Involvement of RAMP1/p38MAPK signaling pathway in osteoblast differentiation in response to mechanical stimulation: a preliminary study.

OBJECTIVE: The present study aimed to investigate the underlying mechanism of mechanical stimulation in regulating osteogenic differentiation. MATERIALS AND METHODS: Osteoblasts were exposed to compressive force (0-4 g/cm2) for 1-3 days or CGRP for 1 or 3 days. Expression of receptor activity modifying protein 1 (RAMP1), the transcription factor RUNX2, osteocalcin, p38 and p-p38 were analyzed by western blotting. Calcium mineralization was analyzed by alizarin red straining. RESULTS: Using compressive force treatments, low magnitudes (1 and 2 g/cm2) of compressive force for 24 h promoted osteoblast differentiation and mineral deposition whereas higher magnitudes (3 and 4 g/cm2) did not produce osteogenic effect. Through western blot assay, we observed that the receptor activity-modifying protein 1 (RAMP1) expression was upregulated, and p38 mitogen-activated protein kinase (MAPK) was phosphorylated during low magnitudes compressive force-promoted osteoblast differentiation. Further investigation of a calcitonin gene-related peptide (CGRP) peptide incubation, a ligand for RAMP1, showed that CGRP at concentration of 25 and 50 ng/ml could increase expression levels of RUNX2 and osteocalcin, and percentage of mineralization, suggesting its osteogenic potential. In addition, with the same conditions, CGRP also significantly upregulated RAMP1 and phosphorylated p38 expression levels. Also, the combination of compressive forces (1 and 2 g/cm2) with 50 ng/ml CGRP trended to increase RAMP1 expression, p38 activity, and osteogenic marker RUNX2 levels, as well as percentage of mineralization compared to compressive force alone. This suggest that RAMP1 possibly acts as an upstream regulator of p38 signaling during osteogenic differentiation. CONCLUSION: These findings suggest that CGRP-RAMP1/p38MAPK signaling implicates in osteoblast differentiation in response to optimal magnitude of compressive force. This study helps to define the underlying mechanism of compressive stimulation and may also enhance the application of compressive stimulation or CGRP peptide as an alternative approach for accelerating tooth movement in orthodontic treatment.

PRDX1 exerts a photoprotection effect by inhibiting oxidative stress and regulating MAPK signaling on retinal pigment epithelium.

BACKGROUND: The purpose of this study was to investigate the photoprotection effect of peroxiredoxin 1 (PRDX1) protein in ultraviolet B (UVB) irradiation-induced damage of retinal pigment epithelium (RPE) and its possible molecular mechanism. METHODS: ARPE-19 cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the PRDX1 expression. The corresponding kits were employed to measure the levels or activities of lactate dehydrogenase (LDH), 8-hydroxy-2-deoxyguanosine (8-OHdG), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD). Western blotting was applied to examine PRDX1 expression and mitogen-activated protein kinase (MAPK) signaling pathway-related proteins. RESULTS: After exposure to 20 mJ/cm2 intensity of UVB irradiation for 24 h, ARPE-19 cells viability was decreased, the leakage degree of LDH and 8-OHdG were increased, and cell apoptosis was elevated. The expression of PRDX1 was significantly down-regulated in UVB-induced ARPE-19 cells. The low expression of PRDX1 was involved in high irradiation intensity. Overexpression of PRDX1 increased cell activity, decreased cell apoptosis, and LDH as well as 8-OHdG leakage in UVB-induced ARPE-19 cells. In addition to alleviating UVB-induced cell damage, PRDX1 overexpression also inhibited UVB-induced oxidative stress (down-regulation of ROS and MDA levels, up-regulation of GSH-Px and SOD activities) and the activation of MAPK signaling pathway in ARPE-19 cells. CONCLUSION: PRDX1 exerts a photoprotection effect on RPE by attenuating UVB-induced cell damage and inhibiting oxidative stress, which can be attributed to the inhibition of MAPK signaling pathway activation.

Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype.

The microtubule-associated protein Tau is a key player in various neurodegenerative conditions, including Alzheimer's disease (AD) and Tauopathies, where its hyperphosphorylation disrupts neuronal microtubular lattice stability. Glaucoma, a neurodegenerative disorder affecting the retina, leads to irreversible vision loss by damaging retinal ganglion cells and the optic nerve, often associated with increased intraocular pressure. Prior studies have indicated Tau expression and phosphorylation alterations in the retina in both AD and glaucoma, yet the causative or downstream nature of Tau protein changes in these pathologies remains unclear. This study investigates the impact of Tau protein modulation on retinal neurons under normal and experimental glaucoma conditions. Employing AAV9-mediated gene therapy for Tau overexpression and knockdown, both manipulations were found to adversely affect retinal structural and functional measures as well as neuroprotective Akt/Erk survival signalling in healthy conditions. In the experimental glaucoma model, Tau overexpression intensified inner retinal degeneration, while Tau silencing provided significant protection against these degenerative changes. These findings underscore the critical role of endogenous Tau protein levels in preserving retinal integrity and emphasize the therapeutic potential of targeting Tau in glaucoma pathology.

CD36 deletion prevents white matter injury by modulating microglia polarization through the Traf5-MAPK signal pathway.

BACKGROUND: White matter injury (WMI) represents a significant etiological factor contributing to neurological impairment subsequent to Traumatic Brain Injury (TBI). CD36 receptors are recognized as pivotal participants in the pathogenesis of neurological disorders, including stroke and spinal cord injury. Furthermore, dynamic fluctuations in the phenotypic polarization of microglial cells have been intimately associated with the regenerative processes within the injured tissue following TBI. Nevertheless, there is a paucity of research addressing the impact of CD36 receptors on WMI and microglial polarization. This investigation aims to elucidate the functional role and mechanistic underpinnings of CD36 in modulating microglial polarization and WMI following TBI. METHODS: TBI models were induced in murine subjects via controlled cortical impact (CCI). The spatiotemporal patterns of CD36 expression were examined through quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunofluorescence staining. The extent of white matter injury was assessed via transmission electron microscopy, Luxol Fast Blue (LFB) staining, and immunofluorescence staining. Transcriptome sequencing was employed to dissect the molecular mechanisms underlying CD36 down-regulation and its influence on white matter damage. Microglial polarization status was ascertained using qPCR, Western blot analysis, and immunofluorescence staining. In vitro, a Transwell co-culture system was employed to investigate the impact of CD36-dependent microglial polarization on oligodendrocytes subjected to oxygen-glucose deprivation (OGD). RESULTS: Western blot and qPCR analyses revealed that CD36 expression reached its zenith at 7 days post-TBI and remained sustained at this level thereafter. Immunofluorescence staining exhibited robust CD36 expression in astrocytes and microglia following TBI. Genetic deletion of CD36 ameliorated TBI-induced white matter injury, as evidenced by a reduced SMI-32/MBP ratio and G-ratio. Transcriptome sequencing unveiled differentially expressed genes enriched in processes linked to microglial activation, regulation of neuroinflammation, and the TNF signaling pathway. Additionally, bioinformatics analysis pinpointed the Traf5-p38 axis as a critical signaling pathway. In vivo and in vitro experiments indicated that inhibition of the CD36-Traf5-MAPK axis curtailed microglial polarization toward the pro-inflammatory phenotype. In a Transwell co-culture system, BV2 cells treated with LPS + IFN-γ exacerbated the damage of post-OGD oligodendrocytes, which could be rectified through CD36 knockdown in BV2 cells. CONCLUSIONS: This study illuminates that the suppression of CD36 mitigates WMI by constraining microglial polarization towards the pro-inflammatory phenotype through the down-regulation of the Traf5-MAPK signaling pathway. Our findings present a potential therapeutic strategy for averting neuroinflammatory responses and ensuing WMI damage resulting from TBI.

Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events.

Understanding the dynamics of intracellular signaling pathways, such as ERK1/2 (ERK) and Akt1/2 (Akt), in the context of cell fate decisions is important for advancing our knowledge of cellular processes and diseases, particularly cancer. While previous studies have established associations between ERK and Akt activities and proliferative cell fate, the heterogeneity of single-cell responses adds complexity to this understanding. This study employed a data-driven approach to address this challenge, developing machine learning models trained on a dataset of growth factor-induced ERK and Akt activity time courses in single cells, to predict cell division events. The most predictive models were developed by applying discrete wavelet transforms (DWTs) to extract low-frequency features from the time courses, followed by using Ensemble Integration, a data integration and predictive modeling framework. The results demonstrated that these models effectively predicted cell division events in MCF10A cells (F-measure=0.524, AUC=0.726). ERK dynamics were found to be more predictive than Akt, but the combination of both measurements further enhanced predictive performance. The ERK model`s performance also generalized to predicting division events in RPE cells, indicating the potential applicability of these models and our data-driven methodology for predicting cell division across different biological contexts. Interpretation of these models suggested that ERK dynamics throughout the cell cycle, rather than immediately after growth factor stimulation, were associated with the likelihood of cell division. Overall, this work contributes insights into the predictive power of intra-cellular signaling dynamics for cell fate decisions, and highlights the potential of machine learning approaches in unraveling complex cellular behaviors.

GPS2 ameliorates cigarette smoking-induced pulmonary vascular remodeling by modulating the ras-Raf-ERK axis.

BACKGROUND: Mitogen-activated protein kinase (MAPK)signaling-mediated smoking-associated pulmonary vascular remodeling (PVR) plays an important role in the pathogenesis of group 3 pulmonary hypertension (PH). And G protein pathway suppressor 2 (GPS2) could suppress G-protein signaling such as Ras and MAPK, but its role in cigarette smoking -induced PVR (CS-PVR) is unclear. METHODS: An in vivo model of smoke-exposed rats was constructed to assess the role of GPS2 in smoking-induced PH and PVR. In vitro, the effects of GPS2 overexpression and silencing on the function of human pulmonary arterial smooth cells (HPASMCs) and the underlying mechanisms were explored. RESULTS: GPS2 expression was downregulated in rat pulmonary arteries (PAs) and HPASMCs after CS exposure. More importantly, CS-exposed rats with GPS2 overexpression had lower right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness (WT%) than those without. And enhanced proliferation and migration of HPASMCs induced by cigarette smoking extract (CSE) can be evidently inhibited by overexpressed GPS2. Besides, GPS2siRNA significantly enhanced the proliferation, and migration of HPASMCs as well as activated Ras and Raf/ERK signaling, while these effects were inhibited by zoledronic acid (ZOL). In addition, GPS2 promoter methylation level in rat PAs and HPASMCs was increased after CS exposure, and 5-aza-2-deoxycytidine (5-aza) inhibited CSE-induced GPS2 hypermethylation and downregulation in vitro. CONCLUSIONS: GPS2 overexpression could improve the CS-PVR, suggesting that GPS2 might serve as a novel therapeutic target for PH-COPD in the future.

Activation of the cGMP/PKG/ERK signaling pathway associated with PDE5Is inhibits fibroblast activation by downregulating autophagy in early progressive benign prostatic hyperplasia.

