Β-catenin Signaling Research Articles

Interplay between Netrin-1 and Norrin controls arteriovenous zonation of blood–retina barrier integrity

The integrity of the blood-retina barrier (BRB) is crucial for phototransduction and vision, by tightly restricting transport of molecules between the blood and surrounding neuronal cells. Breakdown of the BRB leads to the development of retinal diseases. Here, we show that Netrin-1/Unc5b and Norrin/Lrp5 signaling establish a zonated endothelial cell gene expression program that controls BRB integrity. Using single-cell RNA sequencing (scRNA-seq) of postnatal BRB-competent mouse retina endothelial cells (ECs), we identify >100 BRB genes encoding Wnt signaling components, tight junction proteins, and ion and nutrient transporters. We find that BRB gene expression is zonated across arteries, capillaries, and veins and regulated by opposing gradients of the Netrin-1 receptor Unc5b and Lrp5-β-catenin signaling between retinal arterioles and venules. Mice deficient for Ntn1 or Unc5b display more BRB leakage at the arterial end of the vasculature, while Lrp5 loss of function causes predominantly venular BRB leakage. ScRNA-seq of Ntn1 and Unc5b mutant ECs reveals down-regulated β-catenin signaling and BRB gene expression that is rescued by Ctnnb1 overactivation, along with BRB integrity. Mechanistically, we demonstrate that Netrin-1 and Norrin additively enhance β-catenin transcriptional activity and Lrp5 phosphorylation via the Discs large homologue 1 (Dlg1) scaffolding protein, and endothelial Lrp5-Unc5b function converges in protection of capillary BRB integrity. These findings explain how arteriovenous zonation is established and maintained in the BRB and reveal that BRB gene expression is regulated at the level of endothelial subtypes.

Mitochondrial unfolded protein response-dependent β-catenin signaling promotes neuroendocrine prostate cancer.

The mitochondrial unfolded protein response (UPRmt) maintains mitochondrial quality control and proteostasis under stress conditions. However, the role of UPRmt in aggressive and resistant prostate cancer is not clearly defined. We show that castration-resistant neuroendocrine prostate cancer (CRPC-NE) harbored highly dysfunctional oxidative phosphorylation (OXPHOS) Complexes. However, biochemical and protein analyses of CRPC-NE tumors showed upregulation of nuclear-encoded OXPHOS proteins and UPRmt in this lethal subset of prostate cancer suggestive of compensatory upregulation of stress signaling. Genetic deletion and pharmacological inhibition of the main chaperone of UPRmt heat shock protein 60 (HSP60) reduced neuroendocrine prostate cancer (NEPC) growth in vivo as well as reverted NEPC cells to a more epithelial-like state. HSP60-dependent aggressive NEPC phenotypes was associated with upregulation of β-catenin signaling both in cancer cells and in vivo tumors. HSP60 expression rendered enrichment of aggressive prostate cancer signatures and metastatic potential were inhibited upon suppression of UPRmt. We discovered that UPRmt promoted OXPHOS functions including mitochondrial bioenergetics in CRPC-NE via regulation of β-catenin signaling. Mitochondrial biogenesis facilitated cisplatin resistance and inhibition of UPRmt resensitizes CRPC-NE cells to cisplatin. Together, our findings demonstrated that UPRmt promotes mitochondrial health via upregulating β-catenin signaling and UPRmt represents viable therapeutic target for NEPC.

Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous levels.

ZNRF3, a negative regulator of β-catenin signaling, removes Wnt receptors from the membrane. Currently, it is unknown which tumor-associated variants can be considered driver mutations and through which mechanisms they contribute to cancer. Here we show that all truncating mutations analyzed at endogenous levels exhibit loss-of-function, with longer variants retaining partial activity. Regarding missense mutations, we show that 27/82 ZNRF3 variants in the RING and R-Spondin domain structures, lead to (partial) loss-of-function/hyperactivation. Mechanistically, defective R-Spondin domain variants appear to undergo endoplasmic-reticulum-associated degradation due to protein misfolding, leading to reduced protein levels. They fail to reach the membrane correctly, which can be partially restored for several variants by culturing cells at 27 °C. Although RING and R-Spondin domain mutations in RNF43/ZNRF3 are often considered to possess dominant-negative oncogene-like activity in cancers, our findings challenge this notion. When representative variants are heterozygously introduced into endogenous ZNRF3, their impact on β-catenin signaling mirrors that of heterozygous knockout, suggesting that the supposed dominant-negative effect is non-existent. In other words, so-called "hyperactivating" ZNRF3/RNF43 mutations behave as classical loss-of-function mutations at endogenous levels.

