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Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the β-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of β-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/β-catenin signaling during development.

UNLABELLED: AXIN1 is a major component of the β-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to β-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3β, β-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper β-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected β-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on β-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the β-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect β-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other β-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. SIGNIFICANCE: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its β-catenin regulatory function, whereas the majority are passenger mutations.

Objective: To investigate the protective effect of the Chinese herbal formula of Jiedu Huayu decoction (, JHD) on oral mucosa of rats with oral submucosal fibrosis (OSF) and its potential mechanism of action., Methods: Sprague-Dawley male OSF model rats were constructed by injection of betaine and topical rubbing and were randomly grouped and administered by gavage for 4 weeks. Mouth opening and buccal mucosa scores interleukin levels and the expression of Axin and β-catenin proteins or genes were measured before and after drug administration., Results: After treatment with JHD the buccal mucosal lesions of rats were significantly reduced Axin protein and mRNA expression were significantly increased β-catenin protein and mRNA expression were significantly decreased interleukin-1β and interleukin-6 levels were decreased and interleukin-10 levels were increased., Conclusion: The mechanism of action of JHD can effectively alleviate the pathological damage of buccal mucosa in OSF rats which may be related to the promotion of Axin expression and inhibition of β-catenin expression.

Objective: To explore the protective effect of human urine-derived stem cell exosomes (hUSC-Exos) on radiation-induced salivary gland (SG) injuries in Sprague Dawley rats., Methods: Fresh adult urine was collected, and primary hUSCs were isolated and identified. The hUSCs were hypoxia-pretreated with 1% oxygen for 24 h and then transferred to a normoxic culture environment for 24 h. The hUSC-Exos were collected and identified for exosomes. A radiation-induced injury model was established in the rats, and exosomes were introduced by local injection in the SG and tail vein. The submandibular gland was excised for morphological observation 1 week later. Immunohistochemical detection of the glandular tissue was conducted by α-smooth muscle actin (a-SMA), stem cell growth factor receptor (c-Kit) staining, and periodic acid-Schiff staining. Qualitative polymerase chain reaction and western blot analysis were adopted to detect the gene and protein expression of Wnt3a, GSK3β, and Axin., Results: In both the normoxic and hypoxic hUSC-Exo groups, microvesicular structures with bilayer membranes of approximately 80 nm in diameter were detected, and the expressions of CD9 and CD63 were detected by nanoflow cytometry. Compared with the control group, in the radiation-induced injury model group, the expression of a-SMA was significantly higher, the expression of c-Kit was significantly lower, and the expressions of Wnt3a, GSK3β, and Axin were significantly upregulated; the differences were statistically significant (p < 0.05). Compared with the model group, in the normoxic and hypoxic hUSC-Exo groups, the expression of a-SMA was significantly decreased, the expression of c-Kit was significantly increased, and the expressions of Wnt3a, GSK3β, and Axin were significantly upregulated; the differences were statistically significant (p < 0.05)., Conclusion: Hypoxia-pretreated hUSC-Exos could repair radiation-induced SG injuries by activating the Wnt3a/GSK3β pathway to suppress the expressions of a-SMA and c-Kit., (© 2022 Wiley Periodicals LLC.)

R-spondins (RSPOs) are secreted signaling molecules that potentiate the Wnt/β-catenin pathway by cooperating with Wnt ligands. RSPO1 is crucial in tissue development and tissue homeostasis. However, the molecular mechanism by which RSPOs activate Wnt/β-catenin signaling remains elusive. In this study, we found that RSPOs could mediate the degradation of Axin through the ubiquitin-proteasome pathway. The results of Co-IP showed that the recombinant RSPO1 protein promoted the interaction between Axin1 and CK1ε. Either knockout of the CK1ε gene or treatment with the CK1δ/CK1ε inhibitor SR3029 caused an increase in Axin1 protein levels and attenuated RSPO1-induced degradation of the Axin1 protein. Moreover, we observed an increase in the number of associations of LRP6 with CK1ε and Axin1 following RSPO1 stimulation. Overexpression of LRP6 further potentiated Axin1 degradation mediated by RSPO1 or CK1ε. In addition, recombinant RSPO1 and Wnt3A proteins synergistically downregulated the protein expression of Axin1 and enhanced the transcriptional activity of the SuperTOPFlash reporter. Taken together, these results uncover the novel mechanism by which RSPOs activate Wnt/β-catenin signaling through LRP6/CK1ε-mediated degradation of Axin., (© 2024. The Author(s).)

Background: A myriad of therapeutic modalities for alopecia areata are available; however, none is of high level of evidence, creating an immense need for the evaluation of other treatment modalities, of which topical sodium valproate is of potential role via proposed decrease in beta-catenin breakdown, despite its well-known side effect of hair fall as an oral therapy., Objective: Evaluating the efficacy and the safety of sodium valproate (SV)-loaded nanospanlastics, in comparison to topical corticosteroids, this is the currently available gold standard topical treatment for patchy AA., Methodology: A total of 66 patients with patchy AA were randomly assigned to receive either topical mometasone furoate lotion or topical SV applied twice daily to all patches except a control patch, which was left untreated. Clinical, trichoscopic and biochemical assessments of beta-catenin tissue levels and Axin-2 gene expression were carried out at baseline and after 3 months., Results: Both therapeutic modalities were comparable. Potential efficacy was highlighted by significant improvement in the representative patch, the largest treated patch, to the control patch, the smallest untreated patch in both steroid and valproate groups (p = 0.027, 0.003 respectively). Both beta-catenin levels and Axin-2 gene expression were reduced after treatment, pointing to the inhibitory effect of dominating uncontrolled inflammatory milieu. Baseline beta-catenin was found to significantly negatively correlate with improvement in the representative patch in patients with baseline level above 0.42 ng/ml (p = - 0.042)., Conclusion: Both topical SV and steroids are of comparable modest efficacy. Thus, further evaluation of SV is due in combination with intralesional steroids and other anti-inflammatory treatment modalities, together with developing individualized approaches based on baseline beta-catenin level., Gov Identifier: NCT05017454, https://clinicaltrials.gov/ct2/show/NCT05017454 ., (© 2024. The Author(s).)

Colorectal cancer (CRC) is one of the most common neoplasms worldwide. Among the risk factors of CRC, inflammatory bowel disease (IBD) is one of the most important ones leading to the development of colitis-associated CRC (CAC). G-protein coupled receptors (GPCR) are transmembrane receptors that orchestrate a multitude of signaling cascades in response to external stimuli. Because of their functionality, they are promising targets in research on new strategies for CRC diagnostics and treatment. Recently, regulators of G-proteins (RGS) have been attracting attention in the field of oncology. Typically, they serve as negative regulators of GPCR responses to both physiological stimuli and medications. RGS activity can lead to both beneficial and harmful effects depending on the nature of the stimulus. However, the atypical RGS-AXIN uses its RGS domain to antagonize key signaling pathways in CRC development through the stabilization of the β-catenin destruction complex. Since AXIN does not limit the efficiency of medications, it seems to be an even more promising pharmacological target in CRC treatment. In this review, we discuss the current state of knowledge on RGS significance in sporadic CRC and CAC with particular emphasis on the regulation of GPCR involved in IBD-related inflammation comprising opioid, cannabinoid and serotonin receptors.