Wnt Expression Research Articles

Assessment of single and combined administration of ubiquinone and lactoferrin on histopathology, ultrastructure, oxidative stress, and WNT4 expression gene induced by thioacetamide on hepatorenal system of adult male rats

BackgroundHepatorenal syndrome is a life-threatening medical complication of liver cirrhosis. Hepatic cirrhosis is commonly accompanied by rapid failure of renal functions. Thioacetamide (TAA) is a potent hepatotoxin and a class 2-type carcinogen. Ubiquinone (Coq10) and lactoferrin (LF) are potent antioxidants with antifibrotic and antiinflammatory effects. However, whether Coq10 and LF reduce the hepatorenal injury induced by TAA remains unclear. Here, we investigated the potential protective effect of both/or Coq10 and LF in ameliorating TAA-induced hepatorenal injury and the role of WNT4 gene expression in detecting TAA-induced renal injury in rats. Seventy healthy and mature male Sprague Dawley rats, weighting (200 g ± 20 g) and aging (4–6) weeks were randomly divided into seven groups (n = 10): control, Coq10, LF, TAA, TAA + Coq10, TAA + LF, and TAA + Coq10 + LF. The hepatorenal injury was induced through intraperitoneal (i.p.) injection of TAA (150 mg/kg/twice/weekly) for nine weeks. Coq10 (10 mg/kg/day) and LF (200 mg/kg/day) were orally administered for nine weeks.ResultsTAA induced marked hepatorenal damage, evident by the significant increase in the alanine aminotransferase (ALT), aspartate transaminase (AST), serum creatinine (SCr) activities, and the blood urea nitrogen (BUN) level. Besides, the significant increases in concentrations of malondialdehyde (MDA) and nitric oxide (NOx) together with significant decreases in the activities of catalase (CAT) and superoxide dismutase (SOD). The histopathological analysis of the TAA group showed obvious fibrosis, steatosis, and inflammation of the hepatic parenchyma as well as severe glomerular and tubular damage of the renal parenchyma. In addition, TAA induced marked ultrastructural alterations and up-regulation in the expression of the WNT4 gene in the kidney. Meanwhile, the biochemical, histopathological, and ultrastructural alterations were significantly decreased with significant down-regulation in the expression of WNT4 in the groups exposed to TAA and treated with Coq10 and LF.ConclusionOur data suggested that Coq10 and LF could have protective effects on TAA hepatorenal damage, through improving the hepatic and renal functions, reduction of oxidative stress, structural and ultrastructural alterations, besides down-regulation in the expression of WNT4.

Diversity Outbred Mouse Model Recapitulates Human Heterogeneity in the Development of Western Diet-Induced Complications

Background: Diet-induced obesity (DIO) increases risk for cardiovascular disease largely due to compromised gut barrier integrity and systemic inflammation. Animal models for understanding the pathophysiology of complications associated with DIO do not take address the genetic factors, which determine the individual variation in the susceptibility to develop complications. Hypothesis: DO mice recapitulate the heterogeneity of western diet-induced metabolic syndrome, gut barrier disruption and systemic inflammation. Methods: DO mice were fed with either control (CD) or western diet (WD) (Teklad) (n = 10) for 16 weeks. Blood glucose levels, glucose tolerance and the intestinal permeability were determined. Inflammatory cells in the bone marrow or blood were characterized in basal conditions or in response to hindlimb ischemia (HLI). Blood flow recovery following HLI was measured by using Laser Doppler Imaging system and the intestinal-stem-cells (ISCs) were characterized by using markers, Lgr5, Olfm4 and Wnt3. Results: Wide variation in the weight gain, hyperglycemia and glucose tolerance in DO mice was observed following WD (n = 10). Similarly, wide variation was observed in Wnt3 expression and in the density of ISCs in colon of DO mice on WD. Heterogeneity in the ISC layer correlated with the variation in the intestinal permeability induced by WD compared to the CD ( p < 0.05, n = 10). The number of inflammatory cells (monocytes – CD45+Ly6G−Ly6C+CD115+, neutrophils - CD45+Ly6G+Ly6C+CD115−, pro-inflammatory macrophages - CD45+CD11b+Ly6G−Ly6C+F4/80+CCR2+CD80+ and anti-inflammatory macrophages - CD45+CD11b+Ly6G−Ly6C+F4/80+CX3CR1+CD206+) in the BM or blood showed heterogeneity in WD-DO mice in accordance with variability in the glucose tolerance. Accordingly, blood flow recovery following HLI showed wide variation with three mice on WD showing full recovery, which correlated with variability in the mobilization of inflammatory cells (n = 9). Conclusion: DO mouse model depicts human diversity in response to WD-induced changes in gut barrier integrity, inflammation and ischemic vascular repair therefore serves as a suitable model for evaluating pharmacological agents that would be effective in a diverse population. Jackson Laboratory Diversity Outbred Pilot Award National Institute of Aging (AG056881). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Establishment of Anoxia/cytokine-induced Injury and Regeneration in Colonoids

