Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

BACKGROUND: Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. METHODS: In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. RESULTS: In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. CONCLUSIONS: Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.

TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation.

Background/aim: Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Materials and methods: Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. Results: In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. Conclusion: TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.

Xbra modulates the activity of linker region phosphorylated Smad1 during Xenopus development.

The Bmp/Smad1 pathway plays a crucial role in developmental processes and tissue homeostasis. Mitogen-activated protein kinase (Mapk)/Erk mediated phosphorylation of Smad1 in the linker region leads to Smad1 degradation, cytoplasmic retention and inhibition of Bmp/Smad1 signaling. While Fgf/Erk pathway has been documented to inhibit Bmp/Smad1 signaling, several studies also suggests the cooperative interaction between these two pathways in different context. However, the precise role and molecular pathway of this collaborative interaction remain obscure. Here, we identified Xbra induced by Fgf/Erk signaling as a factor in a protective mechanism for Smad1. Xbra physically interacted with the linker region phosphorylated Smad1 to make Xbra/Smad1/Smad4 trimeric complex, leading to Smad1 nuclear localization and protecting it from ubiquitin-mediated proteasomal degradation. This interaction of Xbra/Smad1/Smad4 led to sustained nuclear localization of Smad1 and the upregulation of lateral mesoderm genes, while concurrently suppression of neural and blood forming genes. Taken together, the results suggests Xbra-dependent cooperative interplays between Fgf/Erk and Bmp/Smad1 signaling during lateral mesoderm specification in Xenopus embryos.

has-miR-134-5p inhibits the proliferation and migration of glioma cells by regulating the BDNF/ERK signaling pathway.

Our research investigated the effects of hsa-miR-134-5p on glioma progression, focusing on its interaction with the BDNF/ERK signaling pathway. U251 and U87 cell lines were analyzed post-transfection with hsa-miR-134-5p mimics and inhibitors, confirming the miRNA's binding to BDNF using dual luciferase assays. Q-PCR was employed to measure expression changes, revealing that hsa-miR-134-5p markedly inhibited glioma cell proliferation, migration, and invasion, as evidenced by CCK8, monoclonal formation, and Transwell assays. Scratch tests and Western blotting demonstrated hsa-miR-134-5p's modulation of the BDNF/ERK pathway and associated decrease in MMP2/9 protein levels. Flow cytometry suggested that hsa-miR-134-5p might also block the G0/S phase transition. In vivo studies using nude mice corroborated the tumor-suppressing effects of hsa-miR-134-5p, which were negated by elevated BDNF levels. Comparative protein analysis across groups confirmed the pathway's significance in tumorigenesis. Our findings identify hsa-miR-134-5p as a key molecule impeding glioma cell growth by curtailing the BDNF/ERK pathway, with the reversal by BDNF upregulation pointing to the potential of therapeutically exploiting the hsa-miR-134-5p/BDNF axis in glioma care.

Transcranial direct current stimulation promotes angiogenesis and improves neurological function via the OXA-TF-AKT/ERK signaling pathway in traumatic brain injury.

Traumatic brain injury (TBI) and its resulting complications pose a major challenge to global public health, resulting in increased rates of disability and mortality. Cerebrovascular dysfunction is nearly universal in TBI cases and is closely associated with secondary injury after TBI. Transcranial direct current stimulation (tDCS) shows great potential in the treatment of TBI; however, the exact mechanism remains elusive. In this study, we performed in vivo and in vitro experiments to explore the effects and mechanisms of tDCS in a controlled cortical impact (CCI) rat model simulating TBI. In vivo experiments show that tDCS can effectively reduce brain tissue damage, cerebral edema and neurological deficits. The potential mechanism may be that tDCS improves the neurological function of rats by increasing orexin A (OXA) secretion, upregulating the TF-AKT/ERK signaling pathway, and promoting angiogenesis at the injury site. Cellular experiments showed that OXA promoted HUVEC migration and angiogenesis, and these effects were counteracted by the ERK1/2 inhibitor LY3214996. The results of Matrigel experiment in vivo showed that TNF-a significantly reduced the ability of HUVEC to form blood vessels, but OXA could rescue the effect of TNF-a on the ability of HUVEC to form blood vessels. However, LY3214996 could inhibit the therapeutic effect of OXA. In summary, our preliminary study demonstrates that tDCS can induce angiogenesis through the OXA-TF-AKT/ERK signaling pathway, thereby improving neurological function in rats with TBI.

