DKK1 Protein Research Articles

Antiosteoporotic Activity of Nodakenetin, a Coumarin Compound from Angelica decursiva, by Activation of the Wnt/β-catenin Signaling Pathway

Angelica decursiva (Umbelliferae) is a medicinal plant widely used to treat colds, coughs and fevers in Korea, Japan, and mainland China. The anti-inflammatory activity of nodakenetin, a furano-coumarin compound from A. decursiva, has been reported, although, the antiosteoporotic activity remains unknown. This study sought to investigate the antiosteoporotic activity and precise mechanism of action of nodakenetin in in vitro cell culture and in vivo bone remodeling models. The transcriptional activity of nodakenetin on the Wnt signaling pathway was assessed using the TOPflash/FOPflash assay. The effect of nodakenetin on the osteoblast differentiation was measured using Alizarin red staining and alkaline phosphatase (ALP) activity. Western blotting and real-time RT-PCR were used to assess the effect of nodakenetin on the expression of markers related to Wnt/β-catenin pathway and osteoblast differentiation. The in vivo antiosteoporotic activity of nodakenetin was assessed using an ovariectomized (OVX)-induced bone loss mouse model. Nodakenetin activated the Wnt/β-catenin pathway through regulation of DKK1, β-catenin and other target proteins of the Wnt/β-catenin pathway in HEK293 and MC3T3-E1 cells. Nodakenetin induced the differentiation of MC3T3-E1 cells as shown by enhanced Alizarin red staining and ALP activity. Induction of osteoblast differentiation was related to upregulated expression of bone formation biomarkers such as bone morphogenic proteins and Runx2. Oral administration of nodakenetin in the OVX mouse model effectively protected the deterioration of bone microstructure in OVX mice. Nodakenetin exhibits antiosteoporotic activity in in vitro and in vivo through the activation of the Wnt/β-catenin pathway and subsequent induction of osteoblast differentiation.

The effects of therapeutic hypothermia on the expression of DKK3 and myocardial ischemia-reperfusion injury

BackgroundThe impact and mechanisms of therapeutic hypothermia (TH) on myocardial ischemia–reperfusion (I/R) injury are still unknown. MethodsSprague-Dawley (SD) rat model and primary cardiomyocytes were applied in our study. TTC staining evaluated myocardial infarction, while H&E staining assessed myocardial injury. ELISA quantified lactate dehydrogenase (LDH), IL-1β, IL-6, and TNF-α. Blood serum concentrations of CK, CK-MB, and cTnT were detected using a biochemical assay. TUNEL assay, flow cytometry and western blot were used to detect cell apoptosis. EdU assay was applied to examine cell proliferation. The expression of DKK3 and β-catenin protein was analyzed using Realtime PCR and Western blot. ResultsTH alleviated myocardial infarction size and injury in rat models of I/R, and reduced serum levels of myocardial injury markers and inflammatory factors in animal and cell models. In addition, TH inhibited the increased apoptosis and β-catenin expression as well as decreased DKK3 expression caused by I/R in I/R cell models, while si-DKK3 reversed the changes brought by TH, and XAV939 inhibited the effects of si-DKK3. ConclusionTH may reduce myocardial infarction size, myocardial injury, inflammatory response and apoptosis through DKK3/Wnt/β-catenin pathway, thereby alleviating myocardial I/R injury.

DDIT3 switches osteogenic potential of BMP9 to lipogenic by attenuating Wnt/β-catenin signaling via up-regulating DKK1 in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) functions as a potent inducer of osteogenic differentiation in mesenchymal stem cells (MSCs), holding promise for bone tissue engineering. However, BMP9 also concurrently triggers lipogenic differentiation in MSCs, potentially compromising its osteogenic potential. In this study, we explored the role of DNA damage inducible transcript 3 (DDIT3) in regulating the balance between BMP9-induced osteogenic and lipogenic differentiation in MSCs. Utilizing techniques such as PCR, Western blot, histochemical staining, and in vivo experiments, we analyzed the osteogenic and lipogenic markers induced by BMP9 and delved into the underlying molecular mechanism. We found a significant upregulation of DDIT3 in C3H10T1/2 cells treated with BMP9. This upregulation led to a reduction in BMP9-induced osteogenic markers but an enhancement in lipogenic markers. Conversely, knocking down DDIT3 produced the opposite effects. Furthermore, BMP9-induced bone formation was decreased in the presence of DDIT3, but adipocyte formation was increased. Further investigations demonstrated that BMP9 increased the phosphorylation level of GSK-3β and promoted nuclear translocation of β-catenin, both of which were suppressed by DDIT3. Moreover, DDIT3 decreased the total β-catenin protein level while BMP9 increased the DKK1 protein level, which was further enhanced by DDIT3. Notably, knocking down DKK1 partially reversed the effect of DDIT3 on reducing BMP9-induced osteogenic markers and increasing lipogenic markers. Our findings indicated that DDIT3 enhances lipogenic differentiation by diminishing BMP9's osteogenic potential, possibly through inhibiting Wnt/β-catenin signaling via DKK1 upregulation in MSCs.

Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody

BackgroundMounting experimental evidence has underscored the remarkable role played by the Wnt family of proteins in the spinal cord functioning and therapeutic potential in spinal cord injury (SCI). We aim to provide a therapeutic prospect associated with the modulation of canonical Wnt signaling, examining the spatio-temporal expression pattern of Dickkopf-1 (Dkk1) and its neutralization after SCI. We employ an intraparenchymal injection of the clinically validated Dkk1-blocking antibody, BHQ880, to elucidate its effects in SCI. MethodsA rat model of contusion SCI was used. Histological analyses were performed, wherein Dkk1 protein was sought, and ELISA analyses were employed for Dkk1 detection in cerebrospinal fluid and serum. To ascertain the BHQ880 therapeutic effect, rats were subjected to SCI and then injected with the antibody in the lesion epicenter 24 hours post-injury (hpi). Subsequent evaluation of motor functional recovery extended up to 56 days post-injury (dpi). qRT-PCR and histological analyses were conducted. ResultsWe demonstrate the presence of Dkk1 in the healthy rat spinal cord, with pronounced alterations observed following injury, primarily concentrated in the epicenter regions. Notably, a significative upregulation of Dkk1 was detected at 24 hpi, peaking at 3 dpi and remaining elevated until 42 dpi. Moreover, we revealed that early administration of BHQ880 considerably improved motor functional recovery, promoted preservation of myelinated tissue, and reduced astroglial and microglia/macrophage reactivity. Furthermore, there was a decrease in the acute expression of different inflammatory genes. ConclusionsCollectively, our findings highlight the therapeutic potential of BHQ880 treatment in the context of SCI.

N1F(Improved-Nephropathy 1 Formula) Ameliorates Renal Interstitial Fibrosis via Inhibiting Extracellular Matrix Deposition and Regulating the FGF23/P38MAPK/Wnt Pathway.

Renal fibrosis is the primary pathway in the progression of chronic kidney disease (CKD) towards end-stage renal failure. The currently used drugs currently are ineffective, and their mechanisms of action remain unclear. This study aims to investigate the nephroprotective effect of Improved-Nephropathy 1 Formula (N1F) in a rat model of unilateral ureteral obstruction (UUO) and explore the potential mechanisms of N1F-containing serum in treating TGF-ß1-induced human renal tubular epithelial cells (HK-2). SD rats received 2-week continuous N1F gavage starting on day 2 after UUO. HK-2 cells were pretreated with a P38MAPK inhibitor for 1 h in vitro, followed by induction of the cells with TGF-ß1 and treatment with N1F 48 h later. The chemical composition of N1F was analyzed using high-performance liquid chromatography-Q-Orbitrap high-resolution liquid mass spectrometry. Renal function was assessed by measuring serum creatinine (Scr), blood urea nitrogen (BUN) and urine protein (Upro) levels. Hematoxylin and eosin (HE) and Masson's trichrome (Masson) staining were used to evaluate the extent of renal tissue damage and fibrosis. Western blotting, immunohistochemistry, and immunofluorescence were used to detect the protein levels of relevant indices. The RNA levels of the relevant indices were detected using real-time fluorescence quantitative PCR (RT-qPCR). We identified 361 chemical components in the water extract of N1F. These chemical components of N1F significantly reduced the area associated with interstitial fibrosis in the kidneys of UUO rats and the levels of serum creatinine, urea nitrogen, and urinary protein. Additionally, N1F decreased the protein levels of FGF23, Wnt1, ß-catenin and p-P38MAPK/P38MAPK, along with the expression of renalfibrosis-associated proteins, α-SMA, FN, Collagen III, and Vimentin in the renal tissues of the UUO rats, while enhancing klotho and DKK1 protein levels. In vitro experiments revealed that inhibition of P38MAPK signaling significantly suppressed the expression of proteins related to the Wnt signaling pathway, with a concomitant decrease in the expression of FGF23 and an increase in the expression of Klotho. Notably, the P38MAPK inhibitor (SB203580) had similar effects to N1F in altering the above-mentioned indices in vitro. N1F may exhibit potential therapeutic efficacy against renal fibrosis by inhibiting the FGF23/P38MAPK/Wnt signaling pathway, consequently inhibiting extracellular matrix deposition due to renal injury.

