Inhibition Of Extracellular Signal-regulated Kinase Phosphorylation Research Articles

Polydeoxyribonucleotide exerts opposing effects on ERK activity in human skin keratinocytes and fibroblasts.

Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides. It serves as an anti‑inflammatory and tissue‑regenerating agent. The mitogen‑activated protein kinase pathway modulates cell growth and collagen accumulation. It also regulates inflammation by suppressing the expression of proinflammatory cytokines. In the present study, it was attempted to elucidate the molecular mechanism of PDRN in skin healing by confirming the effects of PDRN treatment on skin keratinocytes and fibroblasts, and by assessing the levels of collagen and inflammatory cytokines regulated by the extracellular signal‑regulated kinase (ERK) pathway. The potential effects of PDRN on skin regeneration were investigated. Fibroblast and keratinocyte proliferation and migration were analyzed using the water‑soluble tetrazolium‑8 and wound healing assays. The upregulation of collagen synthesis by PDRN‑induced ERK activation was analyzed in fibroblasts with or without an ERK inhibitor. Inflammatory cytokine expression levels in keratinocytes were determined using reverse transcription‑quantitative polymerase chain reaction. PDRN promoted the proliferation and migration of keratinocytes and fibroblasts. However, PDRN‑induced ERK phosphorylation differed between keratinocytes and fibroblasts; PDRN increased ERK phosphorylation and collagen accumulation in fibroblasts, while it inhibited matrix metalloproteinase expression. By contrast, PDRN inhibited ERK phosphorylation in keratinocytes, and it decreased inflammatory cytokine expression levels. PDRN affects skin cell proliferation and migration, and collagen and inflammatory cytokine expression levels via ERK signaling. Overall, PDRN exerts a positive effect on skin regeneration, but the mechanism by which it promotes skin regeneration varies among different skin cell types.

HucMSC-Ex Alleviates IBD-Associated Intestinal Fibrosis by Inhibiting ERK Phosphorylation in Intestinal Fibroblasts.

Intestinal fibrosis, one of the complications of inflammatory bowel disease (IBD), is associated with fistula and intestinal stricture formation. There are currently no treatments for fibrosis. Mesenchymal stem cell-derived exosomes have been proven to exert inhibitory and reversal effects in IBD and other organ fibrosis. In this study, we explored the role of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) in IBD-related fibrosis and its associated mechanism to provide new ideas for the prevention and treatment of IBD-related intestinal fibrosis. We established a DSS-induced mouse IBD-related intestinal fibrosis model and observed the effect of hucMSC-Ex on the mouse model. We also used the TGF-induced human intestinal fibroblast CCD-18Co to observe the role of hucMSC-Ex in the proliferation, migration, and activation of intestinal fibroblasts. Having observed that the extracellular-signal-regulated kinase (ERK) pathway in intestinal fibrosis can be inhibited by hucMSC-Ex, we treated intestinal fibroblasts with an ERK inhibitor to emphasize the potential target of ERK phosphorylation in the treatment of IBD-associated intestinal fibrosis. In the animal model of IBD-related fibrosis, hucMSC-Ex alleviated inflammation-related fibrosis as evident in the thinning of the mice's intestinal wall and decreased expression of related molecules. Moreover, hucMSC-Ex inhibited TGF-β-induced proliferation, migration, and activation of human intestinal fibroblasts, and ERK phosphorylation played a key role in IBD-associated fibrosis. The inhibition of ERK decreased the expression of fibrosis-related indicators such as α-SMA, fibronectin, and collagen I. hucMSC-Ex alleviates DSS-induced IBD-related intestinal fibrosis by inhibiting profibrotic molecules and intestinal fibroblast proliferation and migration by decreasing ERK phosphorylation.

KI Essence extract (a spleen-tonifying formula) promotes neurite outgrowth, alleviates oxidative stress and hypomyelination, and modulates microbiome in maternal immune activation offspring.

