Canonical Wnt Pathway Research Articles

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Relationship of Dickkopf-1 With Atherosclerosis and Arterial Stiffness in Renal Transplant Recipients

IntroductionDickkopf wingless (Wnt) signaling pathway inhibitor-1 (DKK-1) is a potent antagonist of the WNT canonical signaling pathway. DKK-1 is a substance that exerts anabolic effects on bone and is also involved in vascular cell regulation. The study aimed to determine the relationship of DKK-1 with atherosclerosis as determined by carotid artery intima-media thickness (CA-IMT) and arterial stiffness (AS) as determined by brachial-ankle pulse wave velocity (baPWV) in renal transplant recipients (RTRs). MethodsA total of 62 (62%) male and 38 (438%) female RTRs with a mean age of 44.22 ± 10.88 years were included in the study. RTRs were compared with 65 healthy individuals. CA-IMT measurement with ultrasonography was used as a marker of atherosclerosis. The presence of AS was detected with the baPWV device. ResultsCreatinine, CA-IMT, and baPWV were higher in the RTRs compared to the healthy subjects. No difference was determined between the two groups regarding log10 DKK-1. No difference was noted in the levels of CA-IMT and baPWV in patients with log10 DKK-1 > 3.83 pg/mL compared to patients with ≤3.83 pg/mL. Correlation and multivariate analyses showed no correlation between log10 DKK-1 and CA-IMT and baPWV. DiscussionIn RTRs, an increased development of atherosclerosis and AS was observed compared to healthy individuals. There was no difference in DKK-1 between the groups based on improved renal function. DKK-1 was not correlated with atherosclerosis and AS.

Wnt5a negatively regulates melanogenesis in primary Arctic fox epidermal melanocytes

Melanocytes, which are mainly found in the epidermis, are responsible for the melanin of skin and hair, and thereby contribute to the appearance of skin and provide protection from damage by ultraviolet radiation. Our previous study revealed that the Wnt5a, one of the many genes that affect melanin production, might be involved in the coat color seasonal change of the Arctic fox by influencing skin melanogenesis. Although the role of Wnt5a in melanocyte lines and melanoma cells has been extensively studied, its role in primary epidermal melanocytes has not been explored. This study aimed to investigate the role and mechanism of the Wnt5a in influencing melanogenesis in Arctic fox primary epidermal melanocytes. We constructed the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout plasmid targeting exons of the Wnt5a and transfected it into primary epidermal melanocytes. The results of the amplification knockout region assay, RT-qPCR assay, and western blot assay showed the success of Wnt5a knockout. RT-qPCR assay and melanin content assay showed that melanin production in melanocytes was significantly increased after Wnt5a knockout, and melanin-related key genes, such as microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1, were significantly elevated. In addition, we also found that the expression of the β-catenin gene of the Wnt canonical pathway was significantly elevated after Wnt5a knockout. In conclusion, our results indicate that the Wnt5a plays a negative regulatory role in melanogenesis in primary epidermal melanocytes, and is presumably involved in antagonizing or inhibiting canonical Wnt signaling.

"Comparative study of adipose-derived stem cells from localized scleroderma patients and healthy donors in treating skin fibrosis".

Localized scleroderma (LoS) is an autoimmune disease characterized by fibrosis of the skin and atrophy of the subcutaneous fat tissue. Adipose-derived mesenchymal stem cells (ASCs) is a promising treatment approach for LoS. However, ASCs from scleroderma patients (LoS-ASCs) have been shown to exhibit altered characteristics compared to ASCs from healthy donors (healthy-ASCs). This study aimed to compare the abilities of LoS-ASCs and healthy-ASCs in treating skin fibrosis. The paracrine ability of ASCs was tested with cytokine array. Bleomycin-challenged mice models received subcutaneous injection of LoS-ASCs and healthy-ASCs. Pathological staining and western blotting of COL1, α-SMA was performed. Fibroblasts derived from LoS lesions (LoS-FB) was co-cultured with ASCs, and subjected to RNA sequencing to further explore the similarities and differences in the treatment mechanism. In vivo comparison revealed that healthy ASCs had a stronger proliferation ability and secreted higher levels of growth factors and cytokines, including VEGFA, PDGFB, and IL-10. Pathological staining of the skin in mice models treated with ASCs demonstrated that healthy ASCs were more effective in reducing dermal thickness and collagen deposition, while increasing microvessel density and the proportion of M2 macrophages. Co-culture with both healthy-ASCs and LoS-ASCs reduced the proliferation and migration abilities of LoS-FB, as well as the protein expression of α-SMA and COL1. RNA sequencing and validation revealed potential difference in the canonical Wnt pathway. Healthy-ASCs exhibited stronger proliferation, paracrine, anti-fibrosis, pro-angiogenesis, and immunomodulation abilities in treating skin fibrosis in scleroderma mouse models. Allogenic ASCs obtained from healthy donors are more efficient in treating skin fibrosis, and could serve as a potential alternative for patients who are not suitable candidates for liposuction surgery in the future.

