DKK1 Expression Research Articles

Enhancing immunotherapy efficacy in colorectal cancer: targeting the FGR-AKT-SP1-DKK1 axis with DCC-2036 (Rebastinib)

This research demonstrates that DCC-2036 (Rebastinib), a potent third-generation tyrosine kinase inhibitor (TKI), effectively suppresses tumor growth in colorectal cancer (CRC) models with functional immune systems. The findings underscore the capacity of DCC-2036 to enhance both the activation and cytotoxic functionality of CD8+ T cells, which are crucial for facilitating anti-tumor immune responses. Through comprehensive multi-omics investigations, significant shifts in both gene and protein expression profiles were detected, notably a marked decrease in DKK1 levels. This reduction in DKK1 was linked to diminished CD8+ T cell effectiveness, correlating with decreased FGR expression. Moreover, our findings identify FGR as a pivotal modulator that influences DKK1 expression via the PI3K-AKT-SP1 signaling cascade. Correlative analysis of clinical specimens supports the experimental data, showing that increased levels of FGR and DKK1 in CRC tissues are associated with inferior clinical outcomes and reduced efficacy of immunotherapeutic interventions. Consequently, targeting the FGR-AKT-SP1-DKK1 pathway with DCC-2036 could potentiate immunotherapy by enhancing CD8+ T cell functionality and their tumor infiltration. This strategy may contribute significantly to the refinement of therapeutic approaches for CRC, potentially improving patient prognoses.

Molecular insights into the therapeutic mechanisms of Bushen-Qiangdu-Zhilv decoction for ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic rheumatic immune disease characterized by high disability rates, significantly affecting patients' quality of life. BuShen-QiangDu-ZhiLv Decoction (BQZD), developed by the renowned traditional Chinese medicine practitioner Jiao Shude, has been traditionally used for AS treatment. However, the bioactive components and the precise mechanisms underlying BQZD's therapeutic effects remain largely unexplored. To investigate the protective effects and elucidate the molecular mechanisms of BQZD in treating ankylosing spondylitis. Ultra Performance Liquid Chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was used to identify active compounds in BQZD. Bulk RNA-seq and Gene Set Variation Analysis (GSVA) were conducted to assess changes in pathway activity in AS patients before and after three months of BQZD treatment. We also utilized network pharmacology and molecular docking analyses to predict potential mechanisms, identifying key target genes such as IL-6, NFATc1, and c-FOS. Animal experiments were performed to validate these findings. UPLC-MS/MS identified 28 active compounds in BQZD capable of entering the bloodstream, with potential anti-inflammatory, immunomodulatory, and bone metabolism-regulating effects. BQZD treatment led to a significant reduction in ESR, CRP, and ASDAS-CRP scores, indicating clinical improvement in AS patients. RNA-seq analysis showed decreased GSVA scores for the ossification pathway, with moderate reductions in inflammatory response and RANKL signaling pathways. Positive correlations were found between pathway activity and clinical indicators. Network pharmacology and transcriptomic analysis identified IL-6, NFATc1, and c-FOS as key targets. In vivo experiments confirmed that BQZD reduced TNF-α and IL-1β levels, inhibited ectopic ossification, and modulated the expression of DKK-1, MMP-9, and OPN in the CAIA model. BQZD exerts therapeutic effects in AS by regulating inflammation and abnormal ossification through multi-pathway, multi-target mechanisms. The identification of key target genes such as IL-6, NFATc1, and c-FOS provides a solid foundation for future research and clinical applications of BQZD in AS management.

