OBJECTIVE: To investigate the impact of hypoxia on microRNA (miRNA) expression profiles in endometrial glandular epithelial cells (EECs) and elucidate potential mechanisms underlying proliferation, migration, and invasion.
METHODS: EECs in the logarithmic growth phase were exposed to normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. MiRNA expression profiles were analyzed using RNA sequencing, and differential expression of hsa-miR-584-3p was confirmed by real-time quantitative PCR (RT-qPCR). Target prediction through TargetScan identified Dickkopf-1 (DKK-1) as a target gene of hsa-miR-584-3p. The interaction between hsa-miR-584-3p and DKK-1 was validated through a double-luciferase reporter gene assay and Western blotting. Cell proliferation, migration, and invasion were assessed using the Cell Counting Kit-8 (CCK-8) assay, wound healing assay, and Transwell invasion assay, respectively.
RESULTS: Hypoxic conditions significantly upregulated the expression of hsa-miR-584-3p in EECs (P<0.001). TargetScan analysis predicted DKK-1 as a downstream target of hsa-miR-584-3p. The double-luciferase reporter gene assay confirmed the binding of hsa-miR-584-3p to the 3\' untranslated region of the DKK-1 gene, leading to reduced DKK-1 protein expression (P<0.001). Functional assays demonstrated decreased proliferation and increased migration and invasion of EECs under hypoxia.
CONCLUSIONS: Hypoxia-induced upregulation of hsa-miR-584-3p suppresses the function of EECs by targeting DKK-1 protein activity, thereby influencing their proliferation, migration, and invasion.

Background and Objectives: Periodontitis is marked by the destruction of alveolar bone. Sclerostin (SOST) and dickkopf-1 (DKK-1) act as inhibitors of the Wingless-type (Wnt) signaling pathway, a key regulator of bone metabolism. Recent studies have suggested that statins play a role in bone resorption and formation by influencing Wnt signaling. The aim of this study was to determine the levels of SOST and DKK-1 in periodontal patients with and without peroral statins treatment in their therapy. Materials and Methods: A total of 79 patients with diagnosed periodontitis were divided into two groups: 39 patients on statin therapy (SP group) and 40 patients without statin therapy as a control group (P group). The periodontal clinical examination probing (pocket) depth (PD) and gingival recession (GR) were measured, and approximal plaque was detected, while vertical and horizontal bone resorption was measured using a panoramic radiograph image. Clinical attachment loss (CAL) values were calculated using PD and GR values. Gingival crevicular fluid (GCF) was collected and used for measuring SOST and DKK-1 levels. A questionnaire was used to assess lifestyle habits and statin intake. Patients\' medical records were used to obtain biochemical parameters. Results: There was no significant difference in sclerostin concentration between the SP and P group. DKK-1 values were significantly higher in the SP group compared to the control group (p = 0.04). Also, PD (p = 0.001) and GR (p = 0.03) were significantly higher in the SP group. The level of DKK-1 had a positive relationship with the PD, the greater the PD, the higher the level of DKK-1 (Rho = 0.350), while there was no significant association with other parameters. Conclusions: Peroral statins in periodontal patients are associated with GCF levels of DKK-1 but not with sclerostin levels.

In this study, it was aimed to assess effects of subclinical hyperthyroidism (SH) on bone metabolism using osteoprotegerin (OPG), sclerostin, Dickkopf-1 (DKK1) and biochemical parameters. This cross-sectional prospective study included 40 patients with SH and 40 euthyroid controls. Serum OPG, sclerostin, DKK-1, type-1 procollagen, C-terminal polypeptide (CTx) and 24-hours urine N-terminal telopeptide (NTx) were measures using ELISA kit. Bone mineral density measurements were performed using dual energy X-ray absorptiometry (DEXA). Risk for 10-years hip and major fracture was estimated by Turkish version of FRAX. No significant difference was detected in age, gender, body mass index, smoking and menopause rates between SH and control groups. The risk for 10-years hip fracture and major osteoporotic fracture were estimated as 4.45% and 0.55% in SH group, respectively. The OPG levels were significantly lower in patients with SH than controls (P = 0.017). No significant difference was detected in other bone formation and degradation parameters. No significant correlation was detected between OPG level and risk for major osteoporotic fracture (P > 0.05); however, a negative correlation was detected between OPG level and risk for hip fracture (rho = 0.233; P = 0.038). Serum OPG is markedly affected in patients with SH. In addition, OPG seemed to be associated with osteoporotic fracture risk. Available data show that SH is significantly associated with risk for fracture; thus, it is important to assess risk for fracture in patients with SH.

