ERK Signaling Cascade Research Articles

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.

Peptidomic analysis reveals novel peptide PDLC promotes cell proliferation in hepatocellular carcinoma via Ras/Raf/MEK/ERK pathway

Hepatocellular carcinoma (HCC) still presents poor prognosis with low overall survival rates and limited therapeutic options available. Recently, attention has been drawn to peptidomic analysis, an emerging field of proteomics for the exploration of new potential peptide drugs for the treatment of various diseases. However, research on the potential function of HCC peptides is lacking. Here, we analyzed the peptide spectrum in HCC tissues using peptidomic techniques and explored the potentially beneficial peptides involved in HCC. Changes in peptide profiles in HCC were examined using liquid chromatography-mass spectrometry (LC–MS/MS). Analyze the physicochemical properties and function of differently expressed peptides using bioinformatics. The effect of candidate functional peptides on HCC cell growth and migration was evaluated using the CCK-8, colony formation, and transwell assays. Transcriptome sequencing analysis and western blot were employed to delve into the mode of action of potential peptide on HCC. Peptidomic analysis of HCC tissue yielded a total of 8683 peptides, of which 452 exhibited up-regulation and 362 showed down-regulation. The peptides that were differentially expressed, according to bioinformatic analysis, were closely linked to carbon metabolism and the mitochondrial inner membrane. The peptide functional validation identified a novel peptide, PDLC (peptide derived from liver cancer), which was found to dramatically boost HCC cell proliferation through the Ras/Raf/MEK/ERK signaling cascade. Our research defined the peptide’s properties and pattern of expression in HCC and identified a novel peptide, PDLC, with a function in encouraging HCC progression, offering an entirely new potential therapeutic target the disease.

Cirsilineol inhibits RANKL-induced osteoclast activity and ovariectomy-induced bone loss via NF-κb/ERK/p38 signaling pathways

BackgroundPostmenopausal osteoporosis is a chronic metabolic bone disease caused by excessive osteoclast formation and function. Targeting osteoclast differentiation and activity can modulate bone resorption and alleviate osteoporosis. Cirsilineol, an active constituent of Vestita Wall, has shown numerous biological activities and has been used to treat many metabolic diseases. However, whether cirsilineol inhibits osteoclast activity and prevents postmenopausal osteoporosis still remain unknown.Materials and methodsPrimary bone marrow macrophages (BMMs) and RAW264.7 cells were used. Osteoclast activity was measured by TRAP staining, F-actin staining, and bone resorption assay after BMMs were treated with cirsilineol at concentrations of 0, 1, 2.5 and 5 µM. RT-PCR and western blotting were performed to evaluate the expression of osteoclast-related genes. In addition, female C57BL/6 mice underwent OVX surgery and were treated with cirsilineol (20 mg/kg) to demonstrate the effect of cirsilineol on osteoporosis.ResultsCirsilineol significantly inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation in a concentration- and time-dependent manner, respectively. Additionally, cirsilineol inhibited F-actin ring formation, thus reducing the activation of bone resorption ability. Cirsilineol suppressed the expression of osteoclast-related genes and proteins via blocking nuclear factor (NF)-κb, ERK, and p38 signaling cascades. More importantly, cirsilineol treatment in mice with osteoporosis alleviated osteoclasts hyperactivation and bone mass loss caused by estrogen depletion.ConclusionIn this study, the protective effect of cirsilineol on osteoporosis has been investigated for the first time. In conclusion, our findings prove the inhibitory effect of cirsilineol on osteoclast activity via NF-κb/ERK/p38 signaling pathways and strongapplication of cirsilineol can be proposed as a potential therapeutic strategy.

AESIS-1, a Rheumatoid Arthritis Therapeutic Peptide, Accelerates Wound Healing by Promoting Fibroblast Migration in a CXCR2-Dependent Manner.

