Downregulation Of c-Fos Research Articles

MEK inhibition prevents CAR-T cell exhaustion and differentiation via downregulation of c-Fos and JunB

Clinical evidence supports the notion that T cell exhaustion and terminal differentiation pose challenges to the persistence and effectiveness of chimeric antigen receptor-T (CAR-T) cells. MEK1/2 inhibitors (MEKIs), widely used in cancer treatment due to their ability to inhibit aberrant MAPK signaling, have shown potential synergistic effects when combined with immunotherapy. However, the impact and mechanisms of MEKIs on CAR-T cells remain uncertain and controversial. To address this, we conducted a comprehensive investigation to determine whether MEKIs enhance or impair the efficacy of CAR-T cells. Our findings revealed that MEKIs attenuated CAR-T cell exhaustion and terminal differentiation induced by tonic signaling and antigen stimulation, thereby improving CAR-T cell efficacy against hematological and solid tumors. Remarkably, these effects were independent of the specific scFvs and costimulatory domains utilized in CARs. Mechanistically, analysis of bulk and single-cell transcriptional profiles demonstrates that the effect of MEK inhibition was related to diminish anabolic metabolism and downregulation of c-Fos and JunB. Additionally, the overexpression of c-Fos or JunB in CAR-T cells counteracted the effects of MEK inhibition. Furthermore, our Cut-and-Tag assay revealed that MEK inhibition downregulated the JunB-driven gene profiles associated with exhaustion, differentiation, anergy, glycolysis, and apoptosis. In summary, our research unveil the critical role of the MAPK-c-Fos-JunB axis in driving CAR-T cell exhaustion and terminal differentiation. These mechanistic insights significantly broaden the potential application of MEKIs to enhance the effectiveness of CAR-T therapy.

An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells.

This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells. Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR. C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05). Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.

Microwave-responsive gadolinium metal-organic frameworks nanosystem for MRI-guided cancer thermotherapy and synergistic immunotherapy

The clinical application of cancer immunotherapy is unsatisfied due to low response rates and systemic immune-related adverse events. Microwave hyperthermia can be used as a synergistic immunotherapy to amplify the antitumor effect. Herein, we designed a Gd-based metal-organic framework (Gd-MOF) nanosystem for MRI-guided thermotherapy and synergistic immunotherapy, which featured high performance in drug loading and tumor tissue penetration. The PD-1 inhibitor (aPD-1) was initially loaded in the porous Gd-MOF (Gd/M) nanosystem. Then, the phase change material (PCM) and the cancer cell membrane were further sequentially modified on the surface of Gd/MP to obtain Gd-MOF@aPD-1@CM (Gd/MPC). When entering the tumor microenvironment (TME), Gd/MPC induces immunogenic death of tumor cells through microwave thermal responsiveness, improves tumor suppressive immune microenvironment and further enhances anti-tumor ability of T cells by releasing aPD-1. Meanwhile, Gd/MPC can be used for contrast-enhanced MRI. Transcriptomics data revealed that the downregulation of MSK2 in cancer cells leads to the downregulation of c-fos and c-jun, and ultimately leads to the apoptosis of cancer cells after treatment. In general, Gd/MPC nanosystem not only solves the problem of system side effect, but also achieves the controlled drug release via PCM, providing a promising theranostic nanoplatform for development of cancer combination immunotherapy.

Antinociceptive effects of vitamin B-complex: A behavioral and histochemical study in rats

B-vitamins have been evaluated as a useful adjuvant therapy to treat pain. In spite of clinical and experimental evidence indicating the analgesic effect of B-vitamins, few studies have investigated their effect on aspects of the inflammatory pain response. In the present study, we investigated the analgesic effect of chronic application of B-complex vitamins (Neurobion) using an inflammatory experimental pain model in rats. Nociceptive behavioral responses were evaluated in male Wistar rats after plantar injection of formalin, comparing the treatment group (TG) with Neurobion pretreatment to the control group (CG) without the pretreatment. In addition, neuronal activity in the central pain pathway was evaluated using c-Fos immunohistochemical reactivity and NADPH-d histochemistry. A highly significant reduction of painful behaviors such as licking and flinching were observed in TG, especially during the secondary phase of the formalin test compared to CG. Results suggest that long-term pre-treatment using Neurobion can have a beneficial effect in reducing the chronic phase of pain. In addition, we observed a downregulation of c-Fos and NADPH-d in dorsal spinal neurons, suggesting that the antinociceptive effect induced by Neurobion could be due to a suppression of nociceptive transmission at the spinal level, particularly in the afferent regions of the dorsal spinal horn, which these neurons utilizing nitric oxide at least as one of their pain neurotransmitters.