Mushrooms and Chinese traditional herbs have bioactive nutraceuticals with multiple therapeutic functions, including antioxidant and antibacterial activities and microbiome modulation properties. Mushroom-derived bioactive compounds are used in medicines for the treatment of neurological disorders with abnormal brain–gut–microbiome axis. This study examined the effects of KI Essence extract, a spleen-tonifying formula, on neurite growth, antioxidant activity, hypomyelination modulation, and the microbiome profile in lipopolysaccharide (LPS)-induced maternal immune activation (MIA) offspring. The KI Essence extract induced PC12 cell neurite growth by increasing extracellular signal–regulated kinase (ERK) phosphorylation, promoting 2,2′-diphenyl-1-picrylhydrazyl radical scavenging activity, reducing the level of tert-butylhydroperoxide–induced lipid peroxidation in brain homogenates, protecting PC12 cells from H2O2-induced cell death (through the inhibition of ERK phosphorylation), alleviating hypomyelination, and downregulating interleukin‐1β through LPS-activated microglia production; moreover, the numbers of Enterobacteriaceae, Actinobacteria, Peptostreptococcaceae, Erysipelotrichaceae, and Bifidobacterium bacteria in MIA offspring increased. In summary, the KI Essence extract promotes neurite outgrowth, alleviates oxidative stress and hypomyelination, and modulates microbiota dysbiosis in MIA offspring.

The Protective Role of Celastrol in Renal Ischemia-Reperfusion Injury by Activating Nrf2/HO-1, PI3K/AKT Signaling Pathways, Modulating NF-\u03bab Signaling Pathways, and Inhibiting ERK Phosphorylation

Celastrol, a natural triterpenoid derived from Tripterygium wilfordii, possesses numerous biological effects. We investigated celastrol’s antioxidant potential through nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) and its effect on phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling, nuclear factor-kappa B (NF-κB) pathways, and extracellular signal-regulated kinase (ERK) activation in kidney ischemia-reperfusion injury (IRI) rat model. Rats were given celastrol 2 mg/kg orally for 1 week before subjection to renal ischemia-reperfusion surgery. Kidney functions, renal MDA, and reduced glutathione were determined; also, renal levels of ERK1/2, HO-1, PI3K, IL-6, TNF-α, IκBα, NF-κB/p65, and cleaved caspase-3 were measured. In addition, gene expression of kidney injury molecule-1 (KIM-1), Nrf-2, and AKT were determined. Celastrol pretreatment attenuated oxidative stress and increased Nrf2 gene expression and HO-1 level. Also, it activated the PI3K/AKT signaling pathway and decreased the p-ERK:t- ERK ratio and NFκBp65 level, with a remarkable decrease in inflammatory cytokines and cleaved caspase-3 levels compared with those in renal IRI rats. Conclusively, celastrol showed a reno-protective potential against renal IRI by suppressing oxidative stress through enhancing the Nrf2/HO-1 pathway, augmenting cell survival PI3K/AKT signaling pathways, and reducing inflammation by inhibiting NF-κB activation.

Bone marrow mesenchymal stem cells overexpressing hepatocyte growth factor ameliorate hypoxic-ischemic brain damage in neonatal rats.

ObjectivesHypoxic–ischemic brain damage (HIBD) is a major cause of brain injury in neonates. Bone marrow mesenchymal stem cells (BMSCs) show therapeutic potential for HIBD, and genetic modification may enhance their neuroprotective effects. The goal of this study was to investigate the neuroprotective effects of hepatocyte growth factor (HGF)-overexpressing BMSCs (BMSCs-HGF) against HIBD and their underlying mechanisms.Methods:BMSCs were transfected with HGF using adenoviral vectors. HIBD models were established and then BMSCs were transplanted into the brains of HIBD rats via intraventricular injection. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volumes. In vitro, primary cultured cortical neurons were co-cultured with BMSCs in a Transwell plate system. Oxygen–glucose deprivation (OGD) was applied to imitate hypoxic–ischemic insult, and PD98059 was added to the culture medium to block the phosphorylation of extracellular signal-regulated kinase (ERK). Cell apoptosis was determined using TUNEL staining. The expression of HGF was measured by immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blots. The expression of phosphorylated ERK (p-ERK) and B-cell lymphoma-2 (Bcl-2) was measured by western blots.ResultsHGF-gene transfection promoted BMSC proliferation. Moreover, BMSCs-HGF decreased HIBD-induced cerebral infarction volumes and enhanced the protective effects of the BMSCs against HIBD. BMSCs-HGF also increased expression of HGF, p-ERK, and Bcl-2 in brain tissues. In vitro, BMSC-HGF protected neurons against OGD-induced apoptosis. Inhibition of ERK phosphorylation abolished the neuroprotective effect of BMSCs-HGF against OGD.ConclusionsBMSCs-HGF is a potential treatment for HIBD and that the ERK/Bcl-2 pathway is involved in the underlying neuroprotective mechanism.

