogenase (G6PDH), and decreasing of the contents of malondialdehyde (MDA), reactive oxygen species (ROS), and nitric oxide (NO). The results of western blotting analysis showed that CGA inhibited cell apoptosis by increasing the levels of Bcl2 apoptosis regulator and decreasing the levels of Bcl2 associated X (Bax), apoptosis regulator and tumor protein P53. Moreover, CGA promoted cell proliferation in TAA-induced zebrafish larvae, as detected using proliferating cell nuclear antigen fluorescence immunostaining. In addition, CGA inhibited the expression of Wnt signaling pathway genes Dkk1 (encoding Dickkopf Wnt signaling pathway inhibitors), and promoted the expression of Lef1 (encoding lymphoid enhancer binding factor 1) and Wnt2bb (encoding wingless-type MMTV integration site family, member 2Bb). When the Wnt signal inhibitor IWR-1 was added, there was no significant change in liver development in the IWR-1+TAA group compared with the IWR-1+TAA+CGA group (p <0.05), which suggested that CGA regulates liver development via Wnt signaling pathway. Overall, our results suggested that CGA might alleviate TAA-induced toxicity in zebrafish and promote liver development through the Wnt signaling pathway, which provides a basis for the therapeutic effect of CGA on liver dysplasia.

The Low-density Lipoprotein Receptor-related Protein 6 Pathway in the Treatment of Intestinal Barrier Dysfunction Induced by Hypoxia and Intestinal Microbiota through the Wnt/β-catenin Pathway

Our study is to explore the key molecular of Low-density lipoprotein receptor-related protein 6 (LRP6) and the related Wnt/β-catenin pathway regulated by LRP6 during the intestinal barrier dysfunction. Colorectal protein profile analysis showed that LRP6 expression was decreased in dextran sulfate sodium (DSS)-induced colitis mice, and mice received fecal bacteria transplantation from stroke patients. Mice with intestinal hypoxia and intestinal epithelial cells cultured in hypoxia showed decreased expression of LRP6. Overexpression of LPR6 or its N-terminus rescued the Wnt/β-catenin signaling pathway which was inhibited by hypoxia and endoplasmic reticulum stress. In mice overexpressing of LRP6, the expression of β-catenin and DKK1 increased, Bcl2 decreased, and Bax increased. Mice with LRP6 knockout showed an opposite trend, and the expression of Claudin2, Occludin and ZO-1 decreased. Two drugs, curcumin and auranofin could alleviate intestinal barrier damage in DSS-induced colitis mice by targeting LRP-6. Therefore, gut microbiota dysbiosis and hypoxia can inhibit the LRP6 and Wnt/β-catenin pathway, and drugs targeting LRP6 can protect the intestinal barrier.

Downregulation of DKK-1 and Upregulation of B - Catenin in Cancer Floating Spheroids is Associated with Chemoresistance to Wnt Signaling Pathway Inhibitors

Objectives: DKK-1, a negative regulator of ?-catenin in the Wnt pathway is commonly upregulated in cancer cells but its expression and association to chemoresistance have not been investigated extensively. Our aim was to evaluate the expression of DKK-1 and ?-catenin as well as the effect of the Wnt pathway modulators iCRT-14 (iCRT) and WAY626611 (WAY) alone or in combination with Nigericin (NIG) on the viability of cancer cells grown under routine conditions (RC) or as floating spheroids (FSs). Methods: RC or FSs from human prostate (PC3 and LnCAP) and breast (MCF-7 and MDA-MB231) cells were treated for 72 hs with WAY or iCRT. Cell viability was determined by the MTT or the CCK-8 assays and protein expression was determined by western blot analysis. Results: DKK-1 was expressed at different basal levels in RC but was undetectable in FSs. ?-catenin expression was increased in FSs. RC of PC3 and MCF-7 were sensitive to both iCRT and WAY but FSs generated from these cell lines were resistant to both drugs. NIG potentiated the effect of both Wnt inhibitors in FSs. Conclusion: Marked changes in the expression of Wnt pathway proteins, DKK-1 and ?-catenin in FSs are associated with resistance to WAY and iCRT. Keywords: Chemoresistance, DKK-1, tumorspheres, stemness, Wnt/?-catenin