MEK Inhibition in CAR-T Cells Prevents Exhaustion and Terminal Differentiation Via Downregulating C-Fos and JunB

Introduction: The effects of MEK inhibitors (MEKIs) in CAR-T cells are poorly understood and remain controversial. Some groups showed that MEKIs could impair CAR-T cells' function in vitro. However, another group reported that MEKIs could combine with GD2 CAR-T cells to provide additional efficacy. Yet, the mechanism for this combined effect is unclear. Considering that the CAR signaling pathway is similar to TCR, both involve the activation of MAPK signaling. We hypothesized that MEKIs might mitigate CAR-T cells' exhaustion and terminal differentiation by lessening redundant CAR signaling. The study aims to systematically evaluate the role and mechanism of MEK inhibitors on CAR-T cells. Methods and Results: To explore whether MEKIs could mitigate the unbeneficial impact of antigen-independent CAR tonic signaling, we added 3 FDA-approved MEKIs: trametinib, cobimetinib, and binimetinib, respectively, to the culture medium of CD19.28z CAR-T cells for 9 days at the concentration approaching their clinically tolerable peak blood concentration. We found that all the 3 MEKIs could reduce CAR-T cells' terminal differentiation and restrain the expression of exhaustion and activation markers. Among them, trametinib was the most potent because it could achieve the comparable effect of cobimetinib and binimetinib at the lowest concentration. Thus, we chose trametinib for further research. We confirmed that compared to concentrations of 7.5nM and 30nM, 15nM was the optimal concentration of trametinib with mild inhibition on the proliferation of CAR-T cells and a potent effect on CAR-T cells' phenotype. Pre-treatment with trametinib didn't affect the in-vitro cytotoxicity of CD19.28z CAR-T cells. The above effects of trametinib were more significant as treatment time and the dose increased. Similar results could be obtained in analogous experiments with CD19.4-1BBz and GD2.28z CAR-T cells. Intriguingly, GD2.28z CAR-T cells cultured with trametinib had better proliferation and killing capacity because they were more exhausted than CD19.28z CAR-T cells. Using the Nalm-6-bearing leukemia xenograft model, we found that compared to DMSO pre-treated CD19.28z CAR-T cells, trametinib pre-treated CD19.28z CAR-T cells exerted more potent anti-leukemia activity, showed a less exhausted and differentiated state, proliferated better, and further extended mice survival (Fig.1). Similar results could be obtained in analogous in-vivo experiments with CD19.4-1BBz CAR-T cells. To evaluate whether trametinib could protect CAR-T cells from the exhaustion and terminal differentiation triggered by antigen stimulation, we cocultured CD19.28z CAR-T cells with Nalm-6 cells in a medium with or without trametinib. We demonstrated under antigen stimulation, trametinib could effectively inhibit CAR-T cells' activation, exhaustion, apoptosis, terminal differentiation, and phosphorylation of ERK and consequently promote the proliferation of total and CD8 CAR-T cells. In addition, after repetitive antigen stimulation, trametinib could rescue CAR-T cells' functional exhaustion and improve CAR-T cells' in-vitro cytotoxicity. Mechanistically, Single-cell and bulk RNA-Seq revealed that the effect of MEK inhibition was associated with the downregulation of AP-1 and exhaustion-associated transcription factors (TFs) and the upregulation of memory-associated TFs (Fig.2). GSEA revealed the upregulation of naive/memory-associated genes and downregulation of genes involved in T cell activation/effector/exhaustion, AP-1 pathway and apoptosis in the trametinib-treated group. Additionally, single-cell transcriptional profiling demonstrated enrichment of memory and Ki67+cycling CAR-T clusters in the trametinib-treated group. Among the AP-1 TFs downregulated by trametinib, c-Fos and JunB are the direct downstream targets of the canonical MAPK signaling pathway, and both of them were reported to implicate T cell exhaustion. Thus we speculated both downregulation of c-Fos and JunB may contribute to the role of MEKIs. Consistent with our hypothesis, overexpression of c-Fos or JunB in CAR-T cells could partially, if not all, abrogated the effects of MEK inhibition. Significance: Our research provides a strategy to optimize CAR-T antitumor efficacy by MEK inhibition and shed light on the role of c-Fos and JunB in driving CAR-T cell exhaustion and terminal differentiation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CXCR4/CX43 Regulate Diabetic Neuropathic Pain via Intercellular Interactions between Activated Neurons and Dysfunctional Astrocytes during Late Phase of Diabetes in Rats and the Effects of Antioxidant N-Acetyl-L-Cysteine.