Bruceantinol works as a CDK2/4/6 inhibitor to inhibit the growth of breast cancer cells.

Bruceantinol (BOL), isolated from the dried fruit of the Brucea javanica (L.) Merr., exhibits cytotoxic effects on breast cancer cells. However, the underlying mechanism remains to be fully addressed. In this paper, the MCF-7 and MDA-MB-231 human breast cancer cell lines were used as experimental models to uncover how BOL inhibits breast cancer cell growth. The effects of BOL on cell growth, proliferation, the cell cycle, and apoptosis were investigated using the MTT assays, EdU incorporation assays, and flow cytometry, respectively. Bioinformatics techniques were applied to predict the key targets of BOL in breast cancer. Subsequent validation of these targets and the anti-breast cancer mechanism of BOL was conducted through Western blotting, RT-PCR, siRNA transfection, and molecular docking analysis. The results demonstrated that BOL dose- and time-dependently reduced the growth of both cell lines, impeded cell proliferation, disrupted the cell cycle, and induced necrosis in MCF-7 cells and apoptosis in MDA-MB-231 cells. Furthermore, CDK2/4/6 were identified as BOL targets, and their knockdown reduced cell sensitivity to BOL. BOL was found to potentially bind with CDK2/4/6 to facilitate protein degradation through the proteasome pathway. Additionally, BOL activated ERK in MDA-MB-231 cells, and this activation was required for BOL's functions in these cells. Collectively, BOL may act as an inhibitor of CDK2/4/6 to exert anti-breast cancer effects. Its effects on cell growth and CDK2/4/6 expression may also depend on ERK activation in HRs-HER2- breast cancer cells. These results suggest the potential of using BOL for treating breast cancer.

Huanglian Jiedu decoction alleviates neurobehavioral damage in mice with chronic alcohol exposure through the RAS-RAF-MEK-ERK pathway.

Objective: Long-term alcohol consumption can cause organic damage to the brain, resulting in mental and nervous system abnormalities and intellectual impairment. Huanglian Jiedu decoction (HLJDD) is the classic representative of clearing heat and detoxifying. This study aimed to explore the effects and possible mechanisms of HLJDD on brain injury in chronic alcohol-exposed mice. Methods: The alcohol-exposed mice were treated with different doses of HLJDD to observe behavioral changes, hippocampal Aß1-42 deposition, number and ultrastructural changes of neurons in the hippocampus and prefrontal cortex, and expressions of synaptic proteins. On this basis, transcriptome sequencing was used to analyze the differentially expressed genes in different treatment groups, and functional enrichment analysis was performed. Then, WB and RT-PCR were used to verify the expression of the pathway. Results: Chronic alcohol exposure reduced body weight in mice, led to motor cognitive impairment, increased Aß1-42 in the hippocampus, decreased the number of neurons in the hippocampus and prefrontal cortex, and the expression of PSD95 and SYN in the hippocampus. HLJDD significantly improved the cognitive dysfunction of mice and alleviated the damage of the hippocampus and prefrontal cortex. Transcriptome sequencing results showed that the regulatory effects of HLJDD on chronic alcohol-exposed mice may be related to the RAS pathway. Further experiments confirmed that chronic alcohol exposure caused a significant increase in protein and gene expressions of the RAS-RAF-MEK-ERK pathway in mouse, and this activation was reversed by HLJDD. Conclusion: HLJDD may ameliorate brain damage caused by chronic alcohol exposure by regulating the RAS-RAF-MEK-ERK pathway.

ZNF692 promotes osteosarcoma cell proliferation, migration, and invasion through TNK2-mediated activation of the MEK/ERK pathway.