Intestinal epithelial cells (IECs) play key roles in nutrient absorption and fluid secretion, providing a barrier to protect cells. IECs, derived from intestinal stem cells (ISCs), are crucial in maintaining intestinal homeostasis and regeneration, which is crucial in gastrointestinal diseases such as inflammatory bowel disease (IBD). To study epithelial injury/regeneration, primary cultured colonoids were utilized as a model system to mimic injury. They retain the composition and differentiation of the tissue, making it a suitable model for studying homeostasis and regeneration of ISCs in vitro. Anoxia and proinflammatory cytokines frequently coexist and exacerbate one another during intestinal injury. However, anoxic conditions have been mainly ignored in previous research. Thus, we developed a regeneration model using cytokines/anoxia injury to better recapitulate intestinal inflammation. Colonoids were treated with cytokines TNF-α and IFN-γ (5 ng/μL) in anoxic conditions for 24 hours to provoke an injury. After 24 hours of injury, the colonoids were exposed to normal oxygen and media conditions for regeneration. Regeneration was observed for 24, 48, and 72 hours and documented using photomicrographs. Morphological analysis detailed that injury was most severe 24 hours post-injury with the combined induction of cytokines and anoxia resulting in decreased colonoid area and number of buds compared to control groups. RT-PCR analysis detailed mRNA expression of stem cell markers ( Lgr5, Bmi1, and Hopx), Wnt ( Axin2, Myc2, and Ascl2), and YAP target genes ( Sca1 and Ctgf) were increased at peak injury and returned to pre-injury levels at 72 hours of recovery. Regeneration of the colonoids is consistent with in vivo observations, thus confirming the validity of the model. This versatile model can be used to study responses to injury and regeneration of intestinal epithelium and the function of ISCs with applications in intestinal disease therapeutics. University of Maryland College of Agriculture and Natural Resources and the Maryland Agricultural Experiment Station Undergraduate Research Grant Grant #4307572 from the Maryland Stem Cell Research Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TM7SF2-induced lipid reprogramming promotes cell proliferation and migration via CPT1A/Wnt/β-Catenin axis in cervical cancer cells

Cervical cancer poses a serious threat to women’s health globally. Our previous studies found that upregulation of TM7SF2, which works as an enzyme involved in the process of cholesterol biosynthesis expression, was highly correlated with cervical cancer. However, the mechanistic basis of TM7SF2 promoting cervical cancer progression via lipid metabolism remains poorly understood. Therefore, quantification of fatty acids and lipid droplets were performed in vitro and in vivo. The protein-protein interaction was verified by Co-IP technique. The mechanism and underlying signaling pathway of TM7SF2 via CPT1A associated lipid metabolism in cervical cancer development were explored using Western blotting, IHC, colony formation, transwell assay, and wound healing assay. This study reported that overexpression of TM7SF2 increased fatty acids content and lipid droplets both in vivo and in vitro experiments. While knockout of TM7SF2 obviously attenuated this process. Moreover, TM7SF2 directly bonded with CPT1A, a key enzyme in fatty acid oxidation, and regulated CPT1A protein expression in cervical cancer cells. Notably, the proliferation and metastasis of cervical cancer cells were elevated when their CPT1A expression was upregulated. Then, rescue assay identified that CPT1A overexpressed could enhance the cell viability and migration in TM7SF2-knockout cells. Furthermore, depletion of TM7SF2 significantly inhibited WNT and β-catenin proteins expression, which was enhanced by CPT1A-overexpressed. The proliferation and migration of cervical cancer cells were reversed in CPT1A-overexpressed cells with the treatment of MSAB, an inhibitor of Wnt/β-Catenin pathway. This study put forward an idea that TM7SF2-induced lipid reprogramming promotes proliferation and migration via CPT1A/Wnt/β-Catenin axis in cervical cancer, underlying the progression of cervical cancer.

