ERK Signaling Pathway Research Articles

Branched-chain amino acids promote hepatic Cyp7a1 expression and bile acid synthesis via suppressing FGF21-ERK pathway.

Branched-chain amino acids (BCAAs) including leucine, isoleucine and valine have been linked with metabolic and cardiovascular diseases. BCAAs homeostasis is tightly controlled by their catabolic pathway. BCKA dehydrogenase (BCKD) complex is the rate-limiting step for BCAA catabolism. Mitochondrial phosphatase 2C (PP2Cm) dephosphorylates the BCKD E1alpha subunit and activates BCKD complex. Deficiency of PP2Cm impairs BCAA catabolism, leading to higher plasma BCAA concentrations. Emerging evidence shows that bile acids are key regulators of glucose, lipid and energy metabolism. In this study, we investigated whether a direct link existed between BCAAs and bile acids metabolism. Wild-type mice were fed with normal-BCAA or high-BCAA diet, while PP2Cm deficiency mice were fed with normal chow for 14 weeks. The mice were fasted for 6 h before tissue harvest to exclude metabolic changes due to immediate food intake. We showed that the bile acids in tissues and feces were significantly elevated in wild-type mice fed with high-BCAA diet as well as in PP2Cm deficiency mice fed with normal chow. These mice displayed significantly increased expression of cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme of bile acid synthesis in liver, and 7α-hydroxy-4-cholesten-3-one (C4), a freely diffusible metabolite downstream of CYP7A1 in plasma. BCAAs induced Cyp7a1 expression in cultured hepatocytes. In mouse liver and cultured hepatocytes, we demonstrated that elevated BCAAs inhibited fibroblast growth factor 21 (FGF21) expression and ERK signaling pathway. Direct inhibition of ERK by U0126 (800 nM) markedly induced Cyp7a1 expression in cultured hepatocytes. Moreover, the induced Cyp7a1 expression and inhibitory effects of BCAAs on ERK signaling pathway were abolished by treatment with recombinant FGF21 protein in mouse liver and cultured hepatocytes. Collectively, this study demonstrates a direct link between BCAAs and bile acid synthesis. BCAAs promotes Cyp7a1 expression and bile acid synthesis in liver via inhibiting FGF21-ERK signaling pathway. BCAAs-regulated bile acid synthesis and homeostasis may contribute to developing novel therapeutic strategies for the treatment of metabolic disorders.

House dust mites stimulate thymic stromal lymphopoietin production in human bronchial epithelial cells and promote airway remodeling through activation of PAR2 and ERK signaling pathway.

House dust mites (HDM) are common aeroallergens linked to airway inflammation and remodeling in asthma. Protease-activated receptor 2 (PAR2) and thymic stromal lymphopoietin (TSLP) may mediate these immune responses. However, how the epithelium influences fibroblasts toward airway remodeling remains unclear. We hypothesize that HDM stimulates human bronchial epithelial cells (HBECs) to produce TSLP via PAR2 activation, driving fibroblasts toward remodeling processes. HBECs were treated with HDM, with or without the PAR2 antagonist FSLLRY-NH2 (FSL), and TSLP expression was measured by qPCR and ELISA. Phosphorylation of MAPKs was assessed by western blotting. Human lung fibroblasts (HLFs) were exposed to recombinant TSLP or conditioned medium (CM) from HDM-stimulated HBECs, with or without anti-TSLP antibodies. Fibroblast proliferation and collagen production were assessed as remodeling markers. HDM increased ERK phosphorylation (not p38 or JNK) and TSLP expression at mRNA and protein levels. FSL preincubation significantly reduced ERK phosphorylation and TSLP production: HDM-stimulated CM induced fibroblast proliferation and collagen production, effects suppressed by anti-TSLP or FSL. Direct treatment with recombinant TSLP also promoted fibroblast proliferation and collagen synthesis. These findings suggest that HDM promotes HBEC-to-HLF paracrine interactions via PAR2-ERK-TSLP axis, participating in airway remodeling. PAR2 antagonists may represent potential therapeutic targets for HDM-induced remodeling processes.

