Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells.

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) ß-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the ß-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG ß-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC50 value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells.

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) ß-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG ß-subunit CTP region (amino acids 115-149) was inserted between the ß-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG ß-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

Effects of site-directed mutagenesis of GLP-1 and glucagon receptors on signal transduction activated by dual and triple agonists.

The paradigm of one drug against multiple targets, known as unimolecular polypharmacology, offers the potential to improve efficacy while overcoming some adverse events associated with the treatment. This approach is best exemplified by targeting two or three class B1 G protein-coupled receptors, namely, glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic polypeptide receptor for treatment of type 2 diabetes and obesity. Some of the dual and triple agonists have already shown initial successes in clinical trials, although the molecular mechanisms underlying their multiplexed pharmacology remain elusive. In this study we employed structure-based site-directed mutagenesis together with pharmacological assays to compare agonist efficacy across two key signaling pathways, cAMP accumulation and ERK1/2 phosphorylation (pERK1/2). Three dual agonists (peptide 15, MEDI0382 and SAR425899) and one triple agonist (peptide 20) were evaluated at GLP-1R and GCGR, relative to the native peptidic ligands (GLP-1 and glucagon). Our results reveal the existence of residue networks crucial for unimolecular agonist-mediated receptor activation and their distinct signaling patterns, which might be useful to the rational design of biased drug leads.

In-vitro effect of diazoxon, a metabolite of diazinon, on proliferation, signal transduction, and death induction in mononuclear cells of Nile tilapia fish (Oreochromis niloticus).

The immune response of teleosts (bonefish) is altered by diazinon (DZN), an organophosphate pesticide. It has been suggested that such alteration is due to the extraneuronal cholinergic system in fish leukocytes that renders these cells a target of pesticides. Diazoxon (DZO), the oxon metabolite of DZN, has been attributed immunotoxic effects. Still, to date there are no reports on the effects of DZO upon parameters involved in the signaling cascade of immune response cells. Therefore, this work evaluated the effect of DZO on key parameters of cell signaling (intracellular Ca2+ flux, ERK 1/2 phosphorylation), cell proliferation, and antiproliferative processes (apoptosis, senescence, mitochondrial membrane potential) in spleen mononuclear cells of Nile tilapia fish. The results obtained show that DZO does not affect cell proliferation but causes a lack of response to stimulation with PMA and ionomycin to release intracellular calcium. In addition, it inhibits ERK 1/2 phosphorylation and causes loss of mitochondrial membrane potential, apoptosis, and senescence. These results suggest that the lack of cell response to release intracytoplasmic Ca2+ inhibits ERK which disrupts the mitochondrial membrane potential, leading to cell apoptosis and senescence. These findings prove that DZO significantly affects key parameters involved in the survival of immune response cells.

Attenuation of the Diffuse Noxious Inhibitory Controls in Chronic Joint Inflammatory Pain Is Accompanied by Anxiodepressive-Like Behaviors and Impairment of the Descending Noradrenergic Modulation.

The noradrenergic system is paramount for controlling pain and emotions. We aimed at understanding the descending noradrenergic modulatory mechanisms in joint inflammatory pain and its correlation with the diffuse noxious inhibitory controls (DNICs) and with the onset of anxiodepressive behaviours. In the complete Freund's adjuvant rat model of Monoarthritis, nociceptive behaviors, DNICs, and anxiodepressive-like behaviors were evaluated. Spinal alpha2-adrenergic receptors (a2-AR), dopamine beta-hydroxylase (DBH), and noradrenaline were quantified concomitantly with a2-AR pharmacologic studies. The phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) were quantified in the Locus coeruleus (LC), amygdala, and anterior cingulate cortex (ACC). DNIC was attenuated at 42 days of monoarthritis while present on days 7 and 28. On day 42, in contrast to day 28, noradrenaline was reduced and DBH labelling was increased. Moreover, spinal a2-AR were potentiated and no changes in a2-AR levels were observed. Additionally, at 42 days, the activation of ERKs1/2 was increased in the LC, ACC, and basolateral amygdala. This was accompanied by anxiety- and depressive-like behaviors, while at 28 days, only anxiety-like behaviors were observed. The data suggest DNIC is attenuated in prolonged chronic joint inflammatory pain, and this is accompanied by impairment of the descending noradrenergic modulation and anxiodepressive-like behaviors.