Editorial Expression of Concern: Alpha-Synuclein Suppresses Retinoic Acid-Induced Neuronal Differentiation by Targeting the Glycogen Synthase Kinase-3β/β-Catenin Signaling Pathway.

Editorial Expression of Concern: Alpha-Synuclein Suppresses Retinoic Acid-Induced Neuronal Differentiation by Targeting the Glycogen Synthase Kinase-3β/β-Catenin Signaling Pathway.

Cell competition drives bronchiolization and pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive respiratory scarring disease arising from the maladaptive differentiation of lung stem cells into bronchial epithelial cells rather than into alveolar type 1 (AT1) cells, which are responsible for gas exchange. Here, we report that healthy lungs maintain their stem cells through tonic Hippo and β-catenin signaling, which promote Yap/Taz degradation and allow for low-level expression of the Wnt target gene Myc. Inactivation of upstream activators of the Hippo pathway in lung stem cells inhibits this tonic β-catenin signaling and Myc expression and promotes their Taz-mediated differentiation into AT1 cells. Vice versa, increased Myc in collaboration with Yap promotes the differentiation of lung stem cells along the basal and myoepithelial-like lineages allowing them to invade and bronchiolize the lung parenchyma in a process reminiscent of submucosal gland development. Our findings indicate that stem cells exhibiting the highest Myc levels become supercompetitors that drive remodeling, whereas loser cells with lower Myc levels terminally differentiate into AT1 cells.

Ailanthone disturbs cross-talk between cancer cells and tumor-associated macrophages via HIF1-α/LINC01956/FUS/β-catenin signaling pathway in glioblastoma

BackgroundAn increasing number of studies have focused on ailanthone (aila) due to its antitumor activity. However, the role of ailanthone in glioblastoma(GBM) has not been investigated before. This study aims to explore the biological function and the underlying mechanism of ailanthone in GBM.MethodsThe microarray analysis was used to screen out down-stream long non-coding RNAs (lncRNAs) targeted by ailanthone. Real-time PCR(RT-PCR) assay was used to examine LINC01956 expression levels. Colony-formation, Methylthiazolyldiphenyl-tetrazolium bromide(MTT), cell-cycle, organoids culture and in-vivo tumorigenesis assays were used to examine cell growth in vitro and in vivo. Boyden assay was used to examine cell invasion ability in vitro. RNA immunoprecipitation and RNA-protein pull-down assays were used to examine the interaction between LINC01956 and FUS protein. Chromatin Immunoprecipitation(ChIP) assay was used to examine HIF1-α-binding sites in the LINC01956 promoter.ResultsAilanthone decreased GBM cell growth in vitro and in vivo via inducing ferroptosis. Ailanthone treatment exhibited blood‒brain barrier(BBB) permeability and specifically targeted the tumor area. LINC01956 was identified as a down-stream target of Ailanthone. LINC01956 exerted as an onco-lncRNA in GBM. M2 polarization of macrophages induced by exosomes derived from glioma cells overexpressing LINC01956 accelerated GBM progression. Mechanistically, we found that LINC01956 bound to FUS and reduced its ubiquitination. LINC01956 evoked nuclear translocation of phosphorylated (p)-β-catenin by recruiting FUS. Furthermore, under hypoxic conditions, LINC01956 was regulated by HIF-1α. Ailanthone decreased the expression of LINC01956 via suppressing HIF-1α.ConclusionTaken together, our data revealed for the first time that ailanthone regulated HIF-1α/LINC01956/FUS/β-catenin signaling pathway and thereby inhibited GBM progression.

Porcine transient receptor potential channel 1 promotes adipogenesis and lipid deposition

Adipose tissue, an important organ involved in energy metabolism and endocrine, is closely related to animal meat quality and human health. Transient receptor potential channel 1 (TRPC1), an ion transporter, is adipocytes’ major Ca2+ entry channel. However, its function in fat deposition is poorly understood, particularly in pigs, which are both an ideal model for human obesity research and a primary meat source for human diets. In the present investigation, our findings demonstrate a prominent expression of TRPC1 within the adipose tissue of pigs with a strong fat deposition ability. Functional analysis showed that TRPC1 promotes primary preadipocyte proliferation and adipogenic differentiation. In vivo, transgenic mice expressing porcine TRPC1 (Tg-pTRPC1) exhibited aggravated high-fat diet-induced obesity, hepatic steatosis, and insulin resistance. Moreover, TRPC1 may facilitate adipogenesis via activating PI3K/AKT and β-catenin signaling pathways. Our research underscores the pivotal role of porcine TRPC1 as a positive regulator in adipogenesis and lipid accumulation processes, providing a potential target for improving animal meat quality and treating obesity-related diseases in humans.