Stimulating Wnt signaling reveals context-dependent genetic effects on gene regulation in primary human neural progenitors.

Gene regulatory effects have been difficult to detect at many non-coding loci associated with brain-related traits, likely because some genetic variants have distinct functions in specific contexts. To explore context-dependent gene regulation, we measured chromatin accessibility and gene expression after activation of the canonical Wnt pathway in primary human neural progenitors (n = 82 donors). We found that TCF/LEF motifs and brain-structure-associated and neuropsychiatric-disorder-associated variants were enriched within Wnt-responsive regulatory elements. Genetically influenced regulatory elements were enriched in genomic regions under positive selection along the human lineage. Wnt pathway stimulation increased detection of genetically influenced regulatory elements/genes by 66%/53% and enabled identification of 397 regulatory elements primed to regulate gene expression. Stimulation also increased identification of shared genetic effects on molecular and complex brain traits by up to 70%, suggesting that genetic variant function during neurodevelopmental patterning can lead to differences in adult brain and behavioral traits.

Role of Wnt5a in modulation of osteoporotic adipose-derived stem cells and osteogenesis.

Osteoporosis, a condition marked by the deterioration of bone microarchitecture and increased facture risk, arises from a disruption in bone metabolism, with osteoclasts surpassing osteoblasts in bone resorption versus formation. The Wnt signalling pathway, a key regulator of bone maintenance, remains partially understood in osteoporosis. Our research delves into the role of Wnt-related molecules in this disease. In osteoporotic adipose-derived stem cells (OP-ASCs), we detected a significant decrease in Ctnnb1 and Frizzled-6 (Fzd6), contrasted by an increase in Gsk-3β and Wnt5a. Activation of the Wnt pathway by LiCl resulted in elevated Ctnnb1 and Fzd6, but decreased Gsk-3β and Wnt5a levels, promoting OP-ASCs' bone-formation capacity. In contrast, inhibition of this pathway by DKK-1 led to diminished Ctnnb1 and Fzd6, and increased Gsk-3β and Wnt5a, adversely affecting osteogenesis. Furthermore, our findings show that overexpressing Wnt5a impedes, while silencing it enhances the bone-forming capability of OP-ASCs. In a cranial bone defect model, the implantation of Wnt5a-silenced OP-ASCs with biphasic calcium phosphate scaffolds significantly promoted new bone formation. These observations indicated a repression of the canonical Wnt pathway and a stimulation of the non-canonical pathway in OP-ASCs. Silencing Wnt5a increased the osteogenic and regenerative abilities of OP-ASCs. Our study suggests targeting Wnt5a could be a promising strategy for enhancing bone regeneration in post-menopausal osteoporosis.

The overexpression of R-spondin 3 affects hair morphogenesis and hair development along with the formation and maturation of the hair follicle stem cells.

Mice hair follicles (HFs) are a valuable model for studying various aspects of hair biology, including morphogenesis, development, and regeneration due to their easily observable phenotype and genetic manipulability. The initiation and progression of hair follicle morphogenesis, as well as the hair follicle cycle, are regulated by various signaling pathways, of which the main role is played by the Wingless-type MMTV integration site family (Wnt) and the Bone Morphogenic Protein (BMP). During the hair follicle cycle, the BMP pathway maintains hair follicle stem cells (HFSCs) in a dormant state while the Wnt pathway activates them for hair growth. Given the pivotal role of the Wnt pathway in hair biology and HFSCs regulation, we investigated the influence of the Wnt modulator - R-spondin 3 (Rspo3), in these processes. For this purpose, we developed a transgenic mice model with the overexpression of Rspo3 (Rspo3GOF) in the whole ectoderm and its derivatives, starting from early morphogenesis. Rspo3GOF mice exhibited a distinct phenotype with sparse hair and visible bald areas, caused by reduced proliferation and increased apoptosis of hair matrix progenitor cells, which resulted in a premature anagen-to-catagen transition with a shortened growth phase and decreased overall length of all hair types. In addition, Rspo3GOF promoted induction of auchene and awl, canonical Wnt-dependent hair type during morphogenesis, but the overall hair amount remained reduced. We also discovered a delay in the pre-bulge formation during morphogenesis and prolonged immaturity of the HFSC population in the bulge region postnatally, which further impaired proper hair regeneration throughout the mice's lifespan. Our data supported that Rspo3 function observed in our model works in HFSCs' formation of pre-bulge during morphogenesis via enhancing activation of the canonical Wnt pathway, whereas in contrast, in the postnatal immature bulge, activation of canonical Wnt signaling was attenuated. In vitro studies on keratinocytes revealed changes in proliferation, migration, and colony formation, highlighting the inhibitory effect of constitutive overexpression of Rspo3 on these cellular processes. Our research provides novel insights into the role of Rspo3 in the regulation of hair morphogenesis and development, along with the formation and maturation of the HFSCs, which affect hair regeneration.