BACKGROUND: Osteosarcoma is a diverse and aggressive bone tumor. Driver genes regulating osteosarcoma initiation and progression remains incompletely defined. Zinc finger protein 692 (ZNF692), a kind of Krüppel C2H2 zinc finger transcription factor, exhibited abnormal expression in different types of malignancies and showed a correlation with the clinical prognosis of patients as well as the aggressive characteristics of cancer cells. Nevertheless, its specific role in osteosarcoma is still not well understood. METHODS: We investigated the dysregulation and clinical significance of ZNF692 in osteosarcoma through bioinformatic method and experimental validation. A range of in vitro assays, including CCK-8, colony formation, EdU incorporation, wound healing, and transwell invasion tests, were conducted to assess the impact of ZNF692 on cell proliferation, migration, and invasion in osteosarcoma. A xenograft mouse model was established to evaluate the effect of ZNF692 on tumor growth in vivo. Western blot assay was used to measure the protein levels of MEK1/2, P-MEK1/2, ERK1/2, and P-ERK1/2 in cells that had been genetically modified to either reduce or increase the expression of ZNF692. The relationship between ZNF692 and tyrosine kinase non-receptor 2 (TNK2) were validated by qRT-PCR, chromatin immunoprecipitation and luciferase reporter assays. RESULTS: Expression of ZNF692 was increased in both human osteosarcoma tissues and cell lines. Furthermore, the expression of ZNF692 served as an independent predictive biomarker in osteosarcoma. The results of the survival analysis indicated that increased expression of ZNF692 was associated with worse outcome. Downregulation of ZNF692 inhibits the proliferation, migration, and invasion of osteosarcoma cells, whereas upregulation of ZNF692 has the opposite impact. Western blot assay indicates that reducing ZNF692 decreases phosphorylation of MEK1/2 and ERK1/2, whereas increasing ZNF692 expression enhances their phosphorylation. U0126, a potent inhibitor specifically targeting the MEK/ERK signaling pathway, partially counteracts the impact of ZNF692 overexpression on the proliferation, migration, and invasion of osteosarcoma cells. In addition, ZNF692 specifically interacts with the promoter region of TNK2 and stimulates the transcription of TNK2 in osteosarcoma cells. Forcing the expression of TNK2 weakens the inhibitory impact of ZNF692 knockdown on P-MEK1/2 and P-ERK1/2. Similarly, partly inhibiting TNK2 counteracts the enhancing impact of ZNF692 overexpression on the phosphorylation of MEK1/2 and ERK1/2. Functional tests demonstrate that the suppressive effects of ZNF692 knockdown on cell proliferation, migration, and invasion are greatly reduced when TNK2 is overexpressed. In contrast, the reduction of TNK2 hinders the ability of ZNF692 overexpression to enhance cell proliferation, migration, and invasion. CONCLUSION: ZNF692 promotes the proliferation, migration, and invasion of osteosarcoma cells via the TNK2-dependent stimulation of the MEK/ERK signaling pathway. The ZNF692-TNK2 axis might potentially function as a possible predictive biomarker and a promising target for novel therapeutics in osteosarcoma.

Involvement of ERK and Oxidative Stress in Airway Exposure to Cadmium Chloride Aggravates Airway Inflammation in Ovalbumin-Induced Asthmatic Mice.

Inhalation represents a significant route of cadmium (Cd) exposure, which is associated with an elevated risk of lung diseases. This research study aims to evaluate the impact of repeated low-dose cadmium inhalation on exacerbating airway inflammation induced by ovalbumin (OVA) in asthma-afflicted mice. Mice were grouped into four categories: control (Ctrl), OVA, cadmium chloride (CdCl2), and OVA + cadmium chloride (OVA + CdCl2). Mice in the OVA group displayed increased airway mucus secretion and peribronchial and airway inflammation characterized by eosinophil cell infiltration, along with elevated levels of Th2 cytokines (IL-4, IL-5, IL-13) in bronchoalveolar lavage fluids (BALFs). These parameters were further exacerbated in the OVA + CdCl2 group. Additionally, the OVA + CdCl2 group exhibited higher levels of the oxidative stress marker malondialdehyde (MDA), greater activity of glutathione peroxidase (GSH-Px), and higher phosphorylation of extracellular regulated kinase (ERK) in lung tissue. Treatment with U0126 (an ERK inhibitor) and α-tocopherol (an antioxidant) in the OVA + CdCl2 group resulted in reduced peribronchial and airway inflammation as well as decreased airway mucus secretion. These findings indicate that CdCl2 exacerbates airway inflammation in OVA-induced allergic asthma mice following airway exposure. ERK and oxidative stress are integral to this process, and the inhibition of these pathways significantly alleviates the adverse effects of CdCl2 on asthma exacerbation.