The evolution of cephalic fins in manta rays and their relatives: functional evidence for initiation of domain splitting and modulation of the Wnt signaling pathway in the pectoral fin AER of the little skate

BackgroundBatoids possess a unique body plan associated with a benthic lifestyle that includes dorsoventral compression and anteriorly expanded pectoral fins that fuse to the rostrum. The family Myliobatidae, including manta rays and their relatives, exhibit further modifications associated with invasion of the pelagic environment, and the evolution of underwater flight. Notably, the pectoral fins are split into two domains with independent functions that are optimized for feeding and oscillatory locomotion. Paired fin outgrowth is maintained during development by Wnt3, while domain splitting is accomplished by expression of the Wnt antagonist Dkk1, which is differentially expressed in the developing anterior pectoral fins of myliobatids, where cephalic fins separate from pectoral fins. We examine the evolution of this unique feature in the cownose ray (Rhinoptera bonasus), a member of the genus that is sister to Mobula.ResultsHere, we provide functional evidence that DKK1 is sufficient to initiate pectoral fin domain splitting. Agarose beads soaked in DKK1 protein were implanted in the pectoral fins of little skate (Leucoraja erinacea) embryos resulting in AER interruption. This disruption arrests fin ray outgrowth, resembling the myliobatid phenotype. In addition, fins that received DKK1 beads exhibit interruption of Axin2 expression, a downstream target of β-catenin-dependent Wnt signaling and a known AER marker. We demonstrate that Msx1 and Lhx2 are also associated with fin expansion at the AER. These results provide functional evidence for the underlying genetic pathway associated with the evolution of a novel paired fin/limb modification in manta rays and their relatives. We introduce the gas/brake pedal model for paired fin remodeling at the AER, which may have been co-opted from domain splitting in pelvic fins of cartilaginous fishes 370 million years earlier.ConclusionsThe pectoral fins of manta rays and their relatives represent a dramatic remodel of the ancestral batoid body plan. The premiere feature of this remodel is the cephalic fins, which evolved via domain splitting of the anterior pectoral fins through inhibition of fin ray outgrowth. Here, we functionally validate the role of Dkk1 in the evolution of this phenotype. We find that introduction of ectopic DKK1 is sufficient to recapitulate the myliobatid pectoral fin phenotype in an outgroup lacking cephalic fins via AER interruption and fin ray truncation. Additional gene expression data obtained via in situ hybridization suggests that cephalic fin development may have evolved as a co-option of the pathway specifying claspers as modifications to the pelvic fins, the only other known example of domain splitting in vertebrate appendages.

Tumor cells induce neural DKK1 expression to promote MDSC infiltration and subsequent T cell suppression.

Nerves are often overlooked as key components of the tumor microenvironment. However, the molecular mechanisms underlying the reciprocal interactions between tumors and nerves remain largely unknown. In this study, we analyzed data from The Cancer Genome Atlas (TCGA) and identified a significant association between DKK1 expression and poor prognosis, as well as a correlation between DKK1 expression and myeloid-derived suppressor cell (MDSC) infiltration in head and neck squamous cell carcinoma (HNSCC) and pancreatic ductal adenocarcinoma (PDAC), two cancer types characterized by pronounced tumor-nerve interactions. Based on these findings, we hypothesize that tumors may induce DKK1 expression in nerves, and that nerve-derived DKK1 may promote MDSC infiltration and immunosuppression. To test this hypothesis, we employed a combination of experimental approaches, including in vitro co-culture of trigeminal ganglia with tumor cells, multiplex immunohistochemistry, and in vivo administration of DKK1 neutralizing antibodies. Our results indicate that tumor cells significantly induce DKK1 expression in ganglia in co-culture experiments. Additionally, in vivo orthotopic tumor models revealed that DKK1 levels were markedly elevated in both the plasma and ganglia of tumor-bearing mice. Neutralization DKK1 in vivo led to a reduction in MDSC levels and impaired MDSC-mediated T cell suppression in both HNSCC and PDAC orthotopic models. Furthermore, conditional deletion of neuronal DKK1 elucidated its role in MDSC infiltration and immune suppression. Our findings establish a novel molecular axis in which tumor cells modulate the immune microenvironment by inducing the expression of secreted proteins in nerves, thereby enriching the research landscape of the tumor microenvironment.

Chidamide and venetoclax synergistically regulate the Wnt/β-catenin pathway by MYCN/DKK3 in B-ALL.