Crosstalk among canonical Wnt and Hippo pathway members in skeletal muscle and at the neuromuscular junction.

Skeletal muscles are essential for locomotion, posture, and metabolic regulation. To understand physiological processes, exercise adaptation, and muscle-related disorders, it is critical to understand the molecular pathways that underlie skeletal muscle function. The process of muscle contraction, orchestrated by a complex interplay of molecular events, is at the core of skeletal muscle function. Muscle contraction is initiated by an action potential and neuromuscular transmission requiring a neuromuscular junction. Within muscle fibers, calcium ions play a critical role in mediating the interaction between actin and myosin filaments that generate force. Regulation of calcium release from the sarcoplasmic reticulum plays a key role in excitation-contraction coupling. The development and growth of skeletal muscle are regulated by a network of molecular pathways collectively known as myogenesis. Myogenic regulators coordinate the differentiation of myoblasts into mature muscle fibers. Signaling pathways regulate muscle protein synthesis and hypertrophy in response to mechanical stimuli and nutrient availability. Several muscle-related diseases, including congenital myasthenic disorders, sarcopenia, muscular dystrophies, and metabolic myopathies, are underpinned by dysregulated molecular pathways in skeletal muscle. Therapeutic interventions aimed at preserving muscle mass and function, enhancing regeneration, and improving metabolic health hold promise by targeting specific molecular pathways. Other molecular signaling pathways in skeletal muscle include the canonical Wnt signaling pathway, a critical regulator of myogenesis, muscle regeneration, and metabolic function, and the Hippo signaling pathway. In recent years, more details have been uncovered about the role of these two pathways during myogenesis and in developing and adult skeletal muscle fibers, and at the neuromuscular junction. In fact, research in the last few years now suggests that these two signaling pathways are interconnected and that they jointly control physiological and pathophysiological processes in muscle fibers. In this review, we will summarize and discuss the data on these two pathways, focusing on their concerted action next to their contribution to skeletal muscle biology. However, an in-depth discussion of the non-canonical Wnt pathway, the fibro/adipogenic precursors, or the mechanosensory aspects of these pathways is not the focus of this review.

Neuroprotective effect of PPAR gamma agonist in rat model of autism spectrum disorder: Role of Wnt/β-catenin pathway

BackgroundThe clinical manifestation of autism spectrum disorder (ASD) is linked to the disruption of fundamental neurodevelopmental pathways. Emerging evidences claim to have an upregulation of canonical Wnt/β-catenin pathway while downregulation of PPARγ pathway in ASD. This study aims to investigate the therapeutic potential of pioglitazone, a PPARγ agonist, in rat model of ASD. The study further explores the possible role of PPARγ and Wnt/β-catenin pathway and their interaction in ASD by using their modulators. Material and methodsPregnant female Wistar rats received 600 mg/kg of valproic acid (VPA) to induce autistic symptoms in pups. Pioglitazone (10 mg/kg) was used to evaluate neurobehaviors, relative mRNA expression of inflammatory (IL-1β, IL-6, IL-10, TNF-α), apoptotic markers (Bcl-2, Bax, & Caspase-3) and histopathology (H&E, Nissl stain, Immunohistochemistry). Effect of pioglitazone was evaluated on Wnt pathway and 4 μg/kg dose of 6-BIO (Wnt modulator) was used to study the PPARγ pathway. ResultsASD model was established in pups as indicated by core autistic symptoms, increased neuroinflammation, apoptosis and histopathological neurodegeneration in cerebellum, hippocampus and amygdala. Pioglitazone significantly attenuated these alterations in VPA-exposed rats. The expression study results indicated an increase in key transcription factor, β-catenin in VPA-rats suggesting an upregulation of canonical Wnt pathway in them. Pioglitazone significantly downregulated the Wnt signaling by suppressing the expression of Wnt signaling-associated proteins. The inhibiting effect of Wnt pathway on PPARγ activity was indicated by downregulation of PPARγ-associated protein in VPA-exposed rats and those administered with 6-BIO. ConclusionIn the present study, upregulation of canonical Wnt/β-catenin pathway was demonstrated in ASD rat model. Pioglitazone administration significantly ameliorated these symptoms potentially through its neuroprotective effect and its ability to downregulate the Wnt/β-catenin pathway. The antagonism between the PPARγ and Wnt pathway offers a promising therapeutic approach for addressing ASD.