In patients with autoimmune disorders such as rheumatoid arthritis (RA), delayed wound healing is often observed. Timely and effective wound healing is a crucial determinant of a patient's quality of life, and novel materials for skin wound repair, such as bioactive peptides, are continuously being studied and developed. One such bioactive peptide, AESIS-1, has been studied for its well-established anti-rheumatoid arthritis properties. In this study, we attempted to use the anti-RA material AESIS-1 as a therapeutic wound-healing agent based on disease-modifying antirheumatic drugs (DMARDs), which can help restore prompt wound healing. The efficacy of AESIS-1 in wound healing was assessed using a full-thickness excision model in diabetic mice; this is a well-established model for studying chronic wound repair. Initial observations revealed that mice treated with AESIS-1 exhibited significantly advanced wound repair compared with the control group. In vitro studies revealed that AESIS-1 increased the migration activity of human dermal fibroblasts (HDFs) without affecting proliferative activity. Moreover, increased HDF cell migration is mediated by upregulating chemokine receptor expression, such as that of CXC chemokine receptor 2 (CXCR2). The upregulation of CXCR2 through AESIS-1 treatment enhanced the chemotactic reactivity to CXCR2 ligands, including CXC motif ligand 8 (CXCL8). AESIS-1 directly activates the ERK and p38 mitogen-activated protein kinase (MAPK) signaling cascades, which regulate the migration and expression of CXCR2 in fibroblasts. Our results suggest that the AESIS-1 peptide is a strong wound-healing substance that increases the movement of fibroblasts and the expression of CXCR2 by turning on the ERK and p38 MAPK signaling cascades.

Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis.

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

Abstract A089: NST-628 is a potent, fully brain-penetrant, RAS/MAPK pathway molecular glue inhibitor with efficacy in CNS tumor models

Abstract Alterations in the RAS/RAF/MEK/ERK signaling cascade are common across multiple solid tumor types and aberrant signaling of the pathway is a driver for RAS- and RAF-driven cancers. Apart from approved mutation selective inhibitors for BRAF Class I and KRAS G12C mutations, other mutations of RAS and RAF are not directly addressable by currently approved inhibitors and there is a need for inhibitors with superior efficacy, durability and tolerability for the MAPK pathway in RAS- and RAF-driven cancers. Currently approved inhibitors of the RAS-MAPK pathway also have limited central nervous system (CNS) exposure and an area of significant unmet patient need exists for patients with primary and metastatic tumors harboring RAS-MAPK pathway alterations. NST-628 has CNS permeability, with a Kpu,u >1, that is far higher than trametinib or VS-6766 (Kpu,u of 0.11 and 0.18 respectively). The predicted effective human half-life of NST-628 (~9h) is amenable to daily dosing in the clinic. In vivo, NST-628 leads to a dose-dependent inhibition of the MAPK pathway in murine brain tissue[KS1] as measured by phospho-ERK. In an intracranial luciferase-tagged SK-MEL-2 xenograft model (NRAS Q61R), NST-628 leads to tumor regressions with a daily dosing regimen (3 mg/kg QD). Tumor regressions as measured by bioluminescent imaging (BLI) were only seen with NST-628 (50%) in the SK-MEL-2 model, whereas trametinib and VS-6766 showed minimal efficacy due to low exposure in the CNS. In the NF-1 mutant MeWo (NF1 Q1336*) melanoma intracranial xenograft model (luciferase tagged), NST-628 led to tumor regressions at two dosing regimens (1, 3 mg/kg QD) and tumor stasis at lower doses (0.3 mg/kg) as measured by BLI. In contrast, the brain penetrant type II RAF inhibitor DAY101 (formerly TAK580) showed no efficacy in the MeWo model and resulted in paradoxical pathway hyper-activation, as determined by enhanced PD response and tumor growth. Only NST-628 effectively inhibited MAPK signaling as measured by DUSP6 transcript in a time- and dose-dependent manner. Overall, survival of mice harboring MeWo intracranial tumors was also significantly increased versus vehicle control and DAY101 treatment groups. These data collectively support best-in-class potential of NST-628 and initiation of clinical trials in patients with solid tumors harboring RAS- and RAF-mutations, including those presenting clinically with brain metastases and primary intracranial tumors. Citation Format: Meagan B Ryan, Chun Li, Yongxin Han, Klaus P Hoeflich, Michael R Hale, Margit Hagel. NST-628 is a potent, fully brain-penetrant, RAS/MAPK pathway molecular glue inhibitor with efficacy in CNS tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A089.