Retraction: Long non-coding RNA SNHG16 regulates cell behaviors through miR-542-3p/HNF4α axis via RAS/RAF/MEK/ERK signaling pathway in pediatric neuroblastoma cells.

Retraction: Long non-coding RNA SNHG16 regulates cell behaviors through miR-542-3p/HNF4α axis via RAS/RAF/MEK/ERK signaling pathway in pediatric neuroblastoma cells.

BIOM-26. ROLE OF EXTRA-DOMAIN B FIBRONECTIN IN MODULATING GLIOBLASTOMA CELL INVASIVE PROPERTIES

Abstract Glioblastoma (GBM) is a highly malignant tumor characterized by its resistance to current treatment modalities and high recurrence rates. Extra-domain B Fibronectin (EDB-FN), a glycoprotein not found in normal tissues but selectively expressed in the tumor microenvironment, was previously identified by our team as a promising molecular marker for brain tumors, offering potential for both prognostic prediction and therapeutic targeting. However, the specific role and mechanism of action of EDB-FN in GBM cells remain unclear. In this study, we sought to elucidate the potential role of EDB-FN and the underlying mechanisms contributing to GBM malignancy through in vitro investigations. Our findings reveal that silencing EDB-FN in GBM cells leads to a decrease in cell migration, invasiveness, extracellular matrix adhesion ability, and stem cell-like properties. Furthermore, we discovered that FAK and ERK signaling pathways play a crucial role in GBM cell progression in response to EDB-FN. This study underscores the potential of EDB-FN as a specific biomarker for GBM treatment, paving the way for future research in this area.

Dayuan Yin alleviates symptoms of HCoV-229E-induced pneumonia and modulates the Ras/Raf1/MEK/ERK pathway

Viral pneumonia is characterized by inflammation in the lungs triggered by respiratory viruses. Dayuan Yin (DYY), a traditional Chinese medicine formula known for treating infectious diseases, is hypothesized to offer therapeutic benefits in treating viral pneumonia, although its specific molecular impacts remain understudied. This study aimed to evaluate the therapeutic effects of DYY in mitigating HCoV-229E virus-induced pneumonia in mice. This study employed an HCoV-229E virus-infected mouse model to investigate the therapeutic potential and underlying molecular mechanisms of DYY on virus-induced pneumonia. The respiratory function and organ indices post-treatment were assessed. Lung tissue and tracheal lesions were evaluated via immunohistochemistry. Spleen immune cell composition was analyzed using flow cytometry. Inflammatory cytokines and viral loads were quantified using hypersensitive multiplex electrochemiluminescence method and PCR analysis, respectively. The expression levels of MAS1, Ras, Raf1, MEK1/2, and ERK1/2 in lung tissues were determined through western blot analysis. DYY significantly improved respiratory function, and reduced organ pathology in infected mice. It effectively decreased viral loads and inflammatory cytokines such as IL-6, IL-1β, and TNF-α in lung tissues. Enhancements in immune response were evidenced by increased CD4/CD8 ratios in the spleen. DYY also notably upregulated MAS1 protein levels and suppressed the activation of the Ras/Raf1/MEK/ERK signaling pathway. DYY enhanced respiratory function and exerted significant antiviral and immunomodulatory effects in mice infected with the HCoV-229E virus, primarily by modulating MAS1 expression and inhibiting the Ras/Raf1/MEK/ERK pathway.Graphical

Impaired cyclin D3 protein degradation contributes to trastuzumab resistance in HER2 positive breast cancer