PURPOSE: Benign prostatic hyperplasia (BPH) is one of the most prevalent diseases affecting aging males. However, approximately, 8% of the BPH patients under 50-year-old experience remarkably early progression, for reasons that remain elusive. Among the various factors implicated in promoting BPH advancement, the activation of fibroblasts and autophagy hold particular importance. Our research endeavors to explore the mechanisms behind the accelerated progression in these patients. METHODS: Immunohistochemistry and immunofluorescence were performed to detect the expression levels of LC3, p62, PDE5, and α-SMA in diverse BPH tissues and prostate stromal cells. The autophagy activator rapamycin, the autophagy suppressor chloroquine, and siRNA transfection were used to identify the impact of autophagy on fibroblast activation. RESULTS: Prostatic stromal fibroblasts in early progressive BPH tissues displayed activation of autophagy with an upregulation of LC3 and a concurrent downregulation of p62. After starvation or rapamycin treatment to a heightened level of autophagy, fibroblasts exhibited activation. Conversely, chloroquine treatment and ATG-7-knockdown effectively suppressed the level of autophagy and fibroblast activation. High expression of PDE5 was found in early progressive BPH stromal cells. The administration of PDE5 inhibitors (PDE5Is) hindered fibroblast activation through suppressing autophagy by inhibiting the ERK signaling pathway. CONCLUSION: Our findings suggest that autophagy plays a pivotal role in promoting BPH progression through fibroblast activation, while PDE5Is effectively suppress autophagy and fibroblast activation via the ERK signaling pathway. Nevertheless, further investigations are warranted to comprehensively elucidate the role of autophagy in BPH progression.

p200CUX1-regulated BMP8B inhibits the progression of acute myeloid leukemia via the MAPK signaling pathway.

The full-length p200CUX1 protein encoded by the homology frame CUT-like protein (CUX1) plays an important role in tumors as a pro-oncogene or oncogene. However, its role and mechanism in acute myeloid leukemia remain unknown. p200CUX1 regulates several pathways, including the MAPK signaling pathway. Our data showed that p200CUX1 is lowly expressed in THP1 and U937 AML cell lines. Lentiviral overexpression of p200CUX1 reduced proliferation and promoted apoptosis and G0/G1 phase blockade, correlating with MAPK pathway suppression. Additionally, p200CUX1 regulated the expression of bone morphogenetic protein 8B (BMP8B), which is overexpressed in AML. Overexpression of p200CUX1 downregulated BMP8B expression and inhibited the MAPK pathway. Furthermore, BMP8B knockdown inhibited AML cell proliferation, enhanced apoptosis and the sensitivity of ATRA-induced cell differentiation, and blocked G0/G1 transition. Our findings demonstrate the pivotal function of the p200CUX1-BMP8B-MAPK axis in maintaining the viability of AML cells. Consequently, targeting p200CUX1 could represent a viable strategy in AML therapy.

Intervention of exogenous VEGF protect brain microvascular endothelial cells from hypoxia-induced injury by regulating PLCγ/RAS/ERK and PI3K/AKT pathways.

Ischemic stroke rapidly increases the expression level of vascular endothelial growth factor (VEGF), which promotes neovascularization during hypoxia. However, the effect and mechanism of VEGF intervention on cerebrovascular formation remain unclear. Therefore, our research discussed the protective effect of exogenous VEGF on cells in hypoxia environment in cerebral microvascular endothelial cells, simulating ischemic stroke in hypoxic environment. Firstly, we detected the proliferation and apoptosis of cerebral microvascular endothelial cells under hypoxia environment, as well the expression levels of VEGF-E, vascular endothelial growth factor re-ceptor-2 (VEGFR-2), BCL2, PRKCE and PINK1. Moreover, immunofluorescence and western blotting were used to verify the regulation of exogenous VEGF-E on VEGFR-2 expression in hypoxic or normal oxygen environment. Lastly, we manipulated the concentration of VEGF-E in the culture medium to investigate its impact on phospholipase Cγ1 (PLCγ1)/extracellular signaling regulatory protein kinase (ERK) -1/2 and protein kinase B (AKT) pathways. Additionally, we employed a PLCγ1 inhibitor (U73122) to investigate its impact on proliferation and PLCγ1/ERK pathways. The results show that hypoxia inhibited the proliferation of cerebral microvascular endothelial cells, promoted cell apoptosis, significantly up-regulated the expression of VEGF-E, VEGFR-2, PRKCE and PINK1, but down-regulated the expression of BCL2. Interference from exogenous VEGF-E activated PLCγ1/ERK-1/2 and AKT pathways, promoting cell proliferation and inhibiting apoptosis of hypoxic brain microvascular endothelial cells. In summary, exogenous VEGF-E prevents hypoxia-induced damage to cerebral microvascular endothelial cells by activating the PLCγ1/ERK and AKT pathways. This action inhibits the apoptosis pathway in hypoxic cerebral microvascular endothelial cells, thereby safeguarding the blood-brain barrier and the nervous system.