SMURF1 leads to the β-catenin signaling-mediated progression of esophageal squamous carcinoma by losing PATZ1-induced CCNG2 transcription

Cyclin G2 (CCNG2), a known inhibitor of cell cycle progression, has been identified as a suppressor for the canonical β-catenin pathway. This study explores the impact of CCNG2 on β-catenin activity and malignant characteristics of esophageal squamous cell carcinoma (ESCC) cells, and the mechanism behind CCNG2 dysregulation. In ESCC tissues and cells, CCNG2 was under-expressed and associated with poor clinical outcomes, whereas β-catenin showed an opposite trend. Inducing CCNG2 overexpression in ESCC cells led to a reduction in β-catenin levels, which in turn suppressed proliferation, cell cycle progression, migration, invasion, stemness, and tumorigenesis. Additionally, it enhanced the cytotoxicity and proliferation of T cells in co-culture systems. However, these beneficial effects were negated by the Wnt signaling agonist BML-284. Furthermore, PATZ1 was found as a transcription factor promoting CCNG2 transcription. However, the PATZ1 protein in ESCC cells was degraded by SMURF1. Silencing of SMURF1 restored CCNG2 expression and inhibited β-catenin, thereby suppressing the malignant phenotype of ESCC cells and reducing T cell exhaustion. Yet, these effects were blocked by further silencing of PATZ1. In summary, this research demonstrates that SMURF1 activates β-catenin signaling by suppressing the PATZ1/CCNG2 axis, thereby promoting the progression of ESCC.

The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling.

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue- specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine-knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.

A Huluwa phosphorylation switch regulates embryonic axis induction

Embryonic axis formation is essential for patterning and morphogenesis in vertebrates and is tightly regulated by the dorsal organizer. Previously, we demonstrated that maternally derived Huluwa (Hwa) acts as a dorsal determinant, dictating axis formation by activating β-catenin signaling in zebrafish and Xenopus. However, the mechanism of activation and fine regulation of the Hwa protein remains unclear. Through candidate screening we identified a mutation at Ser168 in the PPNSP motif of Hwa that dramatically abolishes its axis-inducing activity. Mechanistically, mutating the Ser168 residue reduced its binding affinity to Tankyrase 1/2 and the degradation of the Axin protein, weakening β-catenin signaling activation. We confirmed that Ser168 is phosphorylated and that phosphorylation increases Hwa activity in β-catenin signaling and axis induction. Several kinases including Cdk16, Cdk2, and GSK3β, were found to enhance Ser168 phosphorylation in vitro and in vivo. Both dominant-negative Cdk16 expression and pHwa (Ser168) antibody treatment reduce Hwa function. Lastly, a knock-in allele mutating Ser168 to alanine resulted in embryos lacking body axes, demonstrating that Ser168 is essential to axis formation. In summary, Ser168 acts as a phosphorylation switch in Hwa/β-catenin signaling for embryonic axis induction, regulated by multiple kinases.

Restoration of follicular β-catenin signaling by mesenchymal stem cells promotes hair growth in mice with androgenetic alopecia

BackgroundThe use of mesenchymal stem cells (MSCs) is recognized as a promising strategy for the treatment of androgenetic alopecia (AGA). However, the underlying mechanism remains to be explored. Here, we evaluated the therapeutic effects and potential mechanisms of the use of human umbilical cord mesenchymal stem cells (hUCMSCs) in dihydrotestosterone (DHT)-induced AGA models in vivo and in vitro.MethodsIntradermal transplantation of hUCMSCs was performed in AGA model mice and therapeutic effects were evaluated using histological and immunofluorescence staining. Transwell assays were used for co-culture of hUCMSCs and dermal papilla cells (DPCs), and communication was assessed using RT-qPCR, immunofluorescence, and apoptosis analysis. Interactions between DPCs and hair follicle stem cells (HFSCs) were investigated using RT-qPCR, EdU assays, and cell cycle analysis. ResultsTreatment of AGA mice with hUCMSCs promoted hair growth, HFs density, skin thickness, and anagen phase activation, while inhibiting DPCs apoptosis, and promoting HFSCs proliferation. In vitro, hUCMSCs activated Wnt/β-catenin signaling in DPCs via Wntless (Wls), while stimulating growth factor secretion and HFSCs proliferation. Blocking β-catenin degradation with MSAB increased DPCs apoptosis, reduced growth factor secretion, and retarded HFSCs proliferation.ConclusionhUCMSCs promoted hair regeneration in AGA model mice. This was found to be dependent on reducing DPCs apoptosis, thereby relieving the inhibitory effects of DPCs on the growth of HFSCs. The activation of the Wnt/β-catenin signaling pathway was shown to play a crucial role in the promotion of hair growth by hUCMSCs in AGA mice.