As the first anti-HER2 targeted agent approved by FDA in 1998, Trastuzumab has significantly improved the outcome of patients with HER2 positive metastatic breast cancer. Unfortunately, resistance to trastuzumab is a severe obstacle to its therapeutic efficacy in clinical application, and its mechanism has not yet been fully elucidated. In our study, we found that stabilization of cyclin D3 could be one reason for trastuzumab resistance. Trastuzumab could induce G1/G0 phase arrest by downregulating cyclin D3 protein expression. However, the protein expression of cyclin D3 was not affected in trastuzumab-resistant cells, which might be related to aberrant activation of ERK signaling pathway. Furthermore, degradation of cyclin D3 protein by trastuzumab was mainly resulted from ubiquitin-dependent proteasome mechanism instead of transcriptional regulation. In trastuzumab-resistant breast cancer cells, trastuzumab-induced degradation of cyclin D3 protein was abrogated. When the ubiquitin pathway was inhibited, cells would show a predisposition to resistance to trastuzumab. Further, CDK4/6 inhibitor can inhibit the proliferation of trastuzumab-resistant HER-2 positive breast cancer cells. Therefore, combination of CDK4/6 inhibitors and anti-HER2 targeted therapy may be an alternative and promising strategy to overcome trastuzumab resistance in the future.

CRH upregulates supervillin through ERK and AKT pathways to promote bladder cancer cell migration.

Corticotropin-releasing hormone (CRH) has been well documented playing a role in the regulation of cellular processes, immune responses, and inflammatory processes that can influence the occurrence and development of tumors. Supervillin (SVIL) is a membrane-associated and actin-binding protein, which is actively involved in the proliferation, spread, and migration of cancer cells. This work investigated CRH's influence on bladder cancer cells'migration and relevant mechanisms. By using human bladder cancer cells T24 and RT4 in wound healing experiments and transwell assay, we found that the migration ability of the T24 cells was significantly increased after CRH treatment. In vivo experiments showed that CRH significantly promoted the metastases of T24 cells in cell line-derived xenograft (CDX) mouse model. Interestingly, downregulation of SVIL by SVIL-specifc small hairpin RNAs significantly reduced the promoting effect of CRH on bladder cancer cell migration. Furthermore, CRH significantly increased SVILmessenger RNA and protein expression in T24 cells, accompanied with AKT andERK phosphorylation in T24 cells. Pretreatment with AKT inhibitor (MK2206) blocked the CRH-induced SVIL expression and ERK phosphorylation. Also, inhibition of ERK signaling pathway by U0126 significantly reduced the CRH-induced SVIL expression and AKT phosphorylation. It suggested that cross-talking between AKT and ERK pathways was involved in the effect of CRH on SVIL. Taken together, we demonstrated that CRH induced migration of bladder cancer cells, in which AKT andERK pathways -SVIL played a key role.

LIF Promotes Sec15b-Mediated STAT3 Exosome Secretion to Maintain Stem Cell Pluripotency in Mouse Embryonic Development.

LIF maintains self-renewal growth in mouse embryonic stem cells (mESC) by activating STAT3, which translocates into nucleus for pluripotent gene induction. However, the ERK signaling pathway activated by LIF at large counteract with pluripotent gene induction during self-renewal growth. Here, it is reported that in mESC STAT3 undergoes multivesicular endosomes (MVEs) translocation and subsequent secretion, LIF-activated STAT3 is acetylated on K177/180 and phosphorylated on Y293 residues within the N-terminal coiled-coil domain, which is responsible for the interaction between STAT3 and Secl5b, an exocyst complex component 6B (EXOC6B). STAT3 translocation into MVEs resulted in the downregulation of T202/Y204-ERK1/2 phosphorylation and up-regulation of S9-GSK3β phosphorylation for maintaining mESC self-renewal growth. STAT3 with K177R/K180R or Y293F substitution fails to execute MVEs translocation and Secl5b-dependent secretion. Mice expressing K177RK180R substitution (STAT3mut/mut) are partially embryonic lethal. In STAT3mut/mut embryos, gene expressions related to hematological system function changed significantly and those living ones carry a series of abnormalities in the hematopoietic system. Furthermore, mice with Secl5b knockout exhibit embryonic lethality. Thus, Secl5b mediated STAT3 MVEs translocation regulates the balance of ERK and GSK3β signaling pathways and maintain mESC self-renewal growth, which is involved in regulating the stability of hematopoietic system.

Cyclophilin A knockdown inhibits the proliferation and metastatic ability of AGS gastric cancer stem cells by downregulating CD147/STAT3/AKT/ERK and epithelial‑mesenchymal transition.