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways.

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

Interleukin-1 beta signals through the ERK signalling pathway to modulate human placental trophoblast migration and invasion in the first trimester of pregnancy.

INTRODUCTION: Interleukin-1 beta (IL-1ß) can promote cell migration, invasion and metastasis in various cancer cells. The mechanism of its role in human trophoblast has not been fully investigated. Therefore, we aimed to investigate the expression level of IL-1ß in first trimester decidua and placenta and its potential role in regulation of extravillous trophoblast cell (EVT) invasion and migration. METHODS: First trimester placenta and decidua were collected to study the expression levels of IL-1ß and its receptors by immunohistochemical staining. Primary isolates of first trimester EVT or the HTR-8/SVneo trophoblast like cell line were used to assess migration and invasion. Matrix metalloproteinase levels were assessed by gelatin zymography and ELISA. The phosphorylation profile of signaling pathway proteins was detected with the Proteome Profiler Human Phospho-Kinase Array Kit. Differentially expressed proteins in cells was detected and verified by Western Blot. RESULTS: IL-1ß, its receptors and antagonist are expressed in first trimester placenta and decidua, exogenous IL-1ß stimulates trophoblast cell outgrowth, migration and invasion through the ERK signaling pathway. IL-1ß was significantly increased in the placenta at 6-7 weeks gestation compared with 8-9 weeks gestation (P < 0.0001). Transwell and RTCA assays indicated that IL-1ß stimulates the invasion and migration of EVT. In addition, IL-1ß promoted the phosphorylation of ERK 1/2. It also promoted the expression of MMP2 and MMP9 in EVT as demonstrated by gelatin zymography assay and enzyme linked immunosorbent assay. DISCUSSION: This study demonstrated IL-1ß expression in placenta and decidua, and that it regulates EVT invasion and migration.

Calcium saccharate/DUSP6 suppresses renal cell carcinoma glycolytic metabolism and boosts sunitinib efficacy via the ERK-AKT pathway.

Current therapeutic options for renal cell carcinoma (RCC) are very limited, which is largely due to inadequate comprehension of molecular pathological mechanisms as well as RCC's resistance to chemotherapy. Dual-specificity phosphatase 6 (DUSP6) has been associated with numerous human diseases. However, its role in RCC is not well understood. Here, we show that diminished DUSP6 expression is linked to RCC progression and unfavorable prognosis. Mechanistically, DUSP6 serves as a tumor suppressor in RCC by intervening the TAF10 and BSCL2 via the ERK-AKT pathway. Further, DUSP6 is also transcriptionally regulated by HNF-4a. Moreover, docking experiments have indicated that DUSP6 expression is enhanced when bound by Calcium saccharate, which also inhibits RCC cell proliferation, metabolic rewiring, and sunitinib resistance. In conclusion, our study identifies Calcium saccharate as a prospective pharmacological therapeutic approach for RCC.

Dual-specificity phosphatase 26 protects against kidney injury caused by ischaemia-reperfusion through restraint of TAK1-JNK/p38-mediated apoptosis and inflammation of renal tubular epithelial cells.

