OBJECTIVETo investigate the changes of expression and subcellular location of nuclear growth-induced protein-B(NGFI-B) in cardiomyocytes of stressed rats and its biological effect and to provide scientific evidences for exploring the mechanism underlying myocardium injury induced by stress.

METHODSThe cell model of stress-induced cardiomyocyte injury were established. Western blot method and confocal microscopy method were used to investigate the subcellular location of NGFI-B in cardiomyocytes under stress. The flow-cytometry was selected to detect the apoptotic rate in cardiomyocytes in vitro. Western blot method was used to determine the content of cytochrome C protein in mitochondria and cytoplasm respectively.

RESULTSStress induced the increase of NGFI-B content in the mitochondria of cardiomyocytes and the translocation of NGFI-B from the nucleus to the mitochondria. The translocation of NGFI-B promoted the release of cytochrome C from the mitochondria and the cardiomyocyte apoptosis. Treatment of stressed cardiomyocytes with leptomycin B, a non-specific blocker of nuclear export, resulted in nuclear retention of NGFI-B and abrogated its ability to induce the release of cytochrome C from the mitochondria.

CONCLUSIONStress could induce NGFI-B translocation from the nucleus to the mitochondria in cardiomyocytes, which activated the mitochondrial pathway of cell apoptosis.

Animals ; Animals, Newborn ; Apoptosis ; Cells, Cultured ; Male ; Myocytes, Cardiac ; cytology ; metabolism ; Nuclear Receptor Subfamily 4, Group A, Member 1 ; metabolism ; Rats ; Rats, Wistar ; Stress, Physiological

OBJECTIVE: To analyze the association of Nur77 with overall survival of gastric cancer patients and investigate the role of Nur77 in invasion and migration of gastric cancer cells. METHODS: Oncomine database was used to analyze the expression of Nur77 in gastric cancer and gastric mucosa tissues, and the distribution characteristics of Nur77 protein between gastric cancer and normal tissues were compared using Human Protein Atlas. GEPIA2 was used to analyze the relationship of Nur77 expression and the patients' survival. The expression of Nur77 in gastric cancer cell lines GES-1, AGS and MKN-45 were detected by Western blotting. The regulatory interactions between IL-6 and Nur77 were verified by transfecting the cells with specific Nur-77 siRNA and Nur-77-overexpressing plasmid. The changes in migration ability of the cells following Nur-77 knockdown were assessed with scratch assay. The effect of Nur-77 overexpression or IL-6 knockdown, or their combination, on migration and invasion of the gastric cancer cells were examined using Transwell assay. The effect of Nur77 expression level on NF-κB/IL-6 pathway activation was analyzed using Western blotting. RESULTS: Oncomine database showed that gastric cancer tissues expressed a significantly higher level of Nur77 mRNA than normal tissues (P < 0.05). Nur77 expression was detected mostly in the nucleus, and a high Nur77 expression was associated with a poor survival outcome of the patients (P < 0.05). In gastric cancer cells, the high expression of Nur77 participated in the regulation of IL-6. Nur77 silencing significantly lowered the migration ability of the cells (P < 0.05), and IL-6 silencing significantly attenuated the enhanced migration caused by Nur77 overexpression (P < 0.05). Nur77 participates in the activation of NF-κB/IL-6 signaling pathway by regulating the expression of p-p65, p65, p-Stat3 and Stat3. CONCLUSION A high Nur77 expression is strongly correlated with a poor prognosis of gastric cancer patients. Nur77 promotes the invasion and migration of gastric cancer cells possibly by regulating the NF-κB/IL-6 signaling pathway.
Cell Line, Tumor ; Cell Movement ; Gene Expression Regulation, Neoplastic ; Humans ; Interleukin-6/metabolism* ; NF-kappa B/metabolism* ; Neoplasm Invasiveness/genetics* ; Nuclear Receptor Subfamily 4, Group A, Member 1 ; RNA, Messenger/metabolism* ; RNA, Small Interfering/genetics* ; Stomach Neoplasms/genetics*

Osteoarthritis (OA) is a prevalent joint disease with no effective treatment strategies. Aberrant mechanical stimuli was demonstrated to be an essential factor for OA pathogenesis. Although multiple studies have detected potential regulatory mechanisms underlying OA and have concentrated on developing novel treatment strategies, the epigenetic control of OA remains unclear. Histone demethylase JMJD3 has been reported to mediate multiple physiological and pathological processes, including cell differentiation, proliferation, autophagy, and apoptosis. However, the regulation of JMJD3 in aberrant force-related OA and its mediatory effect on disease progression are still unknown. In this work, we confirmed the upregulation of JMJD3 in aberrant force-induced cartilage injury in vitro and in vivo. Functionally, inhibition of JMJD3 by its inhibitor, GSK-J4, or downregulation of JMJD3 by adenovirus infection of sh-JMJD3 could alleviate the aberrant force-induced chondrocyte injury. Mechanistic investigation illustrated that aberrant force induces JMJD3 expression and then demethylates H3K27me3 at the NR4A1 promoter to promote its expression. Further experiments indicated that NR4A1 can regulate chondrocyte apoptosis, cartilage degeneration, extracellular matrix degradation, and inflammatory responses. In vivo, anterior cruciate ligament transection (ACLT) was performed to construct an OA model, and the therapeutic effect of GSK-J4 was validated. More importantly, we adopted a peptide-siRNA nanoplatform to deliver si-JMJD3 into articular cartilage, and the severity of joint degeneration was remarkably mitigated. Taken together, our findings demonstrated that JMJD3 is flow-responsive and epigenetically regulates OA progression. Our work provides evidences for JMJD3 inhibition as an innovative epigenetic therapy approach for joint diseases by utilizing p5RHH-siRNA nanocomplexes.
Cartilage, Articular/pathology* ; Chondrocytes/metabolism* ; Down-Regulation ; Epigenesis, Genetic ; Humans ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism* ; Osteoarthritis/pathology* ; RNA, Small Interfering/pharmacology*

Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.
Apoptosis ; drug effects ; Cell Line, Tumor ; Enzyme Activation ; drug effects ; Humans ; Ligands ; Molecular Docking Simulation ; Nuclear Receptor Subfamily 4, Group A, Member 1 ; genetics ; metabolism ; Oximes ; metabolism ; pharmacology ; Protein Conformation ; Proto-Oncogene Proteins c-akt ; metabolism ; Pyrazoles ; metabolism ; pharmacology ; Retinoid X Receptor alpha ; chemistry ; genetics ; metabolism ; Thiazoles ; metabolism ; pharmacology ; Transcription, Genetic ; drug effects ; Transcriptional Activation ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism