Diabetic nephropathy (DN) is an important cause of death in diabetes patients, which is mainly due to its complex pathogenesis. Here, we explored the role of N6-methyladenosine (m6A) RNA methylation in DN development. Renal tubular epithelial cells from DN patients and experimental DN mice treated with streptozotocin (STZ) exhibited a considerable increase in METTL14 and WTAP expression as well as overall m6A methylation. Knocking down the expression of METTL14 and WTAP inhibited the migration and proliferation of tubular epithelial cells. MeRIP-seq analysis of the renal tissues of DN patients revealed that the genes with elevated m6A methylation were concentrated in the Wnt/β-Catenin signaling pathway. Dickkopf homolog 3 (DKK3) was screened out as the gene with the most significant increase in m6A methylation. In addition, the expression change pattern of DKK3 under DN circumstances is in line with those of METTL14 and WTAP. DKK3\'s m6A methylation sites were confirmed to be located in the 3\'UTR region, which is how METTL14 and WTAP improved DKK3\'s mRNA stability. Finally, YTHDF1, a m6A reader, was demonstrated to recognize m6A-methylated DKK3 and promote DKK3 expression.

Retinoblastoma (RB) is a pediatric malignancy, typically diagnosed at birth or during early childhood. The pathogenesis of RB is marked by the amplification of the Basic Helix-Loop-Helix (BHLH) Transcription Factor MYCN, which serves as a transcriptional regulator capable of binding to Dickkopf 3 (DKK3). However, the precise role of DKK3 in the malignant progression of RB cells caused by MYCN remains elusive. In the present study, the expression of MYCN was either overexpressed or interfered in RB cells. Subsequently, the expression level of DKK3 was assessed through quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was evaluated using the Cell Counting Kit-8 assay and 5-ethynyl-2\'-deoxyuridine staining, while cell cycle progression and apoptosis were analyzed by flow cytometry and western blot analysis, respectively. Additionally, the expression of proteins involved in the Wnt/β-catenin/Fra-1/p53 signaling pathway was evaluated via western blot analysis. To gain further insights, Wnt agonists and the P53 inhibitor PFT-α were introduced into exploration. The current investigation revealed a negative correlation between the expression levels of MYCN and DKK3 in RB cells. Additionally, DKK3 overexpression inhibited cell proliferation, promoted cell apoptosis, and arrested cell cycle in RB cells with high expression of MYCN. Moreover, enhanced DKK3 expression inhibited proliferation, promoted cell cycle arrest and apoptosis of RB cells by modulating the wnt/βcatenin/Fra-1/p53 signaling pathway. Furthermore, in vivo experiments revealed that overexpression of DKK3 inhibits the growth of RB tumors. Collectively, our findings elucidate that MYCN stimulates the Wnt/β-catenin/Fra-1 pathway by suppressing DKK3 expression, ultimately suppressing p53 activity and contributing to malignant progression of RB.

BACKGROUND: Endothelial barrier dysfunction is critical for the pathogenesis of sepsis-induced acute lung injury (ALI). Lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cells (HPMECs) are widely used as the cell model of sepsis-associated ALI for exploration of endothelial barrier dysfunction. Dickkopf (DKK) family proteins were reported to mediate endothelial functions in various diseases. The present study explored the effect of Dickkopf-3 (DKK3) on endothelial barrier permeability, angiogenesis, and tight junctions in LPS-stimulated HPMECs.
METHODS: RT-qPCR was required for detecting DKK3 and miR-98-3p expression. The angiogenesis of HPMECs was evaluated by tube formation assays. Monolayer permeability of HPMECs was examined by Transwell rhodamine assays. The protein expression of DKK3 and tight junctions in HPMECs was measured via western blotting. Luciferase reporter assay was used to verify the interaction between miR-98-3p and DKK3.
RESULTS: LPS treatment inhibited angiogenetic ability while increasing the permeability of HPMECs. DKK3 expression was upregulated while miR-98-3p level was reduced in LPS-treated HPMECs. DKK3 knockdown alleviated HPMEC injury triggered by LPS stimulation. MiR-98-3p targeted DKK3 in HPMECs. Overexpression of miR-98-3p protects HPMECs from the LPS-induced endothelial barrier dysfunction, and the protective effect was reversed by DKK3 overexpression.
CONCLUSIONS: MiR-98-3p ameliorates LPS-evoked pulmonary microvascular endothelial barrier dysfunction in sepsis-associated ALI by targeting DKK3.