Hair loss improvement effect of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra herbal mixture in human follicle dermal papilla cell and C57BL/6 mice

In today's society, hair is not only used to protect the body and maintain body temperature, but to play an aesthetic role. Therefore, interest in natural products that can improve hair loss is increasing. In this study, the hair loss improvement effect of a mixture of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra (FHH-CZ) was investigated in Human follicle dermal papilla cell (HFDPC) and C57BL/6 mice. ELISA, Western blot, RT-PCR and animal experiments were performed. The results showed that FHH-CZ suppressed the expression of 5α-reductase type 2 and androgen receptor in testosterone-treated HFDPC. In addition, pretreatment with FHH-CZ reduced the Bax/Bcl2 expression ratio through inhibition of Dkk-1 expression in testosterone-treated HFDPC. In animal experiments, FHH-CZ promoted hair growth and increased the number of hair roots in C57BL/6 mice. Therefore, the results of this study suggested that FHH-CZ could be used as a natural material with few side effects to improve hair loss.

Bone adaptation to mechanical loading in mice is affected by circadian rhythms.

Physical forces are critical for successful function of many organs including bone. Interestingly, the timing of exercise during the day alters physiology and gene expression in many organs due to circadian rhythms. Circadian clocks in tissues, such as bone, express circadian clock genes that target tissue-specific genes, resulting in tissue-specific rhythmic gene expression (clock-controlled genes). We hypothesized that the adaptive response of bone to mechanical loading is regulated by circadian rhythms. First, mice were sham loaded and sacrificed 8h later, which amounted to tissues being collected at zeitgeber time (ZT)2, 6, 10, 14, 18, and 22. Cortical bone of the tibiae collected from these mice displayed diurnal expression of core clock genes and key osteocyte and osteoblast-related genes, such as the Wnt-signaling inhibitors Sost and Dkk1, indicating these are clock-controlled genes. Serum bone turnover markers did not display rhythmicity. Second, mice underwent a single bout of in vivo loading at either ZT2 or ZT14 and were sacrificed 1, 8, or 24h after loading. Loading at ZT2 resulted in Sost upregulation, while loading at ZT14 led to Sost and Dkk1 downregulation. Third, mice underwent daily in vivo tibial loading over 2weeks administered either in the morning, (ZT2, resting phase) or evening (ZT14, active phase). In vivo microCT was performed at days 0, 5, 10, and 15 and conventional histomorphometry was performed at day 15. All outcome measures indicated a robust response to loading, but only microCT-based time-lapse morphometry showed that loading at ZT14 resulted in a greater endocortical bone formation response compared to mice loaded at ZT2. The decreased Sost and Dkk1 expression coincident with the modest, but significant time-of-day specific increase in adaptive bone formation, suggests that circadian clocks influence bone mechanoresponse.

MiR-25-3p protects renal tubular epithelial cells from apoptosis induced by renal IRI by targeting DKK3.

Renal ischemia-reperfusion injury (IRI) is one of the main causes of acute kidney injury (AKI). So far, there have been many studies on renal IRI, although an effective treatment method has not been developed. In recent years, growing evidence has shown that small noncoding RNAs play an important regulatory role in renal IRI. This article aims to explore whether microRNA-25-3p (miR-25-3p) plays a role in the molecular mechanism of renal IRI. The results showed that the expression level of miR-25-3p was significantly downregulated in a rat renal IRI model, and this result was confirmed with in vitro experiments. After the hypoxia-reoxygenation treatment, the apoptosis level of NRK-52E cells transfected with miR-25-3p mimics decreased significantly, and this antiapoptotic effect was antagonized by miR-25-3p inhibitors. In addition, we confirmed that DKK3 is a target of miR-25-3p. miR-25-3p exerts its protective effect against apoptosis on NRK-52E cells by inhibiting the expression of DKK3, and downregulating the expression level of miR-25-3p could disrupt this protective effect. In addition, we reconfirmed the role of miR-25-3p in rats. Therefore, we confirmed that miR-25-3p may target DKK3 to reduce renal cell damage caused by hypoxia and that miR-25-3p may be a new potential treatment for renal IRI.

