BTB Domain-containing Research Articles

Quercetin supplementation in metabolic syndrome: nutrigenetic interactions with the Zbtb16 gene variant in rodent models

BackgroundQuercetin is a promising phytochemical in treating abnormalities associated with metabolic syndrome (MetS). This study aimed to explore the morphometric, metabolic, transcriptomic, and nutrigenetic responses to quercetin supplementation using two genetically distinct MetS models that only differ in the variant of the MetS-related Zbtb16 gene (Zinc Finger And BTB Domain Containing 16).ResultsQuercetin supplementation led to a significant reduction in the relative weight of retroperitoneal adipose tissue in both investigated strains. A decrease in visceral (epididymal) fat mass, accompanied by an increase in brown fat mass after quercetin treatment, was observed exclusively in the SHR strain. While the levels of serum triglycerides decreased within both strains, the free fatty acids levels decreased in SHR-Zbtb16-Q rats only. The total serum cholesterol levels were not affected by quercetin in either of the two tested strains. While there were no significant changes in brown adipose tissue transcriptome, quercetin supplementation led to a pronounced gene expression shift in white retroperitoneal adipose tissue, particularly in SHR-Zbtb16-Q.ConclusionQuercetin administration ameliorates certain MetS-related features; however, the efficacy of the treatment exhibits subtle variations depending on the specific variant of the Zbtb16 gene.

E3 ubiquitin ligase BTBD3 inhibits tumorigenesis of colorectal cancer by regulating the TYRO3/Wnt/β-catenin signaling axis

Clinical trials and studies have implicated that E3 ubiquitin ligase BTBD3 (BTB Domain Containing 3) is a cancer-associated gene. However, the role and underlying mechanism of BTBD3 in colorectal cancer (CRC) is not fully understood yet. Herein, our study demonstrated that the mRNA and protein levels of BTBD3 were decreased in CRC tissues and associated with TYPO3 and Wnt/β-catenin pathway. Our results showed that circRAE1 knockdown and TYRO3 overexpression activated Wnt/β-catenin signaling pathway and the EMT process-associated markers, indicating that circRAE1/miR-388-3p/TYRO3 axis exacerbated tumorigenesis of CRC by activating Wnt/β-catenin signaling pathway. In addition, overexpression of BTBD3 reduced CRC cell migration and invasion in vitro and inhibited tumor growth in vivo. Our data demonstrated that BTBD3 suppressed CRC progression through negative regulation of the circRAE1/miR-388-3p/TYRO3 axis and the Wnt/β-catenin pathway. Our data further confirmed that BTBD3 bound and ubiquitinated β-catenin and led to β-catenin degradation, therefore blocked the Wnt/β-catenin pathway and suppressed the CRC tumorigenesis. This study explored the mechanism of BTBD3 involved in CRC tumorigenesis and provided a new theoretical basis for the prevention and treatment of CRC.

Abstract P224: RbFox2 is a Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Pathway Target that Regulates the Actin Cytoskeleton

Blood pressure (BP) is controlled by an array of cellular pathways. Our prior research revealed that endothelial nitric oxide (NO)-dependent vessel tone and BP are regulated through the RhoBTB1-CUL3 mediated proteasomal degradation of Phosphodiesterase 5 (PDE5). RhoBTB1 is an adaptor protein which delivers protein substrates to the CUL3 E3 ubiquitin ligase for proteasomal degradation. However, the range of protein targets of RhoBTB1 remains unknown. We employed the ascorbate peroxidase (APEX2) proximity labelling system coupled with mass spectroscopy to identify RhoBTB1 targets in A7r5 smooth muscle cells. This analysis identified nearly 75 proteins which putatively bind to the C terminal half of RhoBTB1 (known as B1B2C), the region we previously identified was essential for adaptor function. One of these proteins was RNA binding motif protein 9, known as RBM9 and RbFox2, a key regulator of splicing events. Subsequent co-immunoprecipitation (Co-IP) assays validated the interaction of RbFox2 with RhoBTB1 and its B1B2C domains. Expression of Rbfox2 was increased in response to treatment of A7r5 cells with either MG132, an inhibitor of the proteasome, and MLN4924, an inhibitor of the CUL family. Similarly, RbFox2 expression was increased in CRISPR-Cas9 generated CUL3-deficient HEK293 cells, in aorta isolated from SMC-specific CUL3-deficient mice, and in A7r5 cells expressing an siRNA targeting RhoBTB1. Ang-II treatment of mice, which raised blood pressure by 40 mmHg, also increased RbFox2 expression, presumably through down regulation of RhoBTB1. Targeting RbFox2 by siRNA in A7r5 cells resulted in a decrease in actin fiber formation detected by phalloidin staining. In summary, our findings suggest that RbFox2 regulation may occur through the RhoBTB1-CUL3 proteasomal pathway and may potentially play a mechanistic role in actin cytoskeleton organization and thus arterial stiffness associated with hypertension.