High dose statin treatment reduces circulating Dickkopf-1 following acute myocardial infarction

BackgroundSecreted glycoproteins of the Dickkopf (DKK) family modify Wnt signaling and may influence plaque destabilization but their modulation by statins in MI patients is not known. MethodsWe measured plasma DKK-1 and DKK-3 in patients with acute ST-segment elevation MI (STEMI) before percutaneous coronary intervention (PCI) and after 2 and 7 days and 2 months in patients receiving short-term high-dose (40 mg rosuvastatin, given before PCI; n = 25) and moderate dose (20 mg simvastatin, given the day after PCI; n = 34). In vitro modulation of DKK-1 in human umbilical vein endothelial cells (HUVECs) by statins were assessed. Results(i) Patients receiving high dose rosuvastatin had a marked decline in DKK-1 at day 2 which was maintained throughout the study period. However, a more prevalent use of β-blockers in the simvastatin group, that could have contributed to higher DKK-1 levels in these patients. (ii) There was a strong correlation between baseline DKK-1 levels and change in DKK-1 from baseline to day 2 in patients receiving high dose rosuvastatin treatment. (iii) DKK-3 increased at day 2 but returned to baseline levels at 2 months in both treatment groups. (iv) Statin treatment dose-dependently decreased DKK-1 mRNA and protein levels in HUVEC. ConclusionsOur findings suggest that high dose statin treatment with 40 mg rosuvastatin could persistently down-regulate DKK-1 levels, even at 2 months after the initial event in STEMI patients.

DKK3 Expression in Glioblastoma: Correlations with Biomolecular Markers.

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.

Abstract LB009: Unveiling the significance of DARPP-32 and its partners, including GRB7 and DKK1, in breast cancer: Integrative insights from patient data and cell line transcriptomics

Abstract DARPP-32 plays a crucial role in regulating both protein-phosphatase-1 (PP-1) and protein kinase A (PKA), its function being reliant on its phosphorylation status. We have previously demonstrated that low DARPP-32 expression is associated with adverse survival of breast cancer patients, particularly those with oestrogen receptor (ER) positive disease; however, there is limited information on how DARPP-32 may alter breast cancer cell behaviour. We have recently published an integrative approach using patient data and cell line transcriptomics to understand the role of DARPP-32 in breast cancer. We analysed the transcriptome of T47D ER positive breast cancer cells following DARPP-32 knockdown, including treatments in combination with 17β-estradiol or PKA inhibitor fragment (6-22) amide. 202 differentially expressed transcripts were identified in DARPP-32 knock down cells and treatment of these cells with 17β-estradiol or PKA inhibitor fragment (6-22) amide led to the differential expression of 193 and 181 transcripts respectively. Differentially expressed transcripts were compared with genes identified as associated with DARPP-32 expression in 1980 breast cancer patients within the METABRIC cohort using an Artificial Neural Network approach (ANN). Several genes overlapped with those identified from cell line transcriptomics, including PTK7, TRAF5, and KLK6. Both DKK1 and GRB7 were identified as genes associated with DARPP-32 expression in patients using ANN, therefore protein expression was determined in a large cohort of early-stage breast cancer patients (n>1000). We demonstrated that DKK1 and GRB7 protein expression was significantly correlated with DARPP-32 Threonine-34 phosphorylation levels within the same samples (all P<0.05). We have now applied an ANN-based integrative data mining approach to identify genes with the largest effect on other genes resulting in an interactome map, which was based on genes identified within the cell line transcriptomics and original ANN. 1878 genes were identified for inclusion within an interactome map in the total patient cohort, whereas 1003 were identified in ER negative disease alone and 2144 in ER positive disease alone. Pathway enrichment analysis identified the ubiquitin proteosome pathway, the insulin pathway-protein kinase B signalling cascade and the RAS pathway. This study underscores the significance of DARPP-32, a central molecular switch, in breast cancer, elucidating its impact on patient prognosis and highlighting potential molecular targets for therapeutic intervention. The integrative approach combining patient data with cell line transcriptomics provides insights into the complex mechanisms underlying breast cancer progression and opens avenues for further research and targeted interventions. Citation Format: Behnaz Saidy, Richa Vasan, Megan-Rose Greener, Rosie Durrant, Adelynn Immanuel, Andrew Green, Emad Rakha, Ian Ellis, Graham Ball, Stewart Martin, Sarah J. Storr. Unveiling the significance of DARPP-32 and its partners, including GRB7 and DKK1, in breast cancer: Integrative insights from patient data and cell line transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB009.

