Increase In Β-catenin Levels Research Articles

OTUD7B inhibited hepatic injury from NAFLD by inhibiting K48-linked ubiquitination and degradation of β-catenin

Non-alcoholic fatty liver disease (NAFLD) is the prevalent liver disease. Ovarian tumor domain-containing 7B (OTUD7B) is a deubiquitinating enzyme and its role in NAFLD remains unclear. In high-fat diet (HFD)-induced NAFLD mouse model and palmitic acid (PA)-treated HepG2 cells, OTUD7B expression was decreased. Adenoviral overexpression of OTUD7B in mice resulted in reduced body weight and liver injury, with decreased serum aminotransferase (ALT) and aspartate aminotransferase (AST) levels. OTUD7B overexpression attenuated hepatic lipid deposition (serum TG, TC, LDL-C, HDLC, and FFA levels), which might be through the suppression of lipogenesis and β-oxidation-related genes. The contents of hepatic inflammatory factors (TNF-α, IL-6, and IL-1β) were decreased following OTUD7B overexpression in NAFLD mice. A mechanism study indicated that the protective effect of OTUD7B overexpression might be associated with β-catenin signal activation. OTUD7B overexpression promoted PA-induced β-catenin activity in TopFlash assay, and increased total β-catenin and c-myc levels in cells. The increase in β-catenin levels was contributed to the stabilization via inhibiting K48-linked ubiquitination and proteasomal degradation by OTUD7B. NR4A2 role in NASH has been proved. NR4A2 ChIP-seq and RNA-seq data excluded transcriptional regulation of NR4A2 to OTUD7B, and it was experimentally evidenced that NR4A2 bound to OTUD7B promoter region and positively transcriptionally regulate OTUD7B expression. In summary, OTUD7B overexpression ameliorated hepatic inflammation and steatosis in NAFLD. The possible mechanism of OTUD7B might be through the inhibition of β-catenin degradation by removing K48-linked ubiquitination, which might be regulated by NR4A2.

Microsponges-mediated targeted topical delivery of rosemary oil for hair growth promotion: optimization and in-vivo studies

Individuals experiencing hair loss, irrespective of gender, confront significant psychological challenges. This study explores the untapped potential of rosemary oil (ROS) to stimulate hair growth, addressing its limited permeability. The focus is on innovating ROS-loaded microsponges (MS) for enhanced topical application. Utilizing Box-Behnken design (33), the study optimizes ROS-MS compositions by varying solvent volume, polymer mix, and drug concentration. The optimized ROS-MS formulation exhibits noteworthy attributes: a 94% ± 0.04 production yield, 99.6% ± 0.5 encapsulation efficiency, and 96.4% ± 1.6 cumulative ROS release within 24 h. These microsponges exhibit uniformity with a particle size of 14.1 µm ± 4.5. The OPT-ROSMS-gel showcases favorable characteristics in appearance, spreadability, pH, drug content, and extrudability. Ex-vivo skin deposition tests highlight heightened permeability of OPT-ROSMS-gel compared to pure ROS-gel, resulting in three-fold increased follicular retention. In-vivo studies underscore the superior efficacy of OPT-ROSMS-gel, revealing enhanced hair development in length, thickness, and bulb diameter, surpassing ROS-gel and minoxidil by approximately 1.2 and 1.5 times, respectively, along with nearly two-fold increase in β-catenin levels. In conclusion, microsponges emerge as a promising ROS delivery method, effectively addressing hair loss. This research advances hair loss treatments and underscores the significance of this innovative paradigm in fostering hair growth.

Akkermansia muciniphila promotes testosterone-mediated hair growth inhibition in mice.