Growing evidence suggests that the interactions between astrocytes and neurons exert important functions in the central sensitization of the spinal cord dorsal horn in rodents with diabetes and neuropathic pain (DNP). However, it still remains unclear how signal transmission occurs in the spinal cord dorsal horn between astrocytes and neurons, especially in subjects with DNP. Chemokine CXC receptor 4 (CXCR4) plays critical roles in DNP, and connexin 43 (CX43), which is also primarily expressed by astrocytes, contributes to the development of neuropathy. We thus postulated that astrocytic and neuronal CXCR4 induces and produces inflammatory factors under persistent peripheral noxious stimulation in DNP, while intercellular CX43 can transmit inflammatory stimulation signals. The results showed that streptozotocin-induced type 1 diabetic rats developed heat hyperalgesia and mechanical allodynia. Diabetes led to persistent neuropathic pain. Diabetic rats developed peripheral sensitization at the early phase (2 weeks) and central sensitization at the late phase (5 weeks) after diabetes induction. Both CXCR4 and CX43, which are localized and coexpressed in neurons and astrocytes, were enhanced significantly in the dorsal horn of spinal cord in rats undergoing DNP during late phase of diabetes, and the CXCR4 antagonist AMD3100 reduced the expression of CX43. The nociceptive behavior was reversed, respectively, by AMD3100 at the early phase and by the antioxidant N-acetyl-L-cysteine (NAC) at the late phase. Furthermore, rats with DNP demonstrated downregulation of glial fibrillary acidic protein (GFAP) as well as upregulation of c-fos in the spinal cord dorsal horn at the late phase compared to the controls, and upregulation of GFAP and downregulation of c-fos were observed upon treatment with NAC. Given that GFAP and c-fos are, respectively, makers of astrocyte and neuronal activation, our findings suggest that CXCR4 as an inflammatory stimulation protein and CX43 as an intercellular signal transmission protein both may induce neurons excitability and astrocytes dysfunction in developing DNP.

Gfi1-Mediated Repression of C-Fos, Egr1 and Egr2, and Inhibition of ERK1/2 Signaling Contribute to the Role of Gfi1 in Granulopoiesis

Gfi1 is a transcriptional repressor that plays an important role in hematopoiesis. In the myeloid system, Gfi1 promotes granulopoiesis and antagonizes the alternative monocyte development in part through repressing PU.1 and CSF1 , which promote monopoiesis. However, the molecular mechanism by which Gfi1 favors neutrophil over monocyte development remains incompletely understood. G-CSF is a lineage-specific cytokine that plays a dominant role in granulopoiesis. We recently showed that a G-CSFR mutant in which tyrosine 729 was mutated to phenylalanine (Y729F) promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of ERK1/2 and augmented activation of downstream targets c-Fos and Egr1. To address whether Gfi1 may promote granulopoiesis independent of its repression of Csf1 , we established murine myeloid 32D and multipotent FDCP-mix cells that constitutively expressed G-CSFR Y729F and inducibly expressed Gfi1 in response to doxycycline (Dox; 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1). 32D and FDCP-mix cells showed no response to CSF1. In the absence of Dox, 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1 differentiated into monocytes when cultured in G-CSF. Interestingly, addition of Dox to induce Gfi1 expression rescued neutrophil development in both cell lines. Induction of Gfi1 expression with Dox also markedly attenuated the induction of c-Fos, Egr1 and Egr2 expression in response to G-CSF. In contrast, the mRNA levels of c-Fos, Egr1 and Egr2 were dramatically elevated in lineage marker negative (Lin-) bone marrow (BM) cells from Gfi1-/- mice as compared to BM cells from Gfi1+/+ mice. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that Gfi1 bound to and repressed the activities of c-Fos , Egr1 and Egr2 promoters. c-Fos , Egr1 and Egr2 are immediate early genes that are activated by the ERK1/2 pathway. Notably, G-CSF-stimulated activation of ERK1/2 was stronger in Lin- BM cells from Gfi1-/- mice than in cells from Gfi1+/+ mice. It has been shown that BM cells from Gfi1-/- mice are unable to differentiate into mature neutrophils in vitro , but instead give rise to atypical monocytes. Interestingly, the MEK1/2 inhibitor U0126 or PD0325901 reduced the mRNA levels of c-Fos, Egr1 and Egr2, and partially rescued G-CSF-induced neutrophil development in Gfi1-/- BM cells. Together, our data indicate that Gfi1 inhibits the expression of c-Fos, Egr1 and Egr2 through direct transcriptional repression of their genes and indirect inhibition of ERK1/2 signaling pathway, and that Gfi1-mediated downregulation of c-Fos, Egr1 and Egr2 may contribute to the role of Gfi1 in granulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