Abstract A088: NST-628 is a potent, best-in-class MAPK pathway molecular glue that inhibits RAS- and RAF-driven cancers

Abstract Alterations in the RAS/RAF/MEK/ERK signaling cascade are common across multiple solid tumor types and aberrant signaling of the pathway is a driver for RAS- and RAF-driven cancers. Apart from approved mutation selective inhibitors for BRAF Class I and KRAS G12C mutations, other mutations of RAS and RAF are not directly addressable by currently approved inhibitors and there is a need for inhibitors with superior efficacy, durability and tolerability for the MAPK pathway in RAS- and RAF-driven cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents phosphorylation and activation of MEK by RAF, leading to deep durable inhibition of MEK kinase activity and downstream ERK signaling. In a MEK1 immunoprecipitation assay, NST-628 treatment glues ARAF, BRAF, and CRAF with unphosphorylated MEK1, stabilizing an inactive RAF-MEK complex and deeply inhibiting phospho-ERK signaling. In an unbiased cell line panel screen (550 models), NST-628 demonstrates efficacy across multiple tumor types with RAS-MAPK alterations, including melanoma, lung, and pancreatic models. NST-628 is broadly efficacious in models with BRAF Class II/III mutations, KRAS-mutations (G12C, G12D, G12V, G12R, Q61H), and NRAS-mutations (Q61x, G12x) as well as showing anti-proliferative effects in NF1-mutant/deficient models. Cell lines with NRAS Q61 mutations were particularly sensitive to NST-628 inhibition (GI50 average=150 nM). In vivo, NST-628 has a balanced metabolic profile and predicted half-life and exposure in humans is compatible with low dose daily dosing. NST-628 (3-5 mg/kg QD treatment) led to tumor regressions in HCT116 (KRAS G13D colorectal) and IPC-298 (NRAS Q61L melanoma) xenograft models, 53% and 38% respectively, correlating to inhibition of both phospho-MEK and phospho-ERK in tumors. In comparison to the MEK inhibitor trametinib, NST-628 led to deeper tumor responses and on target pathway inhibition in both models as well as significantly greater tolerability as measured by body weight. In a mini-mouse trial utilizing patient derive xenograft (PDX) tumors harboring NF1, KRAS G12D/R, BRAF Class II/III, and NRAS Q61x mutations, NST-628 (3 mg/kg QD) dosing demonstrates broad anti-tumor responses across melanoma, lung, pancreatic, glioma, and ovarian models. These data collectively, together with the predicted effective human half-life of NST-628 being consistent with daily dosing, support best-in-class potential for NST-628 and initiation of clinical trials in patients with solid tumors harboring RAS- and RAF-mutations is planned. Citation Format: Meagan B Ryan, Natasha Schenk, Marshall Zingg, Matthew Koehler, Brooke Swalm, Bradley Quade, Chaoyang Ye, Arvin C Dar, Yongxin Han, Klaus P Hoeflich, Michael R Hale, Margit Hagel. NST-628 is a potent, best-in-class MAPK pathway molecular glue that inhibits RAS- and RAF-driven cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A088.

MAPK/ERK and PI3K/AKT signaling pathways are activated in adolescent and adult acute lymphoblastic leukemia.

The mitogen-activated protein kinase (MAPK)/ERK signaling cascade and the phosphoinosytol-3 phosphate/Akt (PI3K/Akt) pathways are involved in proliferation and differentiation of hematopoietic cells. The frequency of PI3K/Akt and MAPK pathway activation in adult acute lymphoblastic leukemia (ALL) still need to be elucidated. To assess the activity and prognostic implications of MAPK/ERK and PI3K/Akt pathways in adult (ALL). We examined 28 precursor-B-cell ALL and 6 T-cell primary ALL samples. Flow cytometry was employed to analyze the expression levels of phosphorylated ERK and phosphorylated Akt. Ten out of 15 (67%) ALL fresh samples (7 B-cell, 3 T-cell) showed constitutive p-ERK expression. The p-ERK mean fluorescent index ratio (MFI (R)) showed a tendency to be higher in ALL than in normal T lymphocytes (1.26 [0.74-3.10] vs. 1.08 [1.02-1.21], respectively [p = .069]) and was significantly lower than in leukemic cell lines (median MFI (R) 3.83 [3.71-5.97] [p < .001]). Expression of p-Akt was found in 35% (12/34) (10 B-cell, 2 T-cell). The median MFI (R) expression for p-Akt in primary blast cell was 1.13 (0.48-9.90) compared to 1.01 (1.00-1.20) in normal T lymphocytes (p = ns) and lower than in leukemic cell lines (median MFI (R) 2.10 [1.77-3.40] [p = .037]). Moreover, expression of p-ERK was negatively associated with the expression of CD34 (1.22 [0.74-1.33] vs. 1.52 [1.15-3.10] for CD34(+) and CD34(-) group, respectively, p = .009). Our findings suggest that both MAPK/ERK and PI3K/Akt are constitutively activated in adult ALL, indicating a targeted therapy potential for ALL by using inhibitors of these pathways.

Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury

The current study explores the potential function and the underlying mechanisms of endothelial cell-derived R-spondin 3 (RSPO3) neuroprotection against ischemia/reperfusion-induced neuronal cell injury. In both neuronal cells (Neuro-2a) and primary murine cortical neurons, pretreatment with RSPO3 ameliorated oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced neuronal cell death and oxidative injury. In neurons RSPO3 activated the Akt, Erk and β-Catenin signaling cascade, but only Erk inhibitors reversed RSPO3-induced neuroprotection against OGD/R. In mouse embryonic fibroblasts (MEFs) and neuronal cells, RSPO3-induced LGR4-Gab1-Gαi1/3 association was required for Erk activation, and either silencing or knockout of Gαi1 and Gαi3 abolished RSPO3-induced neuroprotection. In mice, middle cerebral artery occlusion (MCAO) increased RSPO3 expression and Erk activation in ischemic penumbra brain tissues. Endothelial knockdown or knockout of RSPO3 inhibited Erk activation in the ischemic penumbra brain tissues and increased MCAO-induced cerebral ischemic injury in mice. Conversely, endothelial overexpression of RSPO3 ameliorated MCAO-induced cerebral ischemic injury. We conclude that RSPO3 activates Gαi1/3-Erk signaling to protect neuronal cells from ischemia/reperfusion injury.

Mountain-cultivated ginseng protects against cognitive impairments in aged GPx-1 knockout mice via activation of Nrf2/ChAT/ERK signaling pathway

BackgroundEscalating evidence shows that ginseng possesses an antiaging potential with cognitive enhancing activity. As mountain cultivated ginseng (MCG) is cultivated without agricultural chemicals, MCG has emerged as a popular herb medicine. However, little is known about the MCG-mediated pharmacological mechanism on brain aging. MethodsAs we demonstrated that glutathione peroxidase (GPx) is important for enhancing memory function in the animal model of aging, we investigated the role of MCG as a GPx inducer using GPx-1 (a major type of GPx) knockout (KO) mice. We assessed whether MCG modulates redox and cholinergic parameters, and memory function in aged GPx-1 knockout KOmice. ResultsRedox burden of aged GPx-1 KO mice was more evident than that of aged wild-type (WT) mice. Alteration of Nrf2 DNA binding activity appeared to be more evident than that of NFκB DNA binding activity in aged GPx-1 KO mice. Alteration in choline acetyltransferase (ChAT) activity was more evident than that in acetylcholine esterase activity. MCG significantly attenuated reductions in Nrf2 system and ChAT level. MCG significantly enhanced the co-localization of Nrf2-immunoreactivity and ChAT-immunoreactivity in the same cell population. Nrf2 inhibitor brusatol significantly counteracted MCG-mediated up-regulation in ChAT level and ChAT inhibition (by k252a) significantly reduced ERK phosphorylation by MCG, suggesting that MCG might require signal cascade of Nrf2/ChAT/ERK to enhance cognition. ConclusionGPx-1 depletion might be a prerequisite for cognitive impairment in aged animals. MCG-mediated cognition enhancement might be associated with the activations of Nrf2, ChAT, and ERK signaling cascade.

