Extracellular Signal-related Kinase Research Articles

Role of MAPKs in TGF-β1-induced maturation and mineralization in human osteoblast-like cells

Objectives: Our study aimed to clarify the role of mitogen-activated protein kinases (MAPKs) in transforming growth factor (TGF)-β1-stimulated mineralization in the human osteoblast-like MG63 cells. MethodsThe viability of MG63 cells under TGF-β1 stimulation was assessed by MTS assay. Western blotting determined TGF-β1-mediated activation of extracellular signal-related protein kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK). Mineralization-related gene expression was examined by quantitative real-time PCR, and mineral deposition levels were evaluated by alizarin red S staining. ResultsTGF-β1 had no effect on MG63 cell proliferation. Activation of p38 was observed at 3 h post TGF-β1 stimulation. Moreover, JNK phosphorylation was upregulated by TGF-β1 from 1 to 6 h post stimulation, but had no activation on ERK phosphorylation throughout the experimental period. Treatment with JNK inhibitor diminished the alizarin red S-stained area in a dose-dependent manner. Mineral deposition was unaffected by MEK inhibitor, whereas p38 inhibitor increased the red-stained area. Gene expression levels of ALP and BSP were significantly decreased under treatment with JNK inhibitor and p38 inhibitor. The MEK inhibitor had no effect on the TGF-β1–mediated upregulation of ALP and BSP. Although all three inhibitors suppressed expression of COL I, none were found to stimulate expression of OCN. ConclusionsHuman osteoblast-like MG63 cells maturation and mineralization are induced through JNK activation of MAPK signaling in response to TGF-β1.

Progestins and breast cancer hallmarks: The role of the ERK1/2 and JNK pathways in estrogen receptor positive breast cancer cells

Progestins used in hormonal contraceptives and menopausal hormone therapy (MHT) have been linked to increased breast cancer risk. Whether the association holds for all progestins is unclear and the underlying mechanisms remain poorly understood. We directly compared the effects of four progestins (medroxyprogesterone acetate (MPA), norethisterone acetate (NET-A), levonorgestrel (LNG) and drospirenone (DRSP)) to each other and the natural progestogen progesterone (P4) on selected cancer hallmarks. To provide mechanistic insight into these effects, we assessed the role of the progesterone receptor (PR), and the extracellular signal-related kinase (ERK1/2) and c-Jun N terminal (JNK) signaling pathways. We showed that the increased proliferation of the luminal T47D breast cancer cell line by P4 and all progestins, albeit to different extents, was inhibited by PR knockdown and inhibition of both the ERK1/2 and JNK pathways. While knockdown of the PR also blocked the upregulation of MKI67 and CCND1 mRNA expression by selected progestogens, only a role for the ERK1/2 pathway could be established in these effects. Similarly, only a role for the ERK1/2 pathway could be confirmed for progestogen-induced colony formation, whereas both the ERK1/2 and JNK pathways were required for cell migration in response to the three older progestins implicated in the etiology of breast cancer, MPA, NET-A and LNG. Together our results show that all the progestins elicit their effects on cell proliferation via a mechanism requiring the PR, ERK1/2 and JNK pathways. While the ERK1/2 and JNK pathways are also required for increased cell migration by the older progestins, only a role for the ERK1/2 pathway could be established in their effects on colony formation. Notably, the cytoplasmic PR was not needed for activation of the ERK1/2 pathway by the progestogens. Given that DRSP showed significantly lower proliferation than MPA and NET-A, and that it had no effect on breast cancer cell migration and colony formation, hormonal formulations containing the newer generation progestin DRSP may provide a better benefit/risk profile towards breast cancer than those containing the older generation progestins.

Phosphorylated p38 MAP kinase expression by leucocytes is increased in allergic humans and associated with IgE responses.