Inhibition of extracellular signal-regulated kinase pathway suppresses tracheal stenosis in a novel mouse model.

Tracheal stenosis is a refractory and recurrent disease induced by excessive cell proliferation within the restricted tracheal space. We investigated the role of extracellular signal-regulated kinase (ERK), which mediates a broad range of intracellular signal transduction processes in tracheal stenosis and the therapeutic effect of the MEK inhibitor which is the upstream kinase of ERK. We histologically analyzed cauterized tracheas to evaluate stenosis using a tracheal stenosis mouse model. Using Western blot, we analyzed the phosphorylation rate of ERK1/2 after cauterization with or without MEK inhibitor. MEK inhibitor was intraperitoneally injected 30 min prior to cauterization (single treatment) or 30 min prior to and 24, 48, 72, and 96 hours after cauterization (daily treatment). We compared the stenosis of non-inhibitor treatment, single treatment, and daily treatment group. We successfully established a novel mouse model of tracheal stenosis. The cauterized trachea increased the rate of stenosis compared with the normal control trachea. The phosphorylation rate of ERK1 and ERK2 was significantly increased at 5 min after the cauterization compared with the normal controls. After 5 min, the rates decreased over time. The daily treatment group had suppressed stenosis compared with the non-inhibitor treatment group. p-ERK1/2 activation after cauterization could play an important role in the tracheal wound healing process. Consecutive inhibition of ERK phosphorylation is a potentially useful therapeutic strategy for tracheal stenosis.

Melochia corchorifolia extract inhibits melanogenesis in B16F10 mouse melanoma cells via activation of the ERK signaling.

Numerous researches have focused on discovering available inhibitors of melanogenesis from natural medicinal plants with stable efficacy and safety to resolve cutaneous hyperpigmentary problems. Melochia corchorifolia Linn. (MC) has been used as folk medicine to treat various diseases. However, the effect of MC on melanogenesis remains unknown. In this study, we investigated the effect of MC extract on melanogenesis and its underlying mechanisms in B16F10 mouse melanoma cells. B16F10 cells were treated with MC extract, and then, cell viability, melanin content, and tyrosinase activity were analyzed. The mRNA and protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF) were evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Phosphorylated or total protein levels in MC extract-induced signaling pathways were analyzed by Western blotting. Treatment of B16F10 cells with MC extract inhibited melanin synthesis and intracellular tyrosinase activity in a dose-dependent manner with no cytotoxicity. Protein and mRNA expressions of tyrosinase and MITF were also significantly decreased by MC extract treatment. In addition, phosphorylated level of extracellular signal-regulated kinase (ERK) was obviously increased by MC extract, but AKT pathway was not activated. Inhibited ERK phosphorylation by pretreatment with a selective ERK inhibitor PD98059 significantly reversed the decreased melanin content induced by treatment with MC extract in B16F10 cells. MC extract inhibits melanogenesis in B16F10 mouse melanoma cells through suppression of MITF-tyrosinase signaling pathway by ERK activation.

Angiotensin II receptor blocker LCZ696 attenuates cardiac remodeling through the inhibition of the ERK signaling pathway in mice with pregnancy-associated cardiomyopathy

Pregnancy-associated cardiomyopathy (PAH) represents a pregnancy-associated myocardial disease that is characterized by the progression of heart failure due to marked left ventricular systolic dysfunction. Compelling evidence has highlighted the potential of angiotensin (Ang) receptor inhibitors as therapeutic targets in PAH treatment. The present study aims to elucidate the molecular mechanisms underlying Ang II receptor inhibitor LCZ696 treatment in PAH. Initially, a PAH mouse model was induced, followed by intraperitoneal injection of LCZ696. Subsequently, cardiomyocytes and fibroblasts were isolated, cultured, and treated with Ang II and LCZ696, followed by detection of the total survival rate, cardiac injury, cardiac fibrosis and apoptosis. Moreover, in order to quantify the cardiac hypertrophy and fibrosis degree of cardiac fibroblasts, the expression levels of markers of cardiac hypertrophy (ANP, βMHC and TIMP2) and markers of fibrosis (collagen I, collagen III and TGF-β) were evaluated. Furthermore, the potential effect of LCZ696 on the extracellular signal-regulated kinase (ERK) signaling pathway was examined. The acquired findings revealed that LCZ696 increased the total survival rate of PAH mice, but decreased cardiac injury, cardiac fibrosis, and apoptosis in vitro. LCZ696 attenuated cardiac injury induced by Ang II through the inhibition the expression of markers of cardiac hypertrophy, fibrosis and apoptosis by inhibiting ERK phosphorylation in vivo and in vitro. Altogether, LCZ676 could potentially alleviate cardiac remodeling in mice with PAH via blockade of the ERK signaling pathway activation. Our findings suggest that LCZ696 could be a potential target for PAH therapy.