Dual-specificity phosphatase 26 (DUSP26) acts as a pivotal player in the transduction of signalling cascades with its dephosphorylating activity. Currently, DUSP26 attracts extensive attention due to its particular function in several pathological conditions. However, whether DUSP26 plays a role in kidney ischaemia-reperfusion (IR) injury is unknown. Aims of the current work were to explore the relevance of DUSP26 in kidney IR damage. DUSP26 levels were found to be decreased in renal tubular epithelial cells following hypoxia-reoxygenation (HR) and kidney samples subjected to IR treatments. DUSP26-overexpressed renal tubular epithelial cells exhibited protection against HR-caused apoptosis and inflammation, while DUSP26-depleted renal tubular epithelial cells were more sensitive to HR damage. Upregulation of DUSP26 in rat kidneys by infecting adenovirus expressing DUSP26 markedly ameliorated kidney injury caused by IR, while also effectively reducing apoptosis and inflammation. The mechanistic studies showed that the activation of transforming growth factor-ß-activated kinase 1 (TAK1)-JNK/p38 MAPK, contributing to kidney injury under HR or IR conditions, was restrained by increasing DUSP26 expression. Pharmacological restraint of TAK1 markedly diminished DUSP26-depletion-exacebated effects on JNK/p38 activation and HR injury of renal tubular cells. The work reported a renal-protective function of DUSP26, which protects against IR-related kidney damage via the intervention effects on the TAK1-JNK/p38 axis. The findings laid a foundation for understanding the molecular pathogenesis of kidney IR injury and provide a prospective target for treating this condition.

TNF-α can promote membrane invasion by activating the MAPK/MMP9 signaling pathway through autocrine in bone-invasive pituitary adenoma.

AIMS: A bone-invasive pituitary adenoma exhibits aggressive behavior, leading to a worse prognosis. We have found that TNF-α promotes bone invasion by facilitating the differentiation of osteoclasts, however, before bone-invasive pituitary adenoma invades bone tissue, it needs to penetrate the dura mater, and this mechanism is not yet clear. METHODS: We performed transcriptome microarrays on specimens of bone-invasive pituitary adenomas (BIPAs) and noninvasive pituitary adenomas (NIPAs) and conducted differential expressed gene analysis and enrichment analysis. We altered the expression of TNF-α through plasmids, then validated the effects of TNF-α on GH3 cells and verified the efficacy of the TNF-α inhibitor SPD304. Finally, the effects of TNF-α were validated in in vivo experiments. RESULTS: Pathway act work showed that the MAPK pathway was significantly implicated in the pathway network. The expression of TNF-α, MMP9, and p-p38 is higher in BIPAs than in NIPAs. Overexpression of TNF-α elevated the expression of MAPK pathway proteins and MMP9 in GH3 cells, as well as promoted proliferation, migration, and invasion of GH3 cells. Flow cytometry indicated that TNF-α overexpression increased the G2 phase ratio in GH3 cells and inhibited apoptosis. The expression of MMP9 was reduced after blocking the P38 MAPK pathway; overexpression of MMP9 promoted invasion of GH3 cells. In vivo experiments confirm that the TNF-α overexpression group has larger tumor volumes. SPD304 was able to suppress the effects caused by TNF-α overexpression. CONCLUSION: Bone-invasive pituitary adenoma secretes higher levels of TNF-α, which then acts on itself in an autocrine manner, activating the MAPK pathway and promoting the expression of MMP9, thereby accelerating the membrane invasion process. SPD304 significantly inhibits the effect of TNF-α and may be applied in the clinical treatment of bone-invasive pituitary adenoma.