Abstract P197: Identification and Characterization of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Proteins in Placenta

Preeclampsia is life threatening condition affecting about 10% of all pregnancies. A potential mechanism is via malfunctioning syncytiotrophoblasts in the placenta. RhoBTB1 is an adapter protein for the CUL3 E3 ubiquitin ligase which delivers protein substrates for proteasomal degradation. An analysis of gene expression databases revealed that the highest level of RhoBTB1 expression is in the placenta. RNAscope in situ hybridization data reveals that RhoBTB1 expression is specifically localized to syncytiotrophoblasts. In blood vessels, RhoBTB1 controls the state of nitric oxide mediated vasodilation because it acts as a substrate adaptor for Phosphodiesterase 5. Thus, we wanted to identify potential protein targets of RhoBTB1 in the placenta to assess if RhoBTB1 plays a role in placental function. To simplify the first identification of RhoBTB1 binding proteins, we employed HTR-8 /SVneo (HTR-8) cells as a model of placental trophoblast cells. We biotinylated the HTR-8 proteome utilizing ascorbate peroxidase (APEX2) proximity labelling system followed by mass spectrometry to identify RhoBTB1 targets. We employed a fusion protein consisting of the C-terminal half of RhoBTB1 (B1B2C), the region required for the binding of other protein substrates, and a control lacking the most C-terminal region (B1B2). We next chose proteins with binding to B1B2C > B1B2 of 2-fold or greater that were expressed in the placenta based on the Human Protein Atlas. Rho GTPase Activating Protein 29 (ARHGAP29), which controls the regulation of small GTP binding proteins, and S-phase Kinase associated Protein 2 (SKP2), a regulator of cell cycle progression was studied further. SKP2 is interesting because it exhibits a tissue specific tropism for placental expression with its highest expression in extravillous trophoblasts, cytotrophoblasts, and syncytiotrophoblasts. Expression of ARHGAP29 and SKP2 in HTR-8 cells were markedly upregulated by MLN4924 suggesting they are regulated by the CUL pathway. They were also modestly upregulated after partial inhibition of RhoBTB1 by siRNA suggesting they may be RhoBTB1-CUL3 targets. Studies are ongoing to validate the physical interaction of ARHGAP29 and SKP2 with RhoBTB1 by co-immunoprecipitation. We are also using RNAscope to assess the co-expression of RhoBTB1, ARHGAP29 and SKP2 in the human placenta from normal and preeclamptic pregnancies.

Polyphyllin I exerts anti-hepatocellular carcinoma activity by targeting ZBTB16 to activate the PPARγ/RXRα signaling pathway

BackgroundStudies have reported that polyphyllin I (PPI) had effective anti-tumor activity against hepatocellular carcinoma (HCC). However, the precise molecular mechanism of this action and the direct target remain unclear. The aim of this study was to discover the molecular targets and the exact mechanism of PPI in the treatment of HCC.MethodsVarious HCC cells and Zebrafish xenotransplantation models were used to examine the efficacy of PPI against HCC. A proteome microarray, surface plasmon resonance (SPR) analysis, small molecule transfection, and molecular docking were conducted to confirm the direct binding targets of PPI. Transcriptome and Western blotting were then used to determine the exact responding mechanism. Finally, the anticancer effect and its precise mechanism, as well as the safety of PPI, were verified using a mouse tumor xenograft study.ResultsThe results demonstrated that PPI had significant anticancer activity against HCC in both in vitro studies of two cells and the zebrafish model. Notably, PPI selectively enhanced the action of the Zinc finger and BTB domain-containing 16 (ZBTB16) protein by directly binding to it. Furthermore, specific knockdown of ZBTB16 markedly attenuated PPI-dependent inhibition of HCC cell proliferation and migration caused by overexpression of the gene. The transcriptome and Western blotting also confirmed that the interaction between ZBTB16 and PPI also activated the PPARγ/RXRα pathway. Finally, the mouse experiments confirmed the efficacy and safety of PPI to treat HCC.ConclusionsOur results indicate that ZBTB16 is a promising drug target for HCC and that PPI as a potent ZBTB16 agonist has potential as a therapeutic agent against HCC by regulating the ZBTB16/PPARγ/RXRα signaling axis.