Mitogen-activated protein kinases (MAPK) activate cascades that regulate cell proliferation, differentiation and death. Phosphorylated (phos-)p38 MAPK is a cell-signalling pathway associated with Th2 cytokine responses, which is required for immunoglobulin (Ig)E production. It is unknown whether MAPK are associated with IgE production. We examine the evidence linking p38 MAPK to inflammatory responses. Phos-p38, extracellular signal-related kinase (ERK) and c-JUN-n terminal (JNK) MAPK expression by blood leucocyte subsets and levels of serum Igs were measured in blood from adults with asthma and/or rhinoconjunctivitis (N = 28) and non-asthma (N = 10) (flow cytometry, microfluorenzymeimmunoassay). Peripheral blood mononuclear cells (PBMC) from allergic subjects were cultured for 10 days ± anti-CD40/recombinant IL-4 ± inhibitor of phos-P38. Culture supernatants were assayed for IgE (ELISA). Phos-p38 MAPK expression by all leucocyte subsets of allergic subjects was associated with serum IgE levels (p ≤ 0.01), after adjusting for cell counts, age, sex, race and smoking status (p ≤ 0.04). Leucocyte expression of phos-ERK and JNK did not correlate with IgE (p = 0.09-0.99). Instead, phos-ERK expression was associated with serum IgG. When PBMC from atopic subjects were cultured for 10 days with anti-CD40/rhIL-4, IgE levels were 26.2 ± 18 ng/mL. Inclusion of SB202190 (5-20 μg/mL), a specific inhibitor of phos-p38 MAPK, in culture suppressed IgE production in dose-dependent manner, with peak suppression obtained with SB202190 at 20 μg/mL (82.1% ± 11.8) (p = 0.0001), with virtually no cytotoxicity (<5%). Different MAPK pathways may be associated with IgE (p38) and IgG (ERK) responses. Phos-p38 MAPK can be a potential anti-allergy drug target.

Testosterone reduces hippocampal synaptic damage in an androgen receptor-independent manner.

Aging-related reduction in androgen levels may be a possible risk factor for neurodegenerative diseases and contribute to cognitive impairment. Androgens may affect synaptic function and cognition in an androgen receptor (AR)-independent manner; however, the mechanisms connecting theses effects are unknown. Therefore, we used testicular feminization mutation (Tfm) male mice, a model with AR mutation, to test the effects of testosterone on synaptic function and cognition. Our results showed that testosterone ameliorated spatial memory deficit and neuronal damage, and increased dendritic spines density and postsynaptic density protein 95 (PSD95) and glutamate receptor 1 (GluA1) expression in the hippocampus of Tfm male mice. And these effects of testosterone were not inhibited by anastrozole, which suppressed conversion of testosterone to estradiol. Mechanistically, testosterone activated the extracellular signal-related kinase 1/2 (Erk1/2) and cyclic adenosine monophosphate response element-binding protein (CREB) in the hippocampus of Tfm male mice. Meanwhile, Erk1/2 inhibitor SCH772984 blocked the upregulation of phospho-CREB, PSD95, and GluA1 induced by testosterone in HT22 cells pretreated with flutamide, an androgen antagonist. Collectively, our data indicate that testosterone may ameliorate hippocampal synaptic damage and spatial memory deficit by activating the Erk1/2-CREB signaling pathway in an AR-independent manner.

ERK signaling waves via body-wall muscles guide planarian whole-body regeneration across long distances

Whole-body regeneration is a multifaceted process that reinstates a body to its initial three-dimension size and structure after resection injury. It is well-known that signaling waves such as calcium and extracellular signal-related kinase (ERK) signaling waves can efficiently transmit information between tissues or cells. However, the mechanisms responsible for coordinating wound responses over long distances are largely unexplored. A recent study has reported that the propagation of ERK signaling waves via longitudinal body-wall muscles play an essential role in wound response and whole-body regeneration in planarians, underscoring the significance of feedback interactions between spatially distinct tissues during whole-body regeneration over long distances. These findings not only address the central questions of regenerative biology but also have potential implications for regenerative medicine.

Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

GLP-1 and GIP receptors signal through distinct β-arrestin 2-dependent pathways to regulate pancreatic β cell function

Glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIPR) receptors are G-protein-coupled receptors involved in glucose homeostasis. Diabetogenic conditions decrease β-arrestin 2 (ARRB2) levels in human islets. In mouse β cells, ARRB2 dampens insulin secretion by partially uncoupling cyclic AMP (cAMP)/protein kinase A (PKA) signaling at physiological doses of GLP-1, whereas at pharmacological doses, the activation of extracellular signal-related kinase (ERK)/cAMP-responsive element-binding protein (CREB) requires ARRB2. In contrast, GIP-potentiated insulin secretion needs ARRB2 in mouse and human islets. The GIPR-ARRB2 axis is not involved in cAMP/PKA or ERK signaling but does mediate GIP-induced F-actin depolymerization. Finally, the dual GLP-1/GIP agonist tirzepatide does not require ARRB2 for the potentiation of insulin secretion. Thus, ARRB2 plays distinct roles in regulating GLP-1R and GIPR signaling, and we highlight (1) its role in the physiological context and the possible functional consequences of its decreased expression in pathological situations such as diabetes and (2) the importance of assessing the signaling pathways engaged by the agonists (biased/dual) for therapeutic purposes.