The testosterone metabolite 3α-androstanediol inhibits oxidative stress-induced ERK phosphorylation and neurotoxicity in SH-SY5Y cells through an MKP3/DUSP6-dependent mechanism

Testosterone exerts neuroprotective effects on the brain, but the mechanisms by which these effects are exerted appear to be different in males and females. While in females they involve local conversion to estradiol, in males they may be androgen receptor-dependent, or mediated through metabolism to neurosteroids such as 5α-androstane-3α,17β-diol (3α-diol), which acts through different mechanisms than testosterone itself. Recently, we demonstrated that 3α-diol can protect neurons and neuronal-like cells against oxidative stress-induced neurotoxicity associated with prolonged phosphorylation of the extracellular signal-regulated kinase (ERK). The mechanism(s) responsible for these effects remain unknown. In the present study, we sought to determine whether the ERK-specific phosphatase, mitogen-activated protein kinase phosphatase 3/dual specificity phosphatase 6 (MKP3/DUSP6), is involved in the cytoprotective effects of 3α-diol in SH-SY5Y human female neuroblastoma cells. 3α-diol inhibited ERK phosphorylation and ameliorated cell death induced by the oxidative stressor hydrogen peroxide (H2O2). These protective effects were significantly reduced by pre-treatment with the MKP3/DUSP6 inhibitor BCI. In addition, H2O2 decreased expression of MKP3/DUSP6, and this was prevented by co-treatment with 3α-diol. These findings suggest that the protective effects of 3α-diol are mediated through regulation of ERK phosphorylation in neurotoxic conditions and indicate that these effects may be exerted through modulation of MKP3/DUSP6. Targeting the regulation of MKP3/DUSP6 may be beneficial in reducing toxicity under conditions of oxidative stress.

Phosphorylation of extracellular signal-regulated kinase as a biomarker for cannabinoid receptor 2 activation

Cannabinoid receptor 2 (CB2R) is a therapeutic target in inflammatory diseases; its activation by agonists provides important clinical information, but there are currently no methods to quantify CB2R activation in humans. Chinese hamster ovary (CHO)-K1 cells and mouse and human whole blood cells were used for experiments. CB2R was activated in cells by treatment with the agonist CP55,940. Cells were also pretreated with proprietary Compound A and B (experimental agonists). We developed our method based on the finding that CB2R ligand binding and activation stimulates acute-phase extracellular signal-regulated kinase (ERK) phosphorylation in human and rodent immune cells, after which CB2R becomes unresponsive to stimulation by a second CB2R agonist CP55940 for a certain time period. We detected ERK phosphorylation as a measure of target engagement in mouse and human whole blood cells by flow cytometry. In cells overexpressing human or mouse CB2R, pretreatment with Compound A dose-dependently inhibited ERK phosphorylation for 2 h, prolonging the time window for measuring ERK phosphorylation. Our method enables measurement of CB2R activation by its agonists in human blood cells based on detection of ERK phosphorylation, which is useful for therapeutic drug monitoring and other clinical applications.

Extracellular signal-regulated kinase 2 has duality in function between neuronal and astrocyte expression following neonatal hypoxic-ischaemic cerebral injury.

