Inositol 1,4,5-trisphosphate receptor type 2 is associated with the bone-vessel axis in chronic kidney disease-mineral bone disorder.

Objective: The pathogenesis of renal osteopathy and cardiovascular disease suggests the disordered bone-vessel axis in chronic kidney disease-mineral bone disorder (CKD-MBD). However, the mechanism of the bone-vessel axis in CKD-MBD remains unclear.Methods: We established a CKD-MBD rat model to observe the pathophysiological phenotype of the bone-vessel axis and performed RNA sequencing of aortas to identify novel targets of the bone-vessel axis in CKD-MBD.Results: The microarchitecture of the femoral trabecular bone deteriorated and alveolar bone loss was aggravated in CKD-MBD rats. The intact parathyroid hormone and alkaline phosphatase levels increased, 1,25-dihydroxyvitamin D3 levels decreased, and intact fibroblast growth factor-23 levels did not increase in CKD-MBD rats at 16 weeks; other bone metabolic parameters in the serum demonstrated dynamic characteristics. With calcium deposition in the abdominal aortas of CKD-MBD rats, RNA sequencing of the aortas revealed a significant decrease in inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) gene levels in CKD-MBD rats. A similar trend was observed in rat aortic smooth muscle cells. As a secretory protein, ITPR2 serum levels decreased at 4 weeks and slightly increased without statistical differences at 16 weeks in CKD-MBD rats. ITPR2 serum levels were significantly increased in patients with vascular calcification, negatively correlated with blood urea nitrogen levels, and positively correlated with serum tartrate-resistant acid phosphatase 5b levels.Conclusion: These findings provide preliminary insights into the role of ITPR2 in the bone-vessel axis in CKD-MBD. Thus, ITPR2 may be a potential target of the bone-vessel axis in CKD-MBD.

Ubiquitin-specific protease 47 is associated with vascular calcification in chronic kidney disease by regulating osteogenic transdifferentiation of vascular smooth muscle cells.

Chronic kidney disease (CKD) has recently become a serious health and social concern. Vascular calcification, a common complication of CKD, is a risk factor that increases the incidence and mortality of cardiovascular events in patients with CKD. However, there are currently no effective therapeutic targets that can facilitate treatment with fewer side effects for vascular calcification in CKD. To identify potential therapeutic targets, we performed label-free quantification (LFQ) analyses of protein samples from rat aortic vascular smooth muscle cells (RASMCs) after high-phosphorus treatment by nano-UPLC-MS/MS. We determined that ubiquitin-specific protease 47 (USP47) may be associated with CKD vascular calcification by regulating the osteogenic transdifferentiation of the vascular smooth muscle cell (VSMC) phenotype, thus suggesting a novel and potentially effective therapeutic target for CKD vascular calcification. USP47 knockdown significantly reduced the expression of ß-transducin repeat-containing protein (BTRC), serine/threonine-protein kinase akt-1 (AKT1), Klotho, fibroblast growth factor (FGF23), and matrix Gla protein (MGP) in RASMCs after high-phosphorus treatment. Consistent with the results of protein-protein interaction (PPI) analyses, USP47 may be involved in regulating osteogenic transdifferentiation markers, such as runt-related transcription factor 2 (RUNX2), Klotho, FGF23, and MGP through the BTRC/AKT1 pathway upon CKD vascular calcification. These data indicate that USP47 may be associated with vascular calcification in CKD by regulating osteogenic differentiation of VSMCs. USP47 may regulate osteogenic transdifferentiation in VSMCs upon CKD vascular calcification through a process involving the BTRC/AKT1 pathway. This study identified a novel potential therapeutic target for the treatment of vascular calcification in CKD.

Sclerostin is involved in osteogenic transdifferentiation of vascular smooth muscle cells in chronic kidney disease-associated vascular calcification with non-canonical Wnt signaling.