Fusobacterium periodonticum BCT protein targeting glucose metabolism to promote the epithelial-mesenchymal transition of esophageal cancer cells by lactic acid

BackgroundThe cancer microbiota was considered the main risk factor for cancer progression. We had proved that Fusobacterium periodonticum (F.p) was higher abundance in Esophageal cancer(EC)tissues. Bioinformation analysis found that BCT was a key virulence protein of F.p. However, little is known about the role and mechanism of BCT in EC. This study aimed to recognize the key virulence protein of F.p and explore the mechanism of BCT in promoting EC.MethodsWe constructed a eukaryotic expression vector and purified the recombinant protein BCT. CCK8 used to analyzed the activity of EC after treated by different concentration of BCT. UPLC-MS/MS and ELISA used to detect the metabonomics and metabolites. The ability of migration and invasion was completed by transwell assay. RT-QPCR, WB used to analyze the expression of relevant genes.ResultsOur data showed that BCT was higher expression in EC tumor tissues (p < 0.05) and BCT in 20 µg/mL promoted the survival, invasion and migration of EC cells (EC109) (p < 0.05). Meanwhile, UPLC-MS/MS results suggested that BCT resulted in an augmentation of hypotaurine metabolism, arachidonic acid metabolism, glycolysis/gluconeogenesis, tryptophan metabolism, citrate cycle activity in EC109. The metabolic changes resulted in decreasing in glucose and pyruvate levels but increase in lactate dehydrogenase (LDH) activity and lactic acid (LA) as well as the expression of glucose transporter 1, Hexokinase 2, LDH which regulated the glycolysis were all changed (p < 0.05). The BCT treatment upregulated the expression of TLR4, Akt, HIF-1α (p < 0.05) which regulated the production of LA. Furthermore, LA stimulation promoted the expression of GPR81, Wnt, and β-catenin (p < 0.05), thereby inducing EMT and metastasis in EC109 cells.ConclusionAltogether, these findings identified that impact of BCT in regulation of glycolysis in EC109 and its involves the TLR4/Akt/HIF-1α pathway. Meanwhile, glycolysis increasing the release of LA and promote the EMT of EC109 by GPR81/Wnt/β-catenin signaling pathway. In summary, our findings underscore the potential of targeting BCT as an innovative strategy to mitigate the development of EC.

P148 CCN3-derived peptides as anti-fibrotic therapies for systemic sclerosis

Abstract Background/Aims Effective therapy for skin fibrosis in scleroderma (systemic sclerosis: SSc) remains an unmet clinical challenge. Persistently activated M2 macrophages are believed to stimulate myofibroblasts in the disease microenvironment creating a pro-fibrotic niche in the skin. Expression of the anti-fibrotic matricellular protein CCN3 is reduced in animal models of fibrosis. We have identified a small peptide based on an amino acid sequence in CCN3, BLR-200, which mimics the anti-fibrotic activity of CCN3, and propose that BLR-200 could be used to treat patients in whom lack of CCN3 activity is permissive for fibrosis. However, whether there is lack of CCN3 activity in SSc and whether BLR-200 has effective anti-fibrotic activity in SSc are unknown. Methods We used ELISAs (R&amp;D Systems) to detect CCN3 and the M2 macrophage marker CD206 in the serum of scleroderma patients with early stage diffuse subset disease (within two years of disease onset), those with late-stage diffuse subset disease (over five years duration) as well as healthy controls (all n = 20). The bleomycin-induced mouse model of skin scleroderma, in which bleomycin is injected subcutaneously every day for 21 days, was used to assess the antifibrotic ability of BLR200 in vivo. Results In scleroderma serum, CCN3 was significantly reduced, relative to matched healthy controls and patients with inactive disease, in early onset scleroderma patients with active disease that show elevated CD206 levels (p &amp;lt; 0.01). In the mouse model of human SSc, compared to injection with a scrambled control peptide and relative to control mice treated with phosphate buffered saline, injection with BLR-200 prevented bleomycin-induced changes in collagen deposition, myofibroblast differentiation and skin thickness as measured by Trichrome stain, indirect immunofluorescence analysis with an anti-a-smooth muscle actin antibody, and morphometric analysis, respectively (N = 8 mice per group, p &amp;lt; 0.05). Similarly, RNA sequencing and real-time polymerase chain reaction analysis revealed that BLR-200 significantly impaired the ability of bleomycin to induce expression of fibrogenic genes such as: integrin alpha 11, CCN2, CCN1, Smad3, wnt4, YAP1 and tenascin-C (all N = 5, p &amp;lt; 0.05). Spatial transcriptomics analysis revealed that BLR-200 impaired bleomycin-induced alterations in skin cell populations, including the activation and expansion of epithelial and reticular fibroblast niches and the induction of Wnt, hippo, focal adhesion and actin cytoskeleton gene expression cluster, all of which are known to be activators of fibrosis and myofibroblast activation and persistence. Conclusion CCN3 expression is reduced in serum of scleroderma patients with active skin disease that show M2 macrophage activation as visualized by elevated CD206 levels. Since BLR-200 has antifibrotic activity in the bleomycin model of skin scleroderma, our data are consistent with the hypothesis that BLR-200 could be used to block fibrosis progression in early onset diffuse scleroderma patients who possess both low CCN3 and high CD206 in serum. Disclosure J. Nguyen: None. K. Zestranjyan: None. S. Xu: None. B.L. Riser: Corporate appointments; CEO of BLR Bio. R.J. Stratton: None. A. Leask: None.