Molecular and pharmacological characterization of poultry (Gallus gallus, Anas platyrhynchos, Anser cygnoides domesticus) and pig (Sus scrofa domestica) melanocortin-5 receptors and their mutants.

The melanocortin-5 receptor (MC5R) is a member of the G protein-coupled receptor superfamily that plays a critical role in lipid production, skeletal muscle fatty acid oxidation, and adipocyte lipolysis. Although multiple functions and important value of MC5R in human beings have been fully demonstrated, however, the potential molecular cloning, pharmacological characteristics and key amino acids in poultry and pig were still not fully understood. Herein, we successfully cloned MC5R genes from chicken (Gallus gallus, cMC5R), duck (Anas platyrhynchos, dMC5R), goose (Anser cygnoides domesticus, gMC5R) and pig (Sus scrofa domestica, pMC5R), and compared their genetic and protein difference with hMC5R through phylogenetic analysis and homology models. Besides, we constructed three alanine-substitution mutants for each of MC5Rs through homologous reorganization, including c/d/gMC5R-D119A/F254A/H257A and pMC5R-D204A/F339A/H342A. Subsequently, we focused our investigation on the pharmacological characterization of four wide-type MC5Rs and their mutants in HEK293T cells, including the intracellular cAMP generation and phosphorylation level of ERK1/2. The results showed that these mutants had decreased cAMP levels under the stimulation of ligands, in spite of enhanced basal activity for c/d/gF254A and pH342A, indicating their important roles in the location and activation of receptors. Notably, these MC5Rs and mutants displayed significant species-specific phenotypes in the activation of pERK1/2 with ligands, which was not completely consistent with hMC5R. These findings demonstrated that presence of interspecies differences for MC5Rs, particularly for the pERK1/2 pathway. Taken together, our study expands current knowledge about the molecular and pharmacological characterization of c/d/g/pMC5Rs, providing preliminary data for MC5R-targeted drug screening or genetic breeding of economic animals in the future.

Increased CD74 binding and EAE treatment efficacy of a modified DRα1 molecular construct.

Multiple sclerosis (MS) is a demyelinating and degenerative disease of the central nervous system (CNS) with a strong inflammatory component that affects more than 2 million people worldwide (and at least 400,000 in the United States). In MS, macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) enhance the inflammatory event as a result of their interaction with their cognate receptor CD74. Therefore, the search for new agents aimed at blocking this interaction is critical for therapeutic purposes and will be of paramount importance for the treatment of MS. DRα1-MOG-35-55 constructs have been demonstrated to be effective in the treatment of experimental autoimmune encephalomyelitis (EAE) a mouse model for MS. This effect is directly correlated with the binding to its cell surface receptor, CD74, apparently preventing or blocking the binding of two inflammatory factors, MIF and D-DT. Here we report that a single amino acid substitution (L50Q) in the DRα1 domain of the human and mouse DRα1-MOG-35-55 constructs (notated as DRhQ and DRmQ, respectively) possessed increased affinity for CD74, a greater capacity to block MIF binding, the ability to inhibit pERK1/2 signaling and increased therapeutic activity in mice with EAE. These data suggest that binding affinity for CD74 could serve as an in vitro indicator of biological potency of DRhQ and thus support its possible clinical utility as an effective therapy for MS and perhaps other diseases in which there is an inflammatory reaction driven by MIF and D-DT.

Neuroendocrine signaling modulates specific neural networks relevant to migraine.