Age- and Sex-Associated Wnt Signaling Dysregulation is Exacerbated from the Early Stages of Neuropathology in an Alzheimer’s Disease Model

Emerging studies suggest that Wnt signaling is dysregulated in the brains of AD patients, suggesting that this pathway may also contribute to disease progression. However, it remains to be determined whether alterations in the Wnt pathway are the cause or consequence of this disease and which elements of Wnt signaling mainly contribute to the appearance of AD histopathological markers early in disease compared to what occurs during normal aging. The present study aimed to describe the status of several canonical Wnt pathway components and the expression of the AD marker p-tau in the hippocampi of female and male 3xTg-AD mice during disease progression compared to those during normal aging. We analyzed the levels of the canonical Wnt components Wnt7a, Dkk-1, LRP6 and GSK3β as well as the levels of p-tau and BDNF at 3, 6, 9–12 and 18 months of age. We found a gradual increase in Dkk-1 levels during aging prior to Wnt7a and LRP5/6 depletion, which was strongly exacerbated in 3xTg-AD mice even at young ages and correlated with GSK3β activation and p-tau-S202/Thr205 expression. Dkk-1 upregulation, as well as the level of p-tau, was significantly greater in females than in males. Our results suggest that Dkk-1 upregulation is involved in the expression of several features of AD at early stages, which supports the possibility of positively modulating the canonical Wnt pathway as a therapeutic tool to delay this disease at early stages.

LIM kinase 2 activates cardiac fibroblasts and exacerbates postinfarction left ventricular remodeling via crosstalk between the canonical and non-canonical Wnt pathways

Ischemic heart failure rates rise despite decreased acute myocardial infarction (MI) mortality. Excessive myofibroblast activation post-MI leads to adverse remodeling. LIM kinases (LIMK1 and LIMK2) regulate cytoskeleton homeostasis and are pro-fibrotic markers in atrial fibrillation. However, their roles and mechanisms in postinfarction fibrosis and ventricular remodeling remain unclear. This study found that the expression of LIMKs elevated in the border zone (BZ) in mice MI models. LIMK1/2 double knockout (DKO) restrained pathological remodeling and reduced mortality by suppressing myofibroblast activation. By using adeno-associated virus (AAV) with a periostin promoter to overexpress LIMK1 or LIMK2, this study found that myofibroblast-specific LIMK2 overexpression diminished these effects in DKO mice, while LIMK1 did not. LIMK2 kinase activity was critical for myofibroblast proliferation by using AAV overexpressing mutant LIMK2 lack of kinase activity. According to phosphoproteome analysis, functional rescue experiments, co-immunoprecipitation, and protein-protein docking, LIMK2 led to the phosphorylation of β-catenin at Ser 552. LIMK2 nuclear translocation also played a role in myofibroblast proliferation after MI with the help of AAV overexpressing mutant LIMK2 without nuclear location signal. Chromatin immunoprecipitation sequencing identified that LIMK2 bound to Lrp6 promoter region in TGF-β treated cardiac fibroblasts, positively regulating Wnt signaling via Wnt receptor internalization. This study demonstrated that LIMK2 promoted myofibroblast proliferation and adverse cardiac remodeling after MI, by enhancing phospho-β-catenin (Ser552) and Lrp6 signaling. This suggested that LIMK2 could be a target for the treatment of postinfarction injury.