Inhibition of NPC1 suppresses cell proliferation and β-catenin signaling activation of liver cancer.

Niemann-Pick Type C1 (NPC1) plays a significant role in the development of liver diseases and liver cancer. Our objective was to investigate the involvement of NPC1 in regulating liver cancer development. We observed that high levels of NPC1 expression in tumor tissues from patients with liver cancer were associated with a poor prognosis. Through in vitro experiments, we found that inhibiting NPC1 expression reduced the proliferation, invasion, and migration of liver cancer cells, while also inducing apoptosis in these cells. Additionally, the inhibition of NPC1 led to decreased activation of Wnt/β-catenin signaling. In vivo studies further supported our findings by demonstrating that the suppression of liver cancer cell growth was effectively achieved through the inhibition of NPC1. Overall, our results strongly indicate that the inhibition of NPC1 suppresses liver cancer cell proliferation. Targeting NPC1 is a promising potential therapeutic strategy for liver cancer.

Decoding β-catenin expression patterns in ovarian serous carcinoma with clinicopathological implications: insights from National Cancer Institute.

This study aimed to examine the immunohistochemical expression of β-catenin in serous ovarian carcinoma and to investigate its relationship with clinicopathological features and disease outcomes. A retrospective analysis was conducted on 67 cases of serous ovarian carcinoma diagnosed at the Pathology Department of the Egyptian National Cancer Institute, Cairo University, between January 1, 2015, and December 31, 2017. The age of the patients ranged from 26 to 76years. Aberrant β-catenin expression was defined by the presence of cytoplasmic staining with or without membranous staining in at least 10% of tumor cells. Of the cases analyzed, thirty exhibited cytoplasmic staining, 18 demonstrated preserved expression on the cell membrane, 15 showed combined cytoplasmic and membranous staining, while four cases were entirely negative. Significant correlations were observed between β-catenin positivity and both tumor grade and p53 expression, with p values of 0.004 for each correlation. Positive β-catenin expression significantly correlated with tumor grade in serous ovarian carcinoma. Most low-grade serous carcinoma cases exhibited membranous β-catenin expression, whereas high-grade serous carcinoma cases predominantly displayed cytoplasmic staining. Therapeutic strategies aimed at inhibiting β-catenin signaling could provide a novel approach to improving outcomes for patients with high-grade ovarian cancer.

Anti-Obesity Effects of Adzuki Bean Saponins in Improving Lipid Metabolism Through Reducing Oxidative Stress and Alleviating Mitochondrial Abnormality by Activating the PI3K/Akt/GSK3β/β-Catenin Signaling Pathway.

Obesity is a chronic and complex disease defined by the excessive deposition of fat and is highly associated with oxidative stress. Adzuki bean saponins (ABS) showed anti-obesity activity in our previous in vivo study; however, the active saponins of adzuki beans and potential mechanisms are still unclear. This research aims to elucidate the anti-obesity effects of ABS in improving lipid metabolism and oxidative stress, exploring the effective ingredients and potential molecular mechanisms through UHPLC-QE-MS analysis, network pharmacology, bioinformatics, and in vitro experiments both in the 3T3-L1 cell line and HepG2 cell line. The results indicate that ABS can improve intracellular lipid accumulation, adipogenesis, oxidative stress, and mitochondrial damage caused by lipid accumulation including ROS generation, abnormal mitochondrial membrane potential, and ATP disorder. Fifteen saponin components were identified with the UHPLC-QE-MS analysis. The network pharmacology and bioinformatics analyses indicated that the PI3K/Akt signaling pathway is associated with the bioactive effect of ABS. Through Western blotting and immunofluorescence analysis, the anti-obesity effect of ABS is achieved through regulation of the PI3K/Akt/GSK3β/β-catenin signaling pathway and activation of downstream transcription factor c-Myc in the lipid accumulation cell model, and regulation of β-catenin signaling and inhibition of downstream transcription factor C/EBPα in the adipocyte cell model. These results illustrate the biological activity of ABS in improving fat metabolism and oxidative stress by restoring mitochondrial function through β-catenin signaling, the PI3K/Akt/GSK3β/β-catenin signaling pathway, laying the foundation for its further development.