Vascular calcification is prominent in patients with chronic kidney disease (CKD) and is a strong predictor of cardiovascular mortality in the CKD population. However, the mechanism underlying CKD-associated vascular calcification remains unclear. To identify potential therapeutic targets, a 5/6 nephrectomy rat model was established by feeding of a high-phosphorous diet as the CKD group and compared with sham group rats at 4 and 16 weeks. Sequencing analyses of the rat aorta revealed 643 upregulated and 1023 downregulated genes at 4 weeks, as well as 899 upregulated and 1185 downregulated genes at 16 weeks in the CKD group compared to the sham group. Bioinformatics analyses suggested that SOST (which encodes sclerostin) and Wnt signaling are involved in CKD-associated vascular calcification. Furthermore, protein-protein interactions analysis revealed interactions between SOST, WNT5A, and WNT5B, that involved runt-related transcription factor 2 (RUNX2) and transgelin (TAGLN). SOST was increased in CKD-associated vascular calcification following reduction of the Wnt signaling, including WNT5A and WNT5B, both in vivo and in vitro. TargetScan was used to predict the microRNAs (miRNAs) targeting WNT5A and WNT5B. The expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, and miR-3568 were significantly reduced, whereas that of miR-742-3p was significantly increased in calcified rat aortic vascular smooth muscle cells (VSMCs). In CKD rat aortas, the expression of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, miR-742-3p, and miR-22-5p were significantly reduced at both 4 and 16 weeks. Altogether, owing to several assessments, potentially diagnostic and prognostic biomarkers for improving common CKD diagnostic tools were identified in this study. Abbreviations: BUN: blood urea nitrogen; CKD: chronic kidney disease; CKD-MBD: chronic kidney disease-mineral bone disorder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GO: the Gene Ontology; HE: hematoxylin-eosin; HRP: horseradish peroxidase; KEGG: Kyoto Encyclopedia of Genes and Genomes; MiRNAs: microRNAs; PAS: periodic acid-Schiff; RUNX2: runt-related transcription factor 2; SCr: serum creatinine; STRING: the Search Tool for the Retrieval of Interacting Genes/Proteins; TAGLN: transgelin; VSMC: vascular smooth muscle cell.

GPER-regulated lncRNA-Glu promotes glutamate secretion to enhance cellular invasion and metastasis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a group of breast cancer with heterogeneity and poor prognosis and effective therapeutic targets are not available currently. TNBC has been recognized as estrogen-independent breast cancer, while the novel estrogen receptor, namely G protein-coupled estrogen receptor (GPER), was claimed to mediate estrogenic actions in TNBC tissues and cell lines. Through mRNA microarrays, lncRNA microarrays, and bioinformatics analysis, we found that GPER is activated by 17ß-estradiol (E2) and GPER-specific agonist G1, which downregulates a novel lncRNA (termed as lncRNA-Glu). LncRNA-Glu can inhibit glutamate transport activity and transcriptional activity of its target gene VGLUT2 via specific binding. GPER-mediated reduction of lncRNA-Glu promotes glutamate transport activity and transcriptional activity of VGLUT2. Furthermore, GPER-mediated activation of cAMP-PKA signaling contributes to glutamate secretion. LncRNA-Glu-VGLUT2 signaling synergizes with cAMP-PKA signaling to increase autologous glutamate secretion in TNBC cells, which activates glutamate N-methyl-D-aspartate receptor (NMDAR) and its downstream CaMK and MEK-MAPK pathways, thus enhancing cellular invasion and metastasis in vitro and in vivo. Our data provide new insights into GPER-mediated glutamate secretion and its downstream signaling NMDAR-CaMK/MEK-MAPK during TNBC invasion. The mechanisms we discovered may provide new targets for clinical therapy of TNBC.

Dimethyl fumarate ameliorates endotoxin-induced acute kidney injury against macrophage oxidative stress.