Key points This study identifies phosphorylated extracellular signal‐regulated kinase (ERK) to be immediately diminished followed by a rapid if transient increase for up to 4 h following hypoxic–ischaemic insult (HI) in the neonatal mouse.Phosphorylated ERK up‐regulation was prevented with systemic injection of the mitogen‐activated protein kinase kinase (MEK) inhibitor SL327. Treatment with SL327 both pre‐ and post‐HI gave a strong reduction in the number of dying cells and microgliosis.By utilising transgenic mouse mutations, we observe that neuronal ERK2 significantly contributes to tissue damage, while ERK1 and astrocytic ERK2 are neuroprotective.Compared to global inactivation, selective cell‐specific interference with ERK activity could result in stronger neuroprotection. Hypoxia–ischaemia (HI) is a major cause of neonatal brain injury resulting in cerebral palsy, epilepsy, cognitive impairment and other neurological disabilities. The role of extracellular signal‐regulated kinase (ERK) isoforms and their mitogen‐activated protein kinase kinase (MEK)‐dependent phosphorylation in HI has previously been explored but remains unresolved at cellular level. This is pertinent given the growing awareness of the role of non‐neuronal cells in neuroprotection. Using a modified Rice–Vannucci model of HI in the neonatal mouse we observed time‐ and cell‐dependent ERK phosphorylation (pERK), with strongly up‐regulated pERK immunoreactivity first in periventricular white matter axons within 15–45 min of HI, followed by forebrain astrocytes and neurons (1–4 h post‐HI), and return to baseline by 16 h. We explored the effects of pharmacological ERK blockade through the MEK inhibitor SL327 on neonatal HI‐brain damage following HI alone (30 or 60 min) or lipopolysaccharide (LPS)‐sensitised HI insult (30 min). Global inhibition of ERK phosphorylation with systemically applied SL327 abolished forebrain pERK immunoreactivity, and significantly reduced cell death and associated microglial activation at 48 h post‐HI. We then explored the effects of cell‐specific ERK2 deletion alone or in combination with global ERK1 knockout under the same conditions of HI insult. Neuronal ERK2 deletion strongly decreased infarct size, neuronal cell death and microglial activation in grey matter following both HI alone or LPS‐sensitised HI. ERK1 deletion attenuated the protective effect of neuronal ERK2 deletion. Removal of astroglial ERK2 produced a reverse response, with a 3‐ to 4‐fold increase in microglial activation and cell death. Our data suggest a cell‐specific and time‐dependent role of ERK in neonatal HI, with a predominant, neurotoxic effect of neuronal ERK2, which is counteracted by neuroprotection by ERK1 and astrocytic ERK2. Overall, global pharmacological inhibition of ERK phosphorylation is strongly neuroprotective.

Systemic 7,8-Dihydroxyflavone Treatment Protects Immature Retinas Against Hypoxic-Ischemic Injury via Müller Glia Regeneration and MAPK/ERK Activation.

Perinatal hypoxic-ischemic (HI) injury causes significant damages in the immature retina. The brain-derived neurotrophic factor is well known for its neuroprotective role but has limited clinical applications. A selective agonist of tyrosine kinase receptor B, 7,8-dihydroxyflavone (DHF), is a powerful therapeutic tool, when administered systemically. However, it remains unclear whether DHF treatment can protect the immature retinas against HI injury. Postnatal (P) day 7 rat pups were intraperitoneally injected with DHF or vehicle 2 hours before and 18 hours after being subjected to HI injury. The outcomes were assessed at various timepoints after injury by electroretinography and histologic examinations. Neurogenesis was assessed by double-labeling of retinal sections with 5-bromo-2'-deoxyuridine and different neuronal markers. At P8, 24-hours postinjury, brain-derived neurotrophic factor mRNA levels in the retina decreased significantly. DHF treatment partially protected immature retinas at both histologic and functional levels between P14 and P30 but did not prevent apoptosis, inflammation, or damage of the blood-retinal barrier (BRB) at P8. On the other hand, DHF treatment promoted the survival of proliferating inner retinal cells, including Müller glia, and enhanced their transdifferentiation to bipolar cells at P17. Moreover, DHF treatment rescued the levels of extracellular signal-regulated kinase (ERK) phosphorylation, which were significantly decreased after injury. The neuroprotective effects of DHF were markedly eliminated by inhibition of ERK phosphorylation. Early systemic DHF treatment has neuroprotective effects against HI injury in immature retinas, possibly via promoting neurogenesis through the tyrosine kinase receptor B/ERK signaling pathway.