Migraine is a disabling brain disorder involving abnormal trigeminovascular activation and sensitization. Fasting or skipping meals is considered a migraine trigger and altered fasting glucose and insulin levels have been observed in migraineurs. Therefore peptides involved in appetite and glucose regulation including insulin, glucagon and leptin could potentially influence migraine neurobiology. We aimed to determine the effect of insulin (10U·kg-1), glucagon (100µg·200µl-1) and leptin (0.3, 1 and 3mg·kg-1) signaling on trigeminovascular nociceptive processing at the level of the trigeminocervical-complex and hypothalamus. Male rats were anesthetized and prepared for craniovascular stimulation. In vivo electrophysiology was used to determine changes in trigeminocervical neuronal responses to dural electrical stimulation, and phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) immunohistochemistry to determine trigeminocervical and hypothalamic neural activity; both in response to intravenous administration of insulin, glucagon, leptin or vehicle control in combination with blood glucose analysis. Blood glucose levels were significantly decreased by insulin (p<0.001) and leptin (p<0.01) whereas glucagon had the opposite effect (p<0.001). Dural-evoked neuronal firing in the trigeminocervical-complex was significantly inhibited by insulin (p<0.001), glucagon (p<0.05) and leptin (p<0.01). Trigeminocervical-complex pERK1/2 cell expression was significantly decreased by insulin and leptin (both p<0.001), and increased by glucagon (p<0.001), when compared to vehicle control. However, only leptin affected pERK1/2 expression in the hypothalamus, significantly decreasing pERK1/2 immunoreactive cell expression in the arcuate nucleus (p<0.05). These findings demonstrate that insulin, glucagon and leptin can alter the transmission of trigeminal nociceptive inputs. A potential neurobiological link between migraine and impaired metabolic homeostasis may occur through disturbed glucose regulation and a transient hypothalamic dysfunction.

pERK1/2 Peripheral Recruitment and Filopodia Protrusion Augment Oligodendrocyte Progenitor Cell Migration: Combined Effects of PDGF-A and Fibronectin.

Oligodendrocyte progenitor cell (OPC) migration is critical for effective myelination of the central nervous system. Not only during normal myelination but also during remyelination, the growth factors (GFs) and extracellular matrix (ECM) protein affect the OPC migration. Studies showed the altered levels of GFs and ECM in the demyelinating lesions. In our earlier studies, we have shown that the effect of platelet-derived growth factor alpha (PDGF-A) on OPC migration is dose- and time-dependent. In that we have shown that the physiological concentration (1 ng/ml) of PDGF-A was unable to induce OPC migration at transient exposure (30 min). However, the involvement of ECM in the regulation of PDGF-A mediated OPC migration was not clear. In the present study, we have used fibronectin (FN) as ECM. PDGF-A and FN have similar and overlapping intracellular signaling pathways including the extracellular regulated kinases 1 and 2 (ERK1/2). Here we demonstrate how physiological concentration of PDGF-A combines with FN to augment OPC migration in vitro. The present study is first of its kind to show the importance of the synergistic effects of PDGF-A and FN on peripheral recruitment of phosphorylated/activated ERK1/2 (pERK1/2), actin-pERK1/2 co-localization, and filopodia formation, which are essential for the enhanced OPC migration. These findings were further confirmed by ERK1/2 inhibition studies, using the pharmacological inhibitor U0126. An understanding of these complex interactions may lead to additional strategies for transplanting genetically modified OPCs to repair widespread demyelinated lesions.

Cartilage oligomeric matrix protein participates in the pathogenesis of liver fibrosis.

BACKGROUND & AIMS: Liver fibrosis is characterized by significant accumulation of extracellular matrix (ECM) proteins, mainly fibrillar collagen-I, as a result of persistent liver injury. Cartilage oligomeric matrix protein (COMP) is largely found in the ECM of skeletal tissue. Increased COMP expression has been associated with fibrogenesis in systemic sclerosis, lung fibrosis, chronic pancreatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that COMP could induce fibrillar collagen-I deposition and participate in matrix remodeling thus contributing to the pathophysiology of liver fibrosis. METHODS: Thioacetamide (TAA) and carbon tetrachloride (CCl4) were used to induce liver fibrosis in wild-type (WT) and Comp-/- mice. In vitro experiments were performed with primary hepatic stellate cells (HSCs). RESULTS: COMP expression was detected in livers from control WT mice and was upregulated in response to either TAA or CCl4-induced liver fibrosis. TAA-treated or CCl4-injected Comp-/- mice showed less liver injury, inflammation and fibrosis compared to their corresponding control WT mice. Challenge of HSCs with recombinant COMP (rCOMP) induced intra- plus extracellular collagen-I deposition and increased matrix metalloproteinases (MMPs) 2, 9 and 13, albeit similar expression of transforming growth factor beta (TGFß) protein, in addition to Tgfß, tumour necrosis factor alpha (Tnfα) and tissue inhibitor of metalloproteinases-1 (Timp1) mRNAs. We demonstrated that COMP binds collagen-I; yet, it does not prevent collagen-I cleavage by MMP1. Last, rCOMP induced collagen-I expression in HSCs via CD36 receptor signaling and activation of the MEK1/2-pERK1/2 pathway. CONCLUSION: These results suggest that COMP contributes to liver fibrosis by regulating collagen-I deposition. LAY SUMMARY: Cartilage oligomeric matrix protein (COMP) induces fibrillar collagen-I deposition via the CD36 receptor signaling and activation of the MEK1/2-pERK1/2 pathway, and participates in extracellular matrix remodeling contributing to the pathophysiology of liver fibrosis.

