TRPV4 activates cytosolic phospholipase A2 via Ca2+‐dependent PKC/ERK1/2 signalling in controlling hypertensive contraction

Activation of TRPV4 (transient receptor potential vanilloid 4) has been reported to result in endothelium‐dependent contraction in the aortae of hypertensive mice. This contraction involved increased cPLA₂ (cytosolic phospholipase A₂) activity. The mechanism by which TRPV4 regulates cPLA₂ activity to induce contraction in hypertension, however, is unknown. Through measurements of arterial tension and protein level, we showed that high‐salt diet induced hypertension increases activity of PKC (protein kinase C) and ERK1/2 (extracellular signal‐regulated kinase 1/2). GSK1016790A, a TRPV4 agonist and ACh (acetylcholine) induced contractions were suppressed by Go6983, a PKC inhibitor and PD98059, an ERK1/2 inhibitor. TRPV4 activation increased activity of PKC and ERK1/2 in endothelial cells from hypertensive mice and this response was suppressed by HC067047, a TRPV4 inhibitor and BAPTA/AM, a Ca²⁺ chelator. PLA₂ assay and western blotting showed that blocking of PKC or ERK1/2 inhibited TRPV4 or ACh‐induced cPLA₂ activity. Enzyme immunoassay showed that GSK1016790A or ACh triggered the release of PGF_2α (prostaglandin F_2α) was reduced by inhibition of PKC or ERK1/2. These data further suggest Ca²⁺/PKC/ERK1/2 axis as a novel mechanism for TRPV4 in the activation of cPLA₂ in hypertension.     

Helioxanthin suppresses the cross talk of COX‐2/PGE2 and EGFR/ERK pathway to inhibit Arecoline‐induced Oral Cancer Cell (T28) proliferation and blocks tumor growth in xenografted nude mice

Helioxanthin, an active compound from Taiwania cryptomerioides Hayata, has been shown to have various biological activities. However, their anticancer effect in oral squamous cell carcinoma has not been well established yet. Helioxanthin inhibited the proliferation of oral squamous cell carcinoma cells in a dose‐dependent manner by inducing G2/M phase arrest. Similarly, helioxanthin inhibited cyclooxygenase‐2, (COX‐2), phosphorylated EGFR, and extracellular‐signal‐regulated kinases (ERK) protein level and further reduced the nuclear accumulation of phosphorylated epidermal growth factor receptor (pEGFR) and activator protein‐1(AP‐1) family protein, c‐fos. Moreover, helioxanthin at the dose of 20 and 30 mg kg^?1 for 15 days reduced the tumor growth in animal model. This study demonstrated that Helioxanthin exerts its anticancer activity against oral cancer cells by downregulating EGFR/ERK/c‐fos signaling pathway to inhibit COX‐2 level and by activating cyclin‐dependent kinase inhibitor (p27) to further induce G2/M cell cycle arrest. This helioxanthin may serve as a novel candidate for oral cancer prevention. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2045–2056, 2016.     

Nonylphenol‐induced apoptotic pathways in SCM1 human gastric cancer cells

Environmental chemicals may affect human health by disrupting endocrine function. Many endocrine disrupting chemicals (EDCs) are estrogen‐like molecules that are classified as xenoestrogens (XEs). One XE, nonylphenol, is used as a surfactant or plasticizer and exhibits biotoxicity when accumulated in the body via the food chain. The aim of the present study was to clarify the role of nonylphenol‐induced SCM1 apoptosis by measuring cultured human gastric cancer cell (SCM1) death. Using WST‐1 reduction and propidium iodide‐staining assays, nonylphenol treatment was found to activate caspase‐3 and mitogen‐activated protein kinases (MAPKs), major markers in apoptotic pathways. Nonylphenol also activated the phosphorylation of extracellular signal‐regulated kinase (ERK), c‐Jun NH_2‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (p38 MAPK). However, only SB203580 (a p38MAPK inhibitor) partially inhibited nonylphenol‐induced apoptosis. Nonylphenol induced a [Ca²⁺]_i rise by causing extracellular Ca²⁺ influx and intracellular Ca²⁺ release from the endoplasmic reticulum, and its effects on SCM1 cell death were prevented by pretreatment with the Ca²⁺ chelator BAPTA/AM. These results suggest that nonylphenol caused Ca²⁺‐dependent apoptosis via the activation of p38 MAPK‐associated caspase‐3 in SCM1 cells. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.     