Predictors of bone mineral density in patients receiving glucocorticoid replacement for Addison's disease.

Patients receiving long-term glucocorticoid (GC) treatment are at risk of osteoporosis, while bone effects of substitution doses in Addison's disease (AD) remain equivocal. The project was aimed to evaluate serum bone turnover markers (BTMs): osteocalcin, type I procollagen N-terminal propeptide (PINP), collagen C-terminal telopeptide (CTX), sclerostin, DKK-1 protein, and alkaline phosphatase (ALP) in relation to bone mineral density (BMD) during GC replacement. Serum BTMs and hormones were assessed in 80 patients with AD (22 males, 25 pre- and 33 postmenopausal females) on hydrocortisone (HC) substitution for ≥3 years. Densitometry with dual-energy X-ray absorptiometry covered the lumbar spine (LS) and femoral neck (FN). Among BTMs, only PINP levels were altered in AD. BMD Z-scores remained negative except for FN in males. Considering T-scores, osteopenia was found in LS in 45.5% males, 24% young and 42.4% postmenopausal females, while osteoporosis in 9.0%, 4.0% and 21.1%, respectively. Lumbar BMD correlated positively with body mass (p = 0.0001) and serum DHEA-S (p = 9.899 × 10-6). Negative correlation was detected with HC dose/day/kg (p = 0.0320), cumulative HC dose (p = 0.0030), patient's age (p = 1.038 × 10-5), disease duration (p = 0.0004), ALP activity (p = 0.0041) and CTX level (p = 0.0105). However, only age, body mass, ALP, serum CTX, and sclerostin remained independent predictors of LS BMD. Standard HC substitution does not considerably accelerate BMD loss in AD patients and their serum BTMs: CTX, osteocalcin, sclerostin, DKK-1, and ALP activity remain within the reference ranges. Independent predictors of low lumbar spine BMD, especially ALP activity, serum CTX and sclerostin, might be monitored during GC substitution.

Dkk1 as a Prognostic Marker for Neoadjuvant Chemotherapy Response in Breast Cancer Patients.

To investigate the role of Dkk1 as a predictor of response to NACT in BC patients. This retrospective monocentric study included 145 women who had undergone NACT followed by breast surgery. Dkk1 protein expression was assessed using immunohistochemistry staining in core needle biopsies and mammary carcinoma specimens. Dkk1 levels were lower in treated BC tumours than in untreated tumours. The outcomes of 68 matched pre- and post-therapy tissues showed that Dkk1 levels in mammary carcinoma tissues were significantly predicted by levels in core needle biopsies and that Dkk1 expression was reduced in 83% of cases. Smaller cT stage, positive Her2 expression, and decreased Dkk1-IRS in core needle biopsy tissues were all independent predictors of regression grade (R4), according to Sinn. However, the percentage of Dkk1 expression differences prior to and following NACT had no effect on PFS or OS. In this study, we demonstrated for the first time that Dkk1 could be identified as an independent predictor of NACT response in BC patients, particularly those with TNBC. Further research with a multicentric expanded (pre-/post-therapy) sample set and better-defined populations in terms of molecular subtypes, therapy modality, and long-term follow-up is recommended to obtain more solid evidence.