Osteoblastic bone metastases are commonly detected in patients with advanced prostate cancer (PCa) and are associated with an increased mortality rate. Dickkopf-1 (DKK-1) antagonizes canonical WNT/β-catenin signaling and plays a complex role in bone metastases. We explored the function of cancer cell-specific DKK-1 in PCa growth, metastasis, and cancer-bone interactions using the osteoblastic canine PCa cell line, Probasco. Probasco or Probasco + DKK-1 (cells transduced with human DKK-1) were injected into the tibia or left cardiac ventricle of athymic nude mice. Bone metastases were detected by bioluminescent imaging in vivo and evaluated by micro-computed tomography and histopathology. Cancer cell proliferation, migration, gene/protein expression, and their impact on primary murine osteoblasts and osteoclasts, were evaluated in vitro. DKK-1 increased cancer growth and stimulated cell migration independent of canonical WNT signaling. Enhanced cancer progression by DKK-1 was associated with increased cell proliferation, up-regulation of NF-kB/p65 signaling, inhibition of caspase-dependent apoptosis by down-regulation of non-canonical WNT/JNK signaling, and increased expression of epithelial-to-mesenchymal transition genes. In addition, DKK-1 attenuated the osteoblastic activity of Probasco cells, and bone metastases had decreased cancer-induced intramedullary woven bone formation. Decreased bone formation might be due to the inhibition of osteoblast differentiation and stimulation of osteoclast activity through a decrease in the OPG/RANKL ratio in the bone microenvironment. The present study indicated that the cancer-promoting role of DKK-1 in PCa bone metastases was associated with increased growth of bone metastases, reduced bone induction, and altered signaling through the canonical WNT-independent pathway. DKK-1 could be a promising therapeutic target for PCa.

Psoriatic arthritis (PsA) is a heterogenous systemic inflammatory disorder that affects peripheral joints and skin, but also causes inflammation at entheseal sites, digits (dactylitis) and the axial skeleton. Despite considerable advances, our understanding of the pathogenesis and management of PsA is hampered by its complex clinical expression. We enrolled patients who met the ClASsification for Psoriatic Arthritis (CASPAR) criteria for PsA (n = 17), and healthy controls (n = 13). The lipid profile, C-reactive protein (CRP) and Dickkopf-related protein 1 (DKK-1) circulating levels were measured for all subjects. For the patients with PsA, (1) the erosive character of the articular disease was assessed by a musculoskeletal ultrasound and (2) the cardiovascular risk was evaluated using the Systematic Coronary Risk Evaluation (SCORE) chart and the ultrasound measurement of the carotid intima-media thickness. A higher titer of serum DKK-1 was associated with the presence of erosions (p < 0.005) and the cIMT correlated with DKK-1 levels in patients with PsA (r = 0.6356, p = 0.0061). Additionally, we observed a positive correlation between increased cIMT and CRP (r = 0.5186, p = 0.0329). Our results suggest that DKK-1 could be used as an early biomarker for the erosive character of the articular disease and for the assessment of the cardiovascular risk in PsA patients.