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

Inhibition of TREM-1 alleviates neuroinflammation by modulating microglial polarization via SYK/p38MAPK signaling pathway after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI), as a major public health problem, is characterized by high incidence rate, disability rate, and mortality rate. Neuroinflammation plays a crucial role in the pathogenesis of TBI. Triggering receptor expressed on myeloid cells-1 (TREM-1) is recognized as an amplifier of the inflammation in diseases of the central nervous system (CNS). However, the function of TREM-1 remains unclear post-TBI. This study aimed to investigate the function of TREM-1 in neuroinflammation induced by TBI. METHODS: Brain water content (BWC), modified neurological severity score (mNSS), and Morris Water Maze (MWM) were measured to evaluate the effect of TREM-1 inhibition on nervous system function and outcome after TBI. TREM-1 expression in vivo was evaluated by Western blotting. The cellular localization of TREM-1 in the damaged region was observed via immunofluorescence staining. We also conducted Western blotting to examine expression of SYK, p-SYK and other downstream proteins. RESULTS: We found that inhibition of TREM-1 reduced brain edema, decreased mNSS and improved neurobehavioral outcomes after TBI. It was further determined that TREM-1 was expressed on microglia and modulated subtype transition of microglia. Inhibition of TREM-1 alleviated neuroinflammation, which was associated with SYK/p38MAPK signaling pathway. CONCLUSIONS: These findings suggest that TREM-1 can be a potential clinical therapeutic target for alleviating neuroinflammation after TBI.

TGF-ß downstream of Smad3 and MAPK signaling antagonistically regulate the viability and partial epithelial-mesenchymal transition of liver progenitor cells.

BACKGROUND: Liver progenitor cells (LPCs) are a subpopulation of cells that contribute to liver regeneration, fibrosis and liver cancer initiation under different circumstances. RESULTS: By performing adenoviral-mediated transfection, CCK-8 analyses, F-actin staining, transwell analyses, luciferase reporter analyses and Western blotting, we observed that TGF-ß promoted cytostasis and partial epithelial-mesenchymal transition (EMT) in LPCs. In addition, we confirmed that TGF-ß activated the Smad and MAPK pathways, including the Erk, JNK and p38 MAPK signaling pathways, and revealed that TGFß-Smad signaling induced growth inhibition and partial EMT, whereas TGFß-MAPK signaling had the opposite effects on LPCs. We further found that the activity of Smad and MAPK signaling downstream of TGF-ß was mutually restricted in LPCs. Mechanistically, we found that TGF-ß activated Smad signaling through serine phosphorylation of both the C-terminal and linker regions of Smad2 and 3 in LPCs. Additionally, TGFß-MAPK signaling inhibited the phosphorylation of Smad3 but not Smad2 at the C-terminus, and it reinforced the linker phosphorylation of Smad3 at T179 and S213. We then found that overexpression of mutated Smad3 at linker phosphorylation sites intensifies TGF-ß-induced cytostasis and EMT, mimicking the effects of MAPK inhibition in LPCs, whereas mutation of Smad3 at the C-terminus caused LPCs to blunt TGF-ß-induced cytostasis and partial EMT. CONCLUSION: These results suggested that TGF-ß downstream of Smad3 and MAPK signaling were mutually antagonistic in regulating the viability and partial EMT of LPCs. This antagonism may help LPCs overcome the cytostatic effect of TGF-ß under fibrotic conditions and maintain partial EMT and progenitor phenotypes.

Abrogating PDK4 activates autophagy-dependent ferroptosis in breast cancer via ASK1/JNK pathway.