Increased phosphorylation of ERK1/2 is associated with worse chemotherapeutic outcome and a poor prognosis in advanced lung adenocarcinoma.

Constitutive activation of extracellular signal-regulated kinase (ERK)1/2 pathway, that is activated by various stimuli including growth factors and oncogenic driver mutations, is observed in various cancers. However, the difference of the activated levels of the pathway is still unclear in clinical significances. The aim of this study was to investigate the effect of different ERK1/2 pathway activation, assessed by the expression levels of phosphorylated (p) ERK1/2, on the prognosis of advanced lung adenocarcinoma patients. Paraffin-embedded lung biopsy samples were obtained from 85 lung adenocarcinoma patients. Correlation between pERK1/2 expression levels that were assessed by immunohistochemistry (IHC) analysis and oncogenic driver mutation status, clinicopathological factors, outcome from standard anticancer therapies, and prognosis was investigated. Varying levels of pERK1/2 expression were observed in 68 (80.0 %) patients. The overall survival was significantly reduced in patients with higher pERK1/2 expression in comparison to those with lower expression levels (P = 0.03). In particular, higher pERK1/2 expression levels correlated with worse performance status and worse clinical outcome. Thus, the IHC analysis of pERK1/2 expression levels may predict patient prognosis in advanced lung adenocarcinoma. Inhibition of ERK1/2 pathway activated by various signals may improve the effects of standard chemotherapies and the clinical condition of patients with advanced cancer.

KYNA analogue SZR72 modifies CFA-induced dural inflammation- regarding expression of pERK1/2 and IL-1ß in the rat trigeminal ganglion.

BACKGROUND: Neurogenic inflammation has for decades been considered an important part of migraine pathophysiology. In the present study, we asked the question if administration of a novel kynurenic acid analogue (SZR72), precursor of an excitotoxin antagonist and anti-inflammatory substance, can modify the neurogenic inflammatory response in the trigeminal ganglion. METHODS: Inflammation in the trigeminal ganglion was induced by local dural application of Complete Freunds Adjuvant (CFA). Levels of phosphorylated MAP kinase pERK1/2 and IL-1ß expression in V1 region of the trigeminal ganglion were investigated using immunohistochemistry and Western blot. FINDINGS: Pretreatment with one dose of SZR72 abolished the CFA-induced pERK1/2 and IL-1ß activation in the trigeminal ganglion. No significant change was noted in case of repeated treatment with SZR72 as compared to a single dose. CONCLUSIONS: This is the first study that demonstrates that one dose of KYNA analog before application of CFA can give anti-inflammatory response in a model of trigeminal activation, opening a new line for further investigations regarding possible effects of KYNA derivates.

Celecoxib alleviates oxaliplatin-induced hyperalgesia through inhibition of spinal ERK1/2 signaling.

Numerous pieces of evidence have revealed that oxaliplatin (OXA) evokes mechanical and cold hypersensitivity. However, the mechanism underlying these bothersome side effects needs to be further investigated. It is well known that cyclooxygenase-2 (COX-2) and extracellular signal-regulated kinases (ERK1/2) signaling play crucial roles in several pain states. Our previous data showed that Akt2 in the dorsal root ganglion (DRG) participated in the regulation of OXA-induced neuropathic pain. But it is still unclear whether spinal ERK1/2 signaling is involved in the regulation of OXA-induced hyperalgesia, and the linkage between COX-2 and ERK1/2 signaling in mediating OXA-induced hyperalgesia also remains unclear. In this research, we investigated the possible mechanism of celecoxib, a COX-2 inhibitor, in OXA-induced neuropathic pain. Our results show that single dose of OXA (12 mg/kg) significantly attenuated both the tail withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) at days 4 after the OXA treatment. Administration of celecoxib (30 mg/kg/day) for 4 and 6 days inhibited the decrease in TWL and MWT, and each was significantly higher than that of the OXA+vehicle group and was equivalent to that of the vehicles group. OXA increased the expression of cyclooxygenase-2 (COX-2) mRNA and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) protein in the lumbar 4-5 (L4-5) spinal cord dorsal horn neurons. Administration of celecoxib for 7 days suppressed the increase in expression of COX-2 and pERK1/2 induced by OXA. Our findings suggested that COX-2 and ERK1/2 signaling in spinal cord contributed to the OXA-induced neuropathic pain.

Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons.

BACKGROUND: The corticotropin-releasing factor is a stress-related neuropeptide that modulates locus coeruleus activity. As locus coeruleus has been involved in pain and stress-related patologies, we tested whether the pain-induced anxiety is a result of the corticotropin-releasing factor released in the locus coeruleus. METHODS: Complete Freund's adjuvant-induced monoarthritis was used as inflammatory chronic pain model. α-Helical corticotropin-releasing factor receptor antagonist was microinjected into the contralateral locus coeruleus of 4-week-old monoarthritic animals. The nociceptive and anxiety-like behaviors, as well as phosphorylated extracellular signal-regulated kinases 1/2 and corticotropin-releasing factor receptors expression, were quantified in the paraventricular nucleus and locus coeruleus. RESULTS: Monoarthritic rats manifested anxiety and increased phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus and paraventricular nucleus, although the expression of corticotropin-releasing factor receptors was unaltered. α-Helical corticotropin-releasing factor antagonist administration reversed both the anxiogenic-like behavior and the phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus. CONCLUSIONS: Pain-induced anxiety is mediated by corticotropin-releasing factor neurotransmission in the locus coeruleus through extracellular signal-regulated kinases 1/2 signaling cascade.

Deficiency of FHL2 attenuates airway inflammation in mice and genetic variation associates with human bronchial hyper-responsiveness.

BACKGROUND: Asthma is an inflammatory disease that involves airway hyper-responsiveness and mucus hypersecretion. The LIM-only protein FHL2 is a crucial modulator of multiple signal transduction pathways and functions as a scaffold in specific protein-protein interactions. OBJECTIVE: We sought to investigate the role of FHL2 in airway inflammation. METHODS: Allergic airway inflammation was induced in WT and FHL2-knock out (FHL2-KO) mice with ovalbumin (OVA). Lung tissue, bronchoalveolar lavage fluid (BALF) and draining lymph node cells were analysed for inflammation. FHL2 loss and gain of function studies were performed in lung epithelial cells. RESULTS: FHL2-deficient mice challenged with OVA show significantly reduced airway inflammation as evidenced by reduced infiltration of inflammatory cells including eosinophils, dendritic cells, B cells and T cells. Furthermore, mucus production was decreased in FHL2-KO mice. In BALF, the levels of IL-5, IL-13, eotaxin-1 and eotaxin-2 were significantly lower in FHL2-KO mice. In addition, draining lymph node cells from FHL2-KO mice show reduced levels of IL-5 and IL-13. Consistent with this, OVA-specific serum IgG and IgE levels were reduced in FHL2-KO mice. We also found that phosphorylation of ERK1/2 is markedly attenuated in FHL2-KO lung. Knock-down of FHL2 in human lung epithelial cells resulted in a striking decrease in ERK1/2 phosphorylation and mRNA levels of inflammatory cytokines and MUC5AC, whereas FHL2 overexpression exhibited opposite effects. Finally, the SNP rs4851765 shows an association with the severity of bronchial hyper-responsiveness. CONCLUSION: These results highlight functional involvement of FHL2 in airway inflammation and identify FHL2 as a novel gene associated with asthma severity in human.

Role of the extracellular signal-regulated kinase 1/2 signaling pathway in the process of thrombin-promoting airway remodeling in ovalbumin-allergic rats.