Salidroside prevents gestational hypertension-induced impairment of offspring learning and memory via Wnt/Skp2 pathway

BackgroundSalidroside (Sal) has been found to protect against multiple impairments caused by diabetes, and we designed this study to investigate the effect of Sal on gestational hypertension (GHP)-induced impairment of offspring learning and memory. MethodsWe established a GHP rat model by intraperitoneal injection of NG-nitro-L-arginine methyl ester (L-NAME), and treated with Sal by daily gavage. We used Morris Water Maze test to evaluate the learning and memory ability of offspring rats. HE staining was used to measured the pathological changes in hippocampus of offspring. Immunohistochemistry, cellular immunofluorescence and western blot were used to detect the protein expression. ResultsThe learning and memory abilities of GHP offspring rats were significantly lower than those of normal rat offspring, while Sal treatment could significantly improve the learning and memory abilities of GHP offspring rats. HE staining did not reveal pathological differences in the hippocampus of normal rats, GHP rats and Sal-treated GHP offspring rats. However, Sal treatment can significantly increase the expression of Wnt1 and Skp2 protein, and decrease the expression of P27kiwf and P21waf1 protein in the hippocampus of GHP offspring rats. In vitro, Sal significantly promoted the proliferation and differentiation on neural stem cell, while Wnt1 knockdown could reverse these promotions by Sal. In the hippocampus of GHP offspring rats, Sal treatment significantly increased the expression of Tuj1, SOX2, Ki67 and DCX protein. ConclusionSalidroside significantly improves the learning and memory impairment of offspring caused by GHP, and its mechanism may be related to the fact that Salidroside promotes the proliferation and differentiation of neural stem cells by activating the Wnt1/Skp2 signaling pathway.

Preparation of Puerarin Long Circulating Liposomes and its Effect on Osteoporosis in Castrated Rats

Osteoporosis is a disease that causes low bone mass and deterioration of bone microarchitecture. Puerarin is a natural isoflavone compound that has been shown to possess anti-inflammatory, antioxidant and ameliorative effects on osteoporosis with less adverse reactions. However, its fast metabolism and low oral bioavailability limit its application. This study aimed to prepare d-α-tocopherol polyethylene glycol 1000 succinate (TPGS)- modified Puerarin Long Circulating Liposomes (TPGS-Puerarin-liposomes), in order to improve the oral bioavailability of puerarin, before evaluation of its pharmacological activity in vitro and in vivo. We employed film dispersion method to develop TPGS-Puerarin-liposomes before appropriate characterizations. Afterwards, we utilized in vivo imaging, pharmacokinetic analysis and in vitro drug release testing to further evaluate the in vivo and in vitro delivery efficiency. In addition, we established a castrated osteoporosis rat model to observe the changes in femur tissue structure and bone micromorphology via hematoxylin-eosin (HE) staining and Micro Computed Tomography (Micro CT). Besides, levels of oxidative stress and inflammatory indicators, as well as expression of wnt/β-catenin pathway-related proteins were detected. In terms of physiochemical properties, the respective mean particle size (PS) and zeta potential (ZP) of TPGS-Puerarin-liposomes were 76.63±0.59 nm and -25.54±0.11 mV. The liposomal formulation exhibited encapsulation efficiency (EE) of 95.08±0.25% and drug loading (DL) of 7.84±0.07%, along with excellent storage stability. Compared with free drugs, the TPGS-Puerarin-liposomes demonstrated a sustained release effect and could increase blood concentration of puerarin in rats, thereby significantly improving its bioavailability. Also, in vivo studies have confirmed potential of the liposomes to promote bone tissue targeting and accumulation of puerarin, coupled with significant improvement of the osteoporotic status. Besides, the liposomes could also reduce levels of oxidative stress and inflammatory factors in serum and bone tissue. Additionally, we discovered that TPGS-Puerarin-liposomes increased Wnt, β-catenin and T-cell factor (TCF) expressions at protein level in the wnt/β-catenin signaling pathway. This study has demonstrated the potential of TPGS-Puerarin-liposomes for treatment of osteoporosis.