BACKGROUND: Characterized by macrophage infiltration, renal inflammation during septic acute kidney injury (AKI) reveals a ubiquitous human health problem. Unfortunately, effective therapies with limited side effects are still lacking. This study is aiming to elucidate the role of Dimethyl fumarate (DMF) in macrophages against oxidative stress of septic AKI. METHODS: Balb/c mice were gavaged by 50 mg/kg DMF then injected with 10 mg/kg LPS by i.p. We examined LPS-induced renal dysfunction and histological features in murine kidneys. Raw264.7 macrophage cells were also treated with DMF and then induced by LPS. The mitotracker staining was used to follow mitochondria integrity by confocal microscopy. Flow cytometry measured the production of ROS by DCF-HDA and the expression of iNOS. Western blot detected the expression of Nrf-2 and Sirt1. Co-IP measured the interaction between Sirt1 and Nrf-2. Confocal microscopy observed the colocalization of Sirt1 and Nrf-2 in LPS-treated Raw264.7 macrophage cells. RESULTS: DMF ameliorated murine LPS nephritis with reduced blood urea nitrogen and serum creatinine, as well as decreased the histological alterations compared to the normal control. DMF significantly inhibited the expression of iNOS and reduced the production of nitrite in Raw264.7 cells following LPS treatment. Our study also revealed the role of DMF in protecting against intracellular ROS accumulation and mitochondria dysfunction in LPS-induced nephritis. DMF facilitated colocalization and interaction between Sirt1 and Nrf-2 in LPS-treated cells. CONCLUSIONS: This study showed that DMF alleviated LPS-induced nephritis, indicating protective effects of DMF on macrophage against oxidative stress induced by LPS potentially involving Nrf-2-mediated pathway.

Isoliquiritigenin attenuates septic acute kidney injury by regulating ferritinophagy-mediated ferroptosis.

Defined differently from apoptosis, necrosis, and autophagy, ferroptosis has been implicated in acute kidney injury (AKI) such as ischemia-reperfusion injury induced AKI, folic acid caused AKI and cisplatin induced AKI. However, whether ferroptosis is involved in LPS induced AKI could be remaining unclear and there is still a lack of therapies associated with ferroptosis in LPS induced AKI without side effects. This study aimed to elucidate the role of isoliquiritigenin (ISL) in ferroptosis of LPS-induced AKI. We used LPS to induce renal tubular injury, followed by treatment with ISL both in vitro and in vivo. Human renal tubular HK2 cells were pretreated with 50 µM or 100 µM ISL for 5 h before stimulation with 2 µg/mL LPS. Mice were administered a single dose of either 50 mg/kg ISL orally or 5 mg/kg ferroptosis inhibitor ferrostatin-1 intraperitoneally before 10 mg/kg LPS injection. We found that LPS could induce mitochondria injury of renal tubular presented as the shape of mitochondria appeared smaller than normal with increased membrane density and are faction or destruction of mitochondrial crista through scanning electron microscope. Ferrostatin-1 significantly protected mice against renal dysfunction and renal tubular damage in LPS-induced AKI. ISL inhibited Fe2+ and lipid peroxidation accumulation in LPS-stimulated HK2 cells. It also increased the expression of GPX4 and xCT, reduced the expression of HMGB1 and NCOA4 then attenuated mitochondria injury in renal tubular following LPS stimulation. These results indicated the potential role of ISL against ferritinophagy-mediated ferroptosis in renal tubular following LPS stimulation.

Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and ß1-integrin signaling pathway in tumor cells.

INTRODUCTION: Acquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. ß1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of ß1-integrin in GPER-mediated tamoxifen resistance in breast cancer. METHODS: The expression of ß1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of ß1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R ß1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis. RESULTS: GPER, ß1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of ß1-integrin and GPER were in metastases. Increased ß1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of ß1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of ß1-integrin. Interestingly, silencing of ß1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5ß1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of ß1-integrin in MCF-7R cells. In addition, the downstream kinases of ß1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts. CONCLUSIONS: GPER/EGFR/ERK signaling upregulates ß1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a ß1-integrin-dependent pattern. Thus, ß1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.

ACSL4-Mediated Membrane Phospholipid Remodeling Induces Integrin ß1 Activation to Facilitate Triple-Negative Breast Cancer Metastasis.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has a poor prognosis and a high propensity to metastasize. Lipid metabolism has emerged as a critical regulator of tumor progression and metastasis in other cancer types. Characterization of the lipid metabolic features of TNBC could provide important insights into the drivers of TNBC metastasis. Here, we showed that metastatic TNBC tumors harbor more unsaturated phospholipids, especially long-chain polyunsaturated fatty acids, at the sn-2 position of phosphatidylcholine and phosphatidylethanolamine compared with primary tumors. Metastatic TNBC tumors upregulated ACSL4, a long-chain polyunsaturated acyl-CoA synthetase that drives the preferential incorporation of polyunsaturated fatty acids into phospholipids, resulting in the alteration of membrane phospholipid composition and properties. Moreover, ACSL4-mediated phospholipid remodeling of the cell membrane induced lipid-raft localization and activation of integrin ß1 in a CD47-dependent manner, which led to downstream focal adhesion kinase phosphorylation that promoted metastasis. Importantly, pharmacologic inhibition of ACSL4 suppressed tumor growth and metastasis and increased chemosensitivity in TNBC models in vivo. These findings indicate that ACSL4-mediated phospholipid remodeling enables TNBC metastasis and can be inhibited as a potential strategy to improve the efficacy of chemotherapy in TNBC. SIGNIFICANCE: ACSL4 upregulation in triple-negative breast cancer alters cell membrane phospholipid composition to increase integrin ß1 activation and drive metastasis, indicating that targeting ACSL4 could potentially block metastasis and improve patient outcomes.