Zbtb11 interacts with Otx2 and patterns the anterior neuroectoderm in Xenopus.

The zinc finger and BTB domain-containing 11 gene (zbtb11) is expressed in the Xenopus anterior neuroectoderm, but the molecular nature of the Zbtb11 protein during embryonic development remains to be elucidated. Here, we show the role of Zbtb11 in anterior patterning of the neuroectoderm and the cooperative action with the transcription factor Otx2. Both overexpression and knockdown of zbtb11 caused similar phenotypes: expanded expression of the posterior gene gbx2 in the neural plate, and later microcephaly with reduced eyes, suggesting that a proper level of zbtb11 expression is necessary for normal patterning of the neuroectoderm, including eye formation. Co-immunoprecipitation assays showed that Zbtb11 formed a complex with itself and with a phosphomimetic and repressive form of Otx2, suggesting that Zbtb11 forms a dimer or oligomer and interacts with Otx2 in a phosphorylation-dependent manner. Reporter analysis further showed that Zbtb11 enhanced the activity of the phosphomimetic Otx2 to repress a silencer element of the posterior gene meis3. These data suggest that Zbtb11 coordinates with phosphorylated Otx2 to specify the anterior neuroectoderm by repressing posterior genes.

Identification and Characterization of Potential Anti-Hypertensive Therapeutic Candidates in Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Axis

Blood pressure (BP) is regulated by a variety of cellular mechanisms. We previously reported the regulation of endothelial nitric oxide (NO)-dependent vessel tone and BP through RhoBTB1-CUL3 mediated proteasomal turnover of Phosphodiesterase 5 (PDE5). We determined that the C-terminal half of RhoBTB1 consisting of two BTB-domains (B1 & B2) and the C-terminal (C) domain of RhoBTB1 can deliver PDE5 to proteasomal pathway. The cellular expression of RhoBTB1 and CUL3 is ubiquitous whereas expression of PDE5 is cell specific. Moreover, vessel tone is regulated through NO-independent mechanisms. Therefore, we hypothesized that RhoBTB1 may have other interacting partners, potentially functioning as regulators of vessel tone and BP in vascular smooth muscle (VSM) cells, through a similar mode of binding as PDE5. Therefore, we utilized ascorbate peroxidase (APEX2) proximity labelling system comprising APEX2 tagged B1B2C domain (B1B2C-APEX2) to identify unknown potential regulators of vasomotor tone in RhoBTB1-CUL3 axis in A7R5 VSM cells. The background of the proteomic screen was minimized by utilizing APEX2-B1B2 which lacks the final C-terminal region of the protein and cannot bind PDE5. We validated the interaction of several identified proteins with RhoBTB1 and the B1B2C domains using co-immunoprecipitation (Co-IP). Our Co-IP data corroborated the proteomic screen as several proteins demonstrated differential interaction with B1B2C and B1B2. Among them, we examined RbFox2, a regulator of mRNA splicing for further investigation. Further data established that RbFox2 is upregulated under the following conditions: 1) pharmacological inhibition of the proteasomal pathway, 2) in CRISPR edited CUL3KO HEK293 cells, 3) in aorta of VSM cell-specific CUL3 deleted mice, 4) by siRNA mediated knock-down of RhoBTB1 in SMCs, and 5) in Ang-II treated SMCs and aorta of Ang-II treated animals. We hypothesized that RbFox2 could modulate actin and actinin-dependent cytoskeletal dynamics, an important regulator of arterial stiffness. First, Co-IP data suggest an interaction between RbFox2 and SMC-specific alpha actinin. Next, siRNA-mediated knock down of RbFox2 altered the actin and actinin-dependent cytoskeletal dynamics in SMCs. Collectively, this establishes the potential regulation of RbFox2 through the RhoBTB1-CUL3 proteasomal pathway and that RbFox2 may play a mechanistic role in arterial stiffness in hypertension. Funding: NIH R35 HL144807 (to Dr. Curt D. Sigmund). 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.