The extracellular matrix protein fibronectin promotes metanephric kidney development

Complex interactions of the branching ureteric bud (UB) and surrounding mesenchymal cells during metanephric kidney development determine the final number of nephrons. Impaired nephron endowment predisposes to arterial hypertension and chronic kidney disease. In the kidney, extracellular matrix (ECM) proteins are usually regarded as acellular scaffolds or as the common histological end-point of chronic kidney diseases. Since only little is known about their physiological role in kidney development, we aimed for analyzing the expression and role of fibronectin. In mouse, fibronectin was expressed during all stages of kidney development with significant changes over time. At embryonic day (E) 12.5 and E13.5, fibronectin lined the UB epithelium, which became less pronounced at E16.5 and then switched to a glomerular expression in the postnatal and adult kidneys. Similar results were obtained in human kidneys. Deletion of fibronectin at E13.5 in cultured metanephric mouse kidneys resulted in reduced kidney sizes and impaired glomerulogenesis following reduced cell proliferation and branching of the UB epithelium. Fibronectin colocalized with alpha 8 integrin and fibronectin loss caused a reduction in alpha 8 integrin expression, release of glial-derived neurotrophic factor and expression of Wnt11, both of which are promoters of UB branching. In conclusion, the ECM protein fibronectin acts as a regulator of kidney development and is a determinant of the final nephron number.

Wnt16 Increases Bone-to-Implant Contact in an Osteopenic Rat Model by Increasing Proliferation and Regulating the Differentiation of Bone Marrow Stromal Cells

Osseointegration is a complex biological cascade that regulates bone regeneration after implant placement. Implants possessing complex multiscale surface topographies augment this regenerative process through the regulation of bone marrow stromal cells (MSCs) that are in contact with the implant surface. One pathway regulating osteoblastic differentiation is Wnt signaling, and upregulation of non-canonical Wnts increases differentiation of MSCs on these titanium substrates. Wnt16 is a non-canonical Wnt shown to regulate bone morphology in mouse models. This study evaluated the role of Wnt16 during surface-mediated osteoblastic differentiation of MSCs in vitro and osseointegration in vivo. MSCs were cultured on Ti substrates with different surface properties and non-canonical Wnt expression was determined. Subsequently, MSCs were cultured on Ti substrates +/−Wnt16 (100 ng/mL) and anti-Wnt16 antibodies (2 μg/mL). Wnt16 expression was increased in cells grown on microrough surfaces that were processed to be hydrophilic and have nanoscale roughness. However, treatment MSCs on these surfaces with exogenous rhWnt16b increased total DNA content and osteoprotegerin production, but reduced osteoblastic differentiation and production of local factors necessary for osteogenesis. Addition of anti-Wnt16 antibodies blocked the inhibitor effects of Wnt16. The response to Wnt16 was likely independent of other osteogenic pathways like Wnt11-Wnt5a signaling and semaphorin 3a signaling. We used an established rat model of cortical and trabecular femoral bone impairment following botox injections (2 injections of 8 units/leg each, starting and maintenance doses) to assess Wnt16 effects on whole bone morphology and implant osseointegration. Wnt16 injections did not alter whole bone morphology significantly (BV/TV, cortical thickness, restoration of trabecular bone) but were effective at increasing cortical bone-to-implant contact during impaired osseointegration in the botox model. The mechanical quality of the increased bone was not sufficient to rescue the deleterious effects of botox. Clinically, these results are important to understand the interaction of cortical and trabecular bone during implant integration. They suggest a role for Wnt16 in modulating bone remodeling by reducing osteoclastic activity. Targeted strategies to temporally regulate Wnt16 after implant placement could be used to improve osseointegration by increasing the net pool of osteoprogenitor cells.