CONTEXT: Although it is recognized that thrombin plays a key role in airway remodeling during chronic asthma. In a previous study, we have proved that thrombin promotes airway remodeling via PAR-1 in OVA-allergic rats, but little is known about intracellular signaling pathway involved in the event. OBJECTIVE: In this study, we intend to explore the impact of pERK1/2 signaling pathway on the process of thrombin-induced airway remodeling in OVA-allergic rats. MATERIALS AND METHODS: A rat model of chronic asthma was set up by systemic sensitization and repeated challenge to OVA. The doses of thrombin, recombinant hirudin, PAR-1 inhibitor ER-112780-06, and pERK1/2 inhibitor PD98059 varied for different groups. The expression of pERK1/2 was analyzed by western blot and RT-PCR. Secretion of TGF-ß1 and IL-6 was detected by ELISA. RESULTS: The expression of pERK1/2 was higher in the airway of asthmatic rats than those of normal rats, and was significantly increased by thrombin treatment but decreased by thrombin-inhibitor treatment. Airway remodeling was enhanced by thrombin but weakened by pERK1/2 inhibitor. Expression of growth factors and IL-6 in asthmatic rats was significantly increased by thrombin treatment and decreased by thrombin-inhibitor treatment and pERK1/2 inhibitor treatment. CONCLUSION: These results suggest that ERK1/2 signaling pathway may play an important role in the process of thrombin-promoting airway remodeling in OVA-allergic rats, and pERK1/2 inhibitor effectively inhibits the process.

Fc receptor-like A promotes malignant behavior in renal cell carcinoma and correlates with tumor immune infiltration.

BACKGROUND: Our study aims to investigate the mechanisms through which Fc receptor-like A (FCRLA) promotes renal cell carcinoma (RCC) and to examine its significance in relation to tumor immune infiltration. MATERIALS AND METHODS: The correlation between FCRLA and data clinically related to RCC was explored using The Cancer Genome Atlas (TCGA), then validated using Gene Expression Omnibus (GEO) gene chip data. Enrichment and protein-protein interaction (PPI) network analyses were performed for FCRLA and its co-expressed genes. FCRLA was knocked down in RCC cell lines to evaluate its impact on biological behavior. Then the potential downstream regulators of FCRLA were determined by western blotting, and rescue experiments were performed for verification. The relevance between FCRLA and various immune cells was analyzed through GSEA, TIMER, and GEPIA tools. TIDE and ESTIMATE algorithms were used to predict the effect of FCRLA in immunotherapy. RESULTS: Fc receptor-like A was associated with clinical and T stages and could predict the M stage (AUC = 0.692) and 1-3- and 5-year survival rates (AUC = 0.823, 0.834, and 0.862) of RCC patients. Higher expression of FCLRA predicted an unfavorable overall survival (OS) in TCGA-RCC and GSE167573 datasets (p = 0.03, p = 0.04). FCRLA promoted the malignant biological behavior of RCC cells through the pERK1/2/-MMP2 pathway and was associated with tumor immune microenvironment in RCC. CONCLUSION: Fc receptor-like A is positively correlated with poor outcomes in RCC patients and plays an oncogenic role in RCC through the pERK1/2-MMP2 pathway. Patients with RCC might benefit from immunotherapy targeting FCRLA.

Exploring the Potential of Enhanced Prognostic Performance of NCCN-IPI in Diffuse Large B-Cell Lymphoma by Integrating Tumor Microenvironment Markers: Stromal FOXC1 and Tumor pERK1/2 Expression.

BACKGROUND: FOXC1 and ERK1-2 are proteins implicated in aggressive biological behavior of various malignancies including lymphomas. MATERIAL AND METHODS: We investigate the additive prognostic value of stromal FOXC1 expression and tumor phosphorylated ERK1-2 (pERK1-2) expression to the established National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI), in 92 diffuse large B-cell lymphoma (DLBCL) cases. Multidimensional analysis using statistics and machine learning (ML) models assessed prognostic value of established clinicopathologic variables with stromal FOXC1 and tumor pERK1-2 expressions. RESULTS: Both high FOXC1 stroma group and high pERK1-2 tumor group were significantly associated with shorter progression-free survival (PFS) and overall survival (OS) compared with low group (p = 0.015, 0.034 and p = 0.025, 0.025 each respectively). In multivariable analysis, high FOXC1 stromal expression was an independent prognostic factor of OS (p = 0.037). The addition of stromal FOXC1 and tumor pERK1-2 to the NCCN-IPI score significantly improved prediction of time to death compared with NCCN-IPI score alone (Harrell's C-index = 0.801 vs. 0.764; p = 0.030). ML models reconfirmed the addition of stromal FOXC1 expression and tumor pERK1-2 to NCCN-IPI score had the highest C-index (0.952) among combinations. Stromal FOXC1 and tumor pERK1-2 were determinants of DLBCL prognosis, whose addition significantly improved prognostic performance of the NCCN-IPI.