Knockdown of KBTBD7 attenuates septic lung injury by inhibiting ferroptosis and improving mitochondrial dysfunction

Lung injury in sepsis is caused by an excessive inflammatory response caused by the entry of pathogenic microorganisms into the body. It is also accompanied by the production of large amounts of ROS. Ferroptosis and mitochondrial dysfunction have also been shown to be related to sepsis. Finding suitable sepsis therapeutic targets is crucial for sepsis research. BTB domain-containing protein 7 (KBTBD7) is involved in regulating inflammatory responses, but its role and mechanism in the treatment of septic lung injury are still unclear. In this study, we evaluated the role and related mechanisms of KBTBD7 in septic lung injury. In in vitro studies, we established an in vitro model by inducing human alveolar epithelial cells with lipopolysaccharide (LPS) and found that KBTBD7 was highly expressed in the in vitro model. KBTBD7 knockdown could reduce the inflammatory response by inhibiting the secretion of pro-inflammatory factors and inhibit the production of ROS, ferroptosis and mitochondrial dysfunction. Mechanistic studies show that KBTBD7 interacts with FOXA1, promotes FOXA1 expression, and indirectly inhibits SLC7A11 transcription. In vivo studies have shown that knocking down KBTBD7 improves lung tissue damage in septic lung injury mice, inhibits inflammatory factors, ROS production and ferroptosis. Taken together, knockdown of KBTBD7 shows an alleviating effect on septic lung injury in vitro and in vivo, providing a potential therapeutic target for the treatment of septic lung injury.

Tanshinone IIA alleviates bleomycin-induced pulmonary fibrosis by inhibiting Zbtb16

Pulmonary fibrosis is a complex disease that can occur in a variety of clinical settings. The Zinc Finger and BTB Domain Containing 16 (Zbtb16) is a transcription factor and has not been studied in pulmonary fibrosis. Lung tissues from rats which were treated with bleomycin and Tanshinone IIA (Tan IIA) were collected for mRNA sequencing. Zbtb16, a differentially expressed gene, was screened. Using adeno-associated virus to knock down Zbtb16 in rats, it was found that the lung index and the content of hydroxyproline in lung tissue were decreased. HE and Masson staining revealed that pathological symptoms of lung histopathology were relieved after Zbtb16 knockdown. Protein expressions of α-SMA, Collagen I and Fibronectin were significantly decreased after Zbtb16 knockdown in vivo and in vitro. Meanwhile, the protein content of TGF-β1 and the phosphorylation of Smad2/3 were inhibited by Zbtb16 knockdown. Conversely, under the treatment of Tan IIA and TGF-β1, overexpression of Zbtb16 improved cell viability, increased the expression of fibrosis-related proteins, and promoted the phosphorylation of Smad 2/3. All above demonstrates that Zbtb16 inhibition ameliorates pulmonary fibrosis and suppresses the TGF-β/Smad pathway. Furthermore, Zbtb16 mediates the inhibitory process of Tan IIA on pulmonary fibrosis. This study provides a novel candidate therapeutic target for pulmonary fibrosis.

Crosstalk between short- and long-term calorie restriction transcriptomic signatures with anxiety-like behavior, aging, and neurodegeneration: implications for drug repurposing

Calorie restriction (CR) is considered an effective intervention for anxiety, aging, and obesity. We investigated the effects of short- and long-term CR on behavior as well as transcriptome profiles in the hypothalamus, amygdala, prefrontal cortex, pituitary, and adrenal glands of Hooded Wistar and Long Evans male rats. A reduction in anxiety-like behavior, as assessed via the elevated plus maze, was observed in both short- and long-term CR. Despite this, short- and long-term CR regulated different sets of genes, leading to distinct transcriptomic signatures. The employed models were able to simultaneously analyze categorical and numerical variables, evaluating the effect of tissue type along with expression data. In all tissues, transcription factors, zinc finger protein 45-like and zinc finger BTB domain-containing two, were the top selected genes by the models in short and long-term CR treatments, respectively. Text mining identified associations between genes of the short-term CR signature and neurodegeneration, stress, and obesity and between genes of the long-term signature and the nervous system. Literature mining-based drug repurposing showed that alongside known CR mimetics such as resveratrol and rapamycin, candidates not typically associated with CR mimetics may be repurposed based on their interaction with transcriptomic signatures of CR. This study goes some way to unravelling the global effects of CR and opens new avenues for treatment for emotional disorders, neurodegeneration, and obesity.

ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein

ABSTRACT Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.

Zbtb14 Promotes Non-Alcoholic Fatty Liver Disease-Associated Fibrosis in Gerbils via the β-Catenin Pathway.

Non-alcoholic fatty liver disease (NAFLD) is a popular chronic liver disorder with high morbidity and with no approved therapeutic drugs. Fibrosis is a crucial drug efficacy indicator for NAFLD. Thus, investigating the mechanisms of NAFLD-associated fibrosis and exploring effective therapeutic targets is imperative. Gerbil NAFLD-associated fibrosis model was constructed by feeding a high-fat and high-cholesterol diet. The hematoxylin and eosin staining and the alanine transaminase (ALT) and aspartate transaminase (AST) assays were used to determine liver tissue injury. Masson staining and hydroxyproline (Hyp) level determination were used to assess liver fibrosis. High-throughput mRNA sequencing was used to screen differentially expressed genes in the NAFLD-associated fibrosis model. Cell Counting Kit-8 was utilized to test cell viability. Liver injury and fibrosis were observed in the gerbil NAFLD-associated fibrosis model with increased ALT, AST, and Hyp levels. The screened differentially expressed genes were mainly enriched in "negative regulation of hemopoiesis", "response to interleukin-1", and "granulocyte migration". Zinc Finger and BTB Domain Containing 14 (Zbtb14) was upregulated in liver tissues of the gerbil NAFLD-associated fibrosis model, patients with liver fibrosis, and hepatic stellate cells (HSCs). Additionally, Zbtb14 regulated primary HSCs activation via the β-catenin pathway. Zbtb14 regulated NAFLD-associated fibrosis via the β-catenin pathway, for the first time, and it serves as the probable target for NAFLD therapy.

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts through regulation of the HOXA6/ZBTB12 axis.

Cancer-associated fibroblasts (CAFs) play critical roles in the tumor microenvironment and exert tumor-promoting or tumor-retarding effects on cancer development. Astragaloside IV has been suggested to rescue the pathological impact of CAFs in gastric cancer. This study aimed to investigate the potential mechanism of astragaloside IV in the regulation of CAF pathological functions in gastric cancer development. Homeobox A6 (HOXA6), and Zinc Finger and BTB Domain Containing 12 (ZBTB12) are highly expressed in gastric CAFs compared with normal fibroblasts (NFs) based on the GSE62740 dataset. We found that astragaloside IV-stimulated CAFs suppressed cell growth, migration, and invasiveness of gastric cancer cells. HOXA6 and ZBTB12 were downregulated after astragaloside IV treatment in CAFs. Further analysis revealed that HOXA6 or ZBTB12 knockdown in CAFs also exerted inhibitory effects on the malignant phenotypes of gastric cells. Additionally, HOXA6 or ZBTB12 overexpression in CAFs enhanced gastric cancer cell malignancy, which was reversed after astragaloside IV treatment. Moreover, based on the hTFtarget database, ZBTB12 is a target gene that may be transcriptionally regulated by HOXA6. The binding between HOXA6 and ZBTB12 promoter in 293T cells and CAFs was further confirmed. HOXA6 silencing also induced the downregulation of ZBTB12 mRNA and protein in CAFs. Astragaloside IV was demonstrated to regulate the expression of ZBTB12 by mediating the transcriptional activity of HOXA6. Our findings shed light on the therapeutic value of astragaloside IV for gastric cancer.

Proteomic approach to explore the role of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin-3 (CUL-3) pathway in identifying potential therapeutic candidates in cardioprotection