UNASSIGNED: Chronic kidney disease (CKD) lacks effective treatments and renal fibrosis (RF) is one of CKD\'s outcomes. Dickkopf 3 (DKK3) has been identified as an agonist in CKD. However, the underlying mechanisms of DKK3 in CKD are not fully understood.
UNASSIGNED: H2O2-treated HK-2 cells and ureteric obstruction (UUO) mice were used as RF models. Biomarkers, Masson staining, PAS staining, and TUNEL were used to assess kidney function and apoptosis. Oxidative stress and mitochondria function were also evaluated. CCK-8 and flow cytometry were utilized to assess cell viability and apoptosis. Western blotting, IHC, and qRT-PCR were performed to detect molecular expression levels. Immunofluorescence was applied to determine the subcellular localization. Dual luciferase assay, MeRIP, RIP, and ChIP were used to validate the m6A level and the molecule interaction.
UNASSIGNED: DKK3 was upregulated in UUO mouse kidney tissue and H2O2-treated HK-2 cells. Knockdown of DKK3 inhibited oxidative stress, maintained mitochondrial homeostasis, and alleviated kidney damage and RF in UUO mice. Furthermore, DKK3 silencing suppressed HK-2 cell apoptosis, oxidative stress, and mitochondria fission. Mechanistically, DKK3 upregulation was related to the high m6A level regulated by METTL3. DKK3 activated TCF4/β-catenin and enhanced MFF transcriptional expression by binding to its promoter. Overexpression of MFF reversed in the inhibitory effect of DKK3 knockdown on cell damage.
UNASSIGNED: Upregulation of DKK3 caused by m6A modification activated the Wnt/β-catenin pathway to increase MFF transcriptional expression, leading to mitochondrial dysfunction and oxidative stress, thereby promoting RF progression.

Glioblastoma is the most common malignant primary tumor of the CNS. The prognosis is dismal, with a median survival of 15 months. Surgical treatment followed by adjuvant therapies such as radiotherapy and chemotherapy characterize the classical strategy. The WNT pathway plays a key role in cellular proliferation, differentiation, and invasion. The DKK3 protein, capable of acting as a tumor suppressor, also appears to be able to modulate the WNT pathway. We performed, in a series of 40 patients, immunohistochemical and Western blot evaluations of DKK3 to better understand how the expression of this protein can influence clinical behavior. We used a statistical analysis, with correlations between the expression of DKK3 and overall survival, age, sex, Ki-67, p53, and MGMT and IDH status. We also correlated our data with information included in the cBioPortal database. In our analyses, DKK3 expression, in both immunohistochemistry and Western blot analyses, was reduced or absent in many cases, showing downregulation. To date, no clinical study exists in the literature that reports a potential correlation between IDH and MGMT status and the WNT pathway through the expression of DKK3. Modulation of this pathway through the expression of DKK3 could represent a new tailored therapeutic strategy in the treatment of glioblastoma.