B-cell acute lymphocytic leukemia (B-ALL) is a malignant proliferative B-lymphocyte disease. Although the outcome of B-ALL has greatly improved with combined chemotherapy, immunotherapy, and hematopoietic stem cell transplantation, some patients still experience drug resistance, relapse and a low long-term survival rate, therefore, finding novel approaches to improve the outcome of adult B-ALL patients is critical. Our previous studies revealed that the selective histone deacetylase inhibitor (HDACi) chidamide can inhibit the Wnt/β-catenin signaling pathway by inhibiting MYCN and increasing the expression of DKK3 in B-ALL cells. Some studies have indicated that histone deacetylase inhibitors (HDACis) can dysregulate the B-cell lymphoma-2 (BCL2) protein family, we speculate that chidamide and BCL2 inhibitor venetoclax synergistically inhibit the Wnt/β-catenin signaling pathway by inhibiting MYCN expression and increasing DKK3 expression. In our study, the in vitro and in vivo experiments confirmed that chidamide and venetoclax synergistically inhibited the expression of MYCN and increased the expression of DKK3 by inhibiting the activity of HDAC and BCL2, inhibiting the Wnt/β-catenin signaling pathway and B-ALL cell proliferation. These findings indicate that the HDACi chidamide and the BCL2 inhibitor venetoclax can be used in combination to treat B-ALL, providing a new method and strategy for treating B-ALL.

TOP2A Promotes Proliferation, Migration, and Inflammatory Response in M5-Treated Keratinocytes by Binding CTBP1 to Activate Wnt/β-Catenin Signaling.

Psoriasis is a chronic cutaneous disease, affecting a significant portion of the global population. Topoisomerase II alpha (TOP2A) is upregulated in psoriasis samples, but the precise molecular mechanism remains unclear. We aimed to clarify the biological contribution of TOP2A in psoriasis progression. An in vitro psoriasis model was established on M5-induced keratinocytes (HaCaT cells) to simulate the psoriasis-like alterations. Following TOP2A knockdown without or with c terminal binding protein 1 (CTBP1) overexpression, CCK-8 and EDU staining were employed to analyze the viability and proliferation of HaCaT cells under M5 conditions. The capacities of HaCaT cell migration and invasion were examined with wound healing- and transwell assays. RT-qPCR and immunoblotting were adopted for evaluation of the inflammation and differentiation of M5-stimualted HaCaT cells. Additionally, the binding between TOP2A and CTBP1 was predicated using bioinformatics tools and detected by Co-IP. Finally, the expression of proteins in Wnt/β-catenin signaling was analyzed with the application of immunoblotting. Results suggested that TOP2A was upregulated in psoriasis skin lesions and M5-induced HaCaT cells. Interference with TOP2A attenuated the proliferation, migration, invasion, and inflammatory response in M5-treated HaCaT cells. In particular, TOP2A bound to CTBP1 and upregulated CTBP1 expression in HaCaT cells. Remarkably, CTBP1 upregulation blocked the impacts of TOP2A depletion on the biological behaviors of M5-treated HaCaT cells. Besides, TOP2A deficiency upregulated DKK1 expression as well as downregulated Wnt1, β-catenin, and c-Myc expression in HaCaT cells exposed to M5, which was restored by further CTBP1 overexpression. In summary, TOP2A binds CTBP1 to activate Wnt/β-catenin signaling, thereby promoting the progression of psoriasis.

Integrated bulk and single-cell profiling characterize sphingolipid metabolism in pancreatic cancer