The beneficial effects of Akkermansia muciniphila (Akk) on gut health and inflammation reduction have been demonstrated; however, scientific evidence of hair growth enhancement by Akk has not been reported. Therefore, this study was undertaken to investigate the effect of Akk on improving testosterone-mediated hair growth inhibition. Hair growth inhibition was induced through subcutaneous injection of testosterone into the shaved dorsal skin of C57BL/6 male mice. Live and pasteurized Akk were orally administered at a concentration of 1 × 108 colony-forming unit. After 5 weeks, hair length and skin tissues were analyzed. The live and pasteurized Akk significantly stimulated hair growth, countering the inhibitory effect of testosterone compared to the testosterone-alone group. Hematoxylin and eosin staining revealed a significant increase in hair follicle size in the Akk-treated group. An increase in β-catenin levels, which are associated with hair growth and cell cycle progression, was also observed. Moreover, the Akk-treated group exhibited increased levels of fibroblast growth factors, including Fgf7, Igf1, Fgf7, Fgf10, and Fgf21. However, no significant difference was observed between the live and pasteurized Akk groups. These results underscore the potential of live and pasteurized Akk in improving testosterone-mediated hair growth inhibition.

Shh-Mediated Increase in β-Catenin Levels Maintains Cerebellar Granule Neuron Progenitors in Proliferation.

Cerebellar granule neuron progenitors (CGNPs) give rise to the cerebellar granule neurons in the developing cerebellum. Generation of large number of these neurons is made possible by the high proliferation rate of CGNPs in the external granule layer (EGL) in the dorsal cerebellum. Here, we show that upregulation of β-catenin can maintain murine CGNPs in a state of proliferation. Further, we show that β-catenin mRNA and protein levels can be regulated by the mitogen Sonic hedgehog (Shh). Shh signaling led to an increase in the level of the transcription factor N-myc. N-mycwas found to bind the β-catenin promoter, and the increase in β-catenin mRNA and protein levels could be prevented by blocking N-myc upregulation downstream of Shh signaling. Furthermore, blocking Wingless-type MMTV integration site (Wnt) signaling by Wnt signaling pathway inhibitor Dickkopf 1 (Dkk-1) in the presence of Shh did not prevent the upregulation of β-catenin. We propose that in culture, Shh signaling regulates β-catenin expression through N-mycand results in increased CGNP proliferation.

Glycogen synthase kinase 3-β inhibition induces lymphangiogenesis through β-catenin-dependent and mTOR-independent pathways.

Lymphatic vessels play an important role in health and in disease. In this study, we evaluated the effects of GSK3-β inhibition on lung lymphatic endothelial cells in vitro. Pharmacological inhibition and silencing of GSK3-β resulted in increased lymphangiogenesis of lung lymphatic endothelial cells. To investigate mechanisms of GSK3-β-mediated lymphangiogenesis, we interrogated the mammalian/mechanistic target of rapamycin pathway and found that inhibition of GSK3-β resulted in PTEN activation and subsequent decreased activation of AKT, leading to decreased p-P70S6kinase levels, indicating inhibition of the mTOR pathway. In addition, consistent with a negative role of GSK3-β in β-catenin stability through protein phosphorylation, we found that GSK3-β inhibition resulted in an increase in β-catenin levels. Simultaneous silencing of β-catenin and inhibition of GSK3-β demonstrated that β-catenin is required for GSK3-β-induced lymphangiogenesis.