Search for predictors of achieving minimal disease activity during tofacitinib therapy in patients with psoriatic arthritis

Objective: to search predictors of achieving minimal disease activity (MDA) during therapy in patients with psoriatic arthritis (PsA).Materials and methods. The study included 41 patients, predominantly men (58.9 %), with a confirmed PsA diagnosis and a disease duration of at least 6 months. In all cases, the diagnosis fulfilled the CASPAR criteria. The mean age of the patients at the time of enrolment in the study was 43.0±10.1 years, the duration of PsA was 7.7±7.1 years, the duration of psoriasis was 18.6±10.4 years, and the DAPSA index was 44.2±17.1. All patients were prescribed tofacitinib at a dose of 5 mg twice daily, followed by a possible dose increase to 10 mg twice daily. In addition to a general clinical examination and a standard rheumatological examination, the level of secreted DKK-1 protein and health-related quality of life (HRQoL, using a special PsAID-12 questionnaire) were determined. Multivariate stepwise discriminant analysis was used to search for predictors for the achievement of MDA in patients with PsA and to calculate the coefficients.Results and discussion. Based on the results obtained, a predictor for the achievement of MDA (PMDA) was developed: PMDA=-1.165 × number of inflamed entheses + DKK-1 level (pmol/l) + 3.086 × PsAID-12 “Skin lesions” scale value (if this indicator was ≤3 points, it was assigned a value of 1, if it was >3 points – 0) + 2.568 × PsAID-12 “Pain” scale (if this indicator was ≤6 points, it was assigned a value of 1, if it was >6 points – 0).The ROC analysis, which reflects the prognostic significance of this index, showed AUC (area under the curve) of 0.803 (95% confidence interval 0.739–0.867; p=0.02). PMDA=3.89 was chosen as the cut-off value; the sensitivity of this indicator was 91 %, the specificity – 79 %. Therefore with a PMDA ≥3.89, the probability of the patient achieving a MDA after 3 months is high; with a PMDA ˂ 3.89, it is low.Conclusion. We identified factors influencing the achievement of MDA in patients with PsA and developed a mathematical model. It allows timely assessment of the quality of treatment and its correction if necessary, thereby slowing disease progression.

Genetic detection of two novel LRP5 pathogenic variants in patients with familial exudative vitreoretinopathy

BackgroundFamilial exudative vitreoretinopathy (FEVR) is a genetic eye disorder that leads to abnormal development of retinal blood vessels, resulting in vision impairment. This study aims to identify pathogenic variants by targeted exome sequencing in 9 independent pedigrees with FEVR and characterize the novel pathogenic variants by molecular dynamics simulation.MethodsClinical data were collected from 9 families with FEVR. The causative genes were screened by targeted next-generation sequencing (TGS) and verified by Sanger sequencing. In silico analyses (SIFT, Polyphen2, Revel, MutationTaster, and GERP + +) were carried out to evaluate the pathogenicity of the variants. Molecular dynamics was simulated to predict protein conformation and flexibility transformation alterations on pathogenesis. Furthermore, molecular docking techniques were employed to explore the interactions and binding properties between LRP5 and DKK1 proteins relevant to the disease.ResultsA 44% overall detection rate was achieved with four variants including c.4289delC: p.Pro1431Argfs*8, c.2073G > T: p.Trp691Cys, c.1801G > A: p.Gly601Arg in LRP5 and c.633 T > A: p.Tyr211* in TSPAN12 in 4 unrelated probands. Based on in silico analysis and ACMG standard, two of them, c.4289delC: p.Pro1431Argfs*8 and c.2073G > T: p.Trp691Cys of LRP5 were identified as novel pathogenic variants. Based on computational predictions using molecular dynamics simulations and molecular docking, there are indications that these two variants might lead to alterations in the secondary structure and spatial conformation of the protein, potentially impacting its rigidity and flexibility. Furthermore, these pathogenic variants are speculated to potentially influence hydrogen bonding interactions and could result in an increased binding affinity with the DKK1 protein.ConclusionsTwo novel genetic variants of the LRP5 gene were identified, expanding the range of mutations associated with FEVR. Through molecular dynamics simulations and molecular docking, the potential impact of these variants on protein structure and their interactions with the DKK1 protein has been explored. These findings provide further support for the involvement of these variants in the pathogenesis of the disease.