Anti-Melanogenic Effects of Lilium lancifolium Root Extract via Downregulation of PKA/CREB and MAPK/CREB Signaling Pathways in B16F10 Cells

Hyperpigmentation disorders causing emotional distress require the topical use of depigmenting agents of natural origin. In this study, the anti-melanogenic effects of the Lilium lancifolium root extract (LRE) were investigated in B16F10 cells. Consequently, a non-cytotoxic concentration of the extract reduced intracellular melanin content and tyrosinase activity in a dose-dependent manner, correlating with the diminished expression of core melanogenic enzymes within cells. LRE treatment also inhibited cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)/microphthalmia-associated transcription factor signaling, which regulates the expression of tyrosinase-related genes. Upon examining these findings from a molecular mechanism perspective, LRE treatment suppressed the phosphorylation of protein kinase A (PKA), p38, and extracellular signal-related kinase (ERK), which are upstream regulators of CREB. In addition, L-phenylalanine and regaloside A, specifically identified within the LRE using liquid chromatography-mass spectrometry, exhibited inhibitory effects on melanin production. Collectively, these results imply that LRE potentially suppresses cAMP-mediated melanogenesis by downregulating PKA/CREB and mitogen-activated protein kinase (MAPK)/CREB signaling pathways. Therefore, it can be employed as a novel therapeutic ingredient of natural origin to ameliorate hyperpigmentation disorders.

Patulin Stimulates Progenitor Leydig Cell Proliferation but Delays Its Differentiation in Male Rats during Prepuberty.

Patulin is a mycotoxin with potential reproductive toxicity. We explored the impact of patulin on Leydig cell (LC) development in male rats. Male Sprague Dawley rats (21 days postpartum) were gavaged patulin at doses of 0.5, 1, and 2 mg/kg/day for 7 days. Patulin markedly lowered serum testosterone at ≥0.5 mg/kg and progesterone at 1 and 2 mg/kg, while increasing LH levels at 2 mg/kg. Patulin increased the CYP11A1+ (cholesterol side-chain cleavage, a progenitor LC biomarker) cell number and their proliferation at 1 and 2 mg/kg. Additionally, patulin downregulated Lhcgr (luteinizing hormone receptor), Scarb1 (high-density lipoprotein receptor), and Cyp17a1 (17α-hydroxylase/17,20-lyase) at 1 and 2 mg/kg. It increased the activation of pAKT1 (protein kinase B), pERK1/2 (extracellular signal-related kinases 1 and 2), pCREB (cyclic AMP response binding protein), and CCND1 (cyclin D1), associated with cell cycle regulation, in vivo. Patulin increased EdU incorporation into R2C LC and stimulated cell cycle progression in vitro. Furthermore, patulin showed a direct inhibitory effect on 11β-HSD2 (11β-hydroxysteroid dehydrogenase 2) activity, which eliminates the adverse effects of glucocorticoids. This study provides insights into the potential mechanisms via which patulin affects progenitor LC development in young male rats.

Direct early growth response-1 knockdown decreases melanoma viability independent of mitogen-activated extracellular signal-related kinase inhibition.

To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 ( EGR1 ) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P < 0.01; MEL624 27%, P < 0.001) and conjunctival (YUARGE 13-3064 33%, P < 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P < 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P = 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P = 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.

Abstract P2023: Mitogen-activated Protein Kinase-directed Modulation Of The Non-sarcomeric Actin Cytoskeleton In Cardiomyocytes

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the widening and lengthening of cardiomyocytes, respectively. Mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinases 1/2 (ERK1/2) are known to control this directional growth of cardiomyocytes, such that activated MEK1 causes concentric hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 MEK1 inhibitor, or control GFP adenovirus. The resulting data pinpointed over 2700 sites on 324 proteins as changing their phosphorylation status with modulation of this signaling cascade. Gene ontology analysis found that cytoskeletal-associated proteins were the largest subset of proteins with phosphorylation changes. Traditional and PhosTag western blots verified that many of these cytoskeletal proteins change expression as well as their phosphorylation status in NRVMs. In vivo experiments showed that cytoplasmic actin expression was upregulated in MEK1 transgenic or ERK1/2 gene-deleted hearts. Furthermore, MEK1 transgenic adult cardiomyocytes had enhanced cytoplasmic actin at lateral membranes. Palladin, an actin nucleating protein, changed both phosphorylation status and isoform expression in response to modulation of MEK1-ERK1/2 signaling. Adeno-associated viruses were used to overexpress different palladin isoforms. Immunostaining for these proteins showed that localization of palladin isoforms moved from the subcortical compartment to the sarcomere, suggesting these isoforms preferentially associate with either cytoskeletal or sarcomeric actin. Future studies will further examine the signaling roles that cytoplasmic actin may play in healthy and diseased cardiomyocytes and delineate preferential binding of actin associated proteins between cytoplasmic and sarcomeric actin in cardiomyocytes.