Protein-protein interaction studies have contributed enormously to understand the fundamentals of human impairments including cardiovascular diseases. We previously identified Phosphodiesterase 5 (PDE5) as an interacting partner of RhoBTB1. The latter can function as an adapter for CUL-3 E3 ring ubiquitin ligase complex delivering PDE5 for ubiquitination and proteasomal degradation. This in turn regulates the nitric oxide response of vascular smooth muscle (VSM) and consequently blood pressure. Ubiquitous expression of RhoBTB1, its link with CUL-3, and PDE5 suggests that RhoBTB1 may have other binding partners in VSM and other cells. The binding sites of these partners on RhoBTB1 might be similar to PDE5. With the notion that PDE5 binding region on RhoBTB1 could be responsible for binding of unknown partners in RhoBTB1-CUL-3 pathway, we truncated full-length RhoBTB1 into its component domains, individually (GTPase, BTB1, BTB2, and C-terminal (CT)) and in combination (e.g. B1B2C, B2C, and PB1B2). HEK293 cells were used to express epitope-tagged RhoBTB1 constructs and PDE5. The interaction between different constructs of RhoBTB1 and PDE5 was determined by co-immunoprecipitation (Co-IP). We showed that RhoBTB1 could bind to PDE5 utilizing the combination of its B1B2C domains. Our data also suggest that the CT domain itself was essential for the interaction between RhoBTB1 and PDE5 but was not sufficient on its own to bind PDE5. The Co-IP data was further corroborated with the findings of immunofluorescence and proximity labeling assay (PLA). Subsequent protein stability and ubiquitination studies revealed that B1B2C is catalytically sufficient for CUL3-dependent proteasomal degradation like full-length RhoBTB1. Furthermore, macromolecular protein-protein docking revealed the binding interface between RhoBTB1 and CUL-3 comprising of 24 amino acids. Additional Co-IP data suggested that Ser363 and Pro353 on RhoBTB1 domain are required for binding to CUL-3 as mutants of RhoBTB1 (Ser363Ala and Pro353Ala) could not deliver PDE5 for proteasomal degradation. To further identify unknown interacting partners in RhoBTB1-CUL-3 axis, we utilized the ascorbate peroxidase (APEX2) proximity labelling system involving B1B2C as “bait.” Proteomic profiling of the biotinylated proteome through mass spectrometry has identified several unknown interacting partners involved in RhoBTB1-CUL-3 pathway. Some of these have demonstrated upregulation on pharmacological inhibition of proteasome and CUL-3 which establishes their potential involvement in RhoBTB1-CUL-3 pathway. Exploring the involvement of such interacting partners in RhoBTB1-CUL-3 axis may open novel therapeutic avenues in cardioprotection. NIH R35 HL144807 (to Dr. Curt D. Sigmund) This is the full abstract presented at the American Physiology Summit 2023 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.

ZBTB16 eases lipopolysaccharide‑elicited inflammation, apoptosis and degradation of extracellular matrix in chondrocytes during osteoarthritis by suppressing GRK2 transcription.

Osteoarthritis (OA) is a chronic degenerative disease of the bone that is a major contributor of disability in the elderly population. Zinc finger and BTB domain-containing 16 (ZBTB16) is a transcription factor that has been previously revealed to be impaired in human OA tissues. The present study was designed to elaborate the potential impact of ZBTB16 on OA and to possibly assess any latent regulatory mechanism. ZBTB16 expression in human OA tissues was examined using the Gene Expression Series (GSE) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE169077) whereas ZBTB16 expression in chondrocytes was examined using reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Cell viability was examined using a Cell Counting Kit-8 assay. A TUNEL assay and western blotting were used to assess cell apoptosis and apoptosis-related markers, including Bcl-2, Bax and cleaved caspase-3. The levels and expression of inflammatory factors, including TNF-α, IL-1β and IL-6, were determined by ELISA and western blotting. RT-qPCR and western blotting were also used to analyze the expression levels of extracellular matrix (ECM)-degrading enzymes, including MMP-13, a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5, aggrecan and collagen type II α1. After the potential binding of ZBTB16 with the G protein coupled receptor kinase type 2 (GRK2) promoter was predicted using the Cistrome DB database, GRK2 expression was confirmed by RT-qPCR and western blotting. Chromatin immunoprecipitation and luciferase reporter assays were then used to determine the potential interaction between ZBTB16 and the GRK2 promoter. Following GRK2 overexpression in ZBTB16-overexpressing chondrocytes by co-transfection of GRK2 and ZBTB16 overexpression plasmids, the aforementioned functional experiments were performed again. ZBTB16 expression was found to be reduced in human OA tissues compared with in normal cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. ZBTB16 overexpression increased cell viability whilst decreasing apoptosis, inflammation and ECM degradation by LPS-treated chondrocytes. In addition, GRK2 expression was found to be increased in LPS-stimulated chondrocytes. ZBTB16 successfully bound to the GRK2 promoter, which negatively modulated GRK2 expression. GRK2 upregulation reversed the effects of ZBTB16 overexpression on the viability, apoptosis, inflammation and ECM degradation by LPS-challenged chondrocytes. In conclusion, these data suggest that ZBTB16 may inhibit the development of OA through the transcriptional inactivation of GRK2.