Human bone marrow mesenchymal stem cell exosome-derived miR-335-5p promotes osteogenic differentiation of human periodontal ligament stem cells to alleviate periodontitis by downregulating DKK1

To observe the effect of miR-335-5p derived from human bone marrow mesenchymal stem cell (hBMMSCs) exosomes on osteogenic differentiation of human periodontal ligament stem cell (PDLSCs) model of periodontitis and explore its mechanism. The exosomes extracted from hBMMSCs were identified by transmission electron microscopy, Western blotting and PKH67 labeling. The human PDLSC model of TNF-α-induced periodontitis were co-cultured with the extracted exosomes, and qRT-PCR was performed to detect the changes in the expressions of miR-335-5p and the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) and the osteogenic marker genes (RunX2, OCN and BMP-2). Alizarin red staining and ALP staining were used to detect the formation of calcium nodules in the treated cells, and the expression level of DKK1 protein was detected with Western blotting. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-335-5p and DKK1. High expressions of CD9 and CD81 were detected in the extracted hBMMSC exosomes (P < 0.05). In TNF-α-induced hPDLSCs, treatment with the extracted exosomes significantly reduced the mRNA expressions of IL-1β, IL-6 and IL-8, enhanced the mRNA expressions of RunX2, OCN, and BMP-2, and promoted the formation of calcium nodules. MiR-335-5p was highly expressed in hBMMSC-derived exosomes, and overexpression of miR-335-5p significantly downregulated DKK1 protein expression, inhibited the mRNA expressions of IL-1β, IL-6 and IL-8, and promoted the mRNA expressions of osteogenic markers and the formation of calcium nodules in hPDLSCs. HBMMSC exosome-derived miR-335-5p promotes osteogenic differentiation of hPDLSCs and inhibits the development of periodontitis by downregulating DKK1.

Characterization of an ablative fractional CO2 laser-induced wound-healing model based on in vitro 3D reconstructed skin.

This study describes the development and characterization of a novel in vitro wound-healing model based on a full-thickness reconstructed skin by exposing the tissue to fractional ablative laser treatment. A 3D full-thickness skin model was fabricated and treated with fractional ablative CO2 laser. Wound-healing process was characterized by HE staining, noninvasive OCT imaging, immunostaining, as well as transepidermal water loss measurement. Cytokines and proteins involved in the inflammatory and dermal remodeling process were studied by ELISA and protein array assays. Fractional ablative CO2 treatment induced a wound zone of 9 mm in diameter, containing 56 micro-wounds with 200 μm diameter and 500-700 μm in depth on reconstructed full-thickness skin model. HE staining revealed a typical wound morphology and healing process with migration of keratinocytes, formation and extrusion of necrotic tissue, and cell inclusion in dermis, which correlates with clinical observations. Based on OCT and TEWL measurements, the re-epithelialization took place over 2 days. Laser-triggered keratinocytes proliferation and differentiation were demonstrated by activated Ki67 and Filaggrin expression respectively. Injury-invoked cytokine ICAM-1 showed instant upregulation on Day 1. Decreased epidermis thickness and depression of IGFBP-2 protein level synergistically indicated the unavoidable thermal side effects from laser treatment. Downregulated DKK-1 protein level and upregulation of α-SMA together implicated the risk of potential fibrosis post-laser treatment. This in vitro laser wounded reconstructed skin model captured the key events of wound-healing process, could be used to investigate the mechanisms of wound-healing triggered by a commonly used beauty procedure, and also provides a valuable tool for evaluating the efficacy of novel actives for the post-procedure application.

Dickkopf-related protein 1/cytoskeleton-associated protein 4 signaling activation by Helicobacter pylori-induced activator protein-1 promotes gastric tumorigenesis via the PI3K/AKT/mTOR pathway