Tris (1,3‐dichloro‐2‐propyl) phosphate induces toxicity by stimulating CaMK2 in PC12 cells

Tris (1,3‐dichloro‐2‐propyl) phosphate (TDCIPP) is one of the widely used organophosphorus flame retardants (OPFRs), which are regarded as suitable substitutes for brominated flame retardants (BFRs). Previously, we have validated the toxicity of TDCIPP in PC12 cells owing to the induced alterations in GAP43, NF‐H, CaMK2a/2b, and tubulin α/β proteins; however, limited information is currently available on the toxicity and mechanism of TDCIPP. In the present study, cytotoxicity effects were evaluated by exposing PC12 cells to different concentrations of TDCIPP (0–50 μM) for 4 days. To explore the possible mechanisms through which cytotoxicity is induced, changes in intracellular [Ca²⁺]_i levels and the activation of calmodulin dependent protein kinase 2 (CaMK2), c‐Jun N‐terminal kinase (JNK), extracellular regulated protein kinases (ERK1/2), and p38 mitogen‐activated protein kinases (MAPK) pathways were evaluated. Furthermore, PC12 cells were pretreated with CaMK2 inhibitor KN93 to investigate the relationship between TDCIPP‐induced phosphorylation of CaMK2 and activation of JNK, ERK1/2, and p38 MAPK pathways. Our results indicate that TDCIPP‐induced toxicity might be associated with the overload of [Ca²⁺]_i levels, increased phosphorylation of CaMK2, and activation of the JNK, ERK1/2, and p38 MAPK pathways, the lattermost of which was further demonstrated to be partially elicited by the CaMK2 phosphorylation.     

Apoptosis induced by para‐phenylenediamine involves formation of ROS and activation of p38 and JNK in chang liver cells

para‐phenylenediamine (p‐PD) is a suspected carcinogen, but it has been widely used as a component in permanent hair dyes. In this study, the mechanism of p‐PD‐induced cell death in normal Chang liver cells was investigated. The results demonstrated that p‐PD decreased cell viability in a dose‐dependent manner. Cell death via apoptosis was confirmed by enhanced DNA damage and increased cell number in the sub‐G1 phase of the cell cycle, using Hoechst 33258 dye staining and flow cytometry analysis. Apoptosis via reactive oxygen species generation was detected by the dichlorofluorescin diacetate staining method. Mitogen‐activated protein kinase (MAPK) activation was assessed by western blot analysis and revealed that p‐PD activated not only stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinases (JNK) and p38 MAPK but also extracellular signal‐regulated kinase (ERK). Cytotoxicity and apoptosis induced by p‐PD were markedly enhanced by ERK activation and selectively inhibited by ERK inhibitor PD98059, thus indicating a negative role of ERK. In contrast, inhibition of p38 MAPK activity with the p38‐specific inhibitor SB203580 moderately inhibited cytotoxicity and apoptosis induction by p‐PD. Similarly, SP600125, an inhibitor of SAPK/JNK, moderately inhibited cytotoxicity and apoptosis induced by p‐PD, thus implying that p38 MAPK and SAPK/JNK had a partial role in p‐PD‐induced apoptosis. Western blot analysis revealed that p‐PD significantly increased phosphorylation of p38 and SAPK/JNK and decreased phosphorylation of ERK. In conclusion, the results demonstrated that SAPK/JNK and p38 cooperatively participate in apoptosis induced by p‐PD and that a decreased ERK signal contributes to growth inhibition or apoptosis. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 981–990, 2014.     