A senescence restriction point acting on chromatin integrates oncogenic signals.

We identify a senescence restriction point (SeRP) as a critical event for cells to commit to senescence. The SeRP integrates the intensity and duration of oncogenic stress, keeps a memory of previous stresses, and combines oncogenic signals acting on different pathways by modulating chromatin accessibility. Chromatin regions opened upon commitment to senescence are enriched in nucleolar-associated domains, which are gene-poor regions enriched in repeated sequences. Once committed to senescence, cells no longer depend on the initial stress signal and exhibit a characteristic transcriptome regulated by a transcription factor network that includes ETV4, RUNX1, OCT1, and MAFB. Consistent with a tumor suppressor role for this network, the levels of ETV4 and RUNX1 are very high in benign lesions of the pancreas but decrease dramatically in pancreatic ductal adenocarcinomas. The discovery of senescence commitment and its chromatin-linked regulation suggests potential strategies for reinstating tumor suppression in human cancers.

Effects of MEK1/2 inhibitor U0126 on FGF10-enhanced buffalo oocyte maturation in vitro.

Fibroblast growth factor 10 (FGF10) plays critical roles in oocyte maturation and embryonic development; however, the specific pathway by which FGF10 promotes in vitro maturation of buffalo oocytes remains elusive. The present study was aimed at investigating the mechanism underlying effects of the FGF10-mediated extracellular regulated protein kinases (ERK) pathway on oocyte maturation and embryonic development in vitro. MEK1/2 (mitogen-activated protein kinase kinase) inhibitor U0126, alone or in combination with FGF10, was added to the maturation culture medium during maturation of the cumulus oocyte complex. Morphological observations, orcein staining, apoptosis detection, and quantitative real-time PCR were performed to evaluate oocyte maturation, embryonic development, and gene expression. U0126 affected oocyte maturation and embryonic development in vitro by substantially reducing the nuclear maturation of oocytes and expansion of the cumulus while increasing the apoptosis of cumulus cells. However, it did not have a considerable effect on glucose metabolism. These findings suggest that blocking the MEK/ERK pathway is detrimental to the maturation and embryonic development potential of buffalo oocytes. Overall, FGF10 may regulate the nuclear maturation of oocytes and cumulus cell expansion and apoptosis but not glucose metabolism through the MEK/ERK pathway. Our findings indicate that FGF10 regulates resumption of meiosis and expansion and survival of cumulus cells via MEK/ERK signaling during in vitro maturation of buffalo cumulus oocyte complexes. Elucidation of the mechanism of action of FGF10 and insights into oocyte maturation should advance buffalo breeding. Further studies should examine whether enhancement of MEK/ERK signaling improves embryonic development in buffalo.

The prenatal use of agmatine prevents social behavior deficits in VPA-exposed mice by activating the ERK/CREB/BDNF signaling pathway.

BACKGROUND: According to reports, prenatal exposure to valproic acid can induce autism spectrum disorder (ASD)-like symptoms in both humans and rodents. However, the exact cause and therapeutic method of ASD is not fully understood. Agmatine (AGM) is known for its neuroprotective effects, and this study aims to explore whether giving agmatine hydrochloride before birth can prevent autism-like behaviors in mouse offspring exposed prenatally to valproic acid. METHODS: In this study, we investigated the effects of AGM prenatally on valproate (VPA)-exposed mice. We established a mouse model of ASD by prenatally administering VPA. From birth to weaning, we evaluated mouse behavior using the marble burying test, open-field test, and three-chamber social interaction test on male offspring. RESULTS: The results showed prenatal use of AGM relieved anxiety and hyperactivity behaviors as well as ameliorated sociability of VPA-exposed mice in the marble burying test, open-field test, and three-chamber social interaction test, and this protective effect might be attributed to the activation of the ERK/CREB/BDNF signaling pathway. CONCLUSION: Therefore, AGM can effectively reduce the likelihood of offspring developing autism to a certain extent when exposed to VPA during pregnancy, serving as a potential therapeutic drug.