POS0409 MOLECULAR PROFILING OF THE REMISSION STATE IN PSORIATIC ARTHRITIS: IS THE FIRE EXTINGUISHED?

Background:The most important goal of therapy in psoriatic arthritis (PsA) is to reach the remission state of disease. Nowadays, data on molecular players of clinical remission and effective disease inactivation are scarce: gene expression analysis could highlight markers characteristic of PsA remission state.Objectives:Our aim was to analyze the gene expression profile of patients with clinically inactive (drug-induced remission) PsA versus healthy controls and PsA active state.Methods:From a cohort of 300 patients affected by PsA according to CASPAR criteria, we selected 40 patients (peripheral arthritis subset) with &gt;1-year remission by TNFα antagonism assessed by DAPSA ≤ 4 (R) and 40 PsA with active disease state by DAPSA &gt; 14 score (no bDMARDs ongoing) (A), and 40 healthy controls (HC) matching for mean age and gender ratio. Both PsA groups were not on corticosteroid treatment. Each condition has been profiled first using pooled RNAs from peripheral blood (biological duplicates) by Affymetrix Human GeneChip HTA 2.0. To identify a transcript as differentially expressed in both duplicates, a fold change (FC) 1.5 and p-value 0.05 has been set. Then, from the comparative list of differential expressed genes (DEGs) in R vs HC state, coding mRNAs were selected and interactions analyzed by STRING software, biological functions and annotations by Gene Onthology (GO). From these analysis, 12 genes were validated in the whole PsA cohort (R+A) and HCs, by quantitative real-time PCR (RT-qPCR) in triplicate (TaqMan chemistry, GAPDH housekeeping, 2–ΔΔCt for relative quantification, FC cutoff ±1.5 for differential analysis).Results:The transcriptomic analysis generated 3 comparative lists of mRNAs (R vs HC= 125; R vs A= 1184; A vs HC= 378 transcripts). Then, we focused on remission state analyzing the mRNAs list R vs HC. Filtering data for coding DEGs, was made a list of 24 genes further analyzed for functions and interactions: STRING analysis created one interactions network and GO highlighted in which biological processes these DEGs are involved (Fig panels 1A, 1B). Validation analysis by rt-qPCR in the PsA cohort (R+A) and HCs confirmed the downregulation of c-FOS and the upregulation of CCDC50 (alias YMER) genes in the R vs HC conditions (pvalue= 0.004). The dysregulation of further genes in other comparisons was measured, as shown in Tab and Fig panel 1C.Conclusion:Observing the amount of DEGs, is evident that the drug-induced remission state is more similar to healthy condition, however this trend of “similarity” does not mean “identical”. The bioinformatic analysis showed that DEGs in the R vs HC conditions are primarily involved in biological processes related to immune system as well as correlates in an interactions network. Our molecular characterization of clinical remission revealed that effective disease inactivation appears to be molecularly-driven by two key genes, c-FOS and CCD50 (YMER), both involved in the NF-κB signalling pathway modulation.Coding DEGs mis-regulated in the remission state. Differentially expressed genes (DEGs) in the Remission (R) state vs Healthy Controls (HC) analyzed for multiple-proteins interaction by STRING software V10.5 (panel A) and biological functions by Gene Set Enrichment Analysis (panel B). DEGs validations by RT-qPCR, confirmed the CCD50 and c-FOS dysregulation in the R state (panel C, RQ= Relative Quantification, t test RvsHC pvalue=0,004).GENE SYMBOLGene chip Array R vs HCRemission vs HCActive vs HCRemission vs ActiveALPL-1.7-1.11.3-1.25ANPEP-1.681.01.1-1.1BPI-1.57-1.11.27-1.5CCDC501.51.5-1.251.8CEACAM8-2.3-1.21.2-1.5CHI3L1-1.59-1.1-1.0-1.0DEFA1B-2.3-1.41.9-2.7FCAR-1.56-1.11.1-1.3FOS-1.51-2.0-1.5-1.4KLRB11.52-1-1.61.6PADI2-1.54-1.11.0-1.1TNFSF14-1.511.11.2-1.1DEGs by RT-qPCR. Validated differentially expressed genes (DEGs) in the remission (R) state vs healthy controls (HC) selected from the microarray analysis comparative list (FC cutoff ±1.5). Differential analysis (40 R vs 40 HC vs 40 Active) was made by FoldChange (FC) cutoff ±1.5 (overexpressed ≥ 1.5; - 1.5 ≥ downregulated).Disclosure of Interests:None declared