BackgroundAbnormal sphingolipid metabolism (SM) is closely linked to the incidence of cancers. However, the role of SM in pancreatic cancer (PC) remains unclear. This study aims to explore the significance of SM in the prognosis, immune microenvironment, and treatment of PC.MethodsSingle-cell and bulk transcriptome data of PC were acquired via TCGA and GEO databases. SM-related genes (SMRGs) were obtained via MSigDB database. Consensus clustering was utilized to construct SM-related molecular subtypes. LASSO and Cox regression were utilized to build SM-related prognostic signature. ESTIMATE and CIBERSORT algorithms were employed to assess the tumour immune microenvironment. OncoPredict package was used to predict drug sensitivity. CCK-8, scratch, and transwell experiments were performed to analyze the function of ANKRD22 in PC cell line PANC-1 and BxPC-3.ResultsA total of 153 SMRGs were acquired, of which 48 were linked to PC patients’ prognosis. Two SM-related subtypes (SMRGcluster A and B) were identified in PC. SMRGcluster A had a poorer outcome and more active SM process compared to SMRGcluster B. Immune analysis revealed that SMRGcluster B had higher immune and stromal scores and CD8 + T cell abundance, while SMRGcluster A had a higher tumour purity score and M0 macrophages and activated dendritic cell abundance. PC with SMRGcluster B was more susceptible to gemcitabine, paclitaxel, and oxaliplatin. Then SM-related prognostic model (including ANLN, ANKRD22, and DKK1) was built, which had a very good predictive performance. Single-cell analysis revealed that in PC microenvironment, macrophages, epithelial cells, and endothelial cells had relatively higher SM activity. ANKRD22, DKK1, and ANLN have relatively higher expression levels in epithelial cells. Cell subpopulations with high expression of ANKRD22, DKK1, and ANLN had more active SM activity. In vitro experiments showed that ANKRD22 knockdown can inhibit the proliferation, migration, and invasion of PC cells.ConclusionThis study revealed the important significance of SM in PC and identified SM-associated molecular subtypes and prognostic model, which provided novel perspectives on the stratification, prognostic prediction, and precision treatment of PC patients.

The uremic toxin indoxyl sulfate decreases osteocyte RANKL/OPG and increases Wnt inhibitor RNA expression that is reversed by PTH.

Renal osteodystrophy (ROD) leads to increased fractures, potentially due to underlying low bone turnover in chronic kidney disease (CKD). We hypothesized that indoxyl sulfate (IS), a circulating toxin elevated in CKD and a ligand for the aryl hydrocarbon receptor (AhR), may target the osteocytes leading to bone cell uncoupling in ROD. The IDG-SW3 osteocytes were cultured for 14days (early) and 35days (mature osteocytes) and incubated with 500μM of IS after dose finding studies to confirm AhR activation. Long-term incubation of IS for 14days led to decreased expression of Tnfsf11/Tnfrsf11b ratio (RANKL/OPG), which would increase osteoclast activity, and increased expression of Wnt inhibitors Sost and Dkk1, which would decrease bone formation in addition to decreased mineralization and alkaline phosphatase (ALP) activity. When osteocytes were incubated with IS and the AhR translocation inhibitor CH223191, mineralization and ALP activity were restored. However, the Tnfsf11/Tnfrsf11b ratio and Sost, Dkk1 expression were not altered compared with IS alone, suggesting more complex signaling. In both early and mature osteocytes, co-culture with parathyroid hormone (PTH) and IS reversed the IS-induced upregulation of Sost and Dkk1, and IS enhanced the PTH-induced increase of the Tnfsf11/Tnfrsf11b ratio. Co-culture of IS with PTH additively enhanced the AhR activity assessed by Cyp1a1 and Cyp1b1 expression. In summary, IS in the absence of PTH increased osteocyte messenger RNA (mRNA) Wnt inhibitor expression in both early and mature osteocytes, decreased mRNA expression ofTnfsf11/Tnfrsf11b ratio and decreased mineralization in early osteocytes. These changes would lead to decreased resorption and formation resulting in low bone remodeling. These data suggest IS may be important in the underlying low turnover bone disease observed in CKD when PTH is not elevated. In addition, when PTH is elevated, IS interacts to further increase Tnfsf11/Tnfrsf11b ratio for osteoclast activity in both early and mature osteocytes, which would worsen bone resorption.

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.

Piperonylic Acid Promotes Hair Growth by Activation of EGFR and Wnt/β-Catenin Pathway.