Downregulation of FHL2 suppressed trophoblast migration, invasion and epithelial–mesenchymal transition in recurrent miscarriage

Research questionIs four and a half LIM domain 2 (FHL2) involved in trophoblast migration, invasion and epithelial–mesenchymal transition (EMT) in recurrent miscarriage? DesignVillus tissue was collected from 24 patients who had experienced recurrent miscarriage and 24 healthy controls. FHL2 mRNA and protein expression in villus specimens were observed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Small interfering RNA and overexpression plasmid were used to change the FHL2 expression. JAR and HTR8/SVneo cell lines were used to conduct scratch-wound assay and transwell assay to detect trophoblast migration and invasion of FHL2. Downstream molecule expression of mRNA and protein and EMT markers were verified by qRT-PCR and Western blot. ResultsSignificantly lower FHL2 mRNA (P = 0.019) and protein (P = 0.0014) expression was found in trophoblasts from the recurrent miscarriage group compared with healthy controls. FHL2 knockdown repressed migration (P = 0.0046), invasion (P < 0.001) and EMT, as shown by significant differences in mRNA and protein expression of the EMT markers N-cadherin, E-cadherin, Vimentin and Snail (all P < 0.05) of extravillus trophoblasts. FHL2 overexpression enhanced migration (P = 0.025), invasion (P < 0.001) and EMT of extravillus trophoblasts (all EMT markers P < 0.05). The positive upstream factor FHL2 in the extracellular signal-related kinase pathway induced JunD expression, thereby promoting trophoblast migration and invasion via matrix metalloproteinase 2. ConclusionsFHL2 is involved in a regulatory pathway of trophoblast migration, invasion and EMT during early pregnancy, and may have a role in recurrent miscarriage pathogenesis, which can serve as a possible target for novel therapeutic development.

Tolerized Microglia Protect Neurons Against Endotoxin-Induced TNF-α Production via an LBP-Dependent Intracellular p38 MAPK Signaling Pathway.

The development of microglial endotoxin tolerance (ET) is a critical event in protecting neurons against excessive immune responses when microglia are administered two consecutive lipopolysaccharide (LPS) challenges. However, the intrinsic mechanisms of microglia shape ET programs and protect neurons remain unclear. This study aimed to determine whether extracellular autocrine cascades or intracellular signaling pathways are involved in ET microglia-mediated tumor necrosis factor-alpha (TNF-α) reduction and neuroprotection. Neuron-glia cultures composed of astroglia, neurons, and microglia were performed in different conditions: with or without serum or LPS-binding proteins (LBP), along with an induction approach of ET. Enzyme-linked immunosorbent assay results revealed that LPS induced TNF-α tolerance of microglia in an LBP-dependent manner. Furthermore, we determined whether the early pro-inflammatory cytokines induced by LPS might contribute to the development of microglial ET. Our data showed that the neutralization of TNF-α using an anti-TNF-α antibody had no change in the TNF-α tolerance of microglia during the ET challenge. Furthermore, pre-incubation of TNF-α, interleukin-1 beta, and prostaglandin E2 failed to induce any TNF-α tolerance in microglia after LPS treatment. Moreover, using three specific chemical inhibitors that respectively blocked the activities of the mitogen-activated protein kinases (MAPKs) namely p38, c-Jun N-terminal kinase and extracellular signal-related kinases revealed that inhibition of p38 MAPK by SB203580 disrupted the tolerated microglia-mediated TNF-α reduction and neuroprotection. In summary, our findings demonstrated that the LPS pre-treatment immediately programmed the microglial ET to prevent endotoxin-induced TNF-α production and neuronal damage through the intracellular p38 MAPK signaling pathway.