Ameliorative effects of olibanum essential oil on learning and memory in Aβ1-42-induced Alzheimer’s disease mouse model

Purpose: To study the effect of olibanum essential oil (OEO) on learning and memory in Alzheimer’s disease (AD) mouse.Methods: Mice were administered the 42-amino acid form of amyloid β-peptide (Aβ1-42) to induce AD and then treated with OEO at 150, 300, and 600 mg/kg, p.o. for two weeks. Following treatment, the AD mice were assessed by step-down test (SDT), dark avoidance test (DAT), and Morris water maze test (MWM). Blood and brain tissues were collected for biochemical assessments. Gas chromatographymass spectroscopy was used to analyze the main constituents of OEO.Results: The main constituents of OEO were limonene, α-pinene, and 4-terpineol. Treatment with OEO prolonged t latency in SDT and DAT, but decreased error times. Escape latency decreased and crossing times were rose in the MWM following OEO treatment (p &lt; 0.5). Treatment with OEO also enhanced the acetylcholine levels and decreased the acetylcholinesterase levels in serum and brain tissue (p &lt; 0.5). Additionally, OEO reduced amyloid plaques in the hippocampus and protected hippocampal neurons from damage. Furthermore, OEO decreased c-fos expression in hippocampus tissues from AD mice (p &lt; 0.5).Conclusion: OEO has significant ameliorative effect AD-induced deterioration in learning and memory in AD mouse induced by Aβ1-42. The mechanisms of these effects are related to increased acetylcholine contents, reduction of amyloid plaques, protection of hippocampal neurons, and downregulation of c-fos in brain tissues. The results justify the need for further investigation of candidate drugs derived from OEO for the management of AD.&#x0D; Keywords: Olibanum, Essential oil, Learning, Memory, AD

Dynamin-related protein 1 positively regulates osteoclast differentiation and bone loss.

Dynamin-related protein 1 (DRP1) is a mitochondrial membrane GTPase and regulates mitochondrial fission. In this study, we found that the cytokine RANKL increased the expression of DRP1 and its receptor proteins, Fis1, Mid49, and Mid 51, during osteoclast formation in mouse bone marrow-derived macrophages. Inactivation of the kinase GSK3β appeared to induce DRP1 expression. DRP1 knockdown or the DRP1 inhibitor Mdivi1 suppressed osteoclast differentiation via downregulation of c-Fos and NFATc1, the key transcription factor for osteoclast formation. Finally, the DRP1 inhibitor suppressed lipopolysaccharide-induced osteoclast formation in a calvarial model and ovariectomy-induced bone loss invivo. Taken together, our data demonstrate that DRP1 positively contributes to RANKL-induced osteoclast differentiation by regulating the c-Fos-NFATc1 axis, suggesting the importance of mitochondrial DRP1 in osteoclastogenesis.

NMDA Receptor Signaling Mediates cFos Expression via Top2β-Induced DSBs in Glioblastoma Cells.

The activation of Ca2+-permeable N-methyl-D-aspartic acid (NMDA) receptor channels (NMDARs) is crucial for the development and survival of neurons, but many cancers use NMDAR-mediated signaling as well, enhancing the growth and invasiveness of tumors. Thus, NMDAR-dependent pathways emerge as a promising target in cancer therapy. Here, we use the LN229 and U-87MG glioblastoma multiforme (GBM) cells and immunofluorescence staining of 53BP1 to analyze NMDAR-induced DNA double-strand breaks (DSBs), which represent an important step in the NMDAR signaling pathway in neurons by facilitating the expression of early response genes. Our results show that NMDAR activation leads to the induction of DSBs in a subpopulation of glioma cells. In a further analogy to neurons, our results demonstrate that the induction of DSBs in LN229 cells is dependent on the activity of topoisomerase IIβ (Top2β). Western blot analysis revealed that the inhibition of NMDARs, cAMP-responsive element binding transcription factor (CREB) and Top2β decreased the expression of the proto-oncogene cFos. Knockdown of Top2β with siRNAs resulted in a downregulation of cFos and increased the radiosensitivity of LN229 cells in clonogenic survival. We also observed impaired cFos expression upon NMDAR and Top2β inhibition in a primary GBM cell line, suggesting that NMDAR signaling may be widely used by GBMs, demonstrating the potential of targeting NMDAR signaling proteins for GBM therapy.