To investigate DNA methylation (DNAm) status of dickkopf-associated protein 1 (DKK-1) in ossified hip capsule synovium and serum among patients with ankylosing spondylitis (AS).
Western blot was applied to detect the level of DKK-1 protein expression in hip joint capsule tissues from four patients with AS as well as four patients with femoral neck fracture (FNF) caused by trauma as control. DKK-1 gene promoter methylation (GPM) was examined by methylation-specific polymerase chain reaction. Reverse transcription-polymerase chain reaction was performed to examine the messenger RNA (mRNA) levels of DKK-1, β-catenin, and Wnt3a in both tissue and serum. The DNAm status of serum DKK-1 was measured among 36 patients with AS and syndesmophytes (AS + syndesmophytes group), 40 patients with AS but no syndesmophyte (AS group), and 42 healthy individuals (control group). Also, the serum levels of DKK-1 were measured by enzyme-linked immunosorbent assay. The modified New York criteria (mNYC) together with the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) were adopted to examine the radiographic progression of AS. The receiver operating characteristic (ROC) curve was applied to investigate the diagnostic value of the methylation rate of DKK-1 with regard to radiographic progression.
The expressions of DKK-1 protein and mRNA in hip joint capsule tissues of AS patients were significantly lower, while DKK-1 GPM rate, β-catenin mRNA, and Wnt3a mRNA were markedly higher when compared with FNF group. For serum samples, the DKK-1 methylation rate was significantly higher in AS+ syndesmophytes group in contrast to AS group and healthy controls. Serum levels of DKK-1 protein and mRNA in AS with syndesmophytes group were markedly decreased, while β-catenin mRNA and Wnt3a mRNA expressions were significantly increased than AS with no syndesmophyte group and the healthy control group. AS patients in Grade 4 showed a significantly higher serum DKK-1 GPM rate than those in Grade 3 based on mNYC. Serum DKK-1 GPM level was markedly and positively correlated with mSASSS. Serum levels of DKK-1 in AS+ syndesmophytes group were markedly lower compared with AS but no syndesmophyte group and healthy controls. ROC curve analysis indicated that serum DKK-1 methylation rate serves as a decent indicator for AS radiographic progression.
DNAm of DKK-1 may correlate with pathological bone formation in AS, which may provide new strategies for the treatment of AS abnormal bone formation.

Brain metastases are the most lethal progression event, in part because the biological processes underpinning brain metastases are poorly understood. There is a paucity of realistic models of metastasis, as current in vivo murine models are slow to manifest metastasis. We set out to delineate metabolic and secretory modulators of brain metastases by utilizing two models consisting of in vitro microfluidic devices: 1) a blood brain niche (BBN) chip that recapitulates the blood-brain-barrier and niche; and 2) a migration chip that assesses cell migration. We report secretory cues provided by the brain niche that attract metastatic cancer cells to colonize the brain niche region. Astrocytic Dkk-1 is increased in response to brain-seeking breast cancer cells and stimulates cancer cell migration. Brain-metastatic cancer cells under Dkk-1 stimulation increase gene expression of FGF-13 and PLCB1. Further, extracellular Dkk-1 modulates cancer cell migration upon entering the brain niche.

The study aims to explore the role of the ERK signaling pathway in the crosstalk between Dkk-1 and TNF-α in MC3T3E1 pre-osteoblasts under cyclic tensile/compressive stress. A forced four-point bending system was used to apply cyclic uniaxial tensile/compressive strain (2000 μ, 0.5 Hz) to MC3T3E1 cells. Dkk-1 and TNF-α expression were upregulated in MC3T3E1 cells under compressive strain. Cell proliferation, the cell cycle, osteogenesis-related gene (Wnt5a, Runx2, Osterix) expression, β-catenin expression, and the p-ERK/ERK ratio were significantly enhanced, whereas apoptosis, the RANKL/OPG ratio, and TNF-α expression were significantly attenuated, by Dkk-1 silencing. Dkk-1 expression increased and the effects of Dkk-1 silencing were reversed when exogenous TNF-α was added. Mechanically, TNF-α crosstalked with Dkk-1 through ERK signaling in MC3T3E1 cells. ERK signaling blockade impaired Dkk-1-induced TNF-α expression and TNF-α-mediated Dkk-1 expression. Dkk-1 and TNF-α crosstalked, partially through ERK signaling, in MC3T3E1 cells under compressive/tensile strain, synergistically modulating various biological behaviors of the cells. These findings not only provide mechanical insight into the cellular events and molecular regulation of orthodontic tooth movement (OTM), but also aid the development of novel strategies to accelerate OTM.