Prostate cancer–induced endothelial-cell-to-osteoblast transition drives immunosuppression in the bone–tumor microenvironment through Wnt pathway–induced M2 macrophage polarization

Immune checkpoint therapy has limited efficacy for patients with bone-metastatic castration-resistant prostate cancer (bmCRPC). To improve immunotherapy for bmCRPC, we aimed to identify the mechanism of bmCRPC-induced changes in the immune microenvironment. Among bmCRPC patients, higher levels of a 32-gene M2-like macrophage signature in bone metastasis samples correlated with shorter overall survival. Immunohistochemistry showed that CD206-positive (CD206+) macrophages were enriched in bmCRPC bone biopsy specimens compared with primary tumors or lymph node metastases. In preclinical osteogenic prostate cancer (Pca) xenograft models, CD206+ macrophages were recruited to areas with tumor-induced bone. RNA sequencing (RNAseq) analysis showed higher expression of an M2-like gene signature, with activated canonical and noncanonical Wnt pathways, in tumor-associated macrophages isolated from osteogenic tumors (bone-TAMs) than in TAMs isolated from nonosteogenic tumors (ctrl-TAMs). Mechanistic studies showed that endothelial cells (ECs) that had undergone EC-to-osteoblast (EC-to-OSB) transition, the precursors of tumor-induced OSBs, produced paracrine factors, including Wnts, CXCL14, and lysyl oxidase, which induced M2 polarization and recruited M2-like TAMs to the bone-tumor microenvironment (bone-TME). Bone-TAMs suppressed CD8+ T cells' proliferation and cytolytic activity, and these effects were partially reversed by treating bone-TAMs with Wnt inhibitors. Genetic or pharmacological inhibition of Pca-induced EC-to-OSB transition reduced the levels of M2-like macrophages in osteogenic tumors. Our study demonstrates that Pca-induced EC-to-OSB transition drives immunosuppression in the bone-TME, suggesting that therapies that reduce Pca-induced bone formation may improve immunotherapeutic outcomes for bmCRPC.

The Interplay among Wnt/β-catenin Family Members in Colorectal Adenomas and Surrounding Tissues.

The colorectal adenoma undergoes neoplastic progression via the normal epithelium-adenoma-adenocarcinoma sequence as reported in the Vogelgram. The hazard of developing a tumor is deeply associated with the number and size of adenomas and their subtype. Adenomatous polyps are histologically categorized as follows: approximately 80-90% are tubular, 5-15% are villous, and 5-10% are tubular/villous. Given the higher risk of a malignant transformation observed in tubular/villous adenomas, patients diagnosed with adenomatous polyposis are at an improved risk of developing CRC. The Wnt/β-catenin pathway plays a key role in the onset of colorectal adenoma; in particular, intestinal cells first acquire loss-of-function mutations in the APC gene that induce the formation of adenomas. Wnt/β-catenin pathway APC, Wnt3a, Wnt5a, LEF1, and BCL9 genes and protein expression analyses were conducted by qRT-PCR and western blot in 68 colonic samples (polyps and adjacent mucosa) from 41 patients, of which 17 were affected by FAP. Ten normal colonic mucosal samples were collected from 10 healthy donors. In this study, both the APC gene and protein were less expressed in the colon tumor compared to the adjacent colonic mucosa. Conversely, the activated β-catenin was more expressed in polyps than in the adjacent mucosa. All results confirmed the literature data on carcinomas. A statistically significant correlation between Wnt3a and BCL9 both in polyps and in the adjacent mucosa underlines that the canonical Wnt pathway is activated in early colon carcinogenesis and that the adjacent mucosa is already altered. This is the first study analyzing the difference in expression of the Wnt/β-catenin pathway in human colorectal adenomas. Understanding the progression from adenomas to colorectal carcinomas is essential for the development of new therapeutic strategies and improving clinical outcomes with the use of APC and β-catenin as biomarkers.