Glioma is the most common brain malignancy, characterized by high morbidity, high mortality, and treatment-resistance. Inverted CCAAT box Binding Protein of 90 kDa (ICBP90) has been reported to be involved in tumor progression and the maintenance of DNA methylation. Herein, we constructed ICBP90 over-expression and knockdown glioma cell lines, and found that ICBP90 knockdown inhibited glioma cell proliferation, migration, and invasion. ICBP90 silencing potentially enhanced cellular sensitivity to cis-platinum (DDP) and exacerbated DDP-induced pyroptosis, manifested by the elevated levels of gasdermin D-N-terminal and cleaved caspase 1; whereas, ICBP90 over-expression exhibited the opposite effects. Consistently, ICBP90 knockdown inhibited tumor growth in an in vivo mouse xenograft study using U251 cells stably expressing sh-ICBP90 and oe-ICBP90. Further experiments found that ICBP90 reduced the expression of Dickkopf 3 homolog (DKK3), a negative regulator of β-catenin, by binding its promoter and inducing DNA methylation. ICBP90 knockdown prevented the nuclear translocation of β-catenin and suppressed the expression of c-Myc and cyclin D1. Besides, DKK3 over-expression restored the effects of ICBP90 over-expression on cell proliferation, migration, invasion, and DDP sensitivity. Our findings suggest that ICBP90 inhibits the expression of DKK3 in glioma by maintaining DKK3 promoter methylation, thereby conducing to ICBP90-mediated carcinogenesis and drug insensitivity.

UNASSIGNED: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, and leads to a steady loss of kidney function in adulthood. The variable course of the disease makes it necessary to identify the patients with rapid disease progression who will benefit the most from targeted therapies and interventions. Currently, magnetic resonance imaging-based volumetry of the kidney is the most commonly used tool for this purpose. Biomarkers that can be easily and quantitatively determined, which allow a prediction of the loss of kidney function, have not yet been established in clinical practice. The glycoprotein Dickkopf 3 (DKK3) which is secreted in the renal tubular epithelium upon stress and contributes to tubulointerstitial fibrosis via the Wnt signaling pathway, was recently described as a biomarker for estimating risk of kidney function loss, but has not been investigated for ADPKD. This study aimed to obtain a first insight into whether DKK3 may indeed improve outcome prediction in ADPKD in the future.
UNASSIGNED: In 184 ADPKD patients from the AD(H)PKD registry and 47 healthy controls, the urinary DKK3 (uDKK3) levels were determined using ELISA. Multiple linear regression was used to examine the potential of these values in outcome prediction.
UNASSIGNED: ADPKD patients showed significantly higher uDKK3 values compared with the controls (mean 1970 ± 5287 vs 112 ± 134.7 pg/mg creatinine). Furthermore, there was a steady increase in uDKK3 with an increase in the Mayo class (A/B 1262 ± 2315 vs class D/E 3104 ± 7627 pg/mg creatinine), the best-established biomarker of progression in ADPKD. uDKK3 also correlated with estimated glomerular filtration rate (eGFR). Patients with PKD1 mutations show higher uDKK3 levels compared with PKD2 patients (PKD1: 2304 ± 5119; PKD2: 506.6 ± 526.8 pg/mg creatinine). Univariate linear regression showed uDKK3 as a significant predictor of future eGFR slope estimation. In multiple linear regression this effect was not significant in models also containing height-adjusted total kidney volume and/or eGFR. However, adding both copeptin levels and the interaction term between copeptin and uDKK3 to the model resulted in a significant predictive value of all these three variables and the highest R2 of all models examined (∼0.5).
UNASSIGNED: uDKK3 shows a clear correlation with the Mayo classification in patients with ADPKD. uDKK3 levels correlated with kidney function, which could indicate that uDKK3 also predicts a disproportionate loss of renal function in this collective. Interestingly, we found an interaction between copeptin and uDKK3 in our prediction models and the best model containing both variables and their interaction term resulted in a fairly good explanation of variance in eGFR slope compared with previous models. Considering the limited number of patients in these analyses, future studies will be required to confirm the results. Nonetheless, uDKK3 appears to be an attractive candidate to improve outcome prediction of ADPKD in the future.