The SLC19A1-AS/miR-1343/WNT11 axis is a novel positive regulatory ceRNA network governing goat granulosa cell proliferation

Long noncoding RNAs (lncRNAs), as competitive endogenous RNAs (ceRNAs), can directly or indirectly affect the proliferation and apoptosis of granulosa cells by regulating microRNA (miRNA) pathways. A ceRNA network of the SLC19A1-AS-miR-1343-WNT11 axis was constructed via comprehensive transcriptome sequencing of ovaries from goats with various fertility levels to further elucidate the function and regulatory mechanism of SLC19A1-AS in modulating miR-1343 and WNT11 during granulosa cell proliferation and apoptosis. Subsequent validation experiments were conducted in vitro using granulosa cells. In these experiments, we performed RNA immunoprecipitation (RIP) and identified SLC19A1-AS as a ceRNA in goat granulosa cells that promoted proliferation. Through bioinformatics prediction, luciferase reporter gene assays, and RNA pulldown assays, we confirmed that SLC19A1-AS acts as a sponge for miR-1343, preventing its binding to WNT11 mRNA and thereby increasing the expression of WNT11. This interaction also influenced the proliferation and apoptosis of granulosa cells. Our study systematically validated the biological function of the lncRNA–miRNA-mRNA ceRNA network in goat ovaries and revealed the potential regulatory mechanism by which SLC19A1-AS functions as a ceRNA in granulosa cells. These findings are expected to provide an important experimental foundation for further elucidating the physiological regulatory network of the ovary and contributing to reproductive health in goats.

Phloroglucinol Enhances Anagen Signaling and Alleviates H2O2-Induced Oxidative Stress in Human Dermal Papilla Cells.

Phloroglucinol (PG) is one of the abundant isomeric benzenetriols in brown algae. Due to its polyphenolic structure, PG exhibits various biological activities. However, the impact of PG on anagen signaling and oxidative stress in human dermal papilla cells (HDPCs) is unknown. In this study, we investigated the therapeutic potential of PG for improving hair loss. A non-cytotoxic concentration of PG increased anagen-inductive genes and transcriptional activities of β-Catenin. Since several anagen-inductive genes are regulated by β-Catenin, further experiments were performed to elucidate the molecular mechanism by which PG upregulates anagen signaling. Various biochemical analyses revealed that PG upregulated β-Catenin signaling without affecting the expression of Wnt. In particular, PG elevated the phosphorylation of protein kinase B (AKT), leading to an increase in the inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9. Treatment with the selective phosphoinositide 3-kinase/AKT inhibitor, LY294002, restored the increased AKT/GSK3β/β-Catenin signaling and anagen-inductive proteins induced by PG. Moreover, conditioned medium from PG-treated HDPCs promoted the proliferation and migration of human epidermal keratinocytes via the AKT signaling pathway. Subsequently, we assessed the antioxidant activities of PG. PG ameliorated the elevated oxidative stress markers and improved the decreased anagen signaling in hydrogen peroxide (H2O2)-induced HDPCs. The senescence-associated β-galactosidase staining assay also demonstrated that the antioxidant abilities of PG effectively mitigated H2O2-induced senescence. Overall, these results indicate that PG potentially enhances anagen signaling and improves oxidative stress-induced cellular damage in HDPCs. Therefore, PG can be employed as a novel therapeutic component to ameliorate hair loss symptoms.

Genome-wide association studies highlight novel risk loci for septal defects and left-sided congenital heart defects

BackgroundCongenital heart defects (CHD) are structural defects of the heart affecting approximately 1% of newborns. They exhibit low penetrance and non-Mendelian patterns of inheritance as varied and complex traits. While genetic factors are known to play an important role in the development of CHD, the specific genetics remain unknown for the majority of patients. To elucidate the underlying genetic risk, we performed a genome wide association study (GWAS) of CHDs in general and specific CHD subgroups using the FinnGen Release 10 (R10) (N > 393,000), followed by functional fine-mapping through eQTL and co-localization analyses using the GTEx database.ResultsWe discovered three genome-wide significant loci associated with general CHD. Two of them were located in chromosome 17: 17q21.32 (rs2316327, intronic: LRRC37A2, Odds ratio (OR) [95% Confidence Interval (CI)] = 1.17[1.12–1.23], p = 1.5 × 10–9) and 17q25.3 (rs1293973611, nearest: BAHCC1, OR[95%CI] = 4.48[2.80–7.17], p = 7.0 × 10–10), respectively, and in addition to general CHD, the rs1293973611 locus was associated with the septal defect subtype. The third locus was in band 1p21.2 (rs35046143, nearest: PALMD, OR[95%CI] = 1.15[1.09–1.21], p = 7.1 × 10–9), and it was associated with general CHD and left-sided lesions. In the subgroup analysis, two additional loci were associated with septal defects (rs75230966 and rs6824295), and one with left-sided lesions (rs1305393195). In the eQTL analysis the variants rs2316327 (general CHD), and rs75230966 (septal defects) both located in 17q21.32 (with a LD r2 of 0.41) were both predicted to significantly associate with the expression of WNT9B in the atrial appendage tissue category. This effect was further confirmed by co-localization analysis, which also implicated WNT3 expression in the atrial appendage. A meta-analysis of general CHD together with the UK Biobank (combined N = 881,678) provided a different genome-wide significant locus in LRRC37A2; rs16941382 (OR[95%CI] = 1.15[1.11–1.20], p = 1.5 × 10–9) which is in significant LD with rs2316327.ConclusionsOur results of general CHD and different CHD subcategories identified a complex risk locus on chromosome 17 near BAHCC1 and LRRC37A2, interacting with the genes WNT9B, WNT3 and MYL4, may constitute potential novel CHD risk associated loci, warranting future experimental tests to determine their role.