BACKGROUND: Targeting ferroptosis mediated by autophagy presents a novel therapeutic approach to breast cancer, a mortal neoplasm on the global scale. Pyruvate dehydrogenase kinase isozyme 4 (PDK4) has been denoted as a determinant of breast cancer metabolism. The target of this study was to untangle the functional mechanism of PDK4 in ferroptosis dependent on autophagy in breast cancer. METHODS: RT-qPCR and western blotting examined PDK4 mRNA and protein levels in breast cancer cells. Immunofluorescence staining appraised light chain 3 (LC3) expression. Fe (2 +) assay estimated total iron level. Relevant assay kits and C11-BODIPY (591/581) staining evaluated lipid peroxidation level. DCFH-DA staining assayed intracellular reactive oxygen species (ROS) content. Western blotting analyzed the protein levels of autophagy, ferroptosis and apoptosis-signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) pathway-associated proteins. RESULTS: PDK4 was highly expressed in breast cancer cells. Knockdown of PDK4 induced the autophagy of breast cancer cells and 3-methyladenine (3-MA), an autophagy inhibitor, countervailed the promoting role of PDK4 interference in ferroptosis in breast cancer cells. Furthermore, PDK4 knockdown activated ASK1/JNK pathway and ASK1 inhibitor (GS-4997) partially abrogated the impacts of PDK4 absence on the autophagy and ferroptosis in breast cancer cells. CONCLUSION: To sum up, deficiency of PDK4 activated ASK1/JNK pathway to stimulate autophagy-dependent ferroptosis in breast cancer.

NUPR1 induces autophagy and promotes the progression of Esophageal squamous cell carcinoma via the MAPK-mTOR pathway.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is a dominant pathological type in China. NUPR1 is a complex molecule implicated in various physiological and biological functions whose expression is upregulated in response to stress. Furthermore, autophagy is a vital physiological mechanism in the onset and metastasis of malignancies. This study aims to uncover the influence of NUPR1 on ESCC occurrence and development by regulating autophagy while also exploring its association with the MAPK signaling pathway. METHODS: First, the differences in NUPR1 between ESCC and normal tissues were analyzed through online databases. Subsequently, the pathological tissues of clinical samples were stained and scored using immunohistochemistry. And NUPR1 expression in ESCC cells was investigated, as was the function of NUPR1 in the modulation of ESCC's malignant behavior. Furthermore, a nude mouse ESCC xenograft model was developed. Finally, RNA sequencing was performed on NUPR1-downregulated ESCC cells, which was verified using WB. RESULTS: Our findings initially uncovered differences in the expression of NUPR1 in ESCC and normal tissues. In vitro experiments demonstrated that NUPR1 downregulation significantly inhibited ESCC cell proliferation, invasion, and migration, as well as promoted their apoptosis. Our xenograft model exhibited significant inhibition of ESCC tumors upon NUPR1 downregulation. Subsequently, RNA sequencing uncovered that NUPR1 regulates its malignant biological behavior through MAPK-mTOR signaling pathway. Finally, we found that NUPR1 downregulation can inhibit autophagic flux in ESCC. CONCLUSION: Collectively, our findings show that NUPR1 enhances the progression of ESCC by triggering autophagy and is associated with the MAPK-mTOR signaling pathway.

circUSP13 facilitates the fast-to-slow myofiber shift via the MAPK/ERK signaling pathway in goat skeletal muscles.

Understanding how skeletal muscle fiber proportions are regulated is essential for understanding muscle function and improving the quality of mutton. While circular RNA (circRNA) has a critical function in myofiber type transformation, the specific mechanisms are not yet fully understood. Prior evidence indicates that circular ubiquitin-specific peptidase 13 (circUSP13) can promote myoblast differentiation by acting as a ceRNA, but its potential role in myofiber switching is still unknown. Herein, we found that circUSP13 enhanced slow myosin heavy chain (MyHC-slow) and suppressed MyHC-fast expression in goat primary myoblasts (GPMs). Meanwhile, circUSP13 evidently enhanced the remodeling of the mitochondrial network while inhibiting the autophagy of GPMs. We obtained fast-dominated myofibers, via treatment with rotenone, and further demonstrated the positive role of circUSP13 in the fast-to-slow transition. Mechanistically, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway significantly impaired the slow-to-fast shift in fully differentiated myotubes, which was restored by circUSP13 or IGF1 overexpression. In conclusion, circUSP13 promoted the fast-to-slow myofiber type transition through MAPK/ERK signaling in goat skeletal muscle. These findings provide novel insights into the role of circUSP13 in myofiber type transition and contribute to a better understanding of the genetic mechanisms underlying meat quality.