Neurosteroid metabolites of testosterone and progesterone differentially inhibit ERK phosphorylation induced by amyloid β in SH-SY5Y cells and primary cortical neurons

Gonadal steroid hormones exert neurotrophic and neuroprotective effects on the brain. Recent work suggests potential neuroprotective roles for the 3α-hydroxy, 5α-reduced metabolites of these hormones. Two such metabolites are 5α-androstane-3α,17β-diol (3α-diol) and 5α-pregnan-3α-ol-20-one (allopregnanolone; Allo), which may contribute to the overall protection conferred by their precursors (testosterone and progesterone, respectively) through mechanisms including potentiation of gamma-aminobutyric acid (GABA)A receptor (GABAAR) activity. We have previously demonstrated that physiological concentrations of 3α-diol inhibit prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and the associated neurotoxicity resulting from amyloid β peptide 1–42 (Aβ42) exposure in vitro. In the present study, we sought to characterize the GABAAR-dependency of 3α-diol’s effects, compared to those of Allo, in SH-SY5Y human female neuroblastoma cells and primary cortical neurons isolated from postnatal day 0–1 mice. Both 3α-diol and Allo prevented Aβ42-mediated ERK phosphorylation in SH-SY5Y cells, with substantially different concentration requirements (10 nM for 3α-diol, 100 nM for Allo). Pharmacological inhibition of GABAAR with picrotoxin did not prevent this effect, indicating that neurosteroid-mediated ERK inhibition in SH-SY5Y cells may be GABAAR-independent. While 10 nM and 100 nM concentrations of both neurosteroids inhibited ERK phosphorylation induced by Aβ42 in primary cortical neurons, which have high expression levels of GABAARs, only the effects of Allo were significantly inhibited by picrotoxin. These results suggest that neurosteroid metabolites of testosterone and progesterone may contribute to neuroprotection by suppressing ERK phosphorylation through both GABAAR-dependent and -independent mechanisms.

Involvement of Notch2 in all‑trans retinoic acid‑induced inhibition of mouse embryonic palate mesenchymal cell proliferation.

Cleft palate is among the most common congenital disorders, and can be induced by exposure to all-trans retinoic acid (atRA) during mice and human embryogenesis. However, the mechanism underlying the implication of atRA in the development of cleft palate has yet to be elucidated. In the present study, atRA administered by gavage resulted in formation of a cleft palate in 99% of treated C57BL/6 mice. Notch2 was revealed to be upregulated in mouse embryonic palate mesenchymal (MEPM) cells in the atRA-treated group compared with untreated control mice between embryonic day (E)12.5 and E14.5. In addition, atRA was demonstrated to mediate Notch2 expression via the activation of RA receptors (RARs). Since Notch2 activation has previously been reported to inhibit the proliferation of MEPM cells, the expression levels of extracellular signal-regulated kinase (ERK), p21, cyclin D1 and Ki-67 were assessed in samples from atRA-treated and control mouse embryos between E12.5 and E14.5. It was demonstrated that Notch2 silencing partially reversed the atRA-induced inhibition of ERK phosphorylation in MEPM cells. In addition, the atRA-induced cyclin D1 downregulation and p21 upregulation were partially reversed following Notch2 silencing, whereas the atRA-induced inhibition of cellular proliferation was also attenuated. Furthermore, it was revealed that Notch2 expression was upregulated, whereas Ki-67 expression was downregulated following atRA exposure, as assessed using resin bead-released atRA in MEPM cells. The present findings suggested that during embryonic development, atRA may enhance the expression of Notch2, which may inhibit cellular proliferation, possibly through ERK signaling.

Altered expression of TRAIL on mouse T cells via ERK phosphorylation by Rhodiola rosea L. and its marker compounds

Rhodiola rosea L. extracts have shown neuroprotective, anti-fatigue, anti-inflammatory and anti-tumor properties. However, the studies on their effect on T cell function are rather scarce. We examined the potential of R. rosea extract and its major constituents – salidroside, rosarin, rosavin and rosin to alter cell growth of human Jurkat T cells, apoptosis of splenic mouse CD3 T cells and expression of the surface markers and phosphorylation of extracellular signal-regulated kinase (ERK). The initial screening for cell viability in Jurkat T cells and for apoptosis of mouse T cells showed the strongest activity for rosavin and rosarin. Rosarin and rosavin did not alter significantly the dynamic of CD69 expression upon stimulation, but altered TNF-related apoptosis-inducing ligand (TRAIL) expression. Rosavin inhibited TRAIL up-regulation, while rosarin showed an opposite effect. Indeed, rosarin increased the frequencies of CD3+TRAIL+ T cells and the fold inhibition of ERK phosphorylation. Our data showed that different effects of rosarin and rosavin on TRAIL expression can involve distinct action on ERK signaling and hence highlighted their potential to manipulate TRAIL as a tool to rescue the resistance to apoptosis in autoimmune diseases and cancer.