UNASSIGNED: Chronic kidney disease (CKD) is a health problem associated with high morbidity and mortality. Mineral and bone disorders are complications of CKD with a risk of fractures and cardiovascular disease. Mesenchymal stem cells can differentiate into osteoblasts and regulate their regulation by a network of cytokines and transcription factors.
UNASSIGNED: Analyzing differences in osteoblastogenesis of adipose mesenchymal stem cells in CKD patients and healthy people.
UNASSIGNED: The study sample was adipose mesenchymal stem cells from CKD patient undergoing hemodialysis and healthy people. Osteoblastogenesis was assessed by measuring the concentrations of transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and (DKK-1) in culture media. The Elisa method measured the concentration of these parameters on days 4, 7, 14, and 21. Data were analyzed using an independent t-test and post hoc test with p-value <0.05.
UNASSIGNED: There was a significant difference in CKD patients with increasing TGF-β1 on day 4 (t = 2.821; 95% CI = 30,498-199,727; p = 0.010) and decreased on day 14. In the BMP-2 parameter, there was an increase on day 7 (t = 4.291; 95% CI = 0.289-0.831; p <0.001). Similar conditions were also found in the DKK-1 parameter, increasing on the 7th day, but there was no significant difference (p = 0.583).
UNASSIGNED: Osteoblastogenesis in adipose mesenchymal stem cells in CKD patients differs from that in healthy individuals. Osteoblasts fail in maturation and cause failure in matrix mineralization.

Patients with Gaucher disease (GD) have progressive bone involvement that clinically presents with debilitating bone pain, structural bone changes, bone marrow infiltration (BMI), Erlenmeyer (EM) flask deformity, and osteoporosis. Pain is referred by the majority of GD patients and continues to persist despite the type of therapy. The pain in GD is described as chronic deep penetrating pain; however, sometimes, patients experience severe acute pain. The source of bone pain is mainly debated as nociceptive pain secondary to bone pathology or neuropathic or inflammatory origins. Osteocytes constitute a significant source of secreted molecules that coordinate bone remodeling. Osteocyte markers, sclerostin (SOST) and Dickkopf-1 (DKK-1), inactivate the canonical Wnt signaling pathway and lead to the inhibition of bone formation. Thus, circulated sclerostin and DKK-1 are potential biomarkers of skeletal abnormalities. This study aimed to assess the circulating levels of sclerostin and DKK-1 in patients with GD and their correlation with clinical bone pathology parameters: pain, bone mineral density (BMD), and EM deformity. Thirty-nine patients with GD were classified into cohorts based on the presence and severity of bone manifestations. The serum levels of sclerostin and DKK-1 were quantified by enzyme-linked immunosorbent assays. The highest level of sclerostin was measured in GD patients with pain, BMI, and EM deformity. The multiparameter analysis demonstrated that 95% of GD patients with pain, BMI, and EM deformity had increased levels of sclerostin. The majority of patients with elevated sclerostin also have osteopenia or osteoporosis. Moreover, circulating sclerostin level increase with age, and GD patients have elevated sclerostin levels when compared with healthy control from the same age group. Pearson\'s linear correlation analysis showed a positive correlation between serum DKK-1 and sclerostin in healthy controls and GD patients with normal bone mineral density. However, the balance between sclerostin and DKK-1 waned in GD patients with osteopenia or osteoporosis. In conclusion, the osteocyte marker, sclerostin, when elevated, is associated with bone pain, BMI, and EM flask deformity in GD patients. The altered sclerostin/DKK-1 ratio correlates with the reduction of bone mineral density. These data confirm that the Wnt signaling pathway plays a role in GD-associated bone disease. Sclerostin and bone pain could be used as biomarkers to assess patients with a high risk of BMI and EM flask deformities.