Exercise training protects against atherosclerotic risk factors through vascular NADPH oxidase,extracellular signal‐regulated kinase 1/2 and stress‐activated protein kinase/c‐Jun N‐terminal kinase downregulation in obese rats

Exercise training reverses atherosclerotic risk factors associated with metabolic syndrome and obesity. The aim of the present study was to determine the molecular anti‐inflammatory, anti‐oxidative and anti‐atherogenic effects in aorta from rats with high‐fat diet‐induced obesity. Male Sprague‐Dawley rats were placed on a high‐fat (HFD) or control (CD) diet for 12 weeks. The HFD rats were then divided into four groups: (i) sedentary HFD‐fed rats (HFD‐S); (ii) exercise trained (motor treadmill 5 days/week, 60 min/day, 12 weeks) HFD‐fed rats (HFD‐Ex); (iii) modified diet (HFD to CD) sedentary rats (HF/CD‐S); and (iv) an exercise‐trained modified diet group (HF/CD‐Ex). Tissue levels of NADPH oxidase (activity and expression), NADPH oxidase (Nox) 1, Nox2, Nox4, p47^phox, superoxide dismutase (SOD)‐1, angiotensin AT₁ and AT₂ receptors, phosphorylated mitogen‐activated protein kinase (MAPK; extracellular signal‐regulated kinase (ERK) 1/2, stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinase (JNK)) and vascular cell adhesion molecule‐1 (VCAM‐1) were determined in the aorta. Plasma cytokines (tumour necrosis factor (TNF)‐α and interleukin (IL)‐6) levels were also measured. Obesity was accompanied by increases in NADPH oxidase activity, p47^phox translocation, Nox4 and VCAM‐1 protein expression, MAPK (ERK1/2, SAPK/JNK) phosphorylation and plasma TNF‐α and IL‐6 levels. Exercise training and switching from the HFD to CD reversed almost all these molecular changes. In addition, training increased aortic SOD‐1 protein expression and decreased ERK1/2 phosphorylation. These findings suggest that protective effects of exercise training on atherosclerotic risk factors induced by obesity are associated with downregulation of NADPH oxidase, ERK1/2 and SAPK/JNK activity and increased SOD‐1 expression.     

Involvement of extracellular Ca2+ influx and epidermal growth factor receptor tyrosine kinase transactivation in endothelin-1-induced arachidonic acid release

1. Endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in vascular smooth muscle cells (VSMCs). These channels can be distinguished by their sensitivity to Ca(2+)-channel blockers, SK&F 96365 and LOE 908. LOE 908 is sensitive to NSCC-1 and NSCC-2, and SK&F 96365 is sensitive to NSCC-2 and SOCC. Moreover, these channels play essential roles in ET-1-induced epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) transactivation. The main purpose of the present study was to demonstrate the involvement of EGFR PTK transactivation in ET-1-induced arachidonic acid release in VSMCs. 2. Both SK&F 96365 and LOE 908 inhibited ET-1-induced arachidonic acid release with the IC(50) values correlated to those of ET-1-induced Ca(2+) influx. Moreover, combined treatment with these blockers abolished ET-1-induced arachidonic acid release. 3. AG1478, a specific inhibitor of EGFR PTK, inhibited ET-1-induced arachidonic acid release and extracellular signal-regulated kinase 1 and 2 (ERK1/2). The IC(50) values of AG1478 for ET-1-induced arachidonic acid release and ERK1/2 correlated well with those for ET-1-induced EGFR PTK transactivation. 4. Mitogen-activated protein kinase kinase inhibitor, PD 98059, inhibited ET-1-induced arachidonic acid release. The IC(50) values of PD 98059 for ET-1-induced arachidonic acid release were similar to those for ET-1-induced ERK1/2 activity. In contrast, PD 98059 failed to inhibit ET-1-induced EGFR PTK transactivation. 5. These results indicate that (1) extracellular Ca(2+) influx through NSCCs and SOCC plays important roles for ET-1-induced arachidonic acid release, (2) EGFR PTK transactivation/ERK1/2 pathways are involved in ET-1-induced arachidonic acid release.     

Lead acetate induces EGFR activation upstream of SFK and PKCα linkage to the Ras/Raf-1/ERK signaling

Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC → ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1^S338 and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Gö6976 or depleting PKCα using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKCα, Ras-GTP, phospho-Raf-1^S338 and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKCα activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKCα activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKCα and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade.     