NK4 Regulates Laryngeal Squamous Cell Carcinoma Cell Properties and Inhibits Tumorigenicity by Modulating the DKK1/Wnt/β-Catenin Axis.

ObjectiveTo investigate the effects of NK4 gene on the properties and tumorigenicity in laryngeal squamous cell carcinoma cell.MethodsHere, we used the attenuated Salmonella carrying the NK4 gene to transfect the AMC-HN-8 cells and detected the expression of NK4 by the real-time quantitative polymerase chain reaction (q RT-PCR). The properties of NK4 gene was determined by MTT method, cell scratch test, and flow cytometry. A nude mouse tumorigenesis model was used to evaluate the effect of NK4 gene on the growth of AMC-HN-8 cells in vivo. While a western blot assay was used to assess the expression of DKK1, Wnt1 and β-Catenin in nude mouse tumors.ResultsqRT-PCR showed that the expression of NK4 in the transfection group was significantly higher than that in the control group (P<0.01), and the expression increased with the time of transfection. MTT results showed NK4 overexpression inhibited the proliferation of AMC-HN-8 cells, and the inhibitory activity no longer increased with increasing dose when 30% expression supernatant was added (P<0.01). Scratch experiment showed that NK4 overexpression decreased the cell migration ability (P<0.01). Annexin V/PI double staining experiment showed that NK4 gene induced AMC-HN-8 cell apoptosis (P<0.01), and cell cycle arrest in S phase (P<0.01). NK4 overexpression inhibited tumor formation ability of AMC-HN-8 cells in vivo (P <0.05). WB detection showed that the expression of DKK1 increased, Wnt1 and β-Catenin protein decreased after the high expression of NK4.ConclusionsNK4 gene inhibit cell proliferation and migration, while promote cell apoptosis, and induce cell cycle arrest in S phase of laryngeal carcinoma AMC-HN-8 cells. NK4 overexpression inhibit the tumorigenesis ability of AMC-HN-8 cells, which may be related to the regulation of DKK1/Wnt1/β-Catenin signal axis.

RYR2 mutation in non-small cell lung cancer prolongs survival via down-regulation of DKK1 and up-regulation of GS1-115G20.1: A weighted gene Co-expression network analysis and risk prognostic models.

RYR2 mutation is clinically frequent in non‐small cell lung cancer (NSCLC) with its function being elusive. We downloaded lung squamous cell carcinoma and lung adenocarcinoma samples from the TCGA database, split the samples into RYR2 mutant group (n = 337) and RYR2 wild group (n = 634), and established Kaplan‐Meier curves. The results showed that RYR2 mutant group lived longer than the wild group (p = 0.027). Weighted gene co‐expression network analysis (WGCNA) of differentially expressed genes (DEGs) yielded prognosis‐related genes. Five mRNAs and 10 lncRNAs were selected to build survival prognostic models with other clinical features. The AUCs of 2 models are 0.622 and 0.565 for predicting survival at 3 years. Among these genes, the AUCs of DKK1 and GS1‐115G20.1 expression levels were 0.607 and 0.560, respectively, which predicted the 3‐year survival rate of NSCLC sufferers. GSEA identified an association of high DKK1 expression with TP53, MTOR, and VEGF expression. Several target miRNAs interacting with GS1‐115G20.1 were observed to show the relationship with the phenotype, treatment, and survival of NSCLC. NSCLC patients with RYR2 mutation may obtain better prognosis by down‐regulating DKK1 and up‐regulating GS1‐115G20.1.

Transient neonatal shoulder paralysis causes early osteoarthritis in a mouse model.

Neonatal brachial plexus palsy (NBPP) occurs in approximately 1.5 of every 1,000 live births. The majority of children with NBPP recover function of the shoulder. However, the long-term risk of osteoarthritis (OA) in this population is unknown. The purpose of this study was to investigate the development of OA in a mouse model of transient neonatal shoulder paralysis. Neonatal mice were injected twice per week for 4 weeks with saline in the right supraspinatus muscle (Saline, control) and botulinum toxin A (BtxA, transient paralysis) in the left supraspinatus muscle, and then allowed to recover for 20 or 36 weeks. Control mice received no injections, and all mice were sacrificed at 24 or 40 weeks. BtxA mice exhibited abnormalities in gait compared to controls through 10 weeks of age, but these differences did not persist into adulthood. BtxA shoulders had decreased bone volume (-9%) and abnormal trabecular microstructure compared to controls. Histomorphometry analysis demonstrated that BtxA shoulders had higher murine shoulder arthritis scale scores (+30%), and therefore more shoulder OA compared to controls. Articular cartilage of BtxA shoulders demonstrated stiffening of the tissue. Compared with controls, articular cartilage from BtxA shoulders had 2-fold and 10-fold decreases in Dkk1 and BMP2 expression, respectively, and 3-fold and 14-fold increases in Col10A1 and BGLAP expression, respectively, consistent with established models of OA. In summary, a brief period of paralysis of the neonatal mouse shoulder was sufficient to generate early signs of OA in adult cartilage and bone.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized β-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII–PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Derivation of a Novel CIHI in Patients with Lung Adenocarcinoma for Estimating Tumor Microenvironment and Clinical Prognosis.

An interaction between hypoxia and immunity has been confirmed in tumor tissue. However, there is no combined biomarker for diagnosis on this basis. Therefore, we developed a scoring formula based on markers of hypoxia and immunity. Firstly, the hypoxia-immune formula of lung adenocarcinoma (LUAD) was derived using LASSO-Cox regression in three cohorts from public database, and the corresponding score was calculated for each patient. The formula is as follows: combined hypoxia and immune index (CIHI) = LDHA expression × 0.2252 + GAPDH expression × 0.0727 + ANGPTL4 expression × 0.0724 + VEGFC expression × 0.1911 + DKK1 expression × 0.1355 + ADM expression × 0.0588 + BTK expression × −0.1659. Meanwhile, patients were divided into groups according to high and low CIHI, and expression profiles of hypoxia markers and immune markers were analyzed in different groups. CIHI was used to confirm that patients with high CIHI represented a state of hypoxiahigh-immunitylow, which had worse overall survival. We also discussed the evaluation value in the immune microenvironment and clinical application of CIHI. In conclusion, this study developed and validated a hypoxia-immune formula that can guide hypoxia modifier treatment and immunotherapy in LUAD.

Hyperleukocytic Acute Leukemia Circulating Exosomes Regulate HSCs and BM-MSCs.

Hyperleukocytic acute leukemia (HLAL) circulating exosomes are delivered to hematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (BM-MSCs), thereby inhibiting the normal hematopoietic process. In this paper, we have evaluated and explored the effects of miR-125b, which is carried by HLAL-derived exosomes, on the hematopoietic function of HSCs and BM-MSCs. For this purpose, we have isolated exosomes from the peripheral blood of HLAL patients and healthy volunteers. Then, we measured the level of miR-125b in exosomes cocultured exosomes with HSCs and BM-MSCs. Moreover, we have used miR-125b inhibitors/mimic for intervention and then measured miR-125b expression and colony forming unit (CFU). Apart from it, HSC and BM-MSC hematopoietic-related factors α-globulin, γ-globulin, CSF2, CRTX4 and CXCL12, SCF, IGF1, and DKK1 expression were measured. Evaluation of the miR-125b and BAK1 targeting relationship, level of miR-125b, and expression of hematopoietic-related genes was performed after patients are treated with miR-125b mimic and si-BAK1. We have observed that miR-125b was upregulated in HLAL-derived exosomes. After HLAL-exosome acts on HSCs, the level of miR-125b is upregulated, reducing CFU and affecting the expression of α-globulin, γ-globulin, CSF2, and CRCX4. For BM-MSCs, after the action of HLAL-exo, the level of miR-125b is upregulated and affected the expression of CXCL12, SCF, IGF1, and DKK1. Exosomes derived from HLAL carry miR-125b to target and regulate BAK1. Further study confirmed that miR-125b and BAK1mimic reduced the expression of miR-125b and reversed the effect of miR-125b mimic on hematopoietic-related genes. These results demonstrated that HLAL-derived exosomes carrying miR-125b inhibit the hematopoietic differentiation of HSC and hematopoietic support function of BM-MSC through BAK1.

The role of Wnt pathway antagonists in early-stage lung adenocarcinoma.