Atorvastatin exerts a preventive effect against steroid-induced necrosis of the femoral head by modulating Wnt5a release.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent form of osteonecrosis in young individuals. More efficacious clinical strategies must be used to prevent and treat this condition. One of the mechanisms through which SONFH operates is the disruption of normal differentiation in bone marrow adipocytes and osteoblasts due to prolonged and extensive use of glucocorticoids (GCs). In vitro, it was observed that atorvastatin (ATO) effectively suppressed the impact of dexamethasone (DEX) on bone marrow mesenchymal stem cells (BMSCs), specifically by augmenting their lipogenic differentiation while impeding their osteogenic differentiation. To investigate the underlying mechanisms further, we conducted transcriptome sequencing of BMSCs subjected to different treatments, leading to the identification of Wnt5a as a crucial gene regulated by ATO. The analyses showed that ATO exhibited the ability to enhance the expression of Wnt5a and modulate the MAPK pathway while regulating the Wnt canonical signaling pathway via the WNT5A/LRP5 pathway. Our experimental findings provide further evidence that the combined treatment of ATO and DEX effectively mitigates the effects of DEX, resulting in the upregulation of osteogenic genes (Runx2, Alpl, Tnfrsf11b, Ctnnb1, Col1a) and the downregulation of adipogenic genes (Pparg, Cebpb, Lpl), meanwhile leading to the upregulation of Wnt5a expression. So, this study offers valuable insights into the potential mechanism by which ATO can be utilized in the prevention of SONFH, thereby holding significant implications for the prevention and treatment of SONFH in clinical settings.

Integrin-linked kinase-frizzled 7 interaction maintains cancer stem cells to drive platinum resistance in ovarian cancer

BackgroundPlatinum-based chemotherapy regimens are a mainstay in the management of ovarian cancer (OC), but emergence of chemoresistance poses a significant clinical challenge. The persistence of ovarian cancer stem cells (OCSCs) at the end of primary treatment contributes to disease recurrence. Here, we hypothesized that the extracellular matrix protects CSCs during chemotherapy and supports their tumorigenic functions by activating integrin-linked kinase (ILK), a key enzyme in drug resistance.MethodsTCGA datasets and OC models were investigated using an integrated proteomic and gene expression analysis and examined ILK for correlations with chemoresistance pathways and clinical outcomes. Canonical Wnt pathway components, pro-survival signaling, and stemness were examined using OC models. To investigate the role of ILK in the OCSC-phenotype, a novel pharmacological inhibitor of ILK in combination with carboplatin was utilized in vitro and in vivo OC models.ResultsIn response to increased fibronectin secretion and integrin β1 clustering, aberrant ILK activation supported the OCSC phenotype, contributing to OC spheroid proliferation and reduced response to platinum treatment. Complexes formed by ILK with the Wnt receptor frizzled 7 (Fzd7) were detected in tumors and correlated with metastatic progression. Moreover, TCGA datasets confirmed that combined expression of ILK and Fzd7 in high grade serous ovarian tumors is correlated with reduced response to chemotherapy and poor patient outcomes. Mechanistically, interaction of ILK with Fzd7 increased the response to Wnt ligands, thereby amplifying the stemness-associated Wnt/β-catenin signaling. Notably, preclinical studies showed that the novel ILK inhibitor compound 22 (cpd-22) alone disrupted ILK interaction with Fzd7 and CSC proliferation as spheroids. Furthermore, when combined with carboplatin, this disruption led to sustained AKT inhibition, apoptotic damage in OCSCs and reduced tumorigenicity in mice.ConclusionsThis “outside-in” signaling mechanism is potentially actionable, and combined targeting of ILK-Fzd7 may lead to new therapeutic approaches to eradicate OCSCs and improve patient outcomes.

Aerobic exercise training engages the canonical wnt pathway to improve pulmonary function and inflammation in COPD

BackgroundWe studied whether the exercise improves cigarette smoke (CS) induced chronic obstructive pulmonary disease (COPD) in mice through inhibition of inflammation mediated by Wnt/β-catenin-peroxisome proliferator-activated receptor (PPAR) γ signaling.MethodsFirstly, we observed the effect of exercise on pulmonary inflammation, lung function, and Wnt/β-catenin-PPARγ. A total of 30 male C57BL/6J mice were divided into the control group (CG), smoke group (SG), low-intensity exercise group (LEG), moderate-intensity exercise group (MEG), and high-intensity exercise group (HEG). All the groups, except for CG, underwent whole-body progressive exposure to CS for 25 weeks. Then, we assessed the maximal exercise capacity of mice from the LEG, MEG, and HEG, and performed an 8-week treadmill exercise intervention. Then, we used LiCl (Wnt/β-catenin agonist) and XAV939 (Wnt/β-catenin antagonist) to investigate whether Wnt/β-catenin-PPARγ pathway played a role in the improvement of COPD via exercise. Male C57BL/6J mice were randomly divided into six groups (n = 6 per group): CG, SG, LiCl group, LiCl and exercise group, XAV939 group, and XAV939 and exercise group. Mice except those in the CG were exposed to CS, and those in the exercise groups were subjected to moderate-intensity exercise training. All the mice were subjected to lung function test, lung histological assessment, and analysis of inflammatory markers in the bronchoalveolar lavage fluid, as well as detection of Wnt1, β-catenin and PPARγ proteins in the lung tissue.ResultsExercise of various intensities alleviated lung structural changes, pulmonary function and inflammation in COPD, with moderate-intensity exercise exhibiting significant and comprehensive effects on the alleviation of pulmonary inflammation and improvement of lung function. Low-, moderate-, and high-intensity exercise decreased β-catenin levels and increased those of PPARγ significantly, and only moderate-intensity exercise reduced the level of Wnt1 protein. Moderate-intensity exercise relieved the inflammation aggravated by Wnt agonist. Wnt antagonist combined with moderate-intensity exercise increased the levels of PPARγ, which may explain the highest improvement of pulmonary function observed in this group.ConclusionsExercise effectively decreases COPD pulmonary inflammation and improves pulmonary function. The beneficial role of exercise may be exerted through Wnt/β-catenin-PPARγ pathway.