Panax notoginseng saponins improve oral submucous fibrosis by inhibiting the Wnt/β-catenin signal pathway

ObjectiveOral submucous fibrosis (OSF) is a chronic, insidious, progressive mucosal disease that may be affected by mutations in the Wnt/β-catenin signaling pathway. Panax notoginseng saponins (PNS) is a powerful anti-fibrosis agent; however, its effect and mechanism in treating OSF remain unclear. This study investigated the effect and mechanism of PNS treatment for OSF. Study DesignArecoline was used to induce OSF models in vivo and in vitro, which were then treated with PNS. Hematoxylin-eosin (HE) and Masson trichrome staining were used to observe histopathology changes; E-cadherin and β-catenin were detected by Immunohistochemical assay, and type Ⅰ collagen (CollA1) and β-catenin were detected by immunofluorescent staining. The Wnt/β-catenin pathway and fibrosis signs were assessed using Western Blot and real-time quantitative polymerase chain reaction (RT-qPCR). ResultsThe expression of CollA1, Wnt1, and β-catenin were increased, and E-cadherin, GSK-3β, and β-catenin expression were decreased in OSF models. PNS and inhibitor intervention increased E-cadherin, Wnt1, and β-catenin, and decreased CollA1 and GSK-3β in a dose-dependent manner. ConclusionPNS can improve OSF by inhibiting the Wnt/β-catenin signal pathway and thus may be used as a potential medicine for the treatment of OSF.

Pcgf5: An important regulatory factor in early embryonic neural induction

Polycomb group RING finger (PCGF) proteins, a crucial subunits of the Polycomb complex, plays an important role in regulating gene expression, embryonic development, and cell fate determination. In our research, we investigated Pcgf5, one of the six PCGF homologs, and its impact on the differentiation of P19 cells into neural stem cells. Our findings revealed that knockdown of Pcgf5 resulted in a significant decrease in the expression levels of the neuronal markers Sox2, Zfp521, and Pax6, while the expression levels of the pluripotent markers Oct4 and Nanog increased. Conversely, Pcgf5 overexpression upregulated the expression of Sox2 and Pax6, while downregulating the expression of Oct4 and Nanog. Additionally, our analysis revealed that Pcgf5 suppresses Wnt3 expression via the activation of Notch1/Hes1, and ultimately governs the differentiation fate of neural stem cells. To further validate our findings, we conducted in vivo experiments in zebrafish. We found that knockdown of pcgf5a using morpholino resulted in the downregulated expression of neurodevelopmental genes such as sox2, sox3, and foxg1 in zebrafish embryos. Consequently, these changes led to neurodevelopmental defects. In conclusion, our study highlights the important role of Pcgf5 in neural induction and the determination of neural cell fate.

Epigenetic Effects of Resveratrol on Oncogenic Signaling in Breast Cancer.

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.

Testosterone, β-estradiol, and hepatocellular carcinoma: stimulation or inhibition? A comparative effect analysis on cell cycle, apoptosis, and Wnt signaling of HepG2 cells.