Gastric cancer stem cells (GCSCs) contribute to the challenging aspects of gastric cancer, such as progression, metastasis, treatment resistance and recurrence. Inhibitors targeting cyclophilin A (CypA) have shown potential in curtailing GCSC growth. Building upon this, the current study delved deeper into understanding the functional role of CypA in controlling the proliferation and metastatic capabilities of GCSCs, employing CypA‑specific small interfering RNA. The results revealed that knockdown of CypA led to significant suppression of the growth and tumorsphere‑forming capacity of GCSCs derived from AGS cells. This effect was mediated by arresting the cell cycle at the G0/G1 and S phases, and promoting apoptosis. Furthermore, silencing of CypA exerted inhibitory effects on the migration and invasion of AGS GCSCs by modulating the process of epithelial‑mesenchymal transition. Notably, the observed antiproliferative and antimetastatic effects of CypA knockdown were associated with the downregulation of critical regulators of gastric cancer stemness, such as CD44, CD133, aldehyde dehydrogenase 1 family member A1, NANOG, OCT4 and SOX2. This regulation occurred through inactivation of the CD147/STAT3/AKT/ERK signaling pathway. Additionally, CypA knockdown effectively curbed in vivo tumor growth of AGS GCSCs in a chorioallantoic membrane assay using chick embryos. These findings underscore the critical role of CypA in promoting the proliferation and metastasis of GCSCs, highlighting its potential as an effective therapeutic target for eradicating GCSCs and improving gastric cancer treatment outcomes.

Extracellular vesicles of Clonorchis sinensis promote the malignant phenotypes of cholangiocarcinoma via NF-κB/EMT axis.

Clonorchis sinensis infection is an important risk factor for cholangiocarcinoma (CCA). It has been reported that extracellular vesicles (EVs) are involved in the parasite-host interaction, and EVs of C. sinensis (CsEVs) can contribute to biliary injuries and inflammation. However, uncertainty surrounds the function of CsEVs in the progression of CCA. In this study, differential ultracentrifugation was used to separate CsEVs from the culture supernatant of C. sinensis adult worms, and they were then identified by transmission electron microscopy, nanoparticle tracking analysis and proteome assays. CCK8, EdU-488 and colony formation assays were used to explore the effect of CsEVs on the proliferation of CCA cells in vitro. Wound healing assays, transwell assays and in vivo lung metastasis model were conducted to evaluate the migration and invasion abilities. Moreover, the involvement of EMT process, as well as NF-κB and ERK signaling pathway was assessed. Results showed that CsEVs were successfully isolated and could be taken up by CCA cells, which promoted proliferation by accelerating cell cycle progression. In addition, CsEVs could facilitate cell metastasis by triggering the epithelial-mesenchymal transition (EMT). Mechanistically, activation of NF-κB signaling pathway was involved in the CsEVs-mediated EMT, which could be reversed partly by BAY 11-7082 (an inhibitor of NF-κB). In conclusion, these findings suggested that CsEVs could induce the aberrant proliferation and metastasis of CCA cells by stimulating the NF-κB/EMT axis, providing a novel theoretical explanation for liver fluke-associated CCA.

Structural Modification and Pharmacological Evaluation of (Thiadiazol-2-yl)pyrazines as Novel Piezo1 Agonists for the Intervention of Disuse Osteoporosis.

Piezo1 plays a pivotal role in regulating bone remodeling and homeostasis and has emerged as a promising target for chemical intervention in disuse osteoporosis. Nevertheless, the development of small-molecule Piezo1 agonists is still in its infancy, and highly efficacious Piezo1 agonists are urgently required. In this study, by shedding light on the structural novelty of the canonical Piezo1 agonist Yoda1, we initiated a structural optimization campaign based on the (thiadiazol-2-yl)pyrazine scaffold. A deuterated compound 12a was identified to be the most potent candidate against Piezo1 with an EC50 value of 2.21 μM, which was over 20-fold more potent than the reference Yoda1. This compound effectively activated Piezo1 and initiated Ca2+ influx in MSCs and promoted MSC osteogenesis via activating the Ca2+-related Erk signaling pathway. Furthermore, compound 12a was found to alleviate disuse osteoporosis with a desirable safety profile in a HU (hindlimb-unloading) rat model, thus warranting it as a potential probe for further investigation.