Retinoprotective Effect of SkQ1, Visomitin Eye Drops, Is Associated with Suppression of P38 MAPK and ERK1/2 Signaling Pathways Activity.

Visomitin eye drops are the first and, so far, the only drug based on SkQ1 - the mitochondria-targeted antioxidant 10-(6'-plastoquinonyl) decyltriphenylphosphonium, developed in the laboratories of Moscow State University under the leadership of Academician V. P. Skulachev. SkQ1 is considered as a potential tool to combat the aging program. We have previously shown that it is able to prevent and/or suppress development of all manifestations of accelerated senescence in OXYS rats, including retinopathy, similar to the age-related macular degeneration (AMD). Here, we assessed the effect of Visomitin instillations on progression of the AMD-like pathology and p38 MAPK and ERK1/2 activity in the OXYS rat retina (from the age of 9 to 12 months). Wistar and OXYS rats treated with placebo (composition identical to Visomitin with the exception of SkQ1) were used as controls. Ophthalmological examination showed that in the OXYS rats receiving placebo, retinopathy progressed and severity of clinical manifestations did not differ from the intact OXYS rats. Visomitin suppressed progression of the AMD-like pathology in the OXYS rats and significantly improved structural and functional parameters of the retinal pigment epithelium cells and state of microcirculation in the choroid, which, presumably, contributed to preservation of photoreceptors, associative and ganglion neurons. It was found that the activity of p38 MAPK and ERK1/2 in the retina of 12-month-old OXYS rats is higher than that of the Wistar rats of the same age, as indicated by the increased content of phosphorylated forms of p38 MAPK and ERK1/2 and their target protein tau (at position T181 and S396). Visomitin decreased phosphorylation of p38 MAPK, ERK1/2, and tau indicating suppression of activity of these MAPK signaling cascades. Thus, Visomitin eye drops are able to suppress progression of the AMD-like pathology in the OXYS rats and their effect is associated with the decrease in activity of the MAPK signaling cascades.

In vitro and in vivo exposure of endothelial cells to dibutyl phthalate promotes monocyte adhesion.

The effect of endothelial cells' exposure to dibutyl phthalate (DBP) on monocyte adhesion is largely unknown. We evaluated monocyte adhesion to DBP-exposed endothelial cells by combining three approaches: short-term exposure (24 h) of EA.hy926 cells to 10-6, 10-5, and 10-4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10-9, 10-8, and 10-7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. Monocyte adhesion to human EA.hy926 and rat aortic endothelial cells, expression of selected cellular adhesion molecules and chemokines, and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) were analyzed. We observed increased monocyte adhesion to DBP-exposed EA.hy926 cells in vitro and to rat aortic endothelium ex vivo. ERK1/2 inhibitor prevented monocyte adhesion to DBP-exposed EA.hy926 cells in short-term exposure experiments. Increased ERK1/2 phosphorylation in rat aortic endothelium and transient decrease in ERK1/2 activation following long-term exposure of EA.hy926 cells to DBP were also observed. In summary, exposure of endothelial cells to DBP promotes monocyte adhesion, thus suggesting a possible role for this phthalate in the development of atherosclerosis. ERK1/2 signaling could be the mediator of monocyte adhesion to DBP-exposed endothelial cells, but only after short-term high-level exposure.

Alnustone promotes megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway.

OBJECTIVES: Thrombocytopenia is a disease in which the number of platelets in the peripheral blood decreases. It can be caused by multiple genetic factors, and numerous challenges are associated with its treatment. In this study, the effects of alnustone on megakaryocytes and platelets were investigated, with the aim of developing a new therapeutic approach for thrombocytopenia. METHODS: Random forest algorithm was used to establish a drug screening model, and alnustone was identified as a natural active compound that could promote megakaryocyte differentiation. The effect of alnustone on megakaryocyte activity was determined using cell counting kit-8. The effect of alnustone on megakaryocyte differentiation was determined using flow cytometry, Giemsa staining, and phalloidin staining. A mouse model of thrombocytopenia was established by exposing mice to X-rays at 4 Gy and was used to test the bioactivity of alnustone in vivo. The effect of alnustone on platelet production was determined using zebrafish. Network pharmacology was used to predict targets and signaling pathways. Western blotting and immunofluorescence staining determined the expression levels of proteins. RESULTS: Alnustone promoted the differentiation and maturation of megakaryocytes in vitro and restored platelet production in thrombocytopenic mice and zebrafish. Network pharmacology and western blotting showed that alnustone promoted the expression of interleukin-17A and enhanced its interaction with its receptor, and thereby regulated downstream MEK/ERK signaling and promoted megakaryocyte differentiation. CONCLUSIONS: Alnustone can promote megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway and thus provides a new therapeutic strategy for the treatment of thrombocytopenia.