Gastric cancer (GC) is a common malignant tumor with high incidence and mortality rates globally, especially in East Asian countries. Helicobacter pylori (H. pylori) infection is a significant and independent risk factor for GC. However, its underlying mechanism of action is not fully understood. Dickkopf-related protein (DKK) 1 is a Wnt signaling antagonist, and cytoskeleton-associated protein (CKAP) 4 is a newly identified DKK1 receptor. Recent studies found that the binding of DKK1 to CAKP4 mediated the procancer signaling of DKK1 inde-pendent of Wnt signaling. We hypothesize that H. pylori-induced activation of DKK1/CKAP4 signaling contributes to the initiation and progression of GC. To investigate the interaction of H. pylori infection, DKK1 and CAKP4 in GC, as well as the underlying molecular mechanisms. RNA sequencing was used to identify differentially expressed genes (DEGs) between H. pylori-infected and uninfected primary GC cells. Gain- and loss-of-function experiments were performed to verify the H. pylori-induced upregulation of activator protein-1 (AP-1) in GC cells. A dual-luciferase reporter assay and co-immunoprecipitation were used to determine the binding of AP-1 to the DKK1 promoter and DKK1 to CKAP4. Western blotting and immunohistochemistry detected the expression of DKK1, CKAP4, and phos-phatidylinositol 3-kinase (PI3K) pathway-related proteins in GC cells and tissues. Functional experiments and tumorigenicity in nude mice detected malignant behavior of GC cells in vitro and in vivo. We identified 32 DEGs between primary GC cells with and without H. pylori infection, including JUN, fos-like antigen-1 (FOSL1), and DKK1, and confirmed that the three proteins and CKAP4 were highly expressed in H. pylori-infected GC cells, H. pylori-infected gerbil gastric tissues, and human GC tissues. JUN and FOSL1 form AP-1 to transcriptionally activate DKK1 expression by binding to the DKK1 promoter. Activated DKK1 bound to CKAP4, but not the most common Wnt coreceptor low-density lipoprotein receptor-related protein 5/6, to promote GC cell growth, colony formation, migration, invasion, and xenograft tumor growth in nude mice. All these effects were driven by activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway. Targeting the PI3K signaling pathway by LY294002 inhibited DKK1-mediated CKAP4/PI3K signaling activity and the malignant behavior of GC cells. H. pylori induces JUN and FOSL1 expression to form AP-1, which transcriptionally activates DKK1. Binding of DKK1 to KAKP4 contributes to gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

The FoxO4/DKK3 axis represses IFN-γ expression by Th1 cells and limits antimicrobial immunity.

Forkhead box O transcriptional factors, especially FoxO1 and FoxO3a, play critical roles in physiologic and pathologic immune responses. However, the function of FoxO4, another main member of the FoxO family, in lymphoid cells is still poorly understood. Here, we showed that loss of FoxO4 in T cells augmented IFN-γ production of Th1 cells in vitro. Correspondingly, conditional deletion of FoxO4 in CD4+ T cells enhanced T cell–specific responses to Listeria monocytogenes infection in vivo. Genome-wide occupancy and transcriptomic analyses identified Dkk3 (encoding the Dickkopf-3 protein) as a direct transcriptional target of FoxO4. Consistent with the FoxO4-DKK3 relationship, recombinant DKK3 protein restored normal levels of IFN-γ production in FoxO4-deficient Th1 cells through the downregulation of lymphoid enhancer–binding factor 1 (Lef1) expression. Together, our data suggest a potential FoxO4/DKK3 axis in Th1 cell differentiation, providing what we believe to be an important insight and supplement for FoxO family proteins in T lymphocyte biology and revealing a promising target for the treatment of immune-related diseases.

QingreHuoxue decoction protects joint and toe bone morphology and structure in rats with active RA through bidirectional regulation of bone metabolism

QingreHuoxue decoction (QRHX) is a well-known traditional Chinese medicine formula for treating rheumatoid arthritis (RA). It is not known how QRHX is involved in bone metabolism regulation and thereby alleviatesbone injury caused by arthritis. In this study, we used collagen-induced arthritis (CIA) rat model to evaluate the effects of QRHX onarthriticdiarthrodial joints and the underlying molecular mechanisms. Our results showed that QRHX significantly reduced foot joint volume, synovial inflammatory infiltration and the arthritis index (AI) score. Micro-CT imaging showed that toe bone and ankle joint were eroded and destroyed progressively during RA process, andcompensatory hyperplasia appeared in late stage RA. QRHX treatment inhibited foot bone erosion of early and mid-stage RA, and improved bone mineral density and trabecular fine structure in late RA.This studyunlocked the whole process details of ankle and toe bone injuryduring RA, as well as the bone metabolic characteristics under QRHX treatmentin CIArats. QRHX was confirmed to inhibite the expression of RANKL and DKK-1 mRNA and protein, enhance the expression of β-catenin. Meanwhile, QRHX reduced the pro-inflammatorycytokines IL-17, IL-1β and TNF-αand upregulated anti-inflammatory IL-4 in serum, improving local and systemic inflammatory microenvironment. These results demonstrated that QRHX had sustained protective effects on diarthrodial joints/bone during RA by a synergistic regulation mechanism, which reverses the imbalance of bone metabolismthroughinhibiting RANKL related osteoclast pathway and promotingWnt/β-catenin related osteoblast activation pathway.