We aimed to examine the expression levels of the genes encoding adenomatous polyposis coli (APC) 1, APC-2, Dickkopf related protein (DKK)-1, DKK-3, secreted frizzled-related protein (SFRP)-2, SFRP-4, and SFRP-5, which play roles in the Wnt signaling pathway, in lung adenocarcinoma and adjacent normal lung tissues and to evaluate their relationships with clinicopathologic factors. The expression levels of genes in formalin-fixed paraffin-embedded samples of tumor tissue and adjacent intact lung tissue from 57 patients who underwent surgery for lung adenocarcinoma between 2011 and 2018 were determined by real-time PCR analysis. The expression levels of the DKK-1 in tumor tissue, especially in stage I-II tumor tissue, were significantly suppressed compared to those in normal tissue (p < 0.025). Whereas DKK-1 expression was suppressed in the tumor tissue of patients with early-stage lung adenocarcinoma, expression of the SFRP-5 in these patients was significantly higher in tumor tissue than in normal tissue (p < 0.039). In our study, opposing regulation was found between the SFRP-5 and DKK-1, which are known to be extracellular antagonists of the Wnt signaling pathway. The SFRP-5 was found to have an oncogenic role in adenocarcinoma development. Studies of the opposing regulation between these genes in early-stage lung adenocarcinoma may shed light on the mechanisms associated with the development of carcinogenesis. The relationships or interactions of these genes may serve as potential therapeutic targets.

MiR-1290 contributes to oncogenesis and angiogenesis via targeting of THBS1, DKK3 and, SCAI.

Introduction: Colorectal cancer (CRC) is the third most common cancer in the world with high mortality, hence, understanding the molecular mechanisms involved in the tumor progression are important for CRC diagnosis and treatment. MicroRNAs (miRNAs) are key gene expression regulators that can function as tumor suppressors or oncogenes in tumor cells, and modulate angiogenesis as a critical process in tumor metastasis. MiR-1290 has been demonstrated as an onco-miRNA in various types of cancer, however, the role of miR-1290 in CRC is not fully understood. This study aimed to investigate the oncogenic and angiogenic potential of miR-1290 in CRC. Methods: Lenti-miR-1290 was transduced into HCT116, SW480, and human umbilical vein endothelial cells (HUVECs). By bioinformatics analysis, we identified thrombospondin 1 (THBS1) as a novel predicted target for miR-1290. Quantitative real-time PCR, western blotting, and luciferase reporter assay were used to demonstrate suppression of miR-1290 target genes including THBS1, Dickkopf Wnt signaling pathway inhibitor 3 (DKK3), and suppressor of cancer cell invasion (SCAI) in HCT116 and HUVECs. Cell cycle analysis, proliferation, migration and, tube formation were determined by flow cytometry, MTT, wound healing, and tube formation assays, respectively. Results: MiR-1290 significantly decreased the expression of THBS1, DKK3, and SCAI. We demonstrated that miR-1290 enhanced proliferation, migration, and angiogenesis partially through suppression of THBS1, DKK3, and SCAI in CRC. Conclusion: These results suggest a novel function of miR-1290 which may contribute to tumorigenesis and angiogenesis in CRC.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Inhibit Osteoporosis via MicroRNA-27a-Induced Inhibition of DKK2-Mediated Wnt/β-Catenin Pathway.

Osteoporosis (OP) is a systemic skeletal disease that promotes bone fragility and the risk of fractures. Recent studies have shown the relevance of microRNAs (miRNAs) in the development of OP. This study aimed to evaluate the possible mechanisms of action underlying miR-27a loaded by mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in OP. Serum samples from OP patients and normal controls were collected for miRNA microarray analysis. The expression of filtered miRNA was upregulated in osteoblasts (OB) and osteoclasts (OCs) for biological activity assessment. After developing OP mice using ovariectomy (OVX) and confirming OP, the miR-27a expression level was upregulated in mice by MSC-EV application. Dual-luciferase assays were conducted to validate the relationship between miR-27a and DKK2 expression. The poor expression of miR-27a was observed in patients with OP. miR-27a increased the expression of OB markers, the number of ALP-positive cells, and the number of calcium nodules in OCs. In OVX mice, miR-27a increased bone density, improved bone structure damage recovery, decreased the levels of bone resorption markers, and decreased OC number. miR-27a transmitted by MSC-EVs interacted with DKK2. MSC-EVs exerted the same protective effects as miR-27a on OP, whereas miR-27a inhibitor abolished the attenuating effects of MSC-EVs. In contrast, DKK2 depletion reversed the stimulatory effects of the miR-27a inhibitor on OP. The Wnt/β-catenin pathway was activated upon MSC-EV application and DKK2 silencing and was impaired upon the downregulation of the expression of miR-27a. MSC-EVs are effective in preventing mouse OP. This mechanism is mediated by the miR-27a/DKK2/Wnt/β-catenin signaling pathway.