Trametinib plus 4-Methylumbelliferone Exhibits Antitumor Effects by ERK Blockade and CD44 Downregulation and Affects PD-1 and PD-L1 in Malignant Pleural Mesothelioma

IntroductionMalignant pleural mesothelioma (MPM) is a highly aggressive malignancy in which the mitogen-activated protein kinase pathway plays a critical role in the regulation of tumorigenesis. Hyaluronic acid (HA) is a major component of the extracellular matrix, and elevated HA levels with a concurrent increase in malignant properties are associated with MPM. MethodsWe evaluated the effects of trametinib, a mitogen-activated protein kinase (MEK) inhibitor, and 4-methylumbelliferone (4-MU), an HA synthesis inhibitor, alone and in combination on MPM cells in vitro and in vivo. We studied the effects of trametinib, 4-MU, and their combination on MPM cells by using cell viability assays, Western blot analysis, and a mouse xenograft model. ResultsTrametinib and 4-MU exhibited antiproliferative activity in MPM cells. Trametinib blocked MEK-dependent extracellular signal-regulated kinase (ERK) phosphorylation and decreased CD44 expression in a concentration-dependent manner. Trametinib inhibited the expression of Fra-1 (the activator protein 1 [AP1] component), inhibited ERK phosphorylation, and decreased CD44 expression. 4-MU inhibited ERK phosphorylation but not CD44 expression. In a mouse xenograft model, trametinib and 4-MU alone suppressed tumor growth compared with a control. The combination had a greater inhibitory effect than either monotherapy. Immunohistochemical analysis showed that trametinib treatment alone significantly reduced expression of programmed cell death 1 ligand 1. Furthermore, the combination of trametinib and 4-MU resulted in higher expression of programmed cell death 1 and programmed cell death 1 ligand 1 than did the 4-MU treatment alone. ConclusionsOur results suggest that trametinib and 4-MU are promising therapeutic agents in MPM and that further study of the combination is warranted.

8‐C‐(E‐phenylethenyl)quercetin from onion/beef soup induces autophagic cell death in colon cancer cells through ERK activation

Quercetin, a flavonoid, widely distributed in edible fruits and vegetables, was reported to effectively inhibit 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) formation in a food model (roast beef patties) with itself being converted into a novel compound 8-C-(E-phenylethenyl)quercetin (8-CEPQ). Here we investigated whether 8-CEPQ could be formed in a real food system, and tested its anticancer activity in human colon cancer cell lines. LC-MS was applied for the determination of 8-CEPQ formation in onion/beef soup. Anticancer activity of 8-CEPQ was evaluated by using cell viability assay and flow cytometry. Results showed that 8-CEPQ suppressed proliferationand caused G2 phase arrest in colon cancer cells. Based on immunofluorescent staining assay, western blot assay, and RNA knockdown data, we found that 8-CEPQ did not cause apoptotic cell death. Instead, it induced autophagic cell death. Moreover, treatment with 8-CEPQ induced phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of ERK phosphorylation by the mitogen-activated protein kinasekinase (MEK)/ERK inhibitor U0126 attenuated 8-CEPQ-induced autophagy and reversed 8-CEPQ-mediated cell growth inhibition. Our results demonstrate that 8-CEPQ, a novel quercetin derivative, could be formed in onion/beef soup. 8-CEPQ inhibited colon cancer cell growth by inducing autophagic cell death through ERK activation.