Highlights of This Issue

In cancer, increased caspase-3 activation linked to apoptotic cell death provides a mechanistic readout of drug response. Nguyen and colleagues establish that noninvasive positron emission tomography imaging with the caspase-3 binding probe [18F]ICMT-11 permits characterization of caspase-3 dynamics in vivo and the early assessment of apoptosis induced by both cytotoxic and mechanism-based drugs. As the development of noninvasive molecular imaging strategies to detect apoptosis continues to gain momentum, the authors demonstrate that this new imaging approach provides essential insights into the pharmacodynamics of anticancer agents and holds promise for predicting anticancer drug response in tumors.Chondrosarcomas are notoriously resistant to chemotherapy, and novel therapeutic targets are needed. Zhang and colleagues show strong S6 phosphorylation in the majority of chondrosarcoma tumors and heterogeneous activation of receptor tyrosine kinases (RTK) in cell lines. Inhibition of phosphoinositide-3-kinase (PI3K) and mTOR, signaling proteins downstream of RTKs and upstream of S6, potently blocked growth of chondrosarcoma cells in vitro and in vivo. A subset of chondrosarcoma was identified to harbor NRAS mutations, and a corresponding cell line responded to MEK inhibition. These findings provide a rationale for clinical exploration of PI3K/mTOR and MAPK pathway inhibitors in this disease.Effective cancer vaccines seem to require robust costimulatory immune activity spatiotemporally associated with the presentation of a broad array of patient-relevant cancer antigens. Conrad and colleagues developed a practical strategy to achieve this by administering a vaccine prepared through ex vivo infection of viable acute lymphocytic leukemia cells with attenuated yet highly immunogenic strains of engineered rhabdovirus. The potent immunotherapeutic [immunotherapy by leukemiaoncotropic virus (iLOV)] induced durable responses in vivo that required cytotoxic T cells to prevent the lethal progression of murine leukemia. This immune effect targeted only cells contained in the vaccine. iLOV represents a feasible approach to eliminate residual disease following remission induction.Studies have identified the presence of bacteria within the tumors and mucin of patients with pseudomyxoma peritonei, an abdominal cancer originating from an appendiceal neoplasm. Semino-Mora and colleagues further demonstrate the important role of bacteria in pseudomyxoma peritonei disease. Antibiotic treatment significantly reduced bacterial density as well as β-catenin levels in the host cell cytoplasm, the cell nuclei, and mucin-associated cells of patients with peritoneal mucinous carcinomatosis. Furthermore, these patients exhibited a significant increase in β-catenin levels within host cell membranes. Both the reduced bacterial density and redistribution of β-catenin highlight the efficacy of antibiotics as a potential treatment for pseudomyxoma peritonei cancer.

Spinal Cord Early Ischemic Preconditioning Activates the Stabilized Fraction of β-Catenin After Thoracoabdominal Aortic Occlusion in Pigs

Paraplegia after thoracoabdominal aortic surgery is a devastating complication attributed to motor neurons loss and dysfunction, due to spinal cord ischemia. β-Catenin is a protein that has been associated with cell survival and healing and many studies have correlated this protein with late ischemic preconditioning (IPC). Herein we investigate the potential contribution of β-catenin in an early IPC animal model, and its relationship with heat shock protein 70 (Hsp70), suggesting a possible role of this protein as a first window of protection. A total of 42 pigs were used in an experimental thoracoabdominal aortic occlusion model. Twelve animals were used for neurologic evaluation and were randomly assigned to 2 groups (A and B). The remaining 30 animals were used in experiments for biologic measurements and innunohistochemical studies, and were randomly assigned to 5 groups (1-5). Western blotting analysis and immunoprecipitations were performed to study the levels of β-catenin and its binding relationship with Hsp70. The cellular distribution of β-catenin at various time-points was investigated by immunohistochemical studies. According to neurologic evaluation, the animals in the IPC+ischemia group had significantly better neurologic scores compared with those in the ischemia group, indicating a protective role for IPC. The biologic measurements demonstrated a significant (P=0.03) increase in β-catenin levels and translocation of the protein in the nucleus at the end of ischemic preconditioning. Our results suggest a significant role of β-catenin in early IPC protection of spinal cord after thoracoabdominal occlusion, as IPC seems to trigger the activation of the β-catenin stabilized fraction and, thus, its survival pathway.

Identification of a pathway by which glucose regulates β-catenin signalling via the cAMP/protein kinase A pathway in β-cell models

Pancreatic β-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the β-catenin signalling pathway plays an important role in regulating β-cell function, but the mechanisms regulating β-catenin signalling in these cells is not well understood. In the present study we show that β-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and β-TC6-F7 β-cell models. We found a glucose-dependent increase in levels of β-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of β-catenin. This was associated with an increase in phosphorylation of β-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on β-catenin signalling. Finally, reducing β-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate β-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in β-cells.

Lenalidomide Suppression of Multiple Myeloma Cell Proliferation Is Associated with Downregulation of LEF/TCF Activity

Abstract 5014Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev’s direct effect on MM cell survival.To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation.These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug’s cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity. Disclosures:No relevant conflicts of interest to declare.