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.

A Necessary Role for Cyclin D2 Induction During Colon Cancer Progression Mediated by L1.

The cell adhesion molecule L1CAM (L1), mainly known for its function in brain cells, is a Wnt/β-catenin signaling target gene in colorectal cancer (CRC) cells, where it promotes invasion and liver metastasis. We interrogated which genes are expressed at increased levels in human CRC tissue and induced in CRC cell lines overexpressing L1. We found increased cyclin D2 levels in CRC tissue and LS 174T and HCT 116 human CRC cells overexpressing L1. Increased cyclin D2 in CRC cells was associated with higher proliferation rates, faster motility, tumorigenesis, and liver metastasis. The suppression of cyclin D2 expression by shRNA to cyclin D2 blocked the increase in these cellular properties of L1-expressing cells. The overexpression of cyclin D2 in the absence of L1 also conferred tumorigenic properties similar to L1 expression. The pathways involved in the elevation of cyclin D2 by L1 include NF-κB, Akt, and β-catenin signaling but not the Erk pathway. We found that in a significant percentage of human CRC tissue samples, cyclin D2 is expressed at high levels in the nuclei of cancer cells. At the same time, the adjacent normal mucosa was negative for cyclin D2 staining. The results suggest that the increased cyclin D2 expression by L1 is required to induce proliferative, motile tumor development in CRC tissue and can serve as a diagnostic marker and a target for CRC therapy.

CRISPR/CasRx-mediated RNA knockdown targeting β-catenin and Ihh signaling alleviates osteoarthritis

Osteoarthritis (OA) is a chronic degenerative joint disease. Currently, OA is incurable. Abnormal activation of canonical Wnt/β-catenin or Indian hedgehog (Ihh) signaling could lead to OA development and progression. This study aimed to determine if targeting β-catenin and Ihh signaling could yield an effective therapeutic intervention for OA disease. CRISPR/CasRx is a new RNA interference tool that can precisely and efficiently cleave single-strand RNAs. In this study, we screened CRISPR-derived RNA (crRNA) targeting Ctnnb1 and Smo in vitro and selected two optimal crRNAs for each gene. CasRx-mediated Ctnnb1 and Smo knockdown showed high efficiency and specificity with no obvious off-target effects in vitro. We then performed intra-articular injection of selected crRNAs driven by the adeno-associated virus into an OA mouse model. Micro-CT, histological, and histomorphometric analyses were conducted to evaluate the efficacy of CasRx approach on OA treatment. We found that the knockdown of Ctnnb1 and Smo decelerated pathological damage in the keen joint of the experimental OA mouse model. Our findings suggest that CasRx-mediated Ctnnb1 and Smo knockdown could be a potential strategy for OA treatment.

Y-27632 targeting ROCK1&2 modulates cell growth, fibrosis and epithelial-mesenchymal transition in hyperplastic prostate by inhibiting β-catenin pathway

Benign prostatic hyperplasia (BPH) is a prevalent condition affecting the male urinary system, with its molecular mechanisms of pathogenesis remaining unclear. Y-27632, a non-isoform-selective Rho kinase inhibitor, has shown therapeutic potential in various diseases but its effects on static factors and fibrosis in BPH remain unexplored. This study investigated human prostate tissues, human prostate cell lines, and BPH rat model using immunofluorescence, flow cytometry, quantitative reverse transcription polymerase chain reaction, western blotting, and cell counting kit-8. ROCK1 and ROCK2 were significantly up-regulated in BPH tissues, correlating with clinical parameters. Y-27632 targeted the inhibition of ROCK1 & ROCK2 expression and inhibited cell proliferation, fibrosis, epithelial-mesenchymal transition (EMT), while induced cell apoptosis in a dose-dependent manner. Moreover, knockdown of either ROCK isoform inhibited fibrosis and EMT, induced apoptosis, while ROCK overexpression had the opposite effects. ROCK downregulation inhibited the β-catenin signaling pathway (such as C-MYC, Snail and Survivin) and decreased β-catenin protein stability, while inhibiting TGF-β/Smad2/3 signaling. At the in vivo level, Y-27632 reversed prostatic hyperplasia and fibrosis in BPH model rats to some extent. Our study sheds light on the therapeutic potential of Y-27632 in regulating prostate cell growth, fibrosis and EMT, and demonstrates for the first time the regulatory effect of ROCK isoforms on prostate cells, providing the basis for future research of ROCK isoform-selective inhibitors.