Homocysteine-induced sustained GluN2A NMDA receptor stimulation leads to mitochondrial ROS generation and neurotoxicity.

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of Pyk2 and Src family kinases (SFK), which in turn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and SFK activity further contributes to the maintenance of this cycle. Additional studies using live cell imaging of neurons expressing a redox sensitive green fluorescent protein (RoGFP) targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species (ROS) generation, which is dependent on GluN2A-NMDAR mediated sustained ERK MAPK activation. This later finding demonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial ROS generation.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

High-Throughput Cell-Based Screening of Small Molecule KRAS Signaling Inhibitors Using a Homogeneous Time-Resolved Fluorescence (HTRF) Assay.

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.

Activation of ERK/NF-kB Pathways Contributes to the Inflammatory Response in Epithelial Cells and Macrophages Following Manganese Exposure.

Different types of metals, including manganese (Mn), are constantly encountered in various environmental matrices due to natural and anthropogenic activities. They induce a sustained inflammatory response in various organs, which is considered to be an important priming event in the pathogenesis of several diseases. Mn-induced neuroinflammation and subsequent neurodegeneration are well recognized. However, emerging data suggest that occupationally and environmentally relevant levels may affect various organs, including the lungs. Therefore, the present study was carried out to investigate the effects of Mn (as Mn2+) exposure on the inflammatory response in human normal bronchial (BEAS-2B) and adenocarcinoma alveolar basal (A549) epithelial cells, as well as in murine macrophages (J774). Mn2+ exposure significantly induced mRNA and protein expression of various pro-inflammatory mediators (cytokines and chemokines) in all cells compared to corresponding vehicle controls. Furthermore, Mn2+ treatment also led to increased phosphorylation of extracellular-signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-kB) p65 in both epithelial cells and macrophages. As expected, cells treated with inhibitors of ERK1/2 (PD98059) and NF-kB p65 (IMD0354) effectively mitigated the expression of various pro-inflammatory mediators induced by Mn2+, suggesting that ERK/NF-kB pathways have a critical role in the Mn2+-induced inflammatory response. Further, in vivo studies are required to confirm these in vitro findings to support clinical translation.

Okadaic acid enhances NfKB, TLR-4, caspase 3, ERK ½, c-FOS, and 8-OHdG signaling pathways activation in brain tissues of zebrafish larvae.

This study was designed to investigate the potential neuronal damage mechanism of the okadaic acid (OA) in the brain tissues of zebrafish embryos by evaluating in terms of immunofluorescence of Nf KB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG signaling pathways. We also evaluated body malformations. For this purpose, zebrafish embryos were exposed to 0.5 µg/ml, 1 µg/ml and 2.5 µg/ml of OA for 5 days. After application, FITC/GFP labeled protein-specific antibodies were used in immunofluorescence assay for NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG respectively. The results indicated that OA caused immunofluorescence positivity of NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG in a dose-dependent manner in the brain tissues of zebrafish embryos. Pericardial edema (PE), nutrient sac edema (YSE) and body malformations, tail malformation, short tail and head malformation (BM) were detected in zebrafish embryos. These results suggest that OA induces neuronal damage by affecting the modulation of DNA damage, apoptotic, and inflammatory activities in the brain tissues of zebrafish embryos. The increase in signaling pathways shows that OA can cause damage in the structure and function of brain nerve cells. Our results provide a new basis for the comprehensive assessment of the neural damage of OA and will offer enable us to better understand molecular the mechanisms underlying the pathophysiology of OA toxicity.