Rolipram impacts on redox homeostasis and cellular signaling in an experimental model of abdominal aortic aneurysm

IntroductionCyclic nucleotide phosphodiesterases (PDEs) of the PDE4 subfamily are responsible for the hydrolysis and subcellular compartmentalization of cAMP, a second messenger that modulates vascular functionality. We had shown that PDE4B is induced in abdominal aortic aneurysms (AAA) and that PDE4 inhibition by rolipram limits experimental aneurysms. In this study we have delved into the mechanisms underlying the beneficial effect of rolipram on AAA. MethodsAAA were induced in ApoE−/− mice by angiotensin II (Ang II) infusion. Aneurysm formation was evaluated by ultrasonography. The expression of enzymes involved in redox homeostasis was analyzed by real-time RT-PCR and the activation of signaling pathways by Western blot. ResultsInduction of PDE4B in human AAA has been confirmed in a second cohort of patients. In Ang II-infused ApoE−/− mice, rolipram increased the percentage of animals free of aneurysms without affecting the percentage of aortic ruptures. Quantitative analyses determined that this drug significantly attenuated aortic collagen deposition. Additionally, rolipram reduced the increased Nox2 expression triggered by Ang II, exacerbated Sod1 induction, and normalized Sod3 expression. Likewise, PDE4 inhibition decreased the activation of both ERK1/2 and the canonical Wnt pathway, while AKT activity was not altered. ConclusionsThe inhibition of PDE4 activity modulates the expression of enzymes involved in redox homeostasis and affects cell signaling pathways involved in the development of AAA.

The contribution of the WNT pathway to the therapeutic effects of montelukast in experimental murine airway inflammation induced by ovalbumin and lipopolysaccharide.

The wingless/integrase-1 (WNT) pathway involved in the pathogenesis of inflammatory airway diseases has recently generated considerable research interest. Montelukast, a leukotriene receptor antagonist, provides therapeutic benefits in allergic asthma involving eosinophils. We aimed to investigate the role of the WNT pathway in the therapeutic actions of montelukast (MT) in a mixed type of allergic-acute airway inflammation model induced by ovalbumin (OVA) and lipopolysaccharide (LPS) in mice. Female mice were sensitized with intraperitoneal OVA-Al(OH)3 administration in the initiation phase and intranasal OVA followed by LPS administration in the challenge phase. The mice were divided into eight groups: control, asthmatic, and control/asthmatic treated with XAV939 (inhibitor of the canonical WNT pathway), LGK-974 (inhibitor of the secretion of WNT ligands), or MT at different doses. The inhibition of the WNT pathway prevented tracheal 5-HT and bradykinin hyperreactivity, while only the inhibition of the canonical WNT pathway partially reduced 5-HT and bradykinin contractions compared to the inflammation group. Therefore, MT treatment hindered 5-HT and bradykinin hyperreactivity associated with airway inflammation. Furthermore, MT prevented the increases in the phosphorylated GSK-3β and WNT5A levels, which had been induced by airway inflammation, in a dose-dependent manner. Conversely, the MT application caused a further increase in the fibronectin levels, while there was no significant alteration in the phosphorylation of the Smad-2 levels in the isolated lungs of the mice. The MT treatment reversed the increase in the mRNA expression levels of interleukin-17A. An increase in eosinophil and neutrophil counts was observed in bronchoalveolar lavage fluid samples obtained from the mice in the inflammation group, which was hampered by the MT treatment. The inhibition of the WNT pathway did not alter inflammatory cytokine expression or cell infiltration. The WNT pathway mediated the therapeutic effects of MT due to the inhibition of GSK-3β phosphorylation as well as the reduction of WNT5A levels in a murine airway inflammation model.