Tyrosine phosphatase STEP61 negatively regulates amyloid ß-mediated ERK/CREB signaling pathways via α7 nicotinic acetylcholine receptors.

Striatal-enriched phosphatase 61 (STEP61 ) plays an essential role in synaptic plasticity and has recently been implicated in neurodegenerative disease. Here we characterized a possible role of STEP61 in Alzheimer's disease (AD) pathology using a mouse model of AD (Tg-APPswe/PSEN1dE9, APP/PS1 mice) and an in vitro model of AD [cortical neurons treated with amyloid ß (Aß)1-42 peptides]. Our data indicate age-related elevation of STEP61 levels and the proportion of dephosphorylated STEP61 (active STEP61 ) in wild-type mice, which was enhanced in APP/PS1 mice. Furthermore, the increased STEP61 levels and active STEP61 were observed in the hippocampus and cortex from 12-month-old APP/PS1 mice and in Aß1-42 -treated cortical neurons. An α7 nicotinic acetylcholine receptors (nAChRs) antagonist, α-bungarotoxin (BTX), inhibited the Aß1-42 -induced increase of STEP61 expression and activation. In addition, extracellular signal-regulated kinase 1/2 (ERK1/2) and cAMP response element binding (CREB) were impaired in Aß1-42 -treated cortical neurons, and knockdown of STEP61 enhanced the activation of ERK1/2 and CREB. Collectively, these findings indicate two alternate pathological pathways effecting STEP61 regulation in AD. First, Aß regulating STEP61 activity is mediated by Aß binding to α7 nAChRs. Second, STEP61 negatively regulates Aß-mediated ERK/CREB pathway, an important signaling cascade involved in memory formation.

Inhibiting the activation of MAPK (ERK1/2) in stressed Müller cells prevents photoreceptor degeneration.

Rationale: Müller cells play an essential role in maintaining the health of retinal photoreceptors. Dysfunction of stressed Müller cells often results in photoreceptor degeneration. However, how these cells communicate under stress and the signalling pathways involved remain unclear. In this study, we inhibited the MAPK (ERK1/2) signalling, mainly activated in Müller cells, evaluated the protective effects on the photoreceptors and further explored the signalling communication between stressed Müller cells and degenerating photoreceptors. Methods: We evaluated the changes of MAPK (ERK1/2) signalling and its downstream targets in human retinal explants treated with PD98059, a specific phosphorylated ERK1/2 inhibitor, by western blot and immunostaining. We further assessed photoreceptor degeneration by TUNEL staining and outer nuclear layer thickness. We also injected PD98059 into the eyes of mice exposed to photo-oxidative stress. We evaluated the protective effects on photoreceptor degeneration by optical coherence tomography (OCT) and electroretinography (ERG). The crosstalk between Müller cells and photoreceptors was further dissected based on the changes of transcription factors by RNA sequencing and protein profiles of multiple signalling pathways. Results: We found that MAPK (ERK1/2) signalling was mainly activated in Müller cells under stress, both ex vivo and in vivo. PD98059 inhibited the phosphorylation of ERK1/2, reduced expression of the gliotic marker glial fibrillary acidic protein (GFAP) in Müller cells and increased levels of the neuroprotective factor, interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors. Inhibition of pERK1/2 also reduced retinal photo-oxidative damage in mice retinas assessed by OCT and ERG. We also identified that the JAK/STAT3 signalling pathway might mediate signalling transduction from Müller cells to photoreceptors. Conclusion: MAPK (ERK1/2) deactivation through chemical inhibition, mainly in stressed Müller cells, can alleviate gliosis in Müller cells and restore the expression of IRBP in photoreceptors, which appears to prevent retinal degeneration. Our findings suggested a new way to prevent photoreceptor degeneration by manipulating the stress response in Müller cells.