Dysregulated extracellular signal-regulated kinase signaling associated with impaired B-cell receptor endocytosis in patients with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by B-cell dysfunction and, in a subgroup, by expansion of CD21(low) B cells. The CD21(low) Bcells display defects in early B-cell receptor (BCR) signaling resembling those of anergic B cells. We sought to investigate whether B cells from patients with CVID, like anergic B cells, have defects in extracellular signal-regulated kinase (ERK) phosphorylation and in endocytic trafficking of the BCR. Using flow cytometry, we evaluated phosphorylated ERK (pERK) expression and internalization of cross-linked BCR in B-cell subsets. The localization of internalized BCR to lysosome-associated membrane protein 1-positive late endosomes was evaluated with confocal microscopy. Constitutive pERK levels were increased in naive and IgM(+) memory B cells of patients with CVID compared with those of healthy donors, whereas the pERK increment induced by BCR cross-linking was relatively reduced. Intravenous immunoglobulin administration enhanced these anomalies, but they appeared to be intrinsic to B cells from patients with CVID. Cross-linking-induced BCR endocytosis was decreased in the IgM(+) memory B cells, especially in those with a CD21(low) phenotype, but not in the naive B cells of patients with CVID with CD21(low) expansion. Internalized BCR localized normally to late endosomes. Pharmacologic inhibition of ERK phosphorylation suppressed BCR endocytosis in B cells of healthy patients and those with CVID. The B cells of patients with CVID with CD21(low) B-cell expansion resemble anergic B cells based on high constitutive pERK expression. The IgM(+) memory B cells of these patients, especially those that are CD21(low), have a defectinBCR endocytosis seemingly caused by dysregulated ERK signaling.

Preventive Effects of a Kampo Medicine, Kakkonto, on Inflammatory Responses via the Suppression of Extracellular Signal-Regulated Kinase Phosphorylation in Lipopolysaccharide-Treated Human Gingival Fibroblasts

Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. The chemical mediator prostaglandin E2 (PGE2) and cytokines such as interleukin- (IL-)6 and IL-8 have been known to play important roles in inflammatory responses and tissue degradation. In the present study, we investigated the effects of a kampo medicine, kakkonto (TJ-1), on the production of prostaglandin E2 (PGE2), IL-6, and IL-8 by human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Kakkonto concentration dependently suppressed LPS-induced PGE2 production but did not alter basal PGE2 levels. In contrast, kakkonto significantly increased LPS-induced IL-6 and IL-8 production. Kakkonto decreased cyclooxygenase- (COX-)1 activity to approximately 70% at 1 mg/mL but did not affect COX-2 activity. Kakkonto did not affect cytoplasmic phospholipase A2 (cPLA2), annexin1, or LPS-induced COX-2 expression. Kakkonto suppressed LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation, which is known to lead to ERK activation and cPLA2 phosphorylation. These results suggest that kakkonto decreased PGE2 production by inhibition of ERK phosphorylation which leads to inhibition of cPLA2 phosphorylation and its activation. Therefore, kakkonto may be useful to improve gingival inflammation in periodontal disease.

Allosteric MEK1/2 Inhibitor Refametinib (BAY 86-9766) in Combination with Sorafenib Exhibits Antitumor Activity in Preclinical Murine and Rat Models of Hepatocellular Carcinoma

The objectives of the study were to evaluate the allosteric mitogen-activated protein kinase kinase (MEK) inhibitor BAY 86-9766 in monotherapy and in combination with sorafenib in orthotopic and subcutaneous hepatocellular carcinoma (HCC) models with different underlying etiologies in two species. Antiproliferative potential of BAY 86-9766 and synergistic effects with sorafenib were studied in several HCC cell lines. Relevant pathway signaling was studied in MH3924a cells. For in vivo testing, the HCC cells were implanted subcutaneously or orthotopically. Survival and mode of action (MoA) were analyzed. BAY 86-9766 exhibited potent antiproliferative activity in HCC cell lines with half-maximal inhibitory concentration values ranging from 33 to 762 nM. BAY 86-9766 was strongly synergistic with sorafenib in suppressing tumor cell proliferation and inhibiting phosphorylation of the extracellular signal-regulated kinase (ERK). BAY 86-9766 prolonged survival in Hep3B xenografts, murine Hepa129 allografts, and MH3924A rat allografts. Additionally, tumor growth, ascites formation, and serum alpha-fetoprotein levels were reduced. Synergistic effects in combination with sorafenib were shown in Huh-7, Hep3B xenografts, and MH3924A allografts. On the signaling pathway level, the combination of BAY 86-9766 and sorafenib led to inhibition of the upregulatory feedback loop toward MEK phosphorylation observed after BAY 86-9766 monotreatment. With regard to the underlying MoA, inhibition of ERK phosphorylation, tumor cell proliferation, and microvessel density was observed in vivo. BAY 86-9766 shows potent single-agent antitumor activity and acts synergistically in combination with sorafenib in preclinical HCC models. These results support the ongoing clinical development of BAY 86-9766 and sorafenib in advanced HCC.