MiR-202-5p Regulates Geese Follicular Selection by Targeting BTBD10 to Regulate Granulosa Cell Proliferation and Apoptosis

The regulation of granulosa cells (GCs) proliferation and apoptosis is the key step in follicular selection which determines the egg production performance of poultry. miR-202-5p has been reported to be involved in regulating the proliferation and apoptosis of mammalian ovarian GCs. However, its role in regulating the proliferation and apoptosis of goose GCs is still unknown. In the present study, the GCs of pre-hierarchical follicles (phGCs, 8-10 mm) and those of hierarchical follicles (hGCs, F2-F4) were used to investigate the role of miR-202-5p in cell proliferation and apoptosis during follicle selection. In phGCs and hGCs cultured in vitro, miR-202-5p was found to negatively regulate cell proliferation and positively regulate cell apoptosis. The results of RNA-seq showed that BTB Domain Containing 10 (BTBD10) is predicted to be a key target gene for miR-202-5p to regulate the proliferation and apoptosis of GCs. Furthermore, it is confirmed that miR-202-5p can inhibit BTBD10 expression by targeting its 3'UTR region, and BTBD10 was revealed to promote the proliferation and inhibit the apoptosis of phGCs and hGCs. Additionally, co-transfection with BTBD10 effectively prevented miR-202-5p mimic-induced cell apoptosis and the inhibition of cell proliferation. Meanwhile, miR-202-5p also remarkably inhibited the expression of Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta (PIK3CB) and AKT Serine/Threonine Kinase 1 (AKT1), while it was significantly restored by BTBD10. Overall, miR-202-5p suppresses the proliferation and promotes the apoptosis of GCs through the downregulation of PIK3CB/AKT1 signaling by targeting BTBD10 during follicular selection. Our study provides a theoretical reference for understanding the molecular mechanism of goose follicular selection, as well as a candidate gene for molecular marker-assisted breeding to improve the geese' egg production performance.

Hepatic ZBTB22-mediated detoxification ameliorates acetaminophen-induced liver injury by inhibiting pregnane X receptor signaling

Overdose acetaminophen (APAP) can cause acute liver injury (ALI), but the underlying mechanism remains undetermined. This study explored the role of hepatic Zinc Finger And BTB Domain Containing 22 (ZBTB22) in defense against APAP-mediated hepatotoxicity. The results showed that hepatic ZBTB22 expression was significantly reduced in patients with ALI and mice. In mouse primary hepatocytes (MPHs), ZBTB22 deletion aggravated APAP overdose-induced ALI, whereas ZBTB22 overexpression attenuated that pathological progression. The results were further verified in ZBTB22 over-express or knockout mice models. In parallel, hepatocyte-specific ZBTB22 knockout also enhanced ALI. Furthermore, ZBTB22 decreased pregnane X receptor (PXR) expression, and the PXR activator pregnane-16α-carbonitrile suppressed the protective effect of ZBTB22 in APAP-induced ZBTB22-overexpressing mice. Collectively, our findings highlight the protective effect of ZBTB22 against APAP-induced ALI and unravel PXR signaling as the potential mechanism. Strategies to increase hepatic ZBTB22 expression represent a promising therapeutic approach for APAP overdose-induced ALI.

GCN5 regulates ZBTB16 through acetylation, mediates osteogenic differentiation, and affects orthodontic tooth movement.

In the process of orthodontic tooth movement (OTM), periodontal ligament fibroblasts (PDLFs) must undergo osteogenic differentiation. OTM increased the expression of Zinc finger and BTB domain-containing 16 (ZBTB16), which is implicated in osteogenic differentiation. Our goal was to investigate the mechanism of PDLF osteogenic differentiation mediated by ZBTB16. The OTM rat model was established, and PDLFs were isolated and exposed to mechanical force. Hematoxylin-eosin staining, Alizarin Red staining, immunofluorescence, and immunohistochemistry were carried out. The alkaline phosphatase (ALP) activity was measured. Dual-luciferase reporter gene assay and chromatin immunoprecipitation assay were conducted. In OTM models, ZBTB16 was significantly expressed. Additionally, there was an uneven distribution of PDLFs in the OTM group, as well as an increase in fibroblasts and inflammatory infiltration. ZBTB16 interference hindered PDLF osteogenic differentiation and decreased Wnt and β-catenin levels. Meanwhile, ZBTB16 activated the Wnt/β-catenin pathway. ZBTB16 also enhanced the expression of the osteogenic molecules osterix, osteocalcin (OCN), osteopontin (OPN), and bone sialo protein (BSP) at mRNA and protein levels. The interactions between Wnt1 and ZBTB16, as well as GCN5 and ZBTB16, were also verified. The adeno-associated virus-shZBTB16 injection also proved to inhibit osteogenic differentiation and reduce tooth movement distance in in vivo tests. ZBTB16 was up-regulated in OTM. Through acetylation modification of ZBTB16, GCN5 regulated the Wnt/β-catenin signaling pathway and further mediated PDLF osteogenic differentiation.