Inhibition of oxidative stress-induced epithelial-mesenchymal transition in retinal pigment epithelial cells of age-related macular degeneration model by suppressing ERK activation

IntroductionEpithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is related to the pathogenesis of various retinopathies including age-related macular degeneration (AMD). Oxidative stress is the major factor that induces degeneration of RPE cells associated with the etiology of AMD. ObjectivesSodium iodate (NaIO3) generates intracellular reactive oxygen species (ROS) and is widely used to establish a model of AMD due to the selective induction of retinal degeneration. This study was performed to clarify the effects of multiple NaIO3-stimulated signaling pathways on EMT in RPE cells. MethodsThe EMT characteristics in NaIO3-treated human ARPE-19 cells and RPE cells of the mouse eyes were analyzed. Multiple oxidative stress-induced modulators were investigated and the effects of pre-treatment with Ca2+ chelator, extracellular signal-related kinase (ERK) inhibitor, or epidermal growth factor receptor (EGFR) inhibitor on NaIO3-induced EMT were determined. The efficacy of post-treatment with ERK inhibitor on the regulation of NaIO3-induced signaling pathways was dissected and its role in retinal thickness and morphology was evaluated by using histological cross-sections and spectral domain optical coherence tomography. ResultsWe found that NaIO3 induced EMT in ARPE-19 cells and in RPE cells of the mouse eyes. The intracellular ROS, Ca2+, endoplasmic reticulum (ER) stress marker, phospho-ERK, and phospho-EGFR were increased in NaIO3-stimulated cells. Our results showed that pre-treatment with Ca2+ chelator, ERK inhibitor, or EGFR inhibitor decreased NaIO3-induced EMT, interestingly, the inhibition of ERK displayed the most prominent effect. Furthermore, post-treatment with FR180204, a specific ERK inhibitor, reduced intracellular ROS and Ca2+ levels, downregulated phospho-EGFR and ER stress marker, attenuated EMT of RPE cells, and prevented structural disorder of the retina induced by NaIO3. ConclusionsERK is a crucial regulator of multiple NaIO3-induced signaling pathways that coordinate EMT program in RPE cells. Inhibition of ERK may be a potential therapeutic strategy for the treatment of AMD.

Macrophage inhibitory cytokine-1 aggravates diet-induced gallstone formation via increased ABCG5/ABCG8 expression.

Macrophage inhibitory cytokine 1 (MIC-1), which is overproduced in various human cancers and associated with cachexia, acts on the hypothalamus to suppress appetite and reduce body weight. We investigated the mechanisms through which MIC-1 affects bile acid metabolism and gallstone formation, which are poorly understood. Over 6 weeks, male C57BL/6 mice fed either standard chow or a lithogenic diet were intraperitoneally injected with phosphate-buffered saline (PBS) or MIC-1 (200 μg/kg/week). Among lithogenic diet-fed mice, MIC-1 treatment resulted in increased gallstone formation compared with PBS treatment. Compared with PBS treatment, MIC-1 treatment decreased hepatic cholesterol and bile acid levels and reduced expression of HMG-CoA reductase (HMGCR), the master cholesterol metabolism regulator sterol regulatory element-binding protein 2, cholesterol 7α-hydroxylase (CYP7A1), mitochondrial sterol 27-hydroxylase, and oxysterol 7α-hydroxylase. Compared with PBS treatment, MIC-1 treatment had no effect on small heterodimer partner, farnesoid X receptor, or pregnane X receptor expression, and extracellular signal-related kinase and c-Jun N-terminal kinase phosphorylation decreased, suggesting that these factors do not contribute to the MIC-1-induced reduction in CYP7A1 expression. Compared with PBS treatment, MIC-1 treatment increased AMP-activated protein kinase (AMPK) phosphorylation. Treatment with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reduced CYP7A1 and HMGCR expression, whereas the AMPK inhibitor Compound C reversed MIC-1-induced reductions in CYP7A1 and HMGCR expression. Furthermore, in MIC-1-treated mice, total biliary cholesterol levels increased together with increased ATP-binding cassette subfamily G (ABCG)5 and ABCG8 expression. Compared with PBS treatment, MIC-1 treatment did not affect expression of liver X receptors α and β, liver receptor homolog 1, hepatocyte nuclear factor 4α, or NR1I3 (also known as constitutive androstane receptor), which are upstream of ABCG5/8; however, MIC-1 treatment increased ABCG5/8 expression and promoter activities. Our study indicates that MIC-1 influences gallstone formation by increasing AMPK phosphorylation, reducing CYP7A1 and HMGCR expression, and increasing ABCG5 and ABCG8 expression.