The SREB (Super-conserved Receptors Expressed in Brain) family of orphan G protein-coupled receptors is highly conserved in vertebrates and consists of three members: SREB1 (orphan designation GPR27), SREB2 (GPR85), and SREB3 (GPR173). SREBs are associated with processes ranging from neuronal plasticity to reproductive control. Relatively little is known about similarities across the entire family, or how mammalian gene expression patterns compare to non-mammalian vertebrates. In fish, this system may be particularly complex, as some species have gained a fourth member (SREB3B) while others have lost genes. To better understand the system, the present study aimed to: 1) use qPCR to characterize sreb and related gene expression patterns in the brains of three fish species with different systems, and 2) identify possible differences in transcriptional regulation among the receptors, using upstream transcription factor binding sites across 70 ray-finned fish genomes. Overall, regional patterns of sreb expression were abundant in forebrain-related areas. However, some species-specific patterns were detected, such as abundant expression of receptors in zebrafish (Danio rerio) hypothalamic-containing sections, and divergence between sreb3a and sreb3b in pufferfish (Dichotomyctere nigroviridis). In addition, a gene possibly related to the system (dkk3a) was spatially correlated with the receptors in all three species. Genomic regions upstream of sreb2 and sreb3b, but largely not sreb1 or sreb3a, contained many highly conserved transcription factor binding sites. These results provide novel information about expression differences and transcriptional regulation across fish that may inform future research to better understand these receptors.

Multiciliated cells contain hundreds of cilia whose directional movement powers the mucociliary clearance of the airways, a vital host defense mechanism. Multiciliated cell specification requires canonical Wnt signaling, which then must be turned off. Next, ciliogenesis and polarized ciliary orientation are regulated by noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling. The mechanistic relationship between the Wnt pathways is unknown. We show that DKK3, a secreted canonical Wnt regulator and WNT4, a noncanonical Wnt ligand act together to facilitate a canonical to noncanonical Wnt signaling switch during multiciliated cell formation. In primary human airway epithelial cells, DKK3 and WNT4 CRISPR knockout blocks, whereas ectopic expression promotes, multiciliated cell formation by inhibiting canonical Wnt signaling. Wnt4 and Dkk3 single-knockout mice also display defective ciliated cells. DKK3 and WNT4 are co-secreted from basal stem cells and act directly on multiciliated cells via KREMEN1 and FZD6, respectively. We provide a novel mechanism that links specification to cilium biogenesis and polarization for proper multiciliated cell formation.

Excessive inflammatory injury is the main cause of the incidence of severe neonatal pneumonia (NP) and associated deaths. Although dickkopf-3 (DKK3) exhibits anti-inflammatory activity in numerous pathological processes, its role in NP is still unknown. In this study, human embryonic lung WI-38 and MRC-5 cells were treated with lipopolysaccharide (LPS) to induce inflammatory injury of NP in vitro. The expression of DKK3 was downregulated in LPS-stimulated WI-38 and MRC-5 cells. DKK3 overexpression decreased LPS-induced inhibition of cell viability, and reduced LPS-induced apoptosis of WI-38 and MRC-5 cells. DKK3 overexpression also reduced LPS-induced production of pro-inflammatory factors such as ROS, IL-6, MCP-1, and TNF-α. Nuclear respiratory factors 1 (NRF1) knockdown was found to upregulate DKK3 and inactivate the GSK-3β/β-catenin pathway in LPS-injured WI-38 and MRC-5 cells. NRF1 knockdown also suppressed LPS-induced inhibition on cell viability, repressed LPS-induced apoptosis, and inhibited the accumulation of ROS, IL-6, MCP-1, and TNF-α in LPS-injured WI-38 and MRC-5 cells. DKK3 knockdown or re-activation of the GSK-3β/β-catenin pathway reversed the inhibitory effects of NRF1 knockdown on LPS-induced inflammatory injury. In conclusion, NRF1 knockdown can alleviate LPS-triggered inflammatory injury by regulating DKK3 and the GSK-3β/β-catenin pathway.