MEDB-62. Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation

Genomic studies in medulloblastoma have identified distinct disease subgroups: wnt/wingless (WNT), sonic hedgehog (SHH), and non-WNT/non-SHH, comprising group 3 and group 4. Alterations in WNT and SHH signalling form the pathogenetic basis for their subgroups, whereas those for non-WNT/non-SHH tumours remain largely elusive. Recent analyses have revealed recurrent in-frame insertions in the E3 ubiquitin ligase adaptor Kelch Repeat and BTB Domain Containing 4 (KBTBD4) in cases of group 3 and group 4 medulloblastoma. Critically, group 3 and 4 tumours with KBTBD4 mutations typically lack other gene-specific alterations, such as MYC amplification, indicating KBTBD4 insertion mutations as the primary genetic driver of these malignancies. Delineating the role of KBTBD4 mutations in medulloblastoma thus offers significant opportunities to understand its pathogenesis and exploit underpinning mechanisms therapeutically, however their function is currently unknown. Here, we show a novel mechanism in cancer pathogenesis whereby indel mutations in KBTBD4 drive its recognition of neo-substrates for degradation. We observe that KBTBD4 mutants promote the recruitment and ubiquitylation of the REST Corepressor (CoREST), which forms a complex to modulate chromatin accessibility and transcriptional programmes. The degradation of CoREST promoted by KBTBD4 mutations diverts epigenetic programmes inducing significant alterations in transcription to promote increased stemness of cancer cells. Transcriptional analysis of >200 human group 3 and 4 medulloblastomas by RNA-seq, highlights the presence of CoREST and stem-like signatures in tumours with KBTBD4 mutations, which extend to a further sub-set of non-mutant tumours, suggesting CoREST alterations as a novel pathogenetic mechanism of wide relevance in group 3 and 4. Our findings uncover KBTBD4 mutation as a novel driver of epigenetic reprogramming in non-WNT/non-SHH medulloblastoma, establishes a novel mode of tumorigenesis through gain-of-function mutations in ubiquitin ligases (neo-substrate recruitment) and identifies both mutant KBTBD4 and CoREST complexes as new druggable targets for improved tumour-specific therapies.

MiR-361-5p promotes proliferation and inhibits apoptosis of fibroblast-like synoviocytes via targeting ZBTB10 in rheumatoid arthritis

Objectives This study is aimed to explore the key role of miR-361-5p in fibroblast-like synovial (FLS) cells of rheumatoid arthritis (RA) and explore the underlying mechanism. Methods First, we performed RT-qPCR to evaluate the expression of miR-361-5p in both synovial tissues of RA patients and cultured RA-FLS cells. Then CCK-8 assay, EdU staining, Western blot, flow cytometry, and ELISA were conducted to estimate the influence of inhibiting miR-361-5p on RA-FLS cells. Moreover, we used bioinformatics analysis to predict the potential targets of miR-361-5p and perform a dual luciferase report assay for verification. Finally, rescue experiments were performed to prove the role of miR-361-5p/Zinc Finger And BTB Domain Containing 10 (ZBTB10) in the proliferation, cell cycle, and apoptosis of RA-FLS. Results We find that the expression of miR-361-5p is increased in both RA tissues and cultured RA-FLS cells. The inhibition of miR-361-5p can not only inhibit proliferation, arrest the cell cycle in G1/G0 phase, and increase apoptosis, but also reduce the inflammatory factors secreted by RA-FLS cells. In addition, ZBTB10 is a direct target for miR-361-5p, over-expression of ZBTB10 reverses the effect of miR-361-5p in RA-FLS. Conclusions MiR-361-5p promotes the progression of rheumatoid arthritis by targeting ZBTB10. Key points The influences of miR-361-5p on RA-FLS cells.