Investigating the contribution of the unfolded protein response to prostate cancer bone metastasis

Zeynep Kaya, John C. Christianson, Ian G. Mills, Srinivasa R. Rao, Claire M. Edwards&#x0D; &#x0D; The majority of deaths from PCa arise following metastasis, particularly to the skeleton. ER stress and the unfolded protein response (UPR) promote primary PCa, however the contribution of the UPR to PCa bone metastasis remains unknown. The aim of this work was to determine the role of the UPR in PCa bone metastasis, focusing on the osteogenic potential of PCa cells, EMT and migration, and PCa-induced bone disease. Using paired cell lines ARCaPE and ARCaPM which differ in their epithelial (E) and mesenchymal (M) characteristics, we found that components of IRE1 and ATF6 pathways are higher in ARCaPE cells than in ARCaPM and decreased upon osteogenic differentiation of ARCaPM cells. Inhibition of the IRE1 or PERK pathway increased ALP activity in ARCaPM cells. Inhibition of specific arms of the UPR produced a varied response in EMT markers with no effect on migration of ARCaPM cells. Increasing ER stress using tunicamycin significantly reduced migration of ARCaPM cells. The bone disease associated with PCa bone metastases is driven by alterations in a complex signaling network, including the RANKL/OPG pathway and Wnt signaling. ER stress, induced by tunicamycin, decreased RANKL and Dkk1 expression and increased OPG expression in ARCaPM cells. This osteolytic response to ER stress was blocked by PERK inhibition. Taken together, my research demonstrates that the UPR has multiple effects in bone metastatic PCa cells, including a reduction in migration and in osteolytic factors following UPR activation, suggesting a novel mechanism by which the UPR may modulate PCa-induced bone disease.

Niacinamide Down-Regulates the Expression of DKK-1 and Protects Cells from Oxidative Stress in Cultured Human Dermal Papilla Cells.

PurposeAn increasing number of people are suffering from hair loss disorders. Niacinamide has long been used as an active ingredient for anti-hair loss preparations but the exact mechanism has not been clearly elucidated yet. The effects of niacinamide were investigated in cultured human dermal papilla cells (hDPCs).MethodsTo investigate the anti-hair loss effect of niacinamide and its molecular mechanisms, Western blot analysis, ELISA, quantitative RT-PCR and immunocytochemistry were performed. To study the protective effects of niacinamide against H2O2-induced oxidative stress, ROS generation and cytotoxicity were evaluated by DCF-DA assay and LDH release assay, respectively. Minoxidil was used as a positive control.ResultsNiacinamide decreased the protein expression level of DKK-1 which promotes regression of hair follicles by inducing catagen. The protein expression levels of cell senescence markers, p21 (CDKN1A) and p16 (CDKN2A) which are related to cell cycle arrest, were decreased. The expression of versican was increased by niacinamide treatment in cultured hDPCs. We have found that niacinamide decreased the H2O2-induced intracellular ROS production in cultured hDPCs. Moreover, niacinamide decreased the protein expression levels of H2O2-induced p21 and p16 and diminished the secretion of H2O2-induced DKK-1.ConclusionOur data demonstrate that niacinamide could enhance hair growth by preventing oxidative stress-induced cell senescence and premature catagen entry of hair follicles.

Adenoviral Transduction of Dickkopf-1 Alleviates Silica-Induced Silicosis Development in Lungs of Mice.