A Phase I Open Label Study of Fostamatinib, a SYK Inhibitor, in Patients with Lower-Risk Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

INTRODUCTION: Ineffective hematopoiesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) have been associated to upregulated innate immune and inflammatory signals. Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is highly expressed in hematopoietic cells and mediates signaling through Toll-like receptors and pattern recognition receptors leading to activation of the ERK, AKT, NF-kB and p38 signaling cascades. Given prior data suggesting that inhibition of these signals might be associated with improved hematopoiesis in MDS we hypothesized SYK inhibition might have therapeutic efficacy in lower-risk MDS and CMML. METHODS: To study this we designed an open label phase I clinical trial of fostamatinib, a SYK inhibitor, for patients with lower-risk MDS or CMML (defined as those with a score <3.5 by the revised International Prognostic Scoring System [IPSS-R]) and no response to prior erythroid stimulating agents or hypomethylating agents (HMAs). Patients with MDS with del(5q) must have received prior therapy with lenalidomide and those with MDS with ring sideroblasts must have received luspatercept. Patients had to be transfusion dependent, defined as prior RBC or platelet transfusions within the past 28 days, or have hemoglobin <10g/dL or platelets <75x109/L. Patients with profound neutropenia (ANC <0.5x109/L) where excluded. The study includes an intra-patient dose escalation design by which patients start therapy at a dose of fostamatinib of 100mg po bid on days 1-28 of a 28-day cycle. If no response to therapy, by IWG 2006 response criteria, is observed after 8 weeks of therapy dose will be escalated to 150mg po bid on days 1-28 of a 28-day cycle and then, in the absence of response after an additional 8 weeks of therapy, to 200mg po bid on days 1-28 of a 28 day cycle. Patients achieving no response after the second dose escalation will be discontinued from study (Figure 1a). The primary endpoint of the study is to assess the safety and tolerability of fostamatinib. Secondary objectives included overall response rates, transfusion independence and survival outcomes. The study includes futility and safety stopping rules. This study is registered at clinicaltrials.gov (NCT05030675). RESULTS: To date, a total of 5 patients have been treated on study as of July 2022 including 3 patients with MDS and ring sideroblasts. Patient characteristics are detailed in Figure 1b. Median age is 71 years (range 59-82). Median number of prior therapies was 5 (range 4-6) including one patient with a prior allogeneic stem-cell transplant. Four (80%) patients had received prior therapy with HMA. Two patients were transfusion dependent of RBC at the time of enrollment with one patient who was eligible based on the presence of thrombocytopenia. At the current data cut off, three patients developed treatment emergent adverse events the main including grade 1-2 diarrhea or constipation. One patient developed COVID-19 infection during the course of therapy and had mild symptomatology. Fostamatinib was not discontinued in this patient. No episodes of expected on-target uncontrolled hypertension or transaminitis have been observed so far. To date, only 4 patients have completed at least 2 cycles of therapy (8 weeks) and are eligible for response, and only 2 patients have dose escalated beyond 100mg po bid: 1 currently at the 150mg po bid dose level, and 1 at the maximum dose of 200mg po bid. With a median follow up of only 2.7 months (95% CI 0-5.6), no responses have been observed. All 5 patients remain on study: 3 (60%) at the 100mg po bid dose, 1 (20%) at 150mg po bid and 1 (20%) at 200mg po bid. CONCLUSIONS: Preliminary data suggests that treatment with fostamatinib at doses of up to 200mg po bid can be safely administered in patients with lower-risk MDS who have failed available therapies. Enrollment is ongoing to determine the potential efficacy of doses of up to 200mg bid. Additional correlative studies to determine predictors of response are underway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prevalence of gastrointestinal disorders in individuals with RASopathies: May RAS/MAP/ERK pathway dysfunctions be a model of neuropathic pain and visceral hypersensitivity?