Astragaloside IV augments anti-PD-1 therapy to suppress tumor growth in lung cancer by remodeling the tumor microenvironment.

Programmed cell death protein-1 (PD-1) inhibitors are increasingly utilized in the treatment of lung cancer (LC). Combination therapy has recently gained popularity in treating LC. This study aimed to assess the efficacy of combining Astragaloside IV (AS-IV) and anti-PD-1 in LC. C57BL/6J mice were subcutaneously injected with Lewis lung carcinoma (LLC) cells. After 3 weeks, the animals were sacrificed, and the tumors were harvested for analysis. Ki-67 immuno-labeling and TUNEL assay were used for evaluating cell proliferation and apoptosis in tumor tissues. In addition, anti-cleaved caspase 3 was used for immunolabelling of apoptotic cells. Immune cell infiltration (macrophages and T cells) and gene expression in tumor tissues were also investigated by using immunofluorescence staining. Compared to treatment with anti-PD-1 or AS-IV, the combination of AS-IV and anti-PD-1 notably reduced tumor volume and weight of LLC-bearing mice. Additionally, the combination treatment strongly induced the apoptosis and suppressed the proliferation in tumor tissues through inactivating PI3K/Akt and ERK signaling pathways, compared to single treatment group. Moreover, the combination treatment elevated levels of the M1 macrophage marker mCD86, reduced levels of the M2 macrophage marker mCD206, as well as upregulated levels of the T cell activation marker mCD69 in tumor tissues. Collectively, the combination treatment effectively inhibited tumor growth in LLC mice through promoting M1 macrophage polarization and T cell activation. These findings showed that combining AS-IV with anti-PD-1 therapy could be a promising therapeutic approach for LC.

Colorectal fibroblasts promote malignant phenotype of colorectal cancer cells by activating the ERK signaling pathway

To investigate the effect of human colorectal fibroblast (CCD-18Co)-conditioned medium (CCD18-Co-CM) on biological behaviors of colorectal cancer (CRC) cells and explore the possible molecular mechanisms. Real-time cellular analysis (RTCA), clone formation assay and wound healing assay were used to analyze the changes in proliferation, clone formation, and migration abilities of CRC cell lines HCT116 and Caco-2 treated with CCD18-Co-CM. Western blotting was used to detect the changes in ATK, ERK and STAT3 signaling pathways in the CRC cells activated by CCD18-Co-CM. The effect of CCD18-Co-CM on spheroidization ability of the cells was assessed with sphere-formation assay, and the changes in expressions of CRC stemness markers were detected using RT-PCR. CCD-18Co-CM significantly promoted proliferation, colony formation, and migration of HCT116 and Caco-2 cells, enhanced sphere-forming ability and expressions of CRC stemness markers, and increased ERK phosphorylation in the cells. Treatment with SCH772984 effectively inhibited CCD-18Co-CM-induced ERK signaling pathway activation, suppressed the malignant phenotype, and lowered the sphere-forming ability and expression of stemness markers of the two CRC cells. Colorectal fibroblasts promote malignant phenotype of CRC cells by activating the ERK signaling pathway.

Bovine Lactoferrin Promotes Neurite Outgrowth in PC12 Cells via the TrkA Receptor.