Silicosis is an occupational disease caused by inhalation of silica dust, which is hallmarked by progressive pulmonary fibrosis associated with poor prognosis. Wnt/β-catenin signaling is implicated in the development of fibrosis and is a therapeutic target for fibrotic diseases. Previous clinical studies of patients with pneumoconiosis, including silicosis, revealed an increased concentration of circulating WNT3A and DKK1 proteins and inflammatory cells in bronchoalveolar lavage compared with healthy subjects. The present study evaluated the effects of adenovirus-mediated transduction of Dickkopf-1 (Dkk1), a Wnt/β-catenin signaling inhibitor, on the development of pulmonary silicosis in mice. Consistent with previous human clinical studies, our experimental studies in mice demonstrated an aberrant Wnt/β-catenin signaling activity coinciding with increased Wnt3a and Dkk1 proteins and inflammation in lungs of silica-induced silicosis mice compared with controls. Intratracheal delivery of adenovirus expressing murine Dkk1 (AdDkk1) inhibited Wnt/β-catenin activity in mouse lungs. The adenovirus-mediated Dkk1 gene transduction demonstrated the potential to prevent silicosis development and ameliorate silica-induced lung fibrogenesis in mice, accompanied by the reduced expression of epithelia--mesenchymal transition markers and deposition of extracellular matrix proteins compared with mice treated with "null" adenoviral vector. Mechanistically, AdDkk1 is able to attenuate the lung silicosis by inhibiting a silica-induced spike in TGF-β/Smad signaling. In addition, the forced expression of Dkk1 suppressed silica-induced epithelial cell proliferation in polarized human bronchial epithelial cells. This study provides insight into the underlying role of Wnt/β-catenin signaling in promoting the pathogenesis of silicosis and is proof-of-concept that targeting Wnt/β-catenin signaling by Dkk1 gene transduction may be an alternative approach in the prevention and treatment of silicosis lung disease.

TGF-Β SIGNALING REGULATES WNT/Β-CATENIN PATHWAY IN UTERINE LEIOMYOMA CELLS

Uterine leiomyomas, also known as uterine fibroids, are the most common benign gynecologic tumor of the female reproductive tract. Despite their high prevalence and heavy burden, their pathogenesis is not clear. Several signaling aberrations have been identified, including Wnt/β-catenin and TGF-β. TGF-β pathway is a crucial mediator of extracellular matrix (ECM) deposition and fibrosis and is upregulated in leiomyoma compared to the myometrium. The Wnt/β-catenin pathway is a conserved pathway implicated in proliferation, differentiation and stem cell renewal and has also been implicated in uterine leiomyoma development. Due to the importance of these two pathways in uterine leiomyoma development, we are interested in studying the interaction between these two pathways to better understand the pathogenesis of these tumors. After obtaining informed consent, primary fibroid and myometrial cells were isolated from hysterectomy surgeries at Johns Hopkins Hospital. Primary cells and immortalized fibroid (HuLM) and myometrial (UtSM) were treated with TGF-β3 (10ng/mL) for 48 hours. The effect of TGF-β3 on the Wnt/β-catenin pathway was examined using western blot through assessing the expression of total β-catenin, non-phosphorylated β-catenin, the active form of β-catenin, and Dickkopf-1 (DKK1), an inhibitor of Wnt pathway. The student’s t-test was used to determine statistically significant differences (P<0.05). As expected, TGF-β3 treatment induced the expression of ECM proteins collagen 1A and fibronectin in both primary and immortalized fibroid and myometrial cells. The expression of total β-catenin and non-phosphorylated β-catenin was higher in leiomyoma cells, and TGF-β3 treatment significantly increased the expression of total β-catenin and non-phosphorylated β-catenin in both myometrial by 66% and 59%, respectively and fibroid cells by 58% and 62%, respectively (p-value <0.05). The expression of the Wnt pathway inhibitor, DKK-1, was significantly lower in primary leiomyoma cells compared to myometrial cells, and TGF-β3 treatment resulted in a significant reduction in DKK-1 expression in both myometrial by 85% and fibroid cells by 61%(p-value <0.05). These results indicate that TGF-β3 and Wnt/β-catenin signal transduction pathways interact in uterine leiomyoma. Further analyses are needed to clarify the interaction and the role of DKK-1 in leiomyoma development.