RASopathies are a group of neurodevelopmental syndromes caused by germline variants in genes of the Ras/MAP/ERK pathway. Growth failure, neurological involvement, and pain represent the main features of these conditions. ERK signaling cascade plays a crucial role in nociception and visceral pain and it is likely implicated in the genesis of neuropathic pain and maintenance of altered pain states. We studied the prevalence of abdominal pain and functional gastrointestinal (GI) disorders in a large sample of individuals with RASopathies. A brief pain inventory questionnaire and semi-structured dedicated interview were used to investigate presence and localization of pain. A Rome IV questionnaire was used to screen for functional GI disorders. Eighty patients with clinical and molecular diagnoses of RASopathy were recruited (42 with Noonan syndrome; 17 with Costello Syndrome and 21 with cardio-facio-cutaneous syndrome). Overall, the prevalence of abdominal pain was 44% and prevalence of functional GI disorders was 78% with constipation, abdominal pain, and aerophagia being the most frequently detected ones. A significant association was found between pain and irritable bowel syndrome, functional constipation and aerophagia. Children with RASopathies have a high prevalence of functional gastrointestinal disorders. These children could represent a good in vivo model to study neuropathic pain, visceral hypersensitivity and gut-brain axis disorders.

Targeting the effect of sofosbuvir on selective oncogenes expression level of hepatocellular carcinoma Ras/Raf/MEK/ERK pathway in Huh7 cell line

Direct acting antiviral agents are emerging line of treatment to eradicate Hepatitis C virus. Recent controversy over whether direct acting antiviral agents increase rate of hepatocellular carcinoma in HCV patients or prevent it, has increased the need to elaborate underlying mechanisms on molecular basis. This work was aimed to investigate the effect of sofosbuvir on the expression of selected oncogenes from the Ras/Raf/MEK/ERK pathway in Huh7 cell line. Results found concrete molecular evidence that sofosbuvir has significantly altered the expression of selected genes when huh7 cell line was treated with sofosbuvir. Nine genes related to HCC were found to be affected by sofosbuvir in a mixed effect manner. The relative expression of growth factors (VEGF, PDGFRB and HGF) was increased in sofosbuvir treated cell lines. The kinase family genes H-RAS, B-RAF, MET except MAPK1 were downregulated. Similarly, DUSP1 was upregulated and SPRY2 was slightly downregulated; both were negative feedback inhibitors of ERK signalling cascade. Sofosbuvir upregulated the growth factors and MAPK1 which suggests it to be a carcinogen. The downregulation of kinases and upregulation of DUSP1 make it an anticancer drug. Hence, the results from this study are important to prove that sofosbuvir neither reduce nor induce hepatocellular carcinoma.

Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation

Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F‐actin assembly. Using oocyte‐specific Erk1/2 knockout mice, we verified that MOS‐ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS‐ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF.

Suppression of Cholangiocarcinoma Cell Growth and Proliferation by Atractylodes lancea (Thunb) DC. through ERK-Signaling Cascade.

Objective:The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells. Materials and methods:The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. Results:The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. Conclusion:The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.

Cordycerebroside A suppresses VCAM-dependent monocyte adhesion in osteoarthritis synovial fibroblasts by inhibiting MEK/ERK/AP-1 signaling

• VCAM1 and CD11b higher levels express in OA compared with healthy controls. • Cordycerebroside A suppressed VCAM-1 mediated-monocyte adhesion in human OASF. • The MEK/ERK/AP-1 signaling regulated cordycerebroside A effects. Osteoarthritis (OA) is characterized by the infiltration and adhesion of monocyte into the joint synovium. Vascular cell adhesion molecule 1 (VCAM-1) is a critical cell adhesion molecule that controls monocyte motility during OA progression. Cordycerebroside A, a cerebroside compound isolated from Cordyceps militaris , inhibits the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in synovial macrophages, but has not yet been investigated in OA. Gene Expression Omnibus (GEO) dataset analysis revealed higher levels of VCAM-1 and CD11b (a monocyte marker) in OA synovial tissue compared with normal healthy tissue. The same results were observed in anterior cruciate ligament transaction (ACLT)-induced OA in rats compared with normal healthy controls. Cordycerebroside A markedly suppressed VCAM-1 expression and monocyte adhesion in human OA synovial fibroblasts. The MEK, ERK and AP-1 signaling cascades regulated cordycerebroside A-induced inhibition of VCAM-1 production. Thus, cordycerebroside A is a promising agent for the treatment of OA.

Signaling Pathways in Proton and Non-proton ASIC1a Activation.