Abstract 5793: Functional DKK1 antibodies binding to cysteine-rich domain 1 or 2 of hDKK1 demonstrate antagonistic activities on tumor suppression or TCF/LEF signaling

Abstract Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of the β-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. DKK1 has been shown to be a potent inhibitor of Wnt signaling via competing Wnt binding to LRP5/6. DKK1 is tumorigenic in multiple cancer types and also immunosuppressive via MDSCs and NK cells. Emerging evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. With Twist’s precision DNA writing technologies, we have created phage display libraries with diversity greater than 1 × 1010, and utilized machine learning model for optimal discovery. In this study, we discovered anti-DKK1 antibodies bind to different cysteine-rich domain (CRD) of hDKK1 by epitope binning and mapping, and block the binding of DKK1 to the receptor. Binding of the antibodies to different CRDs of hDKK1 lead to different activation effect. The in vitro functional assays showed that the interaction of Wnt to its receptor was restored in the presence of anti-DKK1 antibodies binding to DKK1 CRD2, resulting in TCF/LEF signaling upregulation. Moreover, anti-DKK1 antibodies binding to DKK1 CRD1 induced immune cell activation and led to tumor cell cytotoxicity. In vivo study indicated anti-DKK1 antibody lead is potent in tumor regression. Our anti-DKK1 lead shows promising efficacy in cancer immunotherapy without influencing Wnt canonical pathway. Citation Format: Linya Wang, Zhen Han, Marisa Yang, Ana G. Lujan Hernandez, Vishwas Prabhu, Cameron F. Hu, Tammy Htoy, Mouna Villalta, Tom Z. Yuan, Hoa Giang, Aaron Sato. Functional DKK1 antibodies binding to cysteine-rich domain 1 or 2 of hDKK1 demonstrate antagonistic activities on tumor suppression or TCF/LEF signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5793.

Canonical Wnt activator Chir99021 prevents epileptogenesis in the intrahippocampal kainate mouse model of temporal lobe epilepsy

The Wnt signaling pathway mediates the development of dentate granule cell neurons in the hippocampus. These neurons are central to the development of temporal lobe epilepsy and undergo structural and physiological remodeling during epileptogenesis, which results in the formation of epileptic circuits. The pathways responsible for granule cell remodeling during epileptogenesis have yet to be well defined, and represent therapeutic targets for the prevention of epilepsy. The current study explores Wnt signaling during epileptogenesis and for the first time describes the effect of Wnt activation using Wnt activator Chir99021 as a novel anti-epileptogenic therapeutic approach.Focal mesial temporal lobe epilepsy was induced by intrahippocampal kainate (IHK) injection in wild-type and POMC-eGFP transgenic mice. Wnt activator Chir99021 was administered daily, beginning 3 h after seizure induction, and continued up to 21-days. Immature granule cell morphology was quantified in the ipsilateral epileptogenic zone and the contralateral peri-ictal zone 14 days after IHK, targeting the end of the latent period. Bilateral hippocampal electrocorticographic recordings were performed for 28-days, 7-days beyond treatment cessation. Hippocampal behavioral tests were performed after completion of Chir99021 treatment.Consistent with previous studies, IHK resulted in the development of epilepsy after a 14 day latent period in this well-described mouse model. Activation of the canonical Wnt pathway with Chir99021 significantly reduced bilateral hippocampal seizure number and duration. Critically, this effect was retained after treatment cessation, suggesting a durable antiepileptogenic change in epileptic circuitry. Morphological analyses demonstrated that Wnt activation prevented pathological remodeling of the primary dendrite in both the epileptogenic zone and peri-ictal zone, changes in which may serve as a biomarker of epileptogenesis and anti-epileptogenic treatment response in pre-clinical studies. These findings were associated with improved object location memory with Chir99021 treatment after IHK.This study provides novel evidence that canonical Wnt activation prevents epileptogenesis in the IHK mouse model of mesial temporal lobe epilepsy, preventing pathological remodeling of dentate granule cells. Wnt signaling may therefore play a key role in mesial temporal lobe epileptogenesis, and Wnt modulation may represent a novel therapeutic strategy in the prevention of epilepsy.