Vascular inflammaging: Endothelial CEACAM1 expression is upregulated by TNF-α via independent activation of NF-κB and β-catenin signaling.

Chronic inflammation with progressive age, called inflammaging, contributes to the pathogenesis of cardiovascular diseases. Previously, we have shown increased vascular expression of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in aged mice and humans, presumably via mutual upregulation with the pro-inflammatory cytokine TNF-α. CEACAM1 is critical for aging-associated vascular alterations like endothelial dysfunction, fibrosis, oxidative stress, and sustained inflammation and can be regarded as a main contributor to vascular inflammaging. This study was conducted to elucidate the mechanisms underlying endothelial CEACAM1 upregulation by TNF-α in detail. Using wildtype (WT) and TNF-α knockout (Tnf-/-) mice, we confirmed that the aging-related upregulation of endothelial CEACAM1 critically depends on TNF-α. The underlying mechanisms were analyzed in an endothelial cell culture model. TNF-α time-dependently upregulated CEACAM1 invitro. In pharmacological experiments, we identified an early NF-κB- and a delayed β-catenin-mediated response. Involvement of β-catenin was further substantiated by siRNA-mediated knockdown of the β-catenin-targeted transcription factor TCF4. Both signaling pathways acted independent from each other. Elucidating the delayed response, co-immunoprecipitation analysis revealed release of β-catenin from adherens junctions by TNF-α. Finally, TNF-α activated Akt kinase by increasing its Ser473 phosphorylation. Consequently, Akt kinase facilitated β-catenin signaling by inhibiting its degradation via phosphorylation of GSK3β at Ser9 and by increased phosphorylation of β-catenin at Ser552 that augments its transcriptional activity. Taken together, our study provides novel mechanistic insights into the aging-related, inflammation-mediated endothelial upregulation of CEACAM1. Beyond the pathogenesis of cardiovascular diseases, these findings may be significant to all fields of inflammaging.

Low-intensity pulsed ultrasound regulates bone marrow mesenchymal stromal cells differentiation and inhibits bone loss by activating the IL-11-Wnt/β-catenin signaling pathway

BackgroundOsteoporosis (OP) is a common metabolic bone disease. Low-intensity pulsed ultrasound (LIPUS) can effectively promote bone formation and fracture healing. The Wnt/β-catenin signaling pathway is crucial for regulating bone homeostasis and bone diseases, and its downregulation is one of the main mechanisms of osteoporosis pathogenesis. Interleukin-11 (IL-11), which is regulated by mechanical stress, is a key factor in bone remodeling. Here, we investigated the optimal intervention parameters for LIPUS, the relationships among LIPUS, IL-11, and the Wnt/β-catenin signaling pathway, and the effects of LIPUS on bone loss and potential molecular mechanisms in ovariectomized (OVX) mice. MethodsBone marrow mesenchymal stromal cells (BMSCs) were subjected to LIPUS intervention for 0, 10, or 20 min to determine the optimal intervention time. The mediating role of IL-11 in LIPUS intervention was explored through IL-11 knockdown and overexpression. Finally, animal experiments were conducted to investigate the in vivo therapeutic effects of LIPUS. ResultsThe optimal intervention time for LIPUS was 20 min. LIPUS promoted IL-11 expression and upregulated the Wnt/β-catenin signaling pathway, thereby promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs. IL-11 mediates the regulation of the Wnt/β-catenin signaling pathway by LIPUS. Additionally, LIPUS effectively improved the bone microstructure in ovariectomized mice, inhibited bone loss, promoted IL-11 expression in bone tissue, and activated the Wnt/β-catenin signaling pathway in the femur. ConclusionLow-intensity pulsed ultrasound can regulate BMSCs differentiation and inhibit bone loss by promoting IL-11 expression and activating the Wnt/β-catenin signaling pathway.