Black Rice (Oryza sativa L.) Fermented with Lactobacillus casei Attenuates Osteoclastogenesis and Ovariectomy-Induced Osteoporosis.

The aim of the present study was to investigate the antiosteoclastogenic effects of black rice (Oryza sativa L.) fermented with Lactobacillus casei (LAB) in RANKL-induced RAW macrophage cells and its antiosteoporosis activity against ovariectomy-induced osteoporosis in rats. LAB extract (LABE) treatment attenuated receptor activator of nuclear factor-kappa B (NF-κB) ligand-induced osteoclastic differentiation in RAW cells by inhibiting intercellular reactive oxygen species generation and downregulating the activation of mitogen-activated protein kinases and NF-κB, leading to the downregulation of c-Fos and expression of nuclear factor of activated T cells c1. This consequently suppressed the expression of osteoclast-specific genes including those for cathepsin K, tartrate-resistant acid phosphatase, calcitonin receptor, and integrin β3. Oral administration of LABE protected against ovariectomy-induced bone loss by significantly inhibiting bone architecture alterations and improving serum bone turnover markers in ovariectomized rats. The findings suggest that the antiosteoporotic activity of LABE may be derived from its antiosteoclastic and anti-bone-resorptive activities. LABE has potential as a promising functional material or substrate to prepare protective agents for osteoporosis and osteoclast-mediated bone diseases.

Gfi1-Mediated Repression of c-Fos, Egr-1 and Egr-2, and Inhibition of ERK1/2 Signaling Contribute to the Role of Gfi1 in Granulopoiesis

Gfi1 supports neutrophil development at the expense of monopoiesis, but the underlying molecular mechanism is incompletely understood. We recently showed that the G-CSFR Y729F mutant, in which tyrosine 729 was mutated to phenylalanine, promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of Erk1/2 and enhanced activation of c-Fos and Egr-1. We show here that Gfi1 inhibited the expression of c-Fos, Egr-1 and Egr-2, and rescued neutrophil development in cells expressing G-CSFR Y729F. Gfi1 directly bound to and repressed c-Fos and Egr-1, as has been shown for Egr-2, all of which are the immediate early genes (IEGs) of the Erk1/2 pathway. Interestingly, G-CSF- and M-CSF-stimulated activation of Erk1/2 was augmented in lineage-negative (Lin−) bone marrow (BM) cells from Gfi1−/− mice. Suppression of Erk1/2 signaling resulted in diminished expression of c-Fos, Egr-1 and Egr-2, and partially rescued the neutrophil development of Gfi1−/− BM cells, which are intrinsically defective for neutrophil development. Together, our data indicate that Gfi1 inhibits the expression of c-Fos, Egr-1 and Egr-2 through direct transcriptional repression and indirect inhibition of Erk1/2 signaling, and that Gfi1-mediated downregulation of c-Fos, Egr-1 and Egr-2 may contribute to the role of Gfi1 in granulopoiesis.

Inhibition on acid-sensing ion channels and analgesic activities of flavonoids isolated from dragon's blood resin.

Acid-sensing ion channel (ASIC) serves important roles in the transmission of nociceptive information. To confirm the analgesic mechanism of dragon's blood resin, patch-clamp technique, in vivo animal experiments, and immunohistochemical staining were used to observe the effects of the three flavonoids (loureirin B, cochinchinemin A, and cochinchinemin B) isolated from dragon's blood resin on ASIC. Results showed that the three flavonoids exerted various inhibitory effects on ASIC currents in rat dorsal root ganglion (DRG) neurons. The combination of the three flavonoids with total concentration of 6.5μM could decrease (53.8±4.3%) of the peak amplitude and (45.8±4.5%) of the sustained portion of ASIC currents. The combination of the three flavonoids was fully efficacious on complete Freud's adjuvant (CFA)-induced inflammatory thermal hyperalgesia at a dose of 6.5mM similar with amiloride at 10mM. The analgesic effects of the combination could be weakened by an ASIC activator 2-guanidine-4-methylquinazoline. CFA-induced hyperalgesia was accompanied by c-Fos up-regulation in DRG neurons, and the combination rescued thermal hyperalgesia through down-regulation of c-Fos and ASIC3 expression in CFA-induced inflammation. These collective results suggested that the flavonoids isolated from dragon's blood resin could be considered as the chemical compounds that exert analgesic effects on inflammatory thermal pain due to action on ASIC.