Dermal papilla cells (DPCs) are located at the bottom of the hair follicle and play a critical role in hair growth, shape, and cycle. Epidermal growth factor receptor (EGFR) and Wnt/β-catenin signaling pathways are essential in promoting keratinocyte activation as well as hair follicle formation in DPCs. Piperonylic acid is a small molecule that induces EGFR activation in keratinocytes. However, the effects of piperonylic acid on DPCs in regard to the stimulation of hair growth have not been studied. In the present study, piperonylic acid was shown to activate the Wnt/β-catenin signaling pathway in addition to the EGFR signaling pathway in DPCs. Piperonylic acid suppressed DKK1 expression, which presumably promoted the accumulation of β-catenin in the nucleus. In addition, piperonylic acid promoted cyclin D upregulation and cell growth and increased the expression of alkaline phosphatase (ALP), a DPC marker. In a clinical study, the group that applied a formulation containing piperonylic acid had a significantly higher number of hairs per unit area than the placebo group. These results identify piperonylic acid as a promising new candidate for hair loss treatment.

8464 A Unique SERM Reveals An Estrogen Receptor-Dependent Secretion Of DKK1 In Breast Cancer Cells

Abstract Disclosure: K.S. Young: None. G. Hancock: None. S. Kregel: None. S.W. Fanning: Grant Recipient; Self; Olema Oncology. Although Estrogen Receptor positive (ER+) breast cancer has been successfully targeted in primary tumors, the critical limitations of hormone therapies are ultimately revealed as many women often experience disease recurrence. A subset of these patients harbor a distinctive tyrosine to serine missense mutation at position 537 (Y537S) in ER leading to allele-specific transcriptional programs that enhance metastasis. ER is a ligand-dependent master transcriptional regulator. Probing unique ER structural features can reveal new transcriptional activities. We developed a structurally unconventional Selective Estrogen Receptor Modulator (SERM), T6I-29, to better understand these structure-transcriptional relationships. T6I-29 shows effective anti-tumoral activities in Y537S ESR1 breast cancer cells. To examine the anti-tumoral mechanism of T6I-29 action, we performed RNA-sequencing on ESR1 Y537S mutant breast cancer cells. Compared to other clinically relevant compounds, our drug uniquely and significantly downregulated DKK1, a tumor secreted protein known to modulate the Wnt/β-Catenin and PI3K/Akt pathways. Upregulation of DKK1 correlates with metastatic burden across multiple cancers but has not been evaluated in ESR1 mutant breast cancers. We find that DKK1 levels are significantly elevated in the plasma of 108 ER+ breast cancer patients compared to 100 matched healthy donors. Furthermore, in wild-type (WT) PIK3CA breast cancer cell lines, those with Y537S ESR1 mutation show constitutive expression and secretion of DKK1, while DKK1 expression and secretion is estrogen-dependent in WT breast cancer cells. Intriguingly, WT cells show significant surface bound DKK1, which is secreted into the media upon hormone stimulation and blocked by T6I-29. Understanding the ER-dependence and functional importance of DKK1 secretion offers a novel approach to potentially reduce breast cancer metastatic burden. Presentation: 6/3/2024

DKK1 Expression in Osteocytes in Patients with Type 2 Diabetes and CKD-MBD

DKK1 Expression in Osteocytes in Patients with Type 2 Diabetes and CKD-MBD

Andrographolide sensitizes glioma to temozolomide by inhibiting DKK1 expression.

Temozolomide (TMZ) is the first-line chemotherapeutic drug for gliomas treatment. However, the clinical efficacy of TMZ in glioma patients was very limited. Therefore, it is urgently needed to discover a novel approach to increase the sensitivity of glioma cells to TMZ. Western blot, immunohistochemical staining, and qRT-PCR assays were used to explore the mechanisms underlying TMZ promoting DKK1 expression and andrographolide (AND) inhibiting DKK1 expression. HPLC was used to detect the ability of andrographolide (AND) to penetrate the blood-brain barrier. MTT assay, bioluminescence images, magnetic resonance imaging (MRI) and H&E staining were employed to measure the proliferative activity of glioma cells and the growth of intracranial tumors. TMZ can promote DKK1 expression in glioma cells and brain tumors of an orthotopic model of glioma. DKK1 could promote glioma cell proliferation and tumor growth in an orthotopic model of glioma. Mechanistically, TMZ increased EGFR expression and subsequently induced the activation of its downstream MEK-ERK and PI3K-Akt pathways, thereby promoting DKK1 expression in glioma cells. Andrographolide inhibited TMZ-induced DKK1 expression through inactivating MEK-ERK and PI3K-Akt pathways. Andrographolide can cross the blood-brain barrier, the combination of TMZ and andrographolide not only improved the anti-tumor effects of TMZ but also showed a survival benefit in an orthotopic model of glioma. Andrographolide can enhance anti-tumor activity of TMZ against glioma by inhibiting DKK1 expression.

DKK1-SE recruits AP1 to activate the target gene DKK1 thereby promoting pancreatic cancer progression

Super-enhancers are a class of DNA cis-regulatory elements that can regulate cell identity, cell fate, stem cell pluripotency, and even tumorigenesis. Increasing evidence shows that epigenetic modifications play an important role in the pathogenesis of various types of cancer. However, the current research is far from enough to reveal the complex mechanism behind it. This study found a super-enhancer enriched with abnormally active histone modifications in pancreatic ductal adenocarcinoma (PDAC), called DKK1-super-enhancer (DKK1-SE). The major active component of DKK1-SE is component enhancer e1. Mechanistically, AP1 induces chromatin remodeling in component enhancer e1 and activates the transcriptional activity of DKK1. Moreover, DKK1 was closely related to the malignant clinical features of PDAC. Deletion or knockdown of DKK1-SE significantly inhibited the proliferation, colony formation, motility, migration, and invasion of PDAC cells in vitro, and these phenomena were partly mitigated upon rescuing DKK1 expression. In vivo, DKK1-SE deficiency not only inhibited tumor proliferation but also reduced the complexity of the tumor microenvironment. This study identifies that DKK1-SE drives DKK1 expression by recruiting AP1 transcription factors, exerting oncogenic effects in PDAC, and enhancing the complexity of the tumor microenvironment.

The metabolites of Bifidobacterium longum BB536 alleviate DHT-damaged human dermal papilla cells by activating WNT/β-catenin signaling

Probiotics have numerous biological functions and clinical studies have reported that consuming probiotic foods could promote hair growth and hair follicle health. Bifidobacterium longum BB536 was selected to investigate the possibility of treating hair loss with Cell-Free Supernatant (CFS) of B. longum BB536 and the alleviation mechanism of CFS on dihydrotestosterone (DHT)-damaged human dermal papilla cells (HDPCs). The results showed that HDPCs treated with 10% CFS of B. longum BB536 for 24 h could significantly promote cell proliferation, repair cell damage, accelerate the process of cell cycle and increase the secretion of VEGF. The CFS of B. longum BB536 reduced the apoptosis rate of DHT-damaged HDPCs. In addition, it could reduce the level of pro-inflammatory factors and improve the alkaline phosphatase (ALP) activity. Metabolites of B. longum BB536 may increase the protein expression of Wnt10b, β-catenin and LEF-1 by activating Wnt/β-catenin signaling pathway and inhibit the expression of DHT-induced negative feedback factors DKK1 and GSK-3β, which alleviates the damage of DHT on HDPCs, some protein expression levels such as LEF-1 are close to minoxidil (common drugs for hair loss). So, metabolites of B. longum BB536 can be used as new treatment for hair loss due to its safety. This study provides theoretical basis for probiotics to prevent androgenic alopecia.

DKK1 loss promotes endometrial fibrosis via autophagy and exosome-mediated macrophage-to-myofibroblast transition