Lactoferrin (LF) is a multifunctional protein abundant in breast milk that modulates the functions of neural stem cells. Recent studies have demonstrated the efficacy of bovine LF (bLF) in mitigating behavioral changes; however, the molecular mechanisms on the nervous system have not yet been elucidated. The presented study aimed to characterize the molecular mechanisms of bLF on nerve extension in PC12 cells. PC12 cells were treated with 0.01-1000 µg/mL of bLF, and cell viability was determined using the cell counting kit-8 assay after treatment for 24 h. Morphometric evaluation was performed after 24 or 72 h of treatment with 50 ng/mL nerve growth factor (NGF) or 100-500 µg/mL bLF. The molecular mechanisms were investigated using Western blotting and real-time quantitative PCR. Cell viability was significantly decreased after treatment with 600-1000 µg/mL bLF for 24 h compared with the control group. Morphometric evaluation revealed neurite outgrowth after 72 h of NGF treatment, with a significant increase in neurite outgrowth after treatment with 250 µg/mL bLF. The phosphorylated p44/42 expression ratio peaked at 5 min and persisted for up to 10 min. Quantitative real-time PCR revealed a significant decrease in MAP2 expression. Our findings suggested that bLF enhanced PC12 cell neurite outgrowth to a similar extent as NGF. These effects are thought to be mediated via the TrkA receptor and activated by the phosphorylated ERK signaling pathway. Therefore, this study demonstrates that bLF promotes neurite outgrowth via a pathway similar to that of NGF.

Mechanisms of pathogenicity in the hypertrophic cardiomyopathy-associated TNNI3 c.235C > T variant

BackgroundHypertrophic cardiomyopathy (HCM) is typically manifested as a hereditary disorder, with 30 %–60 % of cases linked to cardiac sarcomere gene mutations. Despite numerous identified TNNI3 mutations associated with HCM, their severity, prevalence, and disease progression vary. The link between TNNI3 variants and phenotypes remains largely unexplored. This study aims to elucidate the impact of the TNNI3 c.235C > T mutation on HCM through clinical research and cell experiments and to explore its mechanism in HCM development. MethodsWe screened an HCM family for pathogenic gene mutations using gene sequencing. The proband and family members were assessed through electrocardiography, echocardiography, and cardiac MRI, and a pedigree map was created for disease prediction analysis. Mutant plasmids were constructed with the TNNI3 c.235C > T mutation and transfected into the AC16 human cardiomyocyte cell line to investigate the mutation's effects. ResultsThe TNNI3 c.235C > T mutation was identified as the disease-causing variant in the family. This mutation led to the upregulation of hypertrophy-associated genes ANP, BNP, and MYH7, increased cardiomyocyte size, and activation of the ERK signaling pathway. Further investigations revealed that the TNNI3 c.235C > T mutation impaired mitochondrial function, disrupted cardiomyocyte metabolism, and increased cellular autophagy and apoptosis. ConclusionsThe TNNI3 c.235C > T gene mutation may be a pathogenic factor for HCM, showing heterogeneous features and clinical phenotypes. This mutation induces myocardial hypertrophy, activates the ERK signaling pathway, and exacerbates mitochondrial dysfunction, apoptosis, and autophagy in cardiomyocytes. These findings provide insights into the mechanism of HCM caused by gene mutations and may inform HCM treatment strategies.

Toll-Like Receptor 4 (TLR4) Promotes DRG Regeneration and Repair after Sciatic Nerve Injury via the ERK-NF-kB Pathway.

Previously, we found that the expression of Toll-like receptor 4 (TLR4) is altered after sciatic nerve injury, and its differential expression plays a key role in recovery. However, the mechanisms by which TLR4 affects neuronal function in the dorsal root ganglion (DRG) have not been completely evaluated. The objective is to determine TLR4 expression in DRG tissues after sciatic neural injury and exploring the effects of TLR4 knockdown and overexpression in the DRG on neuronal function and nerve regeneration in rats in vivo and in vitro. We established a model of nerve injury and utilized molecular biology and cell biology experiments to explore the molecular mechanisms by which TLR4 in the DRG affects sciatic nerve restoration and regeneration after injury. Verified the localization of TLR4 in DRG neurons. Investigated pathways that related to apoptosis or nerve regeneration by which TLR4 regulates the function of DRG neurons. TLR4 expression was upregulated in the DRG tissues of rats after sciatic nerve injury. TLR4 overexpression promoted axon regeneration and inhibited apoptosis in DRG neurons. TLR4 promoted the regeneration of axons and the recovery of motor and sensory functions in the sciatic nerve after injury in vivo, and the data showed that TLR4 may regulate the function of DRG neurons and promote nerve repair and regeneration through the ERK and NF-κB signaling pathways in vivo and ex vivo. The study suggests that TLR4 may regulate the function of DRG neurons and promote nerve regeneration by affecting the ERK and NF-κB signaling pathways.