The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Insights into the leveraging of GABAergic signaling in cancer therapy.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain of adult mammals. Several studies have demonstrated that the GABAergic system may regulate tumor development via GABA receptors, downstream cyclic adenosine monophosphate (cAMP) pathway, epithelial growth factor receptor (EGFR) pathway, AKT pathway, mitogen-activated protein kinase (MAPK) or extracellular signal-related kinases (ERK) pathway, and matrix metalloproteinase (MMP) pathway, although the exact mechanism is unclear. Pioneering studies reported that GABA signaling exists and functions in the cancer microenvironment and has an immunosuppressive effect that contributes to metastasis and colonization. This article reviews the molecular structures and biological functions of GABAergic components correlated with carcinogenesis, the mechanisms underlying GABAergic signaling that manipulate the proliferation and invasion of cancer cells, and the potential GABA receptor agonists and antagonists for cancer therapy. These molecules may provide an avenue for the development of specific pharmacological components to prevent the growth and metastasis of various cancers.

Activation of the TLR4-JNK but not the TLR4-ERK pathway induced by indole-3-acetic acid exerts anti-proliferative effects on Caco-2 cells.

We previously found that indole-3-acetic acid (IAA) produced from tryptophan by gut microbiota decreases the expression of tumor necrosis factor alpha (TNFα), which is implicated in the pathogenesis of colorectal cancer (CRC). The present study aimed to determine IAA involvement in the proliferation of CRC-derived Caco-2 cells. Cell proliferation was suppressed by IAA, whereas IAA-induced aryl hydrocarbon receptor (AhR) activation had no impact. IAA activated extracellular signal-related (ERK) and c-Jun N-terminal (JNK) kinases, but not p38. Toll-like receptor 4 (TLR4) may be required to activate ERK and JNK, but only the TLR4-JNK pathway might elicit the anti-proliferative effects of IAA. Thus, IAA may be a ligand for TLR4 that contributes to inhibiting CRC cell proliferation by activating TLR4-mediated JNK. Because IAA did not induce cytotoxicity, inhibiting cell cycle progression might affect the anti-proliferative capacity of IAA. Therefore, colonic IAA accumulation might help to prevent CRC development and progression.

Donkey Oil-Based Ketogenic Diet Prevents Tumor Progression by Regulating Intratumor Inflammation, Metastasis and Angiogenesis in CT26 Tumor-Bearing Mice.

Colon cancer is one of the typical malignant tumors, and its prevalence has increased yearly. The ketogenic diet (KD) is a low-carbohydrate and high-fat dietary regimen that inhibits tumor growth. Donkey oil (DO) is a product with a high nutrient content and a high bioavailability of unsaturated fatty acids. Current research investigated the impact of the DO-based KD (DOKD) on CT26 colon cancer in vivo. Our findings revealed that DOKD administration significantly lowered CT26+ tumor cell growth in mice, and the blood β-hydroxybutyrate levels in the DOKD group was significantly higher than those in the natural diet group. Western blot results showed that DOKD significantly down-regulated Src, hypoxia inducible factor-1α (HIF-1α), extracellular signal-related kinases 1 and 2 (Erk1/2), snail, neural cadherin (N-cadherin), vimentin, matrix metallopeptidase 9 (MMP9), signal transducer and activator of transcription 3 (STAT3), and vascular endothelial growth factor A (VEGFA), and it significantly up-regulated the expressions of Sirt3, S100a9, interleukin (IL)-17, nuclear factor-kappaB (NF-κB) p65, Toll-like receptor 4 (TLR4), MyD88, and tumor necrosis factor-α. Meanwhile, in vitro validation results showed that LW6 (a HIF-1α inhibitor) significantly down-regulated the expressions of HIF-1α, N-cadherin, vimentin, MMP9, and VEGFA, which supported those of the in vivo findings. Furthermore, we found that DOKD inhibited CT26+ tumor cell growth by regulating inflammation, metastasis, and angiogenesis by activating the IL-17/TLR4/NF-κB p65 pathway and inhibiting the activation of the Src/HIF-1α/Erk1/2/Snail/N-cadherin/Vimentin/MMP9 and Erk1/2/HIF-1α/STAT3/VEGFA pathways. Our findings suggest that DOKD may suppress colon cancer progression and help prevent colon cancer cachexia.

Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival.

5-Hydroxytryptamine receptor 1E (5-HTR1E) is reported to activate cyclic AMP (cAMP) and extracellular-signal related kinases (ERK) pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin-induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin-induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gβγ and Gq were not associated with 5-HTR1E activation, while blocking protein kinase A (PKA) inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, β-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockdown studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.