Fas-associated factor 1 antagonizes Wnt signaling by promoting β-catenin degradation

The canonical Wnt pathway plays an important role in the regulation of cell proliferation and differentiation. Activation of this signaling pathway causes disruption of the Axin/adenomatous polyposis coli/glycogen synthase kinase 3β complex, resulting in stabilization of β-catenin and its association with lymphoid enhancer factor/T-cell factor in the nucleus. Here, we identify Fas-associated factor 1 (FAF1) as a negative regulator of Wnt/β-catenin signaling. We found overexpression of FAF1 to strongly inhibit Wnt-induced transcriptional reporter activity and to counteract Wnt-induced β-catenin accumulation. Moreover, knockdown of FAF1 resulted in an increase in β-catenin levels and in activation of Wnt/β-catenin-induced transcription. FAF1 was found to interact with β-catenin upon inhibition of proteasome. Ectopic expression of FAF1 promoted β-catenin degradation by enhancing its polyubiquitination. Functional studies in C2C12 myoblasts and KS483 preosteoblastic cells showed that FAF1 depletion resulted in activation of endogenous Wnt-induced genes and enhanced osteoblast differentiation, whereas FAF1 overexpression had the opposite effect. These results identify FAF1 as a novel inhibitory factor of canonical Wnt signaling pathway.

Exosome‐release of beta‐catenin: A novel mechanism to antagonize Wnt signaling

Expression of the tetraspanins CD82 and CD9 suppresses tumor metastasis. Previous studies have shown that CD82 and CD9 are enriched in exosomes. The functional roles of tetraspanins in exosomes and their roles in the suppression of tumor cell metastasis remain unknown. We find that CD82 and CD9 serve as negative regulators of the Wnt/β‐catenin signaling pathway, whose inappropriate activation is involved in the development and progression of several cancers. CD82 and CD9 expression strongly suppressed Wnt/β‐catenin activity as measured by luciferase reporter assay and induced a significant decrease in β‐catenin protein levels in both cytoslic and nuclear fractions in HEK 293T cells. In addition, RNAi knock down of endogenous CD9 resulted in an increase in β‐catenin levels and Wnt/β‐catenin activity. CD9 and CD82 expression inhibited Wnt/β‐catenin signaling through a mechanism independent of GSK‐3β and of the proteasome and lysosome‐mediated protein degradation pathways. CD82 and CD9 expression enhanced β‐catenin transport into exosomes via a mechanism that was blocked by sphingomyelinase inhibitor, GW4869. CD82 failed to induce exosome release of β‐catenin in CHO cells, which do not express endogenous E‐cadherin. However, β‐catenin and E‐cadherin were detected in exosomes purified from the culture medium of CHO cells that were cotransfected with CD82 and E‐cadherin. Exosome release from dendritic cells generated from CD9 knockout mice was reduced compared to dendritic cells generated from wild‐type mice. Taken together, our results indicate that CD82 and CD9 might suppress tumor metastasis through downregulation of the Wnt signaling pathway via a novel mechanism that leads ultimately to the exosomal discharge of β‐catenin.

Elevated levels of beta-catenin and fibronectin in three-dimensional collagen cultures of Dupuytren's disease cells are regulated by tension in vitro.

BackgroundDupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands. Although the molecular pathology of DD is unknown, recent evidence suggests that β-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in β-catenin and fibronectin levels.MethodsWestern blots of β-catenin and fibronectin levels were determined for control and disease primary cell cultures grown within stressed- and attached-collagen matrices. Collagen contraction was quantified, and immunocytochemistry analysis of filamentous actin performed.ResultsDisease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in β-catenin and fibronectin levels, including a transient increase in β-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and β-catenin levels increased in attached collagen-matrix cultures of disease cells, while control cultures showed only increases in fibronectin levels. Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.ConclusionThree-dimensional collagen matrix cultures of primary disease cell lines are more contractile and express a more extensive filamentous actin network than patient-matched control cultures. The elevated levels of β-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.