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases as well as pain conditions. Classically, ASICs are described as transiently activated by a reduced pH, followed by desensitization; the activation allows sodium influx, and in the case of ASIC1a-composed channels, also calcium to some degree. Several factors are emerging and extensively analyzed as modulators, activating, inhibiting, and potentiating specific channel subunits. However, the signaling pathways triggered by channel activation are only starting to be revealed.The channel has been recently shown to be activated through a mechanism other than proton-mediated. Indeed, the large extracellular loop of these channels opens the possibility that other non-proton ligands might exist. One such molecule discovered was a toxin present in the Texas coral snake venom. The finding was associated with the activation of the channel at neutral pH via the toxin and causing intense and unremitting pain.By using different pharmacological tools, we analyzed the downstream signaling pathway triggered either by the proton and non-proton activation for human, mouse, and rat ASIC1a-composed channels in in vitro models. We show that for all species analyzed, the non-protonic mode of activation determines the activation of the ERK signaling cascade at a higher level and duration compared to the proton mode.This study adds to the growing evidence of the important role ASIC1a channels play in different physiological and pathological conditions and also hints at a possible pathological mechanism for a sustained effect.

Urinary exosomes-based Engineered Nanovectors for Homologously Targeted Chemo-Chemodynamic Prostate Cancer Therapy via abrogating EGFR/AKT/NF-kB/IkB signaling

Multi-functional nanovectors based on exosomes from cancer cell culture supernatants in vitro has been successfully utilized for tumor-specific targeting and immune escape. However, the labor-intensive purification procedures for rich-dose and high-purity homogeneous exosomes without using targeting ligands are still a challenging task. Herein, we developed a nanovector Exo-PMA/Fe-HSA@DOX through cloaked by urinary exosome membrane as a chemo/chemodynamic theranostic nano-platform for targeted homologous prostate cancer therapy which pertain to the abrogation of Epidermal Growth Factor Receptor (EGFR) and its downstream AKT/NF-kB/IkB signaling instead of ERK signaling cascades. Urinary exosomes-based nanovectors own the same urological cancer cell membrane antigen inclusive of E-cadherin, CD 47 and are free from intracellular substance such as Histone 3 and COX Ⅳ. The targeting properties of the homologous cancer cell are well preserved in Exo-PMA/Fe-HSA@DOX nanovectors in high purity. Meanwhile, the nanovectors based on urinary exosomes from prostate patients deeply penetrated into prostate cancer DU145 3D MCTS, and successfully achieve superior synergistic low-dose chemo/chemodynamic performance in vivo. In addition, the blockage of bypassing EGFR/AKT/NF-kB/IkB signaling pathway is greatly enhanced via elevated intracellular PMA/Fe-HSA@DOX nanoparticles (NPs). It is expected that the rich source and high purity of urinary exosomes offer a reliable solution for mass production of such nanovectors in the future. The targeted homologous cancer therapy based on the urinary exosomes from cancer patients exemplifies a novel targeted anticancer scheme with efficient and facile method.

Knockout of the NONO Gene Inhibits Neointima Formation in a Mouse Model of Vascular Injury.

[Figure: see text].

The proliferation effects of fluoxetine and amitriptyline on human breast cancer cells and the underlying molecular mechanisms

Some studies have suggested possible estrogen actions for antidepressants such as fluoxetine. However, the specific molecular mechanisms remain unclear. In this study, the molecular mechanism of fluoxetine-induced the proliferation of breast cancer SKBR3 and MCF-7 cells was evaluated by detecting ERα and GPR30-mediated ERK and PI3K/AKT signals. We found that low concentrations of fluoxetine upregulated the expression of GPR30, ERα, CyclinD1, and C-MYC proteins, as well as elevated the phosphorylation of ERK and AKT. The phosphorylation of ERK and AKT decreased when the cells were pretreated with ERα inhibitor ICI, GPR30 inhibitor G15, and PI3K inhibitor WM prior to fluoxetine exposure. The addition of these inhibitors also attenuated the fluoxetine-induced cell proliferation. These findings indicated that fluoxetine activated the PI3K/AKT and ERK signaling cascades via GPR30 to derive the cell proliferation. It suggests that fluoxetine has the potential to exert estrogen actions via GPR30.