Hexabromocyclododecane facilitates FSH activation of ERK1/2 and AKT through epidermal growth factor receptor in rat granulosa cells

The toxicity of hexabromocyclododecane (HBCDD) has been extensively studied; however, the mechanism and the effects of HBCDD on female reproductive system have been less frequently reported. In this study, we exposed rat granulosa cells to HBCDD during in vitro follicle-stimulating hormone (FSH)-driven cell proliferation and differentiation. Here, we show that HBCDD affects the FSH-driven signal transduction and ovulatory competence of granulosa cells. We found that HBCDD over-activates the FSH-stimulated extracellular-regulated kinase 1/2 (ERK1/2) and protein kinase B (PKB, also known as AKT). Inactivation of the epidermal growth factor receptor (EGFR) kinase activity with AG1478 and the mitogen-regulated kinase activity with U0126 completely prevented ERK1/2 activation in the FSH-stimulated and HBCDD-exposed granulosa cells. Moreover, AG1478 restored the HBCDD-induced AKT activation to the level observed in the FSH-stimulated cells. Western blot shows that HBCDD potentiates FSH-stimulated EGFR phosphorylation in granulosa cells. Real-time PCR demonstrates that HBCDD decreases the FSH-induced luteinizing hormone receptor (Lhr) expression. Inadequate level of LHR in the HBCDD-exposed granulosa cells prevented human chorionic gonadotropin in stimulating expression of the ovulatory genes such as amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr). Addition of U0126 and AG1478 restored Lhr level in the FSH-stimulated and HBCDD-exposed granulosa cells. These results indicate a direct effect of HBCDD on EGFR activation, resulting in over-activation of ERK1/2 and AKT signal transduction pathways in the FSH-treated cells. Increased activity of the EGFR-ERK1/2 pathway above physiological level prevents sufficient acquisition of LHR in proliferating granulosa cells, thus compromising ovulation.     

Nickel‐induced VEGF expression via regulation of Akt,ERK1/2, NFκB,and AMPK pathways in H460 cells

Prospective cohort studies have indicated that a highly nickel‐polluted environment may severely affect human health, resulting in such conditions as respiratory tract cancers. Such exposure can trigger vascular endothelial growth factor (VEGF) expression. However, the signal transduction pathways leading to VEGF induction by nickel compounds are not well understood. This study revealed the occurrence of VEGF induction in human non‐small‐cell lung cancer H460 cells exposed to NiCl₂. Moreover, exposing H460 cells to NiCl₂ activated extracellular signal‐regulated protein kinase (ERK), nuclear factor kappa B (NFκB), and protein kinase B (Akt) as well as downregulated AMP activated protein kinase (AMPK) expression. The mitogen‐activated protein kinase (MAPK) and ERK inhibitor significantly blocked NiCl₂‐induced ERK activation and VEGF production. Pretreating H460 cells with a PI3K/Akt inhibitor substantially inhibited NiCl₂‐induced VEGF expression and reduced Akt, ERK, and NFκB phosphorylation. Furthermore, 5‐aminoimidazole‐4‐carboxamide ribonucleoside‐induced AMPK activation improved VEGF expression in NiCl₂‐treated H460 cells significantly. These results indicate that NiCl₂ induces VEGF production through Akt, ERK, NFκB activation and AMPK suppression and mediates various types of pathophysiological angiogenesis.     

Dopamine D1 receptors induce apoptosis of osteosarcoma cells via changes of MAPK pathway

This study explored the effects and mechanisms of dopamine D1 receptors (DR1) activation on the apoptosis of osteosarcoma cells (OS732).The DR1 agonist SKF‐38393 decreased the viability of OS732 cells and increased their rate of apoptosis, whereas the DR1 antagonist SCH‐23390 abolished the effects of SKF‐38393. In OS732 cells, overexpression of DR1 increased the rate of apoptosis, caspase‐9 and ‐3 expression, and the release of cytochrome c (Cyt c), reduced Bcl‐2 expression, inhibited extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation, and induced phosphorylation of p38 mitogen‐activated protein kinase (p38 MAPK) and c‐Jun N‐terminal kinase (JNK). These results suggest that activation of DR1 induces osteosarcoma cell apoptosis via changes to the MAPK pathway.