The RNA-binding protein PCBP1 represses lung adenocarcinoma progression by stabilizing DKK1 mRNA and subsequently downregulating β-catenin

BackgroundPolyC-RNA-binding protein 1 (PCBP1) functions as a tumour suppressor and RNA regulator that is downregulated in human cancers. Here, we aimed to reveal the biological function of PCBP1 in lung adenocarcinoma (LUAD).MethodsFirst, PCBP1 was identified as an important biomarker that maintains LUAD through The Cancer Genome Atlas (TCGA) project screening and confirmed by immunohistochemistry and qPCR. Via colony formation, CCK8, IncuCyte cell proliferation, wound healing and Transwell assays, we confirmed that PCBP1 was closely related to the proliferation and migration of LUAD cells. The downstream gene DKK1 was discovered by RNA sequencing of PCBP1 knockdown cells. The underlying mechanisms were further investigated using western blot, qPCR, RIP, RNA pulldown and mRNA stability assays.ResultsWe demonstrate that PCBP1 is downregulated in LUAD tumour tissues. The reduction in PCBP1 promotes the proliferation, migration and invasion of LUAD in vitro and in vivo. Mechanistically, the RNA-binding protein PCBP1 represses LUAD by stabilizing DKK1 mRNA. Subsequently, decreased expression of the DKK1 protein relieves the inhibitory effect on the Wnt/β-catenin signalling pathway. Taken together, these results show that PCBP1 acts as a tumour suppressor gene, inhibiting the tumorigenesis of LUAD.ConclusionsWe found that PCBP1 inhibits LUAD development by upregulating DKK1 to inactivate the Wnt/β-catenin pathway. Our findings highlight the potential of PCBP1 as a promising therapeutic target.

Clinical Diagnostic Value of Dkk-1 Level in Ankylosing Spondylitis: Comparison of Test Systems Based on Aptamers and Antibodies.

In ankylosing spondylitis, the pathological metabolism of the bone tissue is regulated by various proteins; of these, Dkk-1 protein, an antagonist of the Wnt-signaling pathway, is of particular interest. We compared the methods of Dkk-1 detection in the blood serum of patients with ankylosing spondylitis (conventional ELISA and aptamer/antibody assay) and analyzed the relationship between Dkk-1 level and structural progression of ankylosing spondylitis and secondary osteoporosis. Dkk-1 levels in patients were significantly increased and depended on the stage of the disease, but not on the presence/absence of osteoporosis. Thus, Dkk-1 is a potential serum marker of progression of ankylosing spondylitis reflecting a tendency to structural progression of the disease before the appearance of radiographic changes. The results obtained by both methods practically coincided, which suggests good prospects for applying DNA aptamer-based test system.

Investigation of the relationship between serum sclerostin and dickkopf-1 protein levels with bone turnover in children and adolescents with type-1 diabetes mellitus

Diabetes mellitus (DM) is widely known to have a detrimental effect on bone health and is associated with increased fracture risk. Recently, the Wnt/beta-catenin signaling pathway and its inhibitors sclerostin and dickkopf-1 (Dkk-1) were found to be involved in the control of bone mass. The present study aimed to measure serum sclerostin and Dkk-1 protein levels in children and adolescents with type-1 DM and compare with other bone turnover markers and bone mineral density (BMD). This study was performed on 40 children and adolescents with type-I DM and 40 healthy children and adolescents. Anthropometric measurements and pubertal examination were done. In addition to laboratory analysis, dickkopf-1, sclerostin, cross-linked N-telopeptides of type I collagen (NTx), bone alkaline phosphatase (bALP), and osteocalcin levels were studied. BMD of the participants was measured by calcaneus ultrasonography. Dickkopf-1 levels of the children and adolescents with type-1 DM were significantly higher, vitamin D, NTx, osteocalcin, and phosphorus levels were significantly lower than those of the controls (p<0.001). Fasting blood glucose, HbA1c, and insulin were significantly higher in the type 1 DM group (p<0.01). Both bone remodeling and its compensatory mechanism bone loss are lower in children and adolescents with type-1 DM than in the controls. Also, higher levels of Dkk-1 play a role in decreased bone turnover in these patients. Since Dkk-1 and sclerostin seem to take a role in treating metabolic bone diseases in the future, we believe that our findings are significant in this respective.