IntroductionIntrauterine adhesions (IUA) manifest as endometrial fibrosis, often causing infertility or recurrent miscarriage; however, their pathogenesis remains unclear.ObjectivesThis study assessed the role of Dickkopf WNT signaling pathway inhibitor 1 (DKK1) and autophagy in endometrial fibrosis, using clinical samples as well as in vitro and in vivo experiments.MethodsImmunohistochemistry, immunofluorescence and western blot were used to determine the localization and expression of DKK1 in endometrium; DKK1 silencing and DKK1 overexpression were used to detect the biological effects of DKK1 silencing or expression in endometrial cells; DKK1 gene knockout mice were used to observe the phenotypes caused by DKK1 gene knockout.ResultsIn patients with IUA, DKK1 and autophagy markers were down-regulated; also, α-SMA and macrophage localization were increased in the endometrium. DKK1 conditional knockout (CKO) mice showed a fibrotic phenotype with decreased autophagy and increased localization of α-SMA and macrophages in the endometrium. In vitro studies showed that DKK1 knockout (KO) suppressed the autophagic flux of endometrial stromal cells. In contrast, ectopic expression of DKK1 showed the opposite phenotype. Mechanistically, we discovered that DKK1 regulates autophagic flux through Wnt/β-catenin and PI3K/AKT/mTOR pathways. Further studies showed that DKK1 KO promoted the secretion of interleukin (IL)-8 in exosomes, thereby promoting macrophage proliferation and metastasis. Also, in DKK1 CKO mice, treatment with autophagy activator rapamycin partially restored the endometrial fibrosis phenotype.ConclusionOur findings indicated that DKK1 was a potential diagnostic marker or therapeutic target for IUA.

Noncoding RNA regulates the expression of Krm1 and Dkk2 to synergistically affect aortic valve lesions

Calcific aortic valve disease (CAVD) is becoming an increasingly important global medical problem, but effective pharmacological treatments are lacking. Noncoding RNAs play a pivotal role in the progression of cardiovascular diseases, but their relationship with CAVD remains unclear. Sequencing data revealed differential expression of many noncoding RNAs in normal and calcified aortic valves, with significant differences in circHIPK3 and miR-182-5p expression. Overexpression of circHIPK3 ameliorated aortic valve lesions in a CAVD mouse model. In vitro experiments demonstrated that circHIPK3 inhibits the osteogenic response of aortic valve interstitial cells. Mechanistically, DEAD-box helicase 5 (DDX5) recruits methyltransferase 3 (METTL3) to promote the N6-methyladenosine (m6A) modification of circHIPK3. Furthermore, m6A-modified circHIPK3 increases the stability of Kremen1 (Krm1) mRNA, and Krm1 is a negative regulator of the Wnt/β-catenin pathway. Additionally, miR-182-5p suppresses the expression of Dickkopf2 (Dkk2), the ligand of Krm1, and attenuates the Krm1-mediated inhibition of Wnt signaling. Activation of the Wnt signaling pathway significantly contributes to the promotion of aortic valve calcification. Our study describes the role of the Krm1-Dkk2 axis in inhibiting Wnt signaling in aortic valves and suggests that noncoding RNAs are upstream regulators of this process.

Hair growth-promoting effects of Enz_MoriL on human dermal papilla cells through modulation of the Wnt/β-Catenin and JAK-STAT signaling pathways.

Enz_MoriL is a naturally occurring substance extracted from the leaves of Morus alba L. through enzymatic conversion. Historically, M. alba L. has been recognized for its potential to promote hair regrowth. However, the precise mechanism by which Enz_MoriL affects human hair follicle dermal papilla cells (hDPCs) remains unclear. The aim of this study was to investigate the molecular basis of Enz_MoriL's effect on hair growth in hDPCs. Interferon-gamma (IFN-γ) was used to examine the effects of Enz_MoriL on hDPCs during the anagen and catagen phases, as well as under conditions mimicking alopecia areata (AA). Enz_MoriL demonstrated the ability to promote cell proliferation in both anagen and catagen stages. It increased the levels of active β-catenin in the catagen stage induced by IFN-γ, leading to its nuclear translocation. This effect was achieved by increasing the phosphorylation of GSK3β and decreasing the expression of DKK-1. This stimulation induced proliferation in hDPCs and upregulated the expression of the Wnt family members 3a, 5a, and 7a at the transcript level. Additionally, Enz_MoriL suppressed JAK1 and STAT3 phosphorylation, contrasting with IFN-γ, which induced them in the catagen stage. In conclusion, Enz_MoriL directly induced signals for anagen re-entry into hDPCs by affecting the Wnt/β-catenin pathway and enhancing the production of growth factors. Furthermore, Enz_MoriL attenuated and reversed the interferon-induced AA-like environment by blocking the JAK-STAT pathway in hDPCs.

Wnt Signaling Inhibition Prevents Postnatal Inflammation and Disease Progression in Mouse Congenital Myxomatous Valve Disease.

Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome includes valve leaflet thickening and progressive valve degeneration. However, the underlying mechanisms by which the disease progresses remain undefined. Mice with Fibrillin 1 gene variant Fbn1C1039G/+ recapitulate histopathologic features of Marfan syndrome, and Wnt (Wingless-related integration site) signaling activity was detected in TCF/Lef-lacZ (T-cell factor/lymphoid enhancer factor-β-galactosidase) reporter mice. Single-cell RNA sequencing was performed from mitral valves of wild-type and Fbn1C1039G/+ mice at 1 month of age. Inhibition of Wnt signaling was achieved by conditional induction of the secreted Wnt inhibitor Dkk1 (Dickkopf-1) expression in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ; Fbn1C1039G/+ mice. Dietary doxycycline was administered for 1 month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for ECM (extracellular matrix) and immune cells were performed. Wnt signaling is activated early in mitral valve disease progression, before immune cell infiltration in Fbn1C1039G/+ mice. Single-cell transcriptomics revealed similar mitral valve cell heterogeneity between wild-type and Fbn1C1039G/+ mice at 1 month of age. Wnt pathway genes were predominantly expressed in valve interstitial cells and valve endothelial cells of Fbn1C1039G/+ mice. Inhibition of Wnt signaling in Fbn1C1039G/+ mice at 1 month of age prevented the initiation of MVD as indicated by improved ECM remodeling and reduced valve leaflet thickness with decreased infiltrating macrophages. However, later, Wnt inhibition starting at 2 months did not prevent the progression of MVD. Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation but is not responsible for later-stage valve disease progression once it has been initiated. Thus, Wnt signaling contributes to MVD progression in a time-dependent manner and provides a promising therapeutic target for the early treatment of congenital MVD in Marfan syndrome.

Inhibition of vascular calcification by Compound Danshen Dripping Pill through multiple mechanisms

BackgroundVascular calcification refers to the abnormal accumulation of calcium in the walls of blood vessels and is a risk factor often overlooked in cardiovascular disease. However, there is currently no specific drug for treating vascular calcification. Compound Danshen Dripping Pill (CDDP) is widely used to treat cardiovascular diseases, but its effect on vascular calcification has not been reported. PurposeWe investigated the effects of CDDP on vascular calcification in ApoE−/− mice and in vitro and elucidated its mechanism of action. Study designFirstly, we found that CDDP has the potential to improve calcification based on network pharmacology analysis. Then, we performed the following experiments: in vivo, ApoE−/− mice were fed a high-fat diet randomly supplemented with CDDP for 16 weeks. Atherosclerosis and vascular calcification were determined. In vitro, human aortic smooth muscle cells (HASMCs), human umbilical vein endothelial cells (HUVECs), and human aortic endothelial cells (HAECs) were used to determine the mechanisms for CDDP-inhibited vascular calcification. ResultsIn this study, we observed that CDDP reduced intimal calcification in atherosclerotic lesions of ApoE-deficient mice fed a high-fat diet, as well as the calcification in cultured SMCs and ECs. Mechanistically, CDDP inhibited the Wnt/β-catenin pathway by up-regulating the expression of DKK1 and LRP6, which are upstream inhibitors of Wnt, leading to a reduction in the expression of osteoblastic transition markers (ALP, OPN, BMP2, and RUNX2). Furthermore, CDDP enhanced the secretion of DKK1, which plays a role in mediating EC-SMC crosstalk in calcification. Additionally, VC contributes to vascular aging by inhibiting Sirt1 and increasing senescence parameters (SA-β-gal, p21, and p16). However, CDDP reversed these changes by activating Sirt1. CDDP also reduced the levels of pro-inflammatory cytokines and the senescence-associated secretory phenotype in vivo and in vitro. ConclusionsOur study suggests that CDDP reduces vascular calcification by regulating the DKK1/LRP6/β-catenin signaling pathway in ECs/SMCs and interactions with the crosstalk of ECs and SMCs. It also reduces the senescence of ECs/SMCs, contributing to the Sirt1 activation, indicating CDDP's novel role in ameliorating vascular calcification.