Amentoflavone maintaining extracellular matrix homeostasis and inhibiting subchondral bone loss in osteoarthritis by inhibiting ERK, JNK and NF-κB signaling pathways

Amentoflavone (AF), a plant biflavone isolated from Selaginella sinensis ethanol extract, is characterized by anti-inflammatory and anti-oxidant properties. According to previous studies, inflammation and oxidative stress are closely related to the pathophysiology of osteoarthritis (OA). However, the effects and mechanisms of AF on OA have not been elucidated.To investigate the inhibitory effects and its molecular mechanism of AF on extracellular matrix (ECM) degradation stimulated by IL-1β as well as subchondral bone loss induced by RANKL in mice chondrocytes. Quantitative PCR was used to detect the mRNA expression of genes related to inflammation, ECM, and osteoclast differentiation. Protein expression level of iNOS, COX-2, MMP13, ADAMTS5, COL2A1, SOX9, NFATc1, c-fos, JNK, ERK, P65, IκBα was measured by western blotting. The levels of TNF-α and IL-6 in the supernatants were measured by ELISA. The amount of ECM in chondrocytes was measured using toluidine blue staining. The levels of Aggrecan and Col2a1 in chondrocytes were measured using immunofluorescence. Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and immunofluorescence were used to detect the effect of AF on osteoclast differentiation and bone resorption. The effect of AF on destabilization of the medial meniscus (DMM)-induced OA mice can be detected in hematoxylin–eosin (H&E) staining, Safranin O green staining and immunohistochemistry.AF might drastically attenuated IL-1β-stimulated inflammation and reduction of ECM formation by blocking ERK and NF-κB signaling pathways in chondrocytes. Meanwhile, AF suppressed the formation of osteoclasts and the resorption of bone function induced by RANKL. In vivo, AF played a protective role by stabilizing cartilage ECM and inhibiting subchondral bone loss in destabilization of the medial meniscus (DMM)-induced OA mice, further proving its protective effect in the development of OA. Our study show that AF alleviated OA by suppressing ERK, JNK and NF-κB signaling pathways in OA models in vitro and DMM-induced OA mice, suggesting that AF might be a potential therapeutic agent in the treatment of OA.

SPROUTY2, a Negative Feedback Regulator of Receptor Tyrosine Kinase Signaling, Associated with Neurodevelopmental Disorders: Current Knowledge and Future Perspectives

Growth-factor-induced cell signaling plays a crucial role in development; however, negative regulation of this signaling pathway is important for sustaining homeostasis and preventing diseases. SPROUTY2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase (RTK) signaling that binds to GRB2 during RTK activation and inhibits the GRB2-SOS complex, which inhibits RAS activation and attenuates the downstream RAS/ERK signaling cascade. SPRY was formerly discovered in Drosophila but was later discovered in higher eukaryotes and was found to be connected to many developmental abnormalities. In several experimental scenarios, increased SPRY2 protein levels have been observed to be involved in both peripheral and central nervous system neuronal regeneration and degeneration. SPRY2 is a desirable pharmaceutical target for improving intracellular signaling activity, particularly in the RAS/ERK pathway, in targeted cells because of its increased expression under pathological conditions. However, the role of SPRY2 in brain-derived neurotrophic factor (BDNF) signaling, a major signaling pathway involved in nervous system development, has not been well studied yet. Recent research using a variety of small-animal models suggests that SPRY2 has substantial therapeutic promise for treating a range of neurological conditions. This is explained by its function as an intracellular ERK signaling pathway inhibitor, which is connected to a variety of neuronal activities. By modifying this route, SPRY2 may open the door to novel therapeutic approaches for these difficult-to-treat illnesses. This review integrates an in-depth analysis of the structure of SPRY2, the role of its major interactive partners in RTK signaling cascades, and their possible mechanisms of action. Furthermore, this review highlights the possible role of SPRY2 in neurodevelopmental disorders, as well as its future therapeutic implications.