The causal relationship between the gut microbiota and acute pancreatitis: A 2-sample Mendelian randomization study.

Several observational studies have reported a correlation between the gut microbiota (GM) and the risk of acute pancreatitis (AP). However, the causal relationship between them remains uncertain. We conducted a 2-sample Mendelian randomization (MR) study using pooled data from genome-wide association studies of 211 taxa (131 genera, 35 families, 20 orders, 16 classes, and 9 phyla) and AP patients. We evaluated the causal relationship between the GM and AP using methods such as inverse-variance weighting, MR-Egger, weighted medians, simple mode, and weighted mode. Cochran Q test, MR-Egger regression intercept analysis, and MR-PRESSO were used to examine the heterogeneity, multipotency, and outlier values of the variables, respectively. The reverse causal relationship between AP and the GM was assessed with reverse MR. In total, 5 gut microbial taxa were significantly associated with AP. The inverse-variance weighting results indicated that Acidaminococcaceae (odds ratio [OR]: 0.81, 95% confidence interval [CI]: 0.66-1.00, P = .045) and Ruminococcaceae UCG004 (OR: 0.85, 95% CI: 0.72-0.99, P = .040) were protective factors against the occurrence of AP. Coprococcus 3 (OR: 1.32, 95% CI: 1.03-1.70, P = .030), Eisenbergiella (OR: 1.13, 95% CI: 1.00-1.28, P = .043), and the Eubacterium fissicatena group (OR: 1.18, 95% CI: 1.05-1.33, P = .006) were risk factors for the development of AP. A comprehensive sensitivity analysis proved our results to be reliable. Reverse MR analysis did not indicate any causal relationship between AP and the GM. This study revealed a complex causal relationship between 5 GM taxa and AP, providing new insights into the diagnostic and therapeutic potential of the GM in AP patients.

HnRNPA2B1 ISGylation Regulates m6A-Tagged mRNA Selective Export via ALYREF/NXF1 Complex to Foster Breast Cancer Development.

Regulating nuclear export precisely is essential for maintaining mRNA homeostasis and impacts tumor progression. However, the mechanisms governing nuclear mRNA export remain poorly elucidated. Herein, it is revealed that the enhanced hypoxic long no-ncoding RNA (lncRNA prostate cancer associated transcript 6 (PCAT6) in breast cancer (BC) promotes the nuclear export of m6A-modified mRNAs, bolstering breast cancer stem cells (BCSCs) stemness and doxorubicin resistance. Clinically, hypoxic PCAT6 correlates with malignant BC features and poor prognosis. Mechanically, PCAT6 functions as a scaffold between interferon-stimulated gene 15 (ISG15) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), leading to ISGylation of hnRNPA2B1, thus protecting hnRNPA2B1 from ubiquitination-mediated proteasomal degradation. Interestingly, as an m6A reader, hnRNPA2B1 selectively mediates m6A-tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex, which promotes stemness-related genes expression. HnRNPA2B1 knockdown or mRNA export inhibition can result in the retention of nuclear m6A-tagged mRNA associated with stemness maintenance, which suppresses BCSCs self-renewal and effectively improves the efficacy of doxorubicin therapy. These findings demonstrate the pivotal role of m6A-modified mRNA nuclear export in BC progression, highlighting that the inhibition of m6A-tagged mRNA and its nuclear export is a potential therapeutic strategy for the amelioration of cancer chemotherapy.