Unlike breast and prostate cancers, which are specifically affected by estrogens or androgens, hepatocellular carcinoma has been reported to be influenced by both sex hormones. Given the coincidental differences of hepatocellular carcinoma in men and women, we investigated the effects of β-estradiol and testosterone on the cell cycle, apoptosis, and Wnt signaling in a model of hepatocellular carcinoma to understand the sex hormone-related etiology. To determine the effective concentration of both hormones, an MTT assay was performed. The effects of β-estradiol and testosterone on cell proliferation and death were evaluated by specific staining and flow cytometry. In addition, gene expression levels of estimated factors involved in GPC3-Wnt survival signaling were analyzed using quantitative real-time polymerase chain reaction. Both hormones inhibited hepatic cell proliferation through arresting the cell cycle at S/G2 and increased the apoptosis rate in HepG2 cells. Both hormones dose-dependently decreased GPC3, Wnt, and DVL expression levels as activators of the Wnt-signaling pathway. In the case of Wnt-signaling inhibitors, the effects of both hormones on WIF were negligible, but they increased DKK1 levels in a dose-dependent manner. In each of the effects mentioned above, β-estradiol was notably more potent than testosterone. In contrast to the primary hypothesis of the project, in which testosterone was considered a stimulating carcinogenic factor in HCC pathogenesis, testosterone inhibited the occurrence of HCC similarly to β-estradiol. However, this inhibitory effect was weaker than that of β-estradiol and requires further study.

Mechanical induction of osteoanabolic Wnt1 promotes osteoblast differentiation via Plat.

Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating β-Catenin. Silencing Wnt1 impairs mechanically induced β-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/β-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively.

Alternative Wnt-signaling axis leads to a break of oncogene-induced senescence

Oncogene-induced senescence (OIS) is an important process that suppresses tumor development, but the molecular mechanisms of OIS are still under investigation. It is known that BRAFV600E-mutated melanocytes can overcome OIS and develop melanoma, but the underlying mechanism is largely unknown. Using an established OIS model of primary melanocytes transduced with BRAFV600E, YAP activity was shown to be induced in OIS as well as in melanoma cells compared to that in normal epidermal melanocytes. This led to the assumption that YAP activation itself is not a factor involved in the disruption of OIS. However, its role and interaction partners potentially change. As Wnt molecules are known to be important in melanoma progression, these molecules were the focus of subsequent studies. Interestingly, activation of Wnt signaling using AMBMP resulted in a disruption of OIS in BRAFV600E-transduced melanocytes. Furthermore, depletion of Wnt6, Wnt10b or β-catenin expression in melanoma cells resulted in the induction of senescence. Given that melanoma cells do not exhibit canonical Wnt/β-catenin activity, alternative β-catenin signaling pathways may disrupt OIS. Here, we discovered that β-catenin is an interaction partner of YAP on DNA in melanoma cells. Furthermore, the β-catenin–YAP interaction changed the gene expression pattern from senescence-stabilizing genes to tumor-supportive genes. This switch is caused by transcriptional coactivation via the LEF1/TEAD interaction. The target genes with binding sites for LEF1 and TEAD are involved in rRNA processing and are associated with poor prognosis in melanoma patients. This study revealed that an alternative YAP-Wnt signaling axis is an essential molecular mechanism leading to OIS disruption in melanocytes.

Investigating the impact of vitrification on bovine ovarian tissue morphology, follicle survival, and transcriptomic signature

PurposeOvarian tissue cryopreservation is vital for fertility preservation, yet its effect on ovarian tissue follicle survival and transcriptomic signature requires further investigation. This study delves into the effects of vitrification on tissue morphology, function, and transcriptomic changes, helping to find possibilities for vitrification protocol improvements.MethodsOvarian cortex from 19 bovine animals were used to conduct pre- and post-vitrification culture followed by histological assessment, immunohistochemistry, and TUNEL assay. Follicles’ functionality was assessed for viability and growth within the tissue and in isolated cultures. RNA-sequencing of ovarian tissue was used to explore the transcriptomic alterations caused by vitrification.ResultsFollicle density, cell proliferation, and DNA damage in ovarian stroma were unaffected by vitrification. However, vitrified cultured tissue exhibited reduced follicle density of primordial/primary and antral follicles, while freshly cultured tissue manifested reduction of antral follicles. Increased stromal cell proliferation and DNA damage occurred in both groups post-culture. Isolated follicles from vitrified tissue exhibited similar viability to fresh follicles until day 4, after which the survival dropped. RNA-sequencing revealed minor effects of vitrification on transcriptomic signatures, while culture induced significant gene expression changes in both groups. The altered expression of WNT and hormonal regulation pathway genes post-vitrification suggests the molecular targets for vitrification protocol refinement.ConclusionVitrification minimally affects tissue morphology, follicle density, and transcriptomic signature post-thawing. However, culture revealed notable changes in vitrified tissue samples, including reduced follicle density, decreased isolated follicle survival, and alteration in WNT signalling and ovarian hormonal regulation pathways, highlighted them as possible limitations of the current vitrification protocol.