Effect of medicated thread moxibustion on apoptosis of hippocampal neurons in rat models of chronic cerebral ischemic vascular dementia

To investigate the effect of medicated thread moxibustion onapoptosis in hippocampal neuronsin a rat model of chronic cerebral ischemic vascular dementia. A total of 40 male Wistar rats were randomly divided into normal group and sham-operated group (7 rats each), and rat model of chronic cerebral ischemic vascular dementia (14 rats). The model group rats were treated with medicated thread moxibustion two weeks after surgery, once a day, with one day break every six days, (24 times in all)and an observation period of 4 weeks. At the end of therapy, H&amp;E staining was used to monitor changes in the neurons in CA1 area of the rat hippocampus. Changes in related indexes such as Bax, Bc1-2 and C-fos of neuron apoptosis in hippocampus CA1 area were determined by immunohistochemistry, while protein expression was semi-quantitatively assayed using imaging analysis technique. There was significant hippocampal neuronal necrosis six weeks after model establishment, but the necrosis was milder in rats in the medicated thread moxibustion group. Bax and C-fos were positively expressed and significantly higher in the hippocampus of chronic cerebral ischemic vascular dementia rats (model group) than in the medicated thread moxibustion group after treatment (p &lt;0.01). The expression of Bcl-2 was increased in the medicated thread group after treatment, and was higher in the model group, but comparable to that in the sham-operated group (p=0.975&gt;0.05). Medicated thread moxibustion alleviates hippocampal neuronal necrosis, inhibits neuron apoptosis in hippocampus CA1 area, protects nerves, and maintains relative equilibrium inBax/Bcl-2 through down-regulation of C-fos and Bax and up-regulation of Bcl-2. Thus, cell apoptosis-related pathway may be one of its mechanisms of action.

Neuroanatomical Localization of Galanin in Zebrafish Telencephalon and Anticonvulsant Effect of Galanin Overexpression.

Galanin is a neuropeptide widely expressed in the nervous system, but it is also present in non-neuronal locations. In the brain, galanin may function as an inhibitory neurotransmitter. Several studies have shown that galanin is involved in seizure regulation and can modulate epileptic activity in the brain. The overall goal of the study was to establish zebrafish as a model to study the antiepileptic effect of galanin. The goal of this study was achieved by (1) determining neuroanatomical localization of galanin in zebrafish lateral pallium, which is considered to be the zebrafish homologue of the mammalian hippocampus, the brain region essential for initiation of seizures, and (2) testing the anticonvulsant effect of galanin overexpression. Whole mount immunofluorescence staining and pentylenotetrazole (PTZ)-seizure model in larval zebrafish using automated analysis of motor function and qPCR were used in the study. Immunohistochemical staining of zebrafish larvae revealed numerous galanin-IR fibers innervating the subpallium, but only scarce fibers reaching the dorsal parts of telencephalon, including lateral pallium. In three-month old zebrafish, galanin-IR innervation of the telencephalon was similar; however, many more galanin-IR fibers reached the dorsal telencephalon, but in the lateral pallium only scarce galanin-IR fibers were visible. qRT-PCR revealed, as expected, a strong increase in the expression of galanin in the Tg(hsp70l:galn) line after heat shock; however, also without heat shock, the galanin expression was several-fold higher than in the control animals. Galanin overexpression resulted in downregulation of c-fos after PTZ treatment. Behavioral analysis showed that galanin overexpression inhibited locomotor activity in PTZ-treated and control larvae. The obtained results show that galanin overexpression reduced the incidence of seizure-like behavior episodes and their intensity but had no significant effect on their duration. The findings indicate that in addition to antiepileptic action, galanin modulates arousal behavior and demonstrates a sedative effect. The current study showed that galanin overexpression correlated with a potent anticonvulsant effect in the zebrafish PTZ-seizure model.