SMYD1 modulates the proliferation of multipotent cardiac progenitor cells derived from human pluripotent stem cells during myocardial differentiation through GSK3β/β-catenin&ERK signaling.

The histone-lysine N-methyltransferase SMYD1, which is specific to striated muscle, plays a crucial role in regulating early heart development. Its deficiency has been linked to the occurrence of congenital heart disease. Nevertheless, the precise mechanism by which SMYD1 deficiency contributes to congenital heart disease remains unclear. We established a SMYD1 knockout pluripotent stem cell line and a doxycycline-inducible SMYD1 expression pluripotent stem cell line to investigate the functions of SMYD1 utilizing an in vitro-directed myocardial differentiation model. Cardiomyocytes lacking SMYD1 displayed drastically diminished differentiation efficiency, concomitant with heightened proliferation capacityof cardiac progenitor cells during the early cardiac differentiation stage. These cellular phenotypes were confirmed through experiments inducing the re-expression of SMYD1. Transcriptome sequencing and small molecule inhibitor intervention suggested that the GSK3β/β-catenin&ERK signaling pathway was involved in the proliferation of cardiac progenitor cells. Chromatin immunoprecipitation demonstrated that SMYD1 acted as a transcriptional activator of GSK3β through histone H3 lysine 4 trimethylation. Additionally, dual-luciferase analyses indicated that SMYD1 could interact with the promoter region of GSK3β, thereby augmenting its transcriptional activity. Moreover, administering insulin and Insulin-like growth factor 1 can enhance the efficacy of myocardial differentiation in SMYD1 knockout cells. Our research indicated that the participation of SMYD1 in the GSK3β/β-catenin&ERK signaling cascade modulated the proliferation of cardiac progenitor cells during myocardial differentiation. This process was partly reliant on the transcription of GSK3β. Our research provided a novel insight into the genetic modification effect of SMYD1 during early myocardial differentiation. The findings were essential to the molecular mechanism and potential interventions for congenital heart disease.

Anti-inflammatory effects of phytosphingosine-regulated cytokines and NF-kB and MAPK mechanism.

Phytosphingosine (PHS) is a major component of the skin barrier and a multifunctional physiologically active substance. This study aimed to investigate the types of cytokines regulated by PHS, their anti-skin inflammatory effects, and their anti-inflammatory mechanisms. RAW264.7 cells stimulated with Lipopolysaccharides (LPS) were treated with PHS to measure inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2), and gene expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX2) were confirmed by q-PCR. Cytokines regulated by PHS against LPS-induced inflammation were found through cytokine array, and each factor was reconfirmed through ELISA. Western blot was performed to confirm anti-inflammatory mechanism of Iκbα and MAPK. To confirm anti-skin inflammatory efficacy, HaCaT cells stimulated with TNF-α/IFN-γ were treated with PHS, and TARC, IL-6, and IL-8 were detected by ELISA. PHS suppressed the gene expression of iNOS and COX2, which were increased by LPS, and suppressed NO and PGE2 production. Through cytokine array, it was confirmed that IL-6, IL-10, IL-27 p28/IL-30, IP-10, I-TAC, MCP-5, and TIMP-1 increased by LPS were decreased by PHS. PHS inhibited NF-κB signaling by inhibiting LPS-induced NF-κB nuclear migration and p-Iκbα-mediated Iκbα degradation, and inhibited p38, ERK, and JNK signaling pathways. PHS reduced the production of TARC, IL-6, and IL-8 increased by TNF-α/IFN-γ. These results indicate PHS has anti-inflammatory effects via the suppression of inflammatory factors and pro-inflammatory cytokines through the NF-κB and MAPK pathways. Moreover, these results may explain beneficial effects of PHS in the treatment of skin inflammatory conditions induced by TNF-α/IFN-γ.