Apolipoprotein E plays crucial roles in maintaining bone mass by promoting osteoblast differentiation via ERK1/2 pathway and by suppressing osteoclast differentiation via c-Fos, NFATc1, and NF-κB pathway

Apolipoprotein E (ApoE) plays crucial roles not only in lipid metabolism but also in bone metabolism. Specifically ApoE4, one of major ApoE isoforms, has been demonstrated to be associated with increased risk of developing osteoporosis compared to another major isoform ApoE3. However, the detailed mechanism of how the different ApoE isoforms affect bone metabolism remains unclear. Micro-CT analyses of distal femora demonstrated severely decreased bone mass in 48-week-old female homozygous ApoE-knockout (ApoE-KO) mice compared to age- and gender-matched wild type C57BL/6 J (WT) mice. Physiological levels of either ApoE3 or ApoE4 protein (1–20 μg/ml) significantly increased the expression of osteoblast-related genes and alkaline phosphatase (ALP) activity of primary calvarial osteoblasts by inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in a dose-dependent manner, and ApoE3 showed greater osteoblastic induction compared to ApoE4. Furthermore, both ApoE3 and ApoE4 protein inhibited osteoclastogenesis and the expression of osteoclast-related genes of mouse bone marrow derived macrophages (BMDM) via down regulation of c-Fos, nuclear factor of activated T-cells 1 (NFATc1) and nuclear factor-kappa B (NF-κB) pathway. Moreover, ApoE3 showed greater inhibition of c-Fos, dendritic cell-specific transmembrane protein (DC-STAMP), and Cathepsin K gene expression compared to ApoE4. Collectively, ApoE plays crucial roles in preserving bone mass, suggesting that targeting ApoE and its isoforms as a promising treatment candidate of both osteoporosis and hyperlipidemia.

The Effects of Aronia melanocarpa 'Viking' Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling.

This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa ‘Viking’ (AM) and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS) are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL) were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK) and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1) protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β3. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB)-mediated c-Fos and NFATc1 signaling pathway.

Inhibitory Effect of Purpurogallin on Osteoclast Differentiation in Vitro through the Downregulation of c-Fos and NFATc1.

Purpurogallin, a benzotropolone-containing natural compound, has been reported to exhibit numerous biological and pharmacological functions, such as antioxidant, anticancer, and anti-inflammatory effects. In this study, we enzymatically synthesized purpurogallin from pyrogallol and investigated its role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Purpurogallin attenuated the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts from bone marrow macrophages (BMMs) without causing cytotoxicity, and suppressed upregulation of osteoclast-specific markers, including TRAP (Acp5), cathepsin K (Ctsk), and dendritic cell-specific transmembrane protein (Dcstamp). However, purpurogallin did not affect the bone resorbing function of mature osteoclasts evident by the resorption pit assay. Activation of mitogen-activated protein kinases, Akt and IkB pathways in RANK signaling were not altered by purpurogallin, whereas the expression of c-Fos and NFATc1, key transcriptional regulators in osteoclastogenesis, was dramatically inhibited by purpurogallin. Purpurogallin also significantly reduced the expression level of B lymphocyte-induced maturation protein-1 (Blimp1) gene (Prdm1). Further, downregulation of Blimp1 led to forced expression of anti-osteoclastogenic genes, including interferon regulatory factor-8 (Irf8) and B-cell lymphoma 6 (Bcl6) genes. Taken together, our data suggested that purpurogallin inhibits osteoclast differentiation via downregulation of c-Fos and NFATc1.

C-Fos downregulation positively regulates EphA5 expression in a congenital hypothyroidism rat model.

The EphA5 receptor is well established as an axon guidance molecule during neural system development and plays an important role in dendritic spine formation and synaptogenesis. Our previous study has showed that EphA5 is decreased in the developing brain of congenital hypothyroidism (CH) and the EphA5 promoter methylation modification participates in its decrease. c-Fos, a well-kown transcription factor, has been considered in association with brain development. Bioinformatics analysis showed that the EphA5 promoter region contained five putative c-fos binding sites. The chromatin immunoprecipitation (ChIP) assays were used to assess the direct binding of c-fos to the EphA5 promoter. Furthermore, dual-luciferase assays showed that these three c-fos protein binding sites were positive regulatory elements for EphA5 expression in PC12 cells. Moreover, We verified c-fos positively regulation for EphA5 expression in CH model. Q-PCR and Western blot showed that c-fos overexpression could upregulate EphA5 expression in hippocampal neurons of rats with CH. Our results suggest that c-fos